WO1995023225A2

WO1995023225A2 - Method and reagent for inhibiting the expression of disease related genes

Info

Publication number: WO1995023225A2
Application number: PCT/IB1995/000156
Authority: WO
Inventors: Dan T. Stinchcomb; Bharat Chowrira; Anthony Direnzo; Kenneth G. Draper; Lech W. Dudycz; Susan Grimm; Alexander Karpeisky; Kevin Kisich; Jasenka Matulic-Adamic; James A. Mcswiggen; Anil Modak; Pamela Pavco; Leonid Beigelman; Sean M. Sullivan; David Sweedler; James D. Thompson; Danuta Tracz; Nassim Usman; Francine E. Wincott; Tod Woolf
Original assignee: Ribozyme Pharmaceuticals, Inc.
Priority date: 1994-02-23
Filing date: 1995-02-23
Publication date: 1995-08-31
Also published as: EP0746614A1; CA2183992A1; AU1821495A; AU706417B2; WO1995023225A3

Abstract

Enzymatic RNA molecules which cleave ICAM-1 mRNA, IL-5 mRNA, rel A mRNA, TNF-α mRNA, RSV mRNA or RSV genomic RNA, or CML associated mRNA, and use of these molecules for the treatment of pathological conditions related to those mRNA-levels; ribonucleosides or nucleotides modified in 2', 3' or 5', methods for their synthesis, purification and deprotection; vectors containing multiple enzymatic nucleic acids, optionally in chimeric form with tRNAs; method for introducing enzymatic nucleic acids into cells by forming a complex with a second nucleic acid, where the complex is capable of taking an R-loop base-paired structure; method for altering a mutant nucleic acid in vivo by hybridization with an oligonucleotide capable of activating dsRNA deaminase, comprising an enzymatic activity or a chemical mutagen. Further are disclosed trans-cleaving or -ligating hairpin ribozymes lacking a substrate RNA moiety, as well as hammerhead ribozymes having an interconnecting loop between base pairs in stem II.

Description

METHOD AND REAGENT FOR INHIBITING THE EXPRESSION OF DISEASE RELATED GENES

Background of the Invention

This invention relates to reagents useful as inhibitors of gene expression relating to diseases such as inflammatory or autoimmune disorders, chronic myelogenous leukemia, or respiratory tract illness. Summary of the Invention

The invention features novel enzymatic RNA molecules, or ribozymes, and methods for their use for inhibiting the expression of disease related genes, e.g., ICAM-1 , IL-5, relA, TNF-α, p210 ^bcr-abl, and respiratory syncytial virus genes. Such ribozymes can be used in a method for treatment of diseases caused by the expression of these genes in man and other animals, including other primates.

Ribozymes are RNA molecules having an enzymatic activity which is able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence specific manner. Such enzymatic RNA molecules can be targeted to virtually any RNA transcript, and efficient cleavage has been achieved in vitro. Kim et al., 84 Proc. Natl. Acad. Sci. USA 8788, 1987; Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research 1371 , 1989.

Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. Table 1 summarizes some of the characteristics of these ribozymes.

Ribozymes act by first binding to a target RNA. Such binding occurs through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA which acts to cleave the target RNA. Thus, the ribozyme first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After a ribozyme has bound and cleaved its RNA target it is released from that RNA to search for another target and can repeatedly bind and cleave new targets. The enzymatic nature of a ribozyme is advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the effective concentration of ribozyme necessary to effect a therapeutic treatment is lower than that of an antisense oligonucleotide. The advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ration of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base pairing. Thus, it is thought that the specificity of action of a ribozyme is greater than that of antisense oligonucleotide binding the same RNA site. With their catalytic activity and increased site specificity, ribozymes represent more potent and safe therapeutic molecules than antisense oligonucleotides. Thus, in a first aspect, this invention relates to ribozymes, or enzymatic

RNA molecules, directed to cleave RNA species encoding ICAM-1 , IL-5, relA, TNF-α, p210^bcr-abl, or RSV proteins. In particular, applicant describes the selection and function of ribozymes capable of cleaving these RNAs and their use to reduce levels of ICAM-1 , IL-5, relA, TNF-α, p210 bor-abl or RSV proteins in various tissues to treat the diseases discussed herein. Such ribozymes are also useful for diagnostic uses.

Applicant indicates that these ribozymes are able to inhibit expression of ICAM-1 , IL-5, rel A, TNF-α, p21θbcr-abl. or RSV genes and that the catalytic activity of the ribozymes is required for their inhibitory effect. Those of ordinary skill in the art, will find that it is clear from the examples described that other ribozymes that cleave target ICAM-1 , IL-5, rel A, TNF-α, p210^{b cr-abl}, or RSV encoding mRNAs may be readily designed and are within the invention.

These chemically or enzymatically synthesized RNA molecules contain substrate binding domains that bind to accessible regions of their target mRNAs. The RNA molecules also contain domains that catalyze the cleavage of RNA. Upon binding, the ribozymes cleave the target encoding mRNAs, preventing translation and protein accumulation. In the absence of the expression of the target gene, a therapeutic effect may be observed.

By "gene" is meant to refer to either the protein coding regions of the cognate mRNA, or any regulatory regions in the RNA which regulate synthesis of the protein or stability of the mRNA; the term also refers to those regions of an mRNA which encode the ORF of a cognate polypeptide product, and the proviral genome.

By "enzymatic RNA molecule" it is meant an RNA molecule which has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity which is active to specifically cleave RNA in that target. That is, the enzymatic RNA molecule is able to intermolecularly cleave RNA and thereby inactivate a target RNA molecule. This complementarity functions to allow sufficient hybridization of the enzymatic RNA molecule to the target RNA to allow the cleavage to occur. One hundred percent complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention. By "equivalent" RNA to a virus is meant to include those naturally occurring viral encoded RNA molecules associated with viral caused diseases in various animals, including humans, cats, simians, and other primates. These viral or viral-encoded RNAs have similar structures and equivalent genes to each other.

By "complementarity" it is meant a nucleaic acid that can form hydrogen bond(s) with other RNA sequence by either traditional WatsonCrick or other non-traditional types (for examplke, Hoogsteen type) of base-paired interactions.

In preferred embodiments of this invention, the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but may also be formed in the motif of a hepatitis delta virus, group I intron or RNaseP RNA (in associateion with an RNA guide sequence) or Neurospora VS RNA. Examples of such hammerhead motifs are described by Rossi et al., 1992, Aids Research and Human Retroviruses , 8,183, of hairpin motifs by Hampel and Tritz, 1989 Biochemistry, 28, 4929, EP 0360257 and Hampel et al., 1990, Nucleic Acids Res. 18,299 and an example of the hepatitis delta virus motif is described by Perotta and Been, 1992 Biochemistry, 31 16 of the RNaseP motif by Guerrier-Takada et al., 1983 Cell, 35 849, cleavage of RNA. Upon binding, the ribozymes cleave the target encoding mRNAs, preventing translation and protein accumulation. In the absence of the expression of the target gene, a therapeutic effect may be observed.

By "complementarity" it is meant a nucleaic acid that can form hydrogen bond(s) with other RNA sequence by either traditional Watson-Crick or other non-traditional types (for examplke, Hoogsteen type) of base-paired interactions.

In preferred embodiments of this invention, the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but may also be formed in the motif of a hepatitis delta virus, group I intron or RNaseP RNA (in associateion with an RNA guide sequence) or Neurospora VS RNA. Examples of such hammerhead motifs are described by Rossi et al., 1992, Aids Research and Human Retroviruses , 8,183, of hairpin motifs by Hampel and Tritz, 1989 Biochemistry, 28, 4929, EP 0360257 and Hampel et al., 1990, Nucleic Acids Res. 18,299 and an example of the hepatitis delta virus motif is described by Perotta and Been, 1992 Biochemistry, 31 16 of the RNaseP motif by Guerrier-Takada et al., 1983 Cell, 35 849, expressed in eukaryotic cells from the appropriate DNA or RNA vector. The activity of such ribozymes can be augmented by their release from the primary transcript by a second ribozyme (Draper et al., PCT W093/23569, and Sullivan et al., PCT WO94/02595, both hereby incorporated in their totality by reference herein; Ohkawa, J., et al., 1992, Nucleic Acids Symp. Ser. 27, 15-6; Taira, K. et al., Nucleic Acids Res., 19, 5125-30; Ventura, M., et al., 1993, Nucleic Acids Res., 21 , 3249-55, Chowrira et al., 1994 J. Biol. Chem., 269. 25856 ).

By "inhibit" is meant that the activity or level of ICAM-1 , Rel A, IL-5, TNF-α, p210^bcr-abl or RSV encoding mRNA is reduced below that observed in the absense of the ribozyme, and preferably is below that level observed in the presence of an inactive RNA molecule able to bind to the same site on the mRNA, but unable to cleave that RNA.

Such ribozymes are useful for the prevention of the diseases and conditions discussed above, and any other diseases or conditions that are related to the level of ICAM-1 , IL-5, Rel A, TNF-α, p210^bcr-abl or RSV protein or activity in a cell or tissue. By "related" is meant that the inhibition of ICAM-1 , IL-5, Rel A, TNF-α, _P210^bcr-abl or RSV mRNA translation, and thus reduction in the level of, ICAM-1 , IL-5, Rel A, TNF-α, p210^bcl-abl or RSV proteins will relieve to some extent the symptoms of the disease or condition.

Ribozymes are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells. The RNA or RNA complexes can be locally administered to relevant tissues through the use of a catheter, infusion pump or stent, with or without their incorporation in biopolymers. In preferred embodiments, the ribozymes have binding arms which are complementary to the sequences in Tables 2,3,6-9, 11 , 13, 15-23, 27, 28, 31 , 33, 34, 36 and 37.

Examples of such ribozymes are shown in Tables 4-8, 10, 12, 14-16, 19-22, 24, 26-28, 30, 32, 34 and 36-38. Examples of such ribozymes consist essentially of sequences defined in these Tables. By "consists essentially of" is meant that the active ribozyme contains an enzymatic center equivalent to those in the examples, and binding arms able to bind mRNA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity. For example, stem-loop II sequence of hammerhead ribozymes listed in the above identified Tables can be altered (substitution, deletion, and/or insertion) to contain any sequences provided a minimum of two base-paired stem structure can form. Similarly, stem-loop IV sequence of hairpin ribozymes listed in the above identified Tables can be altered (substitution, deletion, and/or insertion) to contain any sequence, provided a minimum of two base-paired stem structure can form. The sequence listed in the above identified Tables may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables.

In another aspect of the invention, ribozymes that cleave target molecules and inhibit ICAM-1 , IL-5, Rel A, TNF-α, p210^bcr-abl or RSV gene expression are expressed from transcription units inserted into DNA, RNA, or viral vectors. Another means of accumulating high concentrations of a ribozyme(s) within cells is to incorporate the ribozyme-encoding sequences into a DNA or RNA expression vector. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990 Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gao and Huang 1993 Nucleic Acids Res., 21 2867-72; Lieber et al., 1993 Methods Enzymol., 217, 47-66; Zhou et al., 1990 Mol. Cell. Biol., 10, 4529-37). Several investigators have demonstrated that ribozymes expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992 Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992 Proc. Natl. Acad. Sci. USA, 90, 6340-4; L'Huiller et al., 1992 EMBO J. 11 , 4411-8; Lisziewicz et al., 1993 Proc. Natl. Acad. Sci. U.S.A., 90 8000-4). The above ribozyme transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors).

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Description Of The Preferred Embodiments The drawings will first briefly be described. Drawings:

Figure 1 is a diagrammatic representation of the hammerhead ribozyme domain known in the art. Stem II can be≥ 2 base-pair long.

Figure 2(a) is a diagrammatic representation of the hammerhead ribozyme domain known in the art; Figure 2(b) is a diagrammatic representation of the hammerhead ribozyme as divided by Uhlenbeck (1987, Nature, 327, 596-600) into a substrate and enzyme portion; Figure 2(c) is a similar diagram showing the hammerhead divided by Haseloff and Gerlach (1988, Nature, 334, 585-591) into two portions; and Figure 2(d) is a similar diagram showing the hammerhead divided by Jeffries and Symons (1989, Nυcl. Acids. Res., 17, 1371-1371) into two portions.

Figure 3 is a diagrammatic representation of the general structure of a hairpin ribozyme. Helix 2 (H2) is provided with a least 4 base pairs (i.e., n is 1 ,2,3 or 4) and helix 5 can be optionally provided of length 2 or more bases (preferably 3-20 bases, i.e., m is from 1-20 or more). Helix 2 and helix 5 may be covalently linked by one or more bases (i.e., r is≥ 1 base).

Helix 1 , 4 or 5 may also be extended by 2 or more base pairs (e.g., 4-20 base pairs) to stabilize the ribozyme structure, and preferably is a protein binding site. In each instance, each N and N' independently is any normal or modified base and each dash represents a potential base-pairing interaction. These nucleotides may be modified at the sugar, base or phosphate. Complete base-pairing is not required in the helices, but is preferred. Helix 1 and 4 can be of any size (i.e., o and p is each independently from 0 to any number, e.g. 20) as long as some base-pairing is maintained. Essential bases are shown as specific bases in the structure, but those in the art will recognize that one or more may be modified chemically (abasic, base, sugar and/or phosphate modifications) or replaced with another base without significant effect. Helix 4 can be formed from two separate molecules, i.e., without a connecting loop. The connecting loop when present may be a ribonucleotide with or without modifications to its base, sugar or phosphate, "q" is≥ 2 bases. The connecting loop can also be replaced with a non-nucleotide linker molecule. H refers to bases A, U, or C. Y refers to pyrimidine bases. " " refers to a covalent bond.

Figure 4 is a representation of the general structure of the hepatitis delta virus ribozyme domain known in the art.

Figure 5 is a representation of the general structure of the self-cleaving VS RNA ribozyme domain.

Figure 6 is a diagrammatic representation of the genetic map of RSV strain A2. Figure 7 is a diagrammatic representation of the solid-phase synthesis of RNA.

Figure 8 is a diagrammatic representation of exocyclic amino protecting groups for nucleic acid synthesis.

Figure 9 is a diagrammatic representation of the deprotection of RNA. Figure 10 is a graphical representation of the cleavage of an RNA substrate by ribozymes synthesized, deprotected and purified using the improved methods described herein.

Figure 11 is a schematic representation of a two pot deprotection protocol. Base deprotection is carried out with aqueous methyl amine at 65°C for 10 min. The sample is dried in a speed-vac for 2-24 hours depending on the scale of RNA synthesis. Silyl protecting group at the 2'-hydroxyl position is removed by treating the sample with 1.4 M anhydrous HF at 65°C for 1.5 hours.

Figure 12 is a schematic representation of a one pot deprotection of RNA synthesized using RNA phosphoramidite chemistry. Anhydrous methyl amine is used to deprotect bases at 65°C for 15 min. The sample is allowed to cool for 10 min before adding TEA•3HF reagent, to the same pot, to remove protecting groups at the 2'-hydroxyI position. The deprotection is carried out for 1.5 hours.

Figs. 13a - b is a HPLC profile of a 36 nt long ribozyme, targeted to site B. The RNA is deprotected using either the two pot or the one pot deprotection protocol. The peaks corresponding to full-length RNA is indicated. The sequence for site B is CCUGGGCCAGGGAUUA

AUGGAGAUGCCCACU.

Figure 14 is a graph comparing RNA cleavage activity of ribozymes deprotected by two pot vs one pot deprotection protocols. Figure 15 is a schematic representation of an improved method of synthesizing RNA containing phosphorothioate linkages.

Figure 16 shows RNA cleavage reaction catalyzed by ribozymes containing phosphorothioate linkages. Hammerhead ribozyme targeted to site C is synthesized such that 4 nts at the 5' end contain phosphorothioate linkages. P=O refers to ribozyme without phosphorothioate linkages. P=S refers to ribozyme with phosphorothioate linkages. The sequence for site C is UCAUUUUGGCCAUCUC UUCCUUCAGGCGUGG.

Figure 17 is a schematic representation of synthesis of 2'-N-phtalimido-nucleoside phosphoramidite. Figure 18 is a diagrammatic representation of a prior art method for the solid-phase synthesis of RNA using silyl ethers, and the method of this invention using SEM as a 2'-protecting group.

Figure 19 is a diagrammatic representation of the synthesis of 2'- SEM-protected nucleosides and phosphoramidites useful for the synthesis of RNA. B is any nucleotide base as exemplified in the Figure, P is purine and I is inosine. Standard abbreviations are used throughout this application, well known to those in the art.

Figure 20 is a diagrammatic representation of a prior art method for deprotection of RNA using TBDMS protection of the 2'-hydroxyl group. Figure 21 is a diagrammatic representation of the deprotection of RNA having SEM protection of the 2'-hydroxyl group. Figure 22 is a representation of an HPLC chromatogram of a fully deprotected 10-mer of uridylic acid.

Figs. 23 - 25 are diagrammatic representations of hammerhead, hairpin or hepatitis delta virus ribozyme containing self-processing RNA transcript. Solid arrows indicate self-processing sites. Boxes indicate the sites of nucleotide substitution. Solid lines are drawn to show the binding sites of primers used in a primer-extension assay. Lower case letters indicate vector sequence present in the RNA when transcribed from a HindIII-linearized plasmid. (23) HH Cassette, transcript containing the hammerhead trans-acting ribozyme linked to a 3' cis-acting hammerhead ribozyme. The structure of the hammerhead ribozyme is based on phylogenetic and mutational analysis (reviewed by Symons, 1992 supra). The trans ribozyme domain extends from nucleotide 1 through 49. After 3'-end processing, the trans-ribozyme contains 2 non-ribozyme nucleotides (UC at positions 50 and 51) at its 3' end. The 3' processing ribozyme is comprised of nucleotides 44 through 96. Roman numerals I, II and III, indicate the three helices that contribute to the structure of the 3' cis-acting hammerhead ribozyme (Hertel et al., 1992 Nucleic Acids Res. 20, 3252). Substitution of G70 and A71 to U and G respectively, inactivates the hammerhead ribozyme (Ruffner et al., 1990 Biochemistry 29, 10695) and generates the HH(mutant) construct. (24) HP Cassette, transcript containing the hammerhead trans-acting ribozyme linked to a 3' cis-acting hairpin ribozyme. The structure of the hairpin ribozyme is based on phylogenetic and mutational analysis (Berzal-Herranz et al., 1993 EMBO. J 12, 2567). The trans-ribozyme domain extends from nucleotide 1 through 49. After 3'-end processing, the trans-ribozyme contains 5 non-ribozyme nucleotides (UGGCA at positions 50 to 54) at its 3' end. The 3' cis-acting ribozyme is comprised of nucleotides 50 through 115. The transcript named HP(GU) was constructed with a potential wobble base pair between G52 and U77; HP(GC) has a Watson-Crick base pair between G52 and C77. A shortened helix 1 (5 base pairs) and a stable tetraloop (GAAA) at the end of helix 1 was used to connect the substrate with the catalytic domain of the hairpin ribozyme (Feldstein & Bruening, 1993 Nucleic Acids Res. 21 , 1991 ; Altschuler et al., 1992 supra). (25) HDV Cassette, transcript containing the trans-acting hammerhead ribozyme linked to a 3' cis-acting hepatitis delta virus (HDV) ribozyme. The secondary structure of the HDV ribozyme is as proposed by Been and coworkers (Been et al., 1992 Biochemistry 31 , 11843). The trans-ribozyme domain extends from nucleotides 1 through 48. After 3'-end processing, the trans-ribozyme contains 2 non-ribozyme nucleotides (AA at positions 49 to 50) at its 3' end. The 3' cis-acting HDV ribozyme is comprised of nucleotides 50 through 114. Roman numerals I, II, III & IV, indicate the location of four helices within the 3' cis-acting HDV ribozyme (Perrota & Been, 1991 Nature 350, 434). The ΔHDV transcript contains a 31 nucleotide deletion in the HDV portion of the transcript (nucleotides 84 through 115 deleted). Fig. 26 is a schematic representation of a plasmid containing the insert encoding self-processing cassette. The figure is not drawn to scale.

Fig. 27 demonstrates the effect of 3' flanking sequences on RNA self-processing in vitro. H, Plasmid templates linearized with HindIII restriction enzyme. Transcripts from H templates contain four non-ribozyme nucleotides at the 3' end. N, Plasmid templates linearized with Ndel restriction enzyme. Transcripts from Ν templates contain 220 non-ribozyme nucleotides at the 3' end. R, Plasmid templates linearized with Rcal restriction enzyme. Transcripts from R templates contain 450 non-ribozyme nucleotides at the 3' end. Fig. 28 shows the effect of 3' flanking sequences on the transcleavage reaction catalyzed by a hammerhead ribozyme. A 622 nt internally-labeled RΝA (<10 nM) was incubated with ribozyme (1000 nM) under single turn-over conditions (Herschlag and Cech, 1990 Biochemistry 29, 10159). HH+2, HH+37, and HH+52 are trans-acting ribozymes produced by transcription from the HH, ΔHDV, and HH(mutant) constructs, respectively, and that contain 2, 37 and 52 extra nucleotides on the 3' end. The plot of the fraction of uncleaved substrate versus time was fit to a double exponential curve using the KaleidaGraph graphing program (Synergy Software, Reading, PA). A double exponential curve fit was used because the data points did not fall on a single exponential curve, presumably due to varying conformers of ribozyme and/or substrate RΝA.

Fig. 29 shows RΝA self-processing in OST7-1 cells. In vitro lanes contain full-length, unprocessed transcripts that were added to cellular lysates prior to RΝA extraction. These RΝAs were either pre-incubated with MgCl₂ (+) or with DEPC-treated water (-) prior to being hybridized with 5' end-labeled primers. Cellular lanes contain total cellular RNA from cells transfected with one of the four self-processing constructs. Cellular RNA are probed for ribozyme expression using a sequence specific primer-extension assay. Solid arrows indicate the location of primer extension bands corresponding to Full-Length RNA and 3' Cleavage Products.

Figs. 30,31 are diagrammatic representations of self-processing cassettes that will release trans-acting ribozymes with defined, stable stemloop structures at the 5' and the 3' end following self-processing. 30, shows various permutations of a hammerhead self-processing cassette. 31 , shows various permutations of a hairpin self-processing cassette.

Figs. 32a-b Schematic representation of RNA polymerse III promoter structure. Arrow indicates the transcription start site and the direction of coding region. A, B and C, refer to consensus A, B and C box promoter sequences. I, refers to intermediate cis-acting promoter sequence. PSE, refers to proximal sequence element. DSE, refers to distal sequence element. ATF, refers to activating transcription factor binding element. ?, refers to cis-acting sequence element that has not been fully characterized. EBER, Epstein-Barr-virus-encoded-RNA. TATA is a box well known in the art. Figs. 33a-e Sequence of the primary tRNA_i ^met and Δ3-5 transcripts.

The A and B box are internal promoter regions necessary for pol III transcription. Arrows indicate the sites of endogenous tRNA processing. The Δ3-5 transcript is a truncated version of tRNA wherein the sequence 3' of B box has been deleted (Adeniyi-Jones et al., 1984 supra). This modification renders the Δ 3-5 RNA resistant to endogenous tRNA processing.

Figure 34. Schematic representation of RNA structural motifs inserted into the Δ3-5 RNA. Δ3-5/HHI- a hammerhead (HHI) ribozyme was cloned at the 3' region of Δ3-5 RNA; S3- a stable stem-loop structure was incorporated at the 3' end of the Δ3-5/HHI chimera; S5- stable stem-loop structures were incorporated at the 5' and the 3' ends of Δ3-5/HHI ribozyme chimera; S35- sequence at the 3' end of the Δ3-5/HHI ribozyme chimera was altered to enable duplex formation between the 5' end and a complementary 3' region of the same RNA; S35Plus- in addition to structural alterations of S35, sequences were altered to facilitate additional duplex formation within the non-ribozyme sequence of the Δ3-5/HHI chimera.

Figures 35 and 36. Northern analysis to quantitate ribozyme expression in T cell lines transduced with Δ3-5 vectors. 35) Δ3-5/HHI and its variants were cloned individually into the DC retroviral vector (Sullenger et al., 1990 supra). Northern analysis of ribozyme chimeras expressed in MT-2 cells was performed. Total RNA was isolated from cells (Chomczynski & Sacchi, 1987 Analytical Biochemistry 162, 156-159), and transduced with various constructs described in Fig. 34. Northern analysis was carried out using standard protocols (Curr. Protocols Mol. Biol. 1992, ed. Ausubel et al., Wiley & Sons, NY). Nomenclature is same as in Figure 34. This assay measures the level of expression from the type 2 pol III promoter. 36) Expression of S35 constructs in MT2 cells. S35 (+ribozyme), S35 construct containing HHI ribozyme. S35 (-ribozyme), S35 construct containing no ribozyme.

Figure 37. Ribozyme activity in total RNA extracted from transduced MT-2 cells. Total RNA was isolated from cells transduced with Δ3-5 constructs described in Figs. 35 and 36 In a standard ribozyme cleavage reaction, 5 μg total RNA and trace amounts of 5' terminus-labeled ribozyme target RNA were denatured separately by heating to 90°C for 2 min in the presence of 50 mM Tris-HCI, pH 7.5 and 10 mM MgCl₂. RNAs were renatured by cooling the reaction mixture to 37°C for 10-15 min. Cleavage reaction was initiated by mixing the labeled substrate RNA and total cellular RNA at 37°C. The reaction was allowed to proceed for ~ 18h, following which the samples were resolved on a 20 % urea-polyacrylamide gel. Bands were visualized by autoradiography.

Figures 38 and 39. Ribozyme expression and activity levels in S35-transduced clonal CEM cell lines. 38) Northern analysis of S35-transduced clonal CEM cell lines. Standard curve was generated by spiking known concentrations of in vitro transcribed S5 RNA into total cellular RNA isolated from non-transduced CEM cells. Pool, contains RNA from pooled cells transduced with S35 construct. Pool (-G418 for 3 Mo), contains RNA from pooled cells that were initially selected for resistance to G418 and then grown in the absence of G418 for 3 months. Lanes A through N contain RNA from individual clones that were generated from the pooled cells transduced with S35 construct. tRNA_i ^met, refers to the endogenous tRNA. S35, refers to the position of the ribozyme band. M, marker lane. 39) Activity levels in S35-transduced clonal CEM cell lines. RNA isolation and cleavage reactions were as described in Fig.37. Nomenclature is same as in Figs. 35 and 36 except, S, 5' terminus-labeled substrate RNA. P, 8 nt 5' terminus-labeled ribozyme-mediated RNA cleavage product.

Figures 40 and 41 are proposed secondary structures of S35 and S35 containing a desired RNA (HHI), respectively. The position of HHI ribozyme is indicated in figure 41. Intramolecular stem refers to the stem structure formed due to an intramolecular base-paired interaction between the 3' sequence and the complementary 5' terminus. The length of the stem ranges from 15-16 base-pairs. Location of the A and the B boxes are shown.

Figures 42 and 43 are proposed secondary structures of S35 plus and S35 plus containing HHI ribozyme.

Figures 44, 45, 46 and 47 are the nucleotide base sequences of S35, HHIS35, S35 Plus, and HHIS35 Plus respectively.

Figs. 48a-b is a general formula for pol III RNA of this invention.

Figure 49 is a digrammatic representation of 5T construct. In this construct the desired RNA is located 3' of the intramolecular stem.

Figures 50 and 51 contain proposed secondary structures of 5T construct alone and 5T contruct containing a desired RNA (HHI ribozyme) respectively.

Figure 52 is a diagrammatic representation of TRZ-tRNA chimeras. The site of desired RNA insertion is indicated.

Figure 53 shows the general structure of HHITRZ-A ribozyme chimera. A hammerhead ribozyme targeted to site I is inserted into the stem II region of TRZ-tRNA chimera.

Figure 54 shows the general structure of HPITRZ-A ribozyme chimera. A hairpin ribozyme targeted to site I is cloned into the indicated region of TRZ-tRNA chimera. Figure 55 shows a comparison of RNA cleavage activity of HHITRZ-A, HHITRZ-B and a chemically synthesized HHI hammerhead ribozymes.

Figure 56 shows expression of ribozymes in T cell lines that are stably transduced with viral vectors. M, markers; lane 1 , non-transduced CEM cells; lanes 2 and 3, MT2 and CEM cells transduced with retroviral vectors; lanes 4 and 5, MT2 and CEM cells transduced with AAV vectors.

Figs. 57a-b Schematic diagram of adeno-associated virus and adenovirues vectors for ribozyme delivery. Both vectors utilize one or more ribozyme encoding transcription units (RZ) based on RNA polymerase II or RNA polymerase III promoters. A. Diagram of an AAV-based vector containing minimal AAV sequences comprising the inverted terminal repeats (ITR) at each end of the vector genome, an optional selectable marker (Neo) driven by an exogenous promoter (Pro), a ribozyme transcription unit, and sufficient additional sequences (stuffer) to maintain a vector length suitable for efficient packaging. B. Diagram of ribozyme expressing adenovirus vectors containing deletions of one or more wild type adenoviorus coding regions (cross-hatched boxes marked as E1 , pIX, E3, and E4), and insertion of the ribozyme transcription unit at any or several of those regions of deletions. Fig. 58 is a graph showing the effect of arm length variation on the activity of ligated hammerhead (HH) ribozymes. Nomenclature 5/5, 6/6, 7/7, 8/8 and so on refers to the number of base-pairs being formed between the ribozyme and the target. For example, 5/8 means that the HH ribozyme forms 5 bp on the 5' side and 8 bp on the 3' side of the cleavage site for a total of 13 bp. -ΔG refers to the free energy of binding calculated for base-paired interactions between the ribozyme and the substrate RNA (Turner and Sugimoto, 1988 Ann. Rev. Biophys. Chem. 17, 167). RPI A is a HH ribozyme with 6/6 binding arms.

Figs. 59 and 60 and 61 show cleavage of long substrate (622 nt) by ligated HH ribozymes.

Fig. 62 is a diagrammatic representation of a hammerhead ribozyme (HH-H) targeted against a site termed H. Variants of HH-H are also shown that contain either a 2 base-paired stem II (HH-H1 and HH-H2) or a 3 base- paired stem II (HH-H3 and HH-H4). Figs. 63 and 64 show RNA cleavage activity of HH-I and its variants (see Fig.62). 63) cleavage of matched substrate RNA (15 nt). 64) cleavage of long substrate RNA (613 nt).

Figs. 65a-b is a schematic representation of a method of this invention to synthesize a full length hairpin ribozyme. No splint strand is required for ligation but rather the two fragments hybridize together at helix 4 prior to ligation. The only prerequisite is that the 3' fragment is phosphorylated at its 5' end and that the 3' end of the 5' fragment have a hydroxyl group. The hairpin ribozyme is targeted against site J. H1 and H2 are intermolecular helices formed between the ribozyme and the substrate. H3 and H4 are intramolecular helices formed within the hairpin ribozyme motif. Arrow indicates the cleavage site.

Fig. 66 shows RNA cleavage activity of ligated hairpin ribozymes targeted against site J. Figs. 67a-b is a diagrammatic representation of a Site K Hairpin

Ribozyme (HP-K) showing the proposed secondary structure of the hairpin ribozyme•substrate complex as described in the art (Berzal-Herranz et al., 1993 EMBO. J.12, 2567). The ribozyme has been assembled from two fragments (bimolecular ribozyme; Chowrira and Burke, 1992 Nucleic Acids Res. 20, 2835); #H1 and H2 represent intermolecular helix formation between the ribozyme and the substrate. H3 and H4 represent intramolecular helix formation within the ribozyme (intermolecular helix in the case of bimolecular ribozyme). Left panel (HP-K1) indicates 4 base-paired helix 2 and the right panel (HP-K2) indicates 6 base-paired helix 2. Arrow indicates the site of RNA cleavage. All the ribozymes discussed herein were chemically synthesized by solid phase synthesis using RNA phosphoramadite chemistry, unless otherwise indicated. Those skilled in the art will recognize that these ribozymes could also be made transcriptionally in vitro and in vivo. Figure 68 is a graph showing RNA cleavage by hairpin ribozymes targeted to site K. A plot of fraction of the target RNA uncleaved (fraction uncleaved) as a function of time is shown. HP-K2 (6 bp helix 2) cleaves a 422 target RNA to a greater extent than the HP-K1 (4 bp helix 2). To make internally-labeled substrate RNA for trans-ribozyme cleavage reactions, a 422 nt region (containing hairpin site A) was synthesized by PCR using primers that place the T7 RNA promoter upstream of the amplified sequence. Target RNA was transcribed in a standard transcription buffer in the presence of [α-³²P]CTP (Chowrira & Burke, 1991 supra). The reaction mixture was treated with 15 units of ribonuclease-free DNasel, extracted with phenol followed chloroform:isoamyl alcohol (25:1), precipitated with isopropanol and washed with 70% ethanol. The dried pellet was resuspended in 20 μl DEPC-treated water and stored at -20°C.

Unlabeled ribozyme (1μM) and internally labeled 422 nt substrate RNA (<10 nM) were denatured and renatured separately in a standard cleavage buffer (containing 50 mM Tris·HCl pH 7.5 and 10 mM MgCl₂) by heating to 90°C for 2 min. and slow cooling to 37°C for 10 min. The reaction was initiated by mixing the ribozyme and substrate mixtures and incubating at 37°C. Aliquots of 5 μl were taken at regular time intervals, quenched by adding an equal volume of 2X formamide gel loading buffer and frozen on dry ice. The samples were resolved on 5% polyacrylamide sequencing gel and results were quantitatively analyzed by radioanalytic imaging of gels with a Phosphorlmager (Molecular Dynamics, Sunnyvale, CA).

Figs. 69a-b is the Site L Hairpin Ribozyme (HP-L) showing proposed secondary structure of the hairpin ribozyme•substrate complex. The ribozyme was assembled from two fragments as described above. The nomenclature is the same as above.

Figure 70 shows RNA cleavage by hairpin ribozymes targeted to site L. A. plot of fraction of the target RNA uncleaved (fraction uncleaved) as a function of time is shown. HP-L2 (6 bp helix 2) cleaves a 2 KB target RNA to a greater extent than the HP-L1 (4 bp helix 2). To make internally- labeled substrate RNA for trans- ribozyme cleavage reactions, a 2 kB region (containing hairpin site L) was synthesized by PCR using primers that place the T7 RNA promoter upstream of the amplified sequence. The cleavage reactions were carried out as described above. Figs. 71 a-b shows a Site M Hairpin Ribozyme (HP-M) with the proposed secondary structure of the hairpin ribozyme•substrate complex. The ribozyme was assembled from two fragments as described above.

Figure 72 is a graph showing RNA cleavage by hairpin ribozymes targeted to site M. The ribozymes were tested at both 20°C and at 26°C. To make internally-labeled substrate RNA for trans-ribozyme cleavage reactions, a 1.9 KB region (containing hairpin site M) was synthesized by PCR using primers that place the 77 RNA promoter upstream of the amplified sequence. Cleavage reactions were carried out as described above except that 20°C and at 26°C temperatures were used.

Figs. 73a-d shows various structural modifications of the present invention. A) Hairpin ribozyme lacking helix 5. Nomenclature is same as described under figure 3. B) Hairpin ribozyme lacking helix 4 and helix 5. Helix 4 is replaced by a nucleotide loop wherein q is ≥ 2 bases. Nomenclature is same as described under figure 3. C) Hairpin ribozyme lacking helix 5. Helix 4 loop is replaced by a linker 103"L", wherein L is a non-nucleotide linker molecule (Benseler et al., 1993 J. Am. Chem. Soc. 115, 8483; Jennings et al., WO 94/13688). Nomenclature is same as described under figure 3. D) Hairpin ribozyme lacking helix 4 and helix 5. Helix 4 is replaced by non-nucleotide linker molecule "L" (Benseler et al., 1993 supra; Jennings et al., supra). Nomenclature is same as described under figure 3.

Figs. 74a-b shows Hairpin ribozymes containing nucleotide spacer region "s" at the indicated location, wherein s is ≥ 1 base. Hairpin ribozymes containing spacer region, can be synthesized as one fragment or can be assembled from multiple fragments. Nomenclature is same as described under figure 3.

Figs. 75a-e shows the structures of the 5'-C-alkyl-modified nucleotides. R₁ is as defined above. R is OH, H, O-protecting group, NH, or any group described by the publications discussed above, and those described below. B is as defined in the Figure or any other equivalent nucleotide base. CE is cyanoethyl, DMT is a standard blocking group. Other abbreviations are standard in the art. Figure 76 is a diagrammatic representation of the synthesis of 5'-C-alkyl-D-allose nucleosides and their phosphoramidites.

Figure 77 is a diagrammatic representation of the synthesis of 5'-C-alkyl-L-talose nucleosides and their phosphoramidites. Figure 78 is a diagrammatic representation of hammerhead ribozymes targeted to site O containing 5'-C-methyl-L-talo modifications at various positions.

Figure 79 shows RNA cleavage activity of HH-O ribozymes. Fraction of target RNA uncleaved as a function of time is shown. Figure 80 is a diagrammatic representation of a position numbered hammerhead ribozyme (according to Hertel et al. Nucleic Acids Res. 1 992, 20, 3252) showing specific substitutions.

Figs. 81 a-j shows the structures of various 2'-alkyl modified nucleotides which exemplify those of this invention. R groups are alkyl groups, Z is a protecting group.

Figure 82 is a diagrammatic representation of the synthesis of 2'-C-allyl uridine and cytidine.

Figure 83 is a diagrammatic representation of the synthesis of 2'-C-methylene and 2'-C-difluoromethylene uridine. Figure 84 is a diagrammatic representation of the synthesis of 2'-C-methylene and 2'-C-difluoromethylene cytidine.

Figure 85 is a diagrammatic representation of the synthesis of 2'-C-methylene and 2'-C-difluoromethylene adenosine.

Figure 86 is a diagrammatic representation of the synthesis of 2'-C-carboxymethylidine uridine, 2'-C-methoxycarboxymethylidine uridine and derivatized amidites thereof. X is CH₃ or alkyl as discussed above, or another substituent.

Figure 87 is a diagrammatic representation of a synthesis of nucleoside 5'-deoxy-5'-difluoromethylphosphonates. Figure 88 is a diagrammatic representation of the synthesis of nucleoside 5'-deoxy-5'-difluoromethylphosphonate 3'-phosphoramidites, dimers and solid supported dimers.

Figure 89 is a diagrammatic representation of the synthesis of nucleoside 5'-deoxy-5'-difluoromethylene triphosphates.

Figures 90 and 91 are diagrammatic representations of the synthesis of 3'-deoxy-3'-difluoromethylphosphonates and dimers.

Figure 92 is a schematic representation of synthesizing RNA phosphoramidite of a nucleotide containing a 2'-hydroxyl group modification of the present invention.

Figs. 93a-b describes a method for deprotection of oligonucleotides containing a 2'-hydroxyl group modification of the present invention.

Figure 94 is a diagrammatic representation of a hammerhead ribozyme targeted to site N. Positions of 2'-hydroxyl group substitution is indicated.

Figure 95 shows RNA cleavage activity of ribozymes containing a 2'-hydroxyl group modification of the present invention. All RNA, represents hammerhead ribozyme (HHN) with no 2'-hydroxyl group modifications. U7-ala, represents HHN ribozyme containing 2'-NH-alanine modification at the U7 position. U4/U7-ala, represents HHA containing 2'-NH-alanine modifications at U4 and U7 positions. U4 lys, represents HHA containing 2'-NH-lysine modification at U4 position. U7 lys, represents HHA containing 2'-NH-lysine modification at U7 position. U4/U7-lys, represents HHN containing 2'-NH-lysine modification at U4 and U7 positions. Figures 96 and 97 are schematic representations of synthesizing

(solid-phase synthesis) 3' ends of RNA with modification of the present invention. B, refers to either a base, modified base or an H.

Figure 98 and 99 are schematic representations of synthesizing (solid-phase synthesis) 5' ends of RNA with modification of the present invention. B, refers to either a base, modified base or an H.

Figures 100 and 101 are general schematic representations of the invention. Fig. 102a-d is a schematic representation of a method of the invention.

Fig. 103 is a graph of the results of the experiment diagrammed in figure 104.

Figure 104 is a diagrammatic representation of a fusion mRNA used in the experiment diagrammed in Fig. 102.

Figure 105 is a diagrammatic representation of a method for selection of useful ribozymes of this invention.

Figure 106 generally shows R-loop formation, and an R-loop complex. In addition, it indicates the location at which ligands can be provided to target the R-loop complex to cells using at least three different procedures, such as ligand receptor interaction, lipid or calcium phosphate mediated delivery, or electroporation.

Figure 107 shows a method for use of self-processing ribozymes to generate therapeutic ribozymes of unit length. This method is essentially described by Draper et al., PCT WO 93/23509.

Figure 108 shows a method of linking ligands like folate, carbohydrate or peptides to R-loop forming RNA.

Ribozymes of this invention block to some extent ICAM-1 , IL-5, rel A, TNF-α, p210^bcr-abI, or RSV genes expression and can be used to treat diseases or diagnose such diseases. Ribozymes will be delivered to cells in culture and to tissues in animal models. Ribozyme cleavage of ICAM-1 , II-5, rel A, TNF-α ,p210^bcr-abl, or RSV mRNA in these systems may prevent or alleviate disease symptoms or conditions.

I. Target sites Targets for useful ribozymes can be determined as disclosed in

Draper et al PCT WO93/23509, Sullivan et al., PCT WO94/02595 as well as by Draper et al., PCT/US94/13129 and hereby incorporated by reference herein in totality. Rather than repeat the guidance provided in those documents here, below are provided specific examples of such methods, not limiting to those in the art. Ribozymes to such targets are designed as described in those applications and synthesized to be tested in vitro and in vivo, as also described. Such ribozymes can also be optimized and delivered as described therein. While specific examples to animal and human RNA are provided, those in the art will recognize that the equivalent human RNA targets described can be used as described below. Thus, the same target may be used, but binding arms suitable for targeting human RNA sequences are present in the ribozyme. Such targets may also be selected as described below.

It must be established that the sites predicted by the computer-based RNA folding algorithm correspond to potential cleavage sites. Hammerhead or hairpin ribozymes are designed that could bind and are individually analyzed by computer folding (Jaeger et al., 1989 Proc. Natl. Acad. Sci., USA, 86 7706-7710) to assess whether the ribozyme sequences fold into the appropriate secondary structure. Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA. mRNA is screened for accessible cleavage sites by the method described generally in Draper et al., PCT WO93/23569 hereby incorporated by reference herein. Briefly, DNA oligonucleotides representing potential hammerhead or hairpin ribozyme cleavage sites are synthesized. A polymerase chain reaction is used to generate a substrate for T7 RNA polymerase transcription from cDNA clones. Labeled RNA transcripts are synthesized in vitro from DNA templates. The oligonucleotides and the labeled trascripts are annealed, RNaseH is added and the mixtures are incubated for the designated times at 37°C. Reactions are stopped and RNA separated on sequencing polyacrylamide gels. The percentage of the substrate cleaved is determined by autoradiographic quantitation using a phosphor imaging system. From these data, hammerhead or hairpin ribozynme sites are chosen as the most accessible.

Ribozymes of the hammerhead or hairpin motif are designed to anneal to various sites in the mRNA message. The binding arms are complementary to the target site sequences desribed above. The ribozymes are chemically synthesized. The method of synthesis used follows the procedure for normal RNA synthesis as described in Usman et al., 1987 J. Am. Chem. Soc., 109, 7845 and in Scaringe et al., 1990 Nucleic Acids Res., 18, 5433 and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, phosphoramidites at the 3'-end. The average stepwise coupling yeilds are >98%. Inactive ribozymes are synthesized by substituting a U for G5 and a U for A ₁₄ (numbering from Hertel et al., 1992 Nucleic Acids Res., 20, 3252). Hairpin ribozymes are synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-2840). Ribozymes are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbach, 1989, Methods Enzymol, 180, 51). All ribozymes are modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O-methyl, 2'H (for a review see Usman and Cedergren, 1992 TIBS 17,34). Ribozymes are purified by gel electrophoresis using heneral methods or are purified by high pressure liquid chromatography and are resuspended in water.

Example 1 : ICAM-1

Ribozymes that cleave ICAM-1 mRNA represent a novel therapeutic approach to inflammatory or autoimmune disorders. ICAM-1 function can be blocked therapeutically using monoclonal antibodies. Ribozymes have the advantage of being generally immunologically inert, whereas significant neutralizing anti-IgG responses can be observed with some monoclonal antibody treatments.

The following is a brief description of the physiological role of ICAM-1.

The discussion is not meant to be complete and is provided only for understanding of the invention that follows. This summary is not an admission that any of the work described below is prior art to the claimed invention.

Intercellular adhesion molecule-1 (ICAM-1) is a cell surface protein whose expression is induced by inflammatory mediators. ICAM-1 is required for adhesion of leukocytes to endothelial cells and for several immunological functions including antigen presentation, immunoglobulin production and cytotoxic cell activity. Blocking ICAM-1 function prevents immune cell recognition and activity during transplant rejection and in animal models of rheumatoid arthritis, asthma and reperfusion injury. Cell-cell adhesion plays a pivotal role in inflammatory and immune responses (Springer et al., 1987 Ann. Rev. Immunol. 5, 223-252). Cell adhesion is required for leukocytes to bind to and migrate through vascular endothelial cells. In addition, cell-cell adhesion is required for antigen presentation to T cells, for B cell induction by T cells, as well as for the cytotoxicity activity of T cells, NK cells, monocytes or granulocytes. Intercellular adhesion molecule-1 (ICAM-1) is a 110 kilodalton member of the immunoglobulin superfamily that is involved in all of these cell-cell interactions (Simmons et al., 1988 Nature (London) 331, 624-627). ICAM-1 is expressed on only a limited number of cells and at low levels in the absence of stimulation (Dustin et al., 1986 J. Immunol. 137, 245-254). Upon treatment with a number of inflammatory mediators (lipopolysaccharide, γ-interferon, tumor necrosis factor-α, or interleukin-1), a variety of cell types (endothelial, epithelial, fibroblastic and hematopoietic cells) in a variety of tissues express high levels of ICAM-1 on their surface (Sringer et. al. supra; Dustin et al., supra; and Rothlein et al., 1988 J. Immunol. 141 , 1665-1669). Induction occurs via increased transcription of ICAM-1 mRNA (Simmons et al., supra). Elevated expression is detectable after 4 hours and peaks after 16 - 24 hours of induction. ICAM-1 induction is critical for a number of inflammatory and immune responses. In vitro, antibodies to ICAM-1 block adhesion of leukocytes to cytokine-activated endothelial cells (Boyd,1988 Proc. Natl. Acad. Sci. USA 85, 3095-3099; Dustin and Springer, 1988 J. Cell Biol. 107, 321-331 ). Thus, ICAM-1 expression may be required for the extravasation of immune cells to sites of inflammation. Antibodies to ICAM-1 also block T cell killing, mixed lymphocyte reactions, and T cell-mediated B cell differentiation, suggesting that ICAM-1 is required for these cognate cell interactions (Boyd et al., supra). The importance of ICAM-1 in antigen presentation is underscored by the inability of ICAM-1 defective murine B cell mutants to stimulate antigen-dependent T cell proliferation (Dang et al., 1990 J. Immunol. 144, 4082-4091). Conversely, murine L cells require transfection with human ICAM-1 in addition to HLA-DR in order to present antigen to human T cells (Altmann et al., 1989 Nature (London) 338, 512-514). In summary, evidence in vitro indicates that ICAM-1 is required for cell-cell interactions critical to inflammatory responses, cellular immune responses, and humoral antibody responses. By engineering ribozyme motifs we have designed several ribozymes directed against ICAM-1 mRNA sequences. These have been synthesized with modifications that improve their nuclease resistance. These ribozymes cleave ICAM-1 target sequences in vitro. The sequence of human, rat and mouse ICAM-1 mRNA can be screened for accessible sites using a compter folding algorithm. Regions of the mRNA that did not form secondary folding structures and that contain potential hammerhead or hairpin ribozyme cleavage sites can be identified. These sites are shown in Tables 2, 3, and 6-9. (All sequences are 5' to 3' in the tables) While rat, mouse and human sequences can be screened and ribozymes thereafter designed, the human targeted sequences are of most utility.

The sequences of the chemically synthesized ribozymes useful in this study are shown in Tables 4 - 8 and 10. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity and may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables. The ribozymes will be tested for function in vivo by exogenous delivery to human umbilical vein endothelial cells (HUVEC). Ribozymes will be delivered by incorporation into liposomes, by complexing with cationic lipids, by microinjection, or by expression from DNA or RNA vectors described above. Cytokine-induced ICAM-1 expression will be monitored by ELISA, by indirect immunofluoresence, and/or by FACS analysis. ICAM-1 mRNA levels will be assessed by Northern, by RNAse protection, by primer extension or by quantitative RT-PCR analysis. Ribozymes that block the induction of ICAM-1 protein and mRNA by more than 90% will be identified. As disclosed by Sullivan et al., PCT WO94/02595, incorporated by reference herein, ribozymes and/or genes encoding them will be locally delivered to transplant tissue ex vivo in animal models. Expression of the ribozyme will be monitored by its ability to block ex vivo induction of ICAM-1 mRNA and protein. The effect of the anti-ICAM-1 ribozymes on graft rejection will then be assessed. Similarly, ribozymes will be introduced into joints of mice with collagen-induced arthritis or rabbits with Streptococcal cell wall-induced arthritis. Liposome delivery, cationic lipid delivery, or adeno-associated virus vector delivery can be used. One dose

(or a few infrequent doses) of a stable anti-ICAM-1 ribozyme or a gene construct that constitutively expresses the ribozyme may abrogate inflammatory and immune responses in these diseases.

Uses

ICAM-1 plays a central role in immune cell recognition and function.

Ribozyme inhibition of ICAM-1 expression can reduce transplant rejection and alleviate symptoms in patients with rheumatoid arthritis, asthma or other acute and chronic inflammatory disorders. We have engineered several ribozymes that cleave ICAM-1 mRNA. Ribozymes that efficiently inhibit ICAM-1 expression in cells can be readily found and their activity measured with regard to their ability to block transplant rejection and arthritis symptoms in animal models. These anti-ICAM-1 ribozymes represent a novel therapeutic for the treatment of immunological or inflammatory disorders.

The therapeutic utility of reduction of activity of ICAM-1 function is evident in the following disease targets. The noted references indicate the role of ICAM-1 and the therapeutic potential of ribozymes described herein.

Thus, these targets can be therapeutically treated with agents that reduce

ICAM-1 expression or function. These diseases and the studies that support a critical role for ICAM-1 in their pathology are listed below. This list is not meant to be complete and those in the art will recognize further conditions and diseases that can be effectively treated using ribozymes of the present invention.

• Transplant rejection

ICAM-1 is expressed on venules and capillaries of human cardiac biopsies with histological evidence of graft rejection (Briscoe et al., 1991 Transplantation 51 , 537-539).

Antibody to ICAM-1 blocks renal (Cosimi et al., 1990 J. Immunol. 144, 4604- 4612) and cardiac (Flavin et al., 1991 Transplant. Proc. 23, 533-534) graft rejection in primates. A Phase I clinical trial of a monoclonal anti-ICAM-1 antibody showed significant reduction in rejection and a significant increase in graft function in human kidney transplant patients (Haug, et al., 1993 Transplantation 55, 766-72).

• Rheumatoid arthritis ICAM-1 overexpression is seen on synovial fibroblasts, endothelial cells, macrophages, and some lymphocytes (Chin et al., 1990 Arthritis Rheum 33, 1776-86; Koch et al., 1991 Lab Invest 64, 313-20).

Soluble ICAM-1 levels correlate with disease severity (Mason et al., 1993 Arthritis Rheum 36, 519-27). Anti-ICAM antibody inhibits collagen-induced arthritis in mice (Kakimoto et al.,

1992 Cell Immunol 142, 326-37).

Anti-ICAM antibody inhibits adjuvant-induced arthritis in rats (ligo et al., 1991 J Immunol 147, 4167-71).

• Myocardial ischemia, stroke, and reperfusion injury Anti-ICAM-1 antibody blocks adherence of neutrophils to anoxic endothelial cells (Yoshida et al., 1992 Am J Physiol 262, H1891-8).

Anti-ICAM-1 antibody reduces neurological damage in a rabbit model of cerebral stroke (Bowes et al., 1993 Exp Neurol 119, 215-9).

Anti-ICAM-1 antibody protects against reperfusion injury in a cat model of myocardial ischemia (Ma et al., 1992 Circulation 86, 937-46).

• Asthma

Antibody to ICAM-1 partially blocks eosinophil adhesion to endothelial cells and is overexpressed on inflamed airway endothelium and epithelium in vivo (Wegner et al., 1990 Science 247, 456-9). In a primate model of asthma, anti-ICAM-1 antibody blocks airway eosinophilia

(Wegneret al., supra) and prevents the resurgence of airway inflammation and hyper-responsiveness after dexamethosone treatment (Gundel et al., 1992 Clin Exp Allergy 22, 569-75).

• Psoriasis Surface ICAM-1 and a clipped, soluble version of ICAM-1 is expressed in psoriatic lesions and expression correlates with inflammation (Kellner et al., 1991 Br J Dermatol 125, 211-6; Griffiths 1989 J Am Acad Dermatol 20, 617-29; Schopf et al., 1993 Br J Dermatol 128, 34-7). Anti-ICAM antibody blocks keratinocyte antigen presentation to T cells

(Nickoloff et al., 1993 J Immunol 150, 2148-59 ).

• Kawasaki disease

Surface ICAM-1 expression correlates with the disease and is reduced by effective immunoglobulin treatment (Leung, et al., 1989 Lancet 2, 1298-302). Soluble ICAM levels are elevated in Kawasaki disease patients; particularly high levels are observed in patients with coronary artery lesions (Furukawa et al., 1992 Arthritis Rheum 35, 672-7; Tsuji, 1992 Arerugi 41 , 1507-14).

Circulating LFA-1+ T cells are depleted (presumably due to ICAM-1 mediated extravasation) in Kawasaki disease patients (Furukawa et al., 1993 Scand J Immunol 37, 377-80).

Example 2: IL-5

Ribozymes that cleave IL-5 mRNA represent a novel therapeutic approach to inflammatory disorders like asthma. The invention features use of ribozymes to treat chronic asthma, e.g., by inhibiting the synthesis of IL-5 in lymphocytes and preventing the recruitment and activation of eosinophils.

A number of cytokines besides IL-5 may also be involved in the activation of inflammation in asthmatic patients, including platelet activating factor, IL-1 , IL-3, IL-4, GM-CSF, TNF-α, gamma interferon, VCAM, ILAM-1 , ELAM-1 and NF-κB. In addition to these molecules, it is appreciated that any cellular receptors which mediate the activities of the cytokines are also good targets for intervention in inflammatory diseases. These targets include, but are not limited to, the IL-1 R and TNF-αR on keratinocytes, epithelial and endothelial cells in airways. Recent data suggest that certain neuropeptides may play a role in asthmatic symptoms. These peptides include substance P, neurokinin A and calcitonin-gene-related peptides.

These target genes may have more general roles in inflammatory diseases, but are currently assumed to have a role only in asthma. Ribozymes of this invention block to some extent IL-5 expression and can be used to treat disease or diagnose such disease. Ribozymes will be delivered to cells in culture and to cells or tissues in animal models of asthma (Clutterbuck et al., 1989 supra: Garssen et al., 1991 Am. Rev. Respir. Dis. 144, 931-938; Larsen et al., 1992 J. Clin. Invest. 89, 747-752; Mauser et al., 1993 supra). Ribozyme cleavage of IL-5 mRNA in these systems may prevent inflammatory cell function and alleviate disease symptoms.

The sequence of human and mouse IL-5 mRNA were screened for accessible sites using a computer folding algorithm. Potential hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables 11 , 13, and 14, 15. (All sequences are 5' to 3' in the tables.) While mouse and human sequences can be screened and ribozymes thereafter designed, the human targeted sequences are of most utility. However, mouse targeted ribozymes are useful to test efficacy of action of the ribozyme prior to testing in humans. The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme. (In Table 12, lower case letters indicate positions that are not conserved between the Human and the Mouse IL-5 sequences.) The sequences of the chemically synthesized ribozymes useful in this study are shown in Tables 12, 14 - 16. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity. For example, stem loop II sequence of hammerhead ribozymes listed in Tables 12 and 14 (5'-GGCCGAAAGGCC-3") can be altered (substitution, deletion and/or insertion) to contain any sequence provided, a minimum of two base-paired stem structure can form. Similarly, stem-loop IV sequence of hairpin ribozymes listed in Tables 15 and 16 (5'-CACGUUGUG-3') can be altered (substitution, deletion and/or insertion) to contain any sequence provided, a minimum of two base-paired stem structure can form. The sequences listed in Tables 12, 14 - 16 may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables. By engineering ribozyme motifs we have designed several ribozymes directed against IL-5 mRNA sequences. These ribozymes are synthesized with modifications that improve their nuclease resistance. The ability of ribozymes to cleave IL-5 target sequences in vitro is evaluated.

The ribozymes will be tested for function in vivo by analyzing IL-5 expression levels. Ribozymes will be delivered to cells by incorporation into liposomes, by complexing with cationic lipids, by microinjection, or by expression from DNA or RNA vectors. IL-5 expression will be monitored by biological assays, ELISA, by indirect immunofluoresence, and/or by FACS analysis. IL-5 mRNA levels will be assessed by Northern analysis, RNAse protection or primer extension analysis or quantitative RT-PCR. Ribozymes that block the induction of IL-5 activity and/or IL-5 mRNA by more than 90% will be identified.

Uses

Interleukin 5 (IL-5), a cytokine produced by CD4+ T helper cells and mast cells, was originally termed B cell growth factor II (reviewed by Takatsu et al., 1988 Immunol. Rev. 102, 107). It stimulates proliferation of activated B cells and induces production of IgM and IgA. IL-5 plays a major role in eosinophil function by promoting differentiation (Clutterbuck et al., 1989 Blood 73, 1504-12), vascular adhesion (Walsh et al., 1990 Immunology 71 , 258-65) and in vitro survival of eosinophils (Lopez et al., 1988 J. Exp. Med. 167, 219-24). This cytokine also enhances histamine release from basophils (Hirai et al., 1990 J. Exp. Med. 172, 1525-8). The following summaries of clinical results support the selection of IL-5 as a primary target for the treatment of asthma:

Several studies have shown a direct correlation between the number of activated T cells and the number of eosinophils from asthmatic patients vs. normal patients (Oehling et al., 1992 J. Investig. Allergol. Clin. Immunol. 2, 295-9). Patients with either allergic asthma or intrinsic asthma were treated with corticosteroids. The bronchoalveolar lavage was monitored for eosinophils, activated T helper cells and recovery of pulmonary function over a 28 to 30 day period. The number of eosinophils and activated T helper cells decreased progressively with subsequent improvement in pulmonary function compared to intrinsic asthma patients with no corticosteroid treatment.

Bronchoalveolar lavage cells were screened for production of cytokines using in situ hybridization for mRNA. In situ hybridization signals were detected for IL-2, IL-3, IL-4, IL-5 and GM-CSF. Upregulation of mRNA was observed for IL-4, IL-5 and GM-CSF (Robinson et al., 1993 J. Allergy Clin. Immunol. 92, 313-24). Another study showed that upregulation of IL-5 transcripts from allergen challenged vs. saline challenged asthmatic patients (Krishnaswamy et al., 1993 Am. J. Respir. Cell. Mol. Biol. 9, 279-86).

An 18 patient study was performed to determine a mechanism of action for corticosteroid improvement of asthma symptoms. Improvement was monitored by methacholine responsiveness. A correlation was observed between the methacholine responsiveness, a reduction in the number of eosinophils, a reduction in the number of cells expressing IL-4 and IL-5 mRNA and an increase in number of cells expressing interferon-gamma.

Bronchial biopsies from 15 patients were analyzed 24 hours after allergen challenge (Bentley et al., 1993 Am. J. Respir. Cell. Mol. Biol. 8, 35-42). Increased numbers of eosinophils and IL-2 receptor positive cells were found in the biopsies. No differences in the numbers of total leukocytes, T lymphocytes, elastase-positive neutrophils, macrophages or mast cell subtypes were observed. The number of cells expressing IL-5 and GM-CSF mRNA significantly increased.

In another patient study, the eosinophil phenotype was the same for asthmatic patients and normal individuals. However, eosinophils from asthmatic patients had greater leukotriene C4 producing capacity and migration capacity. There were elevated levels of IL-3, IL-5 and GM-CSF in the circulation of asthmatics but not in normal individuals (Bruijnzeel et al., 1992 Schweiz. Med. Wochenschr. 122, 298-301).

Efficacy of antibody to IL-5 was assessed in a guinea pig asthma model. The animals were challenged with ovalbumin and assayed for eosinophilia and the responsiveness to the bronchioconstriction substance P. A 30 mg/kg dose of antibody administered i.p. blocked ovalbumin- induced increased sensitivity to substance P and blocked increases in bronchoalveolar and lung tissue accumulation of eosinophils (Mauser et al., 1993 Am. Rev. Respir. Dis. 148, 1623-7). In a separate study guinea pigs challenged for eight days with ovalbumin were treated with monoclonal antibody to IL-5. Treatment produced a reduction in the number of eosinophils in bronchoalveolar lavage. No reduction was observed for unchallenged guinea pigs and guinea pigs treated with a control antibody. Antibody treatment completely inhibited the development of hyperreactivity to histamine and arecoline after ovalbumin challenge (van Oosterhout et al., 1993 Am. Rev. Respir. Dis. 147, 548-52)

Results obtained from human clinical analysis and animal studies indicate the role of activated T helper cells, cytokines and eosinophils in asthma. The role of IL-5 in eosinophil development and function makes IL-5 a good candidate for target selection. The antibody studies neutralized IL-5 in the circulation thus preventing eosinophilia. Inhibition of the production of IL-5 will achieve the same goal.

Asthma - a prominent feature of asthma is the infiltration of eosinophils and deposition of toxic eosinophil proteins (e.g. major basic protein, eosinophil-derived neurotoxin) in the lung. A number of T-cell-derived factors like IL-5 are responsible for the activation and maintainance of eosinophils (Kay, 1991 J. Allergy Clin. Immun. 87, 893). Inhibition of IL-5 expression in the lungs can decrease the activation of eosinophils and will help alleviate the symptoms of asthma.

Atopy - is characterized by the developement of type I hypersensitive reactions associated with exposure to certain environmental antigens. One of the common clinical manifestations of atopy is eosinophilia (accumulation of abnormally high levels of eosinophils in the blood). Antibodies against IL-5 have been shown to lower the levels of eosinophils in mice (Cook et al., 1993 in Immunopharmacol. Eosinophils ed. Smith and Cook, pp. 193-216, Academic, London, UK)

Parasitic infection-related eosinophilia- infections with parasites like helminths, can lead to severe eosinophilia (Cook et al., 1993 supra). Animal models for eosinophilia suggest that infection of mice, for example, can lead to blood, peritoneal and/or tissue eosinophilia, all of which seem to be lowered to varying degrees by antibodies directed against IL-5.

Pulmonary infiltration eosinophilia- is characterised by accumulation of high levels of eosinophils in pulmonary parenchyma (Gleich, 1990 J. Allerαv Clin. Immunol. 85, 422). L-Tryptophan-associated eosinophilia-myalgia syndrome

(EMS)- The EMS disease is closely linked to the consumption of L-tryptophan, an essential aminoacid used to treat conditions like insomnia (for review see Varga et al., 1993 J Invest. Dermatol. 100, 97s). Pathologic and histologic studies have demonstrated high levels of eosinophils and mononuclear inflammatory cells in patients with EMS. It appears that IL-5 and transforming growth factor play a significant role in the development of EMS (Varga et al., 1993 supra) by activating eosinophils and other inflammatory cells. Thus, ribozymes of the present invention that cleave IL-5 mRNA and thereby IL-5 activity have many potential therapeutic uses, and there are reasonable modes of delivering the ribozymes in a number of the possible indications. Development of an effective ribozyme that inhibits IL-5 function is described above; available cellular and activity assays are numerous, reproducible, and accurate. Animal models for IL-5 function and for each of the suggested disease targets exist (Cook et al., 1993 supra) and can be used to optimize activity.

Example 3: NF-κB

Ribozymes that cleave rel A mRNA represent a novel therapeutic approach to inflammatory or autoimmune disorders. Inflammatory mediators such as lipopolysaccharide (LPS), interleukin-1 (IL-1) or tumor necrosis factor-a (TNF-α) act on cells by inducing transcription of a number of secondary mediators, including other cytokines and adhesion molecules. In many cases, this gene activation is known to be mediated by the transcriptional regulator, NF-κB. One subunit of NF-κB, the relA gene product (termed RelA or p65) is implicated specifically in the induction of inflammatory responses. Ribozyme therapy, due to its exquisite specificity, is particularly well-suited to target intracellular factors that contribute to disease pathology. Thus, ribozymes that cleave mRNA encoded by rel A or TNF-α may represent novel therapeutics for the treatment of inflammatory and autoimmune disorders.

The nuclear DNA-binding activity, NF-κB, was first identified as a factor that binds and activates the immunoglobulin κ light chain enhancer in B cells. NF-κB now is known to activate transcription of a variety of other cellular genes (e.g., cytokines, adhesion proteins, oncogenes and viral proteins) in response to a variety of stimuli (e.g., phorbol esters, mitogens, cytokines and oxidative stress). In addition, molecular and biochemical characterization of NF-κB has shown that the activity is due to a homodimer or heterodimer of a family of DNA binding subunits. Each subunit bears a stretch of 300 amino acids that is homologous to the oncogene, v-rel. The activity first described as NF-κB is a heterodimer of p49 or p50 with p65. The p49 and p50 subunits of NF-κB (encoded by the nf-κB2 or nf-κB1 genes, respectively) are generated from the precursors NF-κB1 (p105) or NF-κB2 (p100). The p65 subunit of NF-κB (now termed Rel A ) is encoded by the rel A locus.

The roles of each specific transcription-activating complex now are being elucidated in cells (N.D. Perkins, et al., 1992 Proc. Natl Acad. Sci USA 89, 1529-1533). For instance, the heterodimer of NF-κB1 and Rel A (p50/p65) activates transcription of the promoter for the adhesion molecule, VCAM-1 , while NF-κB2/RelA heterodimers (p49/p65) actually inhibit transcription (H.B. Shu, et al., Mol. Cell. Biol. 13, 6283-6289 (1993)). Conversely, heterodimers of NF-κB2/RelA (p49/p65) act with Tat-I to activate transcription of the HIV genome, while NF-κB1/RelA (p50/p65) heterodimers have little effect (J. Liu, N.D. Perkins, R.M. Schmid, G.J. Nabel, J. Virol. 1992 66, 3883-3887). Similarly, blocking rel A gene expression with antisense oligonucleotides specifically blocks embryonic stem cell adhesion; blocking NF-κB1 gene expression with antisense oligonucleotides had no effect on cellular adhesion (Narayanan et al., 1993 Mol. Cell. Biol. 13, 3802-3810). Thus, the promiscuous role initially assigned to NF-κB in transcriptional activation (M.J. Lenardo, D. Baltimore, 1989 Cell 58, 227-229) represents the sum of the activities of the rel family of DNA-binding proteins. This conclusion is supported by recent transgenic "knock-out" mice of individual members of the rel family. Such "knock-outs" show few developmental defects, suggesting that essential transcriptional activation functions can be performed by more than one member of the rel family.

A number of specific inhibitors of NF-κB function in cells exist, including treatment with phosphorothioate antisense oliogonucleotide, treatment with double-stranded NF-κB binding sites, and over expression of the natural inhibitor MAD-3 (an 1κB family member). These agents have been used to show that NF-κB is required for induction of a number of molecules involved in inflammation, as described below.

•NF-κB is required for phorbol ester-mediated induction of IL-6 (I. Kitajima, et al., Science 258, 1792-5 (1992)) and IL-8 (Kunsch and Rosen, 1993 Mol. Cell. Biol. 13, 6137-46).

•NF-κB is required for induction of the adhesion molecules ICAM-1 (Eck, et al., 1993 Mol. Cell. Biol. 13, 6530-6536), VCAM-1 (Shu et al., supra), and E-selectin (Read, et al., 1994 J. Exp. Med. 179, 503-512) on endothelial cells. •NF-κB is involved in the induction of the integrin subunit, CD18, and other adhesive properties of leukocytes (Eck et al., 1993 supra).

The above studies suggest that NF-κB is integrally involved in the induction of cytokines and adhesion molecules by inflammatory mediators. Two recent papers point to another connection between NF-κB and inflammation: glucocorticoids may exert their anti-inflammatory effects by inhibiting NF-κB. The glucocorticoid receptor and p65 both act at NF-κB binding sites in the ICAM-1 promoter (van de Stolpe, et al., 1994 J. Biol. Chem. 269, 6185-6192). Glucocorticoid receptor inhibits NF-κB-mediated induction of IL-6 (Ray and Prefontaine, 1994 Proc. Natl Acad. Sci USA 91 , 752-756). Conversely, overexpression of p65 inhibits glucocorticoid induction of the mouse mammary tumor virus promoter. Finally, protein cross-linking and co-immunoprecipitation experiments demonstrated direct physical interaction between p65 and the glucocorticoid receptor (Id.).

Ribozymes of this invention block to some extent NF-κB expression and can be used to treat disease or diagnose such disease. Ribozymes will be delivered to cells in culture and to cells or tissues in animal models of restenosis, transplant rejection and rheumatoid arthritis. Ribozyme cleavage of relA mRNA in these systems may prevent inflammatory cell function and alleviate disease symptoms. The sequence of human and mouse re/A mRNA can be screened for accessible sites using a computer folding algorithm. Potential hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables 17, 18 and 21-22. (All sequences are 5' to 3' in the tables.) While mouse and human sequences can be screened and ribozymes thereafter designed, the human targetted sequences are of most utility.

The sequences of the chemically synthesized ribozymes useful in this study are shown in Tables 19 - 22. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity and may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables. By engineering ribozyme motifs we have designed several ribozymes directed against rel A mRNA sequences. These ribozymes are synthesized with modifications that improve their nuclease resistance. The ability of ribozymes to cleave relA target sequences in vitro is evaluated.

The ribozymes will be tested for function in vivo by analyzing cytokine-induced VCAM-1 , ICAM-1 , IL-6 and IL-8 expression levels. Ribozymes will be delivered to cells by incorporation into liposomes, by complexing with cationic lipids, by microinjection, or by expression from DNA and RNA vectors. Cytokine-induced VCAM-1 , ICAM-1 , IL-6 and IL-8 expression will be monitored by ELISA, by indirect immunofluoresence, and/or by FACS analysis. Rel A mRNA levels will be assessed by Northern analysis, RNAse protection or primer extension analysis or quantitative RT-PCR. Activity of NF-κB will be monitored by gel-retardation assays. Ribozymes that block the induction of NF-κB activity and/or rel A mRNA by more than 50% will be identified. RNA ribozymes and/or genes encoding them will be locally delivered to transplant tissue ex vivo in animal models. Expression of the ribozyme will be monitored by its ability to block ex vivo induction of VCAM-1 , ICAM-1 , IL-6 and IL-8 mRNA and protein. The effect of the anti-rel A ribozymes on graft rejection will then be assessed. Similarly, ribozymes will be introduced into joints of mice with collagen-induced arthritis or rabbits with Streptococcal cell wall-induced arthritis. Liposome delivery, cationic lipid delivery, or adeno-associated virus vector delivery can be used. One dose (or a few infrequent doses) of a stable anti-relA ribozyme or a gene construct that constitutively expresses the ribozyme may abrogate inflammatory and immune responses in these diseases. Uses

A therapeutic agent that inhibits cytokine gene expression, inhibits adhesion molecule expression, and mimics the anti-inflammatory effects of glucocorticoids (without inducing steroid-responsive genes) is ideal for the treatment of inflammatory and autoimmune disorders. Disease targets for such a drug are numerous. Target indications and the delivery options each entails are summarized below. In all cases, because of the potential immunosuppressive properties of a ribozyme that cleaves rel A mRNA, uses are limited to local delivery, acute indications, or ex vivo treatment. •Rheumatoid arthritis (RA).

Due to the chronic nature of RA, a gene therapy approach is logical. Delivery of a ribozyme to inflamed joints is mediated by adenovirus, retrovirus, or adeno-associated virus vectors. For instance, the appropriate adenovirus vector can be administered by direct injection into the synovium: high efficiency of gene transfer and expression for several months would be expected (B.J. Roessler, E.D. Allen, J.M. Wilson, J.W. Hartman, B. L. Davidson, J. Clin. Invest. 92, 1085-1092 (1993)). It is unlikely that the course of the disease could be reversed by the transient, local administration of an anti-inflammatory agent. Multiple administrations may be necessary. Retrovirus and adeno-associated virus vectors would lead to permanent gene transfer and expression in the joint. However, permanent expression of a potent anti-inflammatory agent may lead to local immune deficiency.

•Restenosis. Expression of NF-κB in the vessel wall of pigs causes a narrowing of the luminal space due to excessive deposition of extracellular matrix components. This phenotype is similar to matrix deposition that occurs subsequent to coronary angioplasty. In addition, NF-κB is required for the expression of the oncogene c-myb (F.A. La Rosa, J.W. Pierce, G.E. Soneneshein, Mol. Cell. Biol. 14, 1039-44 (1994)). Thus NF-κB induces smooth muscle proliferation and the expression of excess matrix components: both processes are thought to contribute to reocclusion of vessels after coronary angioplasty.

•Transplantation. NF-κB is required for the induction of adhesion molecules (Eck et al., supra, K. O'Brien, et al., J. Clin. Invest. 92, 945-951 (1993)) that function in immune recognition and inflammatory responses. At least two potential modes of treatment are possible. In the first, transplanted organs are treated ex vivo with ribozymes or ribozyme expression vectors. Transient inhibition of NF-κB in the transplanted endothelium may be sufficient to prevent transplant-associated vasculitis and may significantly modulate graft rejection. In the second, donor B cells are treated ex vivo with ribozymes or ribozyme expression vectors. Recipients would receive the treatment prior to transplant. Treatment of a recipient with B cells that do not express T cell co-stimulatory molecules (such as ICAM-1 , VCAM-1 , and/or B7 an B7-2) can induce antigen-specific anergy. Tolerance to the donor's histocompatibility antigens could result; potentially, any donor could be used for any transplantation procedure. •Asthma.

Granulocyte macrophage colony stimulating factor (GM-CSF) is thought to play a major role in recruitment of eosinophils and other inflammatory cells during the late phase reaction to asthmatic trauma. Again, blocking the local induction of GM-CSF and other inflammatory mediators is likely to reduce the persistent inflammation observed in chronic asthmatics. Aerosol delivery of ribozymes or adenovirus ribozyme expression vectors is a feasible treatment.

•Gene Therapy.

Immune responses limit the efficacy of many gene transfer techniques. Cells transfected with retrovirus vectors have short lifetimes in immune competent individuals. The length of expression of adenovirus vectors in terminally differentiated cells is longer in neonatal or immunecompromised animals. Insertion of a small ribozyme expression cassette that modulates inflammatory and immune responses into existing adenovirus or retrovirus constructs will greatly enhance their potential.

Thus, ribozymes of the present invention that cleave rel A mRNA and thereby NF-κB activity have many potential therapeutic uses, and there are reasonable modes of delivering the ribozymes in a number of the possible indications. Development of an effective ribozyme that inhibits NF-κB function is described above; available cellular and activity assays are number, reproducible, and accurate. Animal models for NF-κB function (Kitajima, et al., supra) and for each of the suggested disease targets exist and can be used to optimize activity. Example 4: TNF-α

Ribozymes that cleave the specific cites in TNF-α mRNA represent a novel therapeutic approach to inflammatory or autoimmune disorders.

Tumor necrosis factor-α (TNF-α) is a protein, secreted by activated leukocytes, that is a potent mediator of inflammatory reactions. Injection of TNF-α into experimental animals can simulate the symptoms of systemic and local inflammatory diseases such as septic shock or rheumatoid arthritis.

TNF-α was initially described as a factor secreted by activated macrophages which mediates the destruction of solid tumors in mice (Old, 1985 Science 230, 4225-4231). TNF-α subsequently was found to be identical to cachectin, an agent responsible for the weight loss and wasting syndrome associated with tumors and chronic infections (Beutler, et al., 1985 Nature 316, 552-554). The cDNA and the genomic locus for TNF-α have been cloned and found to be related to TNF-β (Shakhov et al., 1990 J. Exp. Med. 171 , 35-47). Both TNF-α and TNF-β bind to the same receptors and have nearly identical biological activities. The two TNF receptors have been found on most cell types examined (Smith, et al., 1990 Science 248, 1019-1023). TNF-α secretion has been detected from monocytes/macrophages, CD4+ and CD8+ T-cells, B-cells, lymphokine activated killer cells, neutrophils, astrocytes, endothelial cells, smooth muscle cells, as well as various non-hematopoietic tumor cell lines ( for a review see Turestskaya et al., 1991 in Tumor Necrosis Factor: Structure. Function, and Mechanism of Action B. B. Aggarwal, J. Vilcek, Eds. Marcel Dekker, Inc., pp. 35-60). TNF-α is regulated transcriptionally and translationally, and requires proteolytic processing at the plasma membrane in order to be secreted (Kriegler et al., 1988 Cell 53, 45-53). Once secreted, the serum half life of TNF-α is approximately 30 minutes. The tight regulation of TNF-α is important due to the extreme toxicity of this cytokine. Increasing evidence indicates that overproduction of TNF-α during infections can lead to severe systemic toxicity and death (Tracey & Cerami, 1992 Am. J. Trop. Med. Hvα. 47, 2-7).

Antisense RNA and Hammerhead ribozymes have been used in an attempt to lower the expression level of TNF-α by targeting specified cleavage sites [Sioud et al., 1992 J. Mol. Biol. 223; 831 ; Sioud WO 94/10301 ; Kisich and co-workers, 1990 abstract (FASEB J. 4. A1860; 1991 slide presentation (J. Leukocyte Biol. sup. 2, 70); December, 1992 poster presentation at Anti-HIV Therapeutics Conference in SanDiego, CA; and "Development of anti-TNF-α ribozymes for the control of TNF-α gene expression"- Kisich, Doctoral Dissertation, 1993 University of California, Davis] listing various TNFα targeted ribozymes.

Ribozymes of this invention block to some extent TNF-α expression and can be used to treat disease or diagnose such disease. Ribozymes will be delivered to cells in culture and to cells or tissues in animal models of septic shock and rheumatoid arthritis. Ribozyme cleavage of TNF-α mRNA in these systems may prevent inflammatory cell function and alleviate disease symptoms.

The sequence of human and mouse TNF-α mRNA can be screened for accessible sites using a computer folding algorithm. Hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables 23, 25, and 27 - 28. (All sequences are 5' to 3' in the tables.) While mouse and human sequences can be screened and ribozymes thereafter designed, the human targeted sequences are of most utility. However, mouse targeted ribozymes are useful to test efficacy of action of the ribozyme prior to testing in humans. The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme. (In Table 24, lower case letters indicate positions that are not conserved between the human and the mouse TNF-α sequences.)

The sequences of the chemically synthesized ribozymes useful in this study are shown in Tables 24, 26 - 28. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity. For example, stem-loop II sequence of hammerhead ribozymes listed in Tables 24 and 26 (5'-GGCCGAAAGGCC-3') can be altered (substitution, deletion, and/or insertion) to contain any sequences provided a minimum of two base-paired stem structure can form. Similarly, stem-loop IV sequence of hairpin ribozymes listed in Tables 27 and 28 (5'-CACGUUGUG-3') can be altered (substitution, deletion, and/or insertion) to contain any sequence, provided a minimum of two base-paired stem structure can form. The sequences listed in Tables 24, 26 - 28 may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables or AAV .

In a preferred embodiment of the invention, a transcription unit expressing a ribozyme that cleaves TNF-α RNA is inserted into a plasmid

DNA vector or an adenovirus DNA viral vector or AAV or alpha virus or retroviris vectors. Viral vectors have been used to transfer genes to the intact vasculature or to joints of live animals (Willard et al., 1992

Circulation. 86, I-473.; Nabel et al., 1990 Science. 249, 1285-1288) and both vectors lead to transient gene expression. The adenovirus vector is delivered as recombinant adenoviral particles. DNA may be delivered alone or complexed with vehicles (as described for RNA above). The DNA,

DNA/vehicle complexes, or the recombinant adenovirus particles are locally administered to the site of treatment, e.g., through the use of an injection catheter, stent or infusion pump or are directly added to cells or tissues ex vivo.

In another preferred embodiment of the invention, a transcription unit expressing a ribozyme that cleaves TNF-α RNA is inserted into a retrovirus vector for sustained expression of ribozyme(s). By engineering ribozyme motifs we have designed several ribozymes directed against TNF-α mRNA sequences. These ribozymes are synthesized with modifications that improve their nuclease resistance. The ability of ribozymes to cleave TNF-α target sequences in vitro is evaluated.

The ribozymes will be tested for function in cells by analyzing bacterial lipopolysaccharide (LPS)-induced TNF-α expression levels. Ribozymes will be delivered to cells by incorporation into liposomes, by complexing with cationic lipids, by microinjection, or by expression from DNA vectors. TNF-α expression will be monitored by ELISA, by indirect immunofluoresence, and/or by FACS analysis. TNF-α mRNA levels will be assessed by Northern analysis, RNAse protection, primer extension analysis or quantitative RT-PCR. Ribozymes that block the induction of TNF-α activity and/or TNF-α mRNA by more than 90% will be identified.

RNA ribozymes and/or genes encoding them will be locally delivered to macrophages by intraperitoneal injection. After a period of ribozyme uptake, the peritoneal macrophages are harvested and induced ex vivo with LPS. The ribozymes that significantly reduce TNF-α secretion are selected. The TNF-α can also be induced after ribozyme treatment with fixed Streptococcus in the peritoneal cavity instead of ex vivo. In this fashion the ability of TNF-α ribozymes to block TNF-α secretion in a localized inflammatory response are evaluated. In addition, we will determine if the ribozymes can block an ongoing inflammatory response by delivering the TNF-α ribozymes after induction by the injection of fixed Streptococcus.

To examine the effect of anti-TNF-α ribozymes on systemic inflammation, the ribozymes are delivered by intravenous injection. The ability of the ribozymes to inhibit TNF-α secretion and lethal shock caused by systemic LPS administration are assessed. Similarly, TNF-α ribozymes can be introduced into the joints of mice with collagen-induced arthritis.

Either free delivery, liposome delivery, cationic lipid delivery, adeno-associated virus vector delivery, adenovirus vector delivery, retrovirus vector delivery or plasmid vector delivery in these animal model experiments can be used to supply ribozymes. One dose (or a few infrequent doses) of a stable anti-TNF-α ribozyme or a gene construct that constitutively expresses the ribozyme may abrogate tissue damage in these inflammatory diseases.

Macrophage isolation.

To produce responsive macrophages 1 ml of sterile fluid thioglycollate broth (Difco, Detroit, MI.) was injected i.p. into 6 week old female C57bl/6NCR mice 3 days before peritoneal lavage. Mice were maintained as specific pathogen free in autoclaved cages in a laminar flow hood and given sterilized water to minimize "spontaneous" activation of macrophages. The resulting peritoneal exudate cells (PEC) were obtained by lavage using Hanks balanced salt solution (HBSS) and were plated at 2.5X10⁵/well in 96 well plates (Costar, Cambridge, MA.) with Eagles minimal essential medium (EMEM) containing 10% heat inactivated fetal bovine serum. After adhering for 2 hours the wells were washed to remove non-adherent cells. The resulting cultures were 97% macrophages as determined by morphology and staining for non-specific esterase.

Transfection of ribozymes into macrophages: The ribozymes were diluted to 2X final concentration, mixed with an equal volume of 11 nM lipofectamine (Life Technologies, Gaithersburg, MD.), and vortexed. 100 ml of lipid:ribozyme complex was then added directly to the cells, followed immediately by 10 ml fetal bovine serum. Three hours after ribozyme addition 100 ml of 1 mg/ml bacterial lipopolysaccaride (LPS) was added to each well to stimulate TNF production.

Quantitation of TNF-α in mouse macrophages:

Supernatants were sampled at 0, 2, 4, 8, and 24 hours post LPS stimulation and stored at -70°C. Quantitation of TNF-α was done by a specific ELISA. ELISA plates were coated with rabbit anti-mouse TNF-α serum at 1 :1000 dilution (Genzyme) followed by blocking with milk proteins and incubation with TNF-α containing supernatants. TNF-α was then detected using a murine TNF-α specific hamster monoclonal antibody (Genzyme). The ELISA was developed with goat anti-hamster IgG coupled to alkaline phosphatase.

Assessment of reagent toxicity:

Following ribozyme/lipid treatment of macrophages and harvesting of supernatants viability of the cells was assessed by incubation of the cells with 5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). This compound is reduced by the mitochondrial dihydrogenases, the activity of which correlates well with cell viability. After 12 hours the absorbance of reduced MTT is measured at 585 nm.

Uses

The association between TNF-α and bacterial sepsis, rheumatoid arthritis, and autoimmune disease make TNF-α an attractive target for therapeutic intervention [Tracy & Cerami 1992 supra: Williams et al., 1992

Proc. Natl. Acad. Sci. USA 89, 9784-9788; Jacob, 1992 J. Autoimmun. 5

(Supp. A), 133-143], Septic Shock

Septic shock is a complication of major surgery, bacterial infection, and polytrauma characterized by high fever, increased cardiac output, reduced blood pressure and a neutrophilic infiltrate into the lungs and other major organs. Current treatment options are limited to antibiotics to reduce the bacterial load and non-steroidal anti-inflammatories to reduce fever. Despite these treatments in the best intensive care settings, mortality from septic shock averages 50%, due primarily to multiple organ failure and disseminated vascular coagulation. Septic shock, with an incidence of 200,000 cases per year in the United States, is the major cause of death in intensive care units. In septic shock syndrome, tissue injury or bacterial products initiate massive immune activation, resulting in the secretion of pro-inflammatory cytokines which are not normally detected in the serum, such as TNF-α, interleukin-1 β (IL-1β), γ-interferon (IFN-γ), interleukin-6 (IL-6), and interleukin-8 (IL-8). Other non-cytokine mediators such as leukotriene b4, prostaglandin E2, C3a and C3d also reach high levels (de Boer et al., 1992 Immunopharmacology 24, 135-148).

TNF-α is detected early in the course of septic shock in a large fraction of patients (de Boer et al., 1992 supra). In animal models, injection of TNF-α has been shown to induce shock-like symptoms similar to those induced by LPS injection (Beutler et al., 1985 Science 229, 869-871); in contrast, injection of IL-1β, IL-6, or IL-8 does not induce shock. Injection of TNF-α also causes an elevation of IL-1β, IL-6, IL-8, PgE2, acute phase proteins, and TxA2 in the serum of experimental animals (de Boer et al., 1992 supra). In animal models the lethal effects of LPS can be blocked by preadministration of anti-TNF-α antibodies. The cumulative evidence indicates that TNF-α is a key player in the pathogenesis of septic shock, and therefore a good candidate for therapeutic intervention.

Rheumatoid Arthritis Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joints leading to bone destruction and loss of joint function. At the cellular level, autoreactive T- lymphocytes and monocytes are typically present, and the synoviocytes often have altered morphology and immunostaining patterns. RA joints have been shown to contain elevated levels of TNF-α, IL-1α and IL-1β, IL-6, GM-CSF, and TGF- β (Abney et al., 1991 Imm. Rev. 119, 105-123), some or all of which may contribute to the pathological course of the disease.

Cells cultured from RA joints spontaneously secrete all of the proinflammatory cytokines detected in vivo. Addition of antisera against TNF-α to these cultures has been shown to reduce IL-1α/β production by these cells to undetectable levels (Abney et al., 1991 Supra). Thus, TNF-α may directly induce the production of other cytokines in the RA joint. Addition of the anti-inflammatory cytokine, TGF-β, has no effect on cytokine secretion by RA cultures. Immunocytochemical studies of human RA surgical specimens clearly demonstrate the production of TNF-α, IL-1α/β, and IL-6 from macrophages near the cartilage/pannus junction when the pannus in invading and overgrowing the cartilage (Chu et al., 1992 Br. J. Rheumatology 31 , 653-661). GM-CSF was shown to be produced mainly by vascular endothelium in these samples. Both TNF-α and TGF-β have been shown to be fibroblast growth factors, and may contribute to the accumulation of scar tissue in the RA joint. TNF-α has also been shown to increase osteoclast activity and bone resorbtion, and may have a role in the bone erosion commonly found in the RA joint (Cooper et al., 1992 Clin. Exp. Immunol. 89, 244-250). Elimination of TNF-α from the rheumatic joint would be predicted to reduce overall inflammation by reducing induction of MHC class II, IL-1α/β, II-6, and GM-CSF, and reducing T-cell activation. Osteoclast activity might also fall, reducing the rate of bone erosion at the joint. Finally, elimination of TNF-α would be expected to reduce accumulation of scar tissue within the joint by removal of a fibroblast growth factor.

Treatment with an anti-TNF-α antibody reduces joint swelling and the histological severity of collagen-induced arthritis in mice (Williams et al., 1992 Proc. Natl. Acad. Sci. USA 89, 9784-9788). In addition, a study of RA patients who have received i.v. infusions of anti-TNF-α monoclonal antibody reports a reduction in the number and severity of inflamed joints after treatment. The benefit of monoclonal antibody treatment in the long term may be limited by the expense and immunogenicity of the antibody.

Psoriasis

Psoriasis is an inflammatory disorder of the skin characterized by keratinocyte hyperproliferation and immune cell infiltrate (Kupper, 1990 J. Clin. Invest. 86, 1783-1789). It is a fairly common condition, affecting 1.5-2.0% of the population. The disorder ranges in severity from mild, with small flaky patches of skin, to severe, involving inflammation of the entire epidermis. The cellular infiltrate of psoriasis includes T-lymphocytes, neutrophils, macrophages, and dermal dendrocytes. The majority of T-lymphocytes are activated CD4+ cells of the T_H-1 phenotype, although some CD8+ and CD4-/CD8- are also present. B lymphocytes are typically not found in abundance in psoriatic plaques.

Numerous hypotheses have been offered as to the proximal cause of psoriasis including auto-antibodies and auto-reactive T-cells, overproduction of growth factors, and genetic predisposition. Although there is evidence to support the involvement of each of these factors in psoriasis, they are neither mutually exclusive nor are any of them necessary and sufficient for the pathogenesis of psoriasis (Reeves, 1991 Semin. Dermatol. 10, 217).

The role of cytokines in the pathogenesis of psoriasis has been investigated. Among those cytokines found to be abnormally expressed were TGF-α , IL-1α, IL-1 β, IL-1 ra, IL-6, IL-8, IFN-γ, and TNF-α . In addition to abnormal cytokine production, elevated expression of ICAM-1 , ELAM-1 , and VCAM has been observed (Reeves, 1991 supra). This cytokine profile is similar to that of normal wound healing, with the notable exception that cytokine levels subside upon healing. Keratinocytes themselves have recently been shown to be capable of secreting EGF, TGF-α, IL-6, and TNF-α, which could increase proliferation in an autocrine fashion (Oxholm et al., 1991 APMIS 99, 58-64).

Nickoloff et al., 1993 (J Dermatol Sci. 6, 127-33) have proposed the following model for the initiation and maintenance of the psoriatic plaque:

Tissue damage induces the wound healing response in the skin. Keratinocytes secrete IL-1 α, IL-1 β, IL-6, IL-8, TNF-α. These factors activate the endothelium of dermal capillaries, recruiting PMNs, macrophages, and T-cells into the wound site.

Dermal dendrocytes near the dermal/epidermal junction remain activated when they should return to a quiescent state, and subsequently secrete cytokines including TNF-α, IL-6, and IL-8. Cytokine expression, in turn, maintains the activated state of the endothelium, allowing extravasation of additional immunocytes, and the activated state of the keratinocytes which secrete TGF-α and IL-8. Keratinocyte IL-8 recruits immunocytes from the dermis into the epidermis. During passage through the dermis, T-cells encounter the activated dermal dendrocytes which efficiently activate the T_H-1 phenotype. The activated T-cells continue to migrate into the epidermis, where they are stimulated by keratinocyte-expressed ICAM-1 and MHC class II. IFN-γ secreted by the T-cells synergizes with the TNF-α from dermal dendrocytes to increase keratinocyte proliferation and the levels of TGF-α, IL-8, and IL-6 production. IFN-γ also feeds back to the dermal dendrocyte, maintaining the activated phenotype and the inflammatory cycle.

Elevated serum titres of IL-6 increases synthesis of acute phase proteins including complement factors by the liver, and antibody production by plasma cells. Increased complement and antibody levels increases the probability of autoimmune reactions.

Maintenance of the psoriatic plaque requires continued expression of all of these processes, but attractive points of therapeutic intervention are

TN F-α expression by the dermal dendrocyte to maintain activated endothelium and keratinocytes, and IFN-γ expression by T-cells to maintain activated dermal dendrocytes.

There are 3 million patients in the United States afflicted with psoriasis. The available treatments for psoriasis are corticosteroids. The most widely prescribed are TEMOVATE (clobetasol propionate), LIDEX (fluocinonide), DIPROLENE (betamethasone propionate), PSORCON (diflorasone diacetate) and TRIAMCINOLONE formulated for topical application. The mechanism of action of corticosteroids is multifactorial. This is a palliative therapy because the underlying cause of the disease remains, and upon discontinuation of the treatment the disease returns. Discontinuation of treatment is often prompted by the appearance of adverse effects such as atrophy, telangiectasias and purpura. Corticosteroids are not recommended for prolonged treatments or when treatment of large and/or inflamed areas is required. Alternative treatments include retinoids, such as etretinate, which has been approved for treatment of severe, refractory psoriasis. Alternative retinoid-based treatments are in advanced clinical trials. Retinoids act by converting keratinocytes to a differentiated state and restoration of normal skin development. Immunosuppressive drugs such as cyclosporine are also in the advanced stages of clinical trials. Due to the nonspecific mechanism of action of corticosteroids, retinoids and immunosuppressives, these treatments exhibit severe side effects and should not be used for extended periods of time unless the condition is life-threatening or disabling. There is a need for a less toxic, effective therapeutic agent in psoriatic patients.

HIV and AIDS

The human immunodeficiency virus (HIV) causes several fundamental changes in the human immune system from the time of infection until the development of full-blown acquired immunodeficiency syndrome (AIDS). These changes include a shift in the ratio of CD4+ to

CD8+ T-cells, sustained elevation of IL-4 levels, episodic elevation of TNF-α and TNF-β levels, hypergammaglobulinemia, and lymphoma/leukemia (Rosenberg & Fauci, 1990 Immun. Today 11 , 176; Weiss 1993 Science

260, 1273). Many patients experience a unique tumor, Kaposi's sarcoma and/or unusual opportunistic infections (e.g. Pneumocystis carinii, cytomegalovirus, herpesviruses, hepatitis viruses, papilloma viruses, and tuberculosis). The immunological dysfunction of individuals with AIDS suggests that some of the pathology may be due to cytokine dysregulation.

Levels of serum TNF-α and IL-6 are often found to be elevated in AIDS patients (Weiss, 1993 supra). In tissue culture, HIV infection of monocytes isolated from healthy individuals stimulates secretion of both TNF-α and IL-6. This response has been reproduced using purified gp120, the viral coat protein responsible for binding to CD-4 (Buonaguro et al., 1992 J. Virol. 66, 7159). It has also been demonstrated that the viral gene regulator, Tat, can directly induce TNF transcription. The ability of HIV to directly stimulate secretion of TNF-α and IL-6 may be an adaptive mechanism of the virus. TNF-α has been shown to upregulate transcription of the LTR of HIV, increasing the number of HIV-specific transcripts in infected cells. IL-6 enhances HIV production, but at a post-transcriptional level, apparently increasing the efficiency with which HIV transcripts are translated into protein. Thus, stimulation of TNF-α secretion by the HIV virus may promote infection of neighboring CD4+ cells both by enhancing virus production from latently infected cells and by driving replication of the virus in newly infected cells. The role of TNF-α in HIV replication has been well established in tissue culture models of infection (Sher et al., 1992 Immun. Rev. 127, 183), suggesting that the mutual induction of HIV replication and TNF-α replication may create positive feedback in vivo. However, evidence for the presence of such positive feedback in infected patients is not abundant. TNF-α levels are found to be elevated in some, but not all patients tested. Children with AIDS who were given zidovudine had reduced levels of TNF-α compared to those not given zidovudine (Cremoni et al., 1993 AIPS 7, 128). This correlation lends support to the hypothesis that reduced viral replication is physiologically linked to TNF-α levels. Furthermore, recently it has been shown that the polyclonal B cell activation associated with HIV infection is due to membrane-bound TNF-α. Thus, levels of secreted TNF-α may not accurately reflect the contribution of this cytokine to AIDS pathogenesis. Chronic elevation of TNF-α has been shown to shown to result in cachexia (Tracey et al., 1992 Am. J. Trop. Med. Hyg. 47, 2-7), increased autoimmune disease (Jacob, 1992 supra), lethargy, and immune suppression in animal models (Aderka et al., 1992 Isr. J. Med. Sci. 28, 126-130). The cachexia associated with AIDS may be associated with chronically elevated TNF-α frequently observed in AIDS patients. Similarly, TNF-α can stimulate the proliferation of spindle cells isolated from Kaposi's sarcoma lesions of AIDS patients (Barillari et al., 1992 J Immunol 149, 3727).

A therapeutic agent that inhibits cytokine gene expression, inhibits adhesion molecule expression, and mimics the anti-inflammatory effects of glucocorticoids (without inducing steroid-responsive genes) is ideal for the treatment of inflammatory and autoimmune disorders. Disease targets for such a drug are numerous. Target indications and the delivery options each entails are summarized below. In all cases, because of the potential immunosuppressive properties of a ribozyme that cleaves the specified sites in TNF-α mRNA, uses are limited to local delivery, acute indications, or ex vivo treatment.

•Septic shock. Exogenous delivery of ribozymes to macrophages can be achieved by intraperitoneal or intravenous injections. Ribozymes will be delivered by incorporation into liposomes or by complexing with cationic lipids.

•Rheumatoid arthritis (RA). Pue to the chronic nature of RA, a gene therapy approach is logical.

Pelivery of a ribozyme to inflamed joints is mediated by adenovirus, retrovirus, or adeno-associated virus vectors. For instance, the appropriate adenovirus vector can be administered by direct injection into the synovium: high efficiency of gene transfer and expression for several months would be expected (B.J. Roessler, E.P. Allen, J.M. Wilson, J.W. Hartman, B. L. Davidson, J. Clin. Invest. 92, 1085-1092 (1993)). It is unlikely that the course of the disease could be reversed by the transient, local administration of an anti-inflammatory agent. Multiple administrations may be necessary. Retrovirus and adeno-associated virus vectors would lead to permanent gene transfer and expression in the joint. However, permanent expression of a potent anti-inflammatory agent may lead to local immune deficiency.

•Psoriasis

The psoriatic plaque is a particularly good candidate for ribozyme or vector delivery. The stratum corneum of the plaque is thinned, providing access to the proliferating keratinocytes. T-cells and dermal dendrocytes can be efficiently targeted by trans-epidermal diffusion .

Organ culture systems for biopsy specimens of psoriatic and normal skin are described in current literature (Nickoloff et al., 1993 Supra). Primary human keratinocytes are easily obtained and will be grown into epidermal sheets in tissue culture. In addition to these tissue culture models, the flaky skin mouse develops psoriatic skin in response to UV light. This model would allow demonstration of animal efficacy for ribozyme treatments of psoriasis. «Gene Therapy.

Immune responses limit the efficacy of many gene transfer techniques. Cells transfected with retrovirus vectors have short lifetimes in immune competent individuals. The length of expression of adenovirus vectors in terminally differentiated cells is longer in neonatal or immunecompromised animals. Insertion of a small ribozyme expression cassette that modulates inflammatory and immune responses into existing adenovirus or retrovirus constructs will greatly enhance their potential. Thus, ribozymes of the present invention that cleave TNF-α mRNA and thereby TNF-α activity have many potential therapeutic uses, and there are reasonable modes of delivering the ribozymes in a number of the possible indications. Development of an effective ribozyme that inhibits TNF-α function is described above; available cellular and activity assays are number, reproducible, and accurate. Animal models for TNF-α function and for each of the suggested disease targets exist and can be used to optimize activity.

Example 5: p210^bcr-abl

Chronic myelogenous leukemia exhibits a characteristic disease course, presenting initially as a chronic granulocytic hyperplasia, and invariably evolving into an acute leukemia which is caused by the clonal expansion of a cell with a less differentiated phenotype (i.e.. the blast crisis stage of the disease). CML is an unstable disease which ultimately progresses to a terminal stage which resembles acute leukemia. This lethal disease affects approximately 16,000 patients a year.

Chemotherapeutic agents such as hydroxyurea or busulfan can reduce the leukemic burden but do not impact the life expectancy of the patient (e.g. approximately 4 years). Consequently, CML patients are candidates for bone marrow transplantation (BMT) therapy. However, for those patients which survive BMT, disease recurrence remains a major obstacle

(Apperley et al., 1988 Br. J. Haematol. 69, 239).

The Philadelphia (Ph) chromosome which results from the translocation of the abl oncogene from chromosome 9 to the bcr gene on chromosome 22 is found in greater than 95% of CML patients and in 10-25% of all cases of acute lymphoblastic leukemia [(ALL); Fourth International Workshop on Chromosomes in Leukemia 1982, Cancer Genet. Cytogenet. 11 , 316]. In virtually all Ph-positive CMLs and approximately 50% of the Ph-positive ALLs, the leukemic cells express bcr-abl fusion mRNAs in which exon 2 (b2-a2 junction) or exon 3 (b3-a2 junction) from the major breakpoint cluster region of the bcr gene is spliced to exon 2 of the abl gene. Heisterkamp et al., 1985 Nature 315, 758; Shtivelman et al., 1987, Blood 69, 971). In the remaining cases of Ph-positive ALL, the first exon of the bcr gene is spliced to exon 2 of the abl gene (Hooberman et al., 1989 Proc. Nat. Acad. Sci. USA 86, 4259; Heisterkamp et al., 1988 Nucleic Acids Res. 16, 10069).

The b3-a2 and b2-a2 fusion mRNAs encode 210 kd bcr-abl fusion proteins which exhibit oncogenic activity (Daley et al., 1990 Science 247, 824; Heisterkamp et al., 1990 Nature 344, 251). The importance of the bcr-abl fusion protein (p210^{bcr-a bl}) in the evolution and maintenance of the leukemic phenotype in human disease has been demonstrated using antisense oligonucleotide inhibition of p210^{bcr-a bl} expression. These inhibitory molecules have been shown to inhibit the in vitro proliferation of leukemic cells in bone marrow from CML patients. Szczylik et al., 1991 Science 253, 562). Reddy, U.S. Patent 5,246,921 (hereby incorporated by reference herein) describes use of ribozymes as therapeutic agents for leukemias, such as chronic myelogenous leukemia (CML) by targeting the specific junction region of bcr-abl fusion transcripts. It indicates causing cleavage by a ribozyme at or near the breakpoint of such a hybrid chromosome, specifically it includes cleavage at the sequence GUX, where X is A, U or G. The one example presented is to cleave the sequence 5' AGC AG AGUU (cleavage site) CAA AAGCCCU-3'.

Scanlon WO 91/18625, WO 91/18624, and WO 91/18913 and Snyder et al., WO93/03141 and WO94/13793 describe a ribozyme effective to cleave oncogenic variants of H-ras RNA. This ribozyme is said to inhibit H-ras expression in response to external stimuli.

The invention features use of ribozymes to inhibit the development or expression of a transformed phenotype in man and other animals by modulating expression of a gene that contributes to the expression of CML. Cleavage of targeted mRNAs expressed in pre-neoplastic and transformed cells elicits inhibition of the transformed state.

The invention can be used to treat cancer or pre-neoplastic conditions. Two preferred administration protocols can be used, either in vivo administration to reduce the tumor burden, or ex vivo treatment to eradicate transformed cells from tissues such as bone marrow prior to reimplantation.

This invention features an enzymatic RNA molecule (or ribozyme) which cleaves mRNA associated with development or maintenance of CML. The mRNA targets are present in the 425 nucleotides surrounding the fusion sites of the bcr and abl sequences in the b2-a2 and b3-a2 recombinant mRNAs. Other sequences in the 5' portion of the bcr mRNA or the 3' portion of the abl mRNA may also be targeted for ribozyme cleavage. Cleavage at any of these sites in the fusion mRNA molecules will result in inhibition of translation of the fusion protein in treated cells.

The invention provides a class of chemical cleaving agents which exhibit a high degree of specificity for the mRNA causative of CML. Such enzymatic RNA molecules can be delivered exogenously or endogenously to afflicted cells. In the preferred hammerhead motif the small size (less than 40 nucleotides, preferably between 32 and 36 nucleotides in length) of the molecule allows the cost of treatment to be reduced.

The smallest ribozyme delivered for any type of treatment reported to date (by Rossi et al.. 1992 supra) is an |n yjtro transcript having a length of 142 nucleotides. Synthesis of ribozymes greater than 100 nucleotides in length is very difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. Delivery of ribozymes by expression vectors is primarily feasible using only ex vivo treatments. This limits the utility of this approach. In this invention, an alternative approach uses smaller ribozyme motifs and exogenous delivery. The simple structure of these molecules also increases the ability of the ribozyme to invade targeted regions of the mRNA structure. Thus, unlike the situation when the hammerhead structure is included within longer transcripts, there are no non-ribozyme flanking sequences to interfere with correct folding of the ribozyme structure, as well as complementary binding of the ribozyme to the mRNA target.

The enzymatic RNA molecules of this invention can be used to treat human CML or precancerous conditions. Affected animals can be treated at the time of cancer detection or in a prophylactic manner. This timing of treatment will reduce the number of affected cells and disable cellular replication. This is possible because the ribozymes are designed to disable those structures required for successful cellular proliferation.

Ribozymes of this invention block to some extent p210^bcr-abl expression and can be used to treat disease or diagnose such disease. Ribozymes will be delivered to cells in culture and to tissues in animal models of CML. Ribozyme cleavage of bcr/abl mRNA in these systems may prevent or alleviate disease symptoms or conditions.

The sequence of human bcr/abl mRNA can be screened for accessible sites using a computer folding algorithm. Regions of the mRNA that did not form secondary folding structures and that contain potential hammerhead or hairpin ribozyme cleavage sites can be identified. These sites are shown in Table 29 (All sequences are 5' to 3' in the tables). The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme. The sequences of the chemically synthesized ribozymes most useful in this study are shown in Table 30. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity. For example, stem-loop II sequence of hammerhead ribozymes listed in Table 30 (5'-GGCCGAAAGGCC-3') can be altered (substitution, deletion, and/or insertion) to contain any sequence provided, a minimum of two base-paired stem structure can form. The sequences listed in Tables 30 may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables.

By engineering ribozyme motifs we have designed several ribozymes directed against bcr-abl mRNA sequences. These have been synthesized with modifications that improve their nuclease resistance as described above. These ribozymes cleave bcr-abl target sequences in vitro. The ribozymes are tested for function in vivo by exogenous delivery to cells expressing bcr-abl. Ribozymes are delivered by incorporation into liposomes, by complexing with cationic lipids, by microinjection, or by expression from DNA vectors. Expression of bcr-abl is monitored by ELISA, by indirect immunofluoresence, and/or by FACS analysis. Levels of bcr-abl mRNA are assessed by Northern analysis, RNase protection, by primer extension analysis or by quantitative RT-PCR techniques. Ribozymes that block the induction of p210^bcr-abl) protein and mRNA by more than 20% are identified. Example 6: RSV

This invention relates to the use of ribozymes as inhibitors of respiratory syncytial virus (RSV) production, and in particular, the inhibition of RSV replication.

RSV is a member of the virus family paramyxoviridae and is classified under the genus Pneumovirus (for a review see McIntosh and Chanock, 1990 in Virology ed. B.N. Fields, pp. 1045, Raven Press Ltd. NY). The infectious virus particle is composed of a nucleocapsid enclosed within an envelope. The nucleocapsid is composed of a linear negative singlestranded non-segmented RNA associated with repeating subunits of capsid proteins to form a compact structure and thereby protect the RNA from nuclease degradation. The entire nucleocapsid is enclosed by the envelope. The size of the virus particle ranges from 150 - 300 nm in diameter. The complete life cycle of RSV takes place in the cytoplasm of infected cells and the nucleocapsid never reaches the nuclear compartment (Hall, 1990 in Principles and Practice of Infectious Diseases ed. Mandell et al., Churchill Livingstone, NY).

The RSV genome encodes ten viral proteins essential for viral production. RSV protein products include two structural glycoproteins (G and F) found in the envelope spikes, two matrix proteins [M and M2 (22K)] found in the inner membrane, three proteins localized in the nucleocapsid (N, P and L), one protein that is present on the surface of the infected cell (SH), and two nonstructural proteins [NS1 (1C) and NS2 (1 B)] found only in the infected cell. The mRNAs for the 10 RSV proteins have similar 5' and 3' ends. UV-inactivation studies suggest that a single promoter is used with multiple transcription initiation sites (Barik et al., 1992 J. Virol. 66, 6813). The order of transcription corresponding to the protein assignment on the genomic RNA is 1C, 1B, N, P, M, SH, G, F, 22K and L genes (Huang et al., 1985 Virus Res. 2, 157) and transcript abundance corresponds to the order of gene assignment (for example the 1C and 1B mRNAs are much more abundant than the L mRNA. Synthesis of viral message begins immediately after RSV infection of cells and reaches a maximum at 14 hours post-infection (McIntosh and Chanock, supra).

There are two antigenic subgroups of RSV, A and B, which can circulate simultaneously in the community in varying proportions in different years (McIntosh and Chanock, supra). Subgroup A usually predominates. Within the two subgroups there are numerous strains. By the limited sequence analysis available it seems that homology at the nucleotide level is more complete within than between subgroups, although sequence divergence has been noted within subgroups as well. Antigenic determinates result primarily from both surface glycoproteins, F and G. For F, at least half of the neutralization epitopes have been stably maintained over a period of 30 years. For G however, A and B subgroups may be related antigenically by as little as a few percent. On the nucleotide level, however, the majority of the divergence in the coding region of G is found in the sequence for the extracellular domain (Johnson et al., 1987, Proc. Natl. Acad. Sci. USA 84, 5625).

Respiratory Syncytial Virus (RSV) is the major cause of lower respiratory tract illness during infancy and childhood (Hall, supra) and as such is associated with an estimated 90,000 hospitalizations and 4500 deaths in the United States alone (Update: respiratory syncytial virus activity - United States, 1993, Mmwr Morb Mortal Wkly Rep, 42, 971). Infection with RSV generally outranks all other microbial agents leading to both pneumonia and bronchitis. While primarily affecting children under two years of age, immunity is not complete and reinfection of older children and adults, especially hospital care givers (McIntosh and Chanock, supra), is not uncommon. Immunocompromised patients are severely affected and RSV infection is a major complication for patients undergoing bone marrow transplantation .

Uneventftl RSV respiratory disease resembles a common cold and recovery is in 7 to 12 days. Initial symptoms (rhinorrhea, nasal congestion, slight fever, etc.) are followed in 1 to 3 days by lower respiratory tract signs of infection that include a cough and wheezing. In severe cases, these mild symptoms quickly progress to tachypnea, cyanosis, and listlessness and hospitalization is required. In infants with underlying cardiac or respiratory disease, the progression of symptoms is especially rapid and can lead to respiratory failure by the second or third day of illness. With modem intensive care however, overall mortality is usually less than 5% of hospitalized patients (McIntosh and Chanock, supra).

At present, neither an efficient vaccine nor a specific antiviral agent is available. An immune response to the viral surface glycoproteins can provide resistance to RSV in a number of experimental animals, and a subunit vaccine has been shown to be effective for up to 6 months in children previously hospitalized with an RSV infection (Tristam et al., 1993, J. Infect. Dis. 167, 191). An attenuated bovine RSV vaccine has also been shown to be effective in calves for a similar length of time (Kubota et al., 1992 J. Vet. Med. Sci. 54, 957). Previously however, a formalin-inactivated RSV vaccine was implicated in greater frequency of severe disease in subsequent natural infections with RSV (Connors et al., 1992 J. Virol. 66, 7444).

The current treatment for RSV infection requiring hospitalization is the use of aerosolized ribavirin, a guanosine analog [Antiviral Agents and Viral Diseases of Man, 3rd edition. 1990. (eds. G.J. Galasso, R.J. Whitley, and T.C. Merigan) Raven Press Ltd., NY.]. Ribavirin therapy is associated with a decrease in the severity of the symptoms, improved arterial oxygen and a decrease in the amount of viral shedding at the end of the treatment period. It is not certain, however, whether ribavirin therapy actually shortens the patients' hospital stay or diminishes the need for supportive therapies (McIntosh and Chanock, supra). The benefits of ribavirin therapy are especially clear for high risk infants, those with the most serious symptoms or for patients with underlying bronchopulmonary or cardiac disease. Inhibition of the viral polymerase complex is supported as the main mechanism for inhibition of RSV by ribavirin, since viral but not cellular polypeptide synthesis is inhibited by ribavirin in RSV-infected cells (Antiviral Agents and Viral Diseases of Man, 3rd edition. 1990. (eds. G.J. Galasso, R.J. Whitley, and T.C. Merigan) Raven Press Ltd., NY]. Since ribavirin is at least partially effective against RSV infection when delivered by aerosolization, it can be assumed that the target cells are at or near the epithelial surface. In this regard, RSV antigen had not spread any deeper than the superficial layers of the respiratory epithelium in autopsy studies of fatal pneumonia (McIntosh and Chanock, supra). Jennings et al., WO 94/13688 indicates that targets for specific types of ribozymes include respiratory syncytical virus. The invention features novel enzymatic RNA molecules, or ribozymes, and methods for their use for inhibiting production of respiratory syncytial virus (RSV). Such ribozymes can be used in a method for treatment of diseases caused by these related viruses in man and other animals. The invention also features cleavage of the genomic RNA and mRNA of these viruses by use of ribozymes. In particular, the ribozyme molecules described are targeted to the NS1 (1C), NS2 (1B) and N viral genes. These genes are known in the art (for a review see McIntosh and Chanock, 1990 supra ). Ribozymes that cleave the specified sites in RSV mRΝAs represent a novel therapeutic approach to respiratory disorders. Applicant indicates that ribozymes are able to inhibit the activity of RSV and that the catalytic activity of the ribozymes is required for their inhibitory effect. Those of ordinary skill in the art, will find that it is clear from the examples described that other ribozymes that cleave these sites in RSV mRΝAs encoding 1C, 1 B and Ν proteins may be readily designed and are within the invention. Also, those of ordinary skill in the art, will find that it is clear from the examples described that ribozymes cleaving other mRΝAs encoded by RSV (P, M, SH, G, F, 22K and L) and the genomic RΝA may be readily designed and are within the invention.

In preferred embodiments, the ribozymes have binding arms which are complementary to the sequences in Tables 31 , 33, 35, 37 and 38. Examples of such ribozymes are shown in Tables 32, 34, 36-38. Examples of such ribozymes consist essentially of sequences defined in these Tables. By "consists essentially of" is meant that the active ribozyme contains an enzymatic center equivalent to those in the examples, and binding arms able to bind mRΝA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage. Ribozymes of this invention block to some extent RSV production and can be used to treat disease or diagnose such disease. Ribozymes will be delivered to cells in culture and to cells or tissues in animal models of respiratory disorders. Ribozyme cleavage of RSV encoded mRΝAs or the genomic RΝA in these systems may alleviate disease symptoms. While all ten RSV encoded proteins (1C, 1B, N, P, M, SH, 22K, F, G, and L) are essential for viral life cycle and are all potential targets for ribozyme cleavage, certain proteins (mRNAs) are more favorable for ribozyme targeting than the others. For example RSV encoded proteins 1C, 1 B, SH and 22K are not found in other members of the family paramyxoviridae and appear to be unique to RSV. In contrast the ectodomain of the G protein and the signal sequence of the F protein show significant sequence divergence at the nucleotide level among various RSV sub-groups (Johnson et al., 1987 supra). RSV proteins 1C, 1 B and N are highly conserved among various subtypes at both the nucleotide and amino acid levels. Also, 1C, 1B and N are the most abundant of all RSV proteins.

The sequence of human RSV mRNAs encoding 1C, 1 B and N proteins are screened for accessible sites using a computer folding algorithm. Hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables 31 , 33, 34, 37 and 38 (All sequences are 5' to 3' in the tables.) The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme. Ribozymes of the hammerhead or hairpin motif are designed to anneal to various sites in the mRNA message. The binding arms are complementary to the target site sequences described above. The ribozymes are chemically synthesized. The method of synthesis used follows the procedure for normal RNA synthesis as described in Usman et al., 1987 J. Am. Chem. Soc, 109, 7845-7854 and in Scaringe et al., 1990 Nucleic Acids Res., 18, 5433-5441 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. The average stepwise coupling yields were >98%. Inactive ribozymes were synthesized by substituting a U for G5 and a U for A14 (numbering from Hertel et al., 1992 Nucleic Acids Res., 20, 3252). Hairpin ribozymes are synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-2840). Hairpin ribozymes are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). All ribozymes are modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-o-methyl, 2'-H (for a review see Usman and Cedergren, 1992 TIBS 17, 34). Ribozymes are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography and are resuspended in water. The sequences of the chemically synthesized ribozymes useful in this study are shown in Tables 32, 34, 36, 37 and 38. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity. For example, stem-loop II sequence of hammerhead ribozymes listed in Tables 32 and 34(5'-GGCCGAAAGGCC-3') can be altered (substitution, deletion, and/or insertion) to contain any sequences provided a minimum of two base-paired stem structure can form. Similarly, stem-loop IV sequence of hairpin ribozymes listed in Tables 37 and 38 (5'-CACGUUGUG-3') can be altered (substitution, deletion, and/or insertion) to contain any sequence, provided a minimum of two base-paired stem structure can form. The sequences listed in Tables 32, 34, 36, 37 and 38 may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables. By engineering ribozyme motifs we have designed several ribozymes directed against RSV encoded mRNA sequences. These ribozymes are synthesized with modifications that improve their nuclease resistance. The ability of ribozymes to cleave target sequences in vitro is evaluated.

Numerous, common cell lines can be infected with RSV for experimental purposes. These include HeLa, Vero and several primary epithelial cell lines. A cotton rat animal model of experimental human RSV infection is also available, and the bovine RSV is quite homologous to the human viruses. Rapid clinical diagnosis is through the use of kits designed for the immunofluorescence staining of RSV-infected cells or an ELISA assay, both of which are adaptable for experimental study. RSV encoded mRNA levels will be assessed by Northern analysis, RNAse protection, primer extension analysis or quantitative RT-PCR. Ribozymes that block the induction of RSV activity and/or 1C, 1 B and N protein encoding mRNAs by more than 90% will be identified. Optimizing Ribozyme Activity

Ribozyme activity can be optimized as described by Draper et al., PCT WO93/23569. The details will not be repeated here, but include altering the length of the ribozyme binding arms or chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991 Science 253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162, as well as Jennings et al., WO 94/13688, which describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules. All these publications are hereby incorporated by reference herein.), modifications which enhance their efficacy in cells, and removal of stem II bases to shorten RNA synthesis times and reduce chemical requirements.

Sullivan, et al., PCT WO94/02595, incorporated by reference herein, describes the general methods for delivery of enzymatic RNA molecules . Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. The RNA/vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stent. Alternative routes of delivery include, but are not limited to, intravenous injection, intramuscular injection, subcutaneous injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Sullivan, et al., supra and Draper, et al., supra which have been incorporated by reference herein.

Another means of accumulating high concentrations of a ribozyme(s) within cells is to incorporate the ribozyme-encoding sequences into a DNA expression vector. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990 Proc. Natl. Acad. Sci. U S A. 87, 6743-7: Gao and Huang 1993 Nucleic Acids Res., 21 , 2867-72; Lieber et al., 1993 Methods Enzymol., 217, 47-66; Zhou et al., 1990 Mol. Cell. Biol.. 10, 4529-37). Several investigators have demonstrated that ribozymes expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992 Antisense Res. Dev., 2, 3-15; Ojwang et al.. 1992 Proc. Natl. Acad. Sci. U S A. 89, 10802-6; Chen et al., 1992 Nucleic Acids Res., 20, 4581-9; Yu et al., 1993 Proc. Natl. Acad. Sci. U S A. 90, 6340-4; L'Huillier et al., 1992 EMBO J. 11. 4411-8; Lisziewicz et al., 1993 Proc. Natl. Acad. Sci. U. S. A., 90, 8000-4). The above ribozyme transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral, or alpha virus vectors).

In a preferred embodiment of the invention, a transcription unit expressing a ribozyme that cleaves target RNA is inserted into a plasmid

DNA vector, a retrovirus DNA viral vector, an adenovirus DNA viral vector or an adeno-associated virus vector or alpha virus vector. These and other vectors have been used to transfer genes to live animals (for a review see

Friedman, 1989 Science 244, 1275-1281 ; Roemer and Friedman, 1992 Eur. J. Biochem. 208, 211-225) and leads to transient or stable gene expression. The vectors are delivered as recombinant viral particles. DNA may be delivered alone or complexed with vehicles (as described for RNA above). The DNA, DNA/vehicle complexes, or the recombinant virus particles are locally administered to the site of treatment, e.g., through the use of a catheter, stent or infusion pump.

Diagnostic uses

Ribozymes of this invention may be used as diagnostic tools to examine genetic drift and mutations within diseased cells. The close relationship between ribozyme activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple ribozymes described in this invention, one may map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with ribozymes may be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets may be defined as important mediators of the disease. These experiments will lead to better treatment of the disease progression by affording the possibility of combinational therapies (e.g.. multiple ribozymes targeted to different genes, ribozymes coupled with known small molecule inhibitors, or intermittent treatment with combinations of ribozymes and/or other chemical or biological molecules). Other in vitro uses of ribozymes of this invention are well known in the art, and include detection of the presence of mRNA associated with ICAM-1 , relA, TNF-α, p210, ^bcr-abl or RSV related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.

In a specific example, ribozymes which can cleave only wild-type or mutant forms of the target RNA are used for the assay. The first ribozyme is used to identify wild-type RNA present in the sample and the second ribozyme will be used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA will be cleaved by both ribozymes to demonstrate the relative ribozyme efficiencies in the reactions and the absence of cleavage of the "non-targeted" RNA species. The cleavage products from the synthetic substrates will also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus each analysis will require two ribozymes, two substrates and one unknown sample which will be combined into six reactions. The presence of cleavage products will be determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., ICAM-1 , rel A, TNFα, p210^bcr-abl or RSV) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios will be correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.

II. Chemical Synthesis Of Ribozymes

There follows the chemical synthesis, deprotection, and purification of

RNA, enzymatic RNA or modified RNA molecules in greater than milligram quantities with high biological activity. Applicant has determined that the synthesis of enzymatically active RNA in high yield and quantity is dependent upon certain critical steps used during its preparation. Specifically, it is important that the RNA phosphoramidites are coupled efficiently in terms of both yield and time, that correct exocyclic amino protecting groups be used, that the appropriate conditions for the removal of the exocyclic amino protecting groups and the alkylsilyl protecting groups on the 2'-hydroxyl are used, and that the correct work-up and purification procedure of the resulting ribozyme be used.

To obtain a correct synthesis in terms of yield and biological activity of a large RNA molecule (i.e., about 30 to 40 nucleotide bases), the protection of the amino functions of the bases requires either amide or substituted amide protecting groups, which must be, on the one hand, stable enough to survive the conditions of synthesis, and on the other hand, removable at the end of the synthesis. These requirements are met by the amide protecting groups shown in Figure 8, in particular, benzoyl for adenosine, isobutyryl or benzoyl for cytidine, and isobutyryl for guanosine, which may be removed at the end of the synthesis by incubating the RNA in NH₃/EtOH (ethanolic ammonia) for 20 h at 65 °C. In the case of the phenoxyacetyl type protecting groups shown in Figure 8 on guanosine and adenosine and acetyl protecting groups on cytidine, an incubation in ethanolic ammonia for 4 h at 65 °C is used to obtain complete removal of these protecting groups. Removal of the alkylsilyl 2'-hydroxyl protecting groups can be accomplished using a tetrahydrofuran solution of TBAF at room temperature for 8-24 h.

The most quantitative procedure for recovering the fully deprotected

RNA molecule is by either ethanol precipitation, or an anion exchange cartridge desalting, as described in Scaringe et al. Nucleic Acids Res. 1990, 18, 5433-5341. The purification of the long RNA sequences may be accomplished by a two-step chromatographic procedure in which the molecule is first purified on a reverse phase column with either the trityl group at the 5' position on or off. This purification is accomplished using an acetonitrile gradient with triethylammonium or bicarbonate salts as the aqueous phase. In the case of the trityl on purification, the trityl group may be removed by the addition of an acid and drying of the partially purified RNA molecule. The final purification is carried out on an anion exchange column, using alkali metal perchlorate salt gradients to elute the fully purified RNA molecule as the appropriate metal salts, e.g. Na⁺, Li⁺ etc. A final de-salting step on a small reverse-phase cartridge completes the purification procedure. Applicant has found that such a procedure not only fails to adversely affect activity of a ribozyme, but may improve its activity to cleave target RNA molecules.

Applicant has also determined that significant (see Tables 39-41) improvements in the yield of desired full length product (FLP) can be obtained by:

1. Using 5-S-alkyltetrazole at a delivered or effective concentration of 0.25-0.5 M or 0.15-0.35 M for the activation of the RNA (or analogue) amidite during the coupling step. (By delivered is meant that the actual amount of chemical in the reaction mix is known. This is possible for large scale synthesis since the reaction vessel is of size sufficient to allow such manipulations. The term effective means that available amount of chemical actually provided to the reaction mixture that is able to react with the other reagents present in the mixture. Those skilled in the art will recognize the meaning of these terms from the examples provided herein.) The time for this step is shortened from 10-15 m, vide supra, to 5-10 m. Alkyl, as used herein, refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, =O, =S, NO₂ or N(CH₃)₂, amino, or SH. The term also includes alkenyl groups which are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, =O, =S, NO₂, halogen, N(CH₃)₂, amino, or SH. The term "alkyl" also includes alkynyl groups which have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, =O, =S, NO₂ or N(CH₃)₂, amino or SH.

Such alkyl groups may also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An "aryl" group refers to an aromatic group which has at least one ring having a conjugated π electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An "alkylaryl" group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above. Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An "amide" refers to an -C(O)-NH-R, where R is either alkyl, aryl, alkylaryl or hydrogen. An "ester" refers to an -C(O)-OR', where R is either alkyl, aryl, alkylaryl or hydrogen. 2. Using 5-S-alkyltetrazole at an effective, or final, concentration of 0.1-0.35 M for the activation of the RNA (or analogue) amidite during the coupling step. The time for this step is shortened from 10-15 m, vide supra, to 5-10 m.

3. Using alkylamine (MA, where alkyl is preferably methyl, ethyl, propyl or butyl) or NH₄OH/alkylamine (AMA, with the same preferred alkyl groups as noted for MA) @ 65 °C for 10-15 m to remove the exocyclic amino protecting groups (vs 4-20 h @ 55-65 °C using NH₄OH/EtOH or NH₃/EtOH, vide supra). Other alkylamines, e.g. ethylamine, propylamine, butylamine etc. may also be used.

4. Using anhydrous triethylamine•hydrogen fluoride (aHF•TEA) @ 65 °C for 0.5-1.5 h to remove the 2'-hydroxyl alkylsilyl protecting group (vs 8 - 24 h using TBAF, vide supra or TEA•3HF for 24 h (Gasparutto et al. Nucleic Acids Res. 1992 , 20, 5159-5166). Other alkylamine•HF complexes may also be used, e.g. trimethylamine or diisopropylethylamine.

5. The use of anion-exchange resins to purify and/or analyze the fully deprotected RNA. These resins include, but are not limited to, quartenary or tertiary amino derivatized stationary phases such as silica or polystyrene. Specific examples include Dionex-NA100^®, Mono-Q^®, Poros-Q^®.

Thus, the invention features an improved method for the coupling of RNA phosphoramidites; for the removal of amide or substituted amide protecting groups; and for the removal of 2'-hydroxyl alkylsilyl protecting groups. Such methods enhance the production of RNA or analogs of the type described above (e.g., with substituted 2'-groups), and allow efficient synthesis of large amounts of such RNA. Such RNA may also have enzymatic activity and be purified without loss of that activity. While specific examples are given herein, those in the art will recognize that equivalent chemical reactions can be performed with the alternative chemicals noted above, which can be optimized and selected by routine experimentation.

In another aspect, the invention features an improved method for the purification or analysis of RNA or enzymatic RNA molecules (e.g. 28-70 nucleotides in length) by passing said RNA or enzymatic RNA molecule over an HPLC, e.g., reverse phase and/or an anion exchange chromatography column. The method of purification improves the catalytic activity of enzymatic RNAs over the gel purification method (see Figure 10). Draper et al., PCT WO93/23569, incorporated by reference herein, disclosed reverse phase HPLC purification. The purification of long RNA molecules may be accomplished using anion exchange chromatography, particularly in conjunction with alkali perchlorate salts. This system may be used to purify very long RNA molecules. In particular, it is advantageous to use a Dionex NucleoPak 100^© or a Pharmacia Mono Q^® anion exchange column for the purification of RNA by the anion exchange method. This anion exchange purification may be used following a reverse-phase purification or prior to reverse phase purification. This method results in the formation of a sodium salt of the ribozyme during the chromatography. Replacement of the sodium alkali earth salt by other metal salts, e.g., lithium, magnesium or calcium perchlorate, yields the corresponding salt of the RNA molecule during the purification.

In the case of the 2-step purification procedure, in which the first step is a reverse phase purification followed by an anion exchange step, the reverse phase purification is best accomplished using polymeric, e.g. polystyrene based, reverse-phase media, using either a 5'-trityl-on or 5'-trityl-off method. Either molecule may be recovered using this reverse-phase method, and then, once detritylated, the two fractions may be pooled and then submitted to an anion exchange purification step as described above.

The method includes passing the enzymatically active RNA molecule over a reverse phase HPLC column; the enzymatically active RNA molecule is produced in a synthetic chemical method and not by an enzymatic process; and the enzymatic RNA molecule is partially blocked, and the partially blocked enzymatically active RNA molecule is passed over a reverse phase HPLC column to separate it from other RNA molecules.

In more preferred embodiments, the enzymatically active RNA molecule, after passage over the reverse phase HPLC column, is deprotected and passed over a second reverse phase HPLC column (which may be the same as the reverse phase HPLC column), to remove the enzymatic RNA molecule from other components. In addition, the column is a silica or organic polymer-based C4, C8 or C18 column having a porosity of at least 125 A, preferably 300 A, and a particle size of at least 2 μm, preferably 5 μm.

Activation

The synthesis of RNA molecules may be accomplished chemically or enzymatically. In the case of chemical synthesis the use of tetrazole as an activator of RNA phosphoramidites is known (Usman et al. J. Am. Chem. Soc. 1987, 109, 7845-7854). In this, and subsequent reports, a 0.5 M solution of tetrazole is allowed to react with the RNA phosphoramidite and couple with the polymer bound 5'-hydroxyl group for 10 m. Applicant has determined that using 0.25-0.5 M solutions of 5-S-alkyltetrazoies for only 5 min gives equivalent or better results. The following exemplifies the procedure.

Example 7: Synthesis of RNA and Ribozymes Using 5-S-Alkyltetrazoles as Activating Agent

The method of synthesis used follows the general procedure for RNA synthesis as described in Usman et al., 1987 supra and in Scaringe et al., Nucleic Acids Res. 1990, 18, 5433-5441 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. The major difference used was the activating agent, 5-S-ethyl or -methyltetrazole @ 0.25 M concentration for 5 min.

All small scale syntheses were conducted on a 394 (ABI) synthesizer using a modified 2.5 μmol scale protocol with a reduced 5 min coupling step for alkylsilyl protected RNA and 2.5 m coupling step for 2'-O-methylated RNA. A 6.5-fold excess (162.5 μL of 0.1 M = 32.5 μmol) of phosphoramidite and a 40-fold excess of S-ethyl tetrazole (400 μL of 0.25 M = 100 μmol) relative to polymer-bound 5'-hydroxyl was used in each coupling cycle. Average coupling yields on the 394, determined by colorimetric quantitation of the trityl fractions, was 97.5-99%. Other oligonucleotide synthesis reagents for the 394: Detritylation solution was 2% TCA in methylene chloride; capping was performed with 16% N-Methyl imidazole in THF and 10% acetic anhydride/10% 2,6-lutidine in THF; oxidation solution was 16.9 mM I₂, 49 mM pyridine, 9% water in THF. Fisher Synthesis Grade acetonitrile was used directly from the reagent bottle. S-Ethyl tetrazole solution (0.25 M in acetonitrile) was made up from the solid obtained from Applied Biosystems.

All large scale syntheses were conducted on a modified (eight amidite port capacity) 390Z (ABI) synthesizer using a 25 μmol scale protocol with a

5-15 min coupling step for alkylsilyl protected RΝA and 7.5 m coupling step for 2'-O-methylated RΝA. A six-fold excess (1.5 mL of 0.1 M = 150 μmol) of phosphoramidite and a forty-five-fold excess of S-ethyl tetrazole (4.5 mL of 0.25 M = 1125 μmol) relative to polymer-bound 5'-hydroxyl was used in each coupling cycle. Average coupling yields on the 390Z, determined by colorimetric quantitation of the trityl fractions, was 95.0-96.7%. Oligonucleotide synthesis reagents for the 390Z: Detritylation solution was 2% DCA in methylene chloride; capping was performed with 16% N-Methyl imidazole in THF and 10% acetic anhydride/10% 2,6-lutidine in THF; oxidation solution was 16.9 mM I₂, 49 mM pyridine, 9% water in THF. Fisher Synthesis Grade acetonitrile was used directly from the reagent bottle. S-Ethyl tetrazole solution (0.25-0.5 M in acetonitrile) was made up from the solid obtained from Applied Biosystems.

Deprotection

The first step of the deprotection of RΝA molecules may be accomplished by removal of the exocyclic amino protecting groups with either ΝH₄OH/EtOH:3/1 (Usman et al. J. Am. Chem. Soc. 1987, 109, 7845-7854) or NH₃/EtOH (Scaringe et al. Nucleic Acids Res. 1990, 18, 5433-5341) for ~20 h @ 55-65 °C. Applicant has determined that the use of methylamine or NH4OH/methylamine for 10-15 min @ 55-65 °C gives equivalent or better results. The following exemplifies the procedure.

Example 8: RNA and Ribozyme Deprotection of Exocyclic Amino

Protecting Groups Using Methylamine (MA) or NH₄OH/Methylamine (AMA)

The polymer-bound oligonucleotide, either trityl-on or off, was suspended in a solution of methylamine (MA) or NH₄OH/methylamine (AMA) @ 55-65 °C for 5-15 min to remove the exocyclic amino protecting groups. The polymer-bound oligoribonucleotide was transferred from the synthesis column to a 4 mL glass screw top vial. NH4OH and aqueous methylamine were pre-mixed in equal volumes. 4 mL of the resulting reagent was added to the vial, equilibrated for 5 m at RT and then heated at 55 or 65 °C for 5-15 min. After cooling to -20 °C, the supernatant was removed from the polymer support. The support was washed with 1.0 mL of EtOH:MeCN:H₂O/3:1 :1 , vortexed and the supernatant was then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, were dried to a white powder. The same procedure was followed for the aqueous methylamine reagent.

Table 40 is a summary of the results obtained using the improvements outlined in this application for base deprotection. The second step of the deprotection of RNA molecules may be accomplished by removal of the 2'-hydroxyl alkylsilyl protecting group using TBAF for 8-24 h (Usman et al. J. Am. Chem. Soc. 1987, 109, 7845-7854). Applicant has determined that the use of anhydrous TEA•HF in N-methylpyrrolidine (ΝMP) for 0.5-1.5 h @ 55-65 °C gives equivalent or better results. The following exemplifies this procedure.

Example 9: RΝA and Ribozyme Deprotection of 2'-Hydroxyl Alkylsilyl Protecting Groups Using Anhydrous TEA•HF

To remove the alkylsilyl protecting groups, the ammonia-deprotected oligoribonucleotide was resuspended in 250 μL of 1.4 M anhydrous HF solution (1.5 mL N-methylpyrrolidine, 750 μL TEA and 1.0 mL TEA•3HF) and heated to 65 °C for 1.5 h. 9 mL of 50 mM TEAB was added to quench the reaction. The resulting solution was loaded onto a Qiagen 500^® anion exchange cartridge (Qiagen Inc.) prewashed with 10 mL of 50 mM TEAB. After washing the cartridge with 10 mL of 50 mM TEAB, the RΝA was eluted with 10 mL of 2 M TEAB and dried down to a white powder.

Table 41 is a summary of the results obtained using the improvements outlined in this application for alkylsilyl deprotection.

Example 10: HPLC Purification, Anion Exchange column

For a small scale synthesis, the crude material was diluted to 5 mL with diethylpyrocarbonate treated water. The sample was injected onto either a Pharmacia Mono Q^® 16/10 or Dionex ΝucleoPac^® column with 100% buffer A (10 mM ΝaClO₄). A gradient from 180-210 mM NaClO₄ at a rate of 0.85 mM/void volume for a Pharmacia Mono Q® anion-exchange column or 100-150 mM NaClO₄ at a rate of 1.7 mM/void volume for a Pionex NucleoPac^® anion-exchange column was used to elute the RNA. Fractions were analyzed by a HP-1090 HPLC with a Dionex NucleoPac^® column. Fractions containing full length product at >80% by peak area were pooled. For a trityl-off large scale synthesis, the crude material was desalted by applying the solution that resulted from quenching of the desilylation reaction to a 53 mL Pharmacia HiLoad 26/10 Q-Sepharose® Fast Flow column. The column was thoroughly washed with 10 mM sodium perchlorate buffer. The oligonucleotide was eluted from the column with 300 mM sodium perchlorate. The eluent was quantitated and an analytical HPLC was run to determine the percent full length material in the synthesis. The eluent was diluted four fold in sterile H₂O to lower the salt concentration and applied to a Pharmacia Mono Q^® 16/10 column. A gradient from 10-185 mM sodium perchlorate was run over 4 column volumes to elute shorter sequences, the full length product was then eluted in a gradient from 185-214 mM sodium perchlorate in 30 column volumes. The fractions of interest were analyzed on a HP-1090 HPLC with a Pionex NucleoPac^® column. Fractions containing over 85% full length material were pooled. The pool was applied to a Pharmacia RPC^® column for desalting.

For a trityl-on large scale synthesis, the crude material was desalted by applying the solution that resulted from quenching of the desilylation reaction to a 53 mL Pharmacia HiLoad 26/10 Q-Sepharose^® Fast Flow column. The column was thoroughly washed with 20 mM NH₄CO₃H/10% CH₃CN buffer. The oligonucleotide was eluted from the column with 1.5 M NH₄CO₃H/10% acetonitrile. The eluent was quantitated and an analytical HPLC was run to determine the percent full length material present in the synthesis. The oligonucleotide was then applied to a Pharmacia Resource RPC column. A gradient from 20-55% B (20 mM NH₄CO₃H/25% CH₃CN, buffer A = 20 mM NH₄CO₃H/10% CH₃CN) was run over 35 column volumes. The fractions of interest were analyzed on a HP-1090 HPLC with a Dionex NucleoPac^® column. Fractions containing over 60% full length material were pooled. The pooled fractions were then submitted to manual detritylation with 80% acetic acid, dried down immediately, resuspended in sterile H₂O, dried down and resuspended in H₂O again. This material was analyzed on a HP 1090-HPLC with a Dionex NucleoPac^® column. The material was purified by anion exchange chromatography as in the trityl-off scheme (vide supra). Example 11 Ribozyme Activity Assay

Purified 5'-end labeled RNA substrates (15-25-mers) and purified 5'-end labeled ribozymes (~36-mers) were both heated to 95 °C, quenched on ice and equilibrated at 37 °C, separately. Ribozyme stock solutions were 1 μM, 200 nM, 40 nM or 8 nM and the final substrate RNA concentrations were - 1 nM. Total reaction volumes were 50 μL. The assay buffer was 50 mM Tris-Cl, pH 7.5 and 10 mM MgCl₂. Reactions were initiated by mixing substrate and ribozyme solutions at t = 0. Aliquots of 5 μL were removed at time points of 1 , 5, 15, 30, 60 and 120 m. Each aliquot was quenched in formamide loading buffer and loaded onto a 15% denaturing polyacrylamide gel for analysis. Quantitative analyses were performed using a phosphorimager (Molecular Dynamics).

Example 12: One pot deprotection of RNA

Applicant has shown that aqueous methyl amine is an efficient reagent to deprotect bases in an RNA molecule. However, in a time consuming step (2-24 hrs), the RNA sample needs to be dried completely prior to the deprotection of the sugar 2'-hydroxyl groups. Additionally, deprotection of RNA synthesized on a large scale (e.g., 100 μmol) becomes challenging since the volume of solid support used is quite large. In an attempt to minimize the time required for deprotection and to simplify the process of deprotection of RNA synthesized on a large scale, applicant describes a one pot deprotection protocol (Fig. 12). According to this protocol, anhydrous methylamine is used in place of aqueous methyl amine. Base deprotection is carried out at 65 °C for 15 min and the reaction is allowed to cool for 10 min. Deprotection of 2'-hydroxyl groups is then carried out in the same container for 90 min in a TEA•3HF reagent. The reaction is quenched with 16 mM TEAB solution.

Referring to Fig. 13. hammerhead ribozyme targeted to site B is synthesized using RNA phosphoramadite chemistry and deprotected using either a two pot or a one pot protocol. Profiles of these ribozymes on an HPLC column are compared. The figure shows that RNAs deprotected by either the one pot or the two pot protocols yield similar full-length product profiles. Applicant has shown that using a one pot deprotection protocol, time required for RNA deprotection can be reduced considerably without compromising the quality or the yield of full length RNA.

Referring to Fig. 14, hammerhead ribozymes targeted to site B (from Fig. 13) are tested for their ability to cleave RNA. As shown in the figure 14. ribozymes that are deprotected using one pot protocol have catalytic activity comparable to ribozymes that are deprotected using a two pot protocol. Example 12a. Improved protocol for the synthesis of phosphorothioate containing RNA and ribozymes using 5-S-Alkyltetrazoles as Activating Agent

The two sulfurizing reagents that have been used to synthesize ribophosphorothioates are tetraethylthiuram disulfide (TETD; Vu and Hirschbein, 1991 Tetrahedron Letter 31 , 3005), and 3H-1 ,2-benzodithiol-3-one 1 ,1-dioxide (Beaucage reagent; Vu and Hirschbein, 1991 supra). TETD requires long sulfurization times (600 seconds for DNA and 3600 seconds for RNA). It has recently been shown that for sulfurization of DNA oligonucleotides, Beaucage reagent is more efficient than TETD (Wyrzykiewicz and Ravikumar, 1994 Bioorganic Med. Chem. 4, 1519). Beaucage reagent has also been used to synthesize phosphorothioate oligonucleotides containing 2'-deoxy-2'-fluoro modifications wherein the wait time is 10 min (Kawasaki et al., 1992 J. Med. Chem). The method of synthesis used follows the procedure for RNA synthesis as described herein and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. The sulfurization step for RNA described in the literature is a 8 second delivery and 10 min wait steps (Beaucage and Iyer, 1991 Tetrahedron 49, 6123). These conditions produced about 95% sulfurization as measured by HPLC analysis (Morvan et al., 1990 Tetrahedron Letter 31 , 7149). This 5% contaminating oxidation could arise from the presence of oxygen dissolved in solvents and/or slow release of traces of iodine adsorbed on the inner surface of delivery lines during previous synthesis.

A major improvement is the use of an activating agent, 5-S-ethyltetrazole or 5-S-methyltetrazole at a concentration of 0.25 M for 5 min. Additionally, for those linkages which are phosporothioate, the iodine solution is replaced with a 0.05 M solution of 3H-1 ,2-benzodithiole-3-one 1 ,1-dioxide (Beaucage reagent) in acetonitrile. The delivery time for the sulfurization step is reduced to 5 seconds and the wait time is reduced to 300 seconds.

RNA synthesis is conducted on a 394 (ABI) synthesizer using a modified 2.5 μmol scale protocol with a reduced 5 min coupling step for alkylsilyl protected RNA and 2.5 min coupling step for 2'-O-methylated

RNA. A 6.5-fold excess (162.5 μL of 0.1 M = 32.5 μmol) of phosphoramidite and a 40-fold excess of S-ethyl tetrazole (400 μL of 0.25 M = 100 μmol) relative to polymer-bound 5'-hydroxyl was used in each coupling cycle. Average coupling yields on the 394 synthesizer, determined by colorimetric quantitation of the trityl fractions, was 97.5-99%. Other oligonucleotide synthesis reagents for the 394 synthesizer: detritylation solution was 2% TCA in methylene chloride; capping was performed with 16% N-Methyl imidazole in THF and 10% acetic anhydride/10% 2,6-lutidine in THF; oxidation solution was 16.9 mM I₂, 49 mM pyridine, 9% water in THF. Fisher Synthesis Grade acetonitrile was used directly from the reagent bottle. S-Ethyl tetrazole solution (0.25 M in acetonitrile) was made up from the solid obtained from Applied Biosystems. Sulfurizing reagent was obtained from Glen Research.

Average sulfurization efficiency (ASE) is determined using the formula: ASE = (PS/Total) ^1/n-1 where, PS = integrated ³¹ P ΝMR values of the P=S diester

Total = integration value of all peaks n = length of oligo

Referring to tables 42 and 43, effects of varying the delivery and the wait time for sulfurization with Beaucage's reagent is described. These data suggest that 5 second wait time and 300 second delivery time is the condition under which ASE is maximum.

Using the above conditions a 36 mer hammerhead ribozyme is synthesized which is targeted to site C. The ribozyme is synthesized to contain phosphorothioate linkages at four positions towards the 5' end. RΝA cleavage activity of this ribozyme is shown in Fig. 16. Activity of the phosphorothioate ribozyme is comparable to the activity of a ribozyme lacking any phosphorothioate linkages.

Example 13: Protocol for the synthesis of 2'-Ν-phtalimido-nucleoside phosphoramidite The 2'-amino group of a 2'-deoxy-2'-amino nucleoside is normally protected with N-(9-flourenylmethoxycarbonyl) (Fmoc; Imazawa and Eckstein, 1979 supra; Pieken et al., 1991 Science 253, 314). This protecting group is not stable in CH3CN solution or even in dry form during prolonged storage at -20 °C. These problems need to be overcome in order to achieve large scale synthesis of RNA.

Applicant describes the use of alternative protecting groups for the 2'-amino group of 2'-deoxy-2'-amino nucleoside. Referring to Figure 17, phosphoramidite 17 was synthesized starting from 2'-deoxy-2'-aminonucleoside (12) using transient protection with Markevich reagent (Markiewicz J. Chem. Res. 1979, S, 24). An intermediate 13 was obtained in 50% yield, however subsequent introduction of N-phtaloyl (Pht) group by Nefken's method (Nefkens, 1960 Nature 185, 306), desilylation (15), dimethoxytrytilation (16) and phosphitylation led to phosphoramidite 17. Since overall yield of this multi-step procedure was low (20%) applicant investigated some alternative approaches, concentrating on selective introduction of N-phtaloyl group without acylation of 5' and 3' hydroxyls.

When 2'-deoxy-2'-amino-nucleoside was reacted with 1.05 equivalents of Nefkens reagent in DMF overnight with subsequent treatment with Et₃N (1 hour) only 10-15% of N and 5'(3')-bis-phtaloyl derivatives were formed with the major component being N-Pht-derivative 15. The N,O-bis by-products could be selectively and quantitively converted to N-Pht derivative 15 by treatment of crude reaction mixture with cat. KCN/MeOH.

A convenient "one-pot" procedure for the synthesis of key intermediate 16 involves selective N-phthaloylation with subsequent dimethoxytrytilation by DMTCI/Et3N and resulting in the preparation of DMT derivative 16 in 85% overall yield as follows. Standard phosphytilation of 16 produced phosphoramidite 17 in 87% yield. One gram of 2'-amino nucleoside, for example 2'-amino uridine (US Biochemicals® part # 77140) was co-evaporated twice from dry dimethyl formamide (Dmf) and dried in vacuo overnight. 50 mis of Aldrich sure-seal Dmf was added to the dry 2'-amino uridine via syringe and the mixture was stirred for 10 minutes to produce a clear solution. 1 .0 grams (1.05 eq.) of N-carbethoxyphthalimide (Nefken's reagent, 98% Jannsen Chimica) was added and the solution was stirred overnight. Thin layer chromatography (TLC) showed 90% conversion to a faster moving products (10% ETOH in CHCl₃) and 57 μl of TEA (0.1 eq.) was added to effect closure of the phthalimide ring. After 1 hour an additional 855 μl (1.5 eq.) of TEA was added followed by the addition of 1.53 grams (1.1 eq.) of DMT-Cl (Lancaster Synthesis^®, 98%). The reaction mixture was left to stir overnight and quenched with ETOH after TLC showed greater than 90% desired product. Pmf was removed under vacuum and the mixture was washed with sodium bicarbonate solution (5% aq., 500 mls) and extracted with ethyl acetate (2× 200 mls). A 25mm × 300mm flash column (75 grams Merck flash silica) was used for purification. Compound eluted at 80 to 85% ethyl acetate in hexanes (yield: 80% purity: >95% by ¹ HNMR). Phosphoramidites were then prepared using standard protocols described above. With phosphoramidite 17 in hand applicant synthesized several ribozymes with 2'-deoxy-2'-amino modifications. Analysis of the synthesis demonstrated coupling efficiency in 97-98% range. RNA cleavage activity of ribozymes containing 2'-deoxy-2'-amino-U modifications at U4 and/or U7 positions (see Figure 1), wherein the 2'-amino positions were either protected with Fmoc or Pht, was identical. Additionally, complete deprotection of 2'-deoxy-2'-amino-Uridine was confirmed by base-composition analysis. The coupling efficiency of phosphoramidite 17 was not effected over prolonged storage (1-2 months) at low temperatures.

Protecting 2' Position with a SEM Group There follows a method using the 2'-(trimethylsilyl)ethoxymethyl protecting group (SEM) in the synthesis of oligoribonucleotides, and in particular those enzymatic molecules described above. For the synthesis of RNA it is important that the 2'-hydroxyl protecting group be stable throughout the various steps of the synthesis and base deprotection. At the same time, this group should also be readily removed when desired. To that end the t-butyldimethylsilyl group has been efficacious (Usman, N.; Ogilvie.K.K.; Jiang, M.-Y.; Cedergren.R.J. J. Am. Chem. Soc. 1987, 109, 7845-7854 and Scaringe, S.A.; Franklyn.C.; Usman, N. Nucl. Acids Res. 1990, 18, 5433-5441 ). However, long exposure times to tetra-n-butylammonium fluoride (TBAF) are generally required to fully remove this protecting group from the 2'-hydroxyl. In addition, the bulky alkyl substituents can prove to be a hindrance to coupling thereby necessitating longer coupling times. Finally, it has been shown that the TBDMS group is base labile and is partially deprotected during treatment with ethanolic ammonia (Scaringe, S.A.; Franklyn.C.; Usman, N. Nucl. Acids Res. 1990, 18, 5433-5441 and Stawinski,J.; Stromberg.R.; Thelin.M.; Westman.E. Nucleic Acids Res. 1988, 16, 9285-9298).

The (trimethylsilyl)ethoxymethyl ether (SEM) seems a suitable substitute. This protecting group is stable to base and all but the harshest acidic conditions. Therefore it is stable under the conditions required for oligonucleotide synthesis. It can be readily introduced and the oxygen carbon bond makes it unable to migrate. Finally, the SEM group can be removed with BF₃•OEt₂ very quickly.

There follows a method for synthesis of RNA by protecting the 2'-position of a nucleotide during RNA synthesis with a

(trimethylsilyl)ethoxymethyl (SEM) group. The method can involve use of standard RNA synthesis conditions as discussed below, or any other equivalent steps. Those in the art are familiar with such steps. The nucleotide used can be any normal nucleotide or may be substituted in various positions by methods well known in the art, e.g., as described by

Eckstein et al., International Publication No. WO 92/07065, Perrault et al.,

Nature 1990, 344, 565-568, Pieken et al., Science 1991 , 253, 314-317,

Usman, N.; Cedergren.R.J. Trends in Biochem. Sci. 1992, 17, 334-339,

Usman et al., PCT W093/15187, and Sproat.B. European Patent Application 92110298.4 .

This invention also features a method for covalently linking a SEM group to the 2'-position of a nucleotide. The method involves contacting a nucleoside with an SEM-containing molecule under SEM bonding conditions. In a preferred embodiment, the conditions are dibutyltin oxide, tetrabutylammonium fluoride and SEM-Cl. Those in the art, however, will recognize that other equivalent conditions can also be used.

In another aspect, the invention features a method for removal of an SEM group from a nucleoside molecule or an oligonucleotide. The method involves contacting the molecule or oligonucleotide with boron trifluoride etherate (BF₃•OEt₂) under SEM removing conditions, e.g., in acetonitrile.

Referring to Figure 18. there is shown the method for solid phase synthesis of RNA. A 2',5'-protected nucleotide is contacted with a solid phase bound nucleotide under RNA synthesis conditions to form a dinucleotide. The protecting group (R) at the 2'-position in prior art methods can be a silyl ether, as shown in the Figure. In the method of the present invention, an SEM group is used in place of the silyl ether. Otherwise RNA synthesis can be performed by standard methodology.

Referring to Figure 19. there is shown the synthesis of 2'-O-SEM protected nucleosides and phosphoramadites. Briefly, a 5'-protected nucleoside (1 ) is protected at the 2'- or 3'-position by contacting with a derivative of SEM under appropriate conditions. Specifically, those conditions include contacting the nucleoside with dibutyltin oxide and SEM chloride. The 2 regioisomers are separated by chromatography and the 2'-protected moiety is converted into a phosphoramidite by standard procedure. The 3'-protected nucleoside is converted into a succinate derivative suitable for derivatization of a solid support.

Referring to Figure 20. a prior art method for deprotection of RNA using silyl ethers is shown. This contrasts with the method shown in Figure 21 in which deprotection of RNA containing an SEM group is performed. In step 1 , the base protecting groups and cyanoethyl groups are removed by standard procedure. The SEM group is then removed as shown in the Figure. The details of the synthesis of phosphoramidites and SEM protected nucleosides and their use in synthesis of oligonucleotides and subsequent deprotection of

Example 14: Synthesis of 2'-O-((trimethylsilyl)ethoxymethyl)-5'-O- Dimethoxytrityl Uridine (2)

Referring to Figure 19. 5'-O-dimethoxytrityl uridine 1 (1.0 g, 1.83 mmol) in CH₃CN (18 mL) was added dibutyltin oxide (1.0 g, 4.03 mmol) and TBAF (1 M, 2.38 mL, 2.38 mmol). The mixture was stirred for 2 h at RT (about 20-25°C) at which time (trimethylsilyl)ethoxymethyl chloride (SEM- Cl) (487 μL, 2.75 mmol) was added. The reaction mixture was stirred overnight and then filtered and evaporated. Flash chromatography (30% hexanes in ethyl acetate) yielded 347 mg (28.0%) of 2'-hydroxyl protected nucleoside 2 and 314 mg (25.3%) of 3'-hydroxyl protected nucleoside 3.

Example 15: Synthesis of 2'-O-((trimethylsilyl)ethoxymethyl) Uridine (4)

Nucleoside 2 was detritylated following standard methods, as shown in Figure 19. Example 16: Synthesis of 2'-O-((trimethylsilyl)ethoxymethyl)-5',3'-O-Acetyl Uridine (5)

Nucleoside 4 was acetylated following standard methods, as shown in Figure 19. Example 17: Synthesis of 5',3'-O-Acetyl Uridine (6)

Referring to Figure 19. the fully protected uridine 5 (32 mg, 0.07 mmol) was dissolved in CH₃CN (700 μL) and BF₃•OEt₂ (17.5 μL, 0.14 mmol) was added. The reaction was stirred 15 m and MeOH was added to quench the reaction. Flash chromatography (5% MeOH in CH₂Cl₂) gave 20 mg (88%) of SEM deprotected nucleoside 6.

Example 18: Synthesis of 2'-O-((trimethylsilyl)ethoxymethyl)-3'-O-Succinyl-5'-O- Dimethoxytrityl Uridine (2)

Nucleoside 3 was succinylated and coupled to the support following standard procedures, as shown in Figure 19. Example 19: Synthesis of 2'-O-((trimethylsilyl)ethoxymethyl)-5'-O- Dimethoxytrityl Uridine 3'-(2-Cyanoethyl N,N-diisopropylphosphoramidite) (8)

Nucleoside 3 was phosphitylated following standard methods, as shown in Figure 19. Example 20: Synthesis of RNA Using 2'-O-SEM Protection

Referring to Figure 18. the method of synthesis used follows the general procedure for RNA synthesis as described in Usman, N.; Ogilvie.K.K.; Jiang, M.-Y.; Cedergren.R.J. J. Am. Chem. Soc. 1987, 109, 7845-7854 and in Scaringe, S.A.; Franklyn.C; Usman, N. Nucl. Acids Res. 1990, 18, 5433-5441. The phosphoramidite 8 was coupled following standard RNA methods to provide a 10-mer of uridylic acid. Syntheses were conducted on a 394 (ABI) synthesizer using a modified 2.5 μmol scale protocol with a 10 m coupling step. A thirteen-fold excess (325 μL of 0.1 M = 32.5 μmol) of phosphoramidite and a 80-fold excess of tetrazole (400 μL of 0.5 M = 200 μmol) relative to polymer-bound 5'-hydroxyl was used in each coupling cycle. Average coupling yields on the 394, determined by colorimetric quantitation of the trityl fractions, were 98-99%. Other oligonucleotide synthesis reagents for the 394: Detritylation solution was 2% TCA in methylene chloride; capping was performed with 16% N- Methyl imidazole in THF and 10% acetic anhydride/10% 2,6-lutidine in THF; oxidation solution was 16.9 mM I₂, 49 mM pyridine, 9% water in THF. Fisher Synthesis Grade acetonitrile was used directly from the reagent bottle. Referring to Figure 21. the homopolymer was base deprotected with

N H3/EtOH at 65 °C The solution was decanted and the support was washed twice with a solution of 1 :1 :1 H₂O:CH₃CN:MeOH. The combined solutions were dried down and then diluted with CH₃CN (1 mL). BF₃•OEt₂ (2.5 μL, 30 μmol) was added to the solution and aliquots were removed at ten time points. The results indicate that after 30 min deprotection is complete, as shown in Figure 22.

IIII Vectors Expressing Ribozymes

There follows a method for expression of a ribozyme in a bacterial or eucaryotic cell, and for production of large amounts of such a ribozyme. In general, the invention features a method for preparing multi-copy cassettes encoding a defined ribozyme structure for production of a ribozyme at a decreased cost. A vector is produced which encodes a plurality of ribozymes which are cleaved at their 3' and 5' ends from an RNA transcript producted from the vector by only one other ribozyme. The system is useful for scaling up production of a ribozyme, which may be either modified or unmodified, in situ or in vitro. Such vector systems can be used to express a desired ribozyme in a specific cell, or can be used in an in vitro system to allow productiuon of large amounts of a desired riboqyne, The vectors of this invention allow a higher yield synthesis of a ribozyme in the form of an RNA transcript which is cleaved in situ or in vitro before or after transcript isolation.

Thus, this invention is distinct from the prior art in that a single ribozyme is used to process the 3' and 5' ends of each therapeutic, transacting or desired ribozyme instead of processing only one end, or only one ribozyme. This allows smaller vectors to be derived with multiple transacting ribozymes released by only one other ribozyme from the mRNA transcript. Applicant has also provided methods by which the activity of such ribozymes is increased compared to those in the art, by designing ribozyme-encoding vectors and the corresponding transcript such that folding of the mRNA does not interfere with processing by the releasing ribozyme.

The stability of the ribozyme produced in this method can be enhanced by provision of sequences at the termini of the ribozymes as described by Draper et al., PCT WO 93/23509, hereby incorporated by reference herein.

The method of this invention is advantageous since it provides high yield synthesis of ribozymes by use of low cost transcription-based protocols, compared to existing chemical ribozyme synthesis, and can use isolation techniques currently used to purify chemically synthesized oligonucleotides. Thus, the method allows synthesis of ribozymes in high yield at low cost for analytical, diagnostic, or therapeutic applications.

The method is also useful for synthesis of ribozymes in vitro for ribozyme structural studies, enzymatic studies, target RNA accessibility studies, transcription inhibition studies and nuclease protection studies, much is described by Draper et al., PCT WO 93/23509 hereby incorporated by reference herein.

The method can also be used to produce ribozymes in situ either to increase the intracellular concentration of a desired therapeutic ribozyme, or to produce a concatameric transcript for subsequent in vitro isolation of unit length ribozyme. The desired ribozyme can be used to inhibit gene expression in molecular genetic analyses or in infectious cell systems, and to test the efficacy of a therapeutic molecule or treat afflicted cells.

Thus, in general, the invention features a vector which includes a bacterial, viral or eucaryotic promoter within a plasmid, cosmid, phagmid, virus, viroid, virusoid or phage vector. Other vectors are equally suitable and include double-stranded, or partially double-stranded DNA, formed by an amplification method such as the polymerase chain reaction, or doublestranded, partially double-stranded or single-stranded RNA, formed by sitedirected homologous recombination into viral or viroid RNA genomes. Such vectors need not be circular. Transcriptionally linked to the promoter region is a first ribozyme-encoding region, and nucleotide sequences encoding a ribozyme cleavage sequence which is placed on either side of a region encoding a therapeutic or otherwise desired second ribozyme. Suitable restriction endonuclease sites can be provided to ease construction of this vector in DNA vectors or in requisite DNA vectors of an RNA expression system. The desired second ribozyme may be any desired type of ribozyme, such as a hammerhead, hairpin , hepatitis delta virus (HDV) or other catalytic center, and can include group I and group II introns, as discussed above. The first ribozyme is chosen to cleave the encoded cleavage sequence, and may also be any desired ribozyme, for example, a Tetrahymena derived ribozyme, which may, for example, include an imbedded restriction endonuclease site in the center of a self-recognition sequence to aid in vector construction. This endonuclease site is useful for construction of the vector, and subsequent analysis of the vector.

When the promoter of such a vector is activated an RNA transcript is produced which includes the first and second ribozyme sequences. The first ribozyme sequence is able to act, under appropriate conditions, to cause cleavage at the cleavage sites to release the second ribozyme sequences. These second ribozyme sequences can then act at their target RNA sites, or can be isolated for later use or analysis.

Thus, in one aspect the invention features a vector which includes a first nucleic acid sequence (encoding a first ribozyme having intramolecular cleaving activity), and a second nucleic acid sequence (encoding a second ribozyme having intermolecular cleaving enzymatic activity) flanked by nucleic acid sequences encoding RNA which is cleaved by the first ribozyme to release the second ribozyme from the RNA transcript encoded by the vector. The second ribozyme may be flanked by the first ribozyme either on the 5' side or 3' side. If desired, the first ribozyme may be encoded on a separate vector and may have intermolecular cleaving activity.

As discussed above, the first ribozyme can be chosen to be any self-cleaving ribozyme, and the second ribozyme may be chosen to be any desired ribozyme. The flanking sequences are chosen to include sequences recognized by the first ribozyme. When the vector is caused to express RNA from these nucleic acid sequences, that RNA has the ability under appropriate conditions to cleave each of the flanking regions and thereby release one or more copies of the second ribozyme. If desired, several different second ribozymes can be produced by the same vector, or several different vectors can be placed in the same vessel or cell to produce different ribozymes.

In preferred embodiments, the vector includes a plurality of the nucleic acid sequences encoding the second ribozyme, each flanked by nucleic acid sequences recognized by the first ribozyme. Most preferably, such a plurality includes at least six to nine or even between 60 - 100 nucleic acid sequences. In other preferred embodiments, the vector includes a promoter which regulates expression of the nucleic acid encoding the ribozymes from the vector; and the vector is chosen from a plasmid, cosmid, phagmid, virus, viroid or phage. In a most preferred embodiment, the plurality of nucleic acid sequences are identical and are arranged in sequential order such that each has an identical end nearest to the promoter. If desired, a poly(A) sequence adjacent to the sequence encoding the first or second ribozyme may be provided to increase stability of the RNA produced by the vector; and a restriction endonuclease site adjacent to the nucleic acid encoding the first ribozyme is provided to allow insertion of nucleic acid encoding the second ribozyme during construction of the vector.

In a second aspect, the invention features a method for formation of a ribozyme expression vector by providing a vector including nucleic acid encoding a first ribozyme, as discussed above, and providing a single-stranded PNA encoding a second ribozyme, as discussed above. The single-stranded PNA is then allowed to anneal to form a partial duplex PNA which can be filled in by a treatment with an appropriate enzyme, such as a PNA polymerase in the presence of dNTPs, to form a duplex DNA which can then be ligated to the vector. Large vectors resulting from this method can then be selected to insure that a high copy number of the single-stranded DNA encoding the second ribozyme is incorporated into the vector. In a further aspect, the invention features a method for production of ribozymes by providing a vector as described above, expressing RNA from that vector, and allowing cleavage by the first ribozyme to release the second ribozyme.

In preferred embodiments, three different ribozyme motifs are used as cis-cleaving ribozymes. The hammerhead, hairpin, and hepatitis delta virus (HDV) ribozyme motifs consist of small, well-defined sequences that rapidly self-cleave in vitro (Symons, 1992 Annu. Rev. Biochem. 61 , 641). While structural and functional differences exist among the three ribozyme motifs, they self-process efficiently in vivo. All three ribozyme motifs self-process to 87-95% completion in the absence of 3' flanking sequences. In vitro, the self-processing constructs described in this invention are significantly more active than those reported by Taira et al., 1990 supra: and Altschuler et al., 1992 Gene 122, 85. The present invention enables the use of cis-cleaving ribozymes to efficiently truncate RNA molecules at specific sites in vivo by ensuring lack of secondary structure which prevents processing.

Isolation of Therapeutic Ribozyme

The preferred method of isolating therapeutic ribozyme is by a chromatographic technique. The HPLC purification methods and reverse HPLC purification methods described by Draper et al., PCT WO 93/23509, hereby incorporated by reference herein, can be used. Alternatively, the attachment of complementary oligonucleotides to cellulose or other chromatography columns allows isolation of the therapeutic second ribozyme, for example, by hybridization to the region between the flanking arms and the enzymatic RNA. This hybridization will select against the short flanking sequences without the desired enzymatic RNA, and against the releasing first ribozyme. The hybridization can be accomplished in the presence of a chaotropic agent to prevent nuclease degradation. The oligonucleotides on the matrix can be modified to minimize nuclease activity, for example, by provision of 2'-0-methyl RNA oligonucleotides. Such modifications of the oligonucleotide attached to the column matrix will allow the multiple use of the column with minimal oligo degradation. Many such modifications are known in the art, but a chemically stable non-reducible modification is preferred. For example, phosphorothioate modifications can also be used.

The expressed ribozyme RNA can be isolated from bacterial or eucaryotic cells by routine procedures such as lysis followed by guanidine isothiocyanate isolation.

The current known self-cleaving site of Tetrahymena can be used in an alternative vector of this invention. If desired, the full-length Tetrahymena sequence may be used, or a shorter sequence may be used. It is preferred that, in order to decrease the superfluous sequences in the self-cleaving site at the 5' cleavage end, the hairpin normally present in the Tetrahymena ribozyme should contain the therapeutic second ribozyme 3' sequence and its complement. That is, the first releasing ribozyme-encoding DNA is provided in two portions, separated by DNA encoding the desired second ribozyme. For example, if the therapeutic second ribozyme recognition sequence is CGGACGA/CGAGGA, then CGAGGA is provided in the self-cleaving site loop such that it is in a stem structure recognized by the Tetrahymena ribozyme. The loop of the stem may include a restriction endonuclease site into which the desired second ribozyme-encoding DNA is placed.

If desired, the vector may be used in a therapeutic protocol by use of the systems described by Lechner, PCT WO 92/13070, hereby incorporated by reference herein, to allow a timed expression of the therapeutic second ribozyme, as well as an appropriate shut off of cell or gene function. Thus, the vector will include a promoter which appropriately expresses enzymatically active RNA only in the presence of an RNA or another molecule which indicates the presence of an undesired organism or state. Such enzymatically active RNA will then kill or harm the cell in which it exists, as described by Lechner, id ., or act to cause reduced expression of a desired protein product.

A number of suitable RNA vectors may also be used in this invention. The vectors include plant viroids, plant viruses which contain single or double-stranded RNA genomes and animal viruses which contain RNA genomes, such as the picornaviruses, myxoviruses, paramyxoviruses, hepatitis A virus, reovirus and retroviruses. In many instances cited, use of these viral vectors also results in tissue specific delivery of the ribozymes.

Example 21 : Design of self-processing cassettes In a preferred embodiment, applicant compared the in vitro and in vivo cis-cleaving activity of three different ribozyme motifs-the hammerhead, the hairpin and the hepatitis delta virus ribozyme-in order to assess their potential to process the ends of transcripts in vivo. To make a direct comparison among the three, however, it is important to design the ribozyme-containing transcripts to be as similar as possible. To this end, all the ribozyme cassettes contained the same trans-acting hammerhead ribozyme followed immediately by one of the three cis-acting ribozymes (Figure 23-25). For simplicity, applicant refers to each cassette by an abbreviation that indicates the downstream cis-cleaving ribozyme only. Thus HH refers to the cis-cleaving cassette containing a hammerhead ribozyme, while HP and HDV refer to the cassettes containing hairpin and hepatitis delta virus cis-cleaving ribozymes, respectively. The general design of the ribozyme cassettes, as well as specific differences among the cassettes, are outlined below. A sequence predicted to form a stable stem-loop structure is included at the 5' end of all the transcripts. The hairpin stem contains the T7 RNA polymerase initiation sequence (Milligan & Uhlenbeck, 1989 Methods Enzymol. 180, 51) and its complement, separated be a stable tetra-loop (Antao et al., 1991 Nucleic Acids Res. 19, 5901). By incorporating the T7 initiation sequence into a stem-loop structure, applicant hoped to avoid nonproductive base pairing interactions with either the trans-acting ribozyme or with the cis-acting ribozyme. The presence of a hairpin at the end of a transcript may also contribute to the stability of the transcript in vivo. These are non-limiting examples. Those in the art will recognize that other embodiments can be readily generated using a variety of promoters, initiator sequences and stem-loop structure combinations generally known in the art.

The trans-acting ribozyme used in this study is targeted to a site B (5'...CUGGAGUC^↓GACCUUC...3'). The 5' binding arm of the ribozyme, 5'-GAAGG UC-3' , and the core of the ribozyme, 5'-CUGAUGAGGCCGAAAGGCCGAA-3', remain constant in all cases. In addition, all transcripts also contain a single nucleotide between the 5' stem-loop and the first nucleotide of the ribozyme. The linker nucleotide was required to obtain the same activity in vitro that was measured with an identical ribozyme lacking the 5' hairpin. Because the three cis-cleaving ribozymes have different requirements at the site of cleavage, slight differences were unavoidable at the 3' end of the processed transcript. The junction between the trans- and cis-acting ribozyme is, however, designed so that there is minimal extraneous sequence left at the 3' end of the trans-cleaving ribozyme once cis-cleavage occurs. The only differences between the constructs lie in the 3' binding arm of the ribozyme, where either 6 or 7 nucleotides, 5'-ACUCCA(+/-G)-3', complementary to the target sequence are present and where, after processing, two to five extra nucleotides remain.

The cis-cleaving hammerhead ribozyme used in the HH cassette is based on the design of Grosshans and Cech, 1991 supra. As shown in Figure 23, the 3' binding arm of the trans-acting ribozyme is included in the required base-pairing interactions of the cis-cleaving ribozyme to form stem I. Two extra nucleotides, UC, were included at the end of the 3' binding arm to form the self-processing hammerhead ribozyme site (Ruffner et al., 1990 supra) which remain on the 3' end of the trans-acting ribozyme following self-processing.

The hairpin ribozyme portion of the HP self-processing construct is based on the minimal wild-type sequence (Hampel & Tritz, 1989 supra). A tetra-loop at the end of helix 1 (3' side of the cleavage site) serves to link the two portions and thus allows a minimal five nucleotides to remain at the end of the released trans-acting ribozyme following self-processing. Two variants of HP were designed: HP(GU) and HP(GC). The HP(GU) was constructed with a G·U wobble base pair in helix 2 (A₅₂G substitution; Figure 24). This slight destabilization of helix 2 was intended to improve self-processing activity by promoting product release and preventing the reverse reaction (Berzal-Herranz et al., 1992 Genes & Dev. 6, 129; Chowrira et al., 1993 Biochemistry 32, 1088). The HP(GC) cassette was constructed as a control for strong base-pairing interactions in helix 2 (U₇₇C and A₅₂G substitution; Figure 24). Another modification to discourage the reverse ligation reaction of the hairpin ribozyme was to shorten helix 1 (Figure 24) by one base pair relative to the wild-type sequence (Chowrira & Burke, 1991 Biochemistry 30, 8518).

The HDV ribozyme self-processes efficiently when the nucleotide 5' to the cleavage site is a pyrimidine, and somewhat less so when adenosine is in that position. No other sequence requirements have been identified upstream of the cleavage site, however, we have observed some decrease in activity when a stem-loop structure was present within 2 nt of the cleavage site. The HDV self-processing construct (Fig 25) was designed to generate the trans-acting hammerhead ribozyme with only two additional nucleotides at its 3' end after self-processing. The HDV sequence used here is based on the anti-genomic sequence (Perrota & Been, 1992 supra) but includes the modifications of Been et al., 1992 (Biochemistry 31 , 11843) in which cis-cleavage activity of the ribozyme was improved by the substitution of a shortened helix 4 for a wild-type stem-loop (Figure 25).

To prepare DNA inserts that encode self-processing ribozyme cassettes, partially overlapping top- and bottom-strand oligonucleotides (60-90 nucleotides) were designed to include sequences for the T7 promoter, the trans-acting ribozyme, the cis-cleaving ribozyme and appropriate restriction sites for use in cloning (see Fig. 26). The singlestrand portions of annealed oligonucleotides were converted to doublestrands using Sequenase® (U.S. Biochemicals). Insert DNA was ligated into EcoR1/HindIII-digested puc18 and transformed into E. coli strain DH5α using standard protocols (Maniatis et al., 1982 in Molecular Cloning Cold Spring Harbor Press). The identity of positive clones was confirmed by sequencing small-scale plasmid preparations. Larger scale preparations of plasmid DNA for use as in vitro transcription templates and in transactions were prepared using the protocol and columns from QIAGEN Inc. (Studio City, CA) except that an additional ethanol precipitation was included as the final step.

Example 22: RNA Processing in vitro

Transcription reactions containing linear plasmid templates were carried out essentially as described (Milligan & Uhlenbeck, 1989 Supra: Chowrira & Burke, 1991 Supra). In order to prepare 5' end-labeled transcripts, standard transcription reactions were carried out in the presence of 10-20 μCi [γ-³²P]GTP, 200 μM each NTP and 0.5 to 1 μg of linearized plasmid template. The concentration of MgCl₂ was maintained at 10 mM above the total nucleotide concentration.

To compare the ability of the different ribozyme cassettes to self-process in vitro, each construct was transcribed and allowed to undergo self-processing under identical conditions at 37°C For these comparisons, equal amounts of linearized DNA templates bearing the various ribozyme cassettes were transcribed in the presence of [γ-³²P]GTP to generate 5' end-labeled transcripts. In this manner only the full-length, unprocessed transcripts and the released trans-ribozymes are visualized by autoradiography. In all reactions, Mg²⁺ was included at 10 mM above the nucleotide concentration so that cleavage by all the ribozyme cassettes would be supported. Transcription templates were linearized at several positions by digestion with different restriction enzymes so that self-processing in the presence of increasing lengths of downstream sequence could be compared (see Fig. 26). The resulting transcripts have either 4-5 non-ribozyme nucleotides at the 3' end (HindIII-digested template), 220 nucleotides (NdeI digested templates) or 454 nucleotides of downstream sequence (Real digested template).

As shown in Figure 27, all four ribozyme cassettes are capable of self-processing and yield RΝA products of expected sizes. Two nucleotides essential for hammerhead ribozyme activity (Ruffner et al., 1990 supra) have been changed in the HH(mutant) core sequence (see Figure 23) and so this transcript is unable to undergo self-processing (Fig. 27). This is evidenced by the lack of a released 5' RΝA in the HH(mutant), although the full-length RΝAs are present . Comparison of the amounts of released trans-ribozyme (Fig. 27) indicate that there are differences in the ability of these ribozymes to self-process in vitro, especially with respect to the presence of downstream sequence. For the two HP constructs, it is clear that HP(GC) is more efficient than the HP(GU) ribozyme, both in the presence and in the absence of extra downstream sequence. In addition, the activity of HP(GU) falls off more dramatically when downstream sequence is present. The stronger G:C base pair likely contributes to the HP(GC) construct's ability to fold correctly (and/or more quickly) into the productive structure, even when as much as 216 extra nucleotides are present downstream. The HH ribozyme construct is also quite efficient at self-processing, and slightly better than the HP(GU) construct even when downstream sequence is present.

Of the three ribozyme motifs, the presence of extra downstream sequence seems to most affect the efficiency of HPV. When no extra sequence is present downstream, HPV is quite efficient and self-processes to approximately the same level as the HH and HP(GC) cassettes. However, when extra downstream sequence is present, the self-processing activity seems to decrease almost as dramatically as is seen with the (sub-optimal) HP(GU) cassette. Example 23: Kinetics of self-processing reaction

HindIII-digested template (250 ng) was used in a standard transcription reaction mixture containing: 50 mM Tris·HCl pH 8.3; 1 mM ATP, GTP and UTP; 50 μM CTP; 40 μCi [α-³²P]CTP; 12 mM MgCl₂; 10 mM DTT. The transcription/self-processing reaction was initiated by the addition of T7 RNA polymerase (15 U/μl). Aliquots of 5 μl were taken at regular time intervals and the reaction was stopped by adding an equal volume of 2x formamide loading buffer (95% formamide, 15 mM EDTA, & dyes) and freezing on dry ice. The samples were resolved on a 10% polyacrylamide sequencing gel and results were quantitated by PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Ribozyme self-cleavage rates were determined from non-linear, least-squares fits (KaleidaGraph, Synergy Software, Reeding, PA) of the data to the equation:

where t represents time and k represents the unimolecular rate constant for cleavage (Long & Uhlenbeck, 1994 Proc. Natl. Acad. Sci. USA 91 , 6977).

Linear templates were prepared by digesting the plasmids with HindIII so that transcripts will contain only four to five vector-derived nucleotides at the 3' end (see Figure 23-25). By comparison of the unimolecular rate constant (k) determined for each construct, it is clear that HH is the most efficient at self-processing (Table 44). The HH transcript self-processes 2-fold faster than HDV and 3-fold faster than HP(GC) transcripts. Although the HP(GU) RNA undergoes self-processing, it is at least 6-fold slower than the HP(GC) construct. This is consistent with previous observations that the stability of helix 2 is essential for self-processing and trans-cleavage activity of the hairpin ribozyme (Hampel et al., 1990 supra: Chowrira & Burke, 1991 supra). The rate of HH self-cleavage during transcription measured here (1.2 min^-1 ) is similar to the rate measured by Long and Uhlenbeck 1994 supra using a HH that has a different stem I and stem III. Self-processing rates during transcription for HP and HDV have not been previously reported. However, self-processing of the HDV ribozyme-as measured here during transcription-is significantly slower than when tested after isolation from a denaturing gel (Been et al., 1992 supra). This decrease likely reflects the difference in protocol as well as the presence of 5' flanking sequence in the HDV construct used here. Example 24: Effect of downstream sequences on trans-cleavage in vitro

Transcripts containing the trans ribozyme with or without 3' flanking sequences were assayed for their ability to cleave their target in trans. To this end, transcripts from three templates were resolved on a preparative gel and bands corresponding both to processed trans-acting ribozymes from the HH transcription reaction, and to full-length HH(mutant) and ΔHDV transcripts were isolated. In all three transcripts the trans-acting ribozyme portion is identical-with the exception of sequences at their 3' ends. The HH trans-acting ribozyme contains only an additional UC at its 3' end, while HH(mutant) and ΔHDV have 52 and 37 nucleotides, respectively, at their 3' ends. A 622 nucleotide, internally-labeled target RNA was incubated, under ribozyme excess conditions, along with the three ribozyme transcripts in a standard reaction buffer.

To make internally-labeled substrate RNA for trans-ribozyme cleavage reactions, a 622 nt region (containing hammerhead site P) was synthesized by PCR using primers that place the T7 RNA promoter upstream of the amplified sequence. Target RNA was transcribed in a standard transcription buffer in the presence of [α-³²P]CTP (Chowrira & Burke, 1991 supra). The reaction mixture was treated with 15 units of ribonuclease-free DNasel, extracted with phenol followed chloroform:isoamyl alcohol (25:1), precipitated with isopropanol and washed with 70% ethanol. The dried pellet was resuspended in 20 μl DEPC-treated water and stored at -20°C.

Unlabeled ribozyme (1μM) and internally labeled 622 nt substrate RNA (<10 nM) were denatured and renatured separately in a standard cleavage buffer (containing 50 mM Tris·HCl pH 7.5 and 10 mM MgCl₂) by heating to 90°C for 2 min. and slow cooling to 37°C for 10 min. The reaction was initiated by mixing the ribozyme and substrate mixtures and incubating at 37°C Aliquots of 5 μl were taken at regular time intervals, quenched by adding an equal volume of 2X formamide gel loading buffer and frozen on dry ice. The samples were resolved on 5% polyacrylamide sequencing gel and results were quantitatively analyzed by radioanalytic imaging of gels with a Phosphorlmager® (Molecular Dynamics, Sunnyvale, CA). The HH trans-acting ribozyme cleaves the target RNA approximately

10-fold faster than the ΔHDV transcript and greater than 20-fold faster than the HH(mutant) transcript (Figure 28). The additional nucleotides at the end of HH(mutant) form 7 base-pairs with the 3' target-binding arm of the trans-acting ribozyme (Figure 23). This interaction must be disrupted (at a cost of 6 kcal/mole) to make the trans-acting ribozyme available for binding the target sequence. In contrast, the additional nucleotides at the end of ΔHDV were not designed to form any strong, alternative base-pairing with the trans-ribozyme. Nevertheless, the ΔHDV sequences are predicted to form multiple structures involving the 3' target-binding arm of the trans ribozyme that have stabilities ranging from 1-2 kcal/mole. Thus, the observed reductions in activity for the ΔHDV and HH(mutant) constructs are consistent with the predicted folded structures, and it reinforces the view that the flanking sequences can decrease the catalytic efficiency of a ribozyme through nonproductive interactions with either the ribozyme or the substrate or both. Example 25: RNA self-processing in vivo

Since three of the constructs (HH, HDV and HP(GC)) self-process efficiently in solution, the affect of the mammalian cellular milieu on ribozyme self-processing was next explored by applicant. A transient expression system was employed to investigate ribozyme activity in vivo. A mouse cell line (OST7-1) that constitutively expresses T7 RNA polymerase in the cytoplasm was chosen for this study (Elroy-Stein and Moss, 1990 Proc. Natl. Acad. Sci. USA 87, 6743). In these cells plasmids containing a ribozyme cassette downstream of the T7 promoter will be transcribed efficiently in the cytoplasm (Elroy-Stein & Moss, 1990 supra). Monolayers of a mouse L9 fibroblast cell line (OST7-1 ; Elroy-Stein and Moss, 1990 supra) were grown in 6-well plates with ~ 5×10⁵ cells/well. Cells were transfected with circular plasmids (5 μg/well) using the calcium phosphate-DNA precipitation method (Maniatis et al., 1982 supra). Cells were lysed (4 hours post-transfection) by the addition of standard lysis buffer (200 μl/well) containing 4M guanadinium isothiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sarkosyl (Chomczynski and Sacchi, 1987 Anal. Biochem. 162, 156), and 50 mM EDTA pH 8.0. The lysate was extracted once with water-saturated phenol followed by one extraction with chloroform:isoamyl alcohol (25:1). Total cellular RNA was precipitated with an equal volume of isopropanol. The RNA pellet was resuspended in 0.2 M ammonium acetate and reprecipitated with ethanol. The pellet was then washed with 70% ethanol and resuspended in DEPC-treated water.

Purified cellular RNA (3 μg/reaction) was first denatured in the presence of a 5' end-labeled DNA primer (100 pmol) by heating to 90°C for 2 min. in the absence of Mg²⁺, and then snap-cooling on ice for at least 15 min. This protocol allows for efficient annealing of the primer to its complementary RNA sequence. The primer was extended using Superscript II reverse transcriptase (8 U/μl; BRL) in a buffer containing 50 mM Tris·HCl pH 8.3; 10 mM DTT; 75 mM KCl; 1 mM MgCl₂; 1 mM each dNTP. The extension reaction was carried out at 42°C for 10 min. The reaction was terminated by adding an equal volume of 2x formamide gel loading buffer and freezing on crushed dry ice. The samples were resolved on a 10% polyacrylamide sequencing gel. The primer sequences are as follows: HH primer, 5'-CTCCAGTTTCGAGCTTT-3'; HDV primer, 5'-A A G T A G C C C A G G T C G G A C C - 3 ' ; H P p ri m e r , 5 ' - ACCAGGTAATATACCACAAC-3'.

As shown in Figure 29. specific bands corresponding to full-length precursor RNA and 3' cleavage products were detected from cells transfected with the self-processing cassettes. All three constructs, in addition to being transcriptionally active, appear to self-process efficiently in the cytoplasm of OST7-1 cells. In particular, the HH and HP(GC) constructs self-process to greater than 95%. The overall extent of self-processing in OST7-1 cells appears to be strikingly similar to the extent of self-processing in vitro (Figure 29 "In Vitro +MgCl₂" vs. "Cellular"). Consistent with the in vitro self-processing results, the HP(GU) cassette self-processed to approximately 50% in OST7-1 cells. As expected, transfection with plasmids containing the HH(mutant) cassette yielded a primer-extension product corresponding to the full-length RNA with no detectable cleavage products (Figure 29). The latter result strongly suggests that the primer extension band corresponding to the 3' cleavage product is not an artifact of reverse transcription.

Applicant was concerned with the possibility that RNA self-processing might occur during cell lysis, RNA isolation and /or the primer extension assay. Two precautions were taken to exclude this possibility. First, 50 mM EDTA was included in the lysis buffer. EDTA is a strong chelator of divalent metal ions such as Mg²⁺ and Ca²⁺ that are necessary for ribozyme activity. Divalent metal ions are therefore unavailable to self-processing RNAs following cell lysis. A second precaution involved using primers in the primer-extension assay that were designed to hybridize to essential regions of the processing ribozyme. Binding of these primers should prevent the 3' cis-acting ribozymes from folding into the conformation essential for catalytic activity.

Two experiments were carried out to further eliminate the possibility that self-processing is occurring either during RNA preparations or during the primer extension analysis. The first experiment involves primer extension analysis on full-length precursor RNAs that were added to non-transfected OST7-1 lysates after cell lysis. Thus, only if self-processing is occurring at some point after lysis would cleavage products be detected. Full-length precursor RNAs were prepared by transcribing under conditions of low Mg²⁺ (5 mM) and high NTP concentration (total 12 mM) in an attempt to eliminate the free Mg²⁺ required for the self-processing reaction (Michel et al. 1992 Genes & Dev. 6, 1373). The full-length precursor RNAs were gel-purified, and a known amount was added to lysates of non-transfected OST7-1 cells. RNA was purified from these lysates and incubated for 1 hr in DEPC-treated water at 37° C prior to the standard primer extension analysis (Figure 29. in vitro "-MgCl₂" control). The predominant RNA detected in all cases corresponds to the primer extension product of full-length precursor RNAs. If, instead, the purified RNA containing the full-length precursor is incubated in 10 mM MgCl₂ prior to the primer extension analysis, most or all of the RNA detected by primer extension analysis undergoes cleavage (Figure 29. in vitro "+MgCl₂" control). These results indicate that the standard RNA isolation and primer extension protocols used here do not provide a favorable environment for RNA self-processing, even though the RNA in question is inherently able to undergo self-cleavage.

In a second experiment to demonstrate lack of self-processing during work up, internally-labeled precursor RNAs were prepared and added to non-transfected OST7-1 lysates as in the previous control. The internally-labeled precursor RNAs were carried through the RNA purification and primer extension reactions (in the presence of unlabeled primers) and analyzed to determine the extent of self-processing. By this analysis, the vast majority of the added full-length RNA remained intact during the entire process of RNA isolation and primer extension.

These two control experiments validate the protocols used and support applicant's conclusion that the self-processing reactions catalyzed by HH, HDV and HP(GC) cassettes are occurring in the cytoplasm of OST7-1 cells.

Sequences in figures 23 through 25 are meant to be non-limiting examples. Those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art. In addition, those in the art will recognize that Applicant provides guidance through the above examples as to how to best design vectors of this invention so that secondary structure of the mRNA allows efficient cleavage by releasing ribozymes. Thus, the specific constructs are not limiting in this invention. Such constructs can be readily tested as described above for such secondary structure, either by computer folding algorithms or empirically. Such constructs will then allow at least 80% completion of release of ribozymes, which can be readily determined as described above or by methods known in the art. That is, any such secondary structure in the RNA does not reduce release of the ribozymes by more than 20%.

IV. Ribozymes Expressed by RNA Polymerase III

Applicant has determined that the level of production of a foreign RNA, using a RNA polymerase III (pol III) based system, can be significantly enhanced by ensuring that the RNA is produced with the 5' terminus and a 3' region of the RNA molecule base-paired together to form a stable intramolecular stem structure. This stem structure is formed by hydrogen bond interactions (either Watson-Crick or non-Watson-Crick) between nucleotides in the 3' region (at least 8 bases) and complementary nucleotides in the 5' terminus of the same RNA molecule. Although the example provided below involves a type 2 pol III gene unit, a number of other pol III promoter systems can also be used, for example, tRNA (Hall et al., 1982 Cell 29, 3-5), 5S RNA (Nielsen et al., 1993, Nucleic Acids Res. 21 , 3631-3636), adenovirus VA RNA (Fowlkes and Shenk, 1980 Cell 22, 405-413), U6 snRNA (Gupta and Reddy, 1990 Nucleic Acids Res. 19, 2073-2075), vault RNA (Kickoefer et al., 1993 J. Biol. Chem. 268, 7868-7873), telomerase RNA (Romero and Blackburn, 1991 Cell 67, 343-353), and others.

The construct described in this invention is able to accumulate RNA to a significantly higher level than other constructs, even those in which 5' and 3' ends are involved in hairpin loops. Using such a construct the level of expression of a foreign RNA can be increased to between 20,000 and 50,000 copies per cell. This makes such constructs, and the vectors encoding such constructs, excellent for use in decoy, therapeutic editing and antisense protocols as well as for ribozyme formation. In addition, the molecules can be used as agonist or antagonist RNAs (affinity RNAs). Generally, applicant believes that the intramolecular base-paired interaction between the 5' terminus and the 3' region of the RNA should be in a double-stranded structure in order to achieve enhanced RNA accumulation.

Thus, in one preferred embodiment the invention features a pol III promoter system (e.g., a type 2 system) used to synthesize a chimeric RNA molecule which includes tRNA sequences and a desired RNA (e.g., a tRNA-based molecule). The following exemplifies this invention with a type 2 pol III promoter and a tRNA gene. Specifically to illustrate the broad invention, the RNA molecule in the following example has an A box and a B box of the type 2 pol III promoter system and has a 5' terminus or region able to base-pair with at least 8 bases of a complementary 3' end or region of the same RNA molecule. This is meant to be a specific example. Those in the art will recognize that this is but one example, and other embodiments can be readily generated using other pol III promoter systems and techniques generally known in the art.

By "terminus" is meant the terminal bases of an RNA molecule, ending in a 3' hydroxyl or 5' phosphate or 5' cap moiety. By "region" is meant a stretch of bases 5' or 3' from the terminus that are involved in base-paired interactions. It need not be adjacent to the end of the RNA. Applicant has determined that base pairing of at least one end of the RNA molecule with a region not more than about 50 bases, and preferably only 20 bases, from the other end of the molecule provides a useful molecule able to be expressed at high levels.

By "3' region" is meant a stretch of bases 3' from the terminus that are involved in intramolecular bas-paired interaction with complementary nucleotides in the 5' terminus of the same molecule. The 3' region can be designed to include the 3' terminus. The 3' region therefore is > 0 nucleotides from the 3' terminus. For example, in the S35 construct described in the present invention (Fig. 40) the 3' region is one nucleotide from the 3' terminus. In another example, the 3' region is ~ 43 nt from 3' terminus. These examples are not meant to be limiting. Those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art. Generally, it is preferred to have the 3' region within 100 bases of the 3' terminus.

By "tRNA molecule" is meant a type 2 pol III driven RNA molecule that is generally derived from any recognized tRNA gene. Those in the art will recognize that DNA encoding such molecules is readily available and can be modified as desired to alter one or more bases within the DNA encoding the RNA molecule and/or the promoter system. Generally, but not always, such molecules include an A box and a B box that consist of sequences which are well known in the art (and examples of which can be found throughout the literature). These A and B boxes have a certain consensus sequence which is essential for a optimal pol III transcription.

By "chimeric tRNA molecule" is meant a RNA molecule that includes a pol III promoter (type 2) region. A chimeric tRNA molecule, for example, might contain an intramolecular base-paired structure between the 3' region and complementary 5' terminus of the molecule, and includes a foreign RNA sequence at any location within the molecule which does not affect the activity of the type 2 pol III promoter boxes. Thus, such a foreign RNA may be provided at the 3' end of the B box, or may be provided in between the A and the B box, with the B box moved to an appropriate location either within the foreign RNA or another location such that it is effective to provide pol III transcription. In one example, the RNA molecule may include a hammerhead ribozyme with the B box of a type 2 pol III promoter provided in stem II of the ribozyme. In a second example, the B box may be provided in stem IV region of a hairpin ribozyme. A specific example of such RNA molecules is provided below. Those in the art will recognize that this is but one example, and other embodiments can be readily generated using techniques generally known in the art.

By "desired RNA" molecule is meant any foreign RNA molecule which is useful from a therapeutic, diagnostic, or other viewpoint. Such molecules include antisense RNA molecules, decoy RNA molecules, enzymatic RNA, therapeutic editing RNA and agonist and antagonist RNA.

By "antisense RNA" is meant a non-enzymatic RNA molecule that binds to another RNA (target RNA) by means of RNA-RNA interactions and alters the activity of the target RNA (Eguchi et al., 1991 Annu. Rev. Biochem. 60, 631-652). By "enzymatic RNA" is meant an RNA molecule with enzymatic activity (Cech, 1988 J. American. Med. Assoc. 260, 3030-3035). Enzymatic nucleic acids (ribozymes) act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA.

By "decoy RNA" is meant an RNA molecule that mimics the natural binding domain for a ligand. The decoy RNA therefore competes with natural binding target for the binding of a specific ligand. For example, it has been shown that over-expression of HIV trans-activation response (TAR) RNA can act as a "decoy" and efficiently binds HIV tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA (Sullenger et al., 1990 Cell 63, 601-608). This is meant to be a specific example. Those in the art will recognize that this is but one example, and other embodiments can be readily generated using techniques generally known in the art.

By "therapeutic editing RNA" is meant an antisense RNA that can bind to its cellular target (RNA or DNA) and mediate the modification of a specific base.

By "agonist RNA" is meant an RNA molecule that can bind to protein receptors with high affinity and cause the stimulation of specific cellular pathways. By "antagonist RNA" is meant an RNA molecule that can bind to cellular proteins and prevent it from performing its normal biological function (for example, see Tsai et al., 1992 Proc. Natl. Acad. Sci. USA 89, 8864-8868). In other aspects, the invention includes vectors encoding RNA molecules as described above, cells including such vectors, methods for producing the desired RNA, and use of the vectors and cells to produce this RNA.

Thus, the invention features a transcribed non-naturally occuring RNA molecule which includes a desired therapeutic RNA portion and an intramolecular stem formed by base-pairing interactions between a 3' region and complementary nucleotides at the 5' terminus in the RNA. The stem preferably includes at least 8 base pairs, but may have more, for example, 15 or 16 base pairs. In preferred embodiments, the 5' terminus of the chimeric tRNA includes a portion of the precursor molecule of the primary tRNA molecule, of which≥ 8 nucleotides are involved in base-pairing interaction with the 3' region; the chimeric tRNA contains A and B boxes; natural sequences 3' of the B box are deleted, which prevents endogenous RNA processing; the desired RNA molecule is at the 3' end of the B box; the desired RNA molecule is between the A and the B box; the desired RNA molecule includes the B box; the desired RNA molecule is selected from the group consisting of antisense RNA, decoy RNA, therapeutic editing RNA, enzymatic RNA, agonist RNA and antagonist RNA; the molecule has an intramolecular stem resulting from a base-paired interaction between the 5' terminus of the RNA and a complementary 3' region within the same RNA, and includes at least 8 bases; and the 5' terminus is able to base pair with at least 15 bases of the 3' region.

In most preferred embodiments, the molecule is transcribed by a RNA polymerase III based promoter system, e.g., a type 2 pol III promoter system; the molecule is a chimeric tRNA, and may have the A and B boxes of a type 2 pol III promoter separated by between 0 and 300 bases; DNA vector encoding the RNA molecule of claim 51. In other related aspects, the invention features an RNA or DNA vector encoding the above RNA molecule, with the portions of the vector encoding the RNA functioning as a RNA pol III promoter; or a cell containing the vector ; or a method to provide a desired RNA molecule in a cell, by introducing the molecule into a cell with an RNA molecule as described above. The cells can be derived from animals, plants or human beings.

In order for RNA-based gene therapy approaches to be effective, sufficient amounts of the therapeutic RNA must accumulate in the appropriate intracellular compartment of the treated cells. Accumulation is a function of both promoter strength of the antiviral gene, and the intracellular stability of the antiviral RNA. Both RNA polymerase II (pol II) and RNA polymerase III (pol III) based expression systems have been used to produce therapeutic RNAs in cells (Sarver & Rossi, 1993 AIDS Res. & Human Retroviruses 9, 483-487; Yu et al., 1993 P.N.A.S. (USA) 90, 6340-6344). However, pol III based expression cassettes are theoretically more attractive for use in expressing antiviral RΝAs for the following reasons. Pol II produces messenger RΝAs located exclusively in the cytoplasm, whereas pol III produces functional RΝAs found in both the nucleus and the cytoplasm. Pol II promoters tend to be more tissue restricted, whereas pol III genes encode tRΝAs and other functional RΝAs necessary for basic "housekeeping" functions in all cell types. Therefore, pol III promoters are likely to be expressed in all tissue types. Finally, pol III transcripts from a given gene accumulate to much greater levels in cells relative to pol II genes. Intracellular accumulation of therapeutic RΝAs is also dependent on the method of gene transfer used. For example, the retroviral vectors presently used to accomplish stable gene transfer, integrate randomly into the genome of target cells. This random integration leads to varied expression of the transferred gene in individual cells comprising the bulk treated cell population. Therefore, for maximum effectiveness, the transferred gene must have the capacity to express therapeutic amounts of the antiviral RΝA in the entire treated cell population, regardless of the integration site. Pol III System

The following is just one non-limiting example of the invention. A pol III based genetic element derived from a human tRNA_i ^met gene and termed Δ3-5 (Fig. 33: Adeniyi-Jones et al., 1984 supra), has been adapted to express antiviral RNAs (Sullenger et al., 1990 Mol. Cell. Biol. 10, 6512-6523). This element was inserted into the DC retroviral vector (Sullenger et al., 1990 Mol. Cell. Biol. 10, 6512-6523) to accomplish stable gene transfer, and used to express antisense RNAs against moloney murine leukemia virus and anti-HIV decoy RNAs (Sullenger et al., 1990 Mol. Cell. Biol. 10, 6512-6523; Sullenger et al., 1990 Cell 63, 601-608; Sullenger et al., 1991 J. Virol. 65, 6811-6816; Lee et al., 1992 The New Biologist 4, 66-74). Clonal lines are expanded from individual cells present in the bulk population, and therefore express similar amounts of the therapeutic RNA in all cells. Development of a vector system that generates therapeutic levels of therapeutic RNA in all treated cells would represent a significant advancement in RNA based gene therapy modalities.

Applicant examined hammerhead (HHI) ribozyme (RNA with enzymatic activity) expression in human T cell lines using the Δ3-5 vector system (These constructs are termed "Δ3-5/HHI"; Fig. 34). On average, ribozymes were found to accumulate to less than 100 copies per cell in the bulk T cell populations. In an attempt to improve expression levels of the Δ3-5 chimera, the applicant made a series of modified Δ3-5 gene units containing enhanced promoter elements to increase transcription rates, and inserted structural elements to improve the intracellular stability of the ribozyme transcripts (Fig. 34). One of these modified gene units, termed S35, gave rise to more than a 100-fold increase in ribozyme accumulation in bulk T cell populations relative to the original Δ3-5/HHI vector system. Ribozyme accumulation in individual clonal lines from the pooled T cell populations ranged from 10 to greater than 100 fold more than those achieved with the original Δ3-5/HHI version of this vector.

The S35 gene unit may be used to express other therapeutic RNAs including, but not limited to, ribozymes, antisense, decoy, therapeutic editing, agonist and antagonist RNAs. Application of the S35 gene unit would not be limited to antiviral therapies, but also to other diseases, such as cancer, in which therapeutic RNAs may be effective. The S35 gene unit may be used in the context of other vector systems besides retroviral vectors, including but not limited to, other stable gene transfer systems such as adeno-associated virus (AAV; Carter, 1992 Curr. Opin. Genet. Dev. 3, 74), as well as transient vector systems such as plasmid delivery and adenoviral vectors (Berkner, 1988 BioTechniques 6, 616-629). As described below, the S35 vector encodes a truncated version of a tRNA wherein the 3' region of the RNA is base-paired to complementary nucleotides at the 5' terminus, which includes the 5' precursor portion that is normally processed off during tRNA maturation. Without being bound by any theory, Applicant believes this feature is important in the level of expression observed. Thus, those in the art can now design equivalent RNA molecules with such high expression levels. Below are provided examples of the methodology by which such vectors and tRNA molecules can be made.

Δ3-5 Vectors The use of a truncated human tRNA_i ^met gene, termed Δ3-5 (Fig. 33:

Adeniyi-Jones et al., 1984 supra), to drive expression of antisense RNAs, and subsequently decoy RNAs (Sullenger et al., 1990 supra) has recently been reported. Because tRNA genes utilize internal pol III promoters, the antisense and decoy RNA sequences were expressed as chimeras containing tRNA_i ^met sequences. The truncated tRNA genes were placed into the U3 region of the 3' moloney murine leukemia virus vector LTR (Sullenger et al., 1990 supra).

Base-Paired Structures

Since the Δ3-5 vector combination has been successfully used to express inhibitory levels of both antisense and decoy RNAs, applicant cloned ribozyme-encoding sequences (termed as "Δ3-5/HHI") into this vector to explore its utility for expressing therapeutic ribozymes. However, low ribozyme accumulation in human T cell lines stably transduced with this vector was observed (Fig. 35). To try and improve accumulation of the ribozyme, applicant incorporated various RNA structural elements (Fig. 34) into one of the ribozyme chimeras (Δ3-5/HHI).

Two strategies were used to try and protect the termini of the chimeric transcripts from exonucleolytic degredation. One strategy involved the incorporation of stem-loop structures into the termini of the transcript. Two such constructs were cloned, S3 which contains a stem-loop structure at the 3' end, and S5 which contains stem-loop structures at both ends of the transcript (Figure 34). The second strategy involved modification of the 3' terminal sequences such that the 5' terminus and the 3' end sequences can form a stable base-paired stem. Two such constructs were made: S35 in which the 3' end was altered to hybridize to the 5' leader and acceptor stem of the tRNA_i ^met domain, and S35Plus which was identical to S35 but included more extensive structure formation within the non-ribozyme portion of the Δ3-5 chimeras (Figure 34). These stem-loop structures are also intended to sequester non-ribozyme sequences in structures that will prevent them from interfering with the catalytic activity of the ribozyme. These constructs were cloned, producer cell lines were generated, and stably-transduced human MT2 (Harada et al., 1985 supra) and CEM (Nara & Fischinger, 1988 supra) cell lines were established (Curr. Protocols Mol. Biol. 1992, ed. Ausubel et al., Wiley & Sons, NY). The RNA sequences and structure of S35 and S35 Plus are provided in Figures 40-47.

Referring to Figure 48. there is provided a general structure for a chimeric RNA molecule of this invention. Each N independently represents none or a number of bases which may or may not be base paired. The A and B boxes are optional and can be any known A or B box, or a consensus sequence as exemplified in the figure. The desired nucleic acid to be expressed can be any location in the molecule, but preferably is on those places shown adjacent to or between the A and B boxes (designated by arrows). Figure 49 shows one example of such a structure in which a desired RNA is provided 3' of the intramolecular stem. A specific example of such a construct is provided in Figures 50 and 51.

Example 26: Cloning of Δ3-5-Ribozyme Chimera

Oligonucleotides encoding the S35 insert that overlap by at least 15 nucleotides were designed (5' GATCCACTCTGCTGTTCTGTTTTTGA 3' and 5' CGCGTCAAAAACAGAACAGCAGAGTG 3'). The oligonucleotides (10 μM each) were denatured by boiling for 5 min in a buffer containing 40 mM Tris.HCl, pH8.0. The oligonucleotides were allowed to anneal by snap cooling on ice for 10-15 min.

The annealed oligonucleotide mixture was converted into a double-stranded molecule using Sequenase® enzyme (US Biochemicals) in a buffer containing 40 mM Tris.HCI, pH7.5, 20 mM MgCl₂, 50 mM NaCl, 0.5 mM each of the four deoxyribonucleotide triphosphates, 10 mM DTT. The reaction was allowed to proceed at 37°C for 30 min. The reaction was stopped by heating to 70°C for 15 min. The double stranded DNA was digested with appropriate restriction endonucleases (BamHI and MluI) to generate ends that were suitable for cloning into the Δ3-5 vector.

The double-stranded insert DNA was ligated to the Δ3-5 vector DNA by incubating at room temperature (about 20°C) for 60 min in a buffer containing 66 mM Tris.HCl, pH 7.6, 6.6 mM MgCl₂, 10 mM DTT, 0.066 μM ATP and 0.1 U/μl T4 DNA Ligase (US Biochemicals).

Competent E. coli bacterial strain was transformed with the recombinant vector DNA by mixing the cells and DNA on ice for 60 min. The mixture was heat-shocked by heating to 37°C for 1 min. The reaction mixture was diluted with LB media and the cells were allowed to recover for 60 min at 37°C The cells were plated on LB agar plates and incubated at 37°C for ~ 18 h.

Plasmid DNA was isolated from an overnight culture of recombinant clones using standard protocols (Ausubel et al., Curr. Protocols Mol. Biology 1990, Wiley & Sons, NY).

The identity of the clones were determined by sequencing the plasmid DNA using the Sequenase DNA sequencing kit (US Biochemicals).

The resulting recombinant Δ3-5 vector contains the S35 sequence. The HHI encoding DNA was cloned into this Δ3-5-S35 containing vector using SaclI and BamHI restriction sites.

Example 27: Northern analysis

RNA from the transduced MT2 cells were extracted and the presence of Δ3-5/ribozyme chimeric transcripts were assayed by Northern analysis (Curr. Protocols Mol. Biol. 1992, ed. Ausubel et al., Wiley & Sons, NY). Northern analysis of RNA extracted from MT2 transductants showed that Δ3-5/ribozyme chimeras of appropriate sizes were expressed (Fig. 35.36). In addition, these results demonstrated the relative differences in accumulation among the different constructs (Figure 35.36). The pattern of expression seen from the Δ3-5/HHI ribozyme chimera was similar to 12 other ribozymes cloned into the Δ3-5 vector (not shown). In MT-2 cell line, Δ3-5/HHI ribozyme chimeras accumulated, on average, to less than 100 copies per cell. Addition of a stem-loop onto the 3' end of Δ3-5/HHI did not lead to increased Δ3-5 levels (S3 in Fig. 35,36). The S5 construct containing both 5' and 3' stem-loop structures also did not lead to increased ribozyme levels (Fig. 35.36).

Interestingly, the S35 construct expression in MT2 cells was about 100-fold more abundant relative to the original Δ3-5/HHI vector transcripts (Fig. 35,36). This may be due to increased stability of the S35 transcript.

Example 28: Cleavage activity

To assay whether ribozymes transcribed in the transduced cells contained cleavage activity, total RNA extracted from the transduced MT2 T cells were incubated with a labeled substrate containing the HHI cleavage site (Figure 37). Ribozyme activity in all but the S35 constructs, was too low to detect. However, ribozyme activity was detectable in S35-transduced T cell RNA. Comparison of the activity observed in the S35-transduced MT2 RNA with that seen with MT2 RNA in which varying amounts of in vitro transcribed S5 ribozyme chimeras, indicated that between 1-3 nM of S35 ribozyme was present in S35-transduced MT2 RNA. This level of activity corresponds to an intracellular concentration of 5,000-15,000 ribozyme molecules per cell.

Example 29: Clonal variation Variation in the ribozyme expression levels among cells making up the bulk population was determined by generating several clonal cell lines from the bulk S35 transduced CEM line (Curr. Protocols Mol. Biol. 1992, ed. Ausubel et al., Wiley & Sons, NY) and the ribozyme expression and activity levels in the individual clones were measured (Figure 38 and 39). All the individual clones were found to express active ribozyme. The ribozyme activity detected from each clone correlated well with the relative amounts of ribozyme observed by Northern analysis. Steady state ribozyme levels among the clones ranged from approximately 1 ,000 molecules per cell in clone G to 11 ,000 molecules per cell in clone H (Fig. 38). The mean accumulation among the clones, calculated by averaging the ribozyme levels of the clones, exactly equaled the level measured in the parent bulk population. This suggests that the individual clones are representative of the variation present in the bulk population. The fact that all 14 clones were found to express ribozyme indicate that the percentage of cells in the bulk population expressing ribozyme is also very high. In addition, the lowest level of expression in the clones was still more than 10-fold that seen in bulk cells transduced with the original Δ3-5 vector. Therefore, the S35 gene unit should be much more effective in a gene therapy setting in which bulk cells are removed, transduced and then reintroduced back into a patient.

Example 30: Stability

Finally, the bulk S35-transduced line, resistant to G418, was propogated for a period of 3 months (in the absence of G418) to determine if ribozyme expression was stable over extended periods of time. This situation mimicks that found in the clinic in which bulk cells are transduced and then reintroduced into the patient and allowed to propogate. There was a modest 30% reduction of ribozyme expression after 3 months. This difference probably arose from cells with varying amount of ribozyme expression and exhibiting different growth rates in the culture becoming slightly more prevalent in the culture. However, ribozyme expression is apparently stable for at least this period of time.

Example 31 : Design and construction of TRZ-tRNA Chimera

A transcription unit, termed TRZ, is designed that contains the S35 motif (Figure 52). A desired RNA (e.g. ribozyme) can be inserted into the indicated region of TRZ tRNA chimera. This construct might provide additional stability to the desired RNA. TRZ-A and TRZ-B are non-limiting examples of the TRZ-tRNA chimera.

Referring to Fig. 53-54. a hammerhead ribozyme targeted to site I (HHITRZ-A; Fig. 53) and a hairpin ribozyme (HPITRZ-A; Fig. 54), also targeted to site I, is cloned individually into the indicated region of TRZ tRNA chimera. The resulting ribozyme trancripts retain full RNA cleavage activity (see for example Fig. 55). Applicant has shown that efficient expression of these TRZ tRNA chimera can be achieved in mammalian cells.

Besides ribozymes, desired RNAs like antisense, therapeutic editing RNAs, decoys, can be readily inserted into the indicated region of TRZ-tRNA chimera to achieve therapeutic levels of RNA expression in mammalian cells.

Sequences listed in Figures 40-47 and 50 - 54 are meant to be non-limiting examples. Those skilled in the art will recognize that variants (mutations, insertions and deletions) of the above examples can be readily generated using techniques known in the art, are within the scope of the present invention.

Example 32: Ribozyme expression in T cell lines

Ribozyme expression in T cell lines stably-transduced with either a retroviral-based or an Adeno-associated virus (AAV)-based ribozyme expression vector (Figure 56). The human T cell lines MT2 and CEM were transduced with either retroviral or AAV vectors encoding a neomycin slelctable marker and a ribozyme (S35/HHI) expressed from pol III met_itRNA-driven promoter. Cells stably-transduced with the vectors were selectivelyt expanded medium containing the neomycin antibiotic derivative, G418 (0.7 mg/ml). Ribozyme expression in the stable cell lines was then alalyzed by Northern analysis. The probe used to detect ribozyme transcripts also cross-hybridized with human met_i tRNA sequences. Refering to Figure 56, S35/HHI RNA accumulates to significant levels in MT2 and CEM cells when transduced with either the retrovirus or the AAV vector.

These are meant to be non-limiting examples, those skilled in the art will recognize that other vectors such as adenovirus vector (Figure 57), plasmid DNA vector, alpha virus vectors and the other derivatives there of, can be readily generated to deliver the desired RNA, using techniques known in the art and are within the scope of this invention. Additionally, the transcription units can be expressed individually or in multiples using pol II and/or pol III promoters.

References cited herein, as well as Draper WO 93/23569, 94/02495, 94/06331 , Sullenger WO 93/12657, Thompson WO 93/04573, and Sullivan WO 94/04609, and 93/11253 describe methods for use of vectors decribed herein, and are incorporated by reference herein. In particular these vectors are useful for administration of antisense and decoy RNA molecules. Example 33: Ligated Ribozymes are catalytically active

The ability of ribozymes generated by ligation methods, described in Praper et al., PCT WO 93/23569, to cleave target RNA was tested on either matched substrate RNA (Fig. 58) or long (622 nt) RNA (Fig. 59, 60 and 61).

Matched substrate RNAs were chemically synthesized using solid-phase RNA synthesis chemistry (Scaringe et al., 1990 Nucleic Acids Res. 18, 5433-5441). Substrate RNA was 5' end-labeled using [γ-³²P] ATP and polynucleotide kinase (Curr. Protocols Mol. Biol. 1992, ed. Ausubel et al., Wiley & Sons, NY). Ribozyme reactions were carried out under ribozyme excess conditions (kcat/KM; Herschlag and Cech, 1990 Biochemistry 29, 10159-10171). Briefly, ribozyme and substrate RNA were denatured and renatured separately by heating to 90°C and snap cooling on ice for 10 min in a buffer containing 50 mM Tris. HCl pH 7.5 and 10 mM MgCl₂. Cleavage reaction was initiated by mixing the ribozyme with the substrate at 37°C Aliquots of 5 μl were taken at regular intervals of time and the reaction was stopped by mixing with equal volume of formamide gel loading buffer (Curr. Protocols Mol. Biol. 1992, ed. Ausubel et al., Wiley & Sons, NY). The samples were resolved on 20 % polyacrylamide-urea gel. Refering to Fig. 58. -ΔG refers to the free energy of binding calculated for base-paired interactions between the ribozyme and the substrate RNA (Turner and Sugimoto, 1988 Supra). RPI A is a HH ribozyme with 6/6 binding arms. This ribozyme was synthesized chemically either as a one piece ribozyme or was synthesized in two fragments followed by ligation to generate a one piece ribozyme. The kcat/KM values for the two ribozymes were comparable. A template containing T7 RNA polymerase promoter upstream of 622 nt long target sequence, was PCR amplified from a DNA clone. The target RNA (containing HH ribozyme cleavage sites B, C and D) was transcribed from this PCR amplified template using T7 RNA polymerase. The transcript was internally labeled during transcription by including [α-³²P] CTP as one of the four ribonucleotide triphosphates. The transcription mixture was treated with DNase-1 , following transcription at 37°C for 2 hours, to digest away the DNA template used in the transcription. RNA was precipitated with Isopropanol and the pellet was washed two times with 70% ethanol to get rid of salt and nucleotides used in the transcription reaction. RNA is resuspended in DEPC-treated water and stored at 4°C Ribozyme cleavage reactions were carried out under ribozyme excess (kcat/KM) conditions [Herschlag and Cech 1990 supra]. Briefly, 1000 nM ribozyme and 10 nM internally labeled target RNA were denatured separately by heating to 90°C for 2 min in the presence of 50 mM Tris.HCl, pH 7.5 and 10 mM MgCl_2. The RNAs were renatured by cooling to 37°C for 10-20 min. Cleavage reaction was initiated by mixing the ribozyme and target RNA at 37°C Aliquots of 5 μl were taken at regular intervals of time and the reaction was quenched by adding equal volume of stop buffer. The samples were resolved on a sequencing gel. Example 34: Hammerhead ribozymes with≥ 2 base-paired stem II are catalytically active

To decrease the cost of chemical synthesis of RNA, applicant was interested in determining whether the length of stem II region of a typical hammerhead ribozyme (≥ 4 bp stem II) can be shortened without decreasing the catalytic efficiency of the HH ribozyme. The length of stem II was systematically shortened by one base-pair at a time. HH ribozymes with three and two base-paired stem II were chemically synthesized using solid-phase RNA phosphoramidite chemistry (Scaringe et al., 1990 supra).

Matched and long substrate RNAs were synthesized and ribozyme assays were carried out as described in example 33. Referring to figures , 62, 63 and 64. data shows that shortening stem II of a hammerhead ribozyme does not significantly alter the catalytic efficiency. It is applicant's opinion that hammerhead ribozymes with≥ 2 base-paired stem II region are catalytically active. Example 35: Synthesis of catalytically active hairpin ribozymes

RNA molecules were chemically synthesized having the nucleotide base sequence shown in Fig. 65 for both the 5' and 3' fragments. The 3' fragments are phosphorylated and ligated to the 5' fragment essentially as described in example 37. As is evident from the Figure 65, the 3' and 5' fragments can hybridize together at helix 4 and are covalently linked via GAAA sequence. When this structure hybridizes to a substrate, a ribozyme•substrate complex structure is formed. While helix 4 is shown as 3 base pairs it may be formed with only 1 or 2 base pairs.

40 nM mixtures of ligated ribozymes were incubated with 1-5 nM 5' end-labeled matched substrates (chemically synthesized by solid-phase synthesis using RNA phosphoramidite chemistry) for different times in 50 mM Tris/HCl pH 7.5, 10 mM MgCl₂ and shown to cleave the substrate efficiently (Fig.66).

The target and the ribozyme sequences shown in Fig. 62 and 65 are meant to be non-limiting examples. Those in the art will recognize that other embodiments can be readily generated using other sequences and techniques generally known in the art.

V. Constructs of Hairpin Ribozymes

There follows an improved trans-cleaving hairpin ribozyme in which a new helix (i.e., a sequence able to form a double-stranded region with another single-stranded nucleic acid) is provided in the ribozyme to base-pair with a 5' region of a separate substrate nucleic acid. This helix is provided at the 3' end of the ribozyme after helix 3 as shown in Figure 3. In addition, at least two extra bases may be provided in helix 2 and a portion of the substrate corresponding to helix 2 may be either directly linked to the 5' portion able to hydrogen bond to the 3' end of the hairpin or may have a linker of atleast one base. By trans-cleaving is meant that the ribozyme is able to act in trans to cleave another RNA molecule which is not covalently linked to the ribozyme itself. Thus, the ribozyme is not able to act on itself in an intramolecular cleavage reaction.

By "base-pair" is meant a nucleic acid that can form hydrogen bond(s) with other RNA sequence by either traditional Watson-Crick or other non-traditional types (for example Hoogsteen type) of interactions.

The increase in length of helix 2 of a hairpin ribozyme (with or without helix 5) has several advantages. These include improved stability of the ribozyme-target complex in vivo . In addition, an increase in the recognition sequence of the hairpin ribozyme improves the specificity of the ribozyme. This also makes possible the targeting of potential hairpin ribozyme sites that would otherwise be inaccessible due to neighboring secondary structure.

The increase in length of helix 2 of a hairpin ribozyme (with or without helix 5) enhances trans-ligation reaction catalyzed by the ribozyme. Trans-ligation reactions catalyzed by the regular hairpin ribozyme (4 bp helix 2) is very inefficient (Komatsu et al., 1993 Nucleic Acids Res. 21 , 185). This is attributed to weak base-pairing interactions between substrate RNAs and the ribozyme. By increasing the length of helix 2 (with or without helix 5) the rate of ligation (in vitro and in vivo) can be enhanced several fold. Results of experiments suggest that the length of H2 can be 6 bp without significantly reducing the activity of the hairpin ribozyme. The H2 arm length variation does not appear to be sequence dependent. HP ribozymes with 6 bp H2 have been designed against five different target RNAs and all five ribozymes efficiently cleaved their cognate target RNA. Additionally, two of these ribozymes were able to successfully inhibit gene expression (e.g., TNF-α) in mammalian cells. Results of these experiments are shown below.

HP ribozymes with 7 and 8 bp H2 are also capable of cleaving target RNA in a sequence-specific manner, however, the rate of the cleavage reaction is lower than those catalyzed by HP ribozymes with 6 bp H2.

Example 36: 4 and 6 base pair H2

Referring to Figures 67-72. HP ribozymes were synthesized as described above and tested for activity. Surprisingly, those with 6 base pairs in H2 were still as active as those with 4 base pairs. VI. Chemical Modification

Oligonucleotides with 5'-C-alkyl Group

The introduction of an alkyl group at the 5'-position of a nucleoside or nucleotide sugar introduces an additional center of chirality into the sugar moiety. Referring to Fig. 75. the general structures of 5'-C-alkylnucleotides belonging to the D-allose, 2, and L-talose, 3, sugar families are shown. The family names are derived from the known sugars D-allose and L-talose (R ₁ = CH₃ in 2 and 3 in Figure 75). Useful specific D-allose and L-talose nucleotide derivatives are shown in Figure 76. 29-32 and Figure 77, 58-61 respectively.

This invention relates to the use of 5'-C-alkylnucleotides in oligonucleotides, which are particularly useful for enzymatic cleavage of RNA or single-stranded DNA, and also as antisense oligonucleotides. As the term is used in this application, 5'-C-alkylnucleotide-containing enzymatic nucleic acids are catalytic nucleic molecules that contain 5'-C-alkylnucleotide components replacing, but not limited to, double stranded stems, single stranded "catalytic core" sequences, single-stranded loops or single-stranded recognition sequences. These molecules are able to cleave (preferably, repeatedly cleave) separate RNA or DNA molecules in a nucleotide base sequence specific manner. Such catalytic nucleic acids can also act to cleave intramolecularly if that is desired. Such enzymatic molecules can be targeted to virtually any RNA transcript. Also within the invention are 5'-C-alkylnucleotides which may be present in enzymatic nucleic acid or even in antisense oligonucleotides. Such nucleotides are useful since they enhance the stability of the antisense or enzymatic molecule, and can be used in locations which do not affect the desired activity of the molecule. That is, while the presence of the 5'-C-alkyl group may reduce binding affinity of the oligonucleotide containing this modification, if that moiety is not in an essential base pair forming region then the enhanced stability that it provides to the molecule is advantageous. In addition, while the reduced binding may reduce enzymatic activity, the enhanced stability may make the loss of activity of less consequence. Thus, for example, if a 5'-C-alkyl-containing molecule has 10% the activity of the unmodified molecule, but has 10-fold higher stability in vivo then it has utility in the present invention. The same analysis is true for antisense oligonucleotides containing such modifications. The invention also relates to novel intermediates useful in the synthesis of such nucleotides and oligonucleotides (examples of which are shown in the Figures), and to methods for their synthesis.

Thus, in one aspect, the invention features 5'-C-alkylnucleosides, that is a nucleotide base having at the 5'-position on the sugar molecule an alkyl moiety. In a related aspect, the invention also features 5'-C-alkylnucleotides, and in preferred embodiments features those where the nucleotide is not uridine or thymidine. That is, the invention preferably includes all those nucleotides useful for making enzymatic nucleic acids or antisense molecules that are not described by the art discussed above. In preferred embodiments, the sugar of the nucleoside or nucleotide is in an optically pure form, as the talose or allose sugar. Examples of various alkyl groups useful in this invention are shown in

Figure 75. where each R₁ group is any alkyl. These examples are not limiting in the invention. Specifically, an "alkyl" group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, =O, =S, NO₂ or N(CH₃)₂, amino, or SH. The term also includes alkenyl groups which are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, =O, =S, NO₂, halogen, N(CH₃)₂, amino, or SH. The term "alkyl" also includes alkynyl groups which have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, =O, =S, NO₂ or N(CH₃)₂, amino or SH.

Such alkyl groups may also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An "aryl" group refers to an aromatic group which has at least one ring having a conjugated π electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An "alkylaryl" group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above. Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An "amide" refers to an -C(O)-NH-R, where R is either alkyl, aryl, alkylaryl or hydrogen. An "ester" refers to an -C(0)-OR', where R is either alkyl, aryl, alkylaryl or hydrogen. In other aspects, also related to those discussed above, the invention features oligonucleotides having one or more 5'-C-alkylnucleotides; e.g. enzymatic nucleic acids having a 5'-C-alkylnucleotide; and a method for producing an enzymatic nucleic acid molecule having enhanced activity to cleave an RNA or single-stranded DNA molecule, by forming the enzymatic molecule with at least one nucleotide having at its 5'-position an alkyl group. In other related aspects, the invention features 5'-C-alkylnucleotide triphosphates. These triphosphates can be used in standard protocols to form useful oligonucleotides of this invention.

The 5'-C-alkyl derivatives of this invention provide enhanced stability to the oligonulceotides containing them. While they may also reduce absolute activity in an in vitro assay they will provide enhanced overall activity in vivo. Below are provided assays to determine which such molecules are useful. Those in the art will recognize that equivalent assays can be readily devised. In another aspect, the invention features a method for conversion of a protected allo sugar to a protected talo sugar. In the method, the protected allo sugar is contacted with triphenyl phosphine, diethylazodicarboxylate, and p-nitrobenzoic acid under inversion causing conditions to provide the protected talo sugar. While one example of such conditions is provided below, those in the art will recognize other such conditions. Applicant has found that such conversion allows for ready synthesis of all types of nucleotide bases as exemplified in the figures.

While this invention is applicable to all oligonucleotides, applicant has found that the modified molecules of this invention are particulary useful for enzymatic RNA molecules. Thus, below is provided examples of such molecules. Those in the art will recognize that equivalent procedures can be used to make other molecules without such enzymatic activity. Specifically, Figure 1 shows base numbering of a hammerhead motif in which the numbering of various nucleotides in a hammerhead ribozyme is provided. This is not to be taken as an indication that the Figure is prior art to the pending claims, or that the art discussed is prior art to those claims. Referring to Figure 1. the preferred sequence of a hammerhead ribozyme in a 5'- to 3'-direction of the catalytic core is CUGANGAG [base paired with] CGAAA. In this invention, the use of 5'-C-alkyl substituted nucleotides that maintain or enhance the catalytic activity and or nuclease resistance of the hammerhead ribozyme is described. Substitutions of any nucleotide with any of the modified nucleotides shown in Figure 75 are possible.

The following are non-limiting examples showing the synthesis of nucleic acids using 5'-C-alkyl-substituted phosphoramidites and the syntheses of the amidites.

Example 37: Synthesis of Hammerhead Ribozymes Containing 5'-C-Alkylnucleotides & Other Modified Nucleotides

The method of synthesis would follow the procedure for normal RNA synthesis as described in Usman, N.; Ogilvie.K.K.; Jiang, M.-Y.; Cedergren.R.J. J. Am. Chem. Soc. 1987, 109, 7845-7854 and in Scaringe, S.A.; Franklyn,C.; Usman, N. Nucleic Acids Res. 1990, 18, 5433-5441 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end (compounds 26-29 and 56-59). These 5'-C-alkyl substituted phosphoramidites may be incorporated not only into hammerhead ribozymes, but also into hairpin, hepatitis delta virus, Group 1 or Group 2 intron catalytic nucleic acids, or into antisense oligonucleotides. They are, therefore, of general use in any nucleic acid structure.

Example 38: Methyl-2,3-O-Isopropylidine-6-Deoxy-β-D-allofuranoside (4) A suspension of L-rhamnose (100 g, 0.55 mol), CuSO₄ (120 g) and cone. H₂SO₄ (4.0 mL) in 1.0 L of dry acetone was mixed for 24 h at RT, then filtered. Cone. NH₄OH (5 mL) was added to the filtrate and the newly formed precipitate was filtered. The residue was concentrated in vacuo, coevaporated with pyridine (2 × 300 mL), dissolved in pyridine (500 mL) and cooled to 0 °C A solution of p-toluenesufonylchloride (107 g , 0.56 mmol) in dry DCE (500 mL) was added dropwise over 0.5 h. The reaction mixture was left for 16 h at RT. The reaction was quenched by adding ice-water (0.5 L) and, after mixing for 0.5 h, was extracted with chloroform (0.75 L). The organic layer was washed with H₂O (2 × 500 mL), 10% H₂SO₄ (2 × 300 mL), water (2 × 300 mL), sat. NaHCO₃ (2 × 300 mL), brine (2 × 300 mL), dried over MgSO₄ and evaporated to dryness. The residue (115 g) was dissolved in dry MeOH (1 L) and treated with NaOMe (23.2 g, 0.42 mmol) in MeOH. The reaction mixture was left for 16 h at 20 °C, neutralized with dry CO₂ and evaporated to dryness. The residue was suspended in chloroform (750 mL), filtered , concentrated to 100 mL and purified by flash chromatography in CHCl₃ to yield 45 g (37%) of compound 4.

Example 39: Methyl-2,3-O-Isopropylidine-5-O-t-Butyldiphenylsilyl-6-Deoxy-β-D-Allofuranoside (5).

To solution of methylfuranoside 4 (12.5 g 62.2 mmol) and AgNO₃ (21.25 g, 125.0 mmol) in dry DMF (300 mL) t-butyldiphenylsilyl chloride (22.2 g , 81 mmol) was added dropwise under Ar over 0.5 h. The reaction mixture was stirred for 4 h at RT, diluted with CHCl₃ (200 mL), filtered and evaporated to dryness (below 40 °C using a high vacuum oil pump). The residue was dissolved in CH₂Cl₂ (300 mL) washed with sat. NaHCO₃ (2 × 50 mL), brine (2 × 50 mL), dried over MgSO₄ and evaporated to dryness. The residue was purified by flash chromatography in CH₂Cl₂ to yield 20.0 g (75%) of compound 5.

Example 40: Methyl-5-O-t-Butyldiphenylsilyl-6-Deoxy-β-D-Allofuranoside (6).

Methylfuranoside 5 (13.5 g, 30.6 mmol) was dissolved in

CF₃COOH:dioxane:H₂O / 2:1 :1 (v/v/v, 200 mL) and stirred at 24 °C for 45 m. The reaction mixture was cooled to -10 °C, neutralized with conc. NH₄OH (140 mL) and extracted with CH₂Cl₂ (500 mL). The organic layer was separated, washed with sat. NaHCO₃ (2 × 75 mL), brine (2 × 75 mL), dried over MgSO₄ and evaporated to dryness. The product 6 was purified by flash chromatography using a 0-10% MeOH gradient in CH₂Cl₂. Yield 9.0 g (76%). Example 41 : Methyl-2,3-di-O-Benzoyl-5-O-t-Butyldiphenylsilyl-6-Deoxy-β-D-Allofuranoside (7).

Methylfuranoside 6 (7.0 g, 17.5 mmol) was coevaporated with pyridine (2 × 100 mL) and dissolved in pyridine (100 mL). Benzoyl chloride (5.4 g, 38.5 mmol) was added and the reaction mixture was left at RT for 16 h. Dry EtOH (50 mL) was added and the reaction mixture was evaporated to dryness after 0.5 h. The residue was dissolved in CH₂Cl₂ (300 mL), washed with sat. NaHCO₃ (2 × 75 mL), brine (2 × 75 mL) dried over MgSO₄ and evaporated to dryness. The product was purified by flash chromatography in CH₂Cl₂ to yield 9.5 g (89%) of compound 7.

Example 42: 1-O-Acetyl-2,3-di-O-benzoyl-5-O-t-Butyldiphenylsilyl-6-Deoxy-β-D-Allofuranose (8). Dibenzoate 7 (4.7 g, 7.7 mmol) was dissolved in a mixture of AcOH (10.0 mL), AC2O (20.0 mL) and EtOAc (30 mL) and the reaction mixture was cooled 0 °C 98% H₂SO₄ (0.15 mL) was then added. The reaction mixture was kept at 0 °C for 16 h, and then poured into a cold 1 :1 mixture of sat. NaHCO₃ and EtOAc (150 mL). After 0.5 h of vigorous stirring the organic phase was separated, washed with brine (2 × 75 mL), dried over MgSO₄, evaporated to dryness and coevaporated with toluene (2 × 50 mL). The product was purified by flash chromatography using a gradient of 0-5% MeOH in CH₂Cl₂. Yield: 4.0 g (82% as a mixture of α and β isomers).

Example 43: 1-(2',3'-di-O-Benzoyl-5'-O-t-Butyldiphenylsilyl-6'-Deoxy-β-D-Allofuranosyl)uracil (9).

Uracil (1.44 g, 11.5 mmol) was suspended in mixture of hexamethyldisilazane (100 mL) and pyridine (50 mL) and boiled under reflux until complete dissolution (3 h) occurred, and then for an additional hour. The reaction mixture was cooled to RT, evaporated to dryness and coevaporated with dry toluene (2 × 50 mL). To the residue was added a solution of acetates 8 (6.36 g, 10.0 mmol) in dry CH₃CN (100 mL), followed by CF₃SO₃SiMe₃ (2.8 g, 12.6 mmol). The reaction mixture was kept at 24 °C for 16 h, concentrated to 1/3 of its original volume, diluted with 100 mL of CH₂Cl₂ and extracted with sat. NaHCO₃ (2 × 50 mL), brine (2 × 50 mL) dried over MgS04, and evaporated to dryness. The product 9 was purified by flash chromatography using a gradient of 0-5% MeOH in CH₂Cl₂. Yield: 5.7 g (80%). Example 44: N⁴-Benzoyl-1-(2',3'-Di-O-Benzoyl-5'-O-t-Butyldiphenylsilyl-6'-Deoxy-β-D-Allofuranosyl)Cytosine (10). N⁴-benzoylcytosine (1.84 g, 8.56 mmol) was suspended in mixture of hexamethyldisilazane (100 mL) and pyridine (50 mL) and boiled under reflux until complete dissolution (3 h) occurred, and then for an additional hour. The reaction mixture was cooled to RT evaporated to dryness and coevaporated with dry toluene (2 × 50 mL). To the residue was added a solution of of acetates 8 (3.6 g, 5.6 mmol) in dry CH₃CN (100 mL), followed by CF₃SO₃SiMe₃ (4.76 g, 21.4 mmol). The reaction mixture was boiled under reflux for 5 h, cooled to RT, concentrated to 1/3 of its original volume, diluted with CH₂Cl₂ (100 mL) and extracted with sat. NaHCO₃ (2 × 50 mL), brine (2 × 50 mL) dried over MgSO₄ and evaporated to dryness. Purification by flash chromatography using a gradient of 0-5% MeOH in CH₂Cl₂ yielded 1.8 g (55%) of compound 10. Example 45: N⁶-Benzoyl-9-(2',3'-di-O-Benzoyl-5'-O-t-Butyldiphenylsilyl-6'-Deoxy-β-D-Allofuranosyl)adenine (11). N⁶-benzoyladenine (2.86 g, 11.86 mmol) was suspended in mixture of hexamethyldisilazane (100 mL) and pyridine (50 mL) and boiled under reflux until complete dissolution (7 h) occurred, and then for an additional hour. The reaction mixture was cooled to RT evaporated to dryness and coevaporated with dry toluene (2 × 50 mL). To the residue was added a solution of of acetates 8 (3.6 g, 5.6 mmol) in dry CH₃CN (100 mL) followed by CF₃SO₃SiMe₃ (6.59 g, 29.7 mmol). The reaction mixture was boiled under reflux for 8 h, cooled to RT, concentrated to 1/3 of its original volume, diluted with CH₂Cl₂ (100 mL) and extracted with sat. NaHCO₃ (2 × 50 mL), brine (2 × 50 mL) dried over MgSO₄ and evaporated to dryness. The product 11 was purified by flash chromatography using a gradient of 0-5% MeOH in CH₂Cl₂. Yield: 2.7 g (60%).

Example 46: N²-Isobutyryl-9-(2',3'-di-O-Benzoyl-5'-O-t-Butyldiphenylsilyl-6'-Deoxy-β-D-Allofuranosyl)guanine (12). N²-lsobutyrylguanine (1.47 g , 11.2 mmol) was suspended in mixture of hexamethyldisilazane (100 mL) and pyridine (50 mL) and boiled under reflux until complete dissolution (6 h) occurred, and then for an additional hour. The reaction mixture was cooled to RT evaporated to dryness and coevaporated with dry toluene (2 × 50 mL). To the residue was added a solution of of acetates 8 (3.4 g, 5.3 mmol) in dry CH₃CN (100 mL) followed by CF₃SO₃SiMe₃ (6.22 g, 28.0 mmol). The reaction mixture was boiled under reflux for 8 h, cooled to RT, concentrated to 1/3 of its original volume, diluted with CH₂Cl₂ (100 mL) and extracted with sat. NaHCO₃ (2 x 50 mL), brine (2 × 50 mL) dried over MgSO₄ and evaporated to dryness. The product 12 was purified by flash chromatography using a gradient of 0-2% MeOH in CH₂Cl₂. Yield: 2.1g (54%).

Example 47: N⁶--Benzoyl-9-(2',3'-di-O-benzoyl-6'-Deoxy-β-D-Allofuranosyl)adenine (15).

Nucleoside 11 (1.65 g, 2.0 mmol) was dissolved in THF (50 mL) and a 1 M solution of TBAF in THF (4 mL) was added. The reaction mixture was kept at RT for 4 h, evaporated to dryness and the product purified by flash chromatography using a gradient of 0-5% MeOH in CH₂Cl₂ to yield 1.0 g (85%) of compound 15. Example 48: N⁶-Benzoyl-9-(2',3'-di-O-Benzoyl-5'-O-Dimethoxytrityl-6'-Deoxy-β-D-Allofuranosyl)-adenine (19).

Nucleoside 15 (0.55 g, 0.92 mmol) was dissolved in dry CH₂Cl₂ (50 mL). AgNO₃ (0.34 g, 2.0 mmol), dimethoxytrityl chloride (0.68 g, 2.0 mmol) and sym-collidine (0.48 g) were added under Ar. The reaction mixture was stirred for 2h, diluted with CH₂Cl₂ (100 mL), filtered, evaporated to dryness and coevaporated with toluene (2 × 50 mL). Purification by flash chromatography using a gradient of 0-5% MeOH in CH₂Cl₂ yielded 0.8 g (97%) of compound 19.

Example 49: N⁶-Benzoyl-9-(-5'-O-Dimethoxytrityl-6'-Deoxy-β-D-Allofuranosyl)adenine (23).

Nucleoside 19 (1.8 g, 2 mmol) was dissolved in dioxane (50 mL), cooled to 0 °C and 2 M NaOH (50 mL) was added. The reaction mixture was kept at 0 °C for 45 m, neutralized with Dowex 50 (Pyr⁺ form), filtered and the resin was washed with MeOH (2 x 50 mL). The filtrate was then evaporated to dryness. Purification by flash chromatography using a gradient of 0-10% MeOH in CH₂Cl₂ yielded 1.1 g (80%) of 23. Example 50: N⁶-Benzoyl-9-(-5'-O-Dimethoxytrityl-2'-O-t-butyldimethylsilyl-6'-Deoxy-β-D-Allofuranosyl)adenine (27).

Nucleoside 23 (1.2 g, 1.8 mmol) was dissolved in dry THF (50 mL). Pyridine (0.50 g, 8 mmol) and AgNO₃ (0.4 g, 2.3 mmol) were added. After the AgNO₃ dissolved (1.5 h), t-butyldimethylsilyl chloride (0.35 g , 2.3 mmol) was added and the reaction mixture was stirred at RT for 16 h. The reaction mixture was diluted with CH₂Cl₂ (100 mL), filtered into sat. NaHCO₃ (50 mL), extracted, the organic layer washed with brine (2 × 50 mL), dried over MgSO₄ and evaporated to dryness. The product 27 was purified by flash chromatography using a hexanes:EtOAc / 7:3 gradient. Yield: 0.7 g (50%).

Example 51 : N⁶-Benzoyl-9-(-5'-O-Dimethoxytrityl-2'-O-t-butyldimethylsilyl-6'-Deoxy-β-D-Allofuranosyl)adenine-3'-(2-Cyanoethyl N,N-diisopropylphosphoramidite) (31).

Standard phosphitylation of 27 according to Scaringe, S. A.;

Franklyn.C; Usman, Ν. Nucleic Acids Res. 1990, 18, 5433-5441 yielded phosphoramidite 31 in 73% yield.

Example 52: Methyl-5-O-p-Νitrobenzoyl-2,3-O-Isopropylidine-6-deoxy-β-L-Tallofuranoside (5)

Methylfuranoside 4 (3.1 g 14.2 mmol) was dissolved in dry dioxane

(200 mL), p-nitrobenzoic acid (10.0 g, 60 mmol) and triphenylphosphine (15.74 g, 60.0 mmol) were added followed by DEAD (10.45 g, 60.0 mmol). The reaction mixture was left at RT for 16 h, EtOH (5 mL) was added, and after 0.5 h the reaction mixture was evaporated to dryness. The residue was dissolved in CH₂Cl₂ (300 mL) washed with sat. NaHCO₃ (2 × 75 mL), brine (2 × 75 mL) dried over MgSO₄ and evaporated to dryness. Purification by flash chromatography using a hexanes:EtOAc / 9:1 gradient yielded 4.1 g (78%) of compound 33. Subsequent debenzoylation (NaOMe/MeOH) and silylation (see preparation of 5) led to L-talofuranoside 34 which was converted to phosphoramidites 58-61 using the same methodology as described above for the preparation of the phosphoramidites of the D-allo-isomers 29-32.

The alkyl substituted nucleotides of this invention can be used to form stable oligonucleotides as discussed above for use in enzymatic cleavage or antisense situations. Such oligonucleotides can be formed enzymatically using triphosphate forms by standard procedure. Administration of such oligonucleotides is by standard procedure. See Sullivan et al., PCT WO 94/ 02595. The ribozymes and the target RNA containing site O were synthesized, deprotected and purified as described above. RNA cleavage assay was carried our at 37°C in the presence of 10 mM MgCl₂ as described above.

Applicant has substituted 5'-C-Me-L-talo nucleotides at positions A6, A9, A9 + G10, C11.1 and C11.1 + G10, as shown in Figure 78 (HH-01 to HH-05). HH-0 1,2,4 and 5 showed almost wild type activity (Figure 79). However, HH-03 demonstrated low catalytic activity. Ribozymes HH-01, 2, 3, 4 and 5 are also extremely resistant to degradation by human serum nucleases. Oligonucleotides with 2'-Deoxy-2'-Alkylnucleotide

This invention uses 2'-deoxy-2'-alkylnucleotides in oligonucleotides, which are particularly useful for enzymatic cleavage of RNA or single-stranded DNA, and also as antisense oligonucleotides. As the term is used in this application, 2'-deoxy-2'-alkylnucleotide-containing enzymatic nucleic acids are catalytic nucleic molecules that contain 2'-deoxy-2'-alkylnucleotide components replacing, but not limited to, double stranded stems, single stranded "catalytic core" sequences, single-stranded loops or single-stranded recognition sequences. These molecules are able to cleave (preferably, repeatedly cleave) separate RNA or DNA molecules in a nucleotide base sequence specific manner. Such catalytic nucleic acids can also act to cleave intramolecularly if that is desired. Such enzymatic molecules can be targeted to virtually any RNA transcript.

Also within the invention are 2'-deoxy-2'-alkylnucleotides which may be present in enzymatic nucleic acid or even in antisense oligonucleotides. Contrary to the findings of De Mesmaeker et al. applicant has found that such nucleotides are useful since they enhance the stability of the antisense or enzymatic molecule, and can be used in locations which do not affect the desired activity of the molecule. That is, while the presence of the 2'-alkyl group may reduce binding affinity of the oligonucleotide containing this modification, if that moiety is not in an essential base pair forming region then the enhanced stability that it provides to the molecule is advantageous. In addition, while the reduced binding may reduce enzymatic activity, the enhanced stability may make the loss of activity of less consequence. Thus, for example, if a 2'-deoxy-2'-alkyl-containing molecule has 10% the activity of the unmodified molecule, but has 10-fold higher stability in vivo then it has utility in the present invention. The same analysis is true for antisense oligonucleotides containing such modifications. The invention also relates to novel intermediates useful in the synthesis of such nucleotides and oligonucleotides (examples of which are shown in the Figures), and to methods for their synthesis.

Thus, in one aspect, the invention features 2'-deoxy-2'-alkylnucleotides, that is a nucleotide base having at the 2'-position on the sugar molecule an alkyl moiety and in preferred embodiments features those where the nucleotide is not uridine or thymidine. That is, the invention preferably includes all those nucleotides useful for making enzymatic nucleic acids or antisense molecules that are not described by the art discussed above.

Examples of various alkyl groups useful in this invention are shown in

Figure 81 , where each R group is any alkyl. The term "alkyl" does not include alkoxy groups which have an "-O-alkyl" group, where "alkyl" is defined as described above, where the O is adjacent the 2'-position of the sugar molecule.

In other aspects, also related to those discussed above, the invention features oligonucleotides having one or more 2'-deoxy-2'-alkylnucleotides (preferably not a 2'-alkyl- uridine or thymidine); e.g. enzymatic nucleic acids having a 2'-deoxy-2'-alkylnucleotide; and a method for producing an enzymatic nucleic acid molecule having enhanced activity to cleave an RNA or single-stranded DNA molecule, by forming the enzymatic molecule with at least one nucleotide having at its 2'-position an alkyl group. In other related aspects, the invention features 2'-deoxy-2'-alkylnucleotide triphosphates. These triphosphates can be used in standard protocols to form useful oligonucleotides of this invention.

The 2'-alkyl derivatives of this invention provide enhanced stability to the oligonulceotides containing them. While they may also reduce absolute activity in an in vitro assay they will provide enhanced overall activity in vivo. Below are provided assays to determine which such molecules are useful. Those in the art will recognize that equivalent assays can be readily devised.

In another aspect, the invention features hammerhead motifs having enzymatic activity having ribonucleotides at locations shown in Figure 80 at 5, 6, 8, 12, and 15.1 , and having substituted ribonucleotides at other positions in the core and in the substrate binding arms if desired. (The term "core" refers to positions between bases 3 and 14 in Figure 80, and the binding arms correspond to the bases from the 3'-end to base 15.1 , and from the 5'-end to base 2). Applicant has found that use of ribonucleotides at these five locations in the core provide a molecule having sufficient enzymatic activity even when modified nucleotides are present at other sites in the motif. Other such combinations of useful ribonucleotides can be determined as described by Usman et al. supra. Figure 80 shows base numbering of a hammerhead motif in which the numbering of various nucleotides in a hammerhead ribozyme is provided. This is not to be taken as an indication that the Figure is prior art to the pending claims, or that the art discussed is prior art to those claims. Referring to Figure 80 the preferred sequence of a hammerhead ribozyme in a 5'- to 3'-direction of the catalytic core is CUGANGAG[base paired with]CGAAA. In this invention, the use of 2'-C-alkyl substituted nucleotides that maintain or enhance the catalytic activity and or nuclease resistance of the hammerhead ribozyme is described. Although substitutions of any nucleotide with any of the modified nucleotides shown in Figure 81 are possible, and were indeed synthesized, the basic structure composed of promarily 2'-O-Me nucleotides weth selected substitutions was chosen to maintain maximal catalytic activity (Yang et al. Biochemistry 1992, 31 , 5005-5009 and Paolella et al. , EMBO J. 1992, 11 , 1913-1919) and ease of synthesis, but is not limiting to this invention. Ribozymes from Figure 80 and Table 45 were synthesized and assayed for catalytic activity and nuclease resistance. With the exception of entries 8 and 17, all of the modified ribozymes retained at lease 1/10 of the wild-type catalytic activity. From Table 45, all 2'-modified ribozymes showed very large and significant increases in stability in human serum (shown) and in the other fluids described below (Example 55, data not shown). The order of most agressive nuclease activity was fetal bovine serum, > human serum >human plasma > human synovial fluid. As an overall measure of the effect of these 2'-substitutions on stability and activity, a ratio β was calculated (Table 45). This β value indicated that all modified ribozymes tested had significant, >100 - >1700 fold, increases in overall stability and activity. These increases in β indicate that the lifetime of these modified ribozymes in vivo are significantly increased which should lead to a more pronounced biological effect.

More general substitutions of the 2'-modified nucleotides from Figure 81 also increased the ^t 1/2 of the resulting modified ribozymes. However the catalytic activity of these ribozymes was decreased > 10-fold.

In Figure 86 compound 37 may be used as a general intermediate to prepare derivatized 2'C-alkyl phosphoramidites, where X is CH₃, or an alkyl, or other group described above. The following are non-limiting examples showing the synthesis of nucleic acids using 2'-C-alkyl substituted phosphoramidites, the syntheses of the amidites, their testing for enzymatic activity and nuclease resistance.

Example 53: Synthesis of Hammerhead Ribozymes Containing 2'-Deoxy-2'-Alkylnucleotides & Other 2'-Modified Nucleotides

The method of synthesis used generally follows the procedure for normal RNA synthesis as described in Usman, N.; Ogilvie.K.K.; Jiang, M.-Y.; Cedergren.R.J. J. Am. Chem. Soc. 1987, 109, 7845-7854 and in Scaringe, S.A.; Franklyn.C; Usman, N. Nucleic Acids Res. 1990, 18, 5433-5441 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end (compounds 10, 12, 17, 22, 31 , 18, 26, 32, 36 and 38). Other 2'-modified phosphoramidites were prepared according to: 3 & 4, Eckstein et al. International Publication No. WO 92/07065; and 5 Kois et al. Nucleosides & Nucleotides 1993, 12, 1093-1109. The average stepwise coupling yields were -98%. The 2'-substituted phosphoramidites were incorporated into hammerhead ribozymes as shown in Figure 80. However, these 2'-alkyl substituted phosphoramidites may be incorporated not only into hammerhead ribozymes, but also into hairpin, hepatitis delta virus, Group I or Group II intron catalytic nucleic acids, or into antisense oligonucleotides. They are, therefore, of general use in any nucleic acid structure.

Example 54: Ribozyme Activity Assay

Purified 5'-end labeled RNA substrates (15-25-mers) and purified 5'-end labeled ribozymes (~36-mers) were both heated to 95 °C, quenched on ice and equilibrated at 37 °C, separately. Ribozyme stock solutions were 1 mM, 200 nM, 40 nM or 8 nM and the final substrate RNA concentrations were - 1 nM. Total reaction volumes were 50 mL. The assay buffer was 50 mM Tris-Cl, pH 7.5 and 10 mM MgCl₂. Reactions were initiated by mixing substrate and ribozyme solutions at t = 0. Aliquots of 5 mL were removed at time points of 1 , 5, 15, 30, 60 and 120 m. Each time point was quenched in formamide loading buffer and loaded onto a 15% denaturing polyacrylamide gel for analysis. Quantitative analyses were performed using a phosphorimager (Molecular Dynamics). Example 55: Stability Assay

500 pmol of gel-purified 5'-end-labeled ribozymes were precipitated in ethanol and pelleted by centrifugation. Each pellet was resuspended in 20 mL of appropriate fluid (human serum, human plasma, human synovial fluid or fetal bovine serum) by vortexing for 20 s at room temperature. The samples were placed into a 37 °C incubator and 2 mL aliquots were withdrawn after incubation for 0, 15, 30, 45, 60, 120, 240 and 480 m. Aliquots were added to 20 mL of a solution containing 95% formamide and 0.5X TBE (50 mM Tris, 50 mM borate, 1 mM EDTA) to quench further nuclease activity and the samples were frozen until loading onto gels. Ribozymes were size-fractionated by electrophoresis in 20% acrylamide/8M urea gels. The amount of intact ribozyme at each time point was quantified by scanning the bands with a phosphorimager (Molecular Dynamics) and the half-life of each ribozyme in the fluids was determined by plotting the percent intact ribozyme vs the time of incubation and extrapolation from the graph.

Example 56: 3',5'-O-(Tetraisopropyl-disiloxane-1 ,3-diyl)-2'-O-Phenoxythiocarbonyl-Uridine (7)

To a stirred solution of 3',5'-O-(tetraisopropyl-disiloxane-1 ,3-diyl)-uridine, 6, (15.1 g, 31 mmol, synthesized according to Nucleic Acid Chemistry, ed. Leroy Townsend, 1986 pp. 229-231) and dimethylaminopyridine (7.57 g, 62 mmol) a solution of phenylchlorothionoformate (5.15 mL, 37.2 mmol) in 50 mL of acetonitrile was added dropwise and the reaction stirred for 8 h. TLC (EtOAc:hexanes / 1 :1) showed disappearance of the starting material. The reaction mixture was evaporated, the residue dissolved in chloroform, washed with water and brine, the organic layer was dried over sodium sulfate, filtered and evaporated to dryness. The residue was purified by flash chromatography on silica gel with EtOAc:hexanes / 2:1 as eluent to give 16.44 g (85%) of 7. Example 57: 3'.5'-O-(Tetraisopropyl-disiloxane-1 ,3-diyl)-2'-C-Allyl -Uridine (8)

To a refluxing, under argon, solution of 3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-2'-O-phenoxythiocarbonyl-uridine, 7, (5 g, 8.03 mmol) and allyltributyltin (12.3 mL, 40.15 mmol) in dry toluene, benzoyl peroxide (0.5 g) was added portionwise during 1 h. The resulting mixture was allowed to reflux under argon for an additional 7-8 h. The reaction was then evaporated and the product 8 purified by flash chromatography on silica gel with EtOAc:hexanes / 1 :3 as eluent. Yield 2.82 g (68.7%).

Example 58: 5'-O-Dimethoxytrityl-2'-C-Allyl-Uridine (9) A solution of 8 (1.25 g, 2.45 mmol) in 10 mL of dry tetrahydrofuran

(THF) was treated with a 1 M solution of tetrabutylammoniumfluoride in THF (3.7 mL) for 10 m at room temperature. The resulting mixture was evaporated, the residue was loaded onto a silica gel column, washed with 1 L of chloroform, and the desired deprotected compound was eluted with chloroform:methanol / 9:1. Appropriate fractions were combined, solvents removed by evaporation, and the residue was dried by coevaporation with dry pyridine. The oily residue was redissolved in dry pyridine, dimethoxytritylchioride (1.2 eq) was added and the reaction mixture was left under anhydrous conditions overnight. The reaction was quenched with methanol (20 mL), evaporated, dissolved in chloroform, washed with 5% aq. sodium bicarbonate and brine. The organic layer was dried over sodium sulfate and evaporated. The residue was purified by flash chromatography on silica gel, EtOAc:hexanes / 1 :1 as eluent, to give 0.85 g (57%) of 9 as a white foam. Example 59: 5'-O-Dimethoxytrityl-2'-C-Allyl-Uridine 3'-(2-Cyanoethyl N,N-diisopropylphosphoramidite) (10)

5'-O-Dimethoxytrityl-2'-C-allyl-uridine (0.64 g, 1.12 mmol) was dissolved in dry dichloromethane under dry argon. N,N-Diisopropylethylamine (0.39 mL, 2.24 mmol) was added and the solution was ice-cooled. 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (0.35 mL, 1.57 mmol) was added dropwise to the stirred reaction solution and stirring was continued for 2 h at RT. The reaction mixture was then ice-cooled and quenched with 12 mL of dry methanol. After stirring for 5 m, the mixture was concentrated in vacuo (40 °C) and purified by flash chromatography on silica gel using a gradient of 10-60% EtOAc in hexanes containing 1% triethylamine mixture as eluent. Yield: 0.78 g (90%), white foam.

Example 60: 3',5'-O-(Tetraisopropyl-disiloxane-1 ,3-diyl)-2'-C-Allyl-N⁴-Acetyl-Cytidine (11) Triethylamine (6.35 mL, 45.55 mmol) was added dropwise to a stirred ice-cooled mixture of 1 ,2,4-triazole (5.66 g, 81.99 mmol) and phosphorous oxychloride (0.86 mL, 9.11 mmol) in 50 mL of anhydrous acetonitrile. To the resulting suspension a solution of 3',5'-O-(tetraisopropyl-disiloxane-1 ,3-diyl)-2'-C-allyl uridine (2.32 g, 4.55 mmol) in 30 mL of acetonitrile was added dropwise and the reaction mixture was stirred for 4 h at room temperature. The reaction was concentrated in vacuo to a minimal volume (not to dryness). The residue was dissolved in chloroform and washed with water, saturated aq. sodium bicarbonate and brine. The organic layer was dried over sodium sulfate and the solvent was removed in vacuo. The resulting foam was dissolved in 50 mL of 1 ,4-dioxane and treated with 29% aq. ΝH₄OH overnight at room temperature. TLC (chloroform:methanol /9:1) showed complete conversion of the starting material. The solution was evaporated, dried by coevaporation with anhydrous pyridine and acetylated with acetic anhydride (0.52 mL, 5.46 mmol) in pyridine overnight. The reaction mixture was quenched with methanol, evaporated, the residue was dissolved in chloroform, washed with sodium bicarbonate and brine. The organic layer was dried over sodium sulfate, evaporated to dryness and purified by flash chromatography on silica gel (3% MeOH in chloroform). Yield 2.3 g (90%) as a white foam. Example 61 : 5'-O-Dimethoxytrityl-2'-C-Allyl-N⁴-Acetyl-Cytidine

This compound was obtained analogously to the uridine derivative 9 in 55% yield.

Example 62: 5'-O-Dimethoxytrityl-2'-C-allyl-N⁴-Acetyl-Cytidine 3'-(2-Cyanoethyl N,N-diisopropylphosphoramidite) (12)

2'-O-Dimethoxytrityl-2'-C-allyl-N⁴-acetyl cytidine (0.8 g, 1.31 mmol) was dissolved in dry dichloromethane under argon. N,N-Diisopropylethylamine (0.46 mL, 2.62 mmol) was added and the solution was ice-cooled. 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (0.38 mL, 1.7 mmol) was added dropwise to a stirred reaction solution and stirring was continued for 2 h at room temperature. The reaction mixture was then ice-cooled and quenched with 12 mL of dry methanol. After stirring for 5 m, the mixture was concentrated in vacuo (40 °C) and purified by flash chromatography on silica gel using chloroform:ethanol / 98:2 with 2% triethylamine mixture as eluent. Yield: 0.91 g (85%), white foam.

Example 63: 2'-Deoxy-2'-Methylene-Uridine

2'-Deoxy-2'-methylene-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-uridine 14 (Hansske.F.; Madej.D.; Robins.M. J. Tetrahedron 1984, 40, 125 and Matsuda.A.; Takenuki.K.; Tanaka.S.; Sasaki.T.; Ueda.T. J. Med. Chem. 1991 , 34, 812) (2.2 g, 4.55 mmol ) dissolved in THF (20 mL) was treated with 1 M TBAF in THF (10 mL) for 20 m and concentrated in vacuo. The residue was triturated with petroleum ether and chromatographed on a silica gel column. 2'-Deoxy-2'-methylene-uridine (1.0 g, 3.3 mmol, 72.5%) was eluted with 20% MeOH in CH₂Cl₂. Example 64: 5'-O-DMT-2'-Deoxy-2'-Methylene-Uridine (15)

2'-Deoxy-2'-methylene-uridine (0.91 g, 3.79 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-Cl in pyridine (10 mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was concentrated in vacuo and the residue taken up in CH₂Cl₂ (100 mL) and washed with sat. ΝaHCO₃, water and brine. The organic extracts were dried over MgSO₄, concentrated in vacuo and purified over a silica gel column using EtOAc:hexanes as eluant to yield 15 (0.43 g, 0.79 mmol, 22%). Example 65: 5'-O-DMT-2'-Deoxy-2'-Methylene-Uridine 3'-(2-Cyanoethyl N,N-diisopropylphosphoramidite) (17)

1-(2'-Deoxy-2'-methylene-5'-O-dimethoxytrityl-β-D-ribofuranosyl)-uracil (0.43 g, 0.8 mmol) dissolved in dry CH₂Cl₂ (15 mL) was placed in a round-bottom flask under Ar. Diisopropylethylamine (0.28 mL, 1.6 mmol) was added, followed by the dropwise addition of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.25 mL, 1.12 mmol). The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL). After 10 m the mixture evaporated to a syrup in vacuo (40 °C). The product (0.3 g, 0.4 mmol, 50%) was purified by flash column chromatography over silica gel using a 25-70% EtOAc gradient in hexanes, containing 1% triethylamine, as eluant. R_f 0.42 (CH₂Cl₂: MeOH / 15:1)

Example 66: 2'-Deoxy-2'-Difluoromethylene-3',5'-O-(Tetraisopropyldisiloxane-1 ,3-diyl)-Uridine 2'-Keto-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)uridine 14 (1.92 g,

12.6 mmol) and triphenylphosphine (2.5 g, 9.25 mmol) were dissolved in diglyme (20 mL), and heated to a bath temperature of 160 °C A warm (60°C) solution of sodium chlorodifluoroacetate in diglyme (50 mL) was added (dropwise from an equilibrating dropping funnel) over a period of -1 h. The resulting mixture was further stirred for 2 h and concentrated in vacuo. The residue was dissolved in CH₂Cl₂ and chromatographed over silica gel. 2'-Deoxy-2'-difluoromethylene-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-uridine (3.1 g, 5.9 mmol, 70%) eluted with 25% hexanes in EtOAc.

Example 67: 2'-Deoxy-2'-Difluoromethylene-Uridine 2'-Deoxy-2'-methylene-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-uridine (3.1 g, 5.9 mmol) dissolved in THF (20 mL) was treated with 1 M TBAF in THF (10 mL) for 20 m and concentrated in vacuo. The residue was triturated with petroleum ether and chromatographed on silica gel column. 2'-Deoxy-2'-difluoromethylene-uridine (1.1 g, 4.0 mmol, 68%) was eluted with 20% MeOH in CH₂Cl₂.

Example 68: 5'-O-DMT-2'-Deoxy-2'-Difluoromethylene-Uridine (16)

2'-Deoxy-2'-difluoromethylene-uridine (1.1 g, 4.0 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-Cl (1.42 g, 4.18 mmol) in pyridine (10 mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was concentrated in vacuo and the residue taken up in CH₂Cl₂ (100 mL) and washed with sat. NaHCO₃, water and brine. The organic extracts were dried over MgSO₄, concentrated in vacuo and purified over a silica gel column using 40% EtOAc:hexanes as eluant to yield 5'-O-DMT-2'-deoxy-2'-difluoromethylene-uridine 16 (1.05 g, 1.8 mmol, 45%).

Example 69: 5'-O-DMT-2'-Deoxy-2'-Difluoromethylene-Uridine 3'-(2-Cyanoethyl N,N-diisopropylphosphoramidite) (18)

1-(2'-Deoxy-2'-difluoromethylene-5'-O-dimethoxytrityl-β-D-ribofuranosyl)-uracil (0.577 g, 1 mmol) dissolved in dry CH₂Cl₂ (15 mL) was placed in a round-bottom flask under Ar. Diisopropylethylamine (0.36 mL, 2 mmol) was added, followed by the dropwise addition of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.44 mL, 1.4 mmol). The reaction mixture was stirred for 2 h at RT and quenched with ethanol (1 mL). After 10 m the mixture evaporated to a syrup in vacuo (40 °C). The product (0.404 g, 0.52 mmol, 52%) was purified by flash chromatography over silica gel using 20-50% EtOAc gradient in hexanes, containing 1% triethylamine, as eluant. Rf 0.48 (CH₂Cl₂: MeOH / 15:1). Example 70: 2'-Deoxy-2'-Methylene-3',5'-O-(Tetraisopropyldisiloxane-1 ,3-diyl)-4-N-Acetyl-Cytidine 20

Triethylamine (4.8 mL, 34 mmol) was added to a solution of POCl₃ (0.65 mL, 6.8 mmol) and 1 ,2,4-triazole (2.1 g, 30.6 mmol) in acetonitrile (20 mL) at 0 °C A solution of 2'-deoxy-2'-methylene-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl) uridine 19 (1.65 g, 3.4 mmol) in acetonitrile (20 mL) was added dropwise to the above reaction mixture and left to stir at room temperature for 4 h. The mixture was concentrated in vacuo, dissolved in CH₂Cl₂ (2 × 100 mL) and washed with 5% ΝaHCO₃ (1 × 100 mL). The organic extracts were dried over Na₂SO₄ concentrated in vacuo, dissolved in dioxane (10 mL) and aq. ammonia (20 mL). The mixture was stirred for 12 h and concentrated in vacuo. The residue was azeotroped with anhydrous pyridine (2 × 20 mL). Acetic anhydride (3 mL) was added to the residue dissolved in pyridine, stirred at RT for 4 h and quenched with sat. NaHCO₃ (5 mL). The mixture was concentrated in vacuo, dissolved in CH₂Cl₂ (2 × 100 mL) and washed with 5% NaHCO₃ (1 × 100 mL). The organic extracts were dried over Na₂SO₄, concentrated in vacuo and the residue chromatographed over silica gel. 2'-Deoxy-2'-methylene-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-4-N-acetyl-cytidine 20 (1.3 g, 2.5 mmol, 73%) was eluted with 20% EtOAc in hexanes. Example 71 : 1-(2'-Deoxy-2'-Methylene-5'-O-Dimethoxytrityl-β-D-ribofuranosyl)-4-N-Acetyl-Cytosine 21

2'-Deoxy-2'-methylene-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-4-N-acetyl-cytidine 20 (1.3 g, 2.5 mmol) dissolved in THF (20 mL) was treated with 1 M TBAF in THF (3 mL) for 20 m and concentrated in vacuo. The residue was triturated with petroleum ether and chromatographed on silica gel column. 2'-Deoxy-2'-methylene-4-N-acetyl-cytidine (0.56 g, 1.99 mmol, 80%) was eluted with 10% MeOH in CH₂Cl₂. 2'-Deoxy-2'-methylene-4-N-acetyl-cytidine (0.56 g, 1.99 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-Cl (0.81 g, 2.4 mmol) in pyridine (10 mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was concentrated in vacuo and the residue taken up in CH₂Cl₂ (100 mL) and washed with sat. ΝaHCO₃ (50 mL), water (50 mL) and brine (50 mL). The organic extracts were dried over MgSO₄, concentrated in vacuo and purified over a silica gel column using EtOAc:hexanes / 60:40 as eluant to yield 21 (0.88 g, 1.5 mmol, 75%).

Example 72: 1-(2'-Deoxy-2,-Methylene-5'-O-Dimethoxytrityl-β-D-ribofuranosyl)-4-N-Acetyl-Cytosine 3'-(2-Cyanoethyl-N,N-diisopropylphosphoramidite) (22) 1-(2'-Deoxy-2'-methylene-5'-O-dimethoxytrityl-β-D-ribofuranosyl)-4-N-acetyl-cytosine 21 (0.88 g, 1.5 mmol) dissolved in dry CH₂Cl₂ (10 mL) was placed in a round-bottom flask under Ar. Diisopropylethylamine (0.8 mL, 4.5 mmol) was added, followed by the dropwise addition of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.4 mL, 1.8 mmol). The reaction mixture was stirred 2 h at room temperature and quenched with ethanol (1 mL). After 10 m the mixture evaporated to a syrup in vacuo (40 °C). The product 22 (0.82 g, 1.04 mmol, 69%) was purified by flash chromatography over silica gel using 50-70% EtOAc gradient in hexanes, containing 1% triethylamine, as eluant. Rf 0.36 (CH₂Cl₂:MeOH / 20:1). Example 73: 2'-Deoxy-2'-Difluoromethylene-3',5'-O-(Tetraisopropyl disiloxane-1 ,3-diyl)-4-N-Acetyl-Cytidine (24)

Et₃Ν (6.9 mL, 50 mmol) was added to a solution of POCl₃ (0.94 mL, 10 mmol) and 1 ,2,4-triazole (3.1 g, 45 mmol) in acetonitrile (20 mL) at 0 °C A solution of 2'-deoxy-2'-difluoromethylene-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)uridine 23 ([described in example 14] 2.6 g, 5 mmol) in acetonitrile (20 mL) was added dropwise to the above reaction mixture and left to stir at RT for 4 h. The mixture was concentrated in vacuo, dissolved in CH₂Cl₂ (2 × 100 mL) and washed with 5% NaHCO₃ (1 × 100 mL). The organic extracts were dried over Na₂SO₄ concentrated in vacuo, dissolved in dioxane (20 mL) and aq. ammonia (30 mL). The mixture was stirred for 12 h and concentrated in vacuo. The residue was azeotroped with anhydrous pyridine (2 × 20 mL). Acetic anhydride (5 mL) was added to the residue dissolved in pyridine, stirred at RT for 4 h and quenched with sat. NaHCO₃ (5mL). The mixture was concentrated in vacuo, dissolved in CH₂Cl₂ (2 × 100 mL) and washed with 5% NaHCO₃ (1 × 100 mL). The organic extracts were dried over Na₂SO₄, concentrated in vacuo and the residue chromatographed over silica gel. 2'-Deoxy-2'-difluoromethylene-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-4-N-acetyl-cytidine 24 (2.2 g, 3.9 mmol, 78%) was eluted with 20% EtOAc in hexanes.

Example 74: 1-(2'-Deoxy-2'-Difluoromethylene-5'-O-Dimethoxytrityl-β-D-ribofuranosyl)-4-N-Acetyl-Cytosine (25)

2'-Deoxy-2'-difluoromethylene-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-4-N-acetyl-cytidine 24 (2.2 g, 3.9 mmol) dissolved in THF (20 mL) was treated with 1 M TBAF in THF (3 mL) for 20 m and concentrated in vacuo. The residue was triturated with petroleum ether and chromatographed on a silica gel column. 2'-Deoxy-2'-difluoromethylene-4-N-acetyl-cytidine (0.89 g, 2.8 mmol, 72%) was eluted with 10% MeOH in CH₂Cl₂. 2'-Deoxy-2'-difluoromethylene-4-N-acetyl-cytidine (0.89 g, 2.8 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-Cl (1.03 g, 3.1 mmol) in pyridine (10 mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was concentrated in vacuo and the residue taken up in CH₂Cl₂ (100 mL) and washed with sat. ΝaHCO₃ (50 mL), water (50 mL) and brine (50 mL). The organic extracts were dried over MgSO₄, concentrated in vacuo and purified over a silica gel column using EtOAc:hexanes / 60:40 as eluant to yield 25 (1.2 g, 1.9 mmol, 68%).

Example 75: 1-(2'-Deoxy-2'-Difluoromethylene-5'-O-Dimethoxytrityl-β-D-ribofuranosyl)-4- N-Acetylcytosine 3'-(2-cyanoethyl-N,N-diisopropylphosphoramidite) (26)

1-(2'-Deoxy-2'-difluoromethylene-5'-O-dimethoxytrityl-β-D-ribofuranosyl)-4-N-acetylcytosine 25 (0.6 g, 0.97 mmol) dissolved in dry CH₂Cl₂ (10 mL) was placed in a round-bottom flask under Ar. Diisopropylethylamine (0.5 mL, 2.9 mmol) was added, followed by the dropwise addition of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.4 mL, 1.8 mmol). The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL). After 10 m the mixture was evaporated to a syrup in vacuo (40 °C). The product 26, a white foam (0.52 g, 0.63 mmol, 65%) was purified by flash chromatography over silica gel using 30-70% EtOAc gradient in hexanes, containing 1% triethylamine, as eluant. Rf 0.48 (CH₂Cl_2:MeOH / 20:1).

Example 76: 2'-Keto-3',5'-O-(Tetraisopropyldisiloxane-1 ,3-diyl)-6-N-(4-t-Butylbenzoyl)-Adenosine (28)

Acetic anhydride (4.6 mL) was added to a solution of 3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-6-N-(4-t-butylbenzoyl)-adenosine (Brown, J.; Christodolou, C.; Jones.S.; Modak.A.; Reese, C; Sibanda,S.; Ubasawa A. J. Chem .Soc. Perkin Trans. I 1989, 1735) (6.2 g, 9.2 mmol) in DMSO (37 mL) and the resulting mixture was stirred at room temperature for 24 h. The mixture was concentrated in vacuo. The residue was taken up in EtOAc and washed with water. The organic layer was dried over MgSO₄ and concentrated in vacuo. The residue was purified on a silica gel column to yield 2'-keto-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-6-N-(4-t-butylbenzoyl)-adenosine 28 (4.8 g, 7.2 mmol, 78%).

Example 77: 2'-Deoxy-2'-methylene-3',5'-O-(Tetraisopropyldisiloxane-1 ,3-diyl)-6-N-(4-t-Butylbenzoyl)-Adenosine (29) Under a pressure of argon, sec-butyllithium in hexanes (11.2 mL, 14.6 mmol) was added to a suspension of triphenylmethylphosphonium iodide (7.07 g,17.5 mmol) in THF (25 mL) cooled at -78 °C The homogeneous orange solution was allowed to warm to -30 °C and a solution of 2'-keto-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-6-N-(4-t-butylbenzoyl)-adenosine 28 (4.87 g, 7.3 mmol) in THF (25 mL) was transferred to this mixture under argon pressure. After warming to RT, stirring was continued for 24 h. THF was evaporated and replaced by CH₂Cl₂ (250 mL), water was added (20 mL), and the solution was neutralized with a cooled solution of 2% HCl. The organic layer was washed with H₂O (20 mL), 5% aqueous NaHCO₃ (20 mL), H₂O to neutrality, and brine (10 mL). After drying (Na₂SO₄), the solvent was evaporated in vacuo to give the crude compound, which was chromatographed on a silica gel column. Elution with light petroleum ether:EtOAc / 7:3 afforded pure 2'-deoxy-2'-methylene-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-6-N-(4-t-butylbenzoyl)-adenosine 29 (3.86 g, 5.8 mmol, 79%).

Example 78: 2'-Deoxy-2'-Methylene-6-N-(4-t-Butylbenzoyl)-Adenosine

2'-Deoxy-2'-methylene-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-6-N-(4-t-butylbenzoyl)-adenosine (3.86 g, 5.8 mmol) dissolved in THF (30 mL) was treated with 1 M TBAF in THF (15 mL) for 20 m and concentrated in vacuo. The residue was triturated with petroleum ether and chromatographed on a silica gel column. 2'-Deoxy-2'-methylene-6-N-(4-t-butylbenzoyl)-adenosine (1.8 g, 4.3 mmol, 74%) was eluted with 10% MeOH in CH₂Cl₂. Example 79: 5'-O-DMT-2'-Deoxy-2'-Methylene-6-N-(4-t-Butylbenzoyl)-Adenosine (29)

2'-Deoxy-2'-methylene-6-N-(4-t-butylbenzoyl)-adenosine (0.75 g, 1.77 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-Cl (0.66 g, 1.98 mmol) in pyridine (10 mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was concentrated in vacuo and the residue taken up in CH₂Cl₂ (100 mL) and washed with sat. ΝaHCO₃, water and brine. The organic extracts were dried over MgSO₄, concentrated in vacuo and purified over a silica gel column using 50% EtOAc:hexanes as an eluant to yield 29 (0.81 g, 1.1 mmol, 62%).

Example 80: 5'-O-DMT-2'-Deoxy-2'-Methylene-6-N-(4-t-Butylbenzoyl)- Adenosine 3'-(2-Cyanoethyl N,N-diisopropylphosphoramidite) (31)

1-(2'-Deoxy-2'-methylene-5'-O-dimethoxytrityl-β-D-ribofuranosyl)-6-N- (4-t-butylbenzoyl)-adenine 29 dissolved in dry CH₂Cl₂ (15 mL) was placed in a round bottom flask under Ar. Diisopropylethylamine was added, followed by the dropwise addition of 2-cyanoethyl N, N-diisopropylchlorophosphoramidite. The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL). After 10 m the mixture was evaporated to a syrup in vacuo (40 °C). The product was purified by flash chromatography over silica gel using 30-50% EtOAc gradient in hexanes, containing 1 % triethylamine, as eluant (0.7 g, 0.76 mmol, 68%). R_f 0.45 (CH₂Cl₂: MeOH / 20:1)

Example 81 : 2'-Deoxy-2'-Difluoromethylene-3',5'-O-(Tetraisopropyldisiloxane-1 ,3-diyl)-6-N-(4-t-Butylbenzoyl)-Adenosine

2'-Keto-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-6-N-(4-t-butylbenzoyl)-adenosine 28 (6.7 g, 10 mmol) and triphenylphosphine (2.9 g, 11 mmol ) were dissolved in diglyme (20 mL), and heated to a bath temperature of 160 °C A warm (60 °C) solution of sodium chlorodifluoroacetate (2.3 g, 15 mmol) in diglyme (50 mL) was added (dropwise from an equilibrating dropping funnel) over a period of ~1 h. The resulting mixture was further stirred for 2 h and concentrated in vacuo. The residue was dissolved in CH₂Cl₂ and chromatographed over silica gel. 2'-Deoxy-2'-difluoromethylene-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-6-N-(4-t-butylbenzoyl)-adenosine (4.1 g, 6.4 mmol, 64%) eluted with 15% hexanes in EtOAc.

Example 82: 2'-Deoxy-2'-Difluoromethylene-6-N-(4-f-Butylbenzoyl)-Adenosine

2'-Deoxy-2'-difluoromethylene-3' 5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-6-N-(4-t-butylbenzoyl)-adenosine (4.1 g, 6.4 mmol) dissolved in THF (20 mL) was treated with 1 M TBAF in THF (10 mL) for 20 m and concentrated in vacuo. The residue was triturated with petroleum ether and chromatographed on a silica gel column. 2'-Deoxy-2'-difluoromethylene-6-N-(4-t-butylbenzoyl)-adenosine (2.3 g, 4.9 mmol, 77%) was eluted with 20% MeOH in CH₂Cl₂.

Example 83: 5'-O-DMT-2'-Deoxy-2'-Difluoromethylene-6-N-(4-t-Butylbenzoyl)-Adenosine (30)

2'-Deoxy-2'-difluoromethylene-6-N-(4-t-butylbenzoyl)-adenosine (2.3 g, 4.9 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-Cl in pyridine (10 mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was concentrated in vacuo and the residue taken up in CH₂Cl₂ (100 mL) and washed with sat. NaHCO₃, water and brine. The organic extracts were dried over MgSO₄, concentrated in vacuo and purified over a silica gel column using 50% EtOAc:hexanes as eluant to yield 30 (2.6 g, 3.41 mmol, 69%).

Example 84: 5'-O-DMT-2'-Deoxy-2'-Difluoromethylene-6-N-(4-t-Butylbenzoyl)-Adenosine 3'-(2-Cyanoethyl N,N-diisopropylphosphoramidite) (32)

1-(2'-Deoxy-2'-difluoromethylene-5'-O-dimethoxytrityl-β-D-ribofuranosyl)-6-N-(4-t-butylbenzoyl)-adenine 30 (2.6 g, 3.4 mmol) dissolved in dry C H ₂Cl₂ (25 mL) was placed in a round bottom flask under Ar. Diisopropylethylamine (1.2 mL, 6.8 mmol) was added, followed by the dropwise addition of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (1.06 mL, 4.76 mmol). The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL). After 10 m the mixture evaporated to a syrup in vacuo (40 °C). 32 (2.3 g, 2.4 mmol, 70%) was purified by flash column chromatography over silica gel using 20-50% EtOAc gradient in hexanes, containing 1% triethylamine, as eluant. R_f 0.52 (CH₂Cl₂: MeOH /15:1).

Example 85: 2'-Deoxy-2'-Methoxycarbonylmethylidine-3',5'-O-(Tetraisopropyldisiloxane-1 ,3-diyl)-Uridine (33)

Methyl(triphenylphosphoranylidine)acetate (5.4 g, 16 mmol) was added to a solution of 2'-keto-3',5'-O-(tetraisopropyl disiloxane-1 ,3-diyl)-uridine 14 in CH₂Cl₂ under argon. The mixture was left to stir at RT for 30 h. CH₂Cl₂ (100 mL) and water were added (20 mL), and the solution was neutralized with a cooled solution of 2% HCl. The organic layer was washed with H₂O (20 mL), 5% aq. ΝaHCO₃ (20 mL), H₂O to neutrality, and brine (10 mL). After drying (Na₂SO₄), the solvent was evaporated in vacuo to give crude product, that was chromatographed on a silica gel column. Elution with light petroleum ether:EtOAc / 7:3 afforded pure 2'-deoxy-2'-methoxycarbonylmethylidine-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-uridine 33 (5.8 g, 10.8 mmol, 67.5%). Example 86: 2'-Deoxy-2'-Methoxycarbonylmethylidine-Uridine (34)

Et₃N•3 HF (3 mL) was added to a solution of 2'-deoxy-2'-methoxycarboxylmethylidine-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-uridine 33 (5 g, 9.3 mmol) dissolved in CH₂CI₂ (20 mL) and Et₃N (15 mL). The resulting mixture was evaporated in vacuo after 1 h and chromatographed on a silica gel column eluting 2'-deoxy-2'-methoxycarbonylmethylidine-uridine 34 (2.4 g, 8 mmol, 86%) with THF:CH₂Cl₂ / 4:1.

Example 87: 5'-O-DMT-2'-Deoxy-2'-Methoxycarbonylmethylidine-Uridine 2'-Deoxy-2'-methoxycarbonylmethylidine-uridine 34 (1.2 g, 4.02 mmol) was dissolved in pyridine (20 mL). A solution of DMT-Cl (1.5 g, 4.42 mmol) in pyridine (10 mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was concentrated in vacuo and the residue taken up in CH₂Cl₂ (100 mL) and washed with sat. NaHCO₃, water and brine. The organic extracts were dried over MgSO₄, concentrated in vacuo and purified over a silica gel column using 2-5% MeOH in CH₂Cl₂ as an eluant to yield 5'-O-DMT-2'-deoxy-2'-methoxycarbonylmethylidine-uridine 35 (2.03 g, 3.46 mmol, 86%). Example 88: 5'-O-DMT-2'-Deoxy-2'-Methoxycarbonylmethylidine-Uridine 3'-(2-cyanoethyl-N,N-diisopropylphosphoramidite) (36)

1-(2'-Deoxy-2'-2'-methoxycarbonylmethylidine-5'-O-dimethoxytrityl-β-D-ribofuranosyl)-uridine 35 (2.0 g, 3.4 mmol) dissolved in dry CH₂Cl₂ (10 mL) was placed in a round-bottom flask under Ar. Diisopropylethylamine (1.2 mL, 6.8 mmol) was added, followed by the dropwise addition of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.91 mL, 4.08 mmol). The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL). After 10 m the mixture was evaporated to a syrup in vacuo (40 °C). 5'-O-DMT-2'-deoxy-2'-methoxycarbonylmethylidine-uridine 3'-(2-cyanoethyl-N,N-diisopropylphosphoramidite) 36 (1.8 g, 2.3 mmol, 67%) was purified by flash column chromatography over silica gel using a 30-60% EtOAc gradient in hexanes, containing 1% triethylamine, as eluant. R_f 0.44 (CH₂Cl₂:MeOH / 9.5:0.5). Example 89: 2'-Deoxy-2'-Carboxymethylidine-3',5'-O-(Tetraisopropyldisiloxane-1 ,3-diyl)-Uridine 37

2'-Peoxy-2'-methoxycarbonylmethylidine-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-uridine 33 (5.0 g, 10.8 mmol) was dissolved in MeOH (50 mL) and 1 N NaOH solution (50 mL) was added to the stirred solution at RT. The mixture was stirred for 2 h and MeOH removed in vacuo. The pH of the aqueous layer was adjusted to 4.5 with 1 N HCl solution, extracted with EtOAc (2 × 100 mL), washed with brine, dried over MgSO₄ and concentrated in vacuo to yield the crude acid. 2'-Deoxy-2'-carboxymethylidine-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-uridine 37 (4.2 g, 7.8 mmol, 73%) was purified on a silica gel column using a gradient of 10-15% MeOH in CH₂Cl₂.

The alkyl substituted nucleotides of this invention can be used to form stable oligonucleotides as discussed above for use in enzymatic cleavage or antisense situations. Such oligonucleotides can be formed enzymatically using triphosphate forms by standard procedure. Administration of such oligonucleotides is by standard procedure. See Sullivan et al. PCT WO 94/02595.

Oligonucleotides with 3' and/or 5' Dihalophosphonate

This invention synthesis and uses 3' and/or 5' dihalophosphonate-, e.g., 3' or 5'-CF₂-phosphonate-, substituted nucleotides that maintain or enhance the catalytic activity and/or nuclease resistance of an enzymatic or antisense molecule.

As the term is used in this application, 5'- and/or 3'-dihalophosphonate nucleotide containing ribozymes, deoxyribozymes (see

Usman et al., PCT/US94/11649, incorporated by reference herein), and chimeras of nucleotides, are catalytic nucleic molecules that contain 5'-and/or 3'-dihalophosphonate nucleotide components replacing, but not limited to, double-stranded stems, single-stranded "catalytic core" sequences, single-stranded loops or single-stranded recognition sequences. These molecules are able to cleave (preferably, repeatedly cleave) separate RNA or DNA molecules in a nucleotide base sequence specific manner. Such catalytic nucleic acids can also act to cleave intramolecularly if that is desired. Such enzymatic molecules can be targeted to virtually any RNA or DNA transcript. This invention concerns nucleic acids formed of standard nucleotides or modified nucleotides, which also contain at least one 5'-dihalophosphonate and/or one 3'-dihalophosphonate group.

The synthesis of 1 -O- Ac-2,3-di-O- Bz-D-ribofuranose 5-d-5+dihalomethylphosphonate in three steps from 1-O-methyl-2,3-O-isopropylidene-β-D-ribofuranose 5-deoxy-5-dihalomethylphosphonate is described (e.g., for the difluoro, in Figure 87). Condensation of this suitably derivatized sugar with silylated pyrimidines and purines affords novel nucleoside 5'-deoxy-5'-dihalomethylphosphonates. These intermediates may be incorporated into catalytic or antisense nucleic acids by either chemical (conversion of the nucleoside 5'-deoxy-5'-dihalomethylphosphonates into suitably protected phosphoramidites 12a or solid supports 12b, e.g., Figure 88) or enzymatic means (conversion of the nucleoside 5'-deoxy-5'-dihalomethylphosphonates into their triphosphates, e.g., 14 Figure 89, for T7 transcription).

Thus, in one aspect the invention features 5' and/or 3'-dihalonucleotides and nucleic acids containing such 5' and/or 3'-dihalonucleotides. The general structure of such molecules is shown below.

where R ₁ is H, OH, or R, where R is a hydroxyl protecting group, e.g., acyl, alkysilyl, or carbonate; each R₂ is separately H, OH, or R; each R₃ is separately a phosphate protecting group, e.g., methyl, ethyl, cyanoethyl, p-nitrophenyl, or chlorophenyl; each X is separately any halogen; and each B is any nucleotide base.

The invention in particular features nucleic acid molecules having such modified nucleotides and enzymatic activity. In a related aspect the invention features a method for synthesis of such nucleoside 5'-deoxy-5'-dihalo and/or 3'-deoxy-3'-dihalophosphonates by condensing a dihalophosphonate-containing sugar with a pyrimidine or a purine under conditions suitable to form a nucleoside 5'-deoxy-5'-dihalophosphonate and/or a 3'-deoxy-3'-dihalophosphonate.

Phosphonic acids may exhibit important biological properties because of their similarity to phosphates (Engel, Chem. Rev. 1977, 77, 349-367). Blackburn and Kent (J. Chem. Soc, Perkin Trans. 1986, 913-917) indicate that based on electronic and steric considerations _-fluoro and _,_-difluoromethylphosphonates might mimic phosphate esters better than the corresponding phosphonates. Analogues of pyro- and triphosphates 1 , where the bridging oxygen atoms are replaced by a difluoromethylene group, have been employed as substrates in enzymatic processes (Blackburn et al., Nucleosides & Nucleotides 1985, 4, 165-167; Blackburn et al., Chem. Scr. 1986, 26, 21 -24). 9-(5,5-Difluoro-5-phosphonopentyl)guanine (2) has been utilized as a multisubstrate analogue inhibitor of purine nucleoside phosphorylase (Halazy et al., J. Am. Chem. Soc. 1991 , 113, 315-317). Oligonucleotides containing methylene groups in place of phosphodiester 5'-oxygens are resistant toward nucleases that cleave phosphodiester linkages between phosphorus and the 5'-oxygen (Breaker et al., Biochemistry 1993, 32, 9125-9128), but can still form stable complexes with complementary sequences. Heinemann et al. (Nucleic Acids Res. 1991 , 19, 427-433) found that a single 3'-methylenephosphonate linkage had a minor influence on the conformation of a DNA octamer double helix.

One common synthetic approach to α,α-difluoro-alkylphosphonates features the displacement of a leaving group from a suitable reactive substrate by diethyl (lithiodifluoromethyl)phosphonate (3) (Obayashi et al., Tetrahedron Lett. 1982, 23, 2323-2326). However, our attempts to synthesize nucleoside 5'-deoxy-5'-difluoro-methylphosphonates from 5'-deoxy-5'-iodonucleosides using 3 were unsuccessful, i.e. starting compounds were quantitatively recovered. The reaction of nucleoside 5'-aldehydes with 3, according to the procedure of Martin et al. (Martin et al., Tetrahedron Lett. 1992, 33, 1839-1842), led to a complex mixture of products. Recently, the synthesis of sugar α,α-difluoroalkylphosphonates from primary sugar triflates using 3 was described (Berkowitz et al., J. Org. Chem. 1993, 58, 6174-6176). Unfortunately, our experience is that nucleoside 5'-triflates are too unstable to be used in these syntheses. The following are non-limiting examples showing the synthesis of nucleoside 5'-deoxy-5'-difluoromethyl-phosphonates. Those in the art will recognize that equivalent methods can be readily devised based upon these examples. These examples demonstrate that it is possible to achieve synthesis of 5'-deoxy-5'-difluoro derivatives in good yield and thus guide those in the art to such equivalent methods. The examples also indicate utility of such synthesis to provide useful oligonucleotides as described above.

Those in the art will recognize that useful modified enzymatic nucleic acids can now be designed, much as described by Draper et al., PCT/US94/13129 hereby incorporated by reference herein (including drawings). Example 90: Synthesis of Nucleoside 5'-Peoxy-5'-difluoromethylphosphonates

Referring to Fig. 87, we synthesized a suitable glycosylating agent from the known D-ribose α,α-difluoromethylphosphonate (4) (Martin et al.,

Tetrahedron Lett. 1992, 33, 1839-1842) which served as a key intermediate for the synthesis of nucleoside 5'-difluoromethylphosphonates.

Methyl 2,3-O - i s o p ro pyl i d e n e- β -D-ribofu ranose a, a-difluoromethylphosphonate (4) was synthesized from the 5-aldehyde according to the procedure of Martin et al. (Tetrahedron Lett. 1992, 33, 1839-1842) (Figure 87). Removal of the isopropylidene group was accomplished under mild conditions (I₂-MeOH, reflux, 18 h (Szarek et al., Tetrahedron Lett. 1986, 27, 3827) or Dowex 50 WX8 (H⁺), MeOH, RT (about 20-25°C), 3 days) in 72% yield. The anomeric mixture thus obtained was benzoylated with benzoyl chloride/pyridine to afford the 2,3-di-O-benzoyl derivative, which was subjected to mild acetolysis conditions (Walczak et al., Synthesis, 1993, 790-792) (Ac₂O, AcOH, H₂SO₄, EtOAc, 0°C The desired 1 -O-acetyl-2,3-di-O- benzoyl-D-ribofu ranose difluoromethylphosphonate (5) was obtained in quantitative yield as an anomeric mixture. These derivatives were used for selective glycosylation of silylated uracil and N⁴-acetylcytosine under Vorbrüggen conditions (Vorbrüggen, Nucleoside Analogs. Chemistry, Biology and Medical Applications, NATO ASI Series A, 26, Plenum Press, New York, London, 1980; pp. 35-69. The use of F₃CSO₂OSi(CH₃)₃ as a glycosylation catalyst is precluded because it is expected to lead to the undesired 1-ethyluracil or 9-ethyladenine byproducts: Podyukova, et al., Tetrahedron Lett. 1987, 28, 3623-3626 and references cited therein) (SnCl₄ as a catalyst, boiling acetonitrile) to yield β-nucleosides (62% 6a, 75% 6b). Glycosylation of silylated N⁶-benzoyladenine under the same conditions yielded a mixture of N-9 isomer 6c and N-7 isomer 7 in 34% and 15% yield, respectively. The above nucleotides were successfully deprotected using trimethylsilylbromide for the cleavage of the ethyl groups, followed by treatment with ammonia-methanol to remove the acyl protecting groups. Nucleoside 5'-deoxy-5'-difluoromethylphosphonates 8 were finally purified on a DEAE Sephadex A-25 (HCO₃-) column using a 0.01-0.25 M TEAB gradient for elution and obtained as their sodium salts (82% 8a; 87% 8b; 82% 8c).

Selected analytical data: ³¹ P-NMR (³¹ P) and ¹ H-NMR (¹ H) were recorded on a Varian Gemini 400. Chemical shifts in ppm refer to H₃PO₄ and TMS, respectively. Solvent was CDCI₃ unless otherwise noted. 5: ¹ H δ 8.07-7.28 (m, Bz), 6.66 (d, J _{1 ,2} 4.5, αH1), 6.42 (s, βH1), 5.74 (d, J_{2 ,3} 4.9, βH2), 5.67 (dd, J_3,2 4.9, J_3,4 6.6, βH3), 5.63 (dd, J_3,2 6.7, J_3,4 3.6, αH3), 5.57 (dd, J_2,1 4.5, J_2,3 6.7, αH2), 4.91 (m, H4), 4.30 (m, CH₂CH₃), 2.64 (m, CH₂CF₂), 2.18 (s, βAc), 2.12 (s, αAc), 1.39 (m, CH₂CH₃). ³¹ P δ 7.82 (t, J_{P ,F} 105.2), 7.67 (t, J_P,F 106.5). 6a: ¹ H δ 9.11 (s, 1 H, NH), 8.01 (m, 11H, Bz, H6), 5.94 (d, J _{1 ',2'} 4.1 , 1 H, H1'), 5.83 (dd, J_5,6 8.1 , 1 H, H5), 5.79 (dd,

J_{2', 1 '} 4.1 , J_2',3' 6.5, 1 H, H2'), 5.71 (dd, J_3',2' 6.5, J_3',4' 6.4, 1 H, H3'), 4.79 (dd, J_4',3. 6.4, J_4',F 11.6, 1 H, H4'), 4.31 (m, 4H, CH₂CH₃), 2.75 (tq, J_{H ,F} 19.6, 2H, CH₂CF₂), 1.40 (m, 6H, CH₂CH₃). ³¹ P δ 7.77 (t, J_P,F 104.0). 8c: ³¹ P (vs DSS) (D₂O) δ 5.71 (t, J_P,F 87.9). Compound 7 was deacylated with methanolic ammonia yielding the product that showed λ_max (H₂O) 271 nm and λ_mjn 233 nm, confirming that the site of glycosylation was N-7.

Example 91 :Synthesis of Nucleic Acids Containing Modified Nucleotide Containing Cores

The method of synthesis used follows the procedure for normal RNA synthesis as described in Usman et al., J. Am. Chem. Soc. 1987, 109, 7845-7854 and in Scaringe et al., Nucleic Acids Res. 1990, 18, 5433-5441 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end (Figure 88 and Janda et al., Science 1989, 244:437-440.). These nucleoside 5'-deoxy-5'-difluoromethylphosphonates may be incorporated not only into hammerhead ribozymes, but also into hairpin, hepatitis delta virus, Group 1 or Group 2 introns, or into antisense oligonucleotides. They are, therefore, of general use in any nucleic acid structure. Example 92: Synthesis of Modified Triphosphate

The triphosphate derivatives of the above nucleotides can be formed as shown in Fig. 89. according to known procedures. Nucleic Acid Chem.,

Leroy B. Townsend, John Wiley & Sons, New York 1991 , pp. 337-340;

Nucleotide Analogs, Karl Heinz Scheit; John Wiley & Sons New York 1980, pp. 211-218.

Equivalent synthetic schemes for 3' dihalophosphonates are shown in Figures 90 and 91 using art recognized nomenclature. The conditions can be optimized by standard procedures.

The nucleoside dihalophosphonates described herein are advantageous as modified nucleotides in any nucleic acid structure, e.g., catalytic or antisense, since they are resistant to exo- and endonucleases that normally degrade unmodified nucleic acids in vivo. They also do not perturb the normal structure of the nucleic acid in which they are incorporated thereby maintaining any activity associated with that structure. These compounds may also be of use as monomers as antiviral and/or antitumor drugs.

Oligonucleotides with Amido or Peptido Modification

This invention replaces 2'-hydroxyl group of a ribonucleotide moiety with a 2'-amido or 2'-peptido moiety. In other embodiments, the 3' and 5' portions of the sugar of a nucleotide may be substituted, or the phosphate group may be substituted with amido or peptido moieties. Generally, such a nucleotide has the general structure shown in Formula I below:

FORMULA I

The base (B) is any one of the standard bases or is a modified nucleotide base known to those in the art, or can be a hydrogen group. In addition, either R₁ or R₂ is H or an alkyl, alkene or alkyne group containing between 2 and 10 carbon atoms, or hydrogen, an amine (primary, secondary or tertiary, e.g., R₃NR₄ where each R₃ and R₄ independently is hydrogen or an alkyl, alkene or alkyne having between 2 and 10 carbon atoms, or is a residue of an amino acid, Le,., an amide), an alkyl group, or an amino acid (D or L forms) or peptide containing between 2 and 5 amino acids. The zigzag lines represent hydrogen, or a bond to another base or other chemical moiety known in the art. Preferably, one of R ₁ , R₂ and R₃ is an H, and the other is an amino acid or peptide.

Applicant has recognized that RNA can assume a much more complex structural form than DNA because of the presence of the 2'-hydroxyl group in RNA. This group is able to provide additional hydrogen bonding with other hydrogen donors, acceptors and metal ions within the

RNA molecule. Applicant now provides molecules which have a modified amine group at the 2' position, such that significantly more complex structures can be formed by the modified oligonucleotide. Such modification with a 2'-amido or peptido group leads to expansion and enrichment of the side-chain hydrogen bonding network. The amide and peptide moieties are responsible for complex structural formation of the oligonucleotide and can form strong complexes with other bases, and interfere with standard base pairing interactions. Such interference will allow the formation of a complex nucleic acid and protein conglomerate. Oligonucleotides of this invention are significantly more stable than existing oligonucleotides and can potentially form biologically active bioconjugates not previously possible for oligonucleotides. They may also be used for in vitro selection of unique aptamers, that is, randomly generated oligonucleotides which can be folded into an effective ligand for a target protein, nucleic acid or polysaccharide.

Thus, in one aspect, the invention features an oligonucleotide containing the modified base shown in Formula I, above.

In other aspects, the oligonucleotide may include a 3' or 5' nucleotide having a 3' or 5' located amino acid or aminoacyl group. In all these aspects, as well as the 2'-modified nucleotide, it will be evident that various standard modifications can be made. For example, an "O" may be replaced with an S, the sugar may lack a base (i.e., abasic) and the phosphate moiety may be modified to include other substitutions (see Sproat, supra).

Example 93: General procedure for the preparation of 2'-aminoacyl-2'-deoxy-2'-aminonucleoside conjugates.

Referring to Fig. 92. to the solution of 2'-deoxy-2'-amino nucleoside (1 mmol) and N-Fmoc L- (or D-) amino acid (1 mmol) in methanol [dimethylformamide (DMF) and tetrahydrofuran (THF) can also be used], 1-ethoxycarbonyl-2-ethoxy-1 ,2-dihydroquinoline (EEDQ) [or 1 -isobutyloxycarbonyl-2-isobutyloxy-1 ,2-dihydroquinoline (IIDQ)] (2 mmol) is added and the reaction mixture is stirred at room temperature or up to 50°C from 3-48 hours. Solvents are removed under reduced pressure and the residual syrup is chromatographed on the column of silica-gel using 1-10 % methanol in dichloromethane. Fractions containing the product are concentrated yielding a white foam with yields ranging from 85 to 95 %. Structures are confirmed by ¹ H NMR spectra of conjugates which show correct chemical shifts for nucleoside and aminoacyl part of the molecule. Further proofs of the structures are obtained by cleaving the aminoacyl protecting groups under appropriate conditions and assigning ¹H NMR resonances for the fully deprotected conjugate.

Partially protected conjugates described above are converted into their 5'-O-dimethoxytrityl derivatives and into 3'-phosphoramidites using standard procedures (Oligonucleotide Synthesis: A Practical Approach, M.J. Gait ed.; IRL Press, Oxford, 1984). Incorporation of these phosphoramidites into RNA was performed using standard protocols (Usman et al., 1987 supra).

A general deprotection protocol for oligonucleotides of the present invention is described in Fig. 93.

The scheme shows synthesis of conjugate of 2'-d-2'-aminouridine.

This is meant to be a non-limiting example, and those skilled in the art will recognize that, variations to the synthesis protocol can be readily generated to synthesize other nucelotides (e.g., adenosine, cytidine, guanosine) and/or abasic moieties.

Example 94: RNA cleavage bv hammerhead ribozymes containing 2'-aminoacyl modifications.

Hammerhead ribozymes targeted to site N (see Fig. 94) are synthesized using solid-phase synthesis, as described above. U4 and U7 positions are modified, individually or in combination, with either 2'-NH-alanine or 2'-NH-lysine.

RNA cleavage assay in vitro: Substrate RNA is 5' end-labeled using [γ-³²P] ATP and T4 polynucleotide kinase (US Biochemicals). Cleavage reactions were carried out under ribozyme "excess" conditions. Trace amount (≤ 1 nM) of 5' end-labeled substrate and 40 nM unlabeled ribozyme are denatured and renatured separately by heating to 90°C for 2 min and snap-cooling on ice for 10 -15 min. The ribozyme and substrate are incubated, separately, at 37°C for 10 min in a buffer containing 50 mM Tris-HCl and 10 mM MgCl₂. The reaction is initiated by mixing the ribozyme and substrate solutions and incubating at 37°C Aliquots of 5 μl are taken at regular intervals of time and the reaction is quenched by mixing with equal volume of 2X formamide stop mix. The samples are resolved on 20 % denaturing polyacrylamide gels. The results are quantified and percentage of target RNA cleaved is plotted as a function of time.

Referring to Fig. 95. hammerhead ribozymes containing 2'-NH-alanine or 2'-NH-lysine modifications at U4 and U7 positions cleave the target RNA efficiently. Sequences listed in Figure 94 and the modifications described in Figure 95 are meant to be non-limiting examples. Those skilled in the art will recognize that variants (base-substitutions, deletions, insertions, mutations, chemical modifications) of the ribozyme and RNA containing other 2'-hydroxyl group modifications, including but not limited to amino acids, peptides and cholesterol, can be readily generated using techniques known in the art, and are within the scope of the present invention.

Example 95: Aminoacylation of 3'-ends of RNA

I. Referring to Fig. 96. 3'-OH group of the nucleotide is converted to succinate as described by Gait, supra. This can be linked with amino-alkyl solid support (for example: CpG). Zig-zag line indicates linkage of 3'OH group with the solid support.

1L Preparation of aminoacyl-derivatized solid support

A) Synthesis of O-Dimethoxytrityl (O-DMT) amino acids

Referring to Fig. 97, to a solution of L- (or P-) serine, tyrosine or threonine (2 mmol) in dry pyridine (15 ml) 4,4'-dimethoxytrityl chloride (3 mmol) is added and the reaction mixture is stirred at RT (about 20-25°C) for 16 h. Methanol (10 ml) is then added and the solution evaporated under reduced pressure. The residual syrup was partitioned between 5% aq. NaHCO₃ and dichloromethane, organic layer was washed with brine, dried (Na₂SO₄) and concentrated in vacuo. The residue is purified by flash silicagel column chromatography using 2-10% methanol in dichloromethane (containing 0.5 % pyridine). Fractions containing product are combined and concentrated in vacuo to yield white foam (75-85 % yield).

B) Preparation of the solid support and its derivatization with amino acids

Referring to Fig. 97, the modified solid support (has an OH group instead of the standard NH₂ end group) was prepared according to Haralambidis et al., Tetrahedron Lett. 1987, 28, 5199, (P denotes aminopropyl CPG or polystyrene type support). O-DMT or NH-monomethoxytrityl (NH-MMT amino acid was attached to the above solid support using standard procedures for derivatization of the solid support (Gait, 1984, supra) creating a base-labile ester bond between amino acids and the support. This support is suitable for the construction of RNA/DNA chain using suitably protected nucleoside phosphoramidites.

Example 96: Aminoacylation of 5'-ends of RNA

I. Referring to Fig. 98. 5'-amino-containing sugar moiety was synthesized as described (Mag and Engels, 1989 Nucleic Acids Res. 17,

5973). Aminoacylation of the 5'-end of the monomer was achieved as described above and RNA phosphoramidite of the 5'-aminoacylated monomer was prepared as described by Usman et al., 1987 supra. The phosphoramidite was then incorporated at the 5'-end of the oligonucleotide using standard solid-phase synthesis protocols described above.

II. Referring to Fig. 99, aminoacyl group(s) is attached to the phosphate group at the 5'-end of the RNA using standard procedures described above.

VII. Reversing Genetic Mutations Modification of existing nucleic acid sequences can be achieved by homologous recombination. In this process a transfected sequence recombines with homologous chromosomal sequences and can replace the endogenous cellular sequence. Boggs, 8 International J. Cell Cloning 80, 1990, describes targeted gene modification. It reviews the use of homologous DNA recombination to correct genetic defects. Banga and Boyd, 89 Proc. Natl. Acad. Sci. U.S.A. 1735, 1992, describe a specific example of in vivo site-directed mutagenesis using a 50 base oligonucleotide. In this methodology a gene or gene segment is essentially replaced by the oligonucleotide used. This invention uses a complementary oligonucleotide to position a nucleotide base changing activity at a particular site on a gene (RNA or genomic DNA), such that the nucleotide modifying activity will change (or revert) a mutation to wild-type, or its equivalent. By reversion or change of a mutation, we refer to reversion in a broad sense, such as when a mutation at a second site which leads to functional reversion to a wild type phenotype. Also, due to the degeneracy of the genetic code, a revertant may be achieved by changing any one of the three codon positions. Additionally, creation of a stop codon in a deleterious gene (or transcript) is defined here as reverting a mutant phenotype to wild-type. An example of this type of reversion is creating a stop codon in a critical HIV proviral gene in a human.

Referring to Figures 100 and 101 , broadly there are two approaches to causing a site directed change in order to revert a mutation to wild-type. In one (Fig. 100) the oligonucleotide is used to target RNA specifically. RNA is provided with a complementary (Watson-crick) oligonucleotide sequence to that in the target molecule. In this case the sequence modifying oligonucleotide would (analogously to an antisense oligonucleotide or ribozyme) have to be continuously present to revert the RNA as it is made by the cell. Such a reversion would be transient and would potentially require continuous addition of more sequence modifying oligonucleotide. The transient nature of this approach is an advantage, in that treatment could be stopped by simply removing the sequence modifying oligonucleotide (as with a traditional drug). A second approach targets DNA (Fig. 101) and has the advantage that changes may be permanently encoded in the target cell's genetic code. Thus, a single course (or several courses) of treatment may lead to permanent reversion of the genetic disease. If inadvertent chromosomal mutations are introduced this may cause cancer, mutate other genes, or cause genetic changes in the germ-line (in patients of reproductive age). However, if the base changing activity is a specific methylation that may modulate gene expression it would not necessarily lead to germ-line transmission. See Lewin, Genes, 1983 John Wilely & Sons, Inc. NY pp 493-496. Complementary base pairing to single-stranded PNA or RNA is one method of directing an oligonucleotide to a particular site of PNA. This could occur by a strand displacement mechanism or by targeting PNA when it is single-stranded (such as during replication, or transcription). Another method is using triple-strand binding (triplex formation) to double-stranded PNA, which is an established technique for binding polypyrimidine tracts, and can be extended to recognize all 4 nucleotides. See Povsic, T., Strobel, S., & Dervan, P. (1992). Sequence-specific double-strand alkylation and cleavage of DNA mediated by triple-helix formation. J. Am. Chem. Soc. 114. 5934-5944 (1992). Knorre, D.G., Valentin, V.V., Valentina, F.Z., Lebedev, AN. & Federova, O.S. Design and targeted reactions of oligonucleotide derivatives 1-366 (CRC Press, Novosibirsk, 1993) describe conjugation of reactive groups or enzyme to oligonucleotides and can be used in the methods described herein.

Recently, antisense oligonucleotides have been used to redirect an incorrect splice into order to obtain correct splicing of a splice mutant globin gene in vitro. Dominski Z; Kole R (1993) Restoration of correct splicing in thalassemia pre-mRNA by antisense oligonucleotides. Proc Natl Acad Sci U S A 90:8673-7. Analogously, in one preferred embodiment of this invention a complementary oligomer is used to correct an existiing mutant RNA, instead of the traditional approach of inhibiting that RNA by antisense.

In either the RNA or DNA mode, after binding to a particular site on the RNA or DNA the oligonucleotide will modify the nucleic acid sequence. This can be accomplished by activating an endogenous enzyme (see Figure 102). by appropriate positioning of an enzyme (or ribozyme) conjugated (or activated by the duplex) to the oligonucleotide, or by appropriate positioning of a chemical mutagen. Specific mutagens, such as nitrous acid which deaminates C to U, are most useful, but others can also be used if inactivation of a harmful RNA is desired.

RNA editing is an naturally occurring event in mammalian cells in which a sequence modifying activity edits a RNA to its proper sequence post-transcriptionally. Higuchi, M.,, Single, F., Kohler, M., Sommer, B., and Seeburg, P. (1993) RNA Editing of AMPA Receptor Subunit GluR-B: A base-paired intron-exon structure determines position and efficiency Cell 75:1361-1370. The machinery involved in RNA editing can be co-opted by a suitable oligonucleotide in order to promote chemical modification.

The changes in the base created by the methods of this invention cause a change in the nucleotide sequence, either directly, or after DNA repair by normal cellular mechanisms. These changes functionally correct a genetic defect or introduce a stop codon. Thus, the invention is distinct from techniques in which an active chemical group (e.g., an alkylator) is attached to an antisense or triple strand oligonucleotide in order to chemically inactivate the target RNA or DNA.

Thus, this invention creates an alteration to an existing base in a nucleic acid molecule so that the base is read in vivo as a different base. This includes correcting a sequence instead of inactivating a gene but can also include inactivating a deleterious gene.

Thus, in one aspect, the invention features a method for altering in vivo the nucleotide base sequence of a naturally occurring mutant nucleic acid molecule. The method includes contacting the nucleic acid molecule in vivo with an oligonucleotide or peptide nucleic acid or other sequence specific binding molecules able to form a duplex or triplex molecule with the nucleic acid molecule. After formation of the duplex or triplex molecule a base modifying activity chemically or enzymatically alters the targeted base directly, or after nucleic acid repair in vivo. This results in the functional alteration of the nucleic acid sequence.

By "alter", as it is used in this context, is meant that one or more chemical moieties in a targeted base, or bases, is altered so that the mutant nucleic acid will be functionally different. Thus, this is distinct from prior methods of correcting defects in DNA, such as homologous recombination, in which an entire segment of the targeted sequence is replaced with a segment of DNA from the transfected nucleic acid. This is also distinct from other methods that use reactive groups to inactivate a RNA or DNA target, in that this method functionally corrects the sequence of the target, instead of merely damaging it, by causing it to be read by a polymerase as a different base from the original base. As noted above, the naturally occurring enzymes in a cell can be utilized to cause the chemical alteration, examples of which are provided below.

By "functionally alter" is meant that the ability of the target nucleic acid to perform its normal function (i.e.., transcription or translation control) is changed. For example, an RNA molecule may be altered so that it can cause production of a desired protein, or a DNA molecule can be altered so that upon DNA repair, the DNA sequence is changed.

By "mutant" it is meant a nucleic acid molecule which is altered in some way compared to equivalent molecules present in a normal individual. Such mutants may be well known in the art, and include, molecules present in individuals with known genetic deficiencies, such as muscular dystrophy, or diabetes and the like. It also includes individuals with diseases or conditions characterized by abnormal expression of a gene, such as cancer, thalassemia's and sickle cell anemia, and cystic fibrosis. It allows modulation of lipid metabolism to reduce artery disease, treatment of integrated AIDS genomes, and AIDs RNA, and Alzeimer's disease. Thus, this invention concerns alteration of a base in a mutant to provide a "wild type" phenotype and/or genotype. For deleterious conditions this involves altering a base to allow expression or prevent expression as is necassary. When treating an infection, such as HIV, it concerns inactivation of a gene in the HIV RNA by mutation of the mutant (i.e., non-human gene) to a wild type (i.e., no production of a non-human protein). Such modification is performed in trans rather than in cis as in prior methods.

In preferred embodiments, the oligonucleotide is of a length (at least 12 bases, preferably 17 - 22) sufficient to activate dsRNA deaminase in vivo to cause conversion of an adenine base to inosine; the oligonucleotide is an enzymatic nucleic acid molecule that is active to chemically modify a base (see below); the nucleic acid molecule is DNA or RNA; the oligonucleotide includes a chemical mutagen, e.g., the mutagen is nitrous acid; and the oligonucleotide causes deamination of 5-methylcytosine to thymidine, cytosine to uracil, or adenine to inosine, or methtylation of cytosine to 5-methylcytosine. In a most preferred embodiment, the invention features correction of a mutation, rather than inactivation of a target by causing a mutation.

Using in vitro directed evolution, it is possible to screen for ribozymes with catalytic activities different than RNA cleavage. Bartel, D. and Szostak, J. (1993) Isolation of new ribozymes from a large pool of random sequences. Science 261 :1411-1418. Using these methods of in vitro directed evolution, an enzymatic nucleic acid molecule, or ribozyme that mutates bases, instead of cleaving the phosphodiester backbone can be selected. This is a convenient method of obtaining an enzyme with the appropriate base sequence modifying activities for use in the present invention.

Sequence modifying activities can change one nucleotide to another

(or modify a nucleotide so that it will be repaired by the cellular machinery to another nucleotide). Sequence modifying activities could also delete or add one or more nucleotides to a sequence. A specific embodiment of adding sequences is described by Sullenger and Cech, PCT/US94/12976 hereby incorporated by reference herein), in which entire exons with wild-type sequence are spliced into a mutant transcript. The present invention features only the addition of a few bases (1 - 3).

Thus, in another aspect, the invention features ribozymes or enzymatic nucleic acid molecules active to change the chemical structure of an existing base in a separate nucleic acid molecule. Applicant is the first to determine that such molecules would be useful, and to provide a description of how such molecules might be isolated.

Molecules used to achieve in situ reversion can be delivered using the existing means employed for delivering antisense molecules and ribozymes, including liposomes and cationic lipid complexes. If the in situ reverting molecule is composed only of RNA, then expression vectors can be used in a gene therapy protocol to produce the reverting molecules endogenously, analogously to antisense or ribozymes expression vectors. There are several advantages of using such an expression vector, rather than simply replacing the gene through standard gene therapy. Firstly, this approach would limit the production of the corrected gene to cells that already express that gene. Furthermore, the corrected gene would be properly regulated by its natural transcriptional promoter. Lastly, reversion can be used when the mutant RNA creates a dominant gain of function protein (e.g., in sickle cell anemia), where correction of the mutant RNA is necessary to stop the production of the deleterious mutant protein, and allow production of the corrected protein.

Endogenous Mammalian RNA Editing System It was observed in the mid-1980s that the sequence of certain cellular

RNAs were different from the DNA sequence that encodes them. By a process called RNA editing, cellular RNA are post-transcriptionally modified to a) create a translation initiation and termination codons, b) enable tRNA and rRNA to fold into a functional conformation (for a review see Bass, B. L. (1993) In The RNA World. R. Gesteland, R. and Atkins, J. eds. (Cold Spring Harbor, New York; CSH Lab. Press) pp. 383-418). The process of RNA editing includes base modification, deletion and insertion of nucleotides.

Although, the RNA editing process is widespread among lower eukaryotes, very few RNAs (four) have been reported to undergo editing in mammals (Bass, supra). The predominant mode of RNA editing in mammalian system is base modification (C → U and A→ G). The mechanism of RNA editing in the mammalian system is postulated to be that C→U conversion is catalyzed by cytidine deaminase. The mechanism of conversion of A→G has recently been reported for glutamate receptor B subunit (gluR-B) in rat PC12 cells (Higuchi, M. et al. (1993) Cell 75, 1361-1370). According to Higuchi gluR-B mRNA precursor attains a structure such that intron 11 and exon 11 can form a stable stem-loop structure. This stem-loop structure is a substrate for a nuclear double strand-specific adenosine deaminase enzyme. The deamination will result in the conversion of A→l. Reverse transcription followed by double strand synthesis will result in the incorporation of G in place of A.

In the present invention, the endogenous deaminase activity or other such activities can be utilized to achieve targeted base modification. The following are examples of the invention to illustrate different methods by which in vivo conversion of a base can be achieved. These are provided only to clarify specific embodiments of the invention and are not limiting to the invention. Those in the art will recognize that equivalent methods can be readily devised within the scope of the claims. Example 97: Exploiting cellular dsRNA dependent Adenine to Inosine converter:

An endogenous activity in most mammalian cells and Xenopus oocytes converts about 50% of adenines to inosines in double stranded RNA. (Bass, B. L., & Weintraub, H. (1988). An unwinding activity that covalently modifies it double-stranded RNA substrate. Cell, 55, 1089-1098.). This activity can be used to cause an in situ reversion of a mutation at the RNA level. Referring to Figures 102 and 104, for demonstration purposes a stop codon is incorporated into the coding region of dystrophin, which is fused to the reporter gene luciferase. This stop codon can be reverted by targeting an antisense RNA which is long enough to activate the dsRNA deaminase, which converts Adenines to Inosines. The A to I transition will be read by the ribosome as an A to G transition in some cases and will thereby functionally revert the stop codon. While other A's in this region may be converted to I's and read as G, converting an A to I (G) cannot create a stop codon. The A to I transitions in the region surrounding the target mutation will create some point mutations, however, the function of the dystrophin protein is rarely inactivated by point mutations.

The reverted mRNA was then translated in a cell lysate and assayed for luciferase activity. As evidenced by the dramatic increase in luciferase counts in the graph in figure 103, the A to I transition was read by the ribosome as an A to G transition and the stop codon has successfully been reverted with the lysate treated complex. As a control, an irrelevant non-complementary RNA oligonucleotide was added to the dystrophin/luciferase mRNA. As expected, in this case no translation (luciferase activity) is observed because of the stop codon. As an additional control, the hybrid was not treated with extract, and again no translation (Iuciferase activity) is observed (Figure 103).

While other A's in the targeted region may have been converted to I's and read as G, converting an A to I (G) cannot create a stop codon, so the ribosome will still read through the region. Dystrophin is not generally sensitive to point mutations if the open reading frame is maintained, so a dystrophin protein made from an mRNA reverted by this method should retain full activity. The following detail specifics of the methodology: RNA oligonucleotides were synthesized on a 394 (ABI) synthesizer using phosphoramidite chemistry. The sequence of the synthetic complementary RNA that binds to the mutant dystrophin sequence is as follows (5' to 3'):

CCCGCGGTAGATCTTTCTGGAGGCTTACAGTTTTCTACAAACCTCC CTTCAAA (Seq. ID No. 1)

Referring to Figure 104, fifty-nine base pairs of a human dystrophin mutant sequence containing a stop codon was fused in frame to the luciferase coding region using standard cloning technology, into the Hind III and Not I sites of pRC-CMV (Invitrogen, San Diego, CA). The AUG of luciferase was deleted. The sequences of the insert from the Hind III site to the start of the Iuciferase coding region is (5' to 3'):

GCCCCTGAGGAGCGATGGAGGCCTTGAAGGGAGGTTTGTGGAAAA CTGTAAGCCTCCAGAAAGATCTACCGCGG (Seq ID No. 2) This corresponds to base pairs 3649-3708 of normal dystrophin (Entrez IP # 311627) with a Sac II site at the 3' end. This plasmid was used as a template for in vitro transcription of mRNA using T7 polymerase with the manufacturers protocol (Promega, Madison, WI). Xenopus nuclear extracts were prepared in 0.5X TGKED buffer (0.5X=

25mM Tris (pH 7.9), 12.5% glycerol, 25 mM KCl, 0.25mM PTT and 0.05mM EDTA), by vortexing nuclei and resuspended in a volume of 0.5X TGKEP equal to total cytoplasm volume of the oocytes. Bass, B.L. & Weintraub, H. Cell 55, 1089-1098 (1988). The target mRNA at 500ng/ul was pre-annealed to 1 micromolar complementary or irrelevant RNA oligonucleotide by heating to 70°C, and allowing it to slowly cool to 37°C over 30 minutes. Fifty nanograms of mRNA pre-annealed to the RNA oligonucleotides was added to 7ul of nuclear extracts containing 1 mM ATP, 15mM EDTA, 1600un/ml RNasin and 12.5mM Tris pH 8 to a total volume of 12ul. Bass, B.L. & Weintraub, H. supra. This mixture, which contains the dsRNA deaminase activity, was incubated for 30 minutes at 25°C Next, 1.5ul of this mixture was added to a rabbit reticulocyte lysate in vitro translation mixture and translated for two hours according to the manufacturers protocol (Life Technologies, Gaithersberg, MP), except that an additional 1.3 mM magnesium acetate was added to compensate for the EPTA carried through from the nuclear extract mixture. Luciferase assays were performed on 15ul of extract with the Promega luciferase assay system (Promega, Madison, WI), and luminescence was detected with a 96 well luminometer, and the results are displayed in the graph in figure 102.

Example 98: Base changing activities

The chemical synthesis of antisense and triple-strand forming oligomers conjugated to reactive groups is well studied and characterized (Knorre, D.G., Valentin, V.V., Valentina, F.Z., Lebedev, AN. & Federova, O.S. Design and targeted reactions of oligonucleotide derivatives 1-366 (CRC Press, Novosibirsk, 1993) and Povsic, T., Strobel, S. & Dervan, P. Sequence-specific double-strand alkylation and cleavage of DNA mediated by triple-helix formation J. Am. Chem. Soc. 114, 5934-5944 (1992). Reactive groups such as alkylators that can modify nucleotide bases in targeted RNA or DNA have been conjugated to oligonucleotides. Additionally enzymes that modify nucleic acids have been conjugated to oligonucleotides. (Knorre, P.G., Valentin, V.V., Valentina, F.Z., Lebedev, AN. & Federova, O.S. Design and targeted reactions of oligonucleotide derivatives 1 -366 (CRC Press, Novosibirsk, 1993). In the past these conjugated chemical groups or enzymes have been used to inactivate DNA or RNA that is specifically targeted by antisense or triple-strand interactions. Below is a list of useful base changing activities that could be used to change the sequence of DNA or RNA targeted by antisense or triple strand interactions, in order to achieve in situ reversion of mutations, as described herein (see figure 100-104).

1. Deamination of 5-methylcytosine to create thymidine

(performed by the enzyme cytidine deaminase (Bass, B.L. in The RNA World (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1993). Also, nitrous acid or related compounds promote oxidative deamination of C to be read at T(Microbial Genetics, David Freifelder, Jones and Bartlett Publishers, Inc., Boston, 1987, PP.226-230.). Additionally hydroxylamine or related compounds can transform C to be read at T (Microbial Genetics, David Freifelder, Jones and Bartlett Publishers, Inc., Boston, 1987, PP.226-230.) 2. Deamination of cytosine to create uracil (performed by the enzyme cytidine deaminase (Bass, B.L. in The RNA World (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1993) or by chemical groups similar to nitrous acid that promote oxidative deamination (Microbial Genetics, David Freifelder, Jones and Bartlett Publishers, Inc., Boston, 1987, PP.226-230.)

3. Deamination of Adenine to be read like G (Inosine) (as done by the adenosine deaminase, AMP deaminase or the dsRNA deaminating activity ( Bass, B.L. in The RNA World (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1993). 4. Methylation of cytosine to 5-methylcytosine

5. Transforming thymidine (or uracil) to O²-methyl thymidine (or

O²-methyl uracil), to be read as cytosine by alkynitrosoureas (Xu, and Swann, Tetrahedron Letters 35:303-306 (1994)). 6. Transforming guanine to 6-O-methyl (or other alkyls) to be read as adenine (Mehta and Ludlum, Biochimica et Biophysica Acta, 521 :770-778 (1978) which can be done with the mutagen ethyl methane sulfonate (EMS) Microbial Genetics, David Freifelder, Jones and Bartlett Publishers, Inc., Boston, 1987, PP.226-230.

7. Amination of uracil to cytosine (as performed by the cellular enzyme CTP synthetase (Bass, B.L. in The RNA World (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1993).

The following are examples of useful chemical modifications that can be utilized in the present invention. There are a few preferred straightforward chemical modifications that can change one base to another base. Appropriate mutagenic chemicals are placed on the targetting oligonucleotide, e.g., nitrous acid, or a suitable protein with such activity. Such chemicals and proteins can be attatched by standard procedures. These include molecules which introduce fundamental chemical changes, that would be useful independent of the particular technical approach. See Lewin, Genes.1983 John Wilely & Sons, Inc. NY pp 42-48.

The following matrix shows that the chemical modifications noted can cause transversion reversions (pyrimidine to pyrimidine, or purine to purine) in RNA or DNA. The transversions (pyrimidine to purine, or purine to pyrimidine) are not preferred because these are more difficult chemical transformations. The footnotes refer to the specific desired chemical transformations. The bold footnotes refer to the reaction on the opposite DNA strand. For example, if one desires to change an A to a G, this can be accomplished at the DNA level by using reaction #5 to change a T to a C in the opposing strand. In this example an A/T base pair goes to A/C , then when the DNA is replicated, or mismatch repair occurs this can become G/C, thus the original A has been converted to a G. ISR matrix

Reverted Base

Mutant base A T(U) C G

1 Deamination of 5-methylcytosine to create thymidine.

2 Deamination of cytosine to create uracil.

3 Deamination of Adenine to be read like G (Inosine).

4 Methylation of cytosine to 5-methylcytosine.

5 Transforming thymidine (or uracil) to O²-methyl thymidine (or O²-methyl uracil), to be read as cytosine (Xu, and Swann, Tetrahedron Letters 35:303-306 (1994)).

6 Transforming guanine to 6-O-methyl (or other alkyls) to be read as adenine (Mehta and Ludlum, Biochimica et Biophysica Acta, 521 :770-778 (1978)).

7. Amination of uracil to cytosine. Bass supra, fig. 6c.

In Vitro Selection Strategy

Referring to Figure 105. there is provided a schematic describing an approach to selecting for a ribozyme with such base changing activity. An

RNA is designed that folds back on itself (this is similar to approaches already used to select for RNA ligases, Bartel, D. and Szostak, J. (1993)

Isolation of new ribozymes from a large pool of random sequences.

Science 261 :1411-1418). A degenerate loop opposing the base to be modified provides for diversity. After incubating this library of molecules in a buffer, the RNA is reverse transcribed into DNA (that is, using standard in vitro evolution protocol. Tuerk and Gold, 249 Science 505, 1990) , and then the DNA is selected for having a base change. A restriction enzyme cleavage and size selection or its equivalent is used to isolate the fraction of DNAs with the appropriate base change. The cycle could then be repeated many times. The in vitro selection (evolution) strategy is similar to approaches developed by Joyce (Beaudry, A. A. and Joyce, G.F. (1992) Science 257, 635-641 ; Joyce, G. F. (1992) Scientific American 267. 90-97) and Szostak (Bartel, D. and Szostak, J. (1993) Science 261 :1411-1418; Szostak, J. W. (1993) TIBS 17, 89-93). Briefly, a random pool of nucleic acids is synthesized wherein, each member contains two domains: a) one domain consists of a region with defined (known) nucleotide sequence; b) the second domain consists of a region with degenerate (random) sequence. The known nucleotide sequence domain enables: 1) the nucleic acid to bind to its target (the region flanking the mutant nucleotide), 2) complimentary DNA (cDNA) synthesis and PCR amplification of molecules selected for their base modifying activity, 3) introduction of restriction endonuclease site for the purpose of cloning. The degenerate domain can be created to be completely random (each of the four nucleotides represented at every position within the random region) or the degeneracy can be partial (Beaudry, A. A. and Joyce, G.F. (1992) Science 257, 635-641). In this invention, the degenerate domain is flanked by regions containing known sequences (see Figure 105), such that the degenerate domain is placed across from the mutant base (the base that is targeted for modification). This random library of nucleic acids is incubated under conditions that ensure folding of the nucleic acids into conformations that facilitate the catalysis of base modification (the reaction protocol may also include certain cofactors like ATP or GTP or an S-adenosyl-methionine (if methylation is desired) in order to make the selection more stringent). Following incubation, nucleic acids are converted into complimentary DNA (if the starting pool of nucleic acids is RNA). Nucleic acids with base modification (at the mutant base position) can be separated from rest of the population of nucleic acids by using a variety of methods. For example, a restriction endonuclease cleavage site can either be created or abolished as a result of base modification. If a restriction endonuclease site is created as a result of base modification, then the library can be digested with the restriction endonuclease (RE). The fraction of the population that is cleaved by the RE is the population that has been able to catalyze the base modification reaction (active pool). A new piece of DNA (containing oligonucleotide primer binding sites for PCR and RE sites for cloning) is ligated to the termini of the active pool to facilitate PCR amplification and subsequent cycles (if necessary) of selection. The final pool of nucleic acids with the best base modifying activity is cloned in to a plasmid vector and transformed into bacterial hosts. Recombinant plasmids can then be isolated from transformed bacteria and the identity of clones can be determined using DNA sequencing techniques.

Base modifying enzymatic nucleic acids (identified via in vitro selection) can be used to cause the chemical modification in vivo.

In addition, the ribozyme could be evolved to specifically bind a protein having an enzymatic base changing acitivity.

Such ribozymes can be used to cause the above chemical modifications in vivo. The ribozymes or above noted antisense-type molecules can be administered by methods discussed in the above referenced art.

VIII. Administration of Nucleic Acids

Applicant has determined that double-stranded nucleic acid lacking a transcription termination signal can be used for continuous expression of the encoded RNA. This is achieved by use of an R-loop, i.e., an RNA molecule non-covalently associated with the double-stranded nucleic acid and which causes localized denaturation ("bubble" formation) within the double stranded nucleic acid (Thomas et al., 1976 Proc. Natl. Acad. Sci. USA 73, 2294). In addition, applicant has determined that that the RNA portion of the R-loop can be used to target the whole R-loop complex to a desirable intracellular or cellular site, and aid in cellular uptake of the complex. Further, applicant indicates that expression of enzymatically active RNA or ribozymes can be significantly enhanced by use of such R-loop complexes. Thus, in one aspect, the invention features a method for introduction of enzymatic nucleic acid into a cell or tissue. A complex of a first nucleic acid encoding the enzymatic nucleic acid and a second nucleic acid molecule is provided. The second nucleic acid molecule has sufficient complementarity with the first nucleic acid to be able to form an R-loop base pair structure under physiological conditions. The R-loop is formed in a region of the first nucleic acid molecule which promotes expression of RNA from the first nucleic acid under physiological conditions. The method further includes contacting the complex with a cell or tissue under conditions in which the enzymatic nucleic acid is produced within the cell or tissue.

By "complex" is simply meant that the two nucleic acid molecules interact by intermolecular bond formation (such as by hydrogen bonding) between two complementary base-paired sequences. The complex will generally be stable under physiological condition such that it is able to cause initiation of transcription from the first nucleic acid molecule.

The first and second nucleic acid molecules may be formed from any desired nucleotide bases, either those naturally occurring (such as adenine, guanine, thymine and cytosine), or other bases well known in the art, or may have modifications at the sugar or phosphate moieties to allow greater stability or greater complex formation to be achieved. In addition, such molecules may contain non-nucleotides in place of nucleotides. Such modifications are well known in the art, see e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991 Science. 253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162, as well as Sproat.B. European Patent Application 92110298.4 which describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules. All these publications are hereby incorporated by reference herein.

By "sufficient complementarity" is meant that sufficient base pairing occurs so that the R-loop base pair structure can be formed under the appropriate conditions to cause transcription of the enzymatic nucleic acid. Those in the art will recognize routine tests by which such sufficient base pairs can be determined. In general, between about 15 - 80 bases is sufficient in this invention.

By "physiological condition" is meant the condition in the cell or tissue to be targeted by the first nucleic acid molecule, although the R-loop complex may be formed under many other conditions. One example is use of a standard physiological saline at 37°C, but it is simply desirable in this invention that the R-loop structure exists to some extent at the site of action so that the expression of the desired nucleic acid will be achieved at that site of action. While it is preferred that the R-loop structure be stable under those conditions, even a minimal amount of formation of the R-loop structure to cause expression will be sufficient. Those in the art will recognize that measurement of such expression is readily achieved, especially in the absence of any promoter or leader sequence on the first nucleic acid molecule (Daube and von Hippel, 1992 Science 258, 1320). Such expression can thus only be achieved if an R-loop structure is truly formed with the second nucleic acid. If a promoter of leader sequence is provided, then it is preferred that the R-loop be formed at a site distant from those regions so that transcription is enhanced. In a related aspect, the invention features a method for introduction of ribonucleic acid within a cell or tissue by forming an R-loop base-paired structure (as described above) with the first nucleic acid molecule lacking any promoter region or transcription termination signal such that once expression is initiated it will continue until the first nucleic acid is degraded. In another related aspect, the invention features a method in which the second nucleic acid is provided with a localization factor, such as a protein, e.g., an antibody, transferin, a nuclear localization peptide, or folate, or other such compounds well known in the art, which will aid in targeting the R-loop complex to a desired cell or tissue. In preferred embodiments, the first nucleic acid is a plasmid, e.g., one without a promoter or a transcription termination signal ; the second nucleic acid is of length between about 40-200 bases and is formed of ribonucleotides at a majority of positions; and the second nucleic is covalently bonded with a ligand such as a nucleic acid, protein, peptide, lipid, carbohydrate, cellular receptor, nuclear localization factor, or is attached to maleimide or a thiol group: the first nucleic acid is an expression plasmid lacking a promoter able to express a desired gene, e.g., it is a double-stranded molecule formed with a majority of deoxyribonucleic acids; the R-loop complex is a RNA/DNA heteroduplex; no promoter or leader region is provided in the first nucleic acid; and the R-loop is adapted to prevent nucleosome assembly and is designed to aid recruitment of cellular transcription machinery.

In other preferred embodiments, the first nucleic acid encodes one or more enzymatic nucleic acids, e.g., it is formed with a plurality of intramolecular and intermolecular cleaving enzymatic nucleic acids to allow release of therapeutic enzymatic nucleic acid in vivo.

In a further related aspect, the invention features a complex of the above first nucleic acid molecules and second nucleic acid molecules. R-loop complex

An R-loop complex is designed to provide a non-integrating plasmid so that, when an RNA polymerase binds to the plasmid, transcription is continuous until the plasmid is degraded. This is achieved by hybridizing an RNA molecule, 40 to 200 nucleotides in length, to a DNA expression plasmid resulting in an R-loop structure (see figure 106). This RNA, when conjugated with a ligand that binds to a cell surface receptor, triggers internalization of the plasmid/RNA-ligand complex. Formation of R-loops in general is described by DeWet, 1987 Methods in Enzymol. 145, 235; Neuwald et al., 1977 J. Virol. 21 ,1019; and Meyer et al., 1986 J. Ult. Mol. Str. Res. 96, 187. Thus, those in the art can readily design complexes of this invention following the teachings of the art.

Promoters placed in retroviral genomes have not always behaved as planned in that the additional promoter will serve as a stop signal or reverses the direction of the polymerase. Applicant was told that creation of an R-loop between the promoter and the reporter gene increased the transfection efficiency. Incubation of an RNA molecule with a double-stranded DNA molecule, containing a region of complementarity with the RNA will result in the formation of a stable RNA-DNA hetroduplex and the DNA strand that has a sequence identical to the RNA will be displaced into a loop-like structure called the R-loop. This displacement of DNA strand occurs because an RNA-DNA duplex is more stable compared to a DNA-DNA duplex. Applicant was also told that an 80 nt long RNA was used to generate a R-loop structure in a plasmid encoding the β-galactosidase gene. The R-loop was initiated either in the promoter region or in the leader sequence. Plasmids containing an R-loop structure were microinjected into the cytoplasm of COS cells and the gene expression was assayed. R-loop formation in the promoter region of the plasmid inhibited expression of the gene. RNA that hybridized to the leader sequence between the promoter and the gene, or directly to the first 80 nucleotides of the mRNA increased the expression levels 8-10 fold. The proposed mechanism is that R-loop formation prevents nucleosome assembly, thus making the DNA more accessible for transcription. Alternatively, the R-loop may resemble a RNA primer promoting either DNA replication or transcription (Daube and von Hippel, 1992, supra). One of the salient features of this invention is to generate R-loops in expression vectors of choice and introduce them into cells to achieve enhanced expression from the expression vector. The presence of an R-loop may aid in the recruitment of cellular transcription machinery. Once an RNA polymerase binds to the plasmid and initiates transcription, the process will continue until a termination signal is reached, or the plasmid is degraded.

This invention will increase the expression of ribozymes inside a cell. The idea is to construct a plasmid with no transcription termination signal, such that a transcript-containing multiple ribozyme units can be generated. In order to liberate unit length ribozymes, self-processing ribozymes can be cloned downstream of each therapeutic ribozyme (see figure 107) as described by Draper supra.

Ligand Targeting

Another salient feature of this invention is that the RNA used to generate R-loop structures can be covalently linked to a ligand (nucleic acid, proteins, peptides, lipids, carbohydrates, etc). Specific ligands can be chosen such that the ligand can bind selectively to a desired cell surface receptor. This ligand-receptor interaction will help internalize a plasmid containing an R-loop. Thus, RNA is used to attach the ligand to the DNA such that localization of the gene to certain regions of the cell is achieved. One of several methods can be used to attach a ligand to RNA. This includes the incorporation of deoxythymidine containing a 6 carbon spacer having a terminal primary amine into the RNA (see figure 108). This amino group can be directly derivatized with the ligand, such as folate (Lee and Low, 1994 J. Biol. Chem. 269, 3198-3204). The RNA containing a 6 carbon spacer with a terminal amine group is mixed with folate and the mixture is reacted with activators like 1 -(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC). This reaction should be carried out in the presence of 1-Hydroxybenzotriazole hydrate (HOBT) to prevent any undesirable side reactions. The RNA can also be derivatized with a heterobifuctional crosslinking agent (or linker) like succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB). The SMPB introduces a maleimide into the RNA. This maleimide can then react with a thiol moiety either in a peptide or in a protein. Thiols can also be introduced into proteins or peptides that lack naturally occurring thiols using succinylacetylthioacetate. The amino linker can be attached at the 5' end or 3' end of the RNA. The RNA can also contain a series of nucleotides that do not hybridize to the DNA and extend the linker away from the RNA/DNA complex, thus increasing the accessibility of the ligand for its receptor and not interfering with the hybridization. These techniques can be used to link peptides such as nuclear localization signal (NLS) peptides (Lanford et al., 1984 Cell 37, 801-813; Kalderon et al., 1984 Cell 39, 499-509; Goldfarb et al., 1986 Nature 322, 641-644)and/or proteins like the transferrin (Curiel et al., 1991 Proc. Natl. Acad. Sci. USA 88, 8850-8854; Wagner et al., 1992 Proc. Natl. Acad. Sci. USA 89, 6099-6103; Giulio et al., 1994 Cell. Signal. 6, 83-90) to the ends of R-loop forming RNA in order to facilitate the uptake and localization of the R-loop-DNA complex. To link a protein to the ends of R-loop forming RNA, an intrinsic thiol can be used to react with the maleimide or the thiols can be introduced into the protein itself using either iminothiolate or succinimidyl acetyl thioacetate (SATA; Duncan et al., 1983 Anal. Biochem 132, 68). The SATA requires an additional deprotection step using 0.5 M hydroxylamine.

In addition liposomes can be used to cause an R-loop complex to be delivered to an appropriate intracellular cite by techniques well known in the art. For example, pH-sensitive liposomes (Connor and Huang, 1986 Cancer Res. 46, 3431-3435) can be used to facilitate DNA transfection.

Calcium phosphate mediated or electroporation-mediated delivery of the R-loop complex in to desired cells can also be readily acomplished. In vitro Selection

In vitro selection strategies can be used to select nucleic acids that a) can form stable R-loops b) selectively bind to specific cell surface receptors. These nucleic acids can then be covalently linked to each other.

This will help internalize the R-loop-containing plasmid efficiently using receptor-mediated endocytosis. The in vitro selection (evolution) strategy is similar to approaches developed by Joyce (Beaudry and Joyce, 1992 Science 257, 635-641 ; Joyce, 1992 Scientific American 267, 90-97) and Szostak (Barrel and Szostak, 1993 Science 261 :1411 -1418; Szostak, 1993 TI BS 17, 89-93). Briefly, a random pool of nucleic acids is synthesized wherein each member contains two domains: a) one domain consists of a region with defined (known) nucleotide sequence; b) the second domain consists of a region with degenerate (random) sequence. The known nucleotide sequence domain enables: 1) the nucleic acid to bind to its target (a specific region of the double strand DNA), 2) complimentary DNA (cDNA) synthesis and PCR amplification of molecules selected for their affinity to form R-loop and/or their ability to bind to a specific receptor, 3) introduction of a restriction endonuclease site for the purpose of cloning. The degenerate domain can be created to be completely random (each of the four nucleotides represented at every position within the random region) or the degeneracy can be partial (Beaudry and Joyce, 1992 Science 257, 635-641). In this invention, the degenerate domain is flanked by regions containing known sequences. This random library of nucleic acids is incubated under conditions that ensure equilibrium binding to either double-stranded DNA or cell surface receptor. Following incubation, nucleic acids are converted into complementary DNA (if the starting pool of nucleic acids is RNA). Nucleic acids with desired characteristics can be separated from the rest of the population of nucleic acids by using a variety of methods (Joyce, 1992 supra). The desired pool of nucleic acids can then be carried through subsequent rounds of selection to enrich the population with the most desired traits. These molecules are then cloned in to appropriate vectors. Recombinant plasmids can then be isolated from transformed bacteria and the identity of clones can be determined using DNA sequencing techniques. Other embodiments are within the following claims. TABLE 1

Characteristics of Ribozymes

Group I Introns

Size: ~200 to >1000 nucleotides.

Requires a U in the target sequence immediately 5' of the cleavage site.

Binds 4-6 nucleotides at 5' side of cleavage site.

Over 75 known members of this class. Found in Tetrahymena thermophila rRNA, fungal mitochondria, chloroplasts, phage T4, blue-green algae, and others.

RNAseP RNA (M1 RNA)

Size: ~290 to 400 nucleotides.

RNA portion of a ribonucleoprotein enzyme. Cleaves tRNA precursors to form mature tRNA.

Roughly 10 known members of this group all are bacterial in origin.

Hammerhead Ribozyme

Size: -13 to 40 nucleotides.

Requires the target sequence UH immediately 5' of the cleavage site.

Binds a variable number nucleotides on both sides of the cleavage site.

14 known members of this class. Found in a number of plant pathogens (virusoids) that use RNA as the infectious agent (Figures 1 and 2)

Hairpin Ribozyme

Size: ~50 nucleotides.

Requires the target sequence GUC immediately 3' of the cleavage site.

Binds 4-6 nucleotides at 5' side of the cleavage site and a variable number to the 3' side of the cleavage site.

Only 3 known member of this class. Found in three plant pathogen

(satellite RNAs of the tobacco ringspot virus, ara bis mosaic virus and chicory yellow mottle virus) which uses RNA as the

infectious agent (Figure 3).

Hepatitis Delta Virus (HDV) Ribozyme

Size: 50 - 60 nucleotides (at present).

Cleavage of target RNAs recently demonstrated.

Sequence requirements not fully determined.

Binding sites and structural requirements not fully determined, although no sequences 5' of cleavage site are required.

Only 1 known member of this class. Found in human HDV (Figure 4).

Neurospora VS RNA Ribozyme

Size: ~144 nucleotides (at present) Cleavage of target RNAs recently demonstrated.

Sequence requirements not fully determined.

Binding sites and structural requirements not fully determined. Only 1 known member of this class. Found in Neurospora VS RNA (Figure 5).

Table 2

Human ICAM HH Target sequence

nt. Position Target Sequences nt. Position Target S Sequence:

11 CCCCAGU C GACGCUG 386 ACCGUGU A CUGGACU

23 CUGAGCU C CUCUGCU 394 CUGGACU C CAGAACG

26 AGCUCCU C UGCUACU 420 CACCCCU C CCCUCUU

31 CUCUGCU A CUCAGAG 425 CUCCCCU C UUGGCAG

34 UGCUACU C AGAGUUG 427 CCCCUCU U GGCAGCC

40 UCAGAGU U GCAACCU 450 AGAACCU U ACCCUAC

48 GCAACCU C AGCCUCG 451 GAACCUU A CCCUACG

54 UCAGCCU C GCUAUGG 456 UUACCCU A CGCUGCC

58 CCUCGCU A UGGCUCC 495 CCAACCU C ACCGUGG

64 UAUGGCU C CCAGCAG 510 UGCUGCU C CGUGGGG

96 CCGCACU C CUGGUCC 564 CUGAGGU C ACGACCA

102 UCCUGGU C CUGCUCG 592 GAGAGAU C ACCAUGG

108 UCCUGCU C GGGGCCC 607 AGCCAAU U UCUCGUG

115 CGGGGCU C UGUUCCC 608 GCCAAUU U CUCGUGC

119 GCUCUGU U CCCAGGA 609 CCAAUUU C UCGUGCC

120 CUCUGUU C CCAGGAC 611 AAUUUCU C GUGCCGC

146 CAGACAU C UGUGUCC 656 GAGCUGU U UGAGAAC

152 UCUGUGU C CCCCUCA 657 AGCUGUU U GAGAACA

158 UCCCCCU C AAAAGUC 668 AACACCU C GGCCCCC

165 CAAAAGU C AUCCUGC 677 GCCCCCU A CCAGCUC

168 AAGUCAU C CUGCCCC 684 ACCAGCU C CAGACCU

185 GGAGGCU C CGUGCUG 692 CAGACCU U UGUCCUG

209 AGCACCU C CUGUGAC 693 AGACCUU U GUCCUGC

227 CCCAAGU U GUUGGGC 696 CCUUUGU C CUGCCAG

230 AAGUUGU U GGGCAUA 709 AGCGACU C CCCCACA

237 UGGGCAU A GAGACCC 720 CACAACU U GUCAGCC

248 ACCCCGU U GCCUAAA 723 AACUUGU C AGCCCCC

253 GUUGCCU A AAAAGGA 735 CCCGGGU C CUAGAGG

263 AAGGAGU U GCUCCUG 738 GGGUCCU A GAGGUGG

267 AGUUGCU C CUGCCUG 765 CCGUGGU C UGUUCCC

293 AAGGUGU A UGAACUG 769 GGUCUGU U CCCUGGA

319 AGAAGAU A GCCAACC 770 GUCUGUU C CCUGGAC

335 AUGUGCU A UUCAAAC 785 GGGCUGU U CCCAGUC

337 GUGCUAU U CAAACUG 786 GGCUGUU C CCAGUCU

338 UGCUAUU C AAACUGC 792 UCCCAGU C UCGGAGG

359 GGGCAGU C AACAGCU 794 CCAGUCU C GGAGGCC

367 AACAGCU A AAACCUU 807 CCCAGGU C CACCUGG

374 AAAACCU U CCUCACC 833 CAGAGGU U GAACCCC

375 AAACCUU C CUCACCG 846 CCACAGU C ACCUAUG

378 CCUUCCU C ACCGUGU 851 GUCACCU A UGGCAAC 863 AACGACU C CUUCUCG 1408 UCGAGAU C UUGAGGG

866 GACUCCU U CUCGGCC 1410 GAGAUCU U GAGGGCA

867 ACUCCUU C UCGGCCA 1421 GGCACCU A CCUCUGU

869 UCCUUCU C GGCCAAG 1425 CCUACCU C UGUCGGG

881 AAGGCCU C AGUCAGU 1429 CCUCUGU C GGGCCAG

885 CCUCAGU C AGUGUGA 1444 GAGCACU C AAGGGGA

933 GUGCAGU A AUACUGG 1455 GGGAGGU C ACCCGCG

936 CAGUAAU A CUGGGGA 1482 AUGUGCU C UCCCCCC

978 UGACCAU C UACAGCU 1484 GUGCUCU C CCCCCGG

980 ACCAUCU A CAGCUUU 1493 CCCCGGU A UGAGAUU

986 UACAGCU U UCCGGCG 1500 AUGAGAU U GUCAUCA

987 ACAGCUU U CCGGCGC 1503 AGAUUGU C AUCAUCA

988 CAGCUUU C CGGCGCC 1506 UUGUCAU C AUCACUG

1005 ACGUGAU U CUGACGA 1509 UCAUCAU C ACUGUGG

1006 CGUGAUU C UGACGAA 1518 CUGUGGU A GCAGCCG

1023 CAGAGGU C UCAGAAG 1530 CCGCAGU C AUAAUGG

1025 GAGGUCU C AGAAGGG 1533 CAGUCAU A AUGGGCA

1066 CCACCCU A GAGCCAA 1551 CAGGCCU C AGCACGU

1092 AUGGGGU U CCAGCCC 1559 AGCACGU A CCUCUAU

1093 UGGGGUU C CAGCCCA 1563 CGUACCU C UAUAACC

1125 CCCAGCU C CUGCUGA 1565 UACCUCU A UAACCGC

1163 CGCAGCU U CUCCUGC 1567 CCUCUAU A ACCGCCA

1164 GCAGCUU C UCCUGCU 1584 GGAAGAU C AAGAAAU

1166 AGCUUCU C CUGCUCU 1592 AAGAAAU A CAGACUA

1172 UCCUGGU C UGCAACC 1599 ACAGACU A CAACAGG

1200 GCCAGCU U AUACACA 1651 CACGCCU C CCUGAAC

1201 CCAGCUU A UACACAA 1661 UGAACCU A UCCCGGG

1203 AGCUUAU A CACAAGA 1663 AACCUAU C CCGGGAC

1227 GGGAGCU U CGUGUCC 1678 AGGGCCU C UUCCUCG

1228 GGAGCUU C GUGUCCU 1680 GGCCUCU U CCUCGGC

1233 UUCGUGU C CUGUAUG 1681 GCCUCUU C CUCGGCC

1238 GUCCUGU A UGGCCCC 1684 UCUUCCU C GGCCUUC

1264 GAGGGAU U GUCCGGG 1690 UCGGCCU U CCCAUAU

1267 GGAUUGU C CGGGAAA 1691 CGGCCUU C CCAUAUU

1294 AGAAAAU U CCCAGCA 1696 UUCCCAU A UUGGUGG

1295 GAAAAUU C CCAGCAG 1698 CCCAUAU U GGUGGCA

1306 GCAGACU C CAAUGUG 1737 AAGACAU A UGCCAUG

1321 CCAGGCU U GGGGGAA 1750 UGCAGCU A CACCUAC

1334 AACCCAU U GCCCGAG 1756 UACACCU A CCGGCCC

1344 CCGAGCU C AAGUGUC 1787 AGGGCAU U GUCCUCA

1351 CAAGUGU C UAAAGGA 1790 GCAUUGU C CUCAGUC

1353 AGUGUCU A AAGGAUG 1793 UUGUCCU C AGUCAGA

1366 UGGCACU U UCCCACU 1797 CCUCAGU C AGAUACA

1367 GGCACUU U CCCACUG 1802 GUCAGAU A CAACAGC

1368 GCACUUU C CCACUGC 1812 ACAGCAU U UGGGGCC

1380 UGCCCAU C GGGGAAU 1813 CAGCAUU U GGGGCCA

1388 GGGGAAU C AGUGACU 1825 CCAUGGU A CCUGCAC

1398 UGACUGU C ACUCGAG 1837 CACACCU A AAACACU

1402 UGUCACU C GAGAUCU 1845 AAACACU A GGCCACG 1856 CACGCAU C UGAUCUG 2189 UAUUUAU U GAGUGUC

1861 AUCUGAU C UGUAGUC 2196 UGAGUGU C UUUUAUG

1865 GAUCUGU A GUCACAU 2198 AGUGUCU U UUAUGDA

1868 CUGUAGU C ACAUGAC 2199 GUGUCUU U UAUGUAG

1877 CAUGACU A AGCCAAG 2200 UGUCUUU U AUGUAGG

1901 CAAGACU C AAGACAU 2201 GUCUUUU A UGUAGGC

1912 ACAUGAU U GAUGGAU 2205 UUUAUGU A GGCUAAA

1922 UGGAUGU U AAAGUCU 2210 GUAGGCU A AAUGAAC

1923 GGAUGUU A AAGUCUA 2220 UGAACAU A GGCCUCU

1928 UUAAAGU C UAGCCUG 2224 CAUAGGU C UCUGGCC

1930 AAAGUCU A GCCUGAU 2226 UAGGUCU C UGGCCUC

1964 GAGACAU A GCCCCAC 2233 CUGGCCU C ACGGAGC

1983 AGGACAU A CAACUGG 2242 CGGAGCU C CCAGUCC

1996 GGGAAAU A CUGAAAC 2248 UCCCAGU C CAUGUCA

2005 UGAAACU U GCUGCCU 2254 UCCAUGU C ACAUUCA

2013 GCUGCCU A UUGGGUA 2259 GUCACAU U CAAGGUC

2015 UGCCUAU U GGGUAUG 2260 UCACAUU C AAGGUCA

2020 AUUGGGU A UGCUGAG 2266 UCAAGGU C ACCAGGU

2039 ACAGACU U ACAGAAG 2274 ACCAGGU A CAGUUGU

2040 CAGACUU A CAGAAGA 2279 GUACAGU U GUACAGG

2057 UGGCCCU C CAUAGAC 2282 CAGUUGU A CAGGUUG

2061 CCUCCAU A GACAUGU 2288 UACAGGU U GUACACU

2071 CAUGUGU A GCAUCAA 2291 AGGUUGU A CACUGCA

2076 GUAGCAU C AAAACAC 2321 AAAAGAU C AAAUGGG

2097 CCACACU U CCUGACG 2338 UGGGACU U CUCAUUG

2098 CACACUU C CUGACGG 2339 GGGACUU C UCAUUGG

2115 GCCAGCU U GGGCACU 2341 GACUUCU C AUUGGCC

2128 CUGCUGU C UACUGAC 2344 UUCUCAU U GGCCAAC

2130 GCUGUCU A CUGACCC 2358 CCUGCCU U UCCCCAG

2145 CAACCCU U GAUGAUA 2359 CUGCCUU U CCCCAGA

2152 UGAUGAU A UGUAUUU 2360 UGCCUUU C CCCAGAA

2156 GAUAUGU A UUUAUUC 2376 GAGUGAU U UUUCUAU

2158 UAUGUAU U UAUUCAU 2377 AGUGAUU U UUCUAUC

2159 AUGUAUU U AUUCAUU 2378 GUGAUUU U UCUAUCG

2160 UGUAUUU A UUCAUUU 2379 UGAUUUU U CUAUCGG

2162 UAUUUAU U CAUUUGU 2380 GAUUUUU C UAUCGGC

2163 AUUUAUU C AUUUGUU 2382 UUUUUCU A UCGGCAC

2166 UAUUCAU U UGUUAUU 2384 UUUCUAU C GGCACAA

2167 AUUCAUU U GUUAUUU 2399 AAGCACU A UAUGGAC

2170 CAUUUGU U AUUUUAC 2401 GCACUAU A UGGACUG

2171 AUUUGUU A UUUUACC 2411 GACUGGU A AUGGUUC

2173 UUGUUAU U UUACCAG 2417 UAAUGGU U CACAGGU

2174 UGUUAUU U UACCAGC 2418 AAUGGUU C ACAGGUU

2175 GUUAUUU U ACCAGCU 2425 CACAGGU U CAGAGAU

2176 UUAUUUU A CCAGCUA 2426 ACAGGUU C AGAGAUU

2183 ACCAGCU A UUUAUUG 2433 CAGAGAU U ACCCAGU

2185 CAGCUAU U UAUUGAG 2434 AGAGAUU A CCCAGUG

2186 AGCUAUU U AUUGAGU 2448 GAGGCCU U AUUCCUC

2187 GCUAUUU A UUGAGUG 2449 AGGCCUU A UUCCUCC 2451 GCCUUAU U CCUCCCU 2750 UAUGUGU A GACAAGC

2452 CCUUAUU C CUCCCUU 2759 ACAAGCU C UCGCUCU

2455 UAUUCCU C CCUUCCC 2761 AAGCUCU C GCUCUGU

2459 CCUCCCU U CCCCCCA 2765 UCUCGCU C UGUCACC

2460 CUCCCUU C CCCCCAA 2769 GCUCUGU C ACCCAGG

2479 GACACCU U UGUUAGC 2797 GUGCAAU C AUGGUUC

2480 ACACCUU U GUUAGCC 2803 UCAUGGU U CACUGCA

2483 CCUUUGU U AGCCACC 2804 CAUGGUU C ACUGCAG

2484 CUUUGUU A GCCACCU 2813 CUGCAGU C UUGACCU

2492 GCCACCU C CCCACCC 2815 GCAGUCU U GACCUUU

2504 CCCACAU A CAUUUCU 2821 UUGACCU U UUGGGCU

2508 CAUACAU U UCUGCCA 2822 UGACCUU U UGGGCUC

2509 AUACAUU U CUGCCAG 2823 GACCUUU U GGGCUCA

2510 UACAUUU C UGCCAGU 2829 UUGGGCU C AAGUGAU

2520 CCAGUGU U CACAAUG 2837 AAGUGAU C CUCCCAC

2521 CAGUGUU C ACAAUGA 2840 UGAUCCU C CCACCUC

2533 UGACACU C AGCGGUC 2847 CCCACCU C AGCCUCC

2540 CAGCGGU C AUGUCUG 2853 UCAGCCU C CUGAGUA

2545 GUCAUGU C UGGACAU 2860 CCUGAGU A GCUGGGA

2568 AGGGAAU A UGCCCAA 2872 GGACCAU A GGCUCAC

2579 CCAAGCU A UGCCUUG 2877 AUAGGCU C ACAACAC

2585 UAUGCCU U GUCCUCU 2899 GGCAAAU U CGAUUUU

2588 GCCUUGU C CUCUUGU 2900 GCAAAUU U GAUUUUU

2591 UUGUCCU C UUGUCCU 2904 AUUUGAU U UUUUUUU

2593 GUCCUCU U GUCCUGU 2905 UUUGAUU U UUUUUUU

2596 CUCUUGU C CUGUUUG 2906 UUGAUUU U UUUUUUU

2601 GUCCUGU U UGCAUUU 2907 UGAUUUU U UUUUUUU

2602 UCCUGUU U GCAUUUC 2908 GAUUUUU U UUUUUUU

2607 UUUGCAU U UCACUGG 2909 AUUUUUU U UUUUUUU

2608 UUGCAUU U CACUGGG 2910 UUUUUUU U UUUUUUU

2609 UGCAUUU C ACUGGGA 2911 UUUUUUU U UUUUUUU

2620 GGGAGCU U GCACUAU 2912 UUUUUUU U UUUUUUC

2626 UUGCACU A UUGCAGC 2913 UUUUUUU U UUUUUCA

2628 GCACUAU U GCAGCUC 2914 UUUUUUU U UUUUCAG

2635 UGCAGCU C CAGUUUC 2915 UUUUUUU U UUUCAGA

2640 CUCCAGU U UCCUGCA 2916 UUUUUUU U UUCAGAG

2641 UCCAGUU U CCUGCAG 2917 UUUUUUU U UCAGAGA

2642 CCAGUUU C CUGCAGU 2918 UUUUUUU U CAGAGAC

2653 CAGUGAU C AGGGUCC 2919 UUUUUUU C AGAGACG

2659 UCAGGGU C CUGCAAG 2931 ACGGGGU C UCGCAAC

2689 CCAAGGU A UUGGAGG 2933 GGGGUCU C GCAACAU

2691 AAGGUAU U GGAGGAC 2941 GCAACAU U GCCCAGA

2700 GAGGACU C CCUCCCA 2951 CCAGACU U CCUUUGU

2704 ACUCCCU C CCAGCUU 2952 CAGACUU C CUUUGUG

2711 CCCAGCU U UGGAAGG 2955 ACUUCCU U UGUGUUA

2712 CCAGCUU U GGAAGGG 2956 CUUCCUU U GUGUUAG

2721 GAAGGGU C AUCCGCG 2961 UUUGUGU U AGUUAAU

2724 GGGUCAU C CGCGUGU 2962 UUGUGUU A GUUAAUA

2744 UGUGUGU A UGUGUAG 2965 UGUUAGU U AAUAAAG 2966 GUUAGUU A AUAAAGC

2969 AGUUAAU A AAGCUUU

2975 UAAAGCU U UCUCAAC

2976 AAAGCUU U CUCAACU

2977 AAGCUUU C UCAACUG

2979 GCUUUCU C AACUGCC

Table 3

Mouse ICAM HH Target Sequence

nt. Position Target Sequence nt. Position Target Sequence 11 CCCugGU C acCGuUG 367 AAugGCU u cAACCcg 23 CaGuGgU u CUCUGCU 374 gAAgCCU U CCUgcCC 26 uGgUuCU C UGCUcCU 375 AAgCCUU C CUgcCCc 31 CUCUGCU c CUCcaca 378 CuaaCaU C ACCGUGU 34 UuCUcaU a AGgGUcG 386 ACCGUGU A uUcGuuU 40 gCAcAcU U GuAgCCU 394 CcGGACU u ucGAuCu 48 aggACCU C AGCCUgG 420 CACaCuU C CCCcCcg 54 UggGCCU C GugAUGG 425 CaCCCCU C ccaGCAG 58 CaUgcCU u UaGCUCC 427 CagCUCU c aGCAGug 64 cAcccCU C CCAGCAG 450 AGgACCU c ACCCUgC 96 CucugCU C CUGGcCC 451 GAAaCcU u uCCUuuG 102 UgCcaGU a CUGCUgG 456 UUACCCU c aGCcaCu 108 cuCUGCU C cuGGCcC 495 CuAcCaU C ACCGUGu 115 uGGuuCU C UGcUCCu 510 UGCUGCU C CGUGGGG 119 GgaaUGU c aCCAGGA 564 CUcAGGU a uCcAuCc 120 CUCUGcU C CugGccC 592 GAaAGAU C ACaugGG 146 CAGuCgU C cGcuUCC 607 AGCCAAU U UCUCaUG 152 UCUGUGU C agCCaCu 608 GCCAAUU U CUCaUGC 158 UCCuguU u AAAAacC 609 CCAAUUU C UCaUGCC 165 CAgAAGU u gUuuUGC 611 AAUUUCU C aUGCCGC 168 AAGcCuU C CUGCCCC 656 aAGCUGU U UGAGcug 185 GGuGGgU C CGUGCaG 657 AGCUGUU U GAGcugA 209 gcCACuU C CUcUGgC 668 cgagCCU a GGCCaCC 227 CagAAGU U GUUuuGC 677 GaCCuCU A CCAGCcu 230 AAGUUGU U uuGCucc 684 uuCAGCU C CgGuCCU 237 UGuGCuU u GAGAaCu 692 CgGACuU U cGauCUu 248 AaCCCaU c uCCUAAA 693 AGgaCcU c acCCUGC 253 ccUGCCU A AggAaGA 696 CCUgUuU C CUGCCuc 263 AgGGuuU c uCUaCUG 709 gGCGgCU C CaCCuCA 267 AGggGCU C CUGCCUa 720 uACAACU U uUCAGCu 293 AAGcUGU u UGAgCUG 723 AACUUuU C AGCuCCg 319 AGgAGAU A cugAgCC 735 aCCaGaU C CUgGAGa 335 cUGUGCU u UgagAAC 738 uGGgCCU c GuGaUGG 337 GUcCaAU U CAcACUG 765 CaGUcGU C cGcUuCC 338 aGCUgUU u gAgCUGa 769 GGcCUGU U uCCUGcc 359 GuGCAGU C guCcGCU 770 uUuUGcU C CCUGGAa 785 GGcCUGU U uCCuGcC 1353 AGUGggU c gAaGgUG 786 GcCUGUU u CCuGcCU 1366 UaaCAgU c UaCaACU 792 UggagGU C UCGGAaG 1367 aGCACcU c CCCACcu 794 CugGgCU u GGAGaCu 1368 GuACUgU a CCACUcu 807 CuCgGaU a uACCUGG 1380 UGCCCAU C GGGGugg 833 CAaAGcU c GAca.CCC 1388 GGaGAcU C AGUGgCU 846 CCcugGU C ACCguUG 1398 UGgCUGU C ACagaAc 851 GagACCU c UacCAgC 1402 UGUgcuU u GAGAaCU 863 AgCcACU u CcUCUgG 1408 gCGAGAU C ggGgaGG

866 GAagCCU U CcuGcCC 1410 GAGgUCU c GgaaGgg

867 AuUCgUU u cCGGagA 1421 ccCACCU A CuUuUGU

869 UCuUcCU C augCAAG 1425 aCUgCCU u gGUaGaG

881 AuGGCuU C AacCcGU 1429 uCUCUaU u GccCCuG

885 CCUugGU a gagGUGA 1444 GAaggCU C AgGaGGA

933 cUauAaU c AUuCUGG 1455 GGaAuGU C ACCaGga

936 uAaUcAU u CUGGuGc 1482 AguUGuU u UgCuCCC

978 UaACagU C UACAaCU 1484 cUGuUCU u CCuCauG

980 ACagUCU A CAaCUUU 1493 CuguGcU u UGAGAac

986 UACAaCU U UuCaGCu 1500 AUGAaAU c aUggUCc

987 ACAaCUU U uCaGCuC 1503 gGAcUaU a AUCAUuc

988 CAaCUUU u CaGCuCC 1506 UUaUguU u AUaACcG

1005 ACcaGAU c CUGgaGA 1509 cuAcCAU C ACcGUGu

1006 uGaGAgU C UGggGAA 1518 ucaUGGU c cCAGgCG

1023 ugGAGGU C UCgGAAG 1530 CuauAaU C AUucUGG

1025 GAGGUCU C gGAAGGG 1533 ugGUCAU u gUGGGCc

1066 CCACuCU c aAaauAA 1551 CAuGCCU u AGCAgcU

1092 AcuGGaU c uCAGgCC 1559 AGCACcU c CCcaccU

1093 UGGaccU u CAGCCaA 1563 CuUAugU u UAUAACC

1125 CCCAaCU C uUcuUGA 1555 UAugUuU A UAACCGC

1163 CGaAGCU U CUuuUGC 1567 ugUuUAU A ACCGCCA

1164 GaAGCUU C UuuUGCU 1584 GaAAGAU C AgGAuAU

1156 AGCUUCU u uUGCUCU 1592 AgGAuAU A CAaguUA

1172 UCCUGuU u aaaAACC 1599 ACAaguU A CAgaAGG

1200 cuCuGCU c cUcCACA 1651 CcCaCCU C CCUGAgC

1201 gCuGCUU u UgaACAg 1661 gaAACCU u UCCuuuG

1203 AcuUUuU u CACcAGu 1663 AACCUuU C CuuuGAa

1227 GGuAcaU a CGUGUgC 1678 AGGaCCU C agCCUgG

1228 GaAGCUU C uUuUgCU 1680 aGCCaCU U CCUCuGg

1233 UUCGUuU C CgGagaG 1681 GCCaCUU C CUCuGgC

1238 GUgCUGU A UGGuCCu 1684 aCUUCCU C uGgCUgu

1264 GAaGGgU c GUgCaaG 1690 cCGGaCU U uCgAUcU

1267 uGAgaGU C uGGGgAA 1691 CGGaCUU u CgAUcUU

1294 AGgAgAU a CugAGCc 1696 UgCCCAU c ggGGUGG

1295 GAggggU C uCAGCAG 1698 CggAUAU a ccUGGag

1306 GCAGACU C ugAaaUG 1737 gAGACcU c UaCCAgc

1321 gaAGGCU c aGGaGgA 1750 gGCgGCU c CACCUca

1334 AACCCAU c uCCuaAa 1756 gAagCCU u CCuGCCC

1344 auGAGCU C gAGaGUg 1787 gaGaCAU U GUCCcCA

1351 ugAaUGU a UAAguuA 1790 GCAUUGU u CUCuaau

1793 UgGUCCU C gGcugGA 2173 UUagagU U UUACCAG

1797 CacCAGU C AcAUAaA 2174 UagagUU U UACCAGC

1802 acCAGAU c CuggAGa 2175 agagUUU U ACCAGCU

1812 ACuGgAU c UcaGGCC 2176 gagUUUU A CCAGCUA

1813 CAGCAUU U acccuCA 2183 ACCAGCU A UUUAUUG

1825 CCAcGcU A CCUcugC 2185 CAGCUAU U UAUUGAG 1837 CAugCCU u uAgCuCc 2186 AGCUAUU U AUUGAGU

1845 cgAgcCU A GGCCACc 2187 GCUAUUU A UUGAGUa 1856 CggaCuU u cGAUCUu 2189 UAUUUAU U GAGUacC

1861 AcaUGAU a UccAGUa 2196 caAcUcU u cUUgAUG

1865 cAcuUGU A GcCuCAg 2198 gcaGcCU c UUAUGUu

1863 CaccAGU C ACAUaAa 2199 GccUCUU a UgUuUAu

1877 CAUGcCU u AGCagcu 2200 UcUuccU c AUGcAaG

1901 uAAaACU C AAGggAc 2201 aagUUUU A UGUcGGC

1912 AuAUagU a GAUcagU 2205 UUUAUGU c GGCcugA

1922 UGaAUGU a uAAGUua 2210 GgAGaCU c AgUGgcu

1923 uGAUGcU c AgGUaUc 2220 cuggCAU u GuUCUCU

1928 UUAgAGU u UuaCCaG 2224 CucAGGU a UCcauCC

1930 AgAGUuU u aCCaGcU 2226 UgGaUCU C aGGCCgC

1964 GAGACAU u GuCCCca 2233 CUGaCCU C cuGGAGg

1983 AGGAuAU A CAAgUua 2242 uGGAGCU a gCgGaCC

1996 aGGAgAU A CUGAgcC 2248 UauCcaU CAUccCA

2005 UGgAgCU a GCgGaCc 2254 UCCAauU C ACAcUgA

2013 GCUauuU A UUGaGUA 2259 aUCACAU u CAcGGUg

2015 UGCCcAU c GGGgugG 2260 UCACAUU C AcGGUgc

2020 ggUGGuU c UuCUGAG 2266 ggAAuGU C ACCAGGa

2039 gCuGgCU a gCAGAgG 2274 ACCAGaU c CuGgaGa

2040 CuGACcU c CuGgAGg 2279 GaAggGU c GUgCAaG

2057 UGcuCCU C CAcAucC 2282 aAGcUGU u ugaGcUG

2061 CuaCCAU c acCgUGU 2288 UAuAaGU U aUggcCU

2071 CAcuUGU A GCcUCAg 2291 caGUgGU u CuCUGCu

2076 GUAGCcU C AgAgCua 2321 gAAAGAU C AcAUGGG

2097 CaACuCU u CuUGAuG 2338 UGaGACU c CUgccUG

2098 CACACUU c CcccCcG 2339 GaaACcU u UCcUUuG

2115 GCCAGCU c GGaggaU 2341 GACcUCU a ccaGcCu

2128 CaGCUaU u UAuUGAg 2344 UUucgAU c uuCCAgC

2130 cCUGUuU c CUGcCuC 2358 CCcagCU c UCagCAG

2145 CAACuCU U cuUGAUg 2359 CUGCuUU U gaaCAGA

2152 UauUaAU u UagAgUU 2360 aaCCUUU C CuuuGAA

2156 uugAUGU A UUUAUUa 2376 agGUGgU U cUUCUga

2158 gAUGUAU U UAUUaAU 2377 gGUGgUU c UUCUgag

2159 AUGUAUU U AUUaAUU 2378 agGgUUU c UCUAcuG

2160 UGUAUUU A UUaAUUU 2379 UGcUUUU c ucAUaaG

2162 UAUUUAU U aAUUUag 2380 aAgUUUU a UgUCGGC

2163 AUgUAUU u AUUaaUU 2382 aUUcUCU A UuGcCcC

2166 acUUCAU U cucUAUU 2384 aUcCagU a GaCACAA

2167 AUguAUU U aUUAaUU 2399 AAaCACU A UgUGGAC

2170 uAUUUaU u AaUUUAg 2401 aagCUgU u UGagCUG

2171 AgUUGUU u UgcUcCC 2411 uACUGGU c AgGaUgC

2417 gAAUGGU a CAuAcGU 2691 AAuGUcU c cGAGGcC

2418 AcUGGaU C uCAGGcc 2700 GAaGcCU u CCUgCCc

2425 CAugGGU c gAGgGuU 2704 gacCuCU a CCAGCcU

2426 AuuaaUU u AGAGuUU 2711 CCCAGCU c UcagcaG

2433 uAGAGuU U uaCCAGc 2712 gagGucU c GGAAGGG

2434 AGAGuUU u aCCAGcu 2721 GAAGGGU C gUgCaaG

2448 GAaGCCU U ccUgCcC 2724 GGuaCAU a CGuGUGc

2449 AaGCCUU c cUgCcCC 2744 gGUGgGU c cGUGcAG 2451 GCCUguU U CCUgCCU 2750 UAUuUaU u GAguAcC

2452 CCUguUU C CUgCCUc 2759 cCggaCU u UCGaUCU

2455 gAagCCU u CCUgCCC 2761 AgGacCU C aCcCUGc

2459 CCaCaCU U CCCCCCc 2765 UuUuGCU C UGcCgCu

2460 CaCaCUU C CCCCCcg 2769 agUCUGU C AaaCAGG

2479 GAgACCU c UaccAGC 2797 aUGaAAU c AUGGUcC

2480 uCACCgU U GUgAuCC 2803 UCAUGGU c CcagGCg

2483 CCaaUGU c AGCCACC 2804 ggUGGgU c cgUGCAG

2484 CUUUuUU c aCCAguc 2813 CUcCgGU c cUGACCc

2492 agCACCU C CCCACCu 2815 aCAGUCU a cAaCUUU

2504 CCCACcU A CuUUUgU 2821 cUGACCU c cUGGagg

2508 uAUcCAU c caUcCCA 2822 gGAgCcU c cGGaCUu

2509 uUAgAgU U uUaCCAG 2823 ugCCUUU a GcuCcCA

2510 UAgAgUU u UaCCAGc 2829 cUGGaCU a uAaUcAU

2520 CuuuUGU U CcCAAUG 2837 AgGUGgU u CUuCuga

2521 CAGcaUU u ACccUcA 2840 UGAgaCU C CugCCUg

2533 UGAugCU C AGguaUC 2847 CCaAugU C AGCCaCC

2540 CAGCaGU C cgcUgUG 2853 gCAGCCU C uUauGUu

2545 GUgcUGU a UGGuCcU 2860 gCcaAGU A aCUGuGA

2568 guGaAgU c UGuCaAA 2872 GGACCuU c aGCcaAg

2579 auAAGuU A UGgCcUG 2877 uUccGCU a cCAuCAC

2585 cugGCaU U GUuCUCU 2899 cGgAcuU U cGAUcUU

2588 GCaUUGU u CUCUaaU 2900 uuAAuUU a GAgUUUU

2591 UgGUuCU C UgcUCCU 2904 AcUUcAU U cUcUaUU

2593 cUuCUuU U GcuCUGc 2905 cUUcAUU c UcUaUUg

2596 CUuUUGU u CccaaUG 2906 UUGAUgU a UUUaUUa

2601 acCgUGU a UuCgUUU 2907 UGuaUUU a UUaaUUU

2602 UCCaGcU a cCAUccC 2908 GAagcUU c UUUUgcU

2607 cUcGgAU a UacCUGG 2909 AgcUUcU U UUgcUcU

2608 caGCAgU c CgCUGuG 2910 UgUaUUU a UUaaUUU

2609 gGaAUgU C ACcaGGA 2911 UgUaUUU a UUaaUUU

2620 aGGAcCU c aCcCUgc 2912 UUgUUcU c UaaUgUC

2626 UUuCgaU c UUcCAGC 2913 UUUcUcU a cUggUCA

2628 GCACacU U GuAGCcu 2914 UgcUUUU c UcaUaAG

2635 UuCAGCU C CgGUccu 2915 aUUUaUU a aUUuAGA

2640 ggCCuGU U UCCUGCc 2916 UaUUcgU U UcCgGAG

2641 cCCAGcU c uCaGCAG 2917 aUUcgUU U cCgGAGA

2642 CCuGUUU C CUGCcuc 2918 UUcgUUU c CgGAGAg

2653 uAcUGgU C AGGaUgC 2919 UUcUcaU a AGgGuCG

2659 gaAGGGU C gUGCAAG 2931 ugGaGGU C UCGgAAg

2689 CuAAuGU c UccGAGG 2933 GaGGUCU C GgAAggg

2941 GagACAU U GuCCccA

2951 CCAcgCU a CCUcUGc

2952 CAGcagU C CgcUGUG

2955 AgUgaCU c UGUGUcA

2956 uUUCCUU U GaaUcAa

2961 UcUGUGU c AGccAcU

2962 aUGUaUU u aUUAAUu

2965 UuUgAaU c AAUAAAG 2966 GcUgGcU A gcAgAGg

2969 AaUcAAU A AAGuUUU

2975 UAgAGuU U UacCAgC

2976 gAgGgUU U CUCuACU

2977 AAGCUgU u UgAgCUG

2979 uCaUUCU C uAuUGCC

Table 4

Human ICAM HH Ribozyme Sequences

nt. Position Ribozyme Sequence

11 CAGCGUC CUGAUGAGGCCGAAAGGCCGAA ACUGGGG

23 AGCAGAG CUGAUGAGGCCGAAAGGCCGAA AGCUCAG

26 AGUAGCA CUGAUGAGGCCGAAAGGCCGAA AGGAGCU

31 CUCUGAG CUGAUGAGGCCGAAAGGCCGAA AGCAGAG

34 CAACUCU CUGAUGAGGCCGAAAGGCCGAA AGUAGCA

40 AGGUUGC CUGAUGAGGCCGAAAGGCCGAA ACUCUGA

48 CGAGGCU CUGAUGAGGCCGAAAGGCCGAA AGGUUGC

54 CCAUAGC CUGAUGAGGCCGAAAGGCCGAA AGGCUGA

58 GGAGCCA CUGAUGAGGCCGAAAGGCCGAA AGCGAGG

54 CUGCUGG CUGAUGAGGCCGAAAGGCCGAA AGCCAUA

96 GGACCAG CUGAUGAGGCCGAAAGGCCGAA AGUGCGG

102 CGAGCAG CUGAUGAGGCCGAAAGGCCGAA ACCAGGA

108 GAGCCCC CUGAUGAGGCCGAAAGGCCGAA AGCAGGA

115 GGGAACA CUGAUGAGGCCGAAAGGCCGAA AGCCCCG

119 UCCUGGG CUGAUGAGGCCGAAAGGCCGAA ACAGAGC

120 GUCCUGG CUGAUGAGGCCGAAAGGCCGAA AACAGAG 146 GGACACA CUGAUGAGGCCGAAAGGCCGAA AUGUCUG 152 UGAGGGG CUGAUGAGGCCGAAAGGCCGAA ACACAGA 158 GACUUUU CUGAUGAGGCCGAAAGGCCGAA AGGGGGA 165 GCAGGAU CUGAUGAGGCCGAAAGGCCGAA ACUUUUG 158 GGGGCAG CUGAUGAGGCCGAAAGGCCGAA AUGACUU 185 CAGCACG CUGAUGAGGCCGAAAGGCCGAA AGCCUCC 209 GUCACAG CUGAUGAGGCCGAAAGGCCGAA AGGUGCU 227 GCCCAAC CUGAUGAGGCCGAAAGGCCGAA ACUUGGG 230 UAUGCCC CUGAUGAGGCCGAAAGGCCGAA ACAACUU 237 GGGUCUC CUGAUGAGGCCGAAAGGCCGAA AUGCCCA 248 UUUAGGC CUGAUGAGGCCGAAAGGCCGAA ACGGGGU 253 UCCUUUU CUGAUGAGGCCGAAAGGCCGAA AGGCAAC 263 CAGGAGC CUGAUGAGGCCGAAAGGCCGAA ACUCCUU 267 CAGGCAG CUGAUGAGGCCGAAAGGCCGAA AGCAACU 293 CAGUUCA CUGAUGAGGCCGAAAGGCCGAA ACACCUU 319 GGUUGGC CUGAUGAGGCCGAAAGGCCGAA AUCUUCU 335 GUUUGAA CUGAUGAGGCCGAAAGGCCGAA AGCACAU

337 CAGUUUG CUGAUGAGGCCGAAAGGCCGAA AUAGCAC

338 GCAGUUU CUGAUGAGGCCGAAAGGCCGAA AAUAGCA 359 AGCUGUU CUGAUGAGGCCGAAAGGCCGAA ACUGCCC 367 AAGGUUU CUGAUGAGGCCGAAAGGCCGAA AGCUGUU

374 GGUGAGG CUGAUGAGGCCGAAAGGCCGAA AGGUUUU

375 CGGUGAG CUGAUGAGGCCGAAAGGCCGAA AAGGUUU 378 ACACGGU CUGAUGAGGCCGAAAGGCCGAA AGGAAGG 386 AGUCCAG CUGAUGAGGCCGAAAGGCCGAA ACACGGU 394 CGUUCUG CUGAUGAGGCCGAAAGGCCGAA AGUCCAG 420 AAGAGGG CUGAUGAGGCCGAAAGGCCCAA AGGGGUG 425 CUGCCAA CUGAUGAGGCCGAAAGGCCGAA AGGGGAG 427 GGCUGCC CUGAUGAGGCCGAAAGGCCGAA AGAGGGG

450 GUAGGGU CUGAUGAGGCCGAAAGGCCGAA AGGUUCU

451 CGUAGGG CUGAUGAGGCCGAAAGGCCGAA AAGGUUC 456 GGCAGCG CUGAUGAGGCCGAAAGGCCGAA AGGGUAA 495 CCACGGU CUGAUGAGGCCGAAAGGCCGAA AGGUUGG 510 CCCCACG CUGAUGAGGCCGAAAGGCCGAA AGCAGCA 564 UGGUCGU CUGAUGAGGCCGAAAGGCCGAA ACCUCAG 592 CCAUGGU CUGAUGAGGCCGAAAGGCCGAA AUCUCUC

607 CACGAGA CUGAUGAGGCCGAAAGGCCGAA AUUGGCU

608 GCACGAG CUGAUGAGGCCGAAAGGCCGAA AAUUGGC

609 GGCACGA CUGAUGAGGCCGAAAGGCCGAA AAAUUGG 611 GCGGCAC CUGAUGAGGCCGAAAGGCCGAA AGAAAUU

656 GUUCUCA CUGAUGAGGCCGAAAGGCCGAA ACAGCUC

657 UGUUCUC CUGAUGAGGCCGAAAGGCCGAA AACAGCU 668 GGGGGCC CUGAUGAGGCCGAAAGGCCGAA AGGUGUU 677 GAGCUGG CUGAUGAGGCCGAAAGGCCGAA AGGGGGC 684 AGGUCUG CUGAUGAGGCCGAAAGGCCGAA AGCUGGU

692 CAGGACA CUGAUGAGGCCGAAAGGCCGAA AGGUCUG

693 GCAGGAC CUGAUGAGGCCGAAAGGCCGAA AAGGUCU 696 CUGGCAG CUGAUGAGGCCGAAAGGCCGAA ACAAAGG 709 UGUGGGG CUGAUGAGGCCGAAAGGCCGAA AGUCGCU 720 GGCUGAC CUGAUGAGGCCGAAAGGCCGAA AGUUGUG 723 GGGGGCU CUGAUGAGGCCGAAAGGCCGAA ACAAGUU 735 CCUCUAG CUGAUGAGGCCGAAAGGCCGAA ACCCGGG 738 CCACCUC CUGAUGAGGCCGAAAGGCCGAA AGGACCC 765 GGGAACA CUGAUGAGGCCGAAAGGCCGAA ACCACGG

769 UCCAGGG CUGAUGAGGCCGAAAGGCCGAA ACAGACC

770 GUCCAGG CUGAUGAGGCCGAAAGGCCGAA AACAGAC

785 GACUGGG CUGAUGAGGCCGAAAGGCCGAA ACAGCCC

786 AGACUGG CUGAUGAGGCCGAAAGGCCGAA AACAGCC 792 CCUCCGA CUGAUGAGGCCGAAAGGCCGAA ACUGGGA 794 GGCCUCC CUGAUGAGGCCGAAAGGCCGAA AGACUGG 807 CCAGGUG CUGAUGAGGCCGAAAGGCCGAA ACCUGGG 833 GGGGUUC CUGAUGAGGCCGAAAGGCCGAA ACCUCUG 846 CAUAGGU CUGAUGAGGCCGAAAGGCCGAA ACUGUGG 851 GUUGCCA CUGAUGAGGCCGAAAGGCCGAA AGGUGAC 863 CGAGAAG CUGAUGAGGCCGAAAGGCCGAA AGUCGUU

866 GGCCGAG CUGAUGAGGCCGAAAGGCCGAA AGGAGUC

867 UGGCCGA CUGAUGAGGCCGAAAGGCCGAA AAGGAGU 869 CUUGGCC CUGAUGAGGCCGAAAGGCCGAA AGAAGGA 881 ACUGACU CUGAUGAGGCCGAAAGGCCGAA AGGCCUU 885 UCACACU CUGAUGAGGCCGAAAGGCCGAA ACUGAGG 933 CCAGUAU CUGAUGAGGCCGAAAGGCCGAA ACUGCAC 936 UCCCCAG CUGAUGAGGCCGAAAGGCCGAA AUUACUG 978 AGCUGUA CUGAUGAGGCCGAAAGGCCGAA AUGGUCA 980 AAAGCUG CUGAUGAGGCCGAAAGGCCGAA AGAUGGU

986 CGCCGGA CUGAUGAGGCCGAAAGGCCGAA AGCUGUA

987 GCGCCGG CUGAUGAGGCCGAAAGGCCGAA AAGCUGU

988 GGCGCCG CUGAUGAGGCCGAAAGGCCGAA AAAGCUG 1005 UCGUCAG CUGAUGAGGCCGAAAGGCCGAA AUCACGU

1006 UUCGUCA CUGAUGAGGCCGAAAGGCCGAA AAUCACG

1023 CUUCUGA CUGAUGAGGCCGAAAGGCCGAA ACCUCUG

1025 CCCUUCU CUGAUGAGGCCGAAAGGCCGAA AGACCUC

1066 UUGGCUC CUGAUGAGGCCGAAAGGCCGAA AGGGUGG

1092 GGGCUGG CUGAUGAGGCCGAAAGGCCGAA ACCCCAU

1093 UGGGCUG CUGAUGAGGCCGAAAGGCCGAA AACCCCA

1125 UCAGCAG CUGAUGAGGCCGAAAGGCCGAA AGCUGGG

1153 GCAGGAG CUGAUGAGGCCGAAAGGCCGAA AGCUGCG

1154 AGCAGGA CUGAUGAGGCCGAAAGGCCGAA AAGCUGC

1156 AGAGCAG CUGAUGAGGCCGAAAGGCCGAA AGAAGCU

1172 GGUUGCA CUGAUGAGGCCGAAAGGCCGAA AGCAGGA

1200 UGUGUAU CUGAUGAGGCCGAAAGGCCGAA AGCUGGC

1201 UUGUGUA CUGAUGAGGCCGAAAGGCCGAA AAGCUGG

1203 UCUUGUG CUGAUGAGGCCGAAAGGCCGAA AUAAGCU

1227 GGACACG CUGAUGAGGCCGAAAGGCCGAA AGCUCCC

1228 AGGACAC CUGAUGAGGCCGAAAGGCCGAA AAGCUCC

1233 CAUACAG CUGAUGAGGCCGAAAGGCCGAA ACACGAA

1238 GGGGCCA CUGAUGAGGCCGAAAGGCCGAA ACAGGAC

1264 CCCGGAC CUGAUGAGGCCGAAAGGCCGAA AUCCCUC

1267 UUUCCCG CUGAUGAGGCCGAAAGGCCGAA ACAAUCC

1294 UGCUGGG CUGAUGAGGCCGAAAGGCCGAA AUUUUCU

1295 CUGCUGG CUGAUGAGGCCGAAAGGCCGAA AAUUUUC

1306 CACAUUG CUGAUGAGGCCGAAAGGCCGAA AGUCUGC

1321 UUCCCCC CUGAUGAGGCCGAAAGGCCGAA AGCCUGG

1334 CUCGGGC CUGAUGAGGCCGAAAGGCCGAA AUGGGUU

1344 GACACUU CUGAUGAGGCCGAAAGGCCGAA AGCUCGG

1351 UCCUUUA CUGAUGAGGCCGAAAGGCCGAA ACACUUG

1353 CAUCCUU CUGAUGAGGCCGAAAGGCCGAA AGACACU

1366 AGUGGGA CUGAUGAGGCCGAAAGGCCGAA AGUGCCA

1367 CAGUGGG CUGAUGAGGCCGAAAGGCCGAA AAGUGCC

1368 GCAGUGG CUGAUGAGGCCGAAAGGCCGAA AAAGUGC

1380 AUUCCCC CUGAUGAGGCCGAAAGGCCGAA AUGGGCA

1388 AGUCACU CUGAUGAGGCCGAAAGGCCGAA AUUCCCC

1398 CUCGAGU CUGAUGAGGCCGAAAGGCCGAA ACAGUCA

1402 AGAUCUC CUGAUGAGGCCGAAAGGCCGAA AGUGACA

1408 CCCUCAA CUGAUGAGGCCGAAAGGCCGAA AUCUCGA

1410 UGCCCUC CUGAUGAGGCCGAAAGGCCGAA AGAUCUC

1421 ACAGAGG CUGAUGAGGCCGAAAGGCCGAA AGGUGCC

1425 CCCGACA CUGAUGAGGCCGAAAGGCCGAA AGGUAGG

1429 CUGGCCC CUGAUGAGGCCGAAAGGCCGAA ACAGAGG

1444 UCCCCUU CUGAUGAGGCCGAAAGGCCGAA AGUGCUC

1455 CGCGGGU CUGAUGAGGCCGAAAGGCCGAA ACCUCCC

1482 GGGGGGA CUGAUGAGGCCGAAAGGCCGAA AGCACAU

1484 CCGGGGG CUGAUGAGGCCGAAAGGCCGAA AGAGCAC

1493 AAUCUCA CUGAUGAGGCCGAAAGGCCGAA ACCGGGG

1500 UGAUGAC CUGAUGAGGCCGAAAGGCCGAA AUCUCAU

1503 UGAUGAU CUGAUGAGGCCGAAAGGCCGAA ACAAUCU

1506 CAGUGAU CUGAUGAGGCCGAAAGGCCGAA AUGACAA 1509 CCACAGU CUGAUGAGGCCGAAAGGCCGAA AUGAUGA

1518 CGGCUGC CUGAUGAGGCCGAAAGGCCGAA ACCACAG

1530 CCAUUAU CUGAUGAGGCCGAAAGGCCGAA ACUGCGG

1533 UGCCCAU CUGAUGAGGCCGAAAGGCCGAA AUGACUG

1551 ACGUGCU CUGAUGAGGCCGAAAGGCCGAA AGGCCUG

1559 AUAGAGG CUGAUGAGGCCGAAAGGCCGAA ACGUGCU

1563 GGUUAUA CUGAUGAGGCCGAAAGGCCGAA AGGUACG

1565 GCGGUUA CUGAUGAGGCCGAAAGGCCGAA AGAGGUA

1567 UGGCGGU CUGAUGAGGCCGAAAGGCCGAA AUAGAGG

1584 AUUUCUU CUGAUGAGGCCGAAAGGCCGAA AUCUCCC

1592 UAGUCUG CUGAUGAGGCCGAAAGGCCGAA AUUUCUU

1599 CCUGUUG CUGAUGAGGCCGAAAGGCCGAA AGUCUGU

1551 GUUCAGG CUGAUGAGGCCGAAAGGCCGAA AGGCGUG

1661 CCCGGGA CUGAUGAGGCCGAAAGGCCGAA AGGUUCA

1663 GUCCCGG CUGAUGAGGCCGAAAGGCCGAA AUAGGUU

1678 CGAGGAA CUGAUGAGGCCGAAAGGCCGAA AGGCCCU

1680 GCCGAGG CUGAUGAGGCCGAAAGGCCGAA AGAGGCC

1681 GGCCGAG CUGAUGAGGCCGAAAGGCCGAA AAGAGGC

1684 GAAGGCC CUGAUGAGGCCGAAAGGCCGAA AGGAAGA

1690 AUAUGGG CUGAUGAGGCCGAAAGGCCGAA AGGCCGA

1691 AAUAUGG CUGAUGAGGCCGAAAGGCCGAA AAGGCCG

1696 CCACCAA CUGAUGAGGCCGAAAGGCCGAA AUGGGAA

1698 UGCCACC CUGAUGAGGCCGAAAGGCCGAA AUAUGGG

1737 CAUGGCA CUGAUGAGGCCGAAAGGCCGAA AUGUCUU

1750 GUAGGUG CUGAUGAGGCCGAAAGGCCGAA AGCUGCA

1756 GGGCCGG CUGAUGAGGCCGAAAGGCCGAA AGGUGUA

1787 UGAGGAC CUGAUGAGGCCGAAAGGCCGAA AUGCCCU

1790 GACUGAG CUGAUGAGGCCGAAAGGCCGAA ACAAUGC

1793 UCUGACU CUGAUGAGGCCGAAAGGCCGAA AGGACAA

1797 UGUAUCU CUGAUGAGGCCGAAAGGCCGAA ACUGAGG

1802 GCUGUUG CUGAUGAGGCCGAAAGGCCGAA AUCUGAC

1812 GGCCCCA CUGAUGAGGCCGAAAGGCCGAA AUGCUGU

1813 UGGCCCC CUGAUGAGGCCGAAAGGCCGAA AAUGCUG

1825 GUGCAGG CUGAUGAGGCCGAAAGGCCGAA ACCAUGG

1837 AGUGUUU CUGAUGAGGCCGAAAGGCCGAA AGGUGUG

1845 CGUGGCC CUGAUGAGGCCGAAAGGCCGAA AGUGUUU

1856 CAGAUCA CUGAUGAGGCCGAAAGGCCGAA AUGCGUG

1861 GACUACA CUGAUGAGGCCGAAAGGCCGAA AUCAGAU

1865 AUGUGAC CUGAUGAGGCCGAAAGGCCGAA ACAGAUC

1868 GUCAUGU CUGAUGAGGCCGAAAGGCCGAA ACUACAG

1877 CUUGGCU CUGAUGAGGCCGAAAGGCCGAA AGUCAUG

1901 AUGUCUU CUGAUGAGGCCGAAAGGCCGAA AGUCUUG

1912 AUCCAUC CUGAUGAGGCCGAAAGGCCGAA AUCAUGU

1922 AGACUUU CUGAUGAGGCCGAAAGGCCGAA ACAUCCA

1923 UAGACUU CUGAUGAGGCCGAAAGGCCGAA AACAUCC

1928 CAGGCUA CUGAUGAGGCCGAAAGGCCGAA ACUUUAA

1930 AUCAGGC CUGAUGAGGCCGAAAGGCCGAA AGACUUU

1964 GUGGGGC CUGAUGAGGCCGAAAGGCCGAA AUGUCUC

1983 CCAGUUG CUGAUGAGGCCGAAAGGCCGAA AUGUCCU 1996 GUUUCAG CUGAUGAGGCCGAAAGGCCGAA AUUUCCC

2005 AGGCAGC CUGAUGAGGCCGAAAGGCCGAA AGUUUCA

2013 UACCCAA CUGAUGAGGCCGAAAGGCCGAA AGGCAGC

2015 CAUACCC CUGAUGAGGCCGAAAGGCCGAA AUAGGCA

2020 CUCAGCA CUGAUGAGGCCGAAAGGCCGAA ACCCAAU

2039 CUUCUGU CUGAUGAGGCCGAAAGGCCGAA AGUCUGU

2040 UCUUCUG CUGAUGAGGCCGAAAGGCCGAA AAGUCUG

2057 GUCUAUG CUGAUGAGGCCGAAAGGCCGAA AGGGCCA

2061 ACAUGUC CUGAUGAGGCCGAAAGGCCGAA AUGGAGG

2071 UUGAUGC CUGAUGAGGCCGAAAGGCCGAA ACACAUG

2076 GUGUUUU CUGAUGAGGCCGAAAGGCCGAA AUGCUAC

2097 CGUCAGG CUGAUGAGGCCGAAAGGCCGAA AGUGUGG

2098 CCGUCAG CUGAUGAGGCCGAAAGGCCGAA AAGUGUG

2115 AGUGCCC CUGAUGAGGCCGAAAGGCCCAA AGCUGGC

2128 GUCAGUA CUGAUGAGGCCGAAAGGCCGAA ACAGCAG

2130 GGGUCAG CUGAUGAGGCCGAAAGGCCGAA AGACAGC

2145 UAUCAUC CUGAUGAGGCCGAAAGGCCGAA AGGGUUG

2152 AAAUACA CUGAUGAGGCCGAAAGGCCGAA AUCAUCA

2156 GAAUAAA CUGAUGAGGCCGAAAGGCCGAA ACAUAUC

2158 AUGAAUA CUGAUGAGGCCGAAAGGCCGAA AUACAUA

2159 AAUGAAU CUGAUGAGGCCGAAAGGCCGAA AAUACAU

2160 AAAUGAA CUGAUGAGGCCGAAAGGCCGAA AAAUACA

2162 ACAAAUG CUGAUGAGGCCGAAAGGCCGAA AUAAAUA

2163 AACAAAU CUGAUGAGGCCGAAAGGCCGAA AAUAAAU

2166 AAUAACA CUGAUGAGGCCGAAAGGCCGAA AUGAAUA

2167 AAAUAAC CUGAUGAGGCCGAAAGGCCGAA AAUGAAU

2170 GUAAAAU CUGAUGAGGCCGAAAGGCCGAA ACAAAUG

2171 GGUAAAA CUGAUGAGGCCGAAAGGCCGAA AACAAAU

2173 CUGGUAA CUGAUGAGGCCGAAAGGCCGAA AUAACAA

2174 GCUGGUA CUGAUGAGGCCGAAAGGCCGAA AAUAACA

2175 AGCUGGU CUGAUGAGGCCGAAAGGCCGAA AAAUAAC

2176 UAGCUGG CUGAUGAGGCCGAAAGGCCGAA AAAAUAA

2183 CAAUAAA CUGAUGAGGCCGAAAGGCCGAA AGCUGGU

2185 CUCAAUA CUGAUGAGGCCGAAAGGCCGAA AUAGCUG

2186 ACUCAAU CUGAUGAGGCCGAAAGGCCGAA AAUAGCU

2187 CACUCAA CUGAUGAGGCCGAAAGGCCGAA AAAUAGC

2189 GACACUC CUGAUGAGGCCGAAAGGCCGAA AUAAAUA

2196 CAUAAAA CUGAUGAGGCCGAAAGGCCGAA ACACUCA

2198 UACAUAA CUGAUGAGGCCGAAAGGCCGAA AGACACU

2199 CUACAUA CUGAUGAGGCCGAAAGGCCGAA AAGACAC

2200 CCUACAU CUGAUGAGGCCGAAAGGCCGAA AAAGACA

2201 GCCUACA CUGAUGAGGCCGAAAGGCCGAA AAAAGAC

2205 UUUAGCC CUGAUGAGGCCGAAAGGCCGAA ACAUAAA

2210 GUUCAUU CUGAUGAGGCCGAAAGGCCGAA AGCCUAC

2220 AGAGACC CUGAUGAGGCCGAAAGGCCGAA AUGUUCA

2224 GGCCAGA CUGAUGAGGCCGAAAGGCCGAA ACCUAUG

2226 GAGGCCA CUGAUGAGGCCGAAAGGCCGAA AGACCUA

2233 GCUCCGU CUGAUGAGGCCGAAAGGCCGAA AGGCCAG

2242 GGACUGG CUGAUGAGGCCGAAAGGCCGAA AGCUCCG 2248 UGACAUG CUGAUGAGGCCGAAAGGCCGAA ACUGGGA

2254 UGAAUGU CUGAUGAGGCCGAAAGGCCGAA ACAUGGA

2259 GACCUUG CUGAUGAGGCCGAAAGGCCGAA AUGUGAC

2260 UGACCUU CUGAUGAGGCCGAAAGGCCGAA AAUGUGA

2266 ACCUGGU CUGAUGAGGCCGAAAGGCCGAA ACCUUGA

2274 ACAACUG CUGAUGAGGCCGAAAGGCCGAA ACCUGGU

2279 CCUGUAC CUGAUGAGGCCGAAAGGCCGAA ACUGUAC

2282 CAACCUG CUGAUGAGGCCGAAAGGCCGAA ACAACUG

2288 AGUGUAC CUGAUGAGGCCGAAAGGCCGAA ACCUGUA

2291 UGCAGUG CUGAUGAGGCCGAAAGGCCGAA ACAACCU

2321 CCCAUUU CUGAUGAGGCCGAAAGGCCGAA AUCUUUU

2338 CAAUGAG CUGAUGAGGCCGAAAGGCCGAA AGUCCCA

2339 CCAAUGA CUGAUGAGGCCGAAAGGCCGAA AAGUCCC

2341 GGCCAAU CUGAUGAGGCCGAAAGGCCGAA AGAAGUC

2344 GUUGGCC CUGAUGAGGCCGAAAGGCCGAA AUGAGAA

2358 CUGGGGA CUGAUGAGGCCGAAAGGCCGAA AGGCAGG

2359 UCUGGGG CUGAUGAGGCCGAAAGGCCGAA AAGGCAG

2360 UUCUGGG CUGAUGAGGCCGAAAGGCCGAA AAAGGCA

2376 AUAGAAA CUGAUGAGGCCGAAAGGCCGAA AUCACUC

2377 GAUAGAA CUGAUGAGGCCGAAAGGCCGAA AAUCACU

2378 CGAUAGA CUGAUGAGGCCGAAAGGCCGAA AAAUCAC

2379 CCGAUAG CUGAUGAGGCCGAAAGGCCGAA AAAAUCA

2380 GCCGAUA CUGAUGAGGCCGAAAGGCCGAA AAAAAUC

2382 GUGCCGA CUGAUGAGGCCGAAAGGCCGAA AGAAAAA

2384 UUGUGCC CUGAUGAGGCCGAAAGGCCGAA AUAGAAA

2399 GUCCAUA CUGAUGAGGCCGAAAGGCCGAA AGUGCUU

2401 CAGUCCA CUGAUGAGGCCGAAAGGCCGAA AUAGUGC

2411 GAACCAU CUGAUGAGGCCGAAAGGCCGAA ACCAGUC

2417 ACCUGUG CUGAUGAGGCCGAAAGGCCGAA ACCAUUA

2418 AAGCUGU CUGAUGAGGCCGAAAGGCCGAA AACCAUU

2425 AUCUCUG CUGAUGAGGCCGAAAGGCCGAA ACCUGUG

2426 AAUCUCU CUGAUGAGGCCGAAAGGCCGAA AACCUGU

2433 ACUGGGU CUGAUGAGGCCGAAAGGCCGAA AUCUCUG

2434 CACUGGG CUGAUGAGGCCGAAAGGCCGAA AAUCUCU

2448 GAGGAAU CUGAUGAGGCCGAAAGGCCGAA AGGCCUC

2449 GGAGGAA CUGAUGAGGCCGAAAGGCCGAA AAGGCCU

2451 AGGGAGG CUGAUGAGGCCGAAAGGCCGAA AUAAGGC

2452 AAGGGAG CUGAUGAGGCCGAAAGGCCGAA AAUAAGG

2455 GGGAAGG CUGAUGAGGCCGAAAGGCCGAA AGGAAUA

2459 UGGGGGG CUGAUGAGGCCGAAAGGCCGAA AGGGAGG

2460 UUGGGGG CUGAUGAGGCCGAAAGGCCGAA AAGGGAG

2479 GCUAACA CUGAUGAGGCCGAAAGGCCGAA AGGUGUC

2480 GGCUAAC CUGAUGAGGCCGAAAGGCCGAA AAGGUGU

2483 GGUGGCU CUGAUGAGGCCGAAAGGCCGAA ACAAAGG

2484 AGGUGGC CUGAUGAGGCCGAAAGGCCGAA AACAAAG

2492 GGGUGGG CUGAUGAGGCCGAAAGGCCGAA AGGUGGC

2504 AGAAAUG CUGAUGAGGCCGAAAGGCCGAA AUGUGGG

2508 UGGCAGA CUGAUGAGGCCGAAAGGCCGAA AUGUAUG

2509 CUGGCAG CUGAUGAGGCCGAAAGGCCGAA AAUGUAU 2510 ACUGGCA CUGAUGAGGCCGAAAGGCCGAA AAAUGUA

2520 CAUUGUG CUGAUGAGGCCGAAAGGCCGAA ACACUGG

2521 UCAUUGU CUGAUGAGGCCGAAAGGCCGAA AACACUG

2533 GACCGCU CUGAUGAGGCCGAAAGGCCGAA AGUGUCA

2540 CAGACAU CUGAUGAGGCCGAAAGGCCGAA ACCGCUG

2545 AUGUCCA CUGAUGAGGCCGAAAGGCCGAA ACAUGAC

2558 UUGGGCA CUGAUGAGGCCGAAAGGCCGAA AUUCCCU

2579 CAAGGCA CUGAUGAGGCCGAAAGGCCGAA AGCUUGG

2585 AGAGGAC CUGAUGAGGCCGAAAGGCCGAA AGGCAUA

2588 ACAAGAG CUGAUGAGGCCGAAAGGCCGAA ACAAGGC

2591 AGGACAA CUGAUGAGGCCGAAAGGCCGAA AGGACAA

2593 ACAGGAC CUGAUGAGGCCGAAAGGCCGAA AGAGGAC

2596 CAAACAG CUGAUGAGGCCGAAAGGCCGAA ACAAGAG

2601 AAAUGCA CUGAUGAGGCCGAAAGGCCGAA ACAGGAC

2602 GAAAUGC CUGAUGAGGCCGAAAGGCCGAA AACAGGA

2607 CCAGUGA CUGAUGAGGCCGAAAGGCCGAA AUGCAAA

2608 CCCAGUG CUGAUGAGGCCGAAAGGCCGAA AAUGCAA

2609 UCCCAGU CUGAUGAGGCCGAAAGGCCGAA AAAUGCA

2620 AUAGUGC CUGAUGAGGCCGAAAGGCCGAA AGCUCCC

2626 GCUGCAA CUGAUGAGGCCGAAAGGCCGAA AGUGCAA

2628 GAGCUGC CUGAUGAGGCCGAAAGGCCGAA AUAGUGC

2635 GAAACUG CUGAUGAGGCCGAAAGGCCGAA AGCUGCA

2640 UGCAGGA CUGAUGAGGCCGAAAGGCCGAA ACUGGAG

2641 CUGCAGG CUGAUGAGGCCGAAAGGCCGAA AACUGGA

2642 ACUGCAG CUGAUGAGGCCGAAAGGCCGAA AAACUGG

2653 GGACCCU CUGAUGAGGCCGAAAGGCCGAA AUCACUG

2659 CUUGCAG CUGAUGAGGCCGAAAGGCCGAA ACCCUGA

2689 CCUCCAA CUGAUGAGGCCGAAAGGCCGAA ACCUUGG

2691 GUCCUCC CUGAUGAGGCCGAAAGGCCGAA AUACCUU

2700 UGGGAGG CUGAUGAGGCCGAAAGGCCGAA AGUCCUC

2704 AAGCUGG CUGAUGAGGCCGAAAGGCCGAA AGGGAGU

2711 CCUUCCA CUGAUGAGGCCGAAAGGCCGAA AGCUGGG

2712 CCCUUCC CUGAUGAGGCCGAAAGGCCGAA AAGCUGG

2721 CGCGGAU CUGAUGAGGCCGAAAGGCCGAA ACCCUUC

2724 ACACGCG CUGAUGAGGCCGAAAGGCCGAA AUGACCC

2744 CUACACA CUGAUGAGGCCGAAAGGCCGAA ACACACA

2750 GCUUGUC CUGAUGAGGCCGAAAGGCCGAA ACACAUA

2759 AGAGCGA CUGAUGAGGCCGAAAGGCCGAA AGCUUGU

2761 ACAGAGC CUGAUGAGGCCGAAAGGCCGAA AGAGCUU

2765 GGUGACA CUGAUGAGGCCGAAAGGCCGAA AGCGAGA

2769 CCUGGGU CUCAUGAGGCCGAAAGGCCGAA ACAGAGC

2797 GAACCAU CUGAUGAGGCCGAAAGGCCGAA AUUGCAC

2803 UGCAGUG CUGAUGAGGCCGAAAGGCCGAA ACCAUGA

2804 CUGCAGU CUGAUGAGGCCGAAAGGCCGAA AACCAUG

2813 AGGUCAA CUGAUGAGGCCGAAAGGCCGAA ACUGCAG

2815 AAAGGUC CUGAUGAGGCCGAAAGGCCGAA AGACUGC

2821 AGCCCAA CUGAUGAGGCCGAAAGGCCGAA AGGUCAA

2822 GAGCCCA CUGAUGAGGCCGAAAGGCCGAA AAGGUCA

2823 UGAGCCC CUGAUGAGGCCGAAAGGCCGAA AAAGGUC 2829 AUCACUU CUGAUGAGGCCGAAAGGCCGAA AGCCCAA

2837 GUGGGAG CUGAUGAGGCCGAAAGGCCGAA AUCACUU

2840 GAGGUGG CUGAUGAGGCCGAAAGGCCGAA AGGAUCA

2847 GGAGGCU CUGAUGAGGCCGAAAGGCCGAA AGGUGGG

2853 UACUCAG CUGAUGAGGCCGAAAGGCCGAA AGGCUGA

2860 UCCCAGC CUGAUGAGGCCGAAAGGCCGAA ACUCAGG

2872 GUGAGCC CUGAUGAGGCCGAAAGGCCGAA AUGGUCC

2877 GUGUUGU CUGAUGAGGCCGAAAGGCCGAA AGCCUAU

2899 AAAAUCA CUGAUGAGGCCGAAAGGCCGAA AUUUGCC

2900 AAAAAUC CUGAUGAGGCCGAAAGGCCGAA AAUUUGC

2904 AAAAAAA CUGAUGAGGCCGAAAGGCCGAA AUCAAAU

2905 AAAAAAA CUGAUGAGGCCGAAAGGCCGAA AAUCAAA

2906 AAAAAAA CUGAUGAGGCCGAAAGGCCGAA AAAUCAA

2907 AAAAAAA CUGAUGAGGCCGAAAGGCCGAA AAAAUCA

2908 AAAAAAA CUGAUGAGGCCGAAAGGCCGAA AAAAAUC

2909 AAAAAAA CUGAUGAGGCCGAAAGGCCGAA AAAAAAU

2910 AAAAAAA CUGAUGAGGCCGAAAGGCCGAA AAAAAAA

2911 AAAAAAA CUGAUGAGGCCGAAAGGCCGAA AAAAAAA

2912 GAAAAAA CUGAUGAGGCCGAAAGGCCGAA AAAAAAA

2913 UGAAAAA CUGAUGAGGCCGAAAGGCCGAA AAAAAAA

2914 CUGAAAA CUGAUGAGGCCGAAAGGCCGAA AAAAAAA

2915 UCUGAAA CUGAUGAGGCCGAAAGGCCGAA AAAAAAA

2916 CUCUGAA CUGAUGAGGCCGAAAGGCCGAA AAAAAAA

2917 UCUCUGA CUGAUGAGGCCGAAAGGCCGAA AAAAAAA

2918 GUCUCUG CUGAUGAGGCCGAAAGGCCGAA AAAAAAA

2919 CGUCUCU CUGAUGAGGCCGAAAGGCCGAA AAAAAAA

2931 GUUGCGA CUGAUGAGGCCGAAAGGCCGAA ACCCCGU

2933 AUGUUGC CUGAUGAGGCCGAAAGGCCGAA AGACCCC

2941 UCUGGGC CUGAUGAGGCCGAAAGGCCGAA AUGUUGC

2951 ACAAAGG CUGAUGAGGCCGAAAGGCCGAA AGUCUGG

2952 CACAAAG CUGAUGAGGCCGAAAGGCCGAA AAGUCUG

2955 UAACACA CUGAUGAGGCCGAAAGGCCGAA AGGAAGU

2956 CUAACAC CUGAUGAGGCCGAAAGGCCGAA AAGGAAG

2961 AUUAACU CUGAUGAGGCCGAAAGGCCGAA ACACAAA

2962 UAUUAAC CUGAUGAGGCCGAAAGGCCGAA AACACAA

2965 CUUUAUU CUGAUGAGGCCGAAAGGCCGAA ACUAACA

2966 GCUUUAU CUGAUGAGGCCGAAAGGCCGAA AACUAAC

2969 AAAGCUU CUGAUGAGGCCGAAAGGCCGAA AUUAACU

2975 GUUGAGA CUGAUGAGGCCGAAAGGCCGAA AGCUUUA

2976 AGUUGAG CUGAUGAGGCCGAAAGGCCGAA AAGCUUU

2977 CAGUUGA CUGAUGAGGCCGAAAGGCCGAA AAAGCUU

2979 GGCAGUU CUGAUGAGGCCGAAAGGCCGAA AGAAAGC Table 5

Mouse ICAM HH Ribozyme Sequence

nt. Position Ribozyme Sequence

11 CAACGGU CUGAUGAGGCCGAAAGGCCGAA ACCAGGG

23 AGCAGAG CUGAUGAGGCCGAAAGGCCGAA ACCACUG

26 AGGAGCA CUGAUGAGGCCGAAAGGCCGAA AGAACCA

31 UGUGGAG CUGAUGAGGCCGAAAGGCCGAA AGCAGAG

34 CGACCCU CUGAUGAGGCCGAAAGGCCGAA AUGAGAA

40 AGGCUAC CUGAUGAGGCCGAAAGGCCGAA AGUGUGC

48 CCAGGCU CUGAUGAGGCCGAAAGGCCGAA AGGUCCU

54 CCAUCAC CUGAUGAGGCCGAAAGGCCGAA AGGCCCA

58 GGAGCUA CUGAUGAGGCCGAAAGGCCGAA AGGCAUG

64 CUGCUGG CUGAUGAGGCCGAAAGGCCGAA AGGGGUG

96 GGGCCAG CUGAUGAGGCCGAAAGGCCGAA AGCAGAG

102 CCAGCAG CUGAUGAGGCCGAAAGGCCGAA ACUGGCA

108 GGGCCAG CUGAUGAGGCCGAAAGGCCGAA AGCAGAG

115 AGGAGCA CUGAUGAGGCCGAAAGGCCGAA AGAACCA

119 UCCUGGU CUGAUGAGGCCGAAAGGCCGAA ACAUUCC

120 GGGCCAG CUGAUGAGGCCGAAAGGCCGAA AGCAGAG 146 GGAAGCG CUGAUGAGGCCGAAAGGCCGAA ACGACUG 152 AGUGGCU CUGAUGAGGCCGAAAGGCCGAA ACACAGA 158 GGUUUUU CUGAUGAGGCCGAAAGGCCGAA AACAGGA 165 GCAAAAC CUGAUGAGGCCGAAAGGCCGAA ACUUCUG 168 GGGGCAG CUGAUGAGGCCGAAAGGCCGAA AAGGCUU 185 CUGCACG CUGAUGAGGCCGAAAGGCCGAA ACCCACC 209 GCCAGAG CUGAUGAGGCCGAAAGGCCGAA AAGUGGC 227 GCAAAAC CUGAUGAGGCCGAAAGGCCGAA ACUUCUG 230 GGAGCAA CUGAUGAGGCCGAAAGGCCGAA ACAACUU 237 AGUUCUC CUGAUGAGGCCGAAAGGCCGAA AAGCACA 248 UUUAGGA CUGAUGAGGCCGAAAGGCCGAA AUGGGUU 253 UCUUCCU CUGAUGAGGCCGAAAGGCCGAA AGGCAGG 263 CAGUAGA CUGAUGAGGCCGAAAGGCCGAA AAACCCU 267 UAGGCAG CUGAUGAGGCCGAAAGGCCGAA AGCCCCU 293 CAGCUCA CUGAUGAGGCCGAAAGGCCGAA ACAGCUU 319 GGCUCAG CUGAUGAGGCCGAAAGGCCGAA AUCUCCU 335 GUUCUCA CUGAUGAGGCCGAAAGGCCGAA AGCACAG

337 CAGUGUG CUGAUGAGGCCGAAAGGCCGAA AUUGGAC

338 UCAGCUC CUGAUGAGGCCGAAAGGCCGAA AACAGCU 359 AGCGGAC CUGAUGAGGCCGAAAGGCCGAA ACUGCAC 367 CGGGUUG CUGAUGAGGCCGAAAGGCCGAA AGCCAUU

374 GGGCAGG CUGAUGAGGCCGAAAGGCCGAA AGGCUUC

375 GGGGCAG CUGAUGAGGCCGAAAGGCCGAA AAGGCUU 378 ACACGGU CUGAUGAGGCCGAAAGGCCGAA AUGGUAG 386 AAACGAA CUGAUGAGGCCGAAAGGCCGAA ACACGGU 394 AGAUCGA CUGAUGAGGCCGAAAGGCCGAA AGUCCGG 420 CGGGGGG CUGAUGAGGCCGAAAGGCCGAA AAGUGUG 425 CUGCUGG CUGAUGAGGCCGAAAGGCCGAA AGGGGUG 427 CACUGCU CUGAUGAGGCCGAAAGGCCGAA AGAGCUG

450 GCAGGGU CUGAUGAGGCCGAAAGGCCGAA AGGUCCU

451 CAAAGGA CUGAUGAGGCCGAAAGGCCGAA AGGUUUC 456 AGUGGCU CUGAUGAGGCCGAAAGGCCGAA AGGGUAA 495 ACACGGU CUGAUGAGGCCGAAAGGCCGAA AUGGUAG 510 CCCCACG CUGAUGAGGCCGAAAGGCCGAA AGGAGCA 554 GGAUGGA CUGAUGAGGCCGAAAGGCCGAA ACCUGAG 592 CCCAUGU CUGAUGAGGCCGAAAGGCCGAA AUCUUUC

607 CAUGAGA CUGAUGAGGCCGAAAGGCCGAA AUUGGCU

608 GCAUGAG CUGAUGAGGCCGAAAGGCCGAA AAUUGGC

609 GGCAUGA CUGAUGAGGCCGAAAGGCCGAA AAAUUGG 611 GCGGCAU CUGAUGAGGCCGAAAGGCCGAA AGAAAUU

656 CAGCUCA CUGAUGAGGCCGAAAGGCCGAA ACAGCUU

657 UCAGCUC CUGAUGAGGCCGAAAGGCCGAA AACAGCU 668 GGUGGCC CUGAUGAGGCCGAAAGGCCGAA AGGCUCG 677 AGGCUGG CUGAUGAGGCCGAAAGGCCGAA AGAGGUC 684 AGGACCG CUGAUGAGGCCGAAAGGCCGAA AGCUGAA

692 AAGAUCG CUGAUGAGGCCGAAAGGCCGAA AAGUCCG

693 GCAGGGU CUGAUGAGGCCGAAAGGCCGAA AGGUCCU 696 GAGGCAG CUGAUGAGGCCGAAAGGCCGAA AAACAGG 709 UGAGGUG CUGAUGAGGCCGAAAGGCCGAA AGCCGCC 720 AGCUGAA CUGAUGAGGCCGAAAGGCCGAA AGUUGUA 723 CGGAGCU CUGAUGAGGCCGAAAGGCCGAA AAAAGUU

735 UCUCCAG CUGAUGAGGCCGAAAGGCCGAA AUCUGGU 738 CCAUCAC CUGAUGAGGCCGAAAGGCCGAA AGGCCCA 765 GGAAGCG CUGAUGAGGCCGAAAGGCCGAA ACGACUG

769 GGCAGGA CUGAUGAGGCCGAAAGGCCGAA ACAGGCC

770 UUCCAGG CUGAUGAGGCCGAAAGGCCGAA AGCAAAA 785 GGCAGGA CUGAUGAGGCCGAAAGGCCGAA ACAGGCC

736 AGGCAGG CUGAUGAGGCCGAAAGGCCGAA AACAGGC 792 CUUCCGA CUGAUGAGGCCGAAAGGCCGAA ACCUCCA 794 AGUCUCC CUGAUGAGGCCGAAAGGCCGAA AGCCCAG 807 CCAGGUA CUGAUGAGGCCGAAAGGCCGAA AUCCGAG 833 GGGUGUC CUGAUGAGGCCGAAAGGCCGAA AGCUUUG 846 CAACGGU CUGAUGAGGCCGAAAGGCCGAA ACCAGGG 851 GCUGGUA CUGAUGAGGCCGAAAGGCCGAA AGGUCUC 863 CCAGAGG CUGAUGAGGCCGAAAGGCCGAA AGUGGCU

866 GGGCAGG CUGAUGAGGCCGAAAGGCCGAA AGGCUUC

867 UCUCCGG CUGAUGAGGCCGAAAGGCCGAA AACGAAU 869 CUUGCAU CUGAUGAGGCCGAAAGGCCGAA AGGAAGA 881 ACGGGUU CUGAUGAGGCCGAAAGGCCGAA AAGCCAU 885 UCACCUC CUGAUGAGGCCGAAAGGCCGAA ACCAAGG 933 CCAGAAU CUGAUGAGGCCGAAAGGCCGAA AUUAUAG 936 GCACCAG CUGAUGAGGCCGAAAGGCCGAA AUGAUUA 978 AGUUGUA CUGAUGAGGCCGAAAGGCCGAA ACUGUUA 980 AAAGUUG CUGAUGAGGCCGAAAGGCCGAA AGACUGU

986 AGCUGAA CUGAUCAGGCCGAAAGGCCGAA AGUUGUA

987 CAGCUCA CUGAUGAGGCCGAAAGGCCGAA AAGUUGU 388 GGAGCUG CUGAUGAGGCCGAAAGGCCGAA AAAGUUG 1005 UCUCCAG CUGAUGAGGCCGAAAGGCCGAA AUCUGGU

1006 UUCCCCA CUGAUGAGGCCGAAAGGCCGAA ACUCUCA

1023 CUUCCGA CUGAUGAGGCCGAAAGGCCGAA ACCUCCA

1025 CCCUUCC CUGAUGAGGCCGAAAGGCCGAA AGACCUC

1066 UUAUUUU CUGAUGAGGCCGAAAGGCCGAA AGAGUGG

1092 GGCCUGA CUGAUGAGGCCGAAAGGCCGAA AUCCAGU

1093 UUGGCUG CUGAUGAGGCCGAAAGGCCGAA AGGUCCA

1125 UCAAGAA CUGAUGAGGCCGAAAGGCCGAA AGUUGGG

1163 GCAAAAG CUGAUGAGGCCGAAAGGCCGAA AGCUUCG

1164 AGCAAAA CUGAUGAGGCCGAAAGGCCGAA AAGCUUC

1166 AGAGCAA CUGAUGAGGCCGAAAGGCCGAA AGAAGCU

1172 GGUUUUU CUGAUGAGGCCGAAAGGCCGAA AACAGGA

1200 UGUGGAG CUGAUGAGGCCGAAAGGCCGAA AGCAGAG

1201 CUGUUCA CUGAUGAGGCCGAAAGGCCGAA AAGCAGC

1203 ACUGGUG CUGAUGAGGCCGAAAGGCCGAA AAAAAGU

1227 GCACACG CUGAUGAGGCCGAAAGGCCGAA AUGUACC

1228 AGCAAAA CUGAUGAGGCCGAAAGGCCGAA AAGCUUC

1233 CUCUCCG CUGAUGAGGCCGAAAGGCCGAA AAACGAA

1238 AGGACCA CUGAUGAGGCCGAAAGGCCGAA ACAGCAC

1264 CUUGCAC CUGAUGAGGCCGAAAGGCCGAA ACCCUUC

1267 UUCCCCA CUGAUGAGGCCGAAAGGCCGAA ACUCUCA

1294 GGCUCAG CUGAUGAGGCCGAAAGGCCGAA AUCUCCU

1295 CUGCUGA CUGAUGAGGCCGAAAGGCCGAA ACCCCUC

1306 CAUUUCA CUGAUGAGGCCGAAAGGCCGAA AGUCUGC

1321 UCCUCCU CUGAUGAGGCCGAAAGGCCGAA AGCCUUC

1334 UUUAGGA CUGAUGAGGCCGAAAGGCCGAA AUGGGUU

1344 CACUCUC CUGAUGAGGCCGAAAGGCCGAA AGCUCAU

1351 UAACUUA CUGAUGAGGCCGAAAGGCCGAA ACAUUCA

1353 CACCUUC CUGAUGAGGCCGAAAGGCCGAA ACCCACU

1366 AGUUGUA CUGAUGAGGCCGAAAGGCCGAA ACUGUUA

1367 AGGUGGG CUGAUGAGGCCGAAAGGCCGAA AGGUGCU

1368 AGAGUGG CUGAUGAGGCCGAAAGGCCGAA ACAGUAC

1380 CCACCCC CUGAUGAGGCCGAAAGGCCGAA AUGGGCA

1388 AGCCACU CUGAUGAGGCCGAAAGGCCGAA AGUCUCC

1398 GUUCUGU CUGAUGAGGCCGAAAGGCCGAA ACAGCCA

1402 AGUUCUC CUGAUGAGGCCGAAAGGCCGAA AAGCACA

1408 CCUCCCC CUGAUGAGGCCGAAAGGCCGAA AUCUCGC

1410 CCCUUCC CUGAUGAGGCCGAAAGGCCGAA AGACCUC

1421 ACAAAAG CUGAUGAGGCCGAAAGGCCGAA AGGUGGG

1425 CUCUACC CUGAUGAGGCCGAAAGGCCGAA AGGCAGU

1429 CAGGGGC CUGAUGAGGCCGAAAGGCCGAA AUAGAGA

1444 UCCUCCU CUGAUGAGGCCGAAAGGCCGAA AGCCUUC

1455 UCCUGGU CUGAUGAGGCCGAAAGGCCGAA ACAUUCC

1482 GGGAGCA CUGAUGAGGCCGAAAGGCCGAA AACAACU

1484 CAUGAGG CUGAUGAGGCCGAAAGGCCGAA AGAACAG

1493 GUUCUCA CUGAUGAGGCCGAAAGGCCGAA AGCACAG

1500 GGACCAU CUGAUGAGGCCGAAAGGCCGAA AUUUCAU

1503 GAAUGAU CUGAUGAGGCCGAAAGGCCGAA AUAGUCC

1506 CGGUUAU CUGAUGAGGCCGAAAGGCCGAA AACAUAA 1509 ACACGGU CUGAUGAGGCCGAAAGGCCGAA AUGGUAG

1518 CGCCUGG CUGAUGAGGCCGAAAGGCCGAA ACCAUGA

1530 CCAGAAU CUGAUGAGGCCGAAAGGCCGAA AUUAUAG

1533 GGCCCAC CUGAUGAGGCCGAAAGGCCGAA AUGACCA

1551 AGCUGCU CUGAUGAGGCCGAAAGGCCGAA AGGCAUG

1559 AGGUGGG CUGAUGAGGCCGAAAGGCCGAA AGGUGCU

1563 GGUUAUA CUGAUGAGGCCGAAAGGCCGAA ACAUAAG

1565 GCGGUUA CUGAUGAGGCCGAAAGGCCGAA AAACAUA

1557 UGGCGGU CUGAUGAGGCCGAAAGGCCGAA AUAAACA

1584 AUAUCCU CUGAUGAGGCCGAAAGGCCGAA AUCUUUC

1592 UAACUUG CUGAUGAGGCCGAAAGGCCGAA AUAUCCU

1599 CCUUCUG CUGAUGAGGCCGAAAGGCCGAA AACUUGU

1651 GCUCAGG CUGAUGAGGCCGAAAGGCCGAA AGGUGGG

1661 CAAAGGA CUGAUGAGGCCGAAAGGCCGAA AGGUUUC

1663 UUCAAAG CUGAUGAGGCCGAAAGGCCGAA AAAGGUU

1678 CCAGGCU CUGAUGAGGCCGAAAGGCCGAA AGGUCCU

1680 CCAGAGG CUGAUGAGGCCGAAAGGCCGAA AGUGGCU

1681 GCCAGAG CUGAUGAGGCCGAAAGGCCGAA AAGUGGC

1684 ACAGCCA CUGAUGAGGCCGAAAGGCCGAA AGGAAGU

1690 AGAUCGA CUCAUGAGGCCGAAAGGCCGAA AGUCCGG

1691 AAGAUCG CUGAUGAGGCCGAAAGGCCGAA AAGUCCG

1696 CCACCCC CUGAUGAGGCCGAAAGGCCGAA AUGGGCA

1698 CUCCAGG CUGAUGAGGCCGAAAGGCCGAA AUAUCCG

1737 GCUGGUA CUGAUGAGGCCGAAAGGCCGAA AGGUCUC

1750 UGAGGUG CUGAUGAGGCCGAAAGGCCGAA AGCCGCC

1756 GGGCAGG CUGAUGAGGCCGAAAGGCCGAA AGGCUUC

1787 UGGGGAC CUGAUGAGGCCGAAAGGCCGAA AUGUCUC

1790 AUUAGAG CUGAUGAGGCCGAAAGGCCGAA ACAAUGC

1793 UCCAGCC CUGAUGAGGCCGAAAGGCCGAA AGGACCA

1797 UUUAUGU CUGAUGAGGCCGAAAGGCCGAA ACUGGUG

1802 UCUCCAG CUGAUGAGGCCGAAAGGCCGAA AUCUGGU

1812 GGCCUGA CUGAUGAGGCCGAAAGGCCGAA AUCCAGU

1813 UGAGGGU CUGAUGAGGCCGAAAGGCCGAA AAUGCUG

1825 GCAGAGG CUGAUGAGGCCGAAAGGCCGAA AGCGUGG

1837 GGAGCUA CUGAUGAGGCCGAAAGGCCGAA AGGCAUG

1845 GGUGGCC CUGAUGAGGCCGAAAGGCCGAA AGGCUCG

1856 AAGAUCG CUGAUGAGGCCGAAAGGCCGAA AAGUCCG

1861 UACUGGA CUGAUGAGGCCGAAAGGCCGAA AUCAUGU

1865 CUGAGGC CUGAUGAGGCCGAAAGGCCGAA ACAAGUG

1868 UUUAUGU CUGAUGAGGCCGAAAGGCCGAA ACUGGUG

1877 AGCUGCU CUGAUGAGGCCGAAAGGCCGAA AGGCAUG

1901 GUCCCUU CUGAUGAGGCCGAAAGGCCGAA AGUUUUA

1912 ACUGAUC CUGAUGAGGCCGAAAGGCCGAA ACUAUAU

1922 UAACUUA CUGAUGAGGCCGAAAGGCCGAA ACAUUCA

1923 GAUACCU CUGAUGAGGCCGAAAGGCCGAA AGCAUCA

1928 CUGGUAA CUGAUGAGGCCGAAAGGCCGAA ACUCUAA

1930 AGCUGGU CUGAUGAGGCCGAAAGGCCGAA AAACUCU

1964 UGGGGAC CUCAUGAGGCCGAAAGGCCCAA AUGUCUC

1383 UAACUUG CUGAUGAGGCCGAAAGGCCGAA AUAUCCU 1996 GGCUCAG CUGAUGAGGCCGAAAGGCCGAA AUCUCCU

2005 GGUCCGC CUGAUGAGGCCGAAAGGCCGAA AGCUCCA

2013 UACUCAA CUGAUGAGGCCGAAAGGCCGAA AAAUAGC

2015 CCACCCC CUGAUGAGGCCGAAAGGCCGAA AUGGGCA

2020 CUCAGAA CUGAUGAGGCCGAAAGGCCGAA AACCACC

2039 CCUCUGC CUGAUGAGGCCGAAAGGCCGAA AGCCAGC

2040 CCUCCAG CUGAUGAGGCCGAAAGGCCGAA AGGUCAG

2057 GGAUGUG CUGAUGAGGCCGAAAGGCCGAA AGGAGCA

2061 ACACGGU CUGAUGAGGCCGAAAGGCCGAA AUGGUAG

2071 CUGAGGC CUGAUGAGGCCGAAAGGCCGAA ACAAGUG

2076 UAGCUCU CUGAUGAGGCCGAAAGGCCGAA AGGCUAC

2097 CAUCAAG CUGAUGAGGCCGAAAGGCCGAA AGAGUUG

2098 CGGGGGG CUGAUGAGGCCGAAAGGCCGAA AAGUGUG

2115 AUCCUCC CUGAUGAGGCCGAAAGGCCGAA AGGUGGC

2128 CUCAAUA CUGAUGAGGCCGAAAGGCCGAA AUAGCUG

2130 GAGGCAG CUGAUGAGGCCGAAAGGCCGAA AAACAGG

2145 CAUCAAG CUGAUGAGGCCGAAAGGCCGAA AGAGUUG

2152 AACUCUA CUGAUGAGGCCGAAAGGCCGAA AUUAAUA

2156 UAAUAAA CUGAUGAGGCCGAAAGGCCGAA ACAUCAA

2158 AUUAAUA CUGAUGAGGCCGAAAGGCCGAA AUACAUC

2159 AAUUAAU CUGAUGAGGCCGAAAGGCCGAA AAUACAU

2160 AAAUUAA CUGAUGAGGCCGAAAGGCCGAA AAAUACA

2162 CUAAAUU CUGAUGAGGCCGAAAGGCCGAA AUAAAUA

2163 AAUUAAU CUGAUGAGGCCGAAAGGCCGAA AAUACAU

2166 AAUAGAG CUGAUGAGGCCGAAAGGCCGAA AUGAAGU

2167 AAUUAAU CUGAUGAGGCCGAAAGGCCGAA AAUACAU

2170 CUAAAUU CUGAUGAGGCCGAAAGGCCGAA AUAAAUA

2171 GGGAGCA CUGAUGAGGCCGAAAGGCCGAA AACAACU

2173 CUGGUAA CUGAUGAGGCCGAAAGGCCGAA ACUCUAA

2174 GCUGGUA CUGAUGAGGCCGAAAGGCCGAA AACUCUA

2175 AGCUGGU CUGAUGAGGCCGAAAGGCCGAA AAACUCU

2176 UAGCUGG CUGAUGAGGCCGAAAGGCCGAA AAAACUC

2183 CAAUAAA CUGAUGAGGCCGAAAGGCCGAA AGCUGGU

2185 CUCAAUA CUGAUGAGGCCGAAAGGCCGAA AUAGCUG

2186 ACUCAAU CUGAUGAGGCCGAAAGGCCGAA AAUAGCU

2187 UACUCAA CUGAUGAGGCCGAAAGGCCGAA AAAUAGC

2189 GGUACUC CUGAUGAGGCCGAAAGGCCGAA AUAAAUA

2196 CAUCAAG CUGAUGAGGCCGAAAGGCCGAA AGAGUUG

2198 AACAUAA CUGAUGAGGCCGAAAGGCCGAA AGGCUGC

2199 AUAAACA CUGAUGAGGCCGAAAGGCCGAA AAGAGGC

2200 CUUGCAU CUGAUGAGGCCGAAAGGCCGAA AGGAAGA

2201 GCCGACA CUGAUGAGGCCGAAAGGCCGAA AAAACUU

2205 UCAGGCC CUGAUGAGGCCGAAAGGCCGAA ACAUAAA

2210 AGCCACU CUGAUGAGGCCGAAAGGCCGAA AGUCUCC

2220 AGAGAAC CUGAUGAGGCCGAAAGGCCGAA AUGCCAG

2224 GGAUGGA CUGAUGAGGCCGAAAGGCCGAA ACCUGAG

2226 GCGGCCU CUGAUGAGGCCCAAAGGCCGAA AGAUCCA

2233 CCUCCAG CUCAUGAGGCCCAAAGGCCCAA AGGUCAG

2242 GGUCCGC CUGAUGAGGCCGAAAGGCCGAA AGCUCCA 2248 UGGGAUG CUGAUGAGGCCGAAAGGCCGAA AUGGAUA

2254 UCAGUGU CUGAUGAGGCCGAAAGGCCGAA AAUUGGA

2259 CACCGUG CUGAUGAGGCCGAAAGGCCGAA AUGUGAU

2260 GCACCGU CUGAUGAGGCCGAAAGGCCGAA AAUGUGA

2266 UCCUGGU CUGAUGAGGCCGAAAGGCCGAA ACAUUCC

2274 UCUCCAG CUGAUGAGGCCGAAAGGCCGAA AUCUGGU

2279 CUUGCAC CUGAUGAGGCCGAAAGGCCGAA ACCCUUC

2282 CAGCUCA CUGAUGAGGCCGAAAGGCCGAA ACAGCUU

2288 AGGCCAU CUGAUGAGGCCGAAAGGCCGAA ACUUAUA

2291 AGCAGAG CUGAUGAGGCCGAAAGGCCGAA ACCACUG

2321 CCCAUGU CUGAUGAGGCCGAAAGGCCGAA AUCUUUC

2338 CAGGCAG CUGAUGAGGCCGAAAGGCCGAA AGUCUCA

2339 CAAAGGA CUGAUGAGGCCGAAAGGCCGAA AGGUUUC

2341 AGGCUGG CUGAUGAGGCCGAAAGGCCGAA AGAGGUC

2344 GCUGGAA CUGAUGAGGCCGAAAGGCCGAA AUCGAAA

2358 CUGCUGA CUGAUGAGGCCGAAAGGCCGAA AGCUGGG

2359 UCUGUUC CUGAUGAGGCCGAAAGGCCGAA AAAGCAG

2360 UUCAAAG CUGAUGAGGCCGAAAGGCCGAA AAAGGUU

2376 UCAGAAG CUGAUGAGGCCGAAAGGCCGAA ACCACCU

2377 CUCAGAA CUGAUGAGGCCGAAAGGCCGAA AACCACC

2378 CAGUAGA CUGAUGAGGCCGAAAGGCCGAA AAACCCU

2379 CUUAUGA CUGAUGAGGCCGAAAGGCCGAA AAAAGCA

2380 GCCGACA CUGAUGAGGCCGAAAGGCCGAA AAAACUU

2382 GGGGCAA CUGAUGAGGCCGAAAGGCCGAA AGAGAAU

2384 UUGUGUC CUGAUGAGGCCGAAAGGCCGAA ACUGGAU

2399 GUCCACA CUGAUGAGGCCGAAAGGCCGAA AGUGUUU

2401 CAGCUCA CUGAUGAGGCCGAAAGGCCGAA ACAGCUU

2411 GCAUCCU CUGAUGAGGCCGAAAGGCCGAA ACCAGUA

2417 ACGUAUG CUGAUGAGGCCGAAAGGCCGAA ACCAUUC

2418 GGCCUGA CUGAUGAGGCCGAAAGGCCGAA AUCCAGU

2425 AACCCUC CUGAUGAGGCCGAAAGGCCGAA ACCCAUG

2426 AAACUCU CUGAUGAGGCCGAAAGGCCGAA AAUUAAU

2433 GCUGGUA CUGAUGAGGCCGAAAGGCCGAA AACUCUA

2434 AGCUGGU CUGAUGAGGCCGAAAGGCCGAA AAACUCU

2448 GGGCAGG CUGAUGAGGCCGAAAGGCCGAA AGGCUUC

2449 GGGGCAG CUGAUGAGGCCGAAAGGCCGAA AAGGCUU

2451 AGGCAGG CUGAUGAGGCCGAAAGGCCGAA AACAGGC

2452 GAGGCAG CUGAUGAGGCCGAAAGGCCGAA AAACAGG

2455 GGGCAGG CUGAUGAGGCCGAAAGGCCGAA AGGCUUC

2459 GGGGGGG CUGAUGAGGCCGAAAGGCCGAA AGUGUGG

2460 CGGGGGG CUGAUGAGGCCGAAAGGCCGAA AAGUGUG

2479 GCUGGUA CUGAUGAGGCCGAAAGGCCGAA AGGUCUC

2480 GGAUCAC CUGAUGAGGCCGAAAGGCCGAA ACGGUGA

2483 GGUGGCU CUGAUGAGGCCGAAAGGCCGAA ACAUUGG

2484 GACUGGU CUGAUGAGGCCGAAAGGCCGAA AAAAAAG

2492 AGGUGGG CUGAUGAGGCCGAAAGGCCGAA AGGUGCU

2504 ACAAAAG CUGAUGAGGCCGAAAGGCCGAA AGGUGGG

2508 UGGGAUG CUGAUGAGGCCCAAAGGCCGAA AUGGAUA

2509 CUGGUAA CUGAUGAGGCCGAAAC-GCCGAA ACUCUAA 2510 GCUGGUA CUGAUGAGGCCGAAAGGCCGAA AACUCUA

2520 CAUUGGG CUGAUGAGGCCGAAAGGCCGAA ACAAAAG

2521 UGAGGGU CUGAUGAGGCCGAAAGGCCGAA AAUGCUG

2533 GAUACCU CUGAUGAGGCCGAAAGGCCGAA AGCAUCA

2540 CACAGCG CUGAUGAGGCCGAAAGGCCGAA ACUGCUG

2545 AGGACCA CUGAUGAGGCCGAAAGGCCGAA ACAGCAC

2568 UUUGACA CUGAUGAGGCCGAAAGGCCGAA ACUUCAC

2579 CAGGCCA CUGAUGAGGCCGAAAGGCCGAA AACUUAU

2585 AGAGAAC CUGAUGAGGCCGAAAGGCCGAA AUGCCAG

2588 AUUAGAG CUGAUGAGGCCGAAAGGCCGAA ACAAUGC

2591 AGGAGCA CUGAUGAGGCCGAAAGGCCGAA AGAACCA

2593 GCAGAGC CUGAUGAGGCCGAAAGGCCGAA AAAGAAG

2596 CAUUGGG CUGAUGAGGCCGAAAGGCCGAA ACAAAAG

2601 AAACGAA CUGAUGAGGCCGAAAGGCCGAA ACACGGU

2602 GGGAUGG CUGAUGAGGCCGAAAGGCCGAA AGCUGGA

2607 CCAGGUA CUGAUGAGGCCGAAAGGCCGAA AUCCGAG

2608 CACAGCG CUGAUGAGGCCGAAAGGCCGAA ACUGCUG

2609 UCCUGGU CUGAUGAGGCCGAAAGGCCGAA ACAUUCC

2620 GCAGGGU CUGAUGAGGCCGAAAGGCCGAA AGGUCCU

2626 GCUGGAA CUGAUGAGGCCGAAAGGCCGAA AUCGAAA

2628 AGGCUAC CUGAUGAGGCCGAAAGGCCGAA AGUGUGC

2635 AGGACCG CUGAUGAGGCCGAAAGGCCGAA AGCUGAA

2640 GGCAGGA CUGAUGAGGCCGAAAGGCCGAA ACAGGCC

2641 CUGCUGA CUGAUGAGGCCGAAAGGCCGAA AGCUGGG

2642 GAGGCAG CUGAUGAGGCCGAAAGGCCGAA AAACAGG

2653 GCAUCCU CUGAUGAGGCCGAAAGGCCGAA ACCAGUA

2659 CUUGCAC CUGAUGAGGCCGAAAGGCCGAA ACCCUUC

2689 CCUCGGA CUGAUGAGGCCGAAAGGCCGAA ACAUUAG

2691 GGCCUCG CUGAUGAGGCCGAAAGGCCGAA AGACAUU

2700 GGGCAGG CUGAUGAGGCCGAAAGGCCGAA AGGCUUC

2704 AGGCUGG CUGAUGAGGCCGAAAGGCCGAA AGAGGUC

2711 CUGCUGA CUGAUGAGGCCGAAAGGCCGAA AGCUGGG

2712 CCCUUCC CUGAUGAGGCCGAAAGGCCGAA AGACCUC

2721 CUUGCAC CUGAUGAGGCCGAAAGGCCGAA ACCCUUC

2724 GCACACG CUGAUGAGGCCGAAAGGCCGAA AUGUACC

2744 CUGCACG CUGAUGAGGCCGAAAGGCCGAA ACCCACC

2750 GGUACUC CUGAUGAGGCCGAAAGGCCGAA AUAAAUA

2759 AGAUCGA CUGAUGAGGCCGAAAGGCCGAA AGUCCGG

2761 GCAGGGU CUGAUGAGGCCGAAAGGCCGAA AGGUCCU

2765 AGCGGCA CUGAUGAGGCCGAAAGGCCGAA AGCAAAA

2769 CCUGUUU CUGAUGAGGCCGAAAGGCCGAA ACAGACU

2797 GGACCAU CUGAUGAGGCCGAAAGGCCGAA AUUUCAU

2803 CGCCUGG CUGAUGAGGCCGAAAGGCCGAA ACCAUGA

2804 CUGCACG CUGAUGAGGCCGAAAGGCCGAA ACCCACC

2813 GGGUCAG CUGAUGAGGCCGAAAGGCCGAA ACCGGAG

2815 AAAGUUG CUGAUGAGGCCGAAAGGCCGAA AGACUGU

2821 CCUCCAG CUGAUGAGGCCGAAAGGCCGAA AGGUCAG

2322 AAGUCCG CUCAUGAGGCCCAAAGGCCGAA AGGCUCC

2323 UGGGAGC CUGAUCAGGCCGAAAGGCCGAA AAAGGCA 2829 AUGAUUA CUGAUGAGGCCGAAAGGCCGAA AGUCCAG

2837 UCAGAAG CUGAUGAGGCCGAAAGGCCGAA ACCACCU

2840 CAGGCAG CUGAUGAGGCCGAAAGGCCGAA AGUCUCA

2847 GGUGGCU CUGAUGAGGCCGAAAGGCCGAA ACAUUGG

2853 AACAUAA CUGAUGAGGCCGAAAGGCCGAA AGGCUGC

2860 UCACAGU CUGAUGAGGCCGAAAGGCCGAA ACUUGGC

2872 CUUGGCU CUGAUGAGGCCGAAAGGCCGAA AAGGUCC

2877 GUGAUGG CUGAUGAGGCCGAAAGGCCGAA AGCGGAA

2899 AAGAUCG CUGAUGAGGCCGAAAGGCCGAA AAGUCCG

2900 AAAACUC CUGAUGAGGCCGAAAGGCCGAA AAAUUAA

2904 AAUAGAG CUGAUGAGGCCGAAAGGCCGAA AUGAAGU

2905 CAAUAGA CUGAUGAGGCCGAAAGGCCGAA AAUGAAG

2906 UAAUAAA CUGAUGAGGCCGAAAGGCCGAA ACAUCAA

2907 AAAUUAA CUGAUGAGGCCGAAAGGCCGAA AAAUACA

2908 AGCAAAA CUGAUGAGGCCGAAAGGCCGAA AAGCUUC

2909 AGAGCAA CUGAUGAGGCCGAAAGGCCGAA AGAAGCU

2910 AAAUUAA CUGAUGAGGCCGAAAGGCCGAA AAAUACA

2911 AAAUUAA CUGAUGAGGCCGAAAGGCCGAA AAAUACA

2912 GACAUUA CUGAUGAGGCCGAAAGGCCGAA AGAACAA

2913 UGACCAG CUGAUGAGGCCGAAAGGCCGAA AGAGAAA

2914 CUUAUGA CUGAUGAGGCCGAAAGGCCGAA AAAAGCA

2915 UCUAAAU CUGAUGAGGCCGAAAGGCCGAA AAUAAAU

2916 CUCCGGA CUGAUGAGGCCGAAAGGCCGAA ACGAAUA

2917 UCUCCGG CUGAUGAGGCCGAAAGGCCGAA AACGAAU

2918 CUCUCCG CUGAUGAGGCCGAAAGGCCGAA AAACGAA

2919 CGACCCU CUGAUGAGGCCGAAAGGCCGAA AUGAGAA

2931 CUUCCGA CUGAUGAGGCCGAAAGGCCGAA ACCUCCA

2933 CCCUUCC CUGAUGAGGCCGAAAGGCCGAA AGACCUC

2941 UGGGGAC CUGAUGAGGCCGAAAGGCCGAA AUGUCUC

2951 GCAGAGG CUGAUGAGGCCGAAAGGCCGAA AGCGUGG

2952 CACAGCG CUGAUGAGGCCGAAAGGCCGAA ACUGCUG

2955 UGACACA CUGAUGAGGCCGAAAGGCCGAA AGUCACU

2956 UUGAUUC CUGAUGAGGCCGAAAGGCCGAA AAGGAAA

2961 AGUGGCU CUGAUGAGGCCGAAAGGCCGAA ACACAGA

2962 AAUUAAU CUGAUGAGGCCGAAAGGCCGAA AAUACAU

2965 CUUUAUU CUGAUGAGGCCGAAAGGCCGAA AUUCAAA

2966 CCUCUGC CUGAUGAGGCCGAAAGGCCGAA AGCCAGC

2969 AAAACUU CUGAUGAGGCCGAAAGGCCGAA AUUGAUU

2975 GCUGGUA CUGAUGAGGCCGAAAGGCCGAA AACUCUA

2976 AGUAGAG CUGAUGAGGCCGAAAGGCCGAA AACCCUC

2977 CAGCUCA CUGAUGAGGCCGAAAGGCCGAA ACAGCUU

2979 GGCAAUA CUGAUGAGGCCGAAAGGCCGAA AGAAUGA Table 6

Human ICAM Hairpin Ribozyme/Substrate Sequences

nt. Hairpin Ribozyme Sequence Substrate Position

70 GGGCCGGG AGAA GCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAGCA GCC CCCGGCCC

86 GGAGUGCG AGAA GCGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCGCU GCC CGCACUCC 343 CCCAUCAG AGAA GUUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AAACU GCC CUGAUGGG 635 GCCCUUGG AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUGCG GCC CCAAGGGC 653 UGUUCUCA AGAA GCUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GAGCU GUU UGAGAACA 782 AGACUGGG AGAA GCCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGGCU GUU CCCAGUCU 920 CUGCACAC AGAA GCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGGCU GAC GUGUGCAG 1301 ACAUUGGA AGAA GCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAGCA GAC UCCAAUGU 1373 CCCCGAUG AGAA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCACU GCC CAUCGGGG 1521 AUGACUGC AGAA GCUA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UAGCA GCC GCAGUCAU 1594 CUGUUGUA AGAA GUAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AUACA GAC UACAACAG 2008 ACCCAAUA AGAA GCAA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUGCU GCC UAUUGGGU 2034 UUCUGUAA AGAA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCACA GAC UUACAGAA 2125 GGUCAGUA AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUGCU GUC UACUGACC 2132 GGGUUGGG AGAA GUAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUACU GAC CCCAACCC 2276 ACCUGUAC AGAA GUAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GUACA GUU GUACAGGU 2810 AAGGUCAA AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUGCA GUC UUGACCUU

Table 7

Mouse ICAM Hairpin Ribozyme/Substrate Sequences

nt. Hairpin Ribozyme Sequence Substrate Position

76 GGGAUCAC AGAA GUGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UCACC GUU GUGAUCCC 164 UGAGGAAG AGAA GUUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GAACU GUU CUUCCUCA 252 UCAGCUCA AGAA GCUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AAGCU GUU UGAGCUGA 284 GCACAGCG AGAA GCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAGCA GUC CGCUGUGC 318 AAGCGGAC AGAA GCAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GUGCA GUC GUCCGCUU 447 AGAGCUGG AGAA GCGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCGCG GAC CCAGCUCU 804 UCUCCUGG AGAA GCAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AUGCC GAC CCAGGAGA 847 UCUACCAA AGAA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCACU GCC UUGGUAGA 913 AGGAUCUG AGAA GCUA ACCAGAGLAAACACACGUUGUGGUACAUUACCUGGUA UAGCG GAC CAGAUCCU 946 AAGUUGUA AGAA GUUA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UAACA GUC UACAACUU 1234 CCCAAGCA AGAA GUCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGACG GAC UGCUUGGG 1275 AUUUCAGA AGAA GCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAGCA GAC UCUGAAAU 1325 UGCCUUCC AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUGCA GAC GGAAGGCA 1350 CCCCGAUG AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUGCU GCC CAUCGGGG 1534 ACAUAAGA AGAA GCCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGGCA GCC UCUUAUGU 1851 GUCCACCG AGAA GUAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUACA GCC CGGUGGAC 1880 AGAAUGAA AGAA GCGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACGCU GAC UUCAUUCU

Table 8

Rat ICAM Hairpin Ribozyme/Substrate Sequences

nt. Hairpin Ribozyme Sequence Substrate

Position

5 AAAGUGCA AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUGCU GCC UGCACUUU

59 GGAGCAGA AGAA GCAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AUGCU GCC UCUGCUCC

84 GGGAUCAC AGAA GCGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UCGCC GUU GUGAUCCC

295 GCACAGUG AGAA GCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAGCA GAC CACUGUGC

329 AAGCCGAG AGAA GCGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACGCA GUC CUCGGCUU

433 UUCCACCA AGAA GCGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCGCU GCC UGGUGGAA

626 CAUUCUUG AGAA GUGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UCACU GUU CAAGAAUG

806 UCUCCAGG AGAA GCAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AUGCU GAC CCUGGAGA

849 UCCACUGA AGAA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCACU GCC UCAGUGGA

915 AGGGUCUG AGAA GCCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGGCG GAC CAGACCCU

1182 ACCUCCAA AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUGCG GCC UUGGAGGU

1307 AUGUAAGA AGAA GCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAGCA GAC UCUUACAU

1357 UGCUUUCC AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUGCA GCC GGAAAGCA

1382 UCCCGAUA AGAA GCGG ACCΛGAGΛAACACACGUUGUGGUACAUUACCUGGUA CCGCU GCC UAUCGGGA

1858 GCCCACCA AGAA GUAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUACA GCC UGGUGGGC

1887 AGAAGGAA AGAA GCCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGGCU GAC UUCCUUCU

2012 GAGUUGGG AGAA GUGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACACU GUC CCCAACUC

2303 AGACUCCA AGAA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCACA GCC UGGAGUCU

2539 CCUCCCAC AGAA GCUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AAGCU GUU GUGGGAGG

Table 9: Rat ICAM HH Ribozyme Target Sequence

nt. HH Target sequence nt. HH Target Sequence Position Position

11 GAUCCAAU U CACACUGA 394 GUGGUGCU U CUGAACAG 23 GCUGACUU C CUUCUCUA 420 GCACCCCU C CCAGCGCA 26 GAACUGCU C UUCCUCUU 425 CCUCGGCU U CUGCCACC 31 CCUCUGCU C CUGGUCCU 427 UCCCUGUU U AAAAACCA 34 CUGAAGCU C AGAUAUAC 450 AAGAACCU C AUCCUGCG 40 CUCAAGGU A CAAGCCCC 451 GGGUACUU C CCCCAGGC 48 GAGAACCU C GGCCUGGG 456 CUCGGCUU C UGCCACCA 54 CCCCGCCU C CCUGAGCC 495 GCCACCAU C ACUUUGUA 58 CCGUGCCU U UAGCUCCC 510 GUGCUGCU C CGUGGGAA 64 CAAUGGCU U CAACCCGU 564 GAAAAUGU U CCAACCAC 96 CCUCUGCU C CUGGUCCU 592 GGGAGUAU C ACCAGGGA 102 CUCCUGGU C CUGGUCGC 607 GAGCCAAU U UCUCAUGC

108 GGACUGCU U GGGGAACU 608 AGCCAAUU U CUCAUGCU

115 UCCUACCU U UGUUCCCA 609 GCCAAUUU C UCAUGCUU

119 GACACUGU C CCCAACUC 611 CAAUUUCU C AUGCUUCA

120 GUUGUGAU C CCCGGGCC 656 GUCACUGU U CAAGAAUG 146 CCAGACCU U GGAACUCC 657 UCACUGUU C AAGAAUGC 152 ACCCGGCU C CACCUCAA 668 GAACUGCU C UUCCUCUU 158 AUUUCUUU C ACGAGUCA 677 GCACCCCU C CCAGCGCA 165 UGAACAGU A CUUCCCCC 684 AGGCAGCU C CGGACUUU 168 GAAGCCUU C CUGCCUCG 692 CCAGACCU U GGAACUCC 185 GGGUGGAU C CGUGCAGG 693 CGGACUUU C GAUCUUCC 209 CAGCCCCU A AUCUGACC 696 GCCUGUUU C CUGCCUCU 227 GACCAAGU A ACUGUGAA 709 CAGCAUUU A CCCCUCAC 230 CAAGCUGU U GUGGGAGG 720 CUACAACU U UUCAGCUC 237 CUGAAGCU C GACACCCC 723 CAACUUUU C AGCUCCCA 248 GGCCCCCU A CCUUAGGA 735 CUCCUGGU C CUGGUCGC 253 CACUGCCU C AGUGGAGG 738 UCCUGCCU C GGGGUGGA 263 GAGCCAAU U UCUCAUGC 765 ACUGUGCU U UGAGAACU 267 GAAGCCUU C CUGCCUCG 769 UCUUGUGU U CCCUGGAA 293 GAAGCUCU U CAAGCUGA 770 CUUGUGUU C CCUGGAAG 319 CGGAGGAU C ACAAACGA 785 AGGCCUGU U UCCUGCCU 335 ACUGUGCU U UGAGAACU 786 GGCCUGUU U CCUGCCUC

337 UGUGCUAU A UGGUCCUC 792 CUCCUGGU C CUGGUCGC

338 AAGCUCUU C AAGCUGAG 794 UCCUGCCU C UGAAGCUA 359 CACGCAGU C CUCGGCUU 807 GCUCAGAU A UACCUGGA 367 CAAUGGCU U CAACCCGU 833 CCUGGGGU U GGAGACUA

374 UUACCCCU C ACCCACCU 846 CUGACAGU U AUUUAUUG

375 AGAAGCCU U CCUGCCUC 851 GCUCACCU U UAGCAGCU 378 ACCCACCU C ACAGGGUA 863 CAAUGGCU U CAACCCGU 386 CGCUGUGU U UUGGAGCU 866 CCAUGCUU C CUCUGACA 867 GACCACCU C CCCACCUA 1421 GGGUACUU C CCCCAGGC 869 CUCUUCCU C UUGCGAAG 1425 ACCCACCU C CUCUGGCU 881 AAUGGCUU C AACCCGUG 1429 AUACUUGU A GCCUCAGG 885 GACCAAGU A ACUGUGAA 1444 AGAAGGCU C AGGAGGAG 933 UGUGUAUU C GUUCCCAG 1455 GGGAGUAU C ACCAGGGA 936 GCAGAGAU U UUGUGUCA 1482 AGGGUACU U CCCCCAGG 978 UUGAGAAU C UACAACUU 1484 ACUGCUCU U CCUCUUGC 980 GAGAAUCU A CAACUUUU 1493 CCUGGGGU U GGAGACUA 986 CUACAACU U UUCAGCUC 1500 CG-UGAAAU U AUGGUCAA 987 UACAACUU U UCAGCUCC 1503 GAAAAUGU U CCAACCAC 988 ACAACUUU U CAGCUCCC 1506 UGGGUCAU A AUUGUUGG 1005 UUCGUGAU c GUGGCGUC 1509 GCCACCAU C ACUFUGUA 1006 GUGGGAGU A UCACCAGG 1518 GUCCUGGU C GCCGUUGU 1023 CCGGAGGU C UCAGAAGG 1530 ACCUGGGU C AUAAUUGU 1025 GGAGGUCU C AGAAGGGG 1533 CUGAUCAU U GCGGGCUU 1066 CCUACCUU U GUUCCCAA 1551 GUGGCCCU C UGCUCGUA 1092 AGAGGGGU C UCAGCAGA 1559 UGGGAAGU C CCUGUUUA 1093 AGGGGAAU C CAGCCCCU 1563 UCCUACCU U UGUUCCCA 1125 CCCCAACU C UUGUUGAU 1565 UUACACCU A UUACCGCC 1163 ACGACGCU U CUUUUGCU 1567 ACACCUAU U ACCGCCAG 1164 CGACGCUU C UUUUGCUC 1584 AGGAAGAU C AGGAUAUA 1166 ACGCUUCU U UUGCUCUG 1592 CAGGAUAU A CAAGUUAC 1172 CUUUUGCU C UGCGGCCU 1599 UACAAGUU A CAGAAGGC 1200 AUCCAAUU C ACACUGAA 1651 CCCCGCCU C CCUGAGCC 1201 UUGGGCUU C UCCACAGG 1661 CUGCACUU U GCCCUGGU 1203 GGGCUUCU C CACAGGUC 1663 GAACAGAU C AAUGGACA 1227 UUGGAACU C CAUGUGCU 1678 GAGAACCU C GGCCUGGG 1228 GCGGGCUU C GUGAUCGU 1680 GGGCUUCU C CACAGGUC 1222 CUCCUGGU C CUGGUCGC 1681 GGCCUGUU U CCUGCCUC 1238 UGUGCUAU A UGGUCCUC 1684 CUGCUCGU A GACCUCUC 1264 GGAAAGAU C AUACGGGU 1690 CCCCACCU A CAUACAUU 1267 GUCACUGU U CAAGAAUG 1691 CCGGACUU U CGAUCUUC 1294 CAGAGAUU UGUGUCAG 1696 CUCCUGGU C CUGGUCGC 1295 AGAGGGGU C UCAGCAGA 1698 UCAGAUAU A CCUGGAGA 1306 AGCAGACU C UUACAUGC 1737 GAUCACAU U CACGGUGC 1321 AACAGAGU C UGGGGAAA 1750 GUCCAUUU A CACCUAUU 1334 GUAUUCGU U CCCAGAGC 1756 CCUCUGCU C CUGGUCCU 1344 UCGGUGCU C AGGUAUCC 1787 GAGAACCU C GGCCUGGG 1351 UCAGGCCU A AGAGGACU 1790 GACACUGU C CCCAACUC 1353 UAGCAGCU C AACAAUGG 1793 AUGGUCCU C ACCUGGAC 1366 AGGGUACU CCCCCAGG 1797 UCCCUGUU U AAAAACCA 1367 GGGUACUU C CCCCAGGC 1802 GCUCAGAU A UACCUGGA 1368 GAUGGUGU C CCGCUGCC 1812 AACAGAGU C UGGGGAAA 1380 CUGCCUAU c GGGAUGGU 1813 GCGGGCUU C GUGAUCGU 1388 UGGAGACU A ACUGGAUG 1825 GCCACCAU C ACUGUGUA 1398 CUGGCUGU C ACAGGACA 1837 ACCCACCU C ACAGGGUA 1402 CUGUGCUU U GAGAACUG 1845 AGAGGACU C GGAGGGGC 1408 UUCGUGAU C GUGGCGUC 1856 C CCCCUAAU C UGACCUGC 1410 CGAACUAU C GAGUGGAC 1861 CCAUGUGCU A UAUGGUCC 1865 UAUCCGGU A GACACAAG 2198 GAAUGUCU C CGAGGUCA

1868 UCACGAGU C AUAUAAAU 2199 AGACUCUU A CAUGCCAG

1877 ACAGUACU U CCCCCAGG 2200 GGGUACUU C CCCCAGGC

1901 CUAAAACU C AAGGUACA 2201 GGGCUUCU C CACAGGUC

1912 GAACAGAU C AAUGGACA 2205 UUUUGUGU C AGCCACUG

1922 AUGUAAGU U AUUGCCUA 2210 UGGAGACU A ACUGGAUG

1923 UGGACGCU C ACCUUUAG 2220 GAGAACCU C GGCCUGGG

1928 GCUCAGAU A UACCUGGA 2224 ACAUACAU U CCUACCUU

1930 UGGAGACU A ACUGGAUG 2226 CUGGACCU C AGGCCACA

1964 AGAGAUUU U GUGUCAGC 2233 UCAUGCUU C ACAGAACU

1983 GAGAACCU C GGCCUGGG 2242 ACACAGCU C UCAGUAGU

1996 UGGAAGCU C UUCAAGCU 2248 CUCCUGGU C CUGGUCGC

2005 AUGUAAGU U AUUGCCUA 2254 AUCCAAUU C ACACUGAA

2013 CGCUGCCU A UCGGGAUG 2259 GAUCACAU U CACGGUGC

2015 CUGCCUAU C GGGADΞGU 2260 AUCACAUU C ACGGUGCU

2020 UAUUGAGU A CCCUGUAC 2266 AUCAGGAU A UACAAGUU 2039 CGGAGGAU C ACAAACGA 2274 GAGCAGGU U AACAUGUA 2040 CCUGACCU C CUGGAGGU 2279 GGAAAGAU C AUACGGGU 2057 CUGGUCCU C CAAUGGCU 2282 ACAGUUAU U UAUUGAGU 2061 GCGUCCAU U UACACCUA 2288 GCCCUGGU C CUCCAAUG 2071 AUACUUGU A GCCUCAGG 2291 CAGGAUAU A CAAGUUAC 2076 CGUAGCCU C AGGCCUAA 2321 GGAAAGAU C AUACGGGU 2097 CCAACUCU U GUUGAUGU 2338 UUGGGCUU C UCCACAGG 2098 CCUGACCU C CUGGAGGU 2339 GGGUACUU C CCCCAGGC 2115 UUCCGACU A GGGUCCUG 2341 GGGCCUGU C GGUGCUCA 2128 AGUGCUGU A CCAUGAUC 2344 CUGCUCGU A GACCUCUC 2130 GCCUGUUU C CUGCCUCU 2358 CCCUGCCU C CUCCCACA 2145 CCAACUCU U GUUGAUGU 2359 CCAUCCAU C CCACAGAA 2152 UUGAGAAU C UACAACUU 2360 CUUGUGUU C CCUGGAAG 2156 UGACAGUU A UUUAUUGA 2376 GAACUGCU C UUCCUCUU 2158 UGAUGUAU U UAUUAAUU 2377 GACUUCCU U CUCUAUUA 2159 GAUGUAUU U AUUAAUUC 2378 GCUGAUUU C UUUCACGA 2160 AUGUAUUU A UUAAUUCA 2379 CUGCUCUU C CUCUUGCG 2162 ACAUUCCU A CCUUUGUU 2380 UGAUUUCU U UCACGAGU 2163 UAUUUAUU A AUUCAGAG 2382 AUUUCUUU C ACGAGUCA 2166 UGAUGUAU U UAUUAAUU 2384 UAUCCGGU A GACACAAG 2167 GAUGUAUU U AUUAAUUC 2399 UAAAUACU A UGUGGACG 2170 GUAUUUAU U AAUUCAGA 2401 UGUGCUAU A UGGUCCUC 2171 CAGUUAUU U AUUGAGUA 2411 CAAUUUCU C AUGCUUCA 2173 UGUGCUAU A UGGUCCUC 2417 AUCAGGAU A UACAAGUU 2174 UCUCUAUU A CCCCUGCU 2418 UCAUGCUU C ACAGAACU 2175 AUUUCUUU C ACGAGUCA 2425 UUAUUAAU U CAGAGUUC 2176 GAAAAUGU U CCAACCAC 2426 CCUGGGGU U GGAGACUA 2183 UGACAGUU A UUUAUUGA 2433 UCAGAGUU C UGACAGUU 2185 ACAGUUAU U UAUUGAGU 2434 CGGAGGAU C ACAAACGA2186 CAGUUAUU U AUUGAGUA 2448 UGAACAGU A CUUCCCCC 2187 AGUUAUUU A UUGAGUAC 2449 GAAGCCUU C CUGCCUCG 2189 UUAUUUAU U GAGUACCC 2451 GGCCUGUU U CCUGCCUC2196 CUGACAGU U AUUUAUUG 2452 GCCUGUUU C CUGCCUCU 2455 ACAUUCCU A CCUUUGUU 2761 CGGACUUU C GAUCUUCC

2459 CCCUGCCU C CUCCCACA 2765 CUUUUGCU C UGCGGCCU

2460 CCUACCUU U GUUCCCAA 2769 UUCUCUAU U ACCCCUGC

2479 UUACACCU A UUACCGCC 2797 CGUGAAAU U AUGGUCAA

2480 GUCGCCGU U GUGAUCCC 2803 CUCAUGCU U CACAGAAC

2483 ACCUUUGU U CCCAAUGU 2804 UCAUGCUU C ACAGAACU

2484 CCUUUGUU C CCAAUGUC 2813 GCUCCCAU C CUGACCCU

2492 GACCACCU C CCCACCUA 2815 CGGACUUU C GAUCUUCC

2504 ACCUACAU A CAUUCCUA 2821 CCUGACCU C CUGGAGGU

2508 ACAUACAU U CCUACCUU 2822 UACAACUU U UCAGCUCC

2509 CAUACAUU C CUACCUUU 2823 CAACUUUU C AGCUCCCA

2510 GUCCAUUU A CACCUAUU 2829 UCGGUGCU C AGGUAUCC

2520 ACCUUUGU U CCCAAUGU 2837 CACAGGGU A CUUCCCCC

2521 CCUUUGUU C CCAAUGUC 2840 GCACCCCU C CCAGCGCA

2533 ACAGCAUU U ACCCCUCA 2847 UUACCCCU C ACCCACCU

2540 UCGGUGCU C AGGUAUCC 2853 UUCGAUCU U CCGACUAG

2545 AGGCAGCU C CGGACUUU 2860 UCUUGUGU U CCCUGGAA

2563 CAGAGAUU U UGUGUCAG 2872 GGGCCUGU C GGUGCUCA

2579 CCUGCACU U UGCCCUGG 2877 UGGAGUCU C CCAGCACC

2585 CUGCUCGU A GACCUCUC 2899 AGGCAGCU C CGGACUUU

2588 UGCCUCCU C CCACAGCC 2900 GGCUGACU U CCUUCUCU

2591 CUCUUCCU C UUGCGAAG 2904 GAACUGCU C UUCCUCUU

2593 UCUCUAUU A CCCCUGCU 2905 GGCUGACU U CCUUCUCU

2596 CUCCUGGU C CUGGUCGC 2906 GUUGAUGU A UUUAUUAA

2601 UGUGCUAU A UGGUCCUC 2907 CUGCUCUU C CUCUUGCG

2602 GUCCUGGU C GCCGUUGU 2908 UGAUGUAU U UAUUAAUU

2607 GUGGGAGU A UCACCAGG 2909 GAACUGCU C UUCCUCUU

2608 CUUUAGCU C CCGUGGGA 2910 ACUUCCUU C UCUAUUAC

2609 UGGAGACU A ACUGGAUG 2911 UUCCUUCU C UAUUACCC

2620 UCAGAGUU C UGACAGUU 2912 AUGUAUUU A UUAAUUCA

2626 CUCUCAGU A GUGCUGCU 2913 UGUGUAUU C GUUCCCAG

2628 UACAACUU U UCAGCUCC 2914 GUAUUUAU U AAUUCAGA

2635 UCACAGAU C CAAUUCAC 2915 UAUUUAUU A AUUCAGAG

2640 GCUCAGGU A UCCAUCCA 2916 CUCUUCCU C UUGCGAAG

2641 CCCCACCU A CAUACAUU 2917 CUUCCUCU U GCGAAGAC

2642 GCCUGUUU C CUGCCUCU 2918 AUUUCUUU C ACGAGUCA

2653 CCACAGGU c AGGGUGCU 2919 UUUUGUGU C AGCCACUG

2659 AGAAGGGU c CUGCAAGC 2931 GAUGGUGU C CCGCUGCC

2689 ACUAGGGU c CUGAAGCU 2933 UGGAGUCU C CCAGCACC

2691 UCAGGCCU A AGAGGACU 2941 CAGUACUU C CCCCAGGC

2700 AGGGUACU U CCCCCAGG 2951 ACCAUGCU U CCUCUGAC

2704 GACCACCU C CCCACCUA 2952 CCGGACUU U CGAUCUUC

2711 CCCUACCU U AGGAAGGU 2955 UGCUUCCU C UGACAUGG

2712 CCUACCUU A GGAAGGUG 2956 CUUUCCUU U GAAUCAAU

2721 GGAAAGAU C AUACGGGU 2961 UUUUGUGU C AGCCACUG

2724 AAGAUCAU A CGGGUUUG 2962 UGUGUAUU C GUUCCCAG

2744 GGGUGGAU C CGUGCAGG 2965 CUUUGAAU C AAUAAAGU

2750 GUCCCUGU U UAAAAACC 2966 UGGAAGCU C UUCAAGCU

2759 GACGAACU A UCGAGUGG 2969 GAAUCAAU A AAGUUUUA 2975 UGGAAGCU C UUCAAGCU

2976 UAUAUGGU C CUCACCUG

2977 GAAGCUCU U CAAGCUGA

Table 10: Rat ICAM HH Ribozyme Sequences

nt. Rat HH Ribozyme Sequence

Position

11 UCAGUGUG CUGAUGAGGCCGAAAGGCCGAA AϋUGGAUC

23 UAGAGAAG CUGAUGAGGCCGAAAGGCCGAA AAGUCAGC

26 AAGAGGAA CUGAUGAGGCCGAAAGGCCGAA AGCAGUUC

31 AGGACCAG CUGAUGAGGCCGAAAGGCCGAA AGCAGAGG

34 GUAUAUCU CUGAUGAJGGCCGAAAGGCCGAA AGCϋUCAG

40 GGGGCUUG CUGAUGAGGCCGAAAGGCCGAA ACCUUGAG

48 CCCAGGCC CUGAUGAGGCCGAAAGGCCGAA AGGUUCUC

54 GGCUCAGG CUGAUGAGGCCGAAAGGCCGAA AGGCGGGG

58 GGGAGCUA CUGAUGAGGCCGAAAGGCCGAA AGGCACGG

64 ACGGGUUG CUGAUGAGGCCGAAAGGCCGAA AGCCAϋUG

96 AGGACCAG CUGAUGAGGCCGAAAGGCCGAA AGCAGAGG

102 GCGACCAG CUGAUGAGGCCGAAAGGCCGAA ACCAGGAG

108 AGUUCCCC CUGAUGAGGCCGAAAGGCCGAA AGCAGUCC

115 UGGGAACA CUGAϋGAC^GCCGAAAGGCCGAA AGGUAGGA

119 GAGUUGGG CUGAUGAGGCCGAAAGGCCGAA ACAGUGUC

120 GGCCCGGG CUGAUGAGGCCGAAAGGCCGAA AUCACAAC 146 GGAGUUCC CUGAUGAGGCCGAAAGGCCGAA AGGUCUGG 152 UUGAGGUG CUGAUGAGGCCGAAAGGCCGAA AGCCGGGU 158 UGACUCGU CUGAUGAGGCCGAAAGGCCGAA AAAGAAAU 165 GGGGGAAG CUGAUGAGGCCGAAAGGCCGAA ACUGUUCA 163 CGAGGCAG CUGAUGAGGCCGAAAGGCCGAA AAGGCUUC 185 CCUGCACG CUGAUGAGGCCGAAAGGCCGAA AUCCACCC 209 GGUCAGAU CUGAUGAGGCCGAAAGGCCGAA AGGGGCUG 227 UUCACAGU CUGAUGAGGCCGAAAGGCCGAA ACUUGGUC 230 CCUCCCAC CUGAUGAGGCCGAAAGGCCGAA ACAGCUUG 237 GGGGUGUC CUGAUGAGGCCGAAAGGCCGAA AGCUUCAG 248 UCCUAAGG CUGAUGAGGCCGAAAGGCCGAA AGGGGGCC 253 CCUCCACU CUGAUGAGGCCGAAAGGCCGAA AGGCAGUG 263 GCAUGAGA CUGAUGAGGCCGAAAGGCCGAA AUUGGCUC 267 CGAGGCAG CUGAUGAGGCCGAAAGGCCGAA AAGGCUUC 293 UCAGCUUG CUGAUGAGGCCGAAAGGCCGAA AGAGCUUC 319 UCGUUUGU CUGAUGAGGCCGAAAGGCCGAA AUCCUCCG 335 AGUUCUCA CUGAUGAGGCCGAAAGGCCGAA AGCACAGU

337 GAGGACCA CUGAUGAGGCCGAAAGGCCGAA AUAGCACA

338 CUCAGCUU CUGAUGAGGCCGAAAGGCCGAA AAGAGCUU 359 AAGCCGAG CUGAUGAGGCCGAAAGGCCGAA ACUGCGUG 367 ACGGGUUG CUGAUGAGGCCGAAAGGCCGAA AGCCAUUG

374 AGGUGGGU CUGAUGAGGCCGAAAGGCCGAA AGGGGUAA

375 GAGGCAGG CUGAUGAGGCCGAAAGGCCGAA AGGCUUCU 378 UACCCUGU CUGAUGAGGCCGAAAGGCCGAA AGGUGGGU 386 AGCUCCAA CUGAUGAGGCCGAAAGGCCGAA ACACAGCG 394 CUGUUCAG CUGAUGAGGCCGAAAGGCCGAA AGCACCAC

420 UGCGCUGG CUGAUGAGGCCGAAAGGCCGAA AGGGGUGC

425 GGUGGCAG CUGAUGAGGCCGAAAGGCCGAA AGCCGAGG

427 UGGUUUUU CUGAUGAGGCCGAAAGGCCGAA AACAGGGA

450 CGCAGGAU CUGAUGAGGCCGAAAGGCCGAA AGGUUCUU

451 GCCUGGGG CUGAUGAGGCCGAAAGGCCGAA AAGUACCC 456 UGGUGGCA CUGAUGAGGCCGAAAGGCCGAA AAGCCGAG 495 UACACAGU CUGAUGAGGCCGAAAGGCCGAA AUGGUGGC 510 UUCCCACG CUGAUGAGGCCGAAAGGCCGAA AGCAGCAC 564 GUGGUUGG CUGAUGAGGCCGAAAGGCCGAA ACAUUUUC 592 UCCCUGGU CUGAUGAGGCCGAAAGGCCGAA AUACUCCC

607 GCAUGAGA CUGAUGAGGCCGAAAGGCCGAA AUUGGCUC

608 AGCAUGAG CUGAUGAGGCCGAAAGGCCGAA AAUUGGCU

609 AAGCAUGA CUGAUGAGGCCGAAAGGCCGAA AAAUUGGC 611 UGAAGCAU CUGAUGAGGCCGAAAGGCCGAA AGAAAUUG

656 CAUUCUUG CUGAUGAGGCCGAAAGGCCGAA ACAGUGAC

657 ACAUUCUU CUGAUGAGGCCGAAAGGCCGAA AACAGUGA 668 AAGAGGAA CUGAUGAGGCCGAAAGGCCGAA AGCAGUUC 677 UGCGCUGG CUGAUGAGGCCGAAAGGCCGAA AGGGGUGC 684 AAAGUCCG CUGAUGAGGCCGAAAGGCCGAA AGCUGCCU

692 GGAGUUCC CUGAUGAGGCCGAAAGGCCGAA AGGUCUGG

693 GGAAGAUC CUGAUGAGGCCGAAAGGCCGAA AAAGUCCG 696 AGAGGCAG CUGAUGAGGCCGAAAGGCCGAA AAACAGGC 709 GUGAGGGG CUGAUGAGGCCGAAAGGCCGAA AAAUGCUG 720 GAGCUGAA CUGAUGAGGCCGAAAGGCCGAA AGUUGUAG 723 UGGGAGCU CUGAUGAGGCCGAAAGGCCGAA AAAAGUUG 735 GCGACCAG CUGAUGAGGCCGAAAGGCCGAA ACCAGGAG 738 UCCACCCC CUGAUGAGGCCGAAAGGCCGAA AGGCAGGA 765 AGUUCUCA CUGAUGAGGCCGAAAGGCCGAA AGCACAGU

769 UUCCAGGG CUGAUGAGGCCGAAAGGCCGAA ACACAAGA

770 CUUCCAGG CUGAUGAGGCCGAAAGGCCGAA AACACAAG

785 AGGCAGGA CUGAUGAGGCCGAAAGGCCGAA ACAGGCCU

786 GAGGCAGG CUGAUGAGGCCGAAAGGCCGAA AACAGGCC 792 GCGACCAG CUGAUGAGGCCGAAAGGCCGAA ACCAGGAG 794 GAGCUUCA CUGAUGAGGCCGAAAGGCCGAA AGGCAGGA 807 UCCAGGUA CUGAUGAGGCCGAAAGGCCGAA AUCUGAGC 833 UAGUCUCC CUGAUGAGGCCGAAAGGCCGAA ACCCCAGG 846 CAAUAAAU CUGAUGAGGCCGAAAGGCCGAA ACUGUCAG 851 AGCUGCUA CUGAUGAGGCCGAAAGGCCGAA AGGUGAGC 863 ACGGGUUG CUGAUGAGGCCGAAAGGCCGAA AGCCAUUG

866 UGUCAGAG CUGAUGAGGCCGAAAGGCCGAA AAGCAUGG

867 UAGGUGGG CUGAUGAGGCCGAAAGGCCGAA AGGUGGUC 869 CUUCGCAA CUGAUGAGGCCGAAAGGCCGAA AGGAAGAG 881 CACGGGUU CUGAUGAGGCCGAAAGGCCGAA AAGCCAUU 885 UUCACAGU CUGAUGAGGCCGAAAGGCCGAA ACUUGGUC 933 CUGGGAAC CUGAUGAGGCCGAAAGGCCGAA AAUACACA 936 UGACACAA CUGAUGAGGCCGAAAGGCCGAA AUCUCUGC 978 AAGUUGUA CUGAUGAGGCCGAAAGGCCGAA AUUCUCAA 980 AAAAGUUG CUGAUGAGGCCGAAAGGCCGAA AGAUUCUC 986 GAGCUGAA CUGAUGAGGCCGAAAGGCCGAA AGUUGUAG

987 GGAGCUGA CUGAUGAGGCCGAAAGGCCGAA AAGUUGUA

988 GGGAGCUG CUGAUGAGGCCGAAAGGCCGAA AAAGUUGU

1005 GACGCCAC CUGAUGAGGCCGAAAGGCCGAA AUCACGAA

1006 CCUGGUGA CUGAUGAGGCCGAAAGGCCGAA ACUCCCAC 1023 CCUUCUGA CUGAUGAGGCCGAAAGGCCGAA ACCUCCGG 1025 CCCCUUCU CUGAUGAGGCCGAAAGGCCGAA AGACCUCC 1066 UUGGGAAC CUGAUGAGGCCGAAAGGCCGAA AAGGUAGG

1092 UCUGCUGA CUGAUGAGGCCGAAAGGCCGAA ACCCCUCU

1093 AGGGGCUG CUGAUGAGGCCGAAAGGCCGAA AUUCCCCU 1125 AUCAACAA CUGAUGAGGCCGAAAGGCCGAA AGUUGGGG 1153 AGCAAAAG CUGAUGAGGCCGAAAGGCCGAA AGCGUCGU 1164 GAGCAAAA CUGAUGAGGCCGAAAGGCCGAA AAGCGUCG 1166 CAGAGCAA CUGAUGAGGCCGAAAGGCCGAA AGAAGCGU 1172 AGGCCGCA CUGAUGAGGCCGAAAGGCCGAA AGCAAAAG

1200 UUCAGUGU CUGAUGAGGCCGAAAGGCCGAA AAUUGGAU

1201 CCUGUGGA CUGAUGAGGCCGAAAGGCCGAA AAGCCCAA 1203 GACCUGUG CUGAUGAGGCCGAAAGGCCGAA AGAAGCCC

1227 AGCACAUG CUGAUGAGGCCGAAAGGCCGAA AGUUCCAA

1228 ACGAUCAC CUGAUGAGGCCGAAAGGCCGAA AAGCCCGC 1233 GCGACCAG CUGAUGAGGCCGAAAGGCCGAA ACCAGGAG 1238 GAGGACCA CUGAUGAGGCCGAAAGGCCGAA AUAGCACA 1264 ACCCGUAU CUGAUGAGGCCGAAAGGCCGAA AUCUUUCC 1267 CAUUCUUG CUGAUGAGGCCGAAAGGCCGAA ACAGUGAC

1294 CUGACACA CUGAUGAGGCCGAAAGGCCGAA AAUCUCUG

1295 UCUGCUGA CUGAUGAGGCCGAAAGGCCGAA ACCCCUCU 1306 GCAUGUAA CUGAUGAGGCCGAAAGGCCGAA AGUCUGCU 1321 UUUCCCCA CUGAUGAGGCCGAAAGGCCGAA ACUCUGUU 1334 GCUCUGGG CUGAUGAGGCCGAAAGGCCGAA ACGAAUAC 1344 GGAUACCU CUGAUGAGGCCGAAAGGCCGAA AGCACCGA 1351 AGUCCUCU CUGAUGAGGCCGAAAGGCCGAA AGGCCUGA 1353 CCAUUGUU CUGAUGAGGCCGAAAGGCCGAA AGCUGCUA

1366 CCUGGGGG CUGAUGAGGCCGAAAGGCCGAA AGUACCCU

1367 GCCUGGGG CUGAUGAGGCCGAAAGGCCGAA AAGUACCC 1363 GGCAGCGG CUGAUGAGGCCGAAAGGCCGAA ACACCAUC 1380 ACCAUCCC CUGAUGAGGCCGAAAGGCCGAA AUAGGCAG 1388 CAUCCAGU CUGAUGAGGCCGAAAGGCCGAA AGUCUCCA 1398 UGUCCUGU CUGAUGAGGCCGAAAGGCCGAA ACAGCCAG 1402 CAGUUCUC CUGAUGAGGCCGAAAGGCCGAA AAGCACAG 1408 GACGCCAC CUGAUGAGGCCGAAAGGCCGAA AUCACGAA 1410 GUCCACUC CUGAUGAGGCCGAAAGGCCGAA AUAGUUCG 1421 GCCUGGGG CUGAUGAGGCCGAAAGGCCGAA AAGUACCC 1425 AGCCAGAG CUGAUGAGGCCGAAAGGCCGAA .AGGUGGGU 1429 CCUGAGGC CUGAUGAGGCCGAAAGGCCGAA ACAAGUAU 1444 CUCCUCCU CUGAUGAGGCCGAAAGGCCGAA AGCCUUCU 1455 UCCCUGGU CUGAUGAGGCCGAAAGGCCGAA AUACUCCC 1482 CCUGGGGG CUGAUGAGGCCGAAAGGCCGAA AGUACCCU 1484 GCAAGAGG CUGAUGAGGCCGAAAGGCCGAA AGAGCAGU 1493 UAGUCUCC CUGAUGAGGCCGAAAGGCCGAA ACCCCAGG 1500 UUGACCAU CUGAUGAGGCCGAAAGGCCGAA AUUUCACG

1503 GUGGUUGG CUGAUGAGGCCGAAAGGCCGAA ACAUUUUC

1506 CCAACAAU CUGAUGAGGCCGAAAGGCCGAA AUGACCCA

1509 UACACAGU CUGAUGAGGCCGAAAGGCCGAA AUGGUGGC

1518 ACAACGGC CUGAUGAGGCCGAAAGGCCGAA ACCAGGAC

1530 ACAAUUAU CUGAUGAGGCCGAAAGGCCGAA ACCCAGGU

1533 AAGCCCGC CUGAUGAGGCCGAAAGGCCGAA AUGAUCAG

1551 UACGAGCA CUGAUGAGGCCGAAAGGCCGAA AGGGCCAC

1559 UAAACAGG CUGAUGAGGCCGAAAGGCCGAA ACUUCCCA

1563 UGGGAACA CUGAUGAGGCCGAAAGGCCGAA AGGUAGGA

1565 GGCGGUAA CUGAUGAGGCCGAAAGGCCGAA AGGUGUAA

1567 CUGGCGGU CUGAUGAGGCCGAAAGGCCGAA AUAGGUGU

1584 UAUAUCCU CUGAUGAGGCCGAAAGGCCGAA AUCUUCCU

1592 GUAACUUG CUGAUGAGGCCGAAAGGCCGAA AUAUCCUG

1599 GCCUUCUG CUGAUGAGGCCGAAAGGCCGAA AACUUGUA

1651 GGCUCAGG CUGAUGAGGCCGAAAGGCCGAA AGGCGGGG

1661 ACCAGGGC CUGAUGAGGCCGAAAGGCCGAA AAGUGCAG

1663 UGUCCAUU CUGAUGAGGCCGAAAGGCCGAA AUCUGUUC

1678 CCCAGGCC CUGAUGAGGCCGAAAGGCCGAA AGGUUCUC

1680 GACCUGUG CUGAUGAGGCCGAAAGGCCGAA AGAAGCCC

1681 GAGGCAGG CUGAUGAGGCCGAAAGGCCGAA AACAGGCC

1684 GAGAGGUC CUGAUGAGGCCGAAAGGCCGAA ACGAGCAG

1690 AAUGUAUG CUGAUGAGGCCGAAAGGCCGAA AGGUGGGG

1691 GAAGAUCG CUGAUGAGGCCGAAAGGCCGAA AAGUCCGG

1696 GCGACCAG CUGAUGAGGCCGAAAGGCCGAA ACCAGGAG

1698 UCUCCAGG CUGAUGAGGCCGAAAGGCCGAA AUAUCUGA

1737 GCACCGUG CUGAUGAGGCCGAAAGGCCGAA AUGUGAUC

1750 AAUAGGUG CUGAUGAGGCCGAAAGGCCGAA AAAUGGAC

1756 AGGACCAG CUGAUGAGGCCGAAAGGCCGAA AGCAGAGG

1787 CCCAGGCC CUGAUGAGGCCGAAAGGCCGAA AGGUUCUC

1790 GAGUUGGG CUGAUGAGGCCGAAAGGCCGAA ACAGUGUC

1793 GUCCAGGU CUGAUGAGGCCGAAAGGCCGAA AGGACCAU

1797 UGGUUUUU CUGAUGAGGCCGAAAGGCCGAA AACAGGGA

1802 UCCAGGUA CUGAUGAGGCCGAAAGGCCGAA AUCUGAGC

1812 UUUCCCCA CUGAUGAGGCCGAAAGGCCGAA ACUCUGUU

1813 ACGAUCAC CUGAUGAGGCCGAAAGGCCGAA AAGCCCGC

1825 UACACAGU CUGAUGAGGCCGAAAGGCCGAA AUGGUGGC

1837 UACCCUGU CUGAUGAGGCCGAAAGGCCGAA AGGUGGGU

1845 GCCCCUCC CUGAUGAGGCCGAAAGGCCGAA AGUCCUCU

1856 GCAGGUCA CUGAUGAGGCCGAAAGGCCGAA AUUAGGGG

1861 GGACCAUA CUGAUGAGGCCGAAAGGCCGAA AGCACAUG

1865 CUUGUGUC CUGAUGAGGCCGAAAGGCCGAA ACCGGAUA

1868 AUUUAUAU CUGAUGAGGCCGAAAGGCCGAA ACUCGUGA

1877 CCUGGGGG CUGAUGAGGCCGAAAGGCCGAA AGUACUGU

1901 UGUACCUU CUGAUGAGGCCGAAAGGCCGAA AGUUUUAG

1912 UGUCCAUU CUGAUGAGGCCGAAAGGCCGAA AUCUGUUC

1922 UAGGCAAU CUGAUGAGGCCGAAAGGCCGAA ACUUACAU

1923 CUAAAGGU CUGAUGAGGCCGAAAGGCCGAA AGCGUCCA

1928 UCCAGGUA CUGAUGAGGCCGAAAGGCCGAA AUCUGAGC 1930 CAUCCAGU CUGAUGAGGCCGAAAGGCCGAA AGUCUCCA

1964 GCUGACAC CUGAUGAGGCCGAAAGGCCGAA AAAUCUCU

1983 CCCAGGCC CUGAUGAGGCCGAAAGGCCGAA AGGUUCUC

1996 AGCUUGAA CUGAUGAGGCCGAAAGGCCGAA AGCUUCCA

2005 UAGGCAAU CUGAUGAGGCCGAAAGGCCGAA ACUUACAU

2013 CAUCCCGA CUGAUGAGGCCGAAAGGCCGAA AGGCAGCG

2015 ACCAUCCC CUGAUGAGGCCGAAAGGCCGAA AUAGGCAG

2020 GUACAGGG CUGAUGAGGCCGAAAGGCCGAA ACUCAAUA

2039 UCGUUUGU CUGAUGAGGCCGAAAGGCCGAA AUCCUCCG

2040 ACCUCCAG CUGAUGAGGCCGAAAGGCCGAA AGGUCAGG

2057 AGCCAUUG CUGAUGAGGCCGAAAGGCCGAA AGGACCAG

2061 UAGGUGUA CUGAUGAGGCCGAAAGGCCGAA AUGGACGC

2071 CCUGAGGC CUGAUGAGGCCGAAAGGCCGAA ACAAGUAU

2076 UUAGGCCU CUGAUGAGGCCGAAAGGCCGAA AGGCUACA

2097 ACAUCAAC CUGAUGAGGCCGAAAGGCCGAA AGAGUUGG

2098 ACCUCCAG CUGAUGAGGCCGAAAGGCCGAA AGGUCAGG

2115 CAGGACCC CUGAUGAGGCCGAAAGGCCGAA AGUCGGAA

2128 GAUCAUGG CUGAUGAGGCCGAAAGGCCGAA ACAGCACU

2130 AGAGGCAG CUGAUGAGGCCGAAAGGCCGAA AAACAGGC

2145 ACAUCAAC CUGAUGAGGCCGAAAGGCCGAA AGAGUUGG

2152 AAGUUGUA CUGAUGAGGCCGAAAGGCCGAA AUUCUCAA

2156 UCAAUAAA CUGAUGAGGCCGAAAGGCCGAA AACUGUCA

2158 AAUUAAUA CUGAUGAGGCCGAAAGGCCGAA AUACAUCA

2159 GAAUUAAU CUGAUGAGGCCGAAAGGCCGAA AAUACAUC

2150 UGAAUUAA CUGAUGAGGCCGAAAGGCCGAA AAAUACAU

2162 AACAAAGG CUGAUGAGGCCGAAAGGCCGAA AGGAAUGU

2153 CUCUGAAU CUGAUGAGGCCGAAAGGCCGAA AAUAAAUA

2166 AAUUAAUA CUGAUGAGGCCGAAAGGCCGAA AUACAUCA

2167 GAAUUAAU CUGAUGAGGCCGAAAGGCCGAA AAUACAUC

2170 UCUGAAUU CUGAUGAGGCCGAAAGGCCGAA AUAAAUAC

2171 UACUCAAU CUGAUGAGGCCGAAAGGCCGAA AAUAACUG

2173 GAGGACCA CUGAUGAGGCCGAAAGGCCGAA AUAGCACA

2174 AGCAGGGG CUGAUGAGGCCGAAAGGCCGAA AAUAGAGA

2175 UGACUCGU CUGAUGAGGCCGAAAGGCCGAA AAAGAAAU

2176 GUGGUUGG CUGAUGAGGCCGAAAGGCCGAA ACAUUUUC

2183 UCAAUAAA CUGAUGAGGCCGAAAGGCCGAA AACUGUCA

2185 ACUCAAUA CUGAUGAGGCCGAAAGGCCGAA AUAACUGU

2186 UACUCAAU CUGAUGAGGCCGAAAGGCCGAA AAUAACUG

2187 GUACUCAA CUGAUGAGGCCGAAAGGCCGAA AAAUAACu

2189 GGGUACUC CUGAUGAGGCCGAAAGGCCGAA AUAAAUAA

2196 CAAUAAAU CUGAUGAGGCCGAAAGGCCGAA ACUGUCAG

2198 UGACCUCG CUGAUGAGGCCGAAAGGCCGAA AGACAUUC

2199 CUGGCAUG CUGAUGAGGCCGAAAGGCCGAA AAGAGUCU

2200 GCCUGGGG CUGAUGAGGCCGAAAGGCCGAA AAGUACCC

2201 GACCUGUG CUGAUGAGGCCGAAAGGCCGAA AGAAGCCC

2205 CAGUGGCU CUGAUGAGGCCGAAAGGCCGAA ACACAAAA

2210 CAUCCAGU CUGAUGAGGCCGAAAGGCCGAA AGUCUCCA

2220 CCCAGGCC CUGAUGAGGCCGAAAGGCCGAA AGGUUCUC

2224 AAGGUAGG CUGAUGAGGCCGAAAGGCCGAA AUGUAUGU 2226 UGUGGCCU CUGAUGAGGCCGAAAGGCCGAA AGGUCCAG

2233 AGUUCUGU CUGAUGAGGCCGAAAGGCCGAA AAGCAUGA

2242 ACUACUGA CUGAUGAGGCCGAAAGGCCGAA AGCUGUGU

2248 GCGACCAG CUGAUGAGGCCGAAAGGCCGAA ACCAGGAG

2254 UUCAGUGU CUGAUGAGGCCGAAAGGCCGAA AAUUGGAU

2259 GCACCGUG CUGAUGAGGCCGAAAGGCCGAA AUGUGAUC

2260 AGCACCGU CUGAUGAGGCCGAAAGGCCGAA AAUGUGAU

2266 AACUUGUA CUGAUGAGGCCGAAAGGCCGAA AUCCUGAU

2274 UACAUGUU CUGAUGAC4GCCGAAAGGCCGAA ACCUGCUC

2279 ACCCGUAU CUGAUGAGGCCGAAAGGCCGAA AUCUUUCC

2282 ACUCAAUA CUGAUGAGGCCGAAAGGCCGAA AUAACUGU

2288 CAUUGGAG CUGAUGAGGCCGAAAGGCCGAA ACCAGGGC

2291 GUAACUUG CUGAUGAGGCCGAAAGGCCGAA AUAUCCUG

2321 ACCCGUAU CUGAUGAGGCCGAAAGGCCGAA AUCUUUCC

2338 CCUGUGGA CUGAUGAGGCCGAAAGGCCGAA AAGCCCAA

2339 GCCUGGGG CUGAUGAGGCCGAAAGGCCGAA AAGUACCC

2341 UGAGCACC CUGAUGAGGCCGAAAGGCCGAA ACAGGCCC

2344 GAGAGGUC CUGAUGAGGCCGAAAGGCCGAA ACGAGCAG

2358 UGUGGGAG CUGAUGAGGCCGAAAGGCCGAA AGGCAGGG

2359 UUCUGUGG CUGAUGAGGCCGAAAGGCCGAA AUGGAUGG

2360 CUUCCAGG CUGAUGAGGCCGAAAGGCCGAA AACACAAG

2376 AAGAGGAA CUGAUGAGGCCGAAAGGCCGAA AGCAGUUC

2377 UAAUAGAG CUGAUGAGGCCGAAAGGCCGAA AGGAAGUC

2378 UCGUGAAA CUGAUGAGGCCGAAAGGCCGAA AAAUCAGC

2379 CGCAAGAG CUGAUGAGGCCGAAAGGCCGAA AAGAGCAG

2380 ACUCGUGA CUGAUGAGGCCGAAAGGCCGAA ACAAAUCA

2382 UGACUCGU CUGAUGAGGCCGAAAGGCCGAA AAAGAAAU

2384 CUUGUGUC CUGAUGAGGCCGAAAGGCCGAA ACCGGAUA

2399 CGUCCACA CUGAUGAGGCCGAAAGGCCGAA AGUAUUUA

2401 GAGGACCA CUGAUGAGGCCGAAAGGCCGAA AUAGCACA

2411 UGAAGCAU CUGAUGAGGCCGAAAGGCCGAA AGAAAUUG

2417 AACUUGUA CUGAUGAGGCCGAAAGGCCGAA AUCCUGAU

2418 AGUUCUGU CUGAUGAGGCCGAAAGGCCGAA AAGCAUGA

2425 GAACUCUG CUGAUGAGGCCGAAAGGCCGAA AUUAAUAA

2426 UAGUCUCC CUGAUGAGGCCGAAAGGCCGAA ACCCCAGG

2433 AACUGUCA CUGAUGAGGCCGAAAGGCCGAA AACUCUGA

2434 UCGUUUGU CUGAUGAGGCCGAAAGGCCGAA AUCCUCCG

2448 GGGGGAAG CUGAUGAGGCCGAAAGGCCGAA ACUGUUCA

2449 CGAGGCAG CUGAUGAGGCCGAAAGGCCGAA AAGGCUUC

2451 GAGGCAGG CUGAUGAGGCCGAAAGGCCGAA AACAGGCC

2452 AGAGGCAG CUGAUGAGGCCGAAAGGCCGAA AAACAGGC

2455 AACAAAGG CUGAUGAGGCCGAAAGGCCGAA AGGAAUGU

2459 UGUGGGAG CUGAUGAGGCCGAAAGGCCGAA AGGCAGGG

2460 UUGGGAAC CUGAUGAGGCCGAAAGGCCGAA AAGGUAGG

2479 GGCGGUAA CUGAUGAGGCCGAAAGGCCGAA AGGUGUAA

2480 GGGAUCAC CUGAUGAGGCCGAAAGGCCGAA ACGGCGAC

2483 ACAUUGGG CUGAUGAGGCCGAAAGGCCGAA ACAAAGGU

2484 GACAUUGG CUGAUGAGGCCGAAAGGCCGAA AACAAAGG

2492 UAGGUGGG CUGAUGAGGCCGAAAGGCCGAA A.GGUGGUC 2504 UAGGAAUG CUGAUGAGGCCGAAAGGCCGAA AUGUAGGU

2508 AAGGUAGG CUGAUGAGGCCGAAAGGCCGAA AUGUAUGU

2509 AAAGGUAG CUGAUGAGGCCGAAAGGCCGAA AAUGUAUG

2510 AAUAGGUG CUGAUGAGGCCGAAAGGCCGAA AAAUGGAC

2520 ACAUUGGG CUGAUGAGGCCGAAAGGCCGAA ACAAAGGU

2521 GACAUUGG CUGAUGAGGCCGAAAGGCCGAA AACAAAGG

2533 UGAGGGGU CUGAUGAGGCCGAAAGGCCGAA AAUGCUGU

2540 GGAUACCU CUGAUGAGGCCGAAAGGCCGAA AGCACCGA

2545 AAAGUCCG CUGAUGAGGCCUAAAGGCCGAA AGCUGCCU

2568 CUGACACA CUGAUGAGGCCGAAAGGCCGAA AAUCUCUG

2579 CCAGGGCA CUGAUGAGGCCGAAAGGCCGAA AGUGCAGG

2585 GAGAGGUC CUGAUGAGGCCGAAAGGCCGAA ACGAGCAG

2588 GGCUGUGG CUGAUGAGGCCGAAAGGCCGAA AGGAGGCA

2591 CUUCGCAA CUGAUGAGGCCGAAAGGCCGAA AGGAAGAG

2593 AGCAGGGG CUGAUGAGGCCGAAAGGCCGAA AAUAGAGA

2596 GCGACCAG CUGAUGAGGCCGAAAGGCCGAA ACCAGGAG

2601 GAGGACCA CUGAUGAGGCCGAAAGGCCGAA AUAGCACA

2602 ACAACGGC CUGAUGAGGCCGAAAGGCCGAA ACCAGGAC

2607 CCUGGUGA CUGAUGAGGCCGAAAGGCCGAA ACUCCCAC

2608 UCCCACGG CUGAUGAGGCCGAAAGGCCGAA AGCUAAAG

2609 CAUCCAGU CUGAUGAGGCCGAAAGGCCGAA AGUCUCCA

2620 AACUGUCA CUGAUGAGGCCGAAAGGCCGAA AACUCUGA

2626 AGCAGCAC CUGAUGAGGCCGAAAGGCCGAA ACUGAGAG

2628 GGAGCUGA CUGAUGAGGCCGAAAGGCCGAA AAGUUGUA

2635 GUGAAUUG CUGAUGAGGCCGAAAGGCCGAA AUCUGUGA

2640 UGGAUGGA CUGAUGAGGCCGAAAGGCCGAA ACCUGAGC

2641 AAUGUAUG CUGAUGAGGCCGAAAGGCCGAA AGGUGGGG

2642 AGAGGCAG CUGAUGAGGCCGAAAGGCCGAA AAACAGGC

2653 AGCACCCU CUGAUGAGGCCGAAAGGCCGAA ACCUGUGG

2659 GCUUGCAG CUGAUGAGGCCGAAAGGCCGAA ACCCUUCU

2689 AGCUUCAG CUGAUGAGGCCGAAAGGCCGAA ACCCUAGU

2691 AGUCCUCU CUGAUGAGGCCGAAAGGCCGAA AGGCCUGA

2700 CCUGGGGG CUGAUGAGGCCGAAAGGCCGAA AGUACCCU

2704 UAGGUGGG CUGAUGAGGCCGAAAGGCCGAA AGGUGGUC

2711 ACCUUCCU CUGAUGAGGCCGAAAGGCCGAA AGGUAGGG

2712 CACCUUCC CUGAUGAGGCCGAAAGGCCGAA AAGGUAGG

2721 ACCCGUAU CUGAUGAGGCCGAAAGGCCGAA AUCUUUCC

2724 CAAACCCG CUGAUGAGGCCGAAAGGCCGAA AUGAUCUU

2744 CCUGCACG CUGAUGAGGCCGAAAGGCCGAA AUCCACCC

2750 GGUUUUUA CUGAUGAGGCCGAAAGGCCGAA ACAGGGAC

2759 CCACUCGA CUGAUGAGGCCGAAAGGCCGAA AGUUCGUC

2761 GGAAGAUC CUGAUGAGGCCGAAAGGCCGAA AAAGUCCG

2765 AGGCCGCA CUGAUGAGGCCGAAAGGCCGAA AGCAAAAG

2769 GCAGGGGU CUGAUGAGGCCGAAAGGCCGAA AUAGAGAA

2797 UUGACCAU CUGAUGAGGCCGAAAGGCCGAA AUUUCACG

2803 GUUCUGUG CUGAUGAGGCCGAAAGGCCGAA AGCAUGAG

2804 AGUUCUGU CUGAUGAGGCCGAAAGGCCGAA AAGCAUGA

2813 AGGGUCAG CUGAUGAGGCCGAAAGGCCGAA AUGGGAGC

2815 GGAAGAUC CUGAUGAGGCCGAAAGGCCGAA AAAGUCCG 2821 ACCUCCAG CUGAUGAGGCCGAAAGGCCGAA AGGUCAGG 2822 GGAGCUGA CUGAUGAGGCCGAAAGGCCGAA AAGUUGUA 2823 UGGGAGCU CUGAUGAGGCCGAAAGGCCGAA AAAAGUUG 2829 GGAUACCU CUGAUGAGGCCGAAAGGCCGAA AGCACCGA 2837 GGGGGAAG CUGAUGAGGCCGAAAGGCCGAA ACCCUGUG 2840 UGCGCUGG CUGAUGAGGCCGAAAGGCCGAA AGGGGUGC 2847 AGGUGGGU CUGAUGAGGCCGAAAGGCCGAA AGGGGUAA 2853 CUAGUCGG CUGAUGAGGCCGAAAGGCCGAA AGAUCGAA 2860 UUCCAGGG CUGAUGAGGCCGAAAGGCCGAA ACACAAGA 2372 UGAGCACC CUGAUGAGGCCGAAAGGCCGAA ACAGGCCC 2877 GGUGCUGG CUGAUGAGGCCGAAAGGCCGAA AGACUCCA 2899 AAAGUCCG CUGAUGAGGCCGAAAGGCCGAA AGCUGCCU 2900 AGAGAAGG CUGAUGAGGCCGAAAGGCCGAA AGUCAGCC 2904 AAGAGGAA CUGAUGAGGCCGAAAGGCCGAA AGCAGUUC 2905 AGAGAAGG CUGAUGAGGCCGAAAGGCCGAA AGUCAGCC 2906 UUAAUAAA CUGAUGAGGCCGAAAGGCCGAA ACAUCAAC 2907 CGCAAGAG CUGAUGAGGCCGAAAGGCCGAA AAGAGCAG 2908 AAUUAAUA CUGAUGAGGCCGAAAGGCCGAA AUACAUCA 2909 AAGAGGAA CUGAUGAGGCCGAAAGGCCGAA AGCAGUUC 2910 GUAAUAGA CUGAUGAGGCCGAAAGGCCGAA AAGGAAGU 2911 GGGUAAUA CUGAUGAGGCCGAAAGGCCGAA AGAAGGAA 2912 UGAAUUAA CUGAUGAGGCCGAAAGGCCGAA AAAUACAU 2913 CUGGGAAC CUGAUGAGGCCGAAAGGCCGAA AAUACACA 2914 UCUGAAUU CUGAUGAGGCCGAAAGGCCGAA AUAAAUAC

2915 CUCUGAAU CUGAUGAGGCCGAAAGGCCGAA AAUAAAUA 2916 CUUCGCAA CUGAUGAGGCCGAAAGGCCGAA AGGAAGAG 2917 GUCUUCGC CUGAUGAGGCCGAAAGGCCGAA AGAGGAAG 2913 UGACUCGU CUGAUGAGGCCGAAAGGCCGAA AAAGAAAU 2919 CAGUGGCU CUGAUGAGGCCGAAAGGCCGAA ACACAAAA 2931 GGCAGCGG CUGAUGAGGCCGAAAGGCCGAA ACACCAUC 2933 GGUGCUGG CUGAUGAGGCCGAAAGGCCGAA AGACUCCA 2941 GCCUGGGG CUGAUGAGGCCGAAAGGCCGAA AAGUACUG 2951 GUCAGAGG CUGAUGAGGCCGAAAGGCCGAA AGCAUGGU 2952 GAAGAUCG CUGAUGAGGCCGAAAGGCCGAA AAGUCCGG 2955 CCAUGUCA CUGAUGAGGCCGAAAGGCCGAA AGGAAGCA 2956 AUUGAUUC CUGAUGAGGCCGAAAGGCCGAA AAGGAAAG 2961 CAGUGGCU CUGAUGAGGCCGAAAGGCCGAA ACACAAAA 2962 CUGGGAAC CUGAUGAGGCCGAAAGGCCGAA AAUACACA 2965 ACUUUAUU CUGAUGAGGCCGAAAGGCCGAA AUUCAAAG 2966 AGCUUGAA CUGAUGAGGCCGAAAGGCCGAA AGCUUCCA 2969 UAAAACUU CUGAUGAGGCCGAAAGGCCGAA AUUGAUUC 2975 AGCUUGAA CUGAUGAGGCCGAAAGGCCGAA AGCUUCCA 2975 CAGGUGAG CUGAUGAGGCCGAAAGGCCGAA ACCAUAUA 2977 UCAGCUUG CUGAUGAGGCCGAAAGGCCGAA AGAGCUUC Table 11: Human IL-5 HH Target Sequence

nt. HH Target Sequence nt. HH Target Sequence Position Position

8 AUGCACU U UCUUUGC 245 AAGAAAU C UUUCAGG

9 UGCACUU U CUUUGCC 247 GAAAUCU U UCAGGGA

10 GCACUUU C UUUGCCA 248 AAAUCUU U CAGGGAA

12 ACUUUCU U UGCCAAA 249 AAUCUUU C AGGGAAU

13 CUUUCUU U GCCAAAG 257 AGGGAAU A GGCACAC

36 AGAACGU U UCAGAGC 273 GGAGAGU C AAACUGU

37 GAACGUU U CAGAGCC 291 AGGGGGU A CUGUGGA

38 AACGUUU C AGAGCCA 305 AAAGACU A UUCAAAA

56 GGAUGCU U CUGCAUU 307 AGACUAU U CAAAAAC

57 GAUGCUU C UGCAUUU 308 GACUAUU C AAAAACU

63 UCUGCAU U UGAGUUU 316 AAAAACU U GUCCUUA

64 CUGCAUU U GAGUUUG 319 AACUUGU C CUUAAUA

69 UUUGAGU U UGCUAGC 322 UUGUCCU U AAUAAAG

70 UUGAGUU U GCUAGCU 323 UGUCCUU A AUAAAGA

74 GUUUGCU A GCUCUUG 326 CCUUAAU A AAGAAAU

78 GCUAGCU C UUGGAGC 334 AAGAAAU A CAUUGAC

80 UAGCUCU U GGAGCUG 338 AAUACAU U GACGGCC

91 GCUGCCU A CGUGUAU 380 GGAGAGU A AACCAAU

97 UACGUGU A UGCCAUC 388 AACCAAU U CCUAGAC

104 AUGCCAU C CCCACAG 389 ACCAAUU C CUAGACU

116 CAGAAAU U CCCACAA 392 AAUUCCU A GACUACC

117 AGAAAUU C CCACAAG 397 CUAGACU A CCUGCAA

130 AGUGCAU U GGUGAAA 409 CAAGAGU U UCUUGGU

145 GAGACCU U GGCACUG 410 AAGAGUU U CUUGGUG

155 CACUGCU U UCUACUC 411 AGAGUUU C UUGGUGU

156 ACUGCUU U CUACUCA 413 AGUUUCU U GGUGUAA

157 CUGCUUU C UACUCAU 419 UUGGUGU A AUGAACA

159 GCUUUCU A CUCAUCG 437 AGUGGAU A AUAGAAA

162 UUCUACU C AUCGAAC 440 GGAUAAU A GAAAGUU

165 UACUCAU C GAACU CU 447 AGAAAGU U GAGACUA

171 UCGAACU C UGCUGAU 454 UGAGACU A AACUGGU

179 UGCUGAU A GCCAAUG 462 AACUGGU U UGUUGCA

192 UGAGACU C UGAGGAU 463 ACUGGUU U GUUGCAG

200 UGAGGAU U CCUGUUC 466 GGUUUGU U GCAGCCA

201 GAGGAUU C CUGUUCC 479 CAAAGAU U UUGGAGG

206 UUCCUGU U CCUGUAC 480 AAAGAUU U UGGAGGA

207 UCCUGUU C CUGUACA 481 AAGAUUU U GGAGGAG

212 UUCCUGU A CAUAAAA 497 AGGACAU U UUACUGC

216 UGUACAU A AAAAUCA 498 GGACAUU U UACUGCA

222 UAAAAAU C ACCAACU 499 GACAUUU U ACUGCAG 500 ACAUUUU A CUGCAGU 684 UACUUUU U UCUUAUU 531 AAAGAGU C AGGCCUU 685 ACUUUUU U CUUAUUU 538 CAGGCCU U AAUUUUC 686 CUUUUUU C UUAUUUA 539 AGGCCUU A AUUUUCA 688 UUUUUCU U AUUUAAC 542 CCUUAAU U UUCAAUA 689 UUUUCUU A UUUAACU 543 CUUAAUU U UCAAUAU 691 UUCUUAU U UAACUUA 544 UUAAUUU U CAAUAUA 692 UCUUAUU U AACUUAA 545 UAAUUUU C AAUAUAA 693 CUUAUUU A ACUUAAC 549 UUUCAAU A UAAUUUA 697 UUUAACU U AACAUUC 551 UCAAUAU A AUUUAAC 698 UUAACUU A ACAUUCU 554 AUAUAAU U UAACUUC 703 UUAACAU U CUGUAAA 555 UAUAAUU U AACUUCA 704 UAACAUU C UGUAAAA 556 AUAAUUU A ACUUCAG 708 AUUCUGU A AAAUGUC 560 UUUAACU U CAGAGGG 715 AAAAUGU C UGUUAAC 561 UUAACUU C AGAGGGA 719 UGUCUGU U AACUUAA 573 GGAAAGU A AAUAUUU 720 GUCUGUU A ACUUAAU 577 AGUAAAU A UUUCAGG 724 GUUAACU U AAUAGUA 579 UAAAUAU U UCAGGCA 725 UUAACUU A AUAGUAU 580 AAAUAUU U CAGGCAU 728 ACUUAAU A GUAUUUA 581 AAUAUUU C AGGCAUA 731 UAAUAGU A UUUAUGA 588 CAGGCAU A CUGACAC 733 AUAGUAU U UAUGAAA 597 UGACACU U UGCCAGA 734 UAGUAUU U AUGAAAU 598 GACACUU U GCCAGAA 735 AGUAUUU A UGAAAUG611 AAAGCAU A AAAUUCU 745 AAAUGGU U AAGAAUU616 AUAAAAU U CUUAAAA 746 AAUGGUU A AGAAUUU 617 UAAAAUU C UUAAAAU 752 UAAGAAU U UGGUAAA619 AAAUUCU U AAAAUAU 753 AAGAAUU U GGUAAAU 620 AAUUCUU A AAAUAUA 757 AUUUGGU A AAUUAGU 625 UUAAAAU A UAUUUCA 761 GGUAAAU U AGUAUUU 627 AAAAUAU A UUUCAGA 762 GUAAAUU A GUAUUUA 629 AAUAUAU U UCAGAUA 765 AAUUAGU A UUUAUUU 630 AUAUAUU U CAGAUAU 767 UUAGUAU U UAUUUAA 631 UAUAUUU C AGAUAUC 768 UAGUAUU U AUUUAAU636 UUCAGAU A UCAGAAU 769 AGUAUUU A UUUAAUG638 CAGAUAU C AGAAUCA 771 UAUUUAU U UAAUGUU644 UCAGAAU C AUUGAAG 772 AUUUAUU U AAUGUUA647 GAAUCAU U GAAGUAU 773 UUUAUUU A AUGUUAU653 UUGAAGU A UUUUCCU 778 UUAAUGU U AUGUUGU655 GAAGUAU U UUCCUCC 779 UAAUGUU A UGUUGUG656 AAGUAUU U UCCUCCA 783 GUUAUGU U GUGUUCU657 AGUAUUU U CCUCCAG 788 GUUGUGU U CUAAUAA658 GUAUUUU C CUCCAGG 789 UUGUGUU C UAAUAAA661 UUUUCCU C CAGGCAA 791 GUGUUCU A AUAAAAC672 GCAAAAU U GAUAUAC 794 UUCUAAU A AAACAAA676 AAUUGAU A UACUUUU 805 CAAAAAU A GACAACU678 UUGAUAU A CUUUUUU

681 AUAUACU U UUUUCUU

682 UAUACUU U UUUCUUA Table 12: Human IL-5 HH Ribozyme Sequences

nt . HH Ribozyme Sequence

Position

8 GCAAAGA CUGAUGAGGCCGAAAGGCCGAA AGUGCAU

9 GGCAAAG CUGAUGAGGCCGAAAGGCCGAA AAGUGCA 10 UGGCAAA CUGAUGAGGCCGAAAGGCCGAA AAAGUGC

12 UUUGGCA CUGAUGAGGCCGAAAGGCCGAA AGAAAGU

13 CUUUGGC CUGAUGAGGCCGAAAGGCCGAA AAGAAAG

36 GCUCUGA CUGAUGAGGCCGAAAGGCCGAA ACGUUCU

37 GGCUCUG CUGAUGAGGCCGAAAGGCCGAA AACGUUC

38 UGGCUCU CUGAUGAGGCCGAAAGGCCGAA AAACGUU

56 AAUGCAG CUGAUGAGGCCGAAAGGCCGAA AGCAUCC

57 AAAUGCA CUGAUGAGGCCGAAAGGCCGAA AAGCAUC

63 AAACUCA CUGAUGAGGCCGAAAGGCCGAA AUGCAGA

64 CAAACUC CUGAUGAGGCCGAAAGGCCGAA AAUGCAG

69 GCUAGCA CUGAUGAGGCCGAAAGGCCGAA ACUCAAA

70 AGCUAGC CUGAUGAGGCCGAAAGGCCGAA AACUCAA 74 CAAGAGC CUGAUGAGGCCGAAAGGCCGAA AGCAAAC 78 GCUCCAA CUGAUGAGGCCGAAAGGCCGAA AGCUAGC 80 CAGCUCC CUGAUGAGGCCGAAAGGCCGAA AGAGCUA 91 AUACACG CUGAUGAGGCCGAAAGGCCGAA AGGCAGC 97 GAUGGCA CUGAUGAGGCCGAAAGGCCGAA ACACGUA

104 CUGUGGG CUGAUGAGGCCGAAAGGCCGAA AUGGCAU

116 UUGUGGG CUGAUGAGGCCGAAAGGCCGAA AUUUCUG

117 CUUGUGG CUGADGAGGCCGAAAGGCCGAA AAUUUCU 130 UUUCACC CUGAUGAGGCCGAAAGGCCGAA AUGCACU 145 CAGUGCC CUGAUGAGGCCGAAAGGCCGAA AGGUCUC

155 GAGUAGA CUGAUGAGGCCGAAAGGCCGAA AGCAGUG

156 UGAGUAG CUGAUGAGGCCGAAAGGCCGAA AAGCAGU

157 AUGAGUA CUGAUGAGGCCGAAAGGCCGAA AAAGCAG 159 CGAUGAG CUGAUGAGGCCGAAAGGCCGAA AGAAAGC 162 GUUCGAU CUGAUGAGGCCGAAAGGCCGAA AGUAGAA 165 AGAGUUC CUGAUGAGGCCGAAAGGCCGAA AUGAGUA 171 AUCAGCA CUGAUGAGGCCGAAAGGCCGAA AGUUCGA 179 CAUUGGC CUGAUGAGGCCGAAAGGCCGAA AUCAGCA 192 AUCCUCA CUGAUGAGGCCGAAAGGCCGAA AGUCUCA

200 GAACAGG CUGAUGAGGCUGAAAGGCCGAA AUCCUCA

201 GGAACAG CUGAUGAGGCCGAAAGGCCGAA AAUCCUC

206 GUACAGG CUGAUGAGGCCGAAAGGCCGAA ACAGGAA

207 UGUACAG CUGAUGAGGCCGAAAGGCCGAA AACAGGA 212 UUUUAUG CUGAUGAGGCCGAAAGGCCGAA ACAGGAA 216 UGAUUUU CUGAUGAGGCCGAAAGGCCGAA AUGUACA 222 AGUUGGU CUGAUGAGGCCGAAAGGCCGAA AUUUUUA 245 CCUGAAA CUGAUGAGGCCGAAAGGCCCAA AUUUCUU 247 UCCCUGA CUGAUGAGGCCGAAAGGCCGAA AGAUUUC

248 UUCCCUG CUGAUGAGGCCGAAAGGCCGAA AAGAUUU

249 AUUCCCU CUGAUGAGGCCGAAAGGCCGAA AAAGAUU 257 GUGUGCC CUGAUGAGGCCGAAAGGCCGAA AUUCCCU 273 ACAGUUU CUGAUGAGGCCGAAAGGCCGAA ACUCUCC 291 UCCACAG CUGAUGAGGCCGAAAGGCCGAA ACCCCCU 305 UUUUGAA CUGAUGAGGCCGAAAGGCCGAA AGUCUUU

307 GUUUUUG CUGAUGAGGCCGAAAGGCCGAA AUAGUCU

308 AGUUUUU CUGAUGAGGCCGAAAGGCCGAA AAUAGUC 315 UAAGGAC CUGAUGAGGCCGAAAGGCCGAA AGUUUUU 319 UAUUAAG CUGAUGAGGCCGAAAGGCCGAA ACAAGUU

322 CUUUAUU CUGAUGAGGCCGAAAGGCCGAA AGGACAA

323 UCUUUAU CUGAUGAGGCCGAAAGGCCGAA AAGGACA 326 AUUUCUU CUGAUGAGGCCGAAAGGCCGAA AUUAAGG 334 GUCAAUG CUGAUGAGGCCGAAAGGCCGAA AUUUCUU 338 GGCCGUC CUGAUGAGGCCGAAAGGCCGAA AUGUAUU 380 AUUGGUU CUGAUGAGGCCGAAAGGCCGAA ACUCUCC

388 GUCUAGG CUGAUGAGGCCGAAAGGCCGAA AUUGGUU

389 AGUCUAGCUGAUGAGGCCGAAAGGCCGAA AAUUGGU 392 GGUAGUC CUGAUGAGGCCGAAAGGCCGAA AGGAAUU 397 UUGCAGG CUGAUGAGGCCGAAAGGCCGAA AGUCUAG

409 ACCAAGA CUGAUGAGGCCGAAAGGCCGAA ACUCUUG

410 CACCAAG CUGAUGAGGCCGAAAGGCCGAA AACUCUU

411 ACACCAA CUGAUGAGGCCGAAAGGCCGAA AAACUCU 413 UUACACCCUGAUGAGGCCGAAAGGCCGAA AGAAACU 419 UGUUCAU CUGAUGAGGCCGAAAGGCCGAA ACACCAA 437 UUUCUAU CUGAUGAGGCCGAAAGGCCGAA AUCCACU 440 AACUUUC CUGAUGAGGCCGAAAGGCCGAA AUUAUCC 447 UAGUCUC CUGAUGAGGCCGAAAGGCCGAA ACUUUCU 454 ACCAGUU CUGAUGAGGCCGAAAGGCCGAA AGUCUCA

462 UGCAACA CUGAUGAGGCCGAAAGGCCGAA ACCAGUU

463 CUGCAAC CUGAUGAGGCCGAAAGGCCGAA AACCAGU 466 UGGCUGC CUGAUGAGGCCGAAAGGCCGAA ACAAACC

479 CCUCCAA CUGAUGAGGCCGAAAGGCCGAA AUCUUUG

480 UCCUCCA CUGAUGAGGCCGAAAGGCCGAA AAUCUUU

481 CUCCUCC CUGAUGAGGCCGAAAGGCCGAA AAAUCUU

497 GCAGUAA CUGAUGAGGCCGAAAGGCCGAA AUGUCCU

498 UGCAGUA CUGAUGAGGCCGAAAGGCCGAA AAUGUCC

499 CUGCAGU CUGAUGAGGCCGAAAGGCCGAA AAAUGUC

500 ACUGCAG CUGAUGAGGCCGAAAGGCCGAA AAAAUGU 531 AAGGCCU CUGAUGAGGCCGAAAGGCCGAA ACUCUUU

538 GAAAAUU CUGAUGAGGCCGAAAGGCCGAA AGGCCUG

539 UGAAAAU CUGAUGAGGCCGAAAGGCCGAA AAGGCCU

542 UAUUGAA CUGAUGAGGCCGAAAGGCCGAA AUUAAGG

543 AUAUUGA CUGAUGAGGCCGAAAGGCCGAA AAUUAAG

544 UAUAUUG CUGAUGAGGCCGAAAGGCCGAA AAAUUAA

545 UUAUAUU CUGAUGAGGCCGAAAGGCCGAA AAAAUUA 549 UAAAUUA CUGAUGAGGCCGAAAGGCCGAA AUUGAAA 551 GUUAAAU CUGAUGAGGCCGAAAGGCCGAA AUAUUGA 554 GAAGUUA CUGAUGAGGCCGAAAGGCCGAA AUUAUAU

555 UGAAGUU CUGAUGAGGCCGAAAGGCCGAA AAUUAUA

556 CUGAAGU CUGAUGAGGCCGAAAGGCCGAA AAAUUAU

560 CCCUCUG CUGAUGAGGCCGAAAGGCCGAA AGUUAAA

561 UCCCUCU CUGAUGAGGCCGAAAGGCCGAA AAGUUAA 573 AAAUAUU CUGAUGAGGCCGAAAGGCCGAA ACUUUCC 577 CCUGAAA CUGAUGAGGCCGAAAGGCCGAA AUUUACU

579 UGCCUGA CUGAUGAGGCCGAAAGGCCGAA AUAUUUA

580 AUGCCUG CUGAUGAGGCCGAAAGGCCGAA AAUAUUU

581 UAUGCCU CUGAUGAGGCCGAAAGGCCGAA AAAUAUU 588 GUGUCAG CUGAUGAGGCCGAAAGGCCGAA AUGCCUG

597 UCUGGCA CUGAUGAGGCCGAAAGGCCGAA AGUGUCA

598 UUCUGGC CUGAUGAGGCCGAAAGGCCGAA AAGUGUC 611 AGAAUUU CUGAUGAGGCCGAAAGGCCGAA AUGCUUU

616 UUUUAAG CUGAUGAGGCCGAAAGGCCGAA AUUUUAU

617 AUUUUAA CUGAUGAGGCCGAAAGGCCGAA AAUUUUA

619 AUAUUUU CUGAUGAGGCCGAAAGGCCGAA AGAAUUU

620 UAUAUUU CUGAUGAGGCCGAAAGGCCGAA AAGAAUU 625 UGAAAUA CUGAUGAGGCCGAAAGGCCGAA AUUUUAA 627 UCUGAAA CUGAUGAGGCCGAAAGGCCGAA AUAUUUU

629 UAUCUGA CUGAUGAGGCCGAAAGGCCGAA AUAUAUU

630 AUAUCUG CUGAUGAGGCCGAAAGGCCGAA AAUAUAU

631 GAUAUCU CUGAUGAGGCCGAAAGGCCGAA AAAUAUA 636 AUUCUGA CUGAUGAGGCCGAAAGGCCGAA AUCUGAA 638 UGAUUCU CUGAUGAGGCCGAAAGGCCGAA AUAUCUG 644 CUUCAAU CUGAUGAGGCCGAAAGGCCGAA AUUCUGA 647 AUACUUC CUGAUGAGGCCGAAAGGCCGAA AUGAUUC 653 AGGAAAA CUGAUGAGGCCGAAAGGCCGAA ACUUCAA

655 GGAGGAA CUGAUGAGGCCGAAAGGCCGAA AUACUUC

656 UGGAGGA CUGAUGAGGCCGAAAGGCCGAA AAUACUU

657 CUGGAGG CUGAUGAGGCCGAAAGGCCGAA AAAUACU

658 CCUGGAG CUGAUGAGGCCGAAAGGCCGAA AAAAUAC 661 UUGCCUG CUGAUGAGGCCGAAAGGCCGAA AGGAAAA 672 GUAUAUC CUGAUGAGGCCGAAAGGCCGAA AUUUUGC 676 AAAAGUA CUGAUGAGGCCGAAAGGCCGAA AUCAAUU 678 AAAAAAG CUGAUGAGGCCGAAAGGCCGAA AUAUCAA

681 AAGAAAA CUGAUGAGGCCGAAAGGCCGAA AGUAUAU

682 UAAGAAA CUGAUGAGGCCGAAAGGCCGAA AAGUAUA

683 AUAAGAA CUGAUGAGGCCGAAAGGCCGAA AAAGUAU

684 AAUAAGA CUGAUGAGGCCGAAAGGCCGAA AAAAGUA

685 AAAUAAG CUGAUGAGGCCGAAAGGCCGAA AAAAAGU

686 UAAAUAA CUGAUGAGGCCGAAAGGCCGAA AAAAAAG

688 GUUAAAU CUGAUGAGGCCGAAAGGCCGAA AGAAAAA

689 AGUUAAA CUGAUGAGGCCGAAAGGCCGAA AAGAAAA

691 UAAGUUA CUGAUGAGGCCGAAAGGCCGAA AUAAGAA

692 UUAAGUU CUGAUGAGGCCGAAAGGCCGAA AAUAAGA

693 GUUAAGU CUGAUGAGGCCGAAAGGCCGAA AAAUAAG

697 GAAUGUU CUGAUGAGGCCGAAAGGCCGAA AGUUAAA

698 AGAAUGU CUGAUGAGGCCGAAAGGCCGAA AAGUUAA 703 UUUACAG CUGAUGAGGCCGAAAGGCCGAA AUGUUAA

704 UUUUACA CUGAUGAGGCCGAAAGGCCGAA AAUGUUA

708 GACAUUU CUGAUGAGGCCGAAAGGCCGAA ACAGAAU

715 GUUAACA CUGAUGAGGCCGAAAGGCCGAA ACAUUUU

719 UUAAGUU CUGAUGAGGCCGAAAGGCCGAA ACAGACA

720 AUUAAGU CUGAUGAGGCCGAAAGGCCGAA AACAGAC

724 UACUAUU CUGAUGAGGCCGAAAGGCCGAA AGUUAAC

725 AUACUAU CUGAUGAGGCCGAAAGGCCGAA AAGUUAA

728 UAAAUAC CUGAUGAGGCCGAAAGGCCGAA AUUAAGU

731 UCAUAAA CUGAUGAGGCCGAAAGGCCGAA ACUAUUA

733 UUUCAUA CUGAUGAGGCCGAAAGGCCGAA AUACUAU

734 AUUUCAU CUGAUGAGGCCGAAAGGCCGAA AAUACUA

735 CAUUUCA CUGAUGAGGCCGAAAGGCCGAA AAAUACU

745 AAUUCUU CUGAUGAGGCCGAAAGGCCGAA ACCAUUU

746 AAAUUCU CUGAUGAGGCCGAAAGGCCGAA AACCAUU

752 UUUACCA CUGAUGAGGCCGAAAGGCCGAA AUUCUUA

753 AUUUACC CUGAUGAGGCCGAAAGGCCGAA AAUUCUU

757 ACUAAUU CUGAUGAGGCCGAAAGGCCGAA ACCAAAU

761 AAAUACU CUGAUGAGGCCGAAAGGCCGAA AUUUACC

762 UAAAUAC CUGAUGAGGCCGAAAGGCCGAA AAUUUAC

765 AAAUAAA CUGAUGAGGCCGAAAGGCCGAA ACUAAUU

767 UUAAAUA CUGAUGAGGCCGAAAGGCCGAA AUACUAA

768 AUUAAAU CUGAUGAGGCCGAAAGGCCGAA AAUACUA

769 CAUUAAA CUGAUGAGGCCGAAAGGCCGAA AAAUACU

771 AACAUUA CUGAUGAGGCCGAAAGGCCGAA AUAAAUA

772 UAACAUU CUGAUGAGGCCGAAAGGCCGAA AAUAAAU

773 AUAACAU CUGAUGAGGCCGAAAGGCCGAA AAAUAAA

778 ACAACAU CUGAUGAGGCCGAAAGGCCGAA ACAUUAA

779 CACAACA CUGAUGAGGCCGAAAGGCCGAA AACAUUA

783 AGAACAC CUGAUGAGGCCGAAAGGCCGAA ACAUAAC

788 UUAUUAG CUGAUGAGGCCGAAAGGCCGAA ACACAAC

789 UUUAUUA CUGAUGAGGCCGAAAGGCCGAA AACACAA

791 GUUUUAU CUGAUGAGGCCGAAAGGCCGAA AGAACAC

794 UUUGUUU CUGAUGAGGCCGAAAGGCCGAA AUUAGAA

805 AGUUGUC CUGAUGAGGCCGAAAGGCCGAA AUUUUUG

Table 13: Mouse IL-5 HH Ribozyme Target Sequence

nt. HH Target Sequence nt. HH Target Sequence Position Position

8 cGCuCUU c CUUUGCu 253 AGGGgcU A GaCAuAC

11 uCUUcCU U UGCugAA 259 UagACAU a CUGaAgA 12 CUUcCUU U GCugAAG 269 GaAGAaU C AAACUGU 36 GAAgacU U CAGAGuC 269 GaAGAaU c AAaCugU 36 GaAgAcU u cAgAGUc 269 GAAgaAU c aAAcUgU 37 AAgacUU C AGAGuCA 287 UGGGGGU A CUGUGGA 43 UcaGaGU c AUGAgaA 301 AAAugCU A UUCcAAA 58 GGAUGCU U CUGCAcU 301 AAAugCU a uUCCaaA 59 GAUGCUU C UGCAcUU 303 AUGCuAU u CCaAaAc 59 gAUGcUU c uGcAcUU 303 AugCUAU U CcAAAAC 66 CUGCAcU U GAGUgUu 304 ugCUAUU C cAAAACc 82 UgAcucU c aGcUGUG 315 AACcUGU C aUUAAUA 91 GcUgUGU c uggGCCA 318 cUGUCaU U AAUAAAG 112 ugGAgAU U CCCAugA 319 UGUCaUU A AUAAAGA 113 gGAgAUU C CCAugAG 322 CaUUAAU A AAGAAAU 141 GAGACCU U GaCACaG 330 AAGAAAU A CAUUGAC 141 GAgACcU U GaCAcAg 334 AAUACAU U GACcGCC 158 gUCcgCU C AcCGAgC 334 AAUaCaU u GACcgCC 167 cCGAgCU C UGuUGAc 384 AggCAgU U CCUgGAu 196 UGAGGcU U CCUGUcC 385 ggCAgUU c CUgGAuU 197 GAGGcUU C CUGUcCC 393 CUgGAuU A CCUGCAA 197 gAGGCuU c CUGuCcC 405 CAAGAGU U cCUUGGU 202 UUCCUGU c CCUacuC 406 AAGAGUU c CUUGGUG 202 UUCCUGU c CcUAcuc 409 AGUUcCU U GGUGUgA 206 UGUCccU a cuCaUAA 481 UcaCAAU u UAAgUUA 212 UACUCAU a aAAaUCa 482 cAcAAUU U AAgUUaA 212 UacuCAU A AAAAUCA 483 AcAAUUU A AgUUaAa 218 UaaAaaU c aCcAGCU 483 AcAAUuU a aGUUAAa 218 UAAAAAU C ACCAgCU 495 AAAUUgU c AAcAgAU 218 uAAAAAU c acCAgCU 553 GCUGuuU c CaUuUAU 232 uaUGCAU U GGaGAAA 557 UuUcCAU U UauaUUU 241 gAGAAAU C UUUCAGG 564 UUauAuU u aUgUCCU 241 gAgAaAU c UUucAGG 564 UUAuaUU u AugUcCU 241 gagAAAU c UUUCAGG 565 uaUAUUU a ugUCCuG 241 gAgAaAU c UUUCAGg 565 UAUAuUU a UgUCcUg 243 gaAAucU U UCAGgGg 569 UUuAUGU c cUGUaGU 243 GAAAUCU U UCAGGGg 569 UUUAUGU c cUGUagU 244 AAAUCUU U CAGGGgc 613 AAAGuGU u uaaCCUU 245 AAUCUUU C AGGGgcU 614 AAgUGuU u aACcUUU 620 UUAACcU u uUuGUAU 1407 cCAgUUU A CUcCAGg 793 caAGgCU u UGuGcAU 1407 ccAgUUU a CUCCAGG 816 CUGagUU a UACUCcc 1410 gUUUaCU C CAGGaAA 818 GAguUAU a cUCCcuC 1434 AUgCUUU U aUuUaAU 825 ACUcCcU c CccCUCA 1434 aUgcUuU U AUUUAAu 825 aCUccCU c CcCcUCa 1434 aUgcuUU u AuUUAAU

839 AuCcucU U cGUUGCA 1435 UgCUUUU a UuUaAUU

840 uCcucUU c GUUGCAu 1435 ugcUUUU a uUUAaUU

863 cAAgUAU U cCAGGCu 1438 UuUUAUU U AAuUcug

864 AAgUAUU c CAGGCug 1438 uUUUAUU U AAUucUg 864 AAGUAUU c caggCug 1439 UUUAUUU A AUucUgU 913 gAaCUCU U GGucCaG 1443 UUUaAuU c UGuaAGa 917 UcUuggU c CAGAuGG 1447 AUUCUGU A AgAUGUu 957 UUagcAU c CUUUcUc 1458 ugUUcaU a UUAUUUA 960 GCAuccU u UcUcCuA 1458 ugUUcAU A uUAUUUA 960 GcaUcCU u uCUCcUa 1460 UucAUAU u AUUUAug 962 AUcCuuU c UCcUaGC 1461 UcAUAuU A UUUAUGA 975 gcccCUU u AgAUAgA 1463 AUAuUAU U UAUGAug 987 aGaOGAU A cuuAAUG 1475 AuGgAUU c aGUAAgU 990 UGAuACU u AAugacU 1479 AUUcaGU A AgUUAaU

1000 UGACuCU c UugCuGA 1483 aGuAAGU u AAUAUUU

1027 CgggGCU U cCUgCUC 1483 aGUAAgU U AaUAUUU

1034 UCCUGcU C CUaUcuA 1484 GUAAgUU A aUAUUUA

1037 UgcUCcU A UcUAACU 1487 agUUAAU a UUuAuUA

1039 cUccuAU c UAACUUC 1487 AgUUAaU A UUUAUUa

1039 cUCcUAU c UAACUUc 1489 UUAAUaU U uAuUAcA

1041 CcUAUcU A ACUUcAa 1489 UUAAuAU u UAUUaCA

1051 UUcAAuU U AAuAccC 1489 UUAaUAU U UAUUacA 1148 uGAcUUU u cUuaUGU 1490 UAAUaUU u AuUAcAc

1213 GCUgGaU u UUGGAaa 1490 UAaUAUU U AUuAcAc

1213 gcUGGAU u uUgGAAA 1490 UAaUAUU U AUUacAc

1214 cugGAUU U UGGAaaA 1491 AAUAUUU a uuaCAcg

1215 ugGAUUU U GGAaaAG 1491 AAUAUuU a UuAcAcg 1234 gGGACAU c UccuUGC 1491 AaUAUUU A UuAcAcG 1236 GACAUcU c cuUGCAG 1491 AaUAUUU A UUacAcG

1275 ugGGCCU U AcUUcUC 1494 AUuUAUU a CAcgUAU

1276 gGGCCUU A cUUcUCc 1502 cACGUaU A UaauAUu 1280 CUUAcUU c UCcgUgU 1502 cAcgUAU a UAAUaUU 1298 UgAACUU a AGAaGcA 1507 AUAUAaU a UUcUaaU 1310 gcAAAGU a aAuACcA 1509 AUAAuAU U CUaAuAA 1310 GCAAAgU a aAUAcca 1509 aUaaUaU U CUAAUAA 1310 GcaAAgU a AAUAccA 1510 UAAuAUU C UaAuAAa 1350 AAAGCAU A AAAUggU 1510 UAAuAUU C UaauAAA 1358 AAAUGGU U ggGAugU 1510 UAAuAuU c UaaUAAA 1370 UgUuaUU C AGgUAUC 1510 UaaUaUU C UAAUAAA 1375 UUCAGgU A UCAGggU 1512 aUaUUCU A AUAAAgC 1377 CAGgUAU C AGggUCA 1515 UUCUAAU A AAgCAgA 1383 UCAGggU C AcUGgAG

1405 cccCAgU U UACUcCA

Table 17

Mouse rel A HH Target sequence

nt. Position HH Target Sequence nt. Position HH Target Sequence

19 AAUGGCU a caCaGgA 467 cCAGGCU c cuguUCg 22 aGCUCcU a cGUgGUG 469 AaGCcAU u AGcCAGC 26 CcUCcaU u GcGgACa 473 UuUgAGU C AGauCAg 93 GAuCUGU U uCCCCUC 481 AGCGaAU C CAGACCA 94 AuCUGUU u CCCCUCA 501 AACCCCU U uCAcGUU 100 UuCCCCU C AUCUUuC 502 ACCCCUU u CAcGUUC 103 CCCUCAU C UUuCCcu 508 UuCAcGU U CCUAUAG 105 CUCAUCU U uCCcuCA 509 uCAcGUU C CUAUAGA 106 UCAUCUU u CCcuCAG 512 cGUUCCU A UAGAgGA 129 CAGGCuU C UGGgCCu 514 UUCCUAU A GAgGAGC 138 GGgCCuU A UGUGGAG 534 GGGGACU A uGACuUG 148 UGGAGAU C AUcGAaC 556 UGCGcCU C UGCUUCC 151 AGAUCAU c GAaCAGC 561 CUCUGCU U CCAGGUG 180 AUGCGaU U CCGCUAu 562 UCUGCUU C CAGGUGA 181 UGCGaUU C CGCUAuA 585 aAgCCAU u AGcCAGc 186 UUCCGCU A uAAaUGC 598 GGCCCCU C CuCCUGa 204 GGGCGCU C aGCGGGC 613 CcCCUGU C CUcuCaC 217 GCAGuAU u CCuGGCG 616 CUGUCCU c uCaCAUC 239 CACAGAU A CCACCAA 617 gucCCUU C CUCAgCC 262 CCACCAU C AAGAUCA 620 CCUUCCU C AgCCaug 268 UCAAGAU C AAUGGCU 623 UCCUgcU u CCAUCUc 276 AAUGGCU A CACAGGA 628 AUCCgAU u UUUGAuA 301 UuCGaAU C UCCCUGG 630 CCgAUuU U UGAuAAc 303 CGaAUCU C CCUGGUC 631 CgAUuUU U GAuAAcC 310 CCCUGGU C ACCAAGG 638 UGgCcAU u GUGuuCC 323 GGcCCCU C CUCcuga 661 CCGAGCU C AAGAUCU 326 uCCaCCU C ACCGGCC 667 UCAAGAU C UGCCGAG 335 CCGGCCU C AuCCaCA 687 CGgAACU C UGGgAGC 349 AuGAaCU U GUgGGgA 700 GCUGCCU C GGUGGGG 352 AGaUcaU c GaAcAGc 715 AUGAGAU C UUCuUgC 375 GAUGGCU a CUAUGAG 717 GAGAUCU U CuUgCUG 376 AUGGucU C UccGgaG 713 AGAUCUU C uUgCUGU 378 GGCUaCU A UGAGGCU 721 UucUCCU c CauUGcG 391 CUGAcCU C UGCCCaG 751 AaGACAU U GAGGUGU 409 GCaGuAU C CAuAGcU 759 GAGGUGU A UUUCACG 416 CCgCAGU a UCCAuAg 761 GGUGUAU U UCACGGG 417 CAuAGcU U CCAGAAC 762 GUGUAUU U CACGGGA 418 AuAGcUU C CAGAACC 763 UGUAUUU C ACGGGAC 433 UGGGgAU C CAGUGUG 792 CGAGGCU C CUUUUCu 795 GGCUCCU U UUCuCAA 1167 GAUGAGU U UuCCcCC 796 GCUCCUU U UCuCAAG 1163 AUGAGUU U uCCcCCA 797 CUCCUUU U CuCAAGC 1169 UGAGUUU u CCcCCAU 798 UCCUUUU C uCAAGCU 1182 AUGcUGU U aCCaUCa 829 UGGCCAU U GUGUUCC 1183 UGcUGUU a CCaUCaG 834 AUUGUGU U CCGGACu 1184 GGccccU C CUcCUGa

835 UUGUGUU C CGGACuC 1187 GUccCuU c CUcaGCc

845 GACuCCU C CgUACGC 1188 UUaCCaU C aGGGCAG

849 CCUCCgU A CGCcGAC 1198 GGgAGuU u AGuCuGa

872 CCAGGCU C CUGUuCG 1209 CAGcCCU a caCCUUc

883 UuCGaGU C UCCAUGC 1215 cuGGCCU U aGCaCCG

885 CGaGUCU C CAUGCAG 1229 GGuCCCU u CCucAGc

905 GCGGCCU U CuGAuCG 1237 CCCAgcU C CUGCCCC

906 CGGCCUU C uGAuCGc 1250 CCAGcCU C CAGgCuC

919 GcGAGCU C AGUGAGC 1268 CCCaGCU C CuGCCcc

936 AUGGAgU U CCAGUAC 1279 CCAUGGU c cCuuCcu

937 UGGAgUU C CAGUACu 1281 gUGGgcU C AGCUgcG

942 UUCCAGU A CuUGCCA 1286 AUgAGuU u UccCCCA

953 GCCucAU c CAcAuGA 1309 CuCCUGU u CgAGUCu

962 AGAuGAU C GcCACCG 1315 cCCCAGU u CUAaCCC

965 CagUacU u gCCaGAc 1318 CAGUuCU A aCCCCgG

973 ACCGGAU U GAaGAGA 1331 gGGuCCU C CcCAGuC

986 GAgACcU u cAAGagu 1334 CuuUuCU C AaGCUGa

996 AGGACcU A UGAGACC 1389 ACGCUGU C gGAaGCC

1005 GAGACCU U CAAGAGu 1413 CUGCAGU U UGAUGcU

1006 AGACCUU C AAGAGuA 1414 UGCAGUU U GAUGcUG

1015 AGAGuAU C AUGAAGA 1437 GGGGCCU U GCUUGGC

1028 GAAGAGU C CUUUCAa 1441 CCUUGCU U GGCAACA

1031 GAGUCCU U UCAauGG 1467 GgaGUGU U CACAGAC

1032 AGUCCUU U CAauGGA 1468 gaGUGUU C ACAGACC

1033 GUCCUUU C AauGGAC 1482 CUGGCAU C uGUgGAC

1058 CCGGCCU C CAaCcCG 1486 CuUCgGU a GggAACU

1064 UaCACCU u GAucCAa 1494 GACAACU C aGAGUUU

1072 GgCGuAU U GCUGUGC 1500 UCaGAGU U UCAGCAG

1082 UGUGCCU a CCCGaAa 1501 CaGAGUU U CAGCAGC

1083 aaGCCUU C CCGaAGu 1502 aGAGUUU C AGCAGCU

1092 CGaAaCU C AaCUUCU 1525 gGuGCAU c CCUGUGu

1097 CUCAaCU U CUGUCCC 1566 AUGGAGU A CCCUGAa

1098 UCAaCUU C UGOCCCC 1577 UGAaGCU A UAACUCG

1102 CUUCUGU C CCCAAGC 1579 AaGCUAU A ACUCGCC

1125 CAGCCCU A caCCUUc 1583 UAUAACU C GCCUgGU

1127 GCCaUAU a gCcUUAC 1588 CUCuCCU A GaGAggG

1131 cAUCCCU c agCacCA 1622 CCCAGCU C CUGCcCC

1132 AcaCCUU c cCagCAU 1628 UCCUGCU u CggUaGG

1133 UCCaUcU c CagCuUC 1648 CGGGGCU U CCCAAUG

1137 UUUACuU u AgCgCgc 1660 cUGaCCU C ugccCAG

1140 cCagCAU C CCUcAGC 1663 cuCUgCU U cCAGGuG

1153 GCACCAU C AACUuUG 1664 uCUgCUU c CAGGuGA

1158 AUCAACU U UGAUGAG 1665 CUCgcUU u cGGAGgU

1680 GAAGACU U CUCCUCC

1681 AAGACUU C UCCUCCA

1683 GACUUCU C CUCCAUU

1686 UUCUCCU C CAUUGCG

1690 CCUCCAU U GCGGACA 1704 AUGGACU U CUCuGCu

1705 UGGACUU C UCuGCuC

1707 GACUUCU C uGCuCUu

1721 UUUGAGU C AGAUCAG

1726 GUCAGAU C AGCUCCU

1731 AUCAGCU C CUAAGGu

1734 AGCUCCU A AGGuGcU

1754 CaGugCU C CCaAGAG

Table 18

Human rel A HH Target Sequences

nt. Position HH Target Sequence nt. Position HH Target Sequence

19 AAUGGCU C GUCUGUA 467 GCAGGCU A UCAGUCA

22 GGCUCGU C UGUAGUG 469 AGGCUAU C AGUCAGC

26 CGUCUGU A GUGCACG 473 UAUCAGU C AGCGCAU

93 GAACUGU U CCCCCUC 481 AGCGCAU C CAGACCA

94 AACUGUU C CCCCUCA 501 AACCCCU U CCAAGUU

100 UCCCCCU C AUCUUCC 502 ACCCCUU C CAAGUUC

103 CCCUCAU C UUCCCGG 508 UCCAAGU U CCUAUAG

105 CUCAUCU U CCCGGCA 509 CCAAGUU C CUAUAGA

106 UCAUCUU C CCGGCAG 512 AGUUCCU A UAGAAGA

129 CAGGCCU C UGGCCCC 514 UUCCUAU A GAAGAGC

138 GGCCCCU A UGUGGAG 534 GGGGACU A CGACCUG

148 UGGAGAU C AUUGAGC 556 UGCGGCU C UGCUUCC

151 AGAUCAU U GAGCAGC 561 CUCUGCU U CCAGGUG

180 AUGCGCU U CCGCUAC 562 UCUGCUU C CAGGUGA

181 UGCGCUU C CGCUACA 585 GACCCAU C AGGCAGG

186 UUCCGCU A CAAGUGC 598 GGCCCCU C CGCCUGC

204 GGGCGCU C CGCGGGC 613 CGCCUGU C CUUCCUC

217 GCAGCAU C CCAGGCG 616 CUGUCCU U CCUCAUC

239 CACAGAU A CCACCAA 617 UGUCCUU C CUCAUCC

262 CCACCAU C AAGAUCA 620 CCUUCCU C AUCCCAU

268 UCAAGAU C AAUGGCU 623 UCCUCAU C CCAUCUU

276 AAUGGCU A CACAGGA 628 AUCCCAU C UUUGACA

301 UGCGCAU C UCCCUGG 630 CCCAUCU U UGACAAU

303 CGCAUCU C CCUGGUC 631 CCAUCUU U GACAAUC

310 CCCUGGU C ACCAAGG 638 UGACAAU C GUGCCCC

323 GGACCCU C CUCACCG 661 CCGAGCU C AAGAUCU

326 CCCUCCU C ACCGGCC 667 UCAAGAU C UGCCGAG

335 CCGGCCU C ACCCCCA 687 CGAAACU C UGGCAGC

349 AGGAGCU U GUAGGAA 700 GCUGCCU C GGUGGGG

352 AGCUUGU A GGAAAGG 715 AUGAGAU C UUCCUAC

375 GAUGGCU U CUAUGAG 717 GAGAUCU U CCUACUG

376 AUGGCUU C UAUGAGG 718 AGAUCUU C CUACUGU

378 GGCUUCU A UGAGGCU 721 UCUUCCU A CUGUGUG

391 CUGAGCU C UGCCCGG 751 AGGACAU U GAGGUGU

409 GCUGCAU C CACAGUU 759 GAGGUGU A UUUCACG

416 CCACAGU U UCCAGAA 761 GGUGUAU U UCACGGG

417 CACAGUU U CCAGAAC 762 GUGUAUU U CACGGGA

418 ACAGUUU C CAGAACC 763 UGUAUUU C ACGGGAC

433 UGGGAAU C CAGUGUG 792 CGAGGCU C CUUUUCG

795 GGCUCCU U UUCGCAA 1167 GAUGAGU U UCCCACC

796 GCUCCUU U UCGCAAG 1168 AUGAGUU U CCCACCA

797 CUCCUUU U CGCAAGC 1169 UGAGUUU C CCACCAU

798 UCCUUUU C GCAAGCU 1182 AUGGUGU U UCCUUCU

829 UGGCCAU U GUGUUCC 1183 UGGUGUU U CCUUCUG

834 AUUGUGU U CCGGACC 1184 GGUGUUU C CUUCUGG 835 UUGUGUU C CGGACCC 1187 GUUUCCU U CUGGGCA

845 GACCCCU C CCUACGC 1188 UUUCCUU C UGGGCAG

849 CCUCCCU A CGCAGAC 1198 GGCAGAU C AGCCAGG

872 GCAGGCU C CUGUGCG 1209 CAGGCCU C GGCCUUG

883 UGCGUGU C UCCAUGC 1215 UCGGCCU U GGCCCCG

885 CGUGUCU C CAUGCAG 1229 GGCCCCU C CCCAAGU

905 GCGGCCU U CCGACCG 1237 CCCAAGU C CUGCCCC

906 CGGCCUU C CGACCGG 1250 CCAGGCU C CAGCCCC

919 GGGAGCU C AGUGAGC 1268 CCCUGCU C CAGCCAU

936 AUGGAAU U CCAGUAC 1279 CCAUGGU A UCAGCUC

937 UGGAAUU C CAGUACC 1281 AUGGUAU C AGCUCUG

942 UUCCAGU A CCUGCCA 1286 AUCAGCU C UGGCCCA

953 GCCAGAU A CAGACGA 1309 CCCCUGU C CCAGUCC

962 AGACGAU C GUCACCG 1315 UCCCAGU C CUAGCCC

965 CGAUCGU C ACCGGAU 1318 CAGUCCU A GCCCCΑG

973 ACCGGAU U GAGGAGA 1331 AGGCCCU C CUCAGGC

986 GAAACGU A AAAGGAC 1334 CCCUCCU C AGGCUGU

996 AGGACAU A UGAGACC 1389 ACGCUGU C AGAGGCC

1005 GAGACCU U CAAGAGC 1413 COGCAGU U UGAUGAU

1006 AGACCUU C AAGAGCA 1414 UGCAGUU U GAUGAUG

1015 AGAGCAU C AUGAAGA 1437 GGGGCCU U GCUUGGC

1028 GAAGAGU C CUUUCAG 1441 CCUUGCU U GGCAACA

1031 GAGUCCU U UCAGCGG 1467 GCUGUGU U CACAGAC

1032 AGUCCUU U CAGCGGA 1468 CUGUGUU C ACAGACC

1033 GUCCUUU C AGCGGAC 1482 CUGGCAU C CGUCGAC

1058 CCGGCCU C CACCUCG 1486 CAUCCGU C GACAACU

1064 UCCACCU C GACGCAU 1494 GACAACU C CGAGUUU

1072 GACGCAU U GCUGUGC 1500 UCCGAGU U UCAGCAG

1082 UGUGCCU U CCCGCAG 1501 CCGAGUU U CAGCAGC

1083 GUGCCUU C CCGCAGC 1502 CGAGUUU C AGCAGCU

1092 CGCAGCU C AGCUUCU 1525 AGGGCAU A CCUGUGG

1097 CUCAGCU U COGUCCC 1566 AUGGAGU A CCCUGAG

1098 UCAGCUU C UGUCCCC 1577 UGAGGCU A UAACUCG

1102 CUUCUGU C CCCAAGC 1579 AGGCUAU A ACUCGCC

1125 CAGCCCU A UCCCUUU 1583 UAUAACU C GCCUAGU

1127 GCCCUAU C CCUUUAC 1588 CUCGCCU A GUGACAG

1131 UAUCCCU U UACGUCA 1622 CCCAGCU C CUGCUCC

1132 AUCCCUU U ACGUCAU 1628 UCCUGCU C CACUGGG

1133 UCCCUUU A CGUCAUC 1648 CGGGGCU C CCCAAUG

1137 UUUACGU C AUCCCUG 1660 AUGGCCU C CUUUCAG

1140 ACGUCAU C CCUGAGC 1663 GCCUCCU U UCAGGAG

1153 GCACCAU C AACUAUG 1664 CCUCCUU U CAGGAGA

1158 AUCAACU A UGAUGAG 1665 CUCCUUU C AGGAGAU

1680 GAAGACU U CUCCUCC

1681 AAGACUU C UCCUCCA

1683 GACUUCU C CUCCAUU

1686 UUCUCCU C CAUUGCG

1690 CCUCCAU U GCGGACA

1704 AUGGACU U CUCAGCC 1705 UGGACUU C UCAGCCC

1707 GACUUCU C AGCCCUG

1721 GCUGAGU C AGAUCAG

1726 GUCAGAU C AGCUCCU

1731 AUCAGCU C CUAAGGG

1734 AGCUCCU A AGGGGGU

1754 CUGCCCU C CCCAGAG

Table 19

Mouse rel A HH -Ribozyme Sequences

nt HH Ribozyme Sequence

Sequence

19 UCCUGUG CUGAUGAGGCCGAAAGGCCGAA AGCCAUU

22 CACCACG CUGAUGAGGCCGAAAGGCCGAA AGGAGCU

26 UGUCCGC CUGAUGAGGCCGAAAGGCCGAA AUGGAGG

93 GAGGGGA CUGAUGAGGCCGAAAGGCCGAA ACAGAUC

94 UGAGGGG CUGAUGAGGCCGAAAGGCCGAA AACAGAU 100 GAAAGAU CUGAUGAGGCCGAAAGGCCGAA AGGGGAA 103 AGGGAAA CUGAUGAGGCCGAAAGGCCGAA AUGAGGG

105 UGAGGGA CUGAUGAGGCCGAAAGGCCGAA AGAUGAG

106 CUGAGGG CUGAUGAGGCCGAAAGGCCGAA AAGAUGA 129 AGGCCCA CUGAUGAGGCCGAAAGGCCGAA AAGCCUG 138 CUCCACA CUGAUGAGGCCGAAAGGCCGAA AAGGCCC 148 GUUCGAU CUGAUGAGGCCGAAAGGCCGAA AUCUCCA 151 GCUGUUC CUGAUGAGGCCGAAAGGCCGAA AUGAUCU

180 AUAGCGG CUGAUGAGGCCGAAAGGCCGAA AUCGCAU

181 UAUAGCG CUGAUGAGGCCGAAAGGCCGAA AAUCGCA 186 GCAUUUA CUGAUGAGGCCGAAAGGCCGAA AGCGGAA 204 GCCCGCU CUGAUGAGGCCGAAAGGCCGAA AGCGCCC 217 CGCCAGG CUGAUGAGGCCGAAAGGCCGAA AUACUGC 239 UUGGUGG CUGAUGAGGCCGAAAGGCCGAA AUCUGUG 262 UGAUCUU CUGAUGAGGCCGAAAGGCCGAA AUGGUGG 268 AGCCAUU CUGAUGAGGCCGAAAGGCCGAA AUCUUGA 276 UCCUGUG CUGAUGAGGCCGAAAGGCCGAA AGCCAUU 301 CCAGGGA CUGAUGAGGCCGAAAGGCCGAA AUUCGAA 303 GACCAGG CUGAUGAGGCCGAAAGGCCGAA AGAUUCG 310 CCUUGGU CUGAUGAGGCCGAAAGGCCGAA ACCAGGG 323 UCAGGAG CUGAUGAGGCCGAAAGGCCGAA AGGGGCC 326 GGCCGGU CUGAUGAGGCCGAAAGGCCGAA AGGUGGA 335 UGUGGAU CUGAUGAGGCCGAAAGGCCGAA AGGCCGG 349 UCCCCAC CUGAUGAGGCCGAAAGGCCGAA AGUUCAU 352 GCUGUUC CUGAUGAGGCCGAAAGGCCGAA AUGAUCU

375 CUCAUAG CUGAUGAGGCCGAAAGGCCGAA AGCCAUC

376 CUCCGGA CUGAUGAGGCCGAAAGGCCGAA AGACCAU 378 AGCCUCA CUGAUGAGGCCGAAAGGCCGAA AGUAGCC 391 CUGGGCA CUGAUGAGGCCGAAAGGCCGAA AGGUCAG 409 AGCUAUG CUGAUGAGGCCGAAAGGCCGAA AUACUGC

416 CUAUGGA CUGAUGAGGCCGAAAGGCCGAA ACUGCGG

417 GUUCUGG CUGAUGAGGCCGAAAGGCCGAA AGCUAUG

418 GGUUCUG CUGAUGAGGCCGAAAGGCCGAA AAGCUAU 433 CACACUG CUGAUGAGGCCGAAAGGCCGAA AUCCCCA 467 CGAACAG CUGAUGAGGCCGAAAGGCCGAA AGCCUGG 469 GCUGGCU CUGAUGAGGCCGAAAGGCCGAA AUGGCUU 473 CUGAUCU CUGAUGAGGCCGAAAGGCCGAA ACUCAAA 481 UGGUCUG CUGAUGAGGCCGAAAGGCCGAA AUUCGCU 501 AACGUGA CUGAUGAGGCCGAAAGGCCGAA AGGGGUU

502 GAACGUG CUGAUGAGGCCGAAAGGCCGAA AAGGGGU

508 CUAUAGG CUGAUGAGGCCGAAAGGCCGAA ACGUGAA

509 UCUAUAG CUGAUGAGGCCGAAAGGCCGAA AACGUGA 512 UCCUCUA CUGAUGAGGCCGAAAGGCCCAA AGGAACG 514 GCUCCUC CUGAUGAGGCCGAAAGGCCGAA AUAGGAA 534 CAAGUCA CUGAUGAGGCCGAAAGGCCGAA AGUCCCC 556 GGAAGCA CUGAUGAGGCCGAAAGGCCGAA AGGCGCA

561 CACCUGG CUGAUGAGGCCGAAAGGCCGAA AGCAGAG

562 UCACCUG CUGAUGAGGCCGAAAGGCCGAA AAGCAGA 585 GCUGGCU CUGAUGAGGCCGAAAGGCCGAA AUGGCUU 598 UCAGGAG CUGAUGAGGCCGAAAGGCCGAA AGGGGCC 613 GUGAGAG CUGAUGAGGCCGAAAGGCCGAA ACAGGGG

616 GAUGUGA CUGAUGAGGCCGAAAGGCCGAA AGGACAG

617 GGCUGAG CUGAUGAGGCCGAAAGGCCGAA AAGGGAC 620 CAUGGCU CUGAUGAGGCCGAAAGGCCGAA AGGAAGG 623 GAGAUGG CUGAUGAGGCCGAAAGGCCGAA AGCAGGA 628 UAUCAAA CUGAUGAGGCCGAAAGGCCGAA AUCGGAU

630 GUUAUCA CUGAUGAGGCCGAAAGGCCGAA AAAUCGG

631 GGUUAUC CUGAUGAGGCCGAAAGGCCGAA AAAAUCG 638 GGAACAC CUGAUGAGGCCGAAAGGCCGAA AUGGCCA 661 AGAUCUU CUGAUGAGGCCGAAAGGCCGAA AGCUCGG 667 CUCGGCA CUGAUGAGGCCGAAAGGCCGAA AUCUUGA 687 GCUCCCA CUGAUGAGGCCGAAAGGCCGAA AGUUCCG 700 CCCCACC CUGAUGAGGCCGAAAGGCCGAA AGGCAGC 715 GCAAGAA CUGAUGAGGCCGAAAGGCCGAA AUCUCAU

717 CAGCAAG CUGAUGAGGCCGAAAGGCCGAA AGAUCUC

718 ACAGCAA CUGAUGAGGCCGAAAGGCCGAA AAGAUCU 721 CGCAAUG CUGAUGAGGCCGAAAGGCCGAA AGGAGAA 751 ACACCUC CUGAUGAGGCCGAAAGGCCGAA AUGUCUU 759 CGUGAAA CUGAUGAGGCCGAAAGGCCGAA ACACCUC

761 CCCGUGA CUGAUGAGGCCGAAAGGCCGAA AUACACC

762 UCCCGUG CUGAUGAGGCCGAAAGGCCGAA AAUACAC

763 GUCCCGU CUGAUGAGGCCGAAAGGCCGAA AAAUACA 792 AGAAAAG CUGAUGAGGCCGAAAGGCCGAA AGCCUCG

795 UUGAGAA CUGAUGAGGCCGAAAGGCCGAA AGGAGCC

796 CUUGAGA CUGAUGAGGCCGAAAGGCCGAA AAGGAGC

797 GCUUGAG CUGAUGAGGCCGAAAGGCCGAA AAAGGAG

798 AGCUUGA CUGAUGAGGCCGAAAGGCCGAA AAAAGGA 829 GGAACAC CUGAUGAGGCCGAAAGGCCGAA AUGGCCA

834 AGUCCGG CUGAUGAGGCCGAAAGGCCGAA ACACAAU

835 GAGUCCG CUGAUGAGGCCGAAAGGCCGAA AACACAA 845 GCGUACG CUGAUGAGGCCGAAAGGCCGAA AGGAGUC 849 GUCGGCG CUGAUGAGGCCGAAAGGCCGAA ACGGAGG 872 CGAACAG CUGAUGAGGCCGAAAGGCCGAA AGCCUGG 883 GCAUGGA CUGAUGAGGCCGAAAGGCCGAA ACUCGAA 885 CUGCAUG CUGAUGAGGCCGAAAGGCCGAA AGACUCG

905 CGAUCAG CUGAUGAGGCCGAAAGGCCGAA AGGCCGC

906 GCGAUCA CUGAUGAGGCCGAAAGGCCGAA AAGGCCG 919 GCUCACU CUGAUGAGGCCGAAAGGCCGAA AGCUCGC

936 GUACUGG CUGAUGAGGCCGAAAGGCCGAA ACUCCAU

937 AGUACUG CUGAUGAGGCCGAAAGGCCGAA AACUCCA 942 UGGCAAG CUGAUGAGGCCGAAAGGCCGAA ACUGGAA 953 UCAUGUG CUGAUGAGGCCGAAAGGCCGAA AUGAGGC 962 CGGUGGC CUGAUGAGGCCGAAAGGCCGAA AUCAUCU 965 GUCUGGC CUGAUGAGGCCGAAAGGCCGAA AGUACUG 973 UCUCUUC CUGAUGAGGCCGAAAGGCCGAA AUCCGGU 986 ACUCUUG CUGAUGAGGCCGAAAGGCCGAA AGGUCUC 996 GGUCUCA CUGAUGAGGCCGAAAGGCCGAA AGGUCCU

1005 ACUCUUG CUGAUGAGGCCGAAAGGCCGAA AGGUCUC

1006 UACUCUU CUGAUGAGGCCGAAAGGCCGAA AAGGUCU 1015 UCUUCAU CUGAUGAGGCCGAAAGGCCGAA AUACUCU 1028 UUGAAAG CUGAUGAGGCCGAAAGGCCGAA ACUCUUC

1031 CCAUUGA CUGAUGAGGCCGAAAGGCCGAA AGGACUC

1032 UCCAUUG CUGAUGAGGCCGAAAGGCCGAA AAGGACU

1033 GUCCAUU CUGAUGAGGCCGAAAGGCCGAA AAAGGAC 1058 CGGGUUG CUGAUGAGGCCGAAAGGCCGAA AGGCCGG 1064 UUGGAUC CUGAUGAGGCCGAAAGGCCGAA AGCUGUA 1072 GCACAGC CUGAUGAGGCCGAAAGGCCGAA AUACGCC

1082 UUUCGGG CUGAUGAGGCCGAAAGGCCGAA AGGCACA

1083 ACUUCGG CUGAUGAGGCCGAAAGGCCGAA AAGGCUU 1092 AGAAGUU CUGAUGAGGCCGAAAGGCCGAA AGUUUCG

1097 GGGACAG CUGAUGAGGCCGAAAGGCCGAA AGUUGAG

1098 GGGGACA CUGAUGAGGCCGAAAGGCCGAA AAGUUGA 1102 GCUUGGG CUGAUGAGGCCGAAAGGCCGAA ACAGAAG 1125 GAAGGUG CUGAUGAGGCCGAAAGGCCGAA AGGGCUG 1127 GUAAGGC CUGAUGAGGCCGAAAGGCCGAA AUAUGGC

1131 UGGUGCU CJGAUGAGGCCGAAAGGCCGAA AGGGAUG

1132 AUGCUGG CUGAUGAGGCCGAAAGGCCGAA AAGGUGU

1133 GAAGCUG CUGAUGAGGCCGAAAGGCCGAA AGAUGGA 1137 GCGCGCU CUGAUGAGGCCGAAAGGCCGAA AAGUAAA 1140 GCUGAGG CUGAUGAGGCCGAAAGGCCGAA AUGCUGG 1153 CAAAGUU CUGAUGAGGCCGAAAGGCCGAA AUGGUGC 1158 CUCAUCA CUGAUGAGGCCGAAAGGCCGAA AGUUGAU

1167 GGGGGAA CUGAUGAGGCCGAAAGGCCGAA ACUCAUC

1168 UGGGGGA CUGAUGAGGCCGAAAGGCCGAA AACUCAU

1169 AUGGGGG CUGAUGAGGCCGAAAGGCCGAA AAACUCA

1182 UGAUGGU CUGAUGAGGCCGAAAGGCCGAA ACAGCAU

1183 CUGAUGG CUGAUGAGGCCGAAAGGCCGAA AACAGCA

1184 UCAGGAG CUGAUGAGGCCGAAAGGCCGAA AGGGGCC

1187 GGCUGAG CUGAUGAGGCCGAAAGGCCGAA AAGGGAC

1188 CUGCCCU CUGAUGAGGCOSAAAGGCCGAA AUGGUAA 1198 UCAGACU CUGAUGAGGCCGAAAGGCCGAA AACUCCC 1209 GAAGGUG CUGAUGAGGCCGAAAGGCCGAA AGGGCUG 1215 CGGUGCU CUGAUGAGGCCGAAAGGCCGAA AGGCCAG 1229 GCUGAGG CUGAUGAGGCCGAAAGGCCGAA AGGGACC 1237 GGGGCAG CUGAUGAGGCCGAAAGGCCGAA AGCUGGG 1250 GAGCCUG CUGAUGAGGCCGAAAGGCCGAA AGGCUGG 1268 GGGGCAG CUGAUGAGGCCGAAAGGCCGAA AGCUGGG

1279 AGGAAGG CUGAUGAGGCCGAAAGGCCGAA ACCAUGG

1281 CGCAGCU CUGAUGAGGCCGAAAGGCCGAA AGCCCAC

1286 UGGGGGA CUGAUGAGGCCGAAAGGCCGAA AACUCAU

1309 AGACUCG CUGAUGAGGCCGAAAGGCCGAA ACAGGAG

1315 GGGUUAG CUGAUGAGGCCGAAAGGCCGAA ACUGGGG

1318 CCGGGGU CUGAUGAGGCCGAAAGGCCGAA AGAACUG

1331 GACUGGG CUGAUGAGGCCGAAAGGCCGAA AGGACCC

1334 UCAGCUU CUGAUGAGGCCGAAAGGCCGAA AGAAAAG

1389 GGCUUCC CUGAUGAGGCCGAAAGGCCGAA ACAGCGU

1413 AGCAUCA CUGAUGAGGCCGAAAGGCCGAA ACUGCAG

1414 CAGCAUC CUGAUGAGGCCGAAAGGCCGAA AACUGCA 1437 GCCAAGC CUGAUGAGGCCGAAAGGCCGAA ΑGGCCCC 1441 UGUUGCC CUGAUGAGGCCGAAAGGCCGAA AGCAAGG

1467 GUCUGUG CUGAUGAGGCCGAAAGGCCGAA ACACUCC

1468 GGUCUGU CUGAUGAGGCCGAAAGGCCGAA AACACUC 1482 GUCCACA CUGAUGAGGCCGAAAGGCCGAA AUGCCAG 1486 AGUUCCC CUGAUGAGGCCGAAAGGCCGAA ACCGAAG 1494 AAACUCU CUGAUGAGGCCGAAAGGCCGAA AGUUGUC

1500 CUGCUGA CUGAUGAGGCCGAAAGGCCGAA ACUCUGA

1501 GCUGCUG CUGAUGAGGCCGAAAGGCCGAA AACUCUG

1502 AGCUGCU CUGAUGAGGCCGAAAGGCCGAA AAACUCU 1525 ACACAGG CUGAUGAGGCCGAAAGGCCGAA AUGCACC 1566 UUCAGGG CUGAUGAGGCCGAAAGGCCGAA ACUCCAU 1577 CGAGUUA CUGAUGAGGCCGAAAGGCCGAA AGCUUCA 1579 GGCGAGU CUGAUGAGGCCGAAAGGCCGAA AUAGCUU 1583 ACCAGGC CUGAUGAGGCCGAAAGGCCGAA AGUUAUA 1588 CCCUCUC CUGAUGAGGCCGAAAGGCCGAA AGGAGAG 1622 GGGGCAG CUGAUGAGGCCGAAAGGCCGAA AGCUGGG 1628 CCUACCG CUGAUGAGGCCGAAAGGCCGAA AGCAGGA 1648 CAUUGGG CUGAUGAGGCCGAAAGGCCGAA AGCCCCG 1660 CUGGGCA CUGAUGAGGCCGAAAGGCCGAA AGGUCAG

1663 CACCUGG CUGAUGAGGCCGAAAGGCCGAA AGCAGAG

1664 UCACCUG CUGAUGAGGCCGAAAGGCCGAA AAGCAGA

1665 ACCUCCG CUGAUGAGGCCGAAAGGCCGAA AAGCGAG

1680 GGAGGAG CUGAUGAGGCCGAAAGGCCGAA AGUCUUC

1681 UGGAGGA CUGAUGAGGCCGAAAGGCCGAA AAGUCUU 1683 AAUGGAG CUGAUGAGGCCGAAAGGCCGAA AGAAGUC 1686 CGCAAUG CUGAUGAGGCCGAAAGGCCGAA AGGAGAA 1690 UGUCCGC CUGAUGAGGCCGAAAGGCCGAA AUGGAGG

1704 AGCAGAG CUGAUGAGGCCGAAAGGCCGAA AGUCCAU

1705 GAGCAGA CUGAUGAGGCCGAAAGGCCGAA AAGUCCA 1707 AAGAGCA CUGAUGAGGCCGAAAGGCCGAA AGAAGUC 1721 CUGAUCU CUGAUGAGGCCGAAAGGCCGAA ACUCAAA 1726 AGGAGCU CUGAUGAGGCCGAAAGGCCGAA AUCUGAC 1731 ACCUUAG CUGAUGAGGCCGAAAGGCCGAA AGCUGAU 1734 AGCACCU CUGAUGAGGCCGAAAGGCCGAA AGGAGCU 1754 CUCUUGG CUGAUGAGGCCGAAAGGCCCAA AGCACUG Table 20

Human rel A HH Ribozyme Sequences

nt. Position HH Ribozyme Sequences

19 UACAGAC CUGAUGAGGCCGAAAGGCCGAA AGCCAUU

22 CACUACA CUGAUGAGGCCGAAAGGCCGAA ACGAGCC

26 CGUGCAC CUGAUGAGGCCGAAAGGCCGAA ACAGACG

93 GAGGGGG CUGAUGAGGCCGAAAGGCCGAA ACAGUUC

94 UGAGGGG CUGAUGAGGCCGAAAGGCCGAA AACAGUU 100 GGAAGAU CUGAUGAGGCCGAAAGGCCGAA AGGGGGA 103 CCGGGAA CUGAUGAGGCCGAAAGGCCGAA AUGAGGG

105 UGCCGGG CUGAUGAGGCCGAAAGGCCGAA AGAUGAG

106 CUGCCGG CUGAUGAGGCCGAAAGGCCGAA AAGAUGA 129 GGGGCCA CUGAUGAGGCCGAAAGGCCGAA AGGCCUG 138 CUCCACA CUGAUGAGGCCGAAAGGCCGAA AGGGGCC 148 GCTCAAU CUGAUGAGGCCGAAAGGCCGAA AUCUCCA 151 GCUGCUC CUGAUGAGGCCGAAAGGCCGAA AUGAUCU

180 GUAGCGG CUGAUGAGGCCGAAAGGCCGAA AGCGCAU

181 UGUAGCG CUGAUGAGGCCGAAAGGCCGAA AAGCGCA 186 GCACUUG CUGAUGAGGCCGAAAGGCCGAA AGCGGAA 204 GCCCGCG CUGAUGAGGCCGAAAGGCCGAA AGCGCCC 217 CGCCUGG CUGAUGAGGCCGAAAGGCCGAA AUGCUGC 239 UUGGUGG CUGAUGAGGCCGAAAGGCCGAA AUCUGUG 262 UGAUCUU CUGAUGAGGCCGAAAGGCCGAA AUGGUGG 268 AGCCAUU CUGAUGAGGCCGAAAGGCCGAA AUCUUGA 276 UCCUGUG CUGAUGAGGCCGAAAGGCCGAA AGCCAUU 301 CCAGGGA CUGAUGAGGCCGAAAGGCCGAA AUGCGCA 303 GACCAGG CUGAUGAGGCCGAAAGGCCGAA AGAUGCG 310 CCUUGGU CUGAUGAGGCCGAAAGGCCGAA ACCAGGG 323 CGGUGAG CUGAUGAGGCCGAAAGGCCGAA AGGGUCC 326 GGCCGGU CUGAUGAGGCCGAAAGGCCGAA AGGAGGG 335 UGGGGGU CUGAUGAGGCCGAAAGGCCGAA AGGCCGG 349 UUCCUAC CUGAUGAGGCCGAAAGGCCGAA AGCUCGU 352 CCUUUCC CUGAUGAGGCCGAAAGGCCGAA ACAAGCU

375 CUCAUAG CUGAUGAGGCCGAAAGGCCGAA AGCCAUC

376 CCUCAUA CUGAUGAGGCCGAAAGGCCGAA AAGCCAU 378 AGCCUCA CUGAUGAGGCCGAAAGGCCGAA AGAAGCC 391 CCGGGCA CUGAUGAGGCCGAAAGGCCGAA AGCUCAG 409 AACUGUG CUGAUGAGGCCGAAAGGCCGAA AUGCAGC

416 UUCUGGA CUGAUGAGGCCGAAAGGCCGAA ACUGUGG

417 GUUCUGG CUGAUGAGGCCGAAAGGCCGAA AACUGUG

418 GGUUCUG CUGAUGAGGCCGAAAGGCCGAA AAACUGU 433 CACACUG CUGAUGAGGCCGAAAGGCCGAA AUUCCCA 467 UGACUGA CUGAUGAGGCCGAAAGGCCGAA AGCCUGC 469 GCUGACU CUGAUGAGGCCGAAAGGCCGAA AUAGCCU 473 AUGCGCU CUGAUGAGGCCGAAAGGCCGAA ACUGAUA 481 UGGUCUG CUGAUGAGGCCGAAAGGCCGAA AUGCGCU 501 AACUUGG CUGAUGAGGCCGAAAGGCCGAA AGGGGUU 502 GAACUUG CUGAUGAGGCCGAAAGGCCGAA AAGGGGU

508 CUAUAGG CUGAUGAGGCCGAAAGGCCGAA ACUUGGA

509 UCUAUAG CUGAUGAGGCCCAAAGGCCGAA AACUUGG 512 UCUUCUA CUGAUGAGGCCGAAAGGCCGAA AGGAACU 514 GCUCUUC CUGAUGAGGCCGAAAGGCCGAA AUAGGAA 534 CAGGUCG CUGAUGAGGCCGAAAGGCCGAA AGUCCCC 556 GGAAGCA CUGAUGAGGCCGAAAGGCCGAA AGCCGCA

561 CACCUGG CUGAUGAGGCCGAAAGGCCGAA AGCAGAG

562 UCACCUG CUGAUGAGGCCGAAAGGCCGAA AAGCAGA 585 CCUGCCU CUGAUGAGGCCGAAAGGCCGAA AUGGGUC 598 GCAGGCG CUGAUGAGGCCGAAAGGCCGAA AGGGGCC 613 GAGGAAG CUGAUGAGGCCGAAAGGCCGAA ACAGGCG

616 GAUGAGG CUGAUGAGGCCGAAAGGCCGAA AGGACAG

617 GGAUGAG CUGAUGAGGCCGAAAGGCCGAA AAGGACA 620 AUGGGAU CUGAUGAGGCCGAAAGGCCGAA AGGAAGG 623 AAGAUGG CUGAUGAGGCCGAAAGGCCGAA AUGAGGA 628 UGUCAAA CUGAUGAGGCCGAAAGGCCGAA AUGGGAU

630 AUUGUCA CUGAUGAGGCCGAAAGGCCGAA AGAUGGG

631 GAUUGUC CUGAUGAGGCCGAAAGGCCGAA AAGAUGG 638 GGGGCAC CUGAUGAGGCCGAAAGGCCGAA AUUGUCA 661 AGAUCUU CUGAUGAGGCCGAAAGGCCGAA AGCUCGG 667 CUCGGCA CUGAUGAGGCCGAAAGGCCGAA AUCUUGA 687 GCUGCCA CUGAUGAGGCCGAAAGGCCGAA AGUUUCG 700 CCCCACC CUGAUGAGGCCGAAAGGCCGAA AGGCAGC 715 GUAGGAA CUGAUGAGGCCGAAAGGCCGAA AUCUCAU

717 CAGUAGG CUGAUGAGGCCGAAAGGCCGAA AGAUCUC

718 ACAGUAG CUGAUGAGGCCGAAAGGCCGAA AAGAUCU 721 CACACAG CUGAUGAGGCCGAAAGGCCGAA AGGAAGA 751 ACACCUC CUGAUGAGGCCGAAAGGCCGAA AUGUCCU 759 CGUGAAA CUGAUGAGGCCGAAAGGCCGAA ACACCUC

761 CCCGUGA CUGAUGAGGCCGAAAGGCCGAA AUACACC

762 UCCCGUG CUGAUGAGGCCGAAAGGCCGAA AAUACAC

763 GUCCCGU CUGAUGAGGCCGAAAGGCCGAA AAAUACA 792 CGAAAAG CUGAUGAGGCCGAAAGGCCGAA AGCCUCG

795 UUGCGAA CUGAUGAGGCCGAAAGGCCGAA AGGAGCC

796 CUUGCGA CUGAUGAGGCCGAAAGGCCGAA AAGGAGC

797 GCUUGCG CUGAUGAGGCCGAAAGGCCGAA AAAGGAG

798 AGCUUGC CUGAUGAGGCCGAAAGGCCGAA AAAAGGA 829 GGAACAC CUGAUGAGGCCGAAAGGCCGAA AUGGCCA

834 GGUCCGG CUGAUGAGGCCGAAAGGCCGAA ACACAAU

835 GGGUCCG CUGAUGAGGCCGAAAGGCCGAA AACACAA 845 GCGUAGG CUGAUGAGGCCGAAAGGCCGAA AGGGGUC 849 GUCUGCG CUGAUGAGGCCGAAAGGCCGAA AGGGAGG 372 CGCACAG CUGAUGAGGCCGAAAGGCCGAA AGCCUGC 883 GCAUGGA CUGAUGAGGCCGAAAGGCCGAA ACACGCA 885 CUGCAUG CUGAUGAGGCCGAAAGGCCGAA AGACACG

905 CGGUCGG CUGAUGAGGCCGAAAGGCCGAA AGGCCGC

906 CCGGUCG CUGAUGAGGCCGAAAGGCCGAA AAGGCCG 919 GCUCACU CUGAUGAGGCCGAAAGGCCGAA AGCUCCC 936 GUACUGG CUGAUGAGGCCGAAAGGCCCAA AUUCCAU

937 GGUACUG CUGAUGAGGCCGAAAGGCCGAA AAUUCCA 942 UGGCAGG CUGAUGAGGCCGAAAGGCCGAA ACUGGAA 953 UCGUCUG CUGAUGAGGCCGAAAGGCCGAA AUCUGGC 962 CGGUGAC CUGAUGAGGCCGAAAGGCCGAA AUCGUCU 965 AUCCGGU CUGAUGAGGCCGAAAGGCCGAA ACGAUCG 973 UCUCCUC CUGAUGAGGCCGAAAGGCCGAA AUCCGGU 986 GUCCUUU CUGAUGAGGCCGAAAGGCCGAA ACGUUUC 996 GGUCUCA CUGAUGAGGCCGAAAGGCCGAA AUGUCCU

1005 GCUCUUG CUGAUGAGGCCGAAAGGCCGAA AGGUCUC

1006 UGCUCUU CUGAUGAGGCCGAAAGGCCGAA AAGGUCU 1015 UCUUCAU CUGAUGAGGCCGAAAGGCCGAA AUGCUCU 1028 CUGAAAG CUGAUGAGGCCGAAAGGCCGAA ACUCUUC

1031 CCGCUGA CUGAUGAGGCCGAAAGGCCGAA AGGACUC

1032 UCCGCUG CUGAUGAGGCCGAAAGGCCGAA AAGGACU

1033 GUCCGCU CUGAUGAGGCCGAAAGGCCGAA AAAGGAC 1058 CGAGGUG CUGAUGAGGCCGAAAGGCCGAA AGGCCGG 1064 AUGCGUC CUGAUGAGGCCGAAAGGCCGAA AGGUGGA 1072 GCACAGC CUGAUGAGGCCGAAAGGCCGAA AUGCGUC

1082 CUGCGGG CUGAUGAGGCCGAAAGGCCGAA AGGCACA

1083 GCUGCGG CUGAUGAGGCCGAAAGGCCGAA AAGGCAC 1092 AGAAGCU CUGAUGAGGCCGAAAGGCCGAA AGCUGCG

1097 GGGACAG CUGAUGAGGCCGAAAGGCCGAA AGCUGAG

1098 GGGGACA CUGAUGAGGCCGAAAGGCCGAA AAGCUGA 1102 GCUUGGG CUGAUGAGGCCGAAAGGCCGAA ACAGAAG 1125 AAAGGGA CUGAUGAGGCCGAAAGGCCGAA AGGGCUG 1127 GUAAAGG CUGAUGAGGCCGAAAGGCCGAA AUAGGGC

1131 UGACGUA CUGAUGAGGCCGAAAGGCCGAA AGGGAUA

1132 AUGACGU CUGAUGAGGCCGAAAGGCCGAA AAGGGAU

1133 GAUGACG CUGAUGAGGCCGAAAGGCCGAA AAAGGGA 1137 CAGGGAU CUGAUGAGGCCGAAAGGCCGAA ACGUAAA 1140 GCUCAGG CUGAUGAGGCCGAAAGGCCGAA AUGACGU 1153 CAUAGUU CUGAUGAGGCCGAAAGGCCGAA AUGGUGC 1158 CUCAUCA CUGAUGAGGCCGAAAGGCCGAA AGUUGAU

1167 GGUGGGA CUGAUGAGGCCGAAAGGCCGAA ACUCAUC

1168 UGGUGGG CUGAUGAGGCCGAAAGGCCGAA AACUCAU

1169 AUGGUGG CUGAUGAGGCCGAAAGGCCGAA AAACUCA

1182 AGAAGGA CUGAUGAGGCCGAAAGGCCGAA ACACCAU

1183 CAGAAGG CUGAUGAGGCCGAAAGGCCGAA AACACCA

1184 CCAGAAG CUGAUGAGGCCGAAAGGCCGAA AAACACC

1187 UGCCCAG CUGAUGAGGCCGAAAGGCCGAA AGGAAAC

1188 CUGCCCA CUGAUGAGGCCGAAAGGCCGAA AAGGAAA 1198 CCUGGCU CUGAUGAGGCCGAAAGGCCGAA AUCUGCC 1209 CAAGGCC CUGAUGAGGCCGAAAGGCCGAA AGGCCUG 1215 CGGGGCC CUGAUGAGGCCGAAAGGCCGAA AGGCCGA 1229 ACUUGGG CUGAUGAGGCCGAAAGGCCGAA AGGGGCC 1237 GGGGCAG CUGAUGAGGCCGAAAGGCCGAA ACUUGGG 1250 GGGGCUG CUGAUGAGGCCGAAAGGCCGAA AGCCUGG 1268 AUGGCUG CUGAUGAGGCCGAAAGGCCGAA AGCAGGG 1279 GAGCUGA CUGAUGAGGCCGAAAGGCCGAA ACCAUGG

1281 CAGAGCU CUGAUGAGGCCGAAAGGCCGAA AUACCAU

1286 UGGGCCA CUGAUGAGGCCGAAAGGCCGAA AGCUGAU

1309 GGACUGG CUGAUGAGGCCGAAAGGCCGAA ACAGGGG

1315 GGGCUAG CUGAUGAGGCCGAAAGGCCGAA ACUGGGA

1318 CUGGGGC CUGAUGAGGCCGAAAGGCCGAA AGGACUG

1331 GCCUGAG CUGAUGAGGCCGAAAGGCCGAA AGGGCCU

1334 ACAGCCU CUGAUGAGGCCGAAAGGCCGAA AGGAGGG

1389 GGCCUCU CUGAUGAGGCCGAAAGGCCGAA ACAGCGU

1413 AUCAUCA CUGAUGAGGCCGAAAGGCCGAA ACUGCAG

1414 CAUCAUC CUGAUGAGGCCGAAAGGCCGAA AACUGCA 1437 GCCAAGC CUGAUGAGGCCGAAAGGCCGAA AGGCCCC 1441 UGUUGCC CUGAUGAGGCCGAAAGGCCGAA AGCAAGG

1467 GUCUGUG CUGAUGAGGCCGAAAGGCCGAA ACACAGC

1468 GGUCUGU CUGAUGAGGCCGAAAGGCCGAA AACACAG 1482 GUCGACG CUGAUGAGGCCGAAAGGCCGAA AUGCCAG I486 AGUUGUC CUGAUGAGGCCGAAAGGCCGAA ACGGAUG 1494 AAACUCG CUGAUGAGGCCGAAAGGCCGAA AGUUGUC

1500 CUGCUGA CUGAUGAGGCCGAAAGGCCGAA ACUCGGA

1501 GCUGCUG CUGAUGAGGCCGAAAGGCCGAA AACUCGG

1502 AGCUGCU CUGAUGAGGCCGAAAGGCCGAA AAACUCG 1525 CCACAGG CUGAUGAGGCCGAAAGGCCGAA AUGCCCU 1566 CUCAGGG CUGAUGAGGCCGAAAGGCCGAA ACUCCAU 1577 CGAGUUA CUGAUGAGGCCGAAAGGCCGAA AGCCUCA 1579 GGCGAGU CUGAUGAGGCCGAAAGGCCGAA AUAGCCU 1583 ACUAGGC CUGAUGAGGCCGAAAGGCCGAA AGUUAUA 1588 CUGUCAC CUGAUGAGGCCGAAAGGCCGAA AGGCGAG 1622 GGAGCAG CUGAUGAGGCCGAAAGGCCGAA AGCUGGG 1628 CCCAGUG CUGAUGAGGCCGAAAGGCCGAA AGCAGGA 1648 CAUUGGG CUGAUGAGGCCGAAAGGCCGAA AGCCCCG 1660 CUGAAAG CUGAUGAGGCCGAAAGGCCGAA AGGCCAU

1663 CUCCUGA CUGAUGAGGCCGAAAGGCCGAA AGGAGGC

1664 UCUCCUG CUGAUGAGGCCGAAAGGCCGAA AAGGAGG

1665 AUCUCCU CUGAUGAGGCCGAAAGGCCGAA AAAGGAG

1680 GGAGGAG CUGAUGAGGCCGAAAGGCCGAA AGUCUUC

1704 GGCUGAG CUGAUGAGGCCGAAAGGCCGAA AGUCCAU

1705 GGGCUGA CUGAUGAGGCCGAAAGGCCGAA AAGUCCA 1707 CAGGGCU CUGAUGAGGCCGAAAGGCCGAA AGAAGUC 1721 CUGAUCU CUGAUGAGGCCGAAAGGCCGAA ACUCAGC 1726 AGGAGCU CUGAUGAGGCCGAAAGGCCGAA AUCUGAC 1731 CCCUUAG CUGAUGAGGCCGAAAGGCCGAA AGCUGAU 1734 ACCCCCU CUGAUGAGGCCGAAAGGCCGAA AGGAGCU 1754 CUCUGGG CUGAUGAGGCCGAAAGGCCGAA AGGGCAG Table 21

Human rel A Hairpin Ribozyme/Tar)

nt. Position HHaaiirrppin Ribozyme sequence Substrate

90 UGAGGGGG AGAA GUUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GAACU GUU CCCCCUCA

156 GCUGCUUG AGAA GCUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GAGCA GCC CAAGCAGC

362 GCCAUCCC AGAA GUCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGACU GCC GGGAUGGC

413 GUUCUGGA AGAA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCACA GUU UCCAGAAC

606 GAAGGACA AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUGCC GCC UGUCCUUC

652 UUGAGCUC AGAA GUGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACACU GCC GAGCUCAA

695 CCCACCGA AGAA GCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAGCU GCC UCGGUGGG

853 AGGCUGGG AGAA GCGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACGCA GAC CCCAGCCU

900 GGUCGGAA AGAA GCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGGCG GCC UUCCGACC

955 UGACGAUC AGAA GUAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AUACA GAC GAUCGUCA

1037 GUCGGUGG AGAA GCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAGCG GAC CCACCGAC

1045 GGCCGGGG AGAA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCACC GAC CCCCGGCC

1410 CAUCAUCA AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUGCA GUU UGAUGAUG

1453 ACAGCUGG AGAA GUGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCACA GAC CCAGCUGU

1471 GAUGCCAG AGAA GUGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UCACA GAC CUGGCAUC

Table 22

Mouse rel A Hairpin Ribozyme/Target Sequences

nt. Position Hairpin Ribozyme sequence Substrate

137 GUUGCUUC AGAA GUUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GAACA GCC GAAGCAAC

273 GAGAUUCG AGAA GUUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GAACA GUU CGAAUCUC

343 GCCAUCCC AGAA GUCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGACU GCC GGGAUGGC

366 GGGCAGAG AGAA GCCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGGCU GAC CUCUGCCC

633 UUGAGCUC AGAA GUGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACACU GCC GAGCUCAA

676 CCCACCGA AGAA GCUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GAGCU GCC UCGGUGGG

834 AGGCUGGG AGAA GCGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACGCC GAC CCCAGCCU

881 GAUCAGAA AGAA GCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGGCG GCC UUCUGAUC

1100 AGGUGUAG AGAA GCGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCGCA GCC CUACACCU

1205 GGGCAGAG AGAA GUGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCACC GUC CUCUGCCC

1361 GGGCUUCC AGAA GCGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACGCU GUC GGAAGCCC

1385 CAGCAUCA AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUGCA GUU UGAUGCUG

1431 ACUCCUGG AGAA GUGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCACA GAC CCAGGAGU

1449 GAUGCCAG AGAA GUGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UCACA GAC CUGGCAUC

1802 AAGUCGGG AGAA GCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAGCU GCC CCCGACUU

2009 UGGCUCCA AGAA GUCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGACA GAC UGGAGCCA

2124 UGGUGUCG AGAA GCAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GUGCU GCC CGACACCA

2233 AUUCUGAA AGAA GCCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGGCC GCC UUCAGAAU

2354 UCAGUAAA AGAA GUCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGACA GCC UUUACUGA

Table 23: Human TNF-α HH Ribozyme Target Sequence

nt. HH Target Sequence nt. HH Target Sequence

Position Position

28 GGCAGGU U CUCUUCC

29 GCAGGUU C UCUUCCU 321 GUCAGAU C AUCUUCU

31 AGGUUCU C UUCCUCU 324 AGAUCAU C UUCUCGA

33 GUUCUCU U CCUCUCA 326 AUCAUCU U CUCGAAC

34 UUCUCUU C CUCUCAC 327 UCAUCUU C UCGAACC

37 UCUUCCU C UCACAUA 329 AUCUUCU C GAACCCC

39 UUCCUCU C ACAUACU 352 AGCCUGU A GCCCAUG

44 CUCACAU A CUGACCC 361 CCCAUGU U GUAGCAA

58 CACGGCU C CACCCUC 364 AUGUUGU A GCAAACC

65 CCACCCU C UCUCCCC 374 AAACCCU C AAGCUGA

67 ACCCUCU C UCCCCUG 391 GGCAGCU C CAGUGGC

69 CCUCUCU C CCCUGGA 421 AUGCCCU C CUGGCCA

106 GCAUGAU C CGGGACG 449 GAGAGAU A ACCAGCU

136 AGGCGCU C CCCAAGA 468 GUGCCAU C AGAGGGC

165 CAGGGCU C CAGGCGG 480 GGCCUGU A CCUCAUC

177 CGGUGCU U GUUCCUC 484 UGUACCU C AUCUACU

180 UGCUUGU U CCUCAGC 487 ACCUCAU C UACUCCC

181 GCUUGUU C CUCAGCC 489 CUCAUCU A CUCCCAG 184 UGUUCCU C AGCCUCU 492 AUCUACU C CCAGGUC

190 UCAGCCU C UUCUCCU 499 CCCAGGU C CUCUUCA

192 AGCCUCU U CUCCUUC 502 AGGUCCU C UUCAAGG

193 GCCUCUU C UCCUUCC 504 GUCCUCU U CAAGGGC

195 CUCUUCU C CUUCCUG 505 UCCUCUU C AAGGGCC

198 UUCUCCU U CCUGAUC 525 UGCCCCU C CACCCAU

199 UCUCCUU C CUGAUCG 538 AUGUGCU C CUCACCC

205 UCCUGAU C GUGGCAG 541 UGCUCCU C ACCCACA

226 CCACGCU C UUCUGCC 553 ACACCAU C AGCCGCA

228 ACGCUCU U CUGCCUG 562 GCCGCAU C GCCGUCU

229 CGCUCUU C UGCCUGC 568 UCGCCGU C UCCUACC

243 CUGCACU U UGGAGUG 570 GCCGUCU C CUACCAG

244 UGCACUU U GGAGUGA 573 GUCUCCU A CCAGACC

253 GAGUGAU C GGCCCCC 586 CCAAGGU C AACCUCC

273 GAAGAGU C CCCCAGG 592 UCAACCU C CUCUCUG

286 GGGACCU C UCUCUAA 595 ACCUCCU C UCUGCCA

288 GACCUCU C UCUAAUC 597 CUCCUCU C UGCCAUC

290 CCUCUCU C UAAUCAG 604 CUGCCAU C AAGAGCC

292 UCUCUCU A AUCAGCC 657 CCCUGGU A UGAGCCC

295 CUCUAAU C AGCCCUC 667 AGCCCAU C UAUCUGG

302 CAGCCCU C UGGCCCA 669 CCCAUCU A UCUGGGA 671 CAUCUAU C UGGGAGG 960 UGGGAUU C AGGAAUG

682 GAGGGGU C UUCCAGC 1001 AACCACU A AGAAUUC

684 GGGGUCU U CCAGCUG 1007 UAAGAAU U CAAACUG

685 GGGUCUU C CAGCUGG 1008 AAGAAUU C AAACUGG

709 ACCGACU C AGCGCUG 1021 GGGGCCU C CAGAACU

721 CUGAGAU C AAUCGGC 1029 CAGAACU C ACUGGGG

725 GAUCAAU C GGCCCGA 1040 GGGGCCU A CAGCUUU

735 CCCGACU A UCUCGAC 1046 UACAGCU U UGAUCCC

737 CGACUAU C UCGACUU 1047 ACAGCUU U GAUCCCU

739 ACUAUCU C GACUUUG 1051 CUUUGAU C CCUGACA

744 CUCGACU U UGCCGAG 1060 CUGACAU C UGGAAUC

745 UCGACUU U GCCGAGU 1067 CUGGAAU C UGGAGAC

753 GCCGAGU C UGGGCAG 1085 GGAGCCU U UGGUUCU

763 GGCAGGU C UACUUUG 1086 GAGCCUU U GGUUCUG

765 CAGGUCU A CUUUGGG 1090 CUUUGGU U CUGGCCA

768 GUCUACU U UGGGAUC 1091 UUUGGUU C UGGCCAG

769 UCUACUU U GGGAUCA 1113 CAGGACU U GAGAAGA

775 UUGGGAU C AUUGCCC 1124 AAGACCU C ACCUAGA

778 GGAUCAU U GCCCUGU 1129 CUCACCU A GAAAUUG

801 CGAACAU C CAACCUU 1135 UAGAAAU U GACACAA

808 CCAACCU U CCCAAAC 1151 UGGACCU U AGGCCUU

809 CAACCUU C CCAAACG 1152 GGACCUU A GGCCUUC

820 AACGCCU C CCCUGCC 1158 UAGGCCU U CCUCUCU

833 CCCCAAU C CCUUUAU 1159 AGGCCUU C CUCUCUC

837 AAUCCCU U UAUUACC 1162 CCUUCCU C UCUCCAG

838 AUCCCUU U AUUACCC 1164 UUCCUCU C UCCAGAU

839 UCCCUUU A UUACCCC 1166 CCUCUCU C CAGAUGU

841 CCUUUAU U ACCCCCU 1174 CAGAUGU U UCCAGAC

842 CUUUAUU A CCCCCUC 1175 AGAUGUU U CCAGACU

849 ACCCCCU C CUUCAGA 1176 GAUGUUU C CAGACUU

852 CCCUCCU U CAGACAC 1183 CCAGACU U CCUUGAG

853 CCUCCUU C AGACACC 1184 CAGACUU C CUUGAGA

863 ACACCCU C AACCUCU 1187 ACUUCCU U GAGACAC

869 UCAACCU C UUCUGGC 1208 CAGCCCU C CCCAUGG

871 AACCUCU U CUGGCUC 1224 GCCAGCU C CCUCUAU

872 ACCUCUU C UGGCUCA 1228 GCUCCCU C UAUUUAU

878 UCUGGCU C AAAAAGA 1230 UCCCUCU A UUUAUGU

890 AGAGAAU U GGGGGCU 1232 CCUCUAU U UAUGUUU

898 GGGGGCU U AGGGUCG 1233 CUCUAUU U AUGUUUG

899 GGGGCUU A GGGUCGG 1234 UCUAUUU A UGUUUGC

904 UUAGGGU C GGAACCC 1238 UUUAUGU U UGCACUU

917 CCAAGCU U AGAACUU 1239 UUAUGUU U GCACUUG

918 CAAGCUU A GAACUUU 1245 UUGCACU U GUGAUUA

924 UAGAACU U UAAGCAA 1251 UUGUGAU U AUUUAUU

925 AGAACUU U AAGCAAC 1252 UGUGAUU A UUUAUUA 926 GAACUUU A AGCAACA 1254 UGAUUAU U UAUUAUU

945 CACCACU U CGAAACC 1255 GAUUAUU U AUUAUUU

946 ACCACUU C GAAACCU 1256 AUUAUUU A UUAUUUA

959 CUGGGAU U CAGGAAU 1258 UAUUUAU U AUUUAUU 1259 AUUUAUU A UUUAUUU 1440 UGUUUUU U AAAAUAU

1261 UUAUUAU U UAUUUAU 1441 GUUUUUU A AAAUAUU

1262 UAUUAUU U AUUUAUU 1446 UUAAAAU A UUAUCUG

1263 AUUAUUU A UUUAUUA 1448 AAAAUAU U AUCUGAU

1265 UAUUUAU U UAUUAUU 1449 AAAUAUU A UCUGAUU

1266 AUUUAUU U AUUAUUU 1451 AUAUUAU C UGAUUAA

1267 UUUAUUU A UUAUUUA 1456 AUCUGAU U AAGUUGU

1269 UAUUUAU U AUUUAUU 1457 UCUGAUU A AGUUGUC

1270 AUUUAUU A UUUAUUU 1461 AUUAAGU U GUCUAAA

1272 UUAUUAU U UAUUUAU 1464 AAGUUGU C UAAACAA

1273 UAUUAUU U AUUUAUU 1466 GUUGUCU A AACAAUG

1274 AUUAUUU A UUUAUUU 1479 UGCUGAU U UGGUGAC

1276 UAUUUAU U UAUUUAC 1480 GCUGAUU U GGUGACC

1277 AUUUAUU U AUUUACA 1494 CAACUGU C ACUCAUU

1278 UUUAUUU A UUUACAG 1498 UGUCACU C AUCGCUG

1280 UAUUUAU U UACAGAU 1501 CACUCAU U GCUGAGG

1281 AUUUAUU U ACAGAUG 1512 GAGGCCU C UGCUCCC

1282 UUUAUUU A CAGAUGA 1517 CUCUGCU C CCCAGGG

1294 UGAAUGU A UUUAUUU 1528 AGGGAGU U GUGCCUG

1296 AAUGUAU U UAUUUGG 1533 GUUGUGU C UGUAAUC

1297 AUGUAUU U AUUUGGG 1537 UGUCUGU A AUCGGCC

1298 UGUAUUU A UUUGGGA 1540 CUGUAAU C GGCCUAC

1300 UAUUUAU U UGGGAGA 1546 UCGGCCU A CUAUUCA

1301 AUUUAUU U GGGAGAC 1549 GCCUACU A UUCAGUG

1315 CCGGGGU A UCCUGGG 1551 CUACUAU U CAGUGGC

1317 GGGGUAU C CUGGGGG 1552 UACUAUU C AGUGGCG

1334 CCAAUGU A GGAGCUG 1566 GAGAAAU A AAGGUUG

1345 GCUGCCU U GGCUCAG 1572 UAAAGGU U GCUUAGG

1350 CUUGGCU C AGACAUG 1576 GGUUGCU U AGGAAAG

1359 GACAUGU U UUCCGUG 1577 GUUGCUU A GGAAAGA

1360 ACAUGUU U UCCGOGA

1361 CAUGUUU U CCGUGAA

1362 AUGUUUU C CGUGAAA

1386 GAACAAU A GGCUGUU

1393 AGGCUGU U CCCAUGU

1394 GGCUGUU C CCAUGUA

1401 CCCAUGU A GCCCCCU

1414 CUGGCCU C UGUGCCU

1422 UGUGCCU U CUUUUGA

1423 GUGCCUU C UUUUGAU

1425 GCCUUCU U UUGAUUA

1426 CCUUCUU U UGAUUAU

1427 CUUCUUU U GAUUAUG

1431 UUUUGAU U AUGUUUU

1432 UUUGAUU A UGUUUUU

1436 AUUAUGU U UUUUAAA

1437 UUAUGUU U UUUAAAA

1438 UAUGUUU U UUAAAAU Table 24: Human TNF-α Hammerhead Ribozyme Sequences

nt . HH Ribozyme Se quence

Position

28 GGAAGAG CUGAUGAGGCCGAAAGGCCGAA ACCUGCC

29 AGGAAGA CUGAUGAGGCCGAAAGGCCGAA AACCUGC

31 AGAGGAA CUGAUGAGGCCGAAAGGCCGAA AGAACCU

33 UGAGAGG CUGAUGAGGCCGAAAGGCCGAA AGAGAAC

34 GUGAGAG CUGAUGAGGCCGAAAGGCCGAA AAGAGAA

37 UAUGUGA CUGAUGAGGCCGAAAGGCCGAA AGGAAGA

39 AGUAUGU CUGAUGAGGCCGAAAGGCCGAA AGAGGAA

44 GGGUCAG CUGAUGAGGCCGAAAGGCCGAA AUGUGAG

58 GAGGGUG CUGAUGAGGCCGAAAGGCCGAA AGCCGUG

65 GGGGAGA CUGAUGAGGCCGAAAGGCCGAA AGGGUGG

67 CAGGGGA CUGAUGAGGCCGAAAGGCCGAA AGAGGGU

69 UCCAGGG CUGAUGAGGCCGAAAGGCCGAA AGAGAGG

106 CGUCCCG CUGAUGAGGCCGAAAGGCCGAA AUCAUGC

136 UCUUGGG CUGAUGAGGCCGAAAGGCCGAA AGCGCCU

165 CCGCCUG CUGAUGAGGCCGAAAGGCCGAA AGCCCUG

177 GAGGAAC CUGAUGAGGCCGAAAGGCCGAA AGCACCG

130 GCUGAGG CUGAUGAGGCCGAAAGGCCGAA ACAAGCA

181 GGCUGAG CUGAUGAGGCCGAAAGGCCGAA AACAAGC

184 AGAGGCU CUGAUGAGGCCGAAAGGCCGAA AGGAACA

190 AGGAGAA CUGAUGAGGCCGAAAGGCCGAA AGGCUGA

192 GAAGGAG CUGAUGAGGCCGAAAGGCCGAA AGAGGCU

193 GGAAGGA CUGAUGAGGCCGAAAGGCCGAA AAGAGGC

195 CAGGAAG CUGAUGAGGCCGAAAGGCCGAA AGAAGAG

198 GAUCAGG CUGAUGAGGCCGAAAGGCCGAA AGGAGAA

199 CGAUCAG CUGAUGAGGCCGAAAGGCCGAA AAGGAGA

205 CUGCCAC CUGAUGAGGCCGAAAGGCCGAA AUCAGGA

226 GGCAGAA CUGAUGAGGCCGAAAGGCCGAA AGCGUGG

228 CAGGCAG CUGAUGAGGCCGAAAGGCCGAA AGAGCGU

229 GCAGGCA CUGAUGAGGCCGAAAGGCCGAA AAGAGCG

243 CACUCCA CUGAUGAGGCCGAAAGGCCGAA AGUGCAG

244 UCACUCC CUGAUGAGGCCGAAAGGCCGAA AAGUGCA

253 GGGGGCC CUGAUGAGGCCGAAAGGCCGAA AUCACUC

273 CCUGGGG CUGAUGAGGCCGAAAGGCCGAA ACUCUUC

286 UUAGAGA CUGAUGAGGCCGAAAGGCCGAA AGGUCCC

288 GAUUAGA CUGAUGAGGCCGAAAGGCCGAA AGAGGUC

290 CUGAUUA CUGAUGAGGCCGAAAGGCCGAA AGAGAGG

292 GGCUGAU CUGAUGAGGCCGAAAGGCCGAA AGAGAGA

295 GAGGGCU CUGAUGAGGCCGAAAGGCCGAA AUUAGAG

302 UGGGCCA CUGAUGAGGCCGAAAGGCCGAA AGGGCUG 321 AGAAGAU CUGAUGAGGCCGAAAGGCCGAA AUCUGAC

324 UCGAGAA CUGAUGAGGCCGAAAGGCCGAA AUGAUCU

326 GUUCGAG CUGAUGAGGCCGAAAGGCCGAA AGAUGAU

327 GGUUCGA CUGAUGAGGCCGAAAGGCCGAA AAGAUGA 329 GGGGUUC CUGAUGAGGCCGAAAGGCCGAA AGAAGAU 352 CAUGGGC CUGAUGAGGCCGAAAGGCCGAA ACAGGCU 361 UUGCUAC CUGAUGAGGCCGAAAGGCCGAA ACAUGGG 364 GGUUUGC CUGAUGAGGCCGAAAGGCCGAA ACAACAU 374 UCAGCUU CUGAUGAGGCCGAAAGGCCGAA AGGGUUU 391 GCCACUG CUGAUGAGGCCGAAAGGCCGAA AGCUGCC 421 UGGCCAG CUGAUGAGGCCGAAAGGCCGAA AGGGCAU 449 AGCUGGU CUGAUGAGGCCGAAAGGCCGAA AUCUCUC 468 GCCCUCU CUGAUGAGGCCGAAAGGCCGAA AUGGCAC 480 GAUGAGG CUGAUGAGGCCGAAAGGCCGAA ACAGGCC 484 AGUAGAU CUGAUGAGGCCGAAAGGCCGAA AGGUACA 487 GGGAGUA CUGAUGAGGCCGAAAGGCCGAA AUGAGGU 489 CUGGGAG CUGAUGAGGCCGAAAGGCCGAA AGAUGAG 492 GACCUGG CUGAUGAGGCCGAAAGGCCGAA AGUAGAU 499 UGAAGAG CUGAUGAGGCCGAAAGGCCGAA ACCUGGG 502 CCUUGAA CUGAUGAGGCCGAAAGGCCGAA AGGACCU

504 GCCCUUG CUGAUGAGGCCGAAAGGCCGAA AGAGGAC

505 GGCCCUU CUGAUGAGGCCGAAAGGCCGAA AAGAGGA 525 AUGGGUG CUGAUGAGGCCGAAAGGCCGAA AGGGGCA 538 GGGUGAG CUGAUGAGGCCGAAAGGCCGAA AGCACAU 541 UGUGGGU CUGAUGAGGCCGAAAGGCCGAA AGGAGCA 553 UGCGGCU CUGAUGAGGCCGAAAGGCCGAA AUGGUGU

562 AGACGGC CUGAUGAGGCCGAAAGGCCGAA AUGCGGC

563 GGUAGGA CUGAUGAGGCCGAAAGGCCGAA ACGGCGA 570 CUGGUAG CUGAUGAGGCCGAAAGGCCGAA AGACGGC 573 GGUCUGG CUGAUGAGGCCGAAAGGCCGAA AGGAGAC 586 GGAGGUU CUGAUGAGGCCGAAAGGCCGAA ACCUUGG 592 CAGAGAG CUGAUGAGGCCGAAAGGCCGAA AGGUUGA 595 UGGCAGA CUGAUGAGGCCGAAAGGCCGAA AGGAGGU 597 GAUGGCA CUGAUGAGGCCGAAAGGCCGAA AGAGGAG 604 GGCUCUU CUGAUGAGGCCGAAAGGCCGAA AUGGCAG 657 GGGCUCA CUGAUGAGGCCGAAAGGCCGAA ACCAGGG 667 CCAGAUA CUGAUGAGGCCGAAAGGCCGAA AUGGGCU 669 UCCCAGA CUGAUGAGGCCGAAAGGCCGAA AGAUGGG 671 CCUCCCA CUGAUGAGGCCGAAAGGCCGAA AUAGAUG 682 GCUGGAA CUGAUGAGGCCGAAAGGCCGAA ACCCCUC

684 CAGCUGG CUGAUGAGGCCGAAAGGCCGAA AGACCCC

685 CCAGCUG CUGAUGAGGCCGAAAGGCCGAA AAGACCC 709 CAGCGCU CUGAUGAGGCCGAAAGGCCGAA AGUCGGU 721 GCCGAUU CUGAUGAGGCCGAAAGGCCGAA AUCUCAG 725 UCGGGCC CUGAUGAGGCCGAAAGGCCGAA AUUGAUC 735 GUCGAGA CUGAUGAGGCCCAAAGGCCGAA AGUCGGG 737 AAGUCGA CUGAUGAGGCCGAAAGGCCGAA AUAGUCG 739 CAAAGUC CUGAUGAGGCCGAAAGGCCGAA AGAUAGU 744 CUCGGCA CUGAUGAGGCCGAAAGGCCGAA AGUCGAG 745 ACUCGGC CUGAUGAGGCCGAAAGGCCGAA AAGUCGA

753 CUGCCCA CUGAUGAGGCCGAAAGGCCGAA ACUCGGC

763 CAAAGUA CUGAUGAGGCCGAAAGGCCGAA ACCUGCC

765 CCCAAAG CUGAUGAGGCCGAAAGGCCGAA AGACCUG

768 GAUCCCA CUGAUGAGGCCGAAAGGCCGAA AGUAGAC

769 UGAUCCC CUGAUGAGGCCGAAAGGCCGAA AAGUAGA 775 GGGCAAU CUGAUGAGGCCGAAAGGCCGAA AUCCCAA 778 ACAGGCC CUGAUGAGGCCGAAAGGCCGAA AUGAUCC 801 AAGGUUG CUGAUGAGGCCGAAAGGCCGAA AUGUUCG

808 GUUUGGG CUGAUGAGGCCGAAAGGCCGAA AGGUUGG

809 CGUUUGG CUGAUGAGGCCGAAAGGCCGAA AAGGUUG 820 GGCAGCG CUGAUGAGGCCGAAAGGCCGAA AGGCCUU 833 AUAAAGG CUGAUGAGGCCGAAAGGCCGAA AUUGGGG

837 GGUAAUA CUGAUGAGGCCGAAAGGCCGAA AGGGAUU

838 GGGUAAU CUGAUGAGGCCGAAAGGCCGAA AAGGGAU

839 GGGGUAA CUGAUGAGGCCGAAAGGCCGAA AAAGGGA

841 AGGGGGU CUGAUGAGGCCGAAAGGCCGAA AUAAAGG

842 GAGGGGG CUGAUGAGGCCGAAAGGCCGAA AAUAAAG 849 UCUGAAG CUGAUGAGGCCGAAAGGCCGAA AGGGGGU

852 GUGUCUG CUGAUGAGGCCGAAAGGCCGAA AGGAGGG

853 GGUGUCU CUGAUGAGGCCGAAAGGCCGAA AAGGAGG 863 AGAGGUU CUGAUGAGGCCGAAAGGCCGAA AGGGUGU 869 GCCAGAA CUGAUGAGGCCGAAAGGCCGAA AGGUUGA

871 GAGCCAG CUGAUGAGGCCGAAAGGCCGAA AGAGGUU

872 UGAGCCA CUGAUGAGGCCGAAAGGCCGAA AAGAGGU 878 UCUUUUU CUGAUGAGGCCGAAAGGCCGAA AGCCAGA 890 AGCCCCC CUGAUGAGGCCGAAAGGCCGAA AUUCUCU

898 CGACCCU CUGAUGAGGCCGAAAGGCCGAA AGCCCCC

899 CCGACCC CUGAUGAGGCCGAAAGGCCGAA AAGCCCC 904 GGGUUCC CUGAUGAGGCCGAAAGGCCGAA ACCCUAA

917 AAGUUCU CUGAUGAGGCCGAAAGGCCGAA AGCUUGG

918 AAAGUUC CUGAUGAGGCCGAAAGGCCGAA AAGCUUG

924 UUGCUUA CUGAUGAGGCCGAAAGGCCGAA AGUUCUA

925 GUUGCUU CUGAUGAGGCCGAAAGGCCGAA AAGUUCU

926 UGUUGCU CUGAUGAGGCCGAAAGGCCGAA AAAGUUC

945 GGUUUCG CUGAUGAGGCCGAAAGGCCGAA AGUGGUG

946 AGGUUUC CUGAUGAGGCCGAAAGGCCGAA AAGUGGU

959 AUUCCUG CUGAUGAGGCCGAAAGGCCGAA AUCCCAG

960 CAUUCCU CUGAUGAGGCCGAAAGGCCGAA AAUCCCA 1001 GAAUUCU CUGAUGAGGCCGAAAGGCCGAA AGUGGUU

1007 CAGUUUG CUGAUGAGGCCGAAAGGCCGAA AUUCUUA

1008 CCAGUUU CUGAUGAGGCCGAAAGGCCGAA AAUUCUU 1021 AGUUCUG CUGAUGAGGCCGAAAGGCCGAA AGGCCCC 1029 CCCCAGU CUGAUGAGGCCGAAAGGCCGAA AGUUCUG 1040 AAAGCUG CUGAUGAGGCCGAAAGGCCGAA AGGCCCC

1046 GGGAUCA CUGAUGAGGCCGAAAGGCCGAA AGCUGUA

1047 AGGGAUC CUGAUGAGGCCGAAAGGCCGAA AAGCUGU 1051 UGUCAGG CUGAUGAGGCCGAAAGGCCGAA AUCAAAG 1060 GAUUCCA CUGAUGAGGCCGAAAGGCCGAA AUGUCAG 1067 GUCUCCA CUGAUGAGGCCGAAAGGCCGAA AUUCCAG

1085 AGAACCA CUGAUGAGGCCGAAAGGCCGAA AGGCUCC

1086 CAGAACC CUGAUGAGGCCGAAAGGCCGAA AAGGCUC

1090 UGGCCAG CUGAUGAGGCCGAAAGGCCGAA ACCAAAG

1091 CUGGCCA CUGAUGAGGCCGAAAGGCCGAA AACCAAA 1113 UCUUCUC CUGAUGAGGCCGAAAGGCCGAA AGUCCUG 1124 UCUAGGU CUGAUGAGGCCGAAAGGCCGAA AGGUCUU 1129 CAAUUUC CUGAUGAGGCCGAAAGGCCGAA AGGUGAG 1135 UUGUGUC CUGAUGAGGCCGAAAGGCCGAA AUUUCUA

1151 AAGGCCU CUGAUGAGGCCGAAAGGCCGAA AGGUCCA

1152 GAAGGCC CUGAUGAGGCCGAAAGGCCGAA AAGGUCC

1158 AGAGAGG CUGAUGAGGCCGAAAGGCCGAA AGGCCUA

1159 GAGAGAG CUGAUGAGGCCGAAAGGCCGAA AAGGCCU 1162 CUGGAGA CUGAUGAGGCCGAAAGGCCGAA AGGAAGG 1164 AUCUGGA CUGAUGAGGCCGAAAGGCCGAA AGAGGAA 1166 ACAUCUG CUGAUGAGGCCGAAAGGCCGAA AGAGAGG

1174 GUCUGGA CUGAUGAGGCCGAAAGGCCGAA ACAUCUG

1175 AGUCUGG CUGAUGAGGCCGAAAGGCCGAA AACAUCU

1176 AAGUCUG CUGAUGAGGCCGAAAGGCCGAA AAACAUC

1183 CUCAAGG CUGAUGAGGCCGAAAGGCCGAA AGUCUGG

1184 UCUCAAG CUGAUGAGGCCGAAAGGCCGAA AAGUCUG 1187 GUGUCUC CUGAUGAGGCCGAAAGGCCGAA AGGAAGU 1208 CCAUGGG CUGAUGAGGCCGAAAGGCCGAA AGGGCUG 1224 AUAGAGG CUGAUGAGGCCGAAAGGCCGAA AGCUGGC 1228 AUAAAUA CUGAUGAGGCCGAAAGGCCGAA AGGGAGC 1230 ACAUAAA CUGAUGAGGCCGAAAGGCCGAA AGAGGGA

1232 AAACAUA CUGAUGAGGCCGAAAGGCCGAA AUAGAGG

1233 CAAACAU CUGAUGAGGCCGAAAGGCCGAA AAUAGAG

1234 GCAAACA CUGAUGAGGCCGAAAGGCCGAA AAAUAGA

1238 AAGUGCA CUGAUGAGGCCGAAAGGCCGAA ACAUAAA

1239 CAAGUGC CUGAUGAGGCCGAAAGGCCGAA AACAUAA 1245 UAAUCAC CUGAUGAGGCCGAAAGGCCGAA AGUGCAA

1251 AAUAAAU CUGAUGAGGCCGAAAGGCCGAA AUCACAA

1252 UAAUAAA CUGAUGAGGCCGAAAGGCCGAA AAUCACA

1254 AAUAAUA CUGAUGAGGCCGAAAGGCCGAA AUAAUCA

1255 AAAUAAU CUGAUGAGGCCGAAAGGCCGAA AAUAAUC

1256 UAAAUAA CUGAUGAGGCCGAAAGGCCGAA AAAUAAU

1258 AAUAAAU CUGAUGAGGCCGAAAGGCCGAA AUAAAUA

1259 AAAUAAA CUGAUGAGGCCGAAAGGCCGAA AAUAAAU

1261 AUAAAUA CUGAUGAGGCCGAAAGGCCGAA AUAAUAA

1262 AAUAAAU CUGAUGAGGCCGAAAGGCCGAA AAUAAUA

1263 UAAUAAA CUGAUGAGGCCGAAAGGCCGAA AAAUAAU

1265 AAUAAUA CUGAUGAGGCCGAAAGGCCGAA AUAAAUA

1266 AAAUAAU CUGAUGAGGCCGAAAGGCCGAA AAUAAAU

1267 UAAAUAA CUGAUGAGGCCGAAAGGCCGAA AAAUAAA

1269 AAUAAAU CUGAUGAGGCCGAAAGGCCGAA AUAAAUA

1270 AAAUAAA CUGAUGAGGCCGAAAGGCCGAA AAUAAAU

1272 AUAAAUA CUGAUGAGGCCGAAAGGCCGAA AUAAUAA

1273 AAUAAAU CUGAUGAGGCCGAAAGGCCGAA AAUAAUA 1274 AAAUAAA CUGAUGAGGCCGAAAGGCCGAA AAAUAAU

1276 GUAAAUA CUGAUGAGGCCGAAAGGCCGAA AUAAAUA

1277 UGUAAAU CUGAUGAGGCCGAAAGGCCGAA AAUAAAU

1278 CUGUAAA CUGAUGAGGCCGAAAGGCCGAA AAAUAAA

1280 AUCUGUA CUGAUGAGGCCGAAAGGCCGAA AUAAAUA

1281 CAUCUGU CUGAUGAGGCCGAAAGGCCGAA AAUAAAU

1282 UCAUCUG CUGAUGAGGCCGAAAGGCCGAA AAAUAAA

1294 AAAUAAA CUGAUGAGGCCGAAAGGCCGAA ACAUUCA

1296 CCAAAUA CUGAUGAGGCCGAAAGGCCGAA AUACAUU

1297 CCCAAAU CUGAUGAGGCCGAAAGGCCGAA AAUACAU

1298 UCCCAAA CUGAUGAGGCCGAAAGGCCGAA AAAUACA

1300 UCUCCCA CUGAUGAGGCCGAAAGGCCGAA AUAAAUA

1301 GUCUCCC CUGAUGAGGCCGAAAGGCCGAA AAUAAAU

1315 CCCAGGA CUGAUGAGGCCGAAAGGCCGAA ACCCCGG

1317 CCCCCAG CUGAUGAGGCCGAAAGGCCGAA AUACCCC

1334 CAGCUCC CUGAUGAGGCCGAAAGGCCCAA ACAUUGG

1345 CUGAGCC CUGAUGAGGCCGAAAGGCCGAA AGGCAGC

1350 CAUGUCU CUGAUGAGGCCGAAAGGCCGAA AGCCAAG

1359 CACGGAA CUGAUGAGGCCGAAAGGCCGAA ACAUGUC

1360 UCACGGA CUGAUGAGGCCGAAAGGCCGAA AACAUGU

1361 UUCACGG CUGAUGAGGCCGAAAGGCCGAA. AAACAUG

1362 UUUCACG CUGAUGAGGCCGAAAGGCCGAA AAAACAU

1386 AACAGCC CUGAUGAGGCCGAAAGGCCGAA AUUGUUC

1393 ACAUGGG CUGAUGAGGCCGAAAGGCCGAA ACAGCCU

1394 UACAUGG CUGAUGAGGCCGAAAGGCCGAA AACAGCC

1401 AGGGGGC CUGAUGAGGCCGAAAGGCCGAA ACAUGGG

1414 AGGCACA CUGAUGAGGCCGAAAGGCCGAA AGGCCAG

1422 UCAAAAG CUGAUGAGGCCGAAAGGCCGAA AGGCACA

1423 AUCAAAA CUGAUGAGGCCGAAAGGCCGAA AAGGCAC

1425 UAAUCAA CUGAUGAGGCCGAAAGGCCGAA AGAAGGC

1426 AUAAUCA CUGAUGAGGCCGAAAGGCCGAA AAGAAGG

1427 CAUAAUC CUGAUGAGGCCGAAAGGCCGAA AAAGAAG

1431 AAAACAU CUGAUGAGGCCGAAAGGCCGAA AUCAAAA

1432 AAAAACA CUGAUGAGGCCGAAAGGCCGAA AAUCAAA

1436 UUUAAAA CUGAUGAGGCCGAAAGGCCGAA ACAUAAU

1437 UUUUAAA CUGAUGAGGCCGAAAGGCCGAA AACAUAA

1438 AUUUUAA CUGAUGAGGCCGAAAGGCCGAA AAACAUA

1439 UAUUUUA CUGAUGAGGCCGAAAGGCCGAA AAAACAU

1440 AUAUUUU CUGAUGAGGCCGAAAGGCCGAA AAAAACA

1441 AAUAUUU CUGAUGAGGCCGAAAGGCCGAA AAAAAAC

1446 CAGAUAA CUGAUGAGGCCGAAAGGCCGAA AUUUUAA

1448 AUCAGAU CUGAUGAGGCCGAAAGGCCGAA AUAUUUU

1449 AAUCAGA CUGAUGAGGCCGAAAGGCCGAA AAUAUUU

1451 UUAAUCA CUGAUGAGGCCGAAAGGCCGAA AUAAUAU

1456 ACAACUU CUGAUGAGGCCGAAAGGCCGAA AUCAGAU

1457 GACAACU CUGAUGAGGCCGAAAGGCCGAA AAUCAGA

1461 UUUAGAC CUGAUGAGGCCGAAAGGCCGAA ACUUAAU

1464 UUGUUUA CUGAUGAGGCCGAAAGGCCGAA ACAACUU

1466 CAUUGUU CUGAUGAGGCCCAAAGGCCGAA AGACAAC 1479 GUCACCA CUGAUGAGGCCGAAAGGCCGAA AUCAGCA

1480 GGUCACC CUGAUGAGGCCGAAAGGCCGAA AAUCAGC 1494 AAUGAGU CUGAUGAGGCCGAAAGGCCGAA ACAGUUG 1498 CAGCAAU CUGAUGAGGCCGAAAGGCCGAA AGUGACA 1501 CCUCAGC CUGAUGAGGCCGAAAGGCCGAA AUGAGUG 1512 GGGAGCA CUGAUGAGGCCGAAAGGCCGAA AGGCCUC 1517 CCCUGGG CUGAUGAGGCCGAAAGGCCGAA AGCAGAG 1528 CAGACAC CUGAUGAGGCCGAAAGGCCGAA ACUCCCU 1533 GAUUACA CUGAUGAGGCCGAAAGGCCGAA ACACAAC 1537 GGCCGAU CUGAUGAGGCCGAAAGGCCGAA ACAGACA 1540 GUAGGCC CUGAUGAGGCCGAAAGGCCGAA AUUACAG 1546 UGAAUAG CUGAUGAGGCCGAAAGGCCGAA AGGCCGA 1549 CACUGAA CUGAUGAGGCCGAAAGGCCGAA AGUAGGC

1551 GCCACUG CUGAUGAGGCCGAAAGGCCGAA AUAGUAG

1552 CGCCACU CUGAUGAGGCCGAAAGGCCGAA AAUAGUA 1566 CAACCUU CUGAUGAGGCCGAAAGGCCGAA AUUUCUC 1572 CCUAAGC CUGAUGAGGCCGAAAGGCCGAA ACCUUUA

1576 CUUUCCU CUGAUGAGGCCGAAAGGCCGAA AGCAACC

1577 UCUUUCC CUGAUGAGGCCGAAAGGCCGAA AAGCAAC

Table 25: Mouse TNF-a HH Target Sequences

nt . HH Target Sequence nt . HH Target Sequenc,

Position Position

66 UgGAAAU a GcucCcA 324 GgGUGAU C GGuCCCC 101 GGCAGGU U CUgUcCC 347 GAGAagU u cCCAaaU 101 GGCAGgU u CuGUccC 364 CCUCcCU C UcAUCAG 102 GCAGGUU C UgUcCCU 366 UCcCUCU c AUCAGuu 102 gCAGgUU c ugUCCCU 366 UcCCUCU C auCAGuU 106 GUUCUgU c CCUuUCA 369 CUCUcAU C AGuuCUa 110 UgUcCCU u UCACucA 376 CAGuuCU a UGGCCCA 111 gUCcCUU u CaCUCAC 390 AgACCCU C AcaCUcA 111 guCCCuU u CACuCAc 396 ucaCAcU C AGAUCAU 112 UcCCUuU C ACucACU 401 cUCAGAU C AUCUUCU 116 UuUCACU C AcUGgcc 404 AGAUCAU C UUCUCaA 137 GCCaCAU C uCCcUCc 406 AUCAUCU U CUCaAAa 139 caCAuCU C CCUCcAg 406 AUcAUcU U cUcaAAA 177 GCAUGAU C CGcGACG 407 UCAUCUU C UCaAAau 207 AGGCaCU C CCCcAaA 409 AUCUUCU C aAAauuC 228 GGGGCuU C CAGAACU 409 AuCuuCU c AaAAUUC 228 GGGGCuU c CAGaacU 409 aUcUUcU c AAAauUc 236 CAGaaCU C CAGGCGG 432 AGCCUGU A GCCCAcG 236 CAGaACU c cAGgcGg

249 GGugCCU a UgUCUcA

249 GGuGCCU a UGucUCa 444 AcGUcGU A GCAAACC

501 AcGCCCU C CUGGCCA

261 UCAGCCU C UUCUCaU 560 gGgUUGU a CCUuguC

261 UCAgCCU C UUCUcau 560 GGguUGU A CCUugUC

263 AGCCUCU U CUCaUUC 564 UGUACCU u gUCUACU

263 AgCCUCU U CUcauUC 567 ACCUugU C UACUCCC

264 GCCUCUU C UCaUUCC 569 CUugUCU A CUCCCAG

264 gCCUCUU C UcauUCc 572 gUCUACU C CCAGGUu

266 CUCUUCU C aUUCCUG 572 GUCUaCU c CCAGguu

269 UUCUCaU U CCUGcUu 572 GuCUacU C CCAgGUu

270 UCUCaUU C CUGcUuG 579 CCCAGGU u CUCUUCA

276 UCCUGcU u GUGGGAG 580 CCAGguU c uCUUcAa

297 CCACGCU C UUCUGuC 580 CCaGGuU c UCuUcaa

299 ACGCUCU U CUGuCUa 582 AGGUUCU C UUCaagg

300 CGCUCUU C UGuCUaC 582 AGGUUCU C UUCAAGG

304 CUuCUgU c uAcUGaa 584 GUuCUCU U CAAGGGa

306 UcUGUcU a cUgAAcU 585 UuCUCUU C AAGGGaC

314 CUGaACU U cGGgGUG 608 CcCGaCU a CgugCUC

315 UGaACUU c GGgGUGA 615 aCgUGcU C CUCAcCC

315 uGaaCUU c GGGguGa 615 ACGUGCU C CUCACCC

324 gGGUGaU c GgUCCcC 618 UGCUCCU C ACCCACA 630 ACACCgU C AGCCGau 940 GuCUACU c cUCAGaG

630 ACACCgU C AgCCgaU 943 UACUccU C AGaGcCc

638 agcCgAU u uGCUaUc 972 UCUaaCU u AgAAAGg

643 aUUUGcU a uCUcAuA 972 ucUaaCU u AGAaAgG

645 UuGCuaU C UCaUACC 973 CUaACuU A GAAAggG

647 GCuaUCU C aUACCAG 984 AGgGgAU U auGGcuc

663 agAAaGU C AACCUCC 984 AGGGgaU U aUGgCUc

669 UCAACCU C CUCUCUG 985 GGGGauU a uGGcUCa

669 UcAAccU C cUcUCUG 997 UcAGAgU c CAAcucu

672 ACCUCCU C UCUGCCg 1010 CuguGCU c AGAgCUU

674 CUCCUCU C UGCCgUC 1017 cAGAgCU U UcAaCAA

681 cUGCCgU C AagaGcC 1018 AGAgCUU U cAaCAAC

681 CUGCCgU C AAGAGCC 1019 GAgCUUU c AaCAACu

681 CUGcCgU C aaGAgcC 1073 UgGGCCU c ucAUgCA

734 CCCUGGU A UGAGCCC 1096 AAGgAcU C AAAugGG

734 CccUGGU a ugaGCCc 1106 aUGGGcU U uccGAAU

744 AGCCCAU a UAcCUGG 1107 UGGGcUU u ccGAAUu

746 CCCAUaU A cCUGGGA 1108 GGgCuUU c cGaaUUC

759 GAgGAGU C uuCCAGc 1115 CcGAAuU C ACUGGaG

759 GAGGaGU C UUCCAGC 1133 CGAAugU C CAuuCcU

761 GGaGUCU U CCAGCUG 1164 gagUGgU c AgGUUGc

762 GaGUCUU C CAGCUGG 1180 UcUgUcU c agaAUGA

786 ACCaACU C AGCGCUG 1203 aaGAuCU c AGGCCUU

798 CUGAGgU C AAUCuGC 1210 cAGGCCU U CCUacCU

802 GgUCAAU C uGCCCaA 1211 AGGCCUU C CUacCUu

812 CCCaAgU A cuUaGAC 1214 CCUUCCU a cCUuCAG

816 AgUAcuU a GACUUUG 1218 CcuACcU u CaGACCu

821 uUaGACU U UGCgGAG 1218 COiaCCU U CAGACcu

822 UaGACUU U GCgGAGU 1213 cCuACcU u cAgACCU

830 GCgGAGU C cGGGCAG 1218 CCUacCU u CAGAccU

840 GGCAGGU C UACUUUG 1219 CuaCCUU C AGACcuu

842 CAGGUCU A CUUUGGa 1219 CuAcCUU c agACcUU

842 CAGgucU a CUUugGA 1226 CagACCU U uCCAgAC

842 cagGuCU a CUUUgGA 1226 CAGACCU U UCCAGAC

845 GUCUACU U UGGagUC 1227 agACCUU u CCAgACu

846 UCUACUU U GGagUCA 1227 AGAccUU U CCAGACU

852 UUGGagU C AUUGCuC 1228 GAccUUU C CAGACUc

855 GagUCAU U GCuCUGU 1238 gACUCuU c cCUGAGG

887 AUCCaUU c ucUACCC 1262 CAGCCuU C CuCAcaG

891 AuucuCU a CCCaGCC 1283 CCCCccU C uaUUUAU

905 CCcCaCU C UgaCCCC 1283 cCcCCCU C UAUUUAU

905 cCCCacU c UgACCCC 1285 CCCCUCU A UUUAUaU

905 CcCCACU c uGAccCC 1287 CcuCUAU u UauAuUU

914 GAcCCcU U uacUCUG 1287 CCUCUAU U UAUaUUU

915 ACCCCuU u acUCuGA 1288 CUCUAUU U AUaUUUG

919 CUUUAcU c ugaCCcC 1289 UCUAUUU A UaUUUGC

928 GACCcCU u UaUugUC 1293 UUUAUaU U UGCACUU

928 gAcCCCU U UAUUguC 1293 uUUaUaU u UGcAcUu

932 CCUUUAU U guCuaCU 1294 UUAUaUU U GCACUUa 1300 UUGCACU U aUuAUUu 1462 aCCuUGU u GCCUCCU

1303 CAcuUaU u AuUuAUU 1470 GccuCcU C UUUUGcU

1304 acUuAUU A UUUAUUA 1472 cuCcUCU u UUGcUUA

1306 UuAUUAU U UAUUAUU 1473 uCcUCUU u UGcUUAU

1307 UAUUAUU U AUUAUUU 1474 CcUCUUU u GcUUAUG

1307 UaUUaUU U AuuAUuU 1478 UUUUGcU u AUGUUUa

1308 AUUAUUU A UUAUUUA 1479 UUUGcUU a UGuuuAa

1310 UauUuAU U AUUUAUU 1479 UUUGcUU A UGUUUaa

1310 UAUUUAU U AUUUAUU 1484 UUAUGUU U aaaAcAA

1310 UAUUUAU U AUUUAUU 1498 AAAuauU U AUCUaAc

1311 AUUUAUU A UUUAUUU 1511 AcccAaU U GUCUuAA

1311 AUUUAUU A UUUAUUU 1514 cAaUUGU c UuAAuAA

1311 AuuUAUU A UuUauUU 1516 aUUGUCU u AAuAAcG

1313 UUAUUAU U UAUUUAU 1529 CgcugAU u UGGuGAC

1313 UUAUUAU U UAUUUAU 1529 cGCUGAU U UGGUGAC

1313 uUAUUAU u UauUUAu 1530 gCUGAUU u gGUgacC

1314 UAUUAUU U AUUUAUU 1530 GCUGAUU U GGUGACC

1314 UAUUAUU U AUUUAUU 1563 UgaAcCU c UGcUCCC

1315 AUUAUUU A UUUAUUA 1563 ugaaCCU C UGCUCCC

1317 UAUUUAU U UAUUAUU 1568 CUCUGCU C CCCAcGG

1318 AUUUAUU U AUUAUUU 1589 UGaCUGU A AUuGcCC

1319 UUUAUUU A UUAUUUA 1592 CUGUAAU u GcCCUAC

1326 AUUAUUU A UUUAUUU 1617 GAGAAAU A AAGaUcG

1328 UAUUUAU U UAUUUgC 1623 UAAAGaU c GCUUAaa

1329 AUUUAUU U AUUUgCu 1633 UUAaaaU a aaAAaCC

1330 UUUAUUU A UUUgCuu 25 AgGgaCU a gCCagGA

1332 UAUUUAU U UgCuuAU

1333 AUUUAUU U gCuuAUG

1337 auUUGCU U AuGAAuG

1338 uUUGCUU A uGAAuGu

1346 UGAAUGU A UUUAUUU

1348 AAUGUAU U UAUUUGG

1349 AUGUAUU U AUUUGGa

1350 UGUAUUU A UUUGGaA

1352 uAUuUAU u UGGaAGG

1352 UAUUUAU U UGGaAGg

1353 AUUUAUU U GGaAGgC

1369 GGGGUgU C CUGGaGG

1398 gCUguCU U cAGACAg

1398 GCUGuCU U cagaCAG

1412 GACAUGU U UUCuGUG

1413 ACAUGUU U UCuGUGA

1414 CAUGUUU U CuGUGAA

1415 AUGUUUU C UGUGAAA

1415 AUGUUUU c UgugAaA

1438 gaGCUGU c CCCAccU

1451 CUGGCCU C UcUaCCU

1453 ggCCUCU C UaCCuUG Table 26: Mouse TNF-α Hammerhead Ribozyme Sequences

nt . Mouse HH Ribozyme Sequence

Position

25 UCCUGGC CUGAUGAGGCCGAAAGGCCGAA AGUCCCU

66 UGGGAGC CUGAUGAGGCCGAAAGGCCGAA AUUUCCA

101 GGGACAG CUGAUGAGGCCGAAAGGCCGAA ACCUGCC

102 AGGGACA CUGAUGAGGCCGAAAGGCCGAA AACCUGC 102 AGGGACA CUGAUGAGGCCGAAAGGCCGAA AACCUGC 106 UGAAAGG CUGAUGAGGCCGAAAGGCCGAA ACAGAAC

110 UGAGUGA CUGAUGAGGCCGAAAGGCCGAA AGGGACA

111 GϋGAGUG CUGAUGAGGCCGAAAGGCCGAA AAGGGAC

111 GUGAGUG CUGAUGAGGCCGAAAGGCCGAA AAGGGAC

112 AGUGAGU CUGAUGAGGCCGAAAGGCCGAA AAAGGGA 116 GGCCAGU CUGAUGAGGCCGAAAGGCCGAA AGUGAAA 137 GGAGGGA CUGAUGAGGCCGAAAGGCCGAA AUGUGGC 139 CUGGAGG CUGAUGAGGCCGAAAGGCCGAA AGAUGUG 177 CGUCGCG CUGAUGAGGCCGAAAGGCCGAA AUCAUGC 207 UUUGGGG CUGAUGAGGCCGAAAGGCCGAA AGUGCCU 228 AGUUCUG CUGAUGAGGCCGAAAGGCCGAA AAGCCCC 228 AGUUCUG CUGAUGAGGCCGAAAGGCCGAA AAGCCCC 236 CCGCCUG CUGAUGAGGCCGAAAGGCCGAA AGUUCUG 236 CCGCCUG CUGAUGAGGCCGAAAGGCCGAA AGUUCUG 249 UGAGACA CUGAUGAGGCCGAAAGGCCGAA AGGCACC 249 UGAGACA CUGAUGAGGCCGAAAGGCCGAA AGGCACC

261 AUGAGAA CUGAUGAGGCCGAAAGGCCGAA AGGCUGA

263 GAAUGAG CUGAUGAGGCCGAAAGGCCGAA AGAGGCU

264 GGAAUGA CUGAUGAGGCCGAAAGGCCGAA AAGAGGC 264 GGAAUGA CUGAUGAGGCCGAAAGGCCGAA AAGAGGC 266 CAGGAAU CUGAUGAGGCCGAAAGGCCGAA AGAAGAG

269 AAGCAGG CUGAUGAGGCCGAAAGGCCGAA AUGAGAA

270 CAAGCAG CUGAUGAGGCCGAAAGGCCGAA AAUGAGA 276 CUGCCAC CUGAUGAGGCCGAAAGGCCGAA AGCAGGA 297 GACAGAA CUGAUGAGGCCGAAAGGCCGAA AGCGUGG

299 UAGACAG CUGAUGAGGCCGAAAGGCCGAA AGAGCGU

300 GUAGACA CUGAUGAGGCCGAAAGGCCGAA AAGAGCG 304 UUCAGUA CUGAUGAGGCCGAAAGGCCGAA ACAGAAG 306 AGUUCAG CUGAUGAGGCCGAAAGGCCGAA AGACAGA

314 CACCCCG CUGAUGAGGCCGAAAGGCCGAA AGUUCAG

315 UCACCCC CUGAUGAGGCCGAAAGGCCGAA AAGUUCA 315 UCACCCC CUGAUGAGGCCGAAAGGCCGAA AAGUUCA

324 GGGGACC CUGAUGAGGCCGAAAGGCCGAA AUCACCC

347 AUUUGGG CUGAUGAGGCCGAAAGGCCGAA ACUUCUC

364 CUGAUGA CUGAUGAGGCCGAAAGGCCGAA AGGGAGG

366 AACUGAU CUGAUGAGGCCGAAAGGCCGAA AGAGGGA

369 UAGAACU CUGAUGAGGCCGAAAGGCCGAA AUGAGAG

376 UGGGCCA CUGAUGAGGCCGAAAGGCCGAA AGAACUG

390 UGAGUGU CUGAUGAGGCCGAAAGGCCGAA AGGGCCU

396 AUGAUCU CUGAUGAGGCCGAAAGGCCGAA AGUGUGA

401 AGAAGAU CUGAUGAGGCCGAAAGGCCGAA AUCUGAG

404 UUGAGAA CUGAUGAGGCCGAAAGGCCGAA AUGACCU

406 UUUUGAG CUGAUGAGGCCGAAAGGCCGAA AGAUGAU

407 AUUUUGA CUGAUGAGGCCGAAAGGCCGAA AAGAUGA 409 GAAUUUU CUGAUGAGGCCGAAAGGCCGAA AGAAGAU 409 GAAUUUU CUGAUGAGGCCGAAAGGCCGAA AGAAGAU 409 GAAUUUU CUGAUGAGGCCGAAAGGCCGAA AGAAGAU 432 CGUGGGC CUGAUGAGGCCGAAAGGCCGAA ACAGGCU

444 GGUUUGC CUGAUGAGGCCGAAAGGCCGAA ACGACGU

501 UGGCCAG CUGAUGAGGCCGAAAGGCCGAA AGGGCGU

560 GACAAGG CUGAUGAGGCCGAAAGGCCGAA ACAACCC

564 AGUAGAC CUGAUGAGGCCGAAAGGCCGAA AGGUACA

567 GGGAGUA CUGAUGAGGCCGAAAGGCCGAA ACAAGGU

569 CUGGGAG CUGAUGAGGCCGAAAGGCCGAA AGACAAG 572 AACCUGC CUGAUGAGGCCGAAAGGCCGAA AGUAGAC

572 AACCUGG CUGAUGAGGCCGAAAGGCCGAA AGUAGAC

579 UGAAGAG CUGAUGAGGCCGAAAGGCCGAA ACCUGGG 530 UUGAAGA CUGAUGAGGCCGAAAGGCCGAA AACCUGG

580 UUGAAGA CUGAUGAGGCCGAAAGGCCGAA AACCUGG 532 CCUUGAA CUGAUGAGGCCGAAAGGCCGAA AGAACCU 532 CCUUGAA CUGAUGAGGCCGAAAGGCCGAA AGAACCU 534 UCCCUUG CUGAUGAGGCCGAAAGGCCGAA AGAGAAC 585 GUCCCUU CUGAUGAGGCCGAAAGGCCGAA AAGAGAA 608 GAGCACG CUGAUGAGGCCGAAAGGCCGAA AGUCGGG 615 GGGUGAG CUGAUGAGGCCGAAAGGCCGAA AGCACGU 615 GGGUGAG CUGAUGAGGCCGAAAGGCCGAA AGCACGU 618 UGUGGGU CUGAUGAGGCCGAAAGGCCGAA AGGAGCA 630 AUCGGCU CUGAUGAGGCCGAAAGGCCGAA ACGGUGU 630 AUCGGCU CUGAUGAGGCCGAAAGGCCGAA ACGGUGU 638 GAUAGCA CUGAUGAGGCCGAAAGGCCGAA AUCGGCU 643 UAUGAGA CUGAUGAGGCCGAAAGGCCGAA AGCAAAU 645 GGUAUGA CUGAUGAGGCCGAAAGGCCGAA AUAGCAA 647 CUGGUAU CUGAUGAGGCCGAAAGGCCGAA AGAUAGC 663 GGAGGUU CUGAUGAGGCCGAAAGGCCGAA ACUUUCU

669 CAGAGAG CUGAUGAGGCCGAAAGGCCGAA AGGUUGA

672 CGGCAGA CUGAUGAGGCCGAAAGGCCGAA AGGAGGU

674 GACGGCA CUGAUGAGGCCGAAAGGCCGAA AGAGGAG

681 GGCUCUU CUGAUGAGGCCGAAAGGCCGAA ACGGCAG

631 GGCUCUU CUGAUGAGGCCGAAAGGCCGAA ACGGCAG

734 GGGCUCA CUGAUGAGGCCGAAAGGCCGAA ACCAGGG

744 CCAGGUA CUGAUGAGGCCGAAAGGCCGAA AUGGGCU

746 UCCCAGG CUGAUGAGGCCGAAAGGCCGAA ACAUGGG

759 GCUGGAA CUGAUGAGGCCGAAAGGCCGAA ACUCCUC

761 CAGCUGG CUGAUGAGGCCGAAAGGCCGAA AGACCCC

762 CCAGCUG CUGAUGAGGCCGAAAGGCCGAA AAGACUC 786 CAGCGCU CUGAUGAGGCCGAAAGGCCGAA AGUUGGU 798 GCAGAUU CUGAUGAGGCCGAAAGGCCGAA ACCUCAG 802 UUGGGCA CUGAUGAGGCCGAAAGGCCGAA AUUGACC 312 GUCUAAG CUGAUGAGGCCGAAAGGCCGAA ACUUGGG 816 CAAAGUC CUGAUGAGGCCGAAAGGCCGAA AAGUACU

821 CUCCGCA CUGAUGAGGCCGAAAGGCCGAA AGUCUAA

822 ACUCCGC CUGAUGAGGCCGAAAGGCCGAA AAGUCUA 830 CUGCCCG CUGAUGAGGCCGAAAGGCCGAA ACUCCGC 840 CAAAGUA CUGAUGAGGCCGAAAGGCCGAA ACCUGCC 842 UCCAAAG CUGAUGAGGCCGAAAGGCCGAA AGACCUG 842 UCCAAAG CUGAUGAGGCCGAAAGGCCGAA AGACCUG 842 UCCAAAG CUGAUGAGGCCGAAAGGCCGAA AGACCUG

845 GACUCCA CUGAUGAGGCCGAAAGGCCGAA AGUAGAC

846 UGACUCC CUGAUGAGGCCGAAAGGCCGAA AAGUAGA 852 GAGCAAU CUGAUGAGGCCGAAAGGCCGAA ACUCCAA 855 ACAGAGC CUGAUGAGGCCGAAAGGCCGAA AUGACUC 887 GGGUAGA CUGAUGAGGCCGAAAGGCCGAA AAUGGAU 891 GGCUGGG CUGAUGAGGCCGAAAGGCCGAA AGAGAAU 905 GGGGUCA CUGAUGAGGCCGAAAGGCCGAA AGUGGGG 905 GGGCUCA CUGAUGAGGCCGAAAGGCCGAA AGUGGGG 905 GGGGUCA CUGAUGAGGCCGAAAGGCCGAA AGUGGGG

914 CAGAGUA CUGAUGAGGCCGAAAGGCCGAA AGGGGUC

915 UCAGAGU CUGAUGAGGCCGAAAGGCCGAA AAGGGGU 919 GGGGUCA CUGAUGAGGCCGAAAGGCCGAA AGUAAAG 928 GACAAUA CUGAUGAGGCCGAAAGGCCGAA AGGGGUC 928 GACAAUA CUGAUGAGGCCGAAAGGCCGAA AGGGGUC 932 AGUAGAC CUGAUGAGGCCGAAAGGCCGAA AUAAAGG 940 CUCUGAG CUGAUGAGGCCGAAAGGCCGAA AGUAGAC 943 GGGCUCU CUGAUGAGGCCGAAAGGCCGAA AGGAGUA 972 CCUUUCU CUGAUGAGGCCGAAAGGCCGAA AGUUAGA

972 CCUUUCU CUGAUGAGGCCGAAAGGCCGAA AGUUAGA

973 CCCUUUC CUGAUGAGGCCGAAAGGCCGAA AAGUUAG 984 GAGCCAU CUGAUGAGGCCGAAAGGCCGAA AUCCCCU 984 GAGCCAU CUGAUGAGGCCGAAAGGCCGAA AUCCCCU

985 UGAGCCA CUGAUGAGGCCGAAAGGCCGAA AAUCCCC

997 AGAGUUG CUGAUGAGGCCGAAAGGCCGAA ACUCUGA

1010 AAGCUCU CUGAUGAGGCCGAAAGGCCGAA AGCACAG

1017 UUGUUGA CUGAUGAGGCCGAAAGGCCGAA AGCUCUG

1018 GUUGUUG CUGAUGAGGCCGAAAGGCCGAA AAGCUCU

1019 AGUUGUU CUGAUGAGGCCGAAAGGCCGAA AAAGGUC

1073 UGCAUGA CUGAUGAGGCCGAAAGGCCGAA AGGCCCA

1096 CCCAUUU CUGAUGAGGCCGAAAGGCCGAA AGUCCUU

1106 AUUCGGA CUGAUGAGGCCGAAAGGCCGAA AGCCCAU

1107 AAUUCGG CUGAUGAGGCCGAAAGGCCGAA AAGCCCA

1108 GAAUUCG CUGAUGAGGCCGAAAGGCCGAA AAAGCCC

1115 CUCCAGU CUGAUGAGGCCGAAAGGCCGAA AAUUCGG

1133 AGGAAUG CUGAUGAGGCCGAAAGGCCGAA ACAUUCG

1164 GCAACCU CUGAUGAGGCCGAAAGGCCGAA ACCACUC

1180 UCAUUCU CUGAUGAGGCCGAAAGGCCGAA AGACAGA

1203 AAGGCCU CUGAUGAGGCCGAAAGGCCGAA AGAUCUU

1210 AGGUAGG CUGAUGAGGCCGAAAGGCCGAA AGGCCUG

1211 AAGGUAG CUGAUGAGGCCGAAAGGCCGAA AAGGCCU

1214 CUGAAGG CUGAUGAGGCCGAAAGGCCGAA AGGAAGG

1218 AGGUCUG CUGAUGAGGCCGAAAGGCCGAA AGGUAGG

1219 AAGCUCU CUGAUGAGGCCGAAAGGCCGAA AAGGUAG

1226 GUCUGGA CUGAUGAGGCCGAAAGGCCGAA AGGUCUG

1227 AGUCUGG CUGAUGAGGCCGAAAGGCCGAA AAGGUCU

1228 GAGUCUG CUGAUGAGGCCGAAAGGCCGAA AAAGGUC

1238 CCUCAGG CUGAUGAGGCCGAAAGGCCGAA AAGAGUC

1262 CUGUGAG CUGAUGAGGCCGAAAGGCCGAA AAGGCUG

1283 AUAAAUA CUGAUGAGGCCGAAAGGCCGAA AGGGGGG

1285 AUAUAAA CUGAUGAGGCCGAAAGGCCGAA AGAGGGG

1287 AAAUAUA CUGAUGAGGCCGAAAGGCCGAA AUAGAGG

1288 CAAAUAU CUGAUGAGGCCGAAAGGCCGAA AAUAGAG

1289 GCAAAUA CUGAUGAGGCCGAAAGGCCGAA AAAUAGA

1293 AAGUGCA CUGAUGAGGCCGAAAGGCCGAA AUAUAAA

1294 UAAGUGC CUGAUGAGGCCGAAAGGCCGAA AAUAUAA

1300 AAAUAAU CUGAUGAGGCCGAAAGGCCGAA AGUGCAA

1303 AAUAAAU CUGAUGAGGCCGAAAGGCCGAA AUAAGUG

1304 UAAUAAA CUGAUGAGGCCGAAAGGCCGAA AAUAAGU.

1306 AAUAAUA CUGAUGAGGCCGAAAGGCCGAA AUAAUAA

1307 AAAUAAU CUGAUGAGGCCGAAAGGCCGAA AAUAAUA

1307 AAAUAAU CUGAUGAGGCCGAAAGGCCGAA AAUAAUA 1308 UAAAUAA CUGAUGAGGCCGAAAGGCCGAA AAAUAAU

1310 AAUAAAU CUGAUGAGGCCGAAAGGCCGAA AUAAAUA

1311 AAAUAAA CUGAUGAGGCCGAAAGGCCGAA AAUAAAU

1313 AUAAAUA CUGAUGAGGCCGAAAGGCCGAA AUAAUAA

1314 AAUAAAU CUGAUGAGGCCGAAAGGCCGAA AAUAAUA

1315 UAAUAAA CUGAUGAGGCCGAAAGGCCGAA AAAUAAU

1317 AAUAAUA CUGAUGAGGCCGAAAGGCCGAA AUAAAUA

1318 AAAUAAU CUGAUGAGGCCGAAAGGCCGAA AAUAAAU

1319 UAAAUAA CUGAUGAGGCCGAAAGGCCGAA AAAUAAA

1325 AAAUAAA CUGAUGAGGCCGAAAGGCCGAA AAAUAAU

1328 GCAAAUA CUGAUGAGGCCGAAAGGCCGAA AUAAAUA

1329 AGCAAAU CUGAUGAGGCCGAAAGGCCGAA AAUAAAU

1330 AAGCAAA CUGAUGAGGCCGAAAGGCCGAA AAAUAAA

1332 AUAAGCA CUGAUGAGGCCGAAAGGCCGAA AUAAAUA

1333 CAUAAGC CUGAUGAGGCCGAAAGGCCGAA AAUAAAU

1337 CAUUCAU CUGAUGAGGCCGAAAGGCCGAA AGCAAAU

1338 ACAUUCA CUGAUGAGGCCGAAAGGCCGAA AAGCAAA

1346 AAAUAAA CUGAUGAGGCCGAAAGGCCGAA ACAUUCA

1348 CCAAAUA CUGAUGAGGCCGAAAGGCCGAA AUACAUU

1349 UCCAAAU CUGAUGAGGCCGAAAGGCCGAA AAUACAU

1350 UUCCAAA CUGAUGAGGCCGAAAGGCCGAA AAAUACA

1352 CCUUCCA CUGAUGAGGCCGAAAGGCCGAA AUAAAUA

1353 GCCUUCC CUGAUGAGGCCGAAAGGCCGAA AAUAAAU

1369 CCUCCAG CUGAUGAGGCCGAAAGGCCGAA ACACCCC

1398 CUGUCUG CUGAUGAGGCCGAAAGGCCGAA AGACAGC

1412 CACAGAA CUGAUGAGGCCGAAAGGCCGAA ACAUGUC

1413 UCACAGA CUGAUGAGGCCGAAAGGCCGAA AACAUGU

1414 UUCACAG CUGAUGAGGCCGAAAGGCCGAA AAACAUG

1415 UUUCACA CUGAUGAGGCCGAAAGGCCGAA AAAACAU

1438 AGGUGGG CUGAUGAGGCCGAAAGGCCGAA ACAGCUC

1451 AGGUAGA CUGAUGAGGCCGAAAGGCCGAA AGGGCAG

1453 CAAGGUA CUGAUGAGGCCGAAAGGCCGAA AGAGGCC

1455 AACAAGG CUGAUGAGGCCGAAAGGCCGAA AGAGAGG

1462 AGGAGGC CUGAUGAGGCCGAAAGGCCGAA ACAAGCU

1470 AGCAAAA CUGAUGAGGCCGAAAGGCCGAA AGGAGGC

1472 UAAGCAA CUGAUGAGGCCGAAAGGCCGAA AGAGGAG

1473 AUAAGCA CUGAUGAGGCCGAAAGGCCGAA AAGAGGA

1474 CAUAAGC CUGAUGAGGCCGAAAGGCCGAA AAAGAGG

1473 UAAACAU CUGAUGAGGCCGAAAGGCCGAA AGCAAAA 1479 UUAAACA CUGAUGAGGCCGAAAGGCCGAA AAGCAAA

1479 UUAAACA CUGAUGAGGCCGAAAGGCCGAA AAGCAAA

1484 UUGUUUU CUGAUGAGGCCGAAAGGCCGAA AACAUAA

1498 GUUAGAU CUGAUGAGGCCGAAAGGCCGAA AAUAUUU

1511 UUAAGAC CUGAUGAGGCCGAAAGGCCGAA AUUGGGU

1514 UUAUUAA CUGAUGAGGCCGAAAGGCCGAA ACAAUUG

1516 CGUUAUU CUGAUGAGGCCCAAAGGCCGAA AGACAAU

1529 GUCACCA CUGAUGAGGCCGAAAGGCCGAA AUCAGCG

1530 GGUCACC CUGAUGAGGCCGAAAGGCCGAA AAUCAGC 1530 GGUCACC CUGAUGAGGCCGAAAGGCCGAA AAUCAGC 1563 GGGAGCA CUGAUGAGGCCGAAAGGCCGAA AGGUUCA 1563 GGGAGCA CUGAUGAGGCCGAAAGGCCGAA AGGUUCA 1563 CCGUGGG CUGAUGAGGCCGAAAGGCCGAA AGCAGAG 1589 GGGCAAU CUGAUGAGGCCGAAAGGCCGAA ACAGUCA 1592 GUAGGGC CUGAUGAGGCCGAAAGGCCGAA AUUACAG 1617 CGAUCUU CUGAUGAGGCCGAAAGGCCGAA AUUUCUC 1623 UUUAAGC CUGAUGAGGCCGAAAGGCCGAA AUCUUUA 1633 GGUUUUU CUGAUGAGGCCGAAAGGCCGAA AUUUUAA

Table 29: Human bcr/abl HH Target Sequence

Sequence HH Target Sequence

ID NO.

b2 -a2

Junction

20 UGACCAUCA AUA AGGAAGAAGCC

21 GAAGAAGCC CUU CAGCGGGCAGU 22 AAGAAGCCC UUC AGCGGCCAGUA b3-a2

Junction

23 UAAGCAGAG UUC AAAAGCCCUUC 24 UCAAAAGCC CUU CAGCGGCCAGU 25 CAAAAGCCC UUC AGCGGCCAGUA

Table 30: Human bcr-abl HH Ribozyme Sequences

Sequence HH Ribozyme Sequence

ID No .

26 GGCUUCUUCCU CUGAUGAGGCCGAAAGGCCGAA AUUGAUGGUCA

27 ACUGGCCGCUG CUGAUGAGGCCGAAAGGCCGAA AGGGCUUCUUC

28 UACUGGCCGCU CUGAUGAGGCCGAAAGGCCGAA AAGGGCUUCUU

29 GAAGGGCUUUU CUGAUGAGGCCGAAAGGCCGAA AACUCUGCUUA

30 ACUGGCCGCUG CUGAUGAGGCCGAAAGGCCGAA AGGGCUUUUGA

31 UACUGGCCGCU CUGAUGAGGCCGAAAGGCCGAA AAGGGCUUUUG

Table 31: RSV (1B) HH Target Sequence

nt. HH Target Sequence nt. HH Target Sequence Position Position

10 GGCAAAU A AAUCAAU 276 AAAAUAU A CUGAAUA

14 AAUAAAU C AAUUCAG 283 ACUGAAU A CAACACA

18 AAUCAAU U CAGCCAA 295 ACAAAAU A UGGCACU

19 AUCAAUU C AGCCAAC 303 UGGCACU U UCCCUAU

54 CAAUGAU A AUACACC 304 GGCACUU U CCCUAUG

57 UGAUAAU A CACCACA 305 GCACUUU C CCUAUGC

77 UGAUGAU C ACAGACA 309 UUUCCCU A UGCCAAU

94 AGACCGU U GUCACUU 317 UGCCAAU A UUCAUCA

97 CCGUUGU C ACUUGAG 319 CCAAUAU U CAUCAAU

101 UGUCACU U GAGACCA 320 CAAUAUU C AUCAAUC

110 AGACCAU A AUAACAU 323 UAUUCAU C AAUCAUG

113 CCAUAAU A ACAUCAC 327 CAUCAAU C AUGAUGG

118 AUAACAU C ACUAACC 337 GAUGGGU U CUUAGAA

122 CADCACU A ACCAGAG 338 AUGGGUU C UUAGAAU

134 GAGACAU C AUAACAC 340 GGGUUCU U AGAAUGC 137 ACAUCAU A ACAGACA 341 GGUUCUU A GAAUGCA

148 CACAAAU U UAUAUAC 350 AAUGCAU U GGCAUUA 149 ACAAAUU U AUAUACU 356 UUGGCAU U AAGCCUA 150 CAAAUUU A UAUACUU 357 UGGCAUU A AGCCUAC 152 AAUUUAU A UACUUGA 363 UAAGCCU A CAAAGCA 154 UUUAUAU A CUUGAUA 372 AAAGCAU A CUCCCAU 157 AUAUACU U GAUAAAU 375 GCAUACU C CCAUAAU 161 ACUUGAU A AAUCAUG 380 CUCCCAU A AUAUACA 165 GAUAAAU C AUGAAUG 383 CCAUAAU A UACAAGU 176 AAUGCAU A GUGAGAA 385 AUAAUAU A CAAGUAU 188 GAAAACU U GAUGAAA 391 UACAAGU A UGAUCUC 208 GCCACAU U UACAUUC 396 GUAUGAU C UCAAUCC 209 CCACAUU U ACAUUCC 398 AUGAUCU C AAUCCAU 210 CACAUUU A CAUUCCU 402 UCUCAAU C CAUAAAU 214 UUUACAU U CCUGGUC 406 AAUCCAU A AAUUUCA 215 UUACAUU C CUGGUCA 410 CAUAAAU U UCAACAC 221 UCCUGGU C AACUAUG 411 AUAAAUU U CAACACA 226 GUCAACU A UGAAAUG 412 UAAAUUU C AACACAA 239 UGAAACU A UUACACA 421 ACACAAU A UUCACAC 241 AAACUAU U ACACAAA 423 ACAAUAU U CACACAA 242 AACUAUU A CACAAAG 424 CAAUAUU C ACACAAU 251 ACAAAGU A GGAAGCA 432 ACACAAU C UAAAACA 261 AAGCACU A AAUAUAA 434 ACAAUCU A AAACAAC 265 ACUAAAU A UAAAAAA 446 AACAACU C UAUGCAU 267 UAAAUAU A AAAAAUA 448 CAACUCU A UGCAUAA 274 AAAAAAU A UACUGAA 454 UAUGCAU A ACUAUAC 458 CAUAACU A UACUCCA

460 UAACUAU A CUCCAUA

463 CUAUACU C CAUAGUC

467 ACUCCAU A GUCCAGA

470 CCAUAGU C CAGAUGG

489 UGAAAAU U AUAGUAA

490 GAAAAUU A UAGUAAU

492 AAAUUAU A GUAAUUU

495 UUAUAGU A AUUUAAA

Table 32: RSV (IB) HH Ribozyme Sequence nt. HH Ribozyme Sequence

Position

10 AUUGAUU CUGAUGAGGCCGAAAGGCCGAA AUUUGCC

14 CUGAAUU CUGAUGAGGCCGAAAGGCCGAA AUUUAUU

18 UUGGCUG CUGAUGAGGCCGAAAGGCCGAA AUUGAUU

19 GUUGGCU CUGAUGAGGCCGAAAGGCCGAA AAUUGAU 54 GGUGUAU CUGAUGAGGCCGAAAGGCCGAA AUCAUUG 57 UGUGGUG CUGAUGAGGCCGAAAGGCCGAA AUUAUCA 77 UGUCUGU CUGAUGAGGCCGAAAGGCCGAA AUCAUCA 94 AAGUGAC CUGAUGAGGCCGAAAGGCCGAA ACGGUCU 97 CUCAAGU CUGAUGAGGCCGAAAGGCCGAA ACAACGG

101 UGGUCUC CUGAUGAGGCCGAAAGGCCGAA AGUGACA

110 AUGUUAU CUGAUGAGGCCGAAAGGCCGAA AUGGUCU

113 GUGAUGU CUGAUGAGGCCGAAAGGCCGAA AUUAUGG

118 GGUUAGU CUGAUGAGGCCGAAAGGCCGAA AUGUUAU

122 CUCUGGU CUGAUGAGGCCGAAAGGCCGAA AGUGAUG

134 GUGUUAU CUGAUGAGGCCGAAAGGCCGAA AUGUCUC

137 UGUGUGU CUGAUGAGGCCGAAAGGCCGAA AUGAUGU

148 GUAUAUA CUGAUGAGGCCGAAAGGCCGAA AUUUGUG

149 AGUAUAU CUGAUGAGGCCGAAAGGCCGAA AAUUUGU

150 AAGUAUA CUGAUGAGGCCGAAAGGCCGAA AAAUUUG 152 UCAAGUA CUGAUGAGGCCGAAAGGCCGAA AUAAAUU 154 UAUCAAG CUGAUGAGGCCGAAAGGCCGAA AUAUAAA 157 AUUUAUC CUGAUGAGGCCGAAAGGCCGAA AGUAUAU 161 CAUGAUU CUGAUGAGGCCGAAAGGCCGAA AUCAAGU 165 CAUUCAU CUGAUGAGGCCGAAAGGCCGAA AUUUAUC 176 UUCUCAC CUGAUGAGGCCGAAAGGCCGAA AUGCAUU 188 UUUCAUC CUGAUGAGGCCGAAAGGCCGAA AGUUUUC

208 GAAUGUA CUGAUGAGGCCGAAAGGCCGAA AUGUGGC

209 GGAAUGU CUGAUGAGGCCGAAAGGCCGAA AAUGUGG

210 AGGAAUG CUGAUGAGGCCGAAAGGCCGAA AAAUGUG

214 GACCAGG CUGAUGAGGCCGAAAGGCCGAA AUGUAAA

215 UGACCAG CUGAUGAGGCCGAAAGGCCGAA AAUGUAA 221 CAUAGUU CUGAUGAGGCCGAAAGGCCGAA ACCAGGA 226 CAUUUCA CUGAUGAGGCCGAAAGGCCGAA AGUUGAC 239 UGUGUAA CUGAUGAGGCCGAAAGGCCGAA AGUUUCA

241 UUUGUGU CUGAUGAGGCCGAAAGGCCGAA AUAGUUU

242 CUUUGUG CUGAUGAGGCCGAAAGGCCGAA AAUAGUU 251 UGCUUCC CUGAUGAGGCCGAAAGGCCGAA ACUUUGU 261 UUAUAUU CUGAUGAGGCCGAAAGGCCGAA AGUGCUU 265 UUUUUUA CUGAUGAGGCCGAAAGGCCGAA AUUUAGU 267 UAUUUUU CUGAUGAGGCCGAAAGGCCGAA AUAUUUA 74 UUCAGUA CUGAUGAGGCCGAAAGGCCGAA AUUUUUU 276 UAUUCAG CUGAUGAGGCCGAAAGGCCGAA AUAUUUU 283 UGUGUUG CUGAUGAGGCCGAAAGGCCGAA AUUCAGU

295 AGUGCCA CUGAUGAGGCCGAAAGGCCGAA AUUUUGU

303 AUAGGGA CUGAUGAGGCCGAAAGGCCGAA AGUGCCA

304 CAUAGGG CUGAUGAGGCCGAAAGGCCGAA AAGUGCC

305 GCAUAGG CUGAUGAGGCCGAAAGGCCGAA AAAGUGC 309 AUUGGCA CUGAUGAGGCCGAAAGGCCGAA AGGGAAA 317 UGAUGAA CUGAUGAGGCCGAAAGGCCGAA AUUGGCA

319 AUUGAUG CUGAUGAGGCCGAAAGGCCGAA AUAUUGG

320 GAUUGAU CUGAUGAGGCCGAAAGGCCGAA AAUAUCG 323 CAUGAUU CUGAUGAGGCCGAAAGGCCGAA AUGAAUA 327 CCAUCAU CUGAUGAGGCCGAAAGGCCGAA AUUGAUG

337 UUCUAAG CUGAUGAGGCCGAAAGGCCGAA ACCCAUC

338 AUUCUAA CUGAUGAGGCCGAAAGGCCGAA AACCCAU

340 GCAUUCU CUGAUGAGGCCGAAAGGCCGAA AGAACCC

341 UGCAUUC CUGAUGAGGCCGAAAGGCCGAA AAGAACC 350 UAAUGCC CUGAUGAGGCCGAAAGGCCGAA AUGCAUU

356 UAGGCUU CUGAUGAGGCCGAAAGGCCGAA AUGCCAA

357 GUAGGCU CUGAUGAGGCCGAAAGGCCGAA AAUGCCA 363 UGCUUUG CUGAUGAGGCCGAAAGGCCGAA AGGCUUA 372 AUGGGAG CUGAUGAGGCCGAAAGGCCGAA AUGCUUU 375 AUUAUGG CUGAUGAGGCCGAAAGGCCGAA AGUAUGC 380 UGUAUAU CUGAUGAGGCCGAAAGGCCGAA AUGGGAG 383 ACUUGUA CUGAUGAGGCCGAAAGGCCGAA AUUAUGG 385 AUACUUG CUGAUGAGGCCGAAAGGCCGAA AUAUUAU 391 GAGAUCA CUGAUGAGGCCGAAAGGCCGAA ACUUGUA 396 GGAUUGA CUGAUGAGGCCGAAAGGCCGAA AUCAUAC 398 AUGGAUU CUGAUGAGGCCGAAAGGCCGAA AGAUCAU 402 AUUUAUG CUGAUGAGGCCGAAAGGCCGAA AUUGAGA 406 UGAAAUU CUGAUGAGGCCGAAAGGCCGAA AUGGAUU

410 GUGUUGA CUGAUGAGGCCGAAAGGCCGAA AUUUAUG

411 UGUGUUG CUGAUGAGGCCGAAAGGCCGAA AAUUUAU

412 UUGUGUU CUGAUGAGGCCGAAAGGCCGAA AAAUUUA 421 GUGUGAA CUGAUGAGGCCGAAAGGCCGAA AUUGUGU

423 UUGUGUG CUGAUGAGGCCGAAAGGCCGAA AUAUUGU

424 AUUGUGU CUGAUGAGGCCGAAAGGCCGAA AAUAUUG 432 UGUUUUA CUGAUGAGGCCGAAAGGCCGAA AUUGUGU 434 GUUGUUU CUGAUGAGGCCGAAAGGCCGAA AGAUUGU 446 AUGGAUA CUGAUGAGGCCGAAAGGCCGAA AGUUGUU 448 UUAUGCA CUGAUGAGGCCGAAAGGCCGAA AGAGUUG 454 GUAUAGU CUGAUGAGGCCGAAAGGCCGAA AUGCAUA 458 UGGAGUA CUGAUGAGGCCGAAAGGCCGAA AGUUAUG 460 UAUGGAG CUGAUGAGGCCGAAAGGCCGAA AUAGUUA 463 GACUAUG CUGAUGAGGCCGAAAGGCCGAA AGUAUAG 467 UCUGGAC CUGAUGAGGCCGAAAGGCCGAA AUGGAGU 470 CCAUCUG CUGAUGAGGCCGAAAGGCCGAA ACUAUGG

489 UUACUAU CUGAUGAGGCCGAAAGGCCGAA AUUUUCA

490 AUUACUA CUGAUGAGGCCGAAAGGCCGAA AAUUUUC 492 AAAUUAC CUGAUGAGGCCGAAAGGCCGAA AUAAUUU 495 UUUAAAU CUGAUGAGGCCGAAAGGCCGAA ACUAUAA Table 33 : RSV (1C) HH target Sequence

nt. Target Sequence nt. Target Sequence Position Position

10 GGCAAAU A AGAAUUU 165 UACAUUU A ACUAACG 16 UAAGAAU U UGAUAAG 169 UUUAACU A ACGCUUU 17 AAGAAUU U GAUAAGU 175 UAACGCU U UGGCUAA 21 AUUUGAU A AGUACCA 176 AACGCUU U GGCUAAG 25 GAUAAGU A CCACUUA 131 UUUGGCU A AGGCAGU 31 UACCACU U AAAUUUA 192 CAGCGAU A CAUACAA 32 ACCACUU A AAUUUAA 196 GAUACAU A CAAUCAA 36 CUUAAAU U UAACUCC 201 AUACAAU C AAAUUGA 37 UUAAAUU U AACUCCC 206 AUCAAAU U GAAUGGC 38. UAAAUUU A ACUCCCU 216 AUGGCAU U GUGUUUG 42 UUUAACU C CCUUGGU 221 AUUGUGU U UGCGCAU 46 ACUCCCU U GGUUAGA 222 UUGUGUU U GUGCAUG 50 CCUUGGU U AGAGAUG 231 UGCAUGU U AUUACAA 51 CUUGGUU A GAGAUGG 232 GCAUGUU A UUACAAG 67 CAGCAAU U CAUUGAG 234 AUGUUAU U ACAAGUA 68 AGCAAUU C AUUGAGU 235 UGUUAUU A CAAGUAG 71 AAUUCAU U GAGUAUG 241 UACAAGU A GUGAUAU 76 AUUGAGU A UGAUAAA 247 UAGUGAU A UUUGCCC 81 GUAUGAU A AAAGUUA 249 GUGAUAU U UGCCCUA 87 UAAAAGU U AGAUUAC 250 UGAUAUU U GCCCUAA 88 AAAAGUU A GAUUACA 256 UUGCCCU A AUAAUAA 92 GUUAGAU U ACAAAAU 259 CCCUAAU AUAAUAU 93 UUAGAUU A CAAAAUU 262 UAAUAAU AUAUUGU 100 ACAAAAU U UGUUUGA 265 UAAUAAU UUGUAGU 101 CAAAAUU U GUUUGAC 267 AUAAUAU GUAGUAA 104 AAUUUGU U UGACAAU 270 AUAUUGU GUAAAAU 105 AUUUGUU U GACAAUG 273 UUGUAGU A AAAUCCA 120 AUGAAGU A GCAUUGU 278 GUAAAAU C CAAUUUC 125 GUAGCAU U GUUAAAA 283 AUCCAAU U UCACAAC 128 GCAUUGU U AAAAAUA 284 UCCAAUU U CACAACA 129 CAUUGUU A AAAAUAA 285 CCAAUUU C ACAACAA 135 UAAAAAU A ACAUGCU 300 UGCCAGU A CUACAAA 143 ACAUGCU A UACUGAU 303 CAGUACU A CAAAAUG 145 AUGCUAU A CUGAUAA 316 UGGAGGU U AUAUAUG 151 UACUGAU A AAUUAAU 317 GGAGGUU A UAUAUGG 155 GAUAAAU U AAUACAU 319 AGGUUAU A UAUGGGA 156 AUAAAUU A AUACAUU 321 GUUAUAU A UGGGAAA 159 AAUUAAU A CAUUUAA 338 AUGGAAU U AACACAU 163 AAUACAU U UAACUAA 339 UGGAAUU A ACACAUU 164 AUACAUU U AACUAAC 346 AACACAU U GCUCUCA 350 CAUUGCU C UCAACCU

352 UUGCUCU C AACCUAA

358 UCAACCU A AUGGUCU

364 UAAUGGU C UACUAGA

366 AUGGUCU A CUAGAUG

369 GUCUACU A GAUGACA

379 UGACAAU U GUGAAAU

387 GUGAAAU U AAAUUCU

388 UGAAAUU A AAUUCUC

392 AUUAAAU U CUCCAAA

393 UUAAAUU C UCCAAAA

395 AAAUUCU C CAAAAAA

405 AAAAACU A AGUGAUU

412 AAGUGAU U CAACAAU

413 AGUGAUU C AACAAUG

427 GACCAAU U AUAUGAA

428 ACCAAUU A UAUGAAU

430 CAAUUAU A UGAAUCA

436 UAUGAAU C AAUUAUC

440 AAUCAAU U AUCUGAA

441 AUCAAUU A UCUGAAU

443 CAAUUAU C UGAAUUA

449 UCUGAAU U ACUUGGA

450 CUGAAUU A CUUGGAU

453 AAUUACU U GGAUUUG

458 CUUGGAU U UGAUCUU

459 UUGGAUU U GAUCUUA

463 AUUUGAU C UUAAUCC

465 UUGAUCU U AAUCCAU

466 UGAUCUU A AUCCAUA

469 UCUUAAU C CAUAAAU

473 AAUCCAU A AAUUAUA

477 CAUAAAU U AUAAUUA

478 AUAAAUU A UAAUUAA

480 AAAUUAU A AUUAAUA

483 UUAUAAU U AAUAUCA

484 UAUAAUU A AUAUCAA

487 AAUUAAU A UCAACUA

489 UUAAUAU C AACUAGC

494 AUCAACU A GCAAAUC

501 AGCAAAU C AAUGUCA

507 UGAAUGU C ACUAACA

511 UGUCACU A ACACCAU

519 ACACCAU U AGUUAAU

520 CACCAUU A GUUAAUA

523 CAUUAGU U AAUAUAA

524 AUUAGUU A AUAUAAA Table 34: RSV (IC) HH Ribozyme Sequence

nt . HH Ribozyme Sequence Position

10 AAAUUCU CUGAUGAGGCCGAAAGGCCGAA AUUUGCC

16 CUUAUCA CUGAUGAGGCCGAAAGGCCGAA AUCCUCA

17 ACUUAUC CUGAUGAGGCCGAAAGGCCGAA AAUUCUU 21 UGGUACU CUGAUGAGGCCGAAAGGCCGAA AUCAAAU 25 UAAGUGG CUGAUGAGGCCGAAAGGCCGAA ACUUAUC

31 UAAAUUU CUGAUGAGGCCGAAAGGCCGAA AGUGGUA

32 UUAAAUU CUGAUGAGGCCGAAAGGCCGAA AAGUGGU

36 GGAGUUA CUGAUGAGGCCGAAAGGCCGAA AUUUAAG

37 GGGAGUU CUGAUGAGGCCGAAAGGCCGAA AAUUUAA

38 AGGGAGU CUGAUGAGGCCGAAAGGCCGAA AAAUUUA 42 ACCAAGG CUGAUGAGGCCGAAAGGCCGAA AGUUAAA 46 UCUAACC CUGAUGAGGCCGAAAGGCCGAA AGGGAGU

50 CAUCUCU CUGAUGAGGCCGAAAGGCCGAA ACCAAGG

51 CCAUCUC CUGAUGAGGCCGAAAGGCCGAA AACCAAG

67 CUCAAUG CUGAUGAGGCCGAAAGGCCGAA AUUGCUG

68 ACUCAAU CUGAUGAGGCCGAAAGGCCGAA AAUUGCU 71 CAUACUC CUGAUGAGGCCGAAAGGCCGAA AUGAAUU 76 UUUAUCA CUGAUGAGGCCGAAAGGCCGAA ACCCAAU 81 UAACUUU CUGAUGAGGCCGAAAGGCCGAA AUCAUAC 37 GUAAUCU CUGAUGAGGCCGAAAGGCCGAA ACUUUUA 88 UGUAAUC CUGAUGAGGCCGAAAGGCCGAA AACUUUU

92 AUUUUGU CUGAUGAGGCCGAAAGGCCGAA AUCUAAC

93 AAUUUUG CUGAUGAGGCCGAAAGGCCGAA AAUCUAA

100 UCAAACA CUGAUGAGGCCGAAAGGCCGAA AUUUUGU

101 GUCAAAC CUGAUGAGGCCGAAAGGCCGAA AAUUUUG

104 AUUGUCA CUGAUGAGGCCGAAAGGCCGAA ACAAAUU

105 CAUUGUC CUGAUGAGGCCGAAAGGCCGAA AACAAAU 120 ACAAUGC CUGAUGAGGCCGAAAGGCCGAA ACUUCAU 125 UUUUAAC CUGAUGAGGCCGAAAGGCCGAA AUGCUAC

128 UAUUUUU CUGAUGAGGCCGAAAGGCCGAA ACAAUGC

129 UUAUUUU CUGAUGAGGCCGAAAGGCCGAA AACAAUG 135 AGCAUGU CUGAUGAGGCCGAAAGGCCGAA AUUUUUA 143 AUCAGUA CUGAUGAGGCCGAAAGGCCGAA AGCAUGU 145 UUAUCAG CUGAUGAGGCCGAAAGGCCGAA AUAGCAU 151 AUUAAUU CUGAUGAGGCCGAAAGGCCGAA AUCAGUA

155 AUGUAUU CUGAUGAGGCCGAAAGGCCGAA AUUUAUC

156 AAUGUAU CUGAUGAGGCCGAAAGGCCGAA AAUUUAU 159 UUAAAUG CUGAUGAGGCCGAAAGGCCGAA AUUAAUU

163 UUAGUUA CUGAUGAGGCCGAAAGGCCGAA AUGUAUU

164 GUUAGUU CUGAUGAGGCCGAAAGGCCGAA AAUGUAU

165 CGUUAGU CUGAUGAGGCCGAAAGGCCGAA AAAUGUA 169 AAAGCGU CUGAUGAGGCCGAAAGGCCGAA AGUUAAA

175 UUAGCCA CUGAUGAGGCCGAAAGGCCGAA AGCGUUA

176 CUUAGCC CUGAUGAGGCCGAAAGGCCGAA AAGCGUU 181 ACUGCCU CUGAUGAGGCCGAAAGGCCGAA AGCCAAA 192 UUGUAUG CUGAUGAGGCCGAAAGGCCGAA AUCACUG 196 UUGAUUG CUGAUGAGGCCGAAAGGCCGAA AUGUAUC

201 UCAAUUU CUGAUGAGGCCGAAAGGCCGAA AUUGUAU

206 GCCAUUC CUGAUGAGGCCGAAAGGCCGAA AUUUGAU

216 CAAACAC CUGAUGAGGCCGAAAGGCCGAA AUGCCAU

221 AUGCACA CUGAUGAGGCCGAAAGGCCGAA ACACAAU

222 CAUGCAC CUGAUGAGGCCGAAAGGCCGAA AACACAA

231 UUGUAAU CUGAUGAGGCCGAAAGGCCGAA ACAUGCA

232 CUUGUAA CUGAUGAGGCCGAAAGGCCGAA AACAUGC

234 UACUUGU CUGAUGAGGCCGAAAGGCCGAA AUAACAU

235 CUACUUG CUGAUGAGGCCGAAAGGCCGAA AAUAACA 241 AUAUCAC CUGAUGAGGCCGAAAGGCCGAA ACUUGUA 247 GGGCAAA CUGAUGAGGCCGAAAGGCCGAA AUCACUA

249 UAGGGCA CUGAUGAGGCCGAAAGGCCGAA AUAUCAC

250 UUAGGGC CUGAUGAGGCCGAAAGGCCGAA AAUAUCA 256 UUAUUAU CUGAUGAGGCCGAAAGGCCGAA AGGGCAA 259 AUAUUAU CUGAUGAGGCCGAAAGGCCGAA AUUAGGG 262 ACAAUAU CUGAUGAGGCCGAAAGGCCGAA AUUAUUA 265 ACUACAA CUGAUGAGGCCGAAAGGCCGAA AUUAUUA 267 UUACUAC CUGAUGAGGCCGAAAGGCCGAA AUAUUAU 270 AUUUUAC CUGAUGAGGCCGAAAGGCCGAA ACAAUAU 273 UGGAUUU CUGAUGAGGCCGAAAGGCCGAA ACUACAA 278 GAAAUUG CUGAUGAGGCCGAAAGGCCGAA AUUUUAC

283 GUUGUGA CUGAUGAGGCCGAAAGGCCGAA AUUGGAU

284 UGUUGUG CUGAUGAGGCCGAAAGGCCGAA AAUUGGA

285 UUGUUGU CUGAUGAGGCCGAAAGGCCGAA AAAUUGG 300 UUUGUAG CUGAUGAGGCCGAAAGGCCGAA ACUGGCA 303 CAUUUUG CUGAUGAGGCCGAAAGGCCGAA AGUACUG

316 CAUAUAU CUGAUGAGGCCGAAAGGCCGAA ACCUCCA

317 CCAUAUA CUGAUGAGGCCGAAAGGCCGAA AACCUCC 319 UCCCAUA CUGAUGAGGCCGAAAGGCCGAA AUAACCU 321 UUCCCCA CUGAUGAGGCCGAAAGGCCGAA AUAUAAC

338 ACGUGUU CUGAUGAGGCCGAAAGGCCGAA AUUCCAU

339 AAUGUGU CUGAUGAGGCCGAAAGGCCGAA AAUUCCA 346 UGAGAGC CUGAUGAGGCCGAAAGGCCGAA AUGUGUU 350 AGGUUGA CUGAUGAGGCCGAAAGGCCGAA AGCAAUG 352 UUAGGUU CUGAUGAGGCCGAAAGGCCGAA AGAGCAA 358 AGACCAU CUGAUGAGGCCGAAAGGCCGAA AGGUUGA 364 UCUAGUA CUGAUGAGGCCGAAAGGCCGAA ACCAUUA 366 CAUCUAG CUGAUGAGGCCGAAAGGCCGAA AGACCAU 369 UGUCAUC CUGAUGAGGCCGAAAGGCCGAA AGUAGAC 379 AUUUCAC CUGAUGAGGCCGAAAGGCCGAA AUUGUCA

387 AGAAUUU CUGAUGAGGCCGAAAGGCCGAA AUUUCAC

388 GAGAAUU CUGAUGAGGCCGAAAGGCCGAA AAUUUCA 392 UUUGGAG CUGAUGAGGCCGAAAGGCCGAA AUUUAAU 393 UUUUGGA CUGAUGAGGCCGAAAGGCCGAA AAUUUAA

395 UUUUUUG CUGAUGAGGCCGAAAGGCCGAA AGAAUUU

405 AAUCACU CUGAUGAGGCCGAAAGGCCGAA AGUUUUU

412 AUUGUUG CUGAUGAGGCCGAAAGGCCGAA AUCACUU

413 CAUUGUU CUGAUGAGGCCGAAAGGCCGAA AAUCACU

427 UUCAUAU CUGAUGAGGCCGAAAGGCCGAA AUUGGUC

428 AUUCAUA CUGAUGAGGCCGAAAGGCCGAA AAUUGGU 430 UGAUUCA CUGAUGAGGCCGAAAGGCCGAA AUAAUOG 436 GAUAAUU CUGAUGAGGCCGAAAGGCCGAA AUCCAUA

440 CUCAGAU CUGAUGAGGCCGAAAGGCCGAA AUUGAUU

441 AUUCAGA CUGAUGAGGCCGAAAGGCCGAA AAUUGAU 443 UAAUUCA CUGAUGAGGCCGAAAGGCCGAA AUAAUUG

449 UCCAAGU CUGAUGAGGCCGAAAGGCCGAA AUUCAGA

450 AUCCAAG CUGAUGAGGCCGAAAGGCCGAA AAUUCAG 453 CAAAUCC CUGAUGAGGCCGAAAGGCCGAA AGUAAUU

458 AAGAUCA CUGAUGAGGCCGAAAGGCCGAA AUCCAAG

459 UAAGAUC CUGAUGAGGCCGAAAGGCCGAA AAUCCAA 463 GGAUUAA CUGAUGAGGCCGAAAGGCCGAA AUCAAAU

465 AUGGAUU CUGAUGAGGCCGAAAGGCCGAA AGAUCAA

466 UAUGGAU CUGAUGAGGCCGAAAGGCCGAA AAGAUCA 469 AUUUAUG CUGAUGAGGCCGAAAGGCCGAA AUUAAGA 473 UAUAAUU CUGAUGAGGCCGAAAGGCCGAA AUGGAUU

477 UAAUUAU CUGAUGAGGCCGAAAGGCCGAA AUUUAUG

478 UUAAUUA CUGAUGAGGCCGAAAGGCCGAA AAUUUAU 480 UAUUAAU CUGAUGAGGCCGAAAGGCCGAA AUAAUUU

483 UGAUAUU CUGAUGAGGCCGAAAGGCCGAA AUUAUAA

484 UUGAUAU CUGAUGAGGCCGAAAGGCCGAA AAUUAUA 487 UAGUUGA CUGAUGAGGCCGAAAGGCCGAA AUUAAUU 489 GCUAGUU CUGAUGAGGCCGAAAGGCCGAA AUAUUAA 494 GAUUUGC CUGAUGAGGCCGAAAGGCCGAA AGUUGAU 501 UGACAUU CUGAUGAGGCCGAAAGGCCGAA AUUUGCU 507 UGUUAGU CUGAUGAGGCCGAAAGGCCGAA ACAUOGA 511 AUGGUGU CUGAUGAGGCCGAAAGGCCGAA AGUGACA

519 AUUAACU CUGAUGAGGCCGAAAGGCCGAA AUGGUGU

520 UAUUAAC CUGAUGAGGCCGAAAGGCCGAA AAUGGUG

523 UUAUAUU CUGAUGAGGCCGAAAGGCCGAA ACUAAUG

524 UUUAUAU CUGAUGAGGCCGAAAGGCCGAA AACUAAU

Table 35: RSV (N) HH Target Sequence

nt. HH Target Sequence nt. HH Target Sequence Position Position

9 GGCAAAU A CAAAGAU 217 GGUAUGU U AUAUGCG

21 GAUGGCU C UUAGCAA 218 GUAUGUU A UAUGCGA 23 UGGCUCU U AGCAAAG 220 AUGUUAU A UGCGAUG 24 GGCUCUU A GCAAAGU 229 GCGAUGU C UAGGCCA 32 GCAAAGU C AAGUUGA 231 GAUGUCU A GGUUAGG 37 GUCAAGU U GAAUGAU 235 UCUAGGU U AGGAAGA 45 GAAUGAU A CACUCAA 236 CUAGGUU A GGAAGAG 50 AUACACU C AACAAAG 254 ACACCAU A AAAAUAC 60 CAAAGAU C AACUUCU 260 UAAAAAU A CUCAGAG 65 AUCAACU U CUGUCAU 263 AAAUACU C AGAGAUG 66 UCAACUU C UGUCAUC 277 GCGGGAU A UCAUGUA 70 CUUCUGU C AUCCAGC 279 GGGAUAU C AUGUAAA 73 CUGUCAU C CAGCAAA 284 AUCAUGU A AAAGCAA 82 AGCAAAU A CACCAUC 299 AUGGAGU A GAUGUAA 89 ACACCAU C CAACGGA 305 UAGAUGU A ACAACAC 108 AGGAGAU A GUAUUGA 315 AACACAU C GUCAAGA 111 AGAUAGU A UUGAUAC 318 ACAUCGU AAGACAU 113 AUAGUAU U GAUACUC 326 AAGACAU AAUGGAA 117 UAUUGAU A CUCCUAA 327 AGAGAUU AUGGAAA 120 UGAUACU C CUAAUUA 346 AUGAAAU UGAAGUG 123 UACUCCU A AUUAUGA 347 UGAAAUU GAAGUGU 126 UCCUAAU U AUGAUGU 355 GAAGUGU U AACA 27 UUG 1 CCUAAUU A UGAUGUG 356 AAGUGUU A ACA 146 UUGG

AACACAU C AAUAAGU 361 UUAACAU U GGCAAGC 150 CAUCAAU A AGCUAUG 370 GCAAGCU U AACAACU 154 AAUAAGU U AUGUGGC 371 CAAGCUU A ACAACCG 155 AUAAGUU A UGUGGCA 383 CUGAAAU U CAAAUCA 166 GGCAUGU U AUUAAUC 384 UGAAAUU C AAA

167 UCAA

GCAUGUU A UUAAUCA 389 UUCAAAU C AACAUUG 169 AUGUUAU U AAUCACA 395 UCAACAU U GAGAUAG 170 UGUUAUU A AUCACAG 401 UUGAGAU A GAA

173 UCUA

UAUUAAU C ACAGAAG 406 AUAGAAU UAGAAAA 186 AGAUGCU A AUCAUAA 408 AGAAUCU GAAAAUC 189 UGCUAAU C AUAAAUU 415 AGAAAAU CUACAAA 192 UAAUCAU A AAUUCAC 418 AAAUCCU CAAAAAA 196 CAUAAAU U CACUGGG 431 AAAUGCU AAAGAAA 197 AUAAAUU C ACUGGGU 449 GAGAGGU GCUCCAG 205 ACUGGGU U AAUAGGU 453 GGUAGCU CAGAAUA 206 CUGGGUU A AUAGGUA 460 CCAGAAU CAGGCAU 209 GGUUAAU A GGUAUGU 472 CAUGACU UCCUGAU 213 AAUAGGU A UGUUAUA 474 UGACUCU CUGAUUG 480 UCCUGAU U GUGGGAU 696 UUUUGGU A UAGCACA

491 GGAUGAU A AUAUUAU 698 UUGGUAU A GCACAAU

494 UGAUAAU A UUAUGUA 706 GCACAAU C UUCUACC

496 AUAAUAU U AUGUAUA 708 ACAAUCU U CUACCAG

497 UAAUAUU A UGUAUAG 709 CAAUCUU C UACCAGA

501 AUUAUGU A UAGCAGC 711 AUCUUCU A CCAGAGG

503 UAUGUAU A GCAGCAU 726 UGGCAGU A GAGUUGA

511 GCAGCAU U AGUAAUA 731 GUAGAGU U GAAGGGA

512 CAGCAUU A GUAAUAA 740 AAGGGAU U UUUGCAG

515 CAUUAGU A AUAACUA 741 AGGGAUU U UUGCAGG

518 UAGUAAU A ACUAAAU 742 GGGAUUU U UGCAGGA

522 AAUAACU A AAUUAGC 743 GGAUUUU U GCAGGAU

526 ACUAAAU U AGGAGCA 751 GCAGGAU U GUUUAUG

527 CUAAAUU A GCAGCAG 754 GGAUUGU U UAUGAAU

544 GACAGAU C UGGUCUU 755 GAUUGUU U AUGAAUG

549 AUCUGGU C UUACAGC 756 AUUGUUU A UGAAUGC

551 CUGGUCU U ACAGCCG 766 AAUGCCU A UGGUGCA

552 UGGUCUU A CAGCCGU 787 GUGAUGU U ACGGUGG

563 CCGUGAU U AGGAGAG 788 UGAUGUU A CGGUGGG

564 CGUGAUU A GGAGAGC 800 GGGGAGU C UUAGCAA

573 GAGAGCU A AUAAUGU 802 GGAGUCU U AGCAAAA

576 AGCUAAU A AUGUCCU 803 GAGUCUU A GCAAAAU

581 AUAAUGU C CUAAAAA 811 GCAAAAU C AGUUAAA

584 AUGUCCU A AAAAAUG 815 AAUCAGU U AAAAAUA

603 GAAACGU U AGAAAGG 816 AUCAGUU A AAAAUAU

604 AAACGUU A CAAAGGC 822 UAAAAAU A UUAUGUU

613 AAAGGCU U ACUACCC 824 AAAAUAU U AUGUUAG

614 AAGGCUU A CUACCCA 825 AAAUAUU A UGUUAGG

617 GCUUACU A CCCAAGG 829 AUUAUGU U AGGACAU

629 AGGACAU A GCCAACA 830 UUAUGUU A GGACAUG

640 AACAGCU U CUAUGAA 840 ACAUGCU A GUGUGCA

641 ACAGCUU C UAUGAAG 866 AACAAGU U GUUGAGG

643 AGCUCCU A UGAAGUG 869 AAGUUGU U GAGGUUU

652 GAAGUGU U UGAAAAA 875 UUGAGGU U UAUGAAU

653 AAGUGUU U GAAAAAC 876 UGAGGUU U AUGAAUA

663 AAAACAU C CCCACUU 877 GAGGUUU A UGAAUAU

670 CCCCACU U UAUAGAU 883 UAUGAAU A UGCCCAA

671 CCCACUU U AUAGAUG 895 CAAAAAU U GGGUGGU

672 CCACUUU A UAGAUGU 913 GCAGGAU U CUACCAU

674 ACUUUAU A GAUGUUU 914 CAGGAUU C UACCAUA

680 UAGAUGU U UUUGUUC 916 GGAUUCU A CCAUAUA

681 AGAUGUU U UUGUUCA 921 CUACCAU A UAUUGAA

682 GAUGUUU U UGUUCAU 923 ACCAUAU A UUGAACA

683 AUGUUUU U GUUCAUU 925 CAUAUAU U GAACAAC

686 UUUUUGU U CAUUUUG 943 AAAGCAU C AUUAUUA

687 UUUUGUU C AUUUUGG 946 GCAUCAU U AUUAUCU

690 UGUUCAU U UUGGUAU 947 CAUCAUU A UUAUCUU

691 GUUCAUU U UGGUAUA 949 UCAUUAU U AUCUUUG

692 UUCAUUU U GGUAUAG 950 CAUUAUU A UCUUUGA 952 UUAUUAU C UUUGACU

954 AUUAUCU U UGACUCA

955 UUAUCUU U GACUCAA

960 UUUGACU C AAUUUCC

964 ACUCAAU U UCCUCAC

965 CUCAAUU U CCUCACU

966 UCAAUUU C CUCACUU

969 AUUUCCU C ACUUCUC

973 CCUCACU U CUCCAGU

974 CUCACUU C UCCAGUG

976 CACUUCU C CAGUGUA

983 CCAGUGU A GUAUCAG

986 GUGUAGU A UUAGGCA

988 GUAGUAU U AGGCAAU

989 UAGUAUU A GGCAAUG

1007 CUGGCCU A GGCAUAA

1013 UAGGCAU A AUGGGAG

1024 GGAGAGU A CAGAGGU

1032 CAGAGGU A CACCGAG

1044 GAGGAAU C AAGAUCU

1050 UCAAGAU C UAUAUGA

1052 AAGAUCU A UAUGAUG

1054 GAUCUAU A UGAUGCA

1072 AAGGCAU A UGCUGAA

1085 AACAACU C AAAGAAA

1103 GOGUGAU U AACUACA

1104 UGUGAUU A ACUACAG

1108 AUUAACU A CAGUGUA

1115 ACAGUGU A CUAGACU

1118 GUGUACU A GACUCGA

1123 CUAGACU U GACAGCA

1139 AAGAACU A GAGGCUA

1146 AGAGGCU A UCAAACA

1148 AGGCUAU C AAACAUC

1155 CAAACAU C AGCUUAA

1160 AUCAGCU U AAUCCAA

1151 UCAGCUU A AUCCAAA

1154 GCUUAAU C CAAAAGA

1173 AAAAGAU A AUGAUGU

1181 AUGAUGU A GAGCUUU

1187 UAGAGCU U UGAGUUA

1188 AGAGCUU U GAGUUAA

1193 UUUGAGU U AAUAAAA

1194 UUGAGUU A AUAAAAA Table 36: RSV (N) HH Ribozyme Sequence

nt . HH Ribozyme Sequence

Position

9 AUCUUUG CUGAUGAGGCCGAAAGGCCGAA AUUUGCC

21 UUGCUAA CUGAUGAGGCCGAAAGGCCGAA AGCCAUC

23 CUUUGCU CUGAUGAGGCCGAAAGGCCGAA AGAGCCA

24 ACUUUGC CUGAUGAGGCCGAAAGGCCGAA AAGAGCC 32 UCAACUU CUGAUGAGGCCGAAAGGCCGAA ACUUUGC 37 AUCAUUC CUGAUGAGGCCGAAAGGCCGAA ACUUGAC 45 UUGAGUG CUGAUGAGGCCGAAAGGCCGAA AUCAUUC 50 CUUUGUU CUGAUGAGGCCGAAAGGCCGAA AGUGUAU 60 AGAAGUC CUGAUGAGGCCGAAAGGCCGAA AUCUUUG

65 AUGACAG CUGAUGAGGCCGAAAGGCCGAA AGUUGAU

66 GAUGACA CUGAUGAGGCCGAAAGGCCGAA AAGUUGA 70 GCUGGAU CUGAUGAGGCCGAAAGGCCGAA ACAGAAG 73 UUUGCUG CUGAUGAGGCCGAAAGGCCGAA AUGACAG 82 GAUGGUG CUGAUGAGGCCGAAAGGCCGAA AUUUGCU 89 UCCGUUG CUGAUGAGGCCGAAAGGCCGAA AUGGUGU

108 UCAAUAC CUGAUGAGGCCGAAAGGCCGAA AUCUCCU

111 GUAUCAA CUGAUGAGGCCGAAAGGCCGAA ACUAUCU

113 GAGUAUC CUGAUGAGGCCGAAAGGCCGAA AUACUAU

117 UUAGGAG CUGAUGAGGCCGAAAGGCCGAA AUCAAUA

120 UAAUUAG CUGAUGAGGCCGAAAGGCCGAA AGUAUCA

123 UCAUAAU CUGAUGAGGCCGAAAGGCCGAA AGGAGUA

126 ACAUCAU CUGAUGAGGCCGAAAGGCCGAA AUUAGGA

127 CACAUCA CUGAUGAGGCCGAAAGGCCGAA AAUUAGG 146 ACUUAUU CUGAUGAGGCCGAAAGGCCGAA AUGUGUU 150 CAUAACU CUGAUGAGGCCGAAAGGCCGAA AUUGAUG

154 GCCACAU CUGAUGAGGCCGAAAGGCCGAA ACUUAUU

155 UGCCACA CUGAUGAGGCCGAAAGGCCGAA AACUUAU

166 GAUUAAU CUGAUGAGGCCGAAAGGCCGAA ACAUGCC

167 UGAUUAA CUGAUGAGGCCGAAAGGCCGAA AACAUGC

169 UGUGAUU CUGAUGAGGCCGAAAGGCCGAA AUAACAU

170 CUGUGAU CUGAUGAGGCCGAAAGGCCGAA AAUAACA 173 CUUCUGU CUGAUGAGGCCGAAAGGCCGAA AUUAAUA 186 UUAUGAU CUGAUGAGGCCGAAAGGCCGAA AGCAUCU 189 AAUUUAU CUGAUGAGGCCGAAAGGCCGAA AUUAGCA 192 GUGAAUU CUGAUGAGGCCGAAAGGCCGAA AUGAUUA

196 CCCAGUG CUGAUGAGGCCGAAAGGCCGAA AUUUAUG

197 ACCCAGC CCGAUGAGGCCGAAAGGCCGAA AAUUUAU

205 ACCUAUU CUGAUGAGGCCGAAAGGCCGAA ACCCAGU

206 UACCUAU CUGAUGAGGCCGAAAGGCCGAA AACCCAG 209 ACAUACC CUGAUGAGGCCGAAAGGCCGAA AUUAACC 213 UAUAACA CUGAUGAGGCCGAAAGGCCGAA ACCUAUU 217 CGCAUAU CUGAUGAGGCCGAAAGGCCGAA ACAUACC

218 UCGCAUA CUGAUGAGGCCGAAAGGCCGAA AACAUAC 220 CAUCGCA CUGAUGAGGCCGAAAGGCCGAA AUAACAU 229 UAACCUA CUGAUGAGGCCGAAAGGCCGAA ACAUCGC 231 CCUAACC CUGAUGAGGCCGAAAGGCCGAA AGACAUC

235 UCUUCCU CUGAUGAGGCCGAAAGGCCGAA ACCUAGA

236 CUCUUCC CUGAUGAGGCCGAAAGGCCGAA AACCUAG 254 GUAUUUU CUGAUGAGGCCGAAAGGCCGAA AUGGUGU 260 CUCUGAG CUGAUGAGGCCGAAAGGCCGAA AUUUUUA 263 CACCUCU CUGAUGAGGCCGAAAGGCCGAA AGUAUUU 277 UACAUGA CUGAUGAGGCCGAAAGGCCGAA AUCCCGC 279 UUUACAU CUGAUGAGGCCGAAAGGCCGAA AUAUCCC 284 UUGCUUU CUGAUGAGGCCGAAAGGCCGAA AGAUGAU 299 UUACAUC CUGAUGAGGCCGAAAGGCCGAA ACUCCAU 305 GUGUUGU CUGAUGAGGCCGAAAGGCCGAA ACAUCUA 315 UCUUGAC CUGAUGAGGCCGAAAGGCCGAA AUGUGUU 318 AUGUCUU CUGAUGAGGCCGAAAGGCCGAA ACGAUGU

326 UUCCAUU CUGAUGAGGCCGAAAGGCCGAA AUGUCUU

327 UUUCCAU CUGAUGAGGCCGAAAGGCCGAA AAUGUCU

346 CACCUCA CUGAUGAGGCCGAAAGGCCGAA AUUUCAU

347 ACACUUC CUGAUGAGGCCGAAAGGCCGAA AAUUUCA

355 CAAUGUU CUGAUGAGGCCGAAAGGCCGAA ACACUUC

356 CCAAUGU CUGAUGAGGCCGAAAGGCCGAA AACACUU 361 GCUUGCC CUGAUGAGGCCGAAAGGCCGAA AUGUUAA

370 AGUUGUU CUGAUGAGGCCGAAAGGCCGAA AGCUUGC

371 CAGUUGU CUGAUGAGGCCGAAAGGCCGAA AAGCUUG

383 UGAUUUG CUGAUGAGGCCGAAAGGCCGAA AUUUCAG

384 UUGAUUU CUGAUGAGGCCGAAAGGCCGAA AAUUUCA 389 CAAUGUU CUGAUGAGGCCGAAAGGCCGAA AUUUGAA 395 CUAUCUC CUGAUGAGGCCGAAAGGCCGAA AUGUUGA 401 UAGAUUC CUGAUGAGGCCGAAAGGCCGAA AUCUCAA 406 UUUUCUA CUGAUGAGGCCGAAAGGCCGAA AUUCUAU 408 GAUUUUC CUGAUGAGGCCGAAAGGCCGAA AGAUUCU 415 UUUGUAG CUGAUGAGGCCGAAAGGCCGAA AUUUUCU 418 UCUUUUG CUGAUGAGGCCGAAAGGCCGAA AGGAUUU 431 UUUCUUU CUGAUGAGGCCGAAAGGCCGAA AGCAUUU 449 CCGGAGC CUGAUGAGGCCGAAAGGCCGAA ACCUCUC 453 UAUUCUG CUGAUGAGGCCGAAAGGCCGAA AGCUACC 460 AUGCCUG CUGAUGAGGCCGAAAGGCCGAA AUUCUGG 472 AUCAGGA CUGAUGAGGCCGAAAGGCCGAA AGUCAUG 474 CAAUCAG CUGAUGAGGCCGAAAGGCCGAA AGAGUCA 480 AUCCCAC CUGAUGAGGCCGAAAGGCCGAA AUCAGGA 491 AUAAUAU CUGAUGAGGCCGAAAGGCCGAA AUCAUCC 494 UACAUAA CUGAUGAGGCCGAAAGGCCGAA AUUACCA

496 UAUACAU CUGAUGAGGCCGAAAGGCCGAA AUAUUAU

497 CUAUACA CCGAUGAGGCCGAAAGGCCGAA AAUAUUA 501 GCUGCUA CUGAUGAGGCCGAAAGGCCGAA ACAUAAU 503 AUGCUGC CUGAUGAGGCCGAAAGGCCGAA AUACAUA 511 UAUUACU CUGAUGAGGCCGAAAGGCCGAA AUGCUGC 512 UUAUUAC CUGAUGAGGCCGAAAGGCCGAA AAUGCUG 515 UAGUUAU CUGAUGAGGCCGAAAGGCCGAA ACUAAUG 518 AUUUAGU CUGAUGAGGCCGAAAGGCCGAA AUUACUA 522 GCUAAUU CUGAUGAGGCCGAAAGGCCGAA AGUUAUU 526 UGCUGCU CUGAUGAGGCCGAAAGGCCGAA AUUUAGU 527 CUGCUGC CUGAUGAGGCCGAAAGGCCGAA AAUUUAG 544 AAGACCA CUGAUGAGGCCGAAAGGCCGAA AUCUGUC 549 GCUGUAA CUGAUGAGGCCGAAAGGCCGAA ACCAGAU 551 CGGCUGU CCGAUGAGGCCGAAAGGCCGAA AGACCAG 552 ACGGCUG CUGAUGAGGCCGAAAGGCCGAA AAGACCA 563 CUCUCCU CUGAUGAGGCCGAAAGGCCGAA AUCACGG 564 GCUCUCC CUGAUGAGGCCGAAAGGCCGAA AAUCACG 573 ACACUAU CUGAUGAGGCCGAAAGGCCGAA AGCUCUC 576 AGGACAU CUGAUGAGGCCGAAAGGCCGAA AUUAGCU 581 UUUUUAG CUGAUGAGGCCGAAAGGCCGAA ACAUUAU 584 CAUUUUU CUGAUGAGGCCGAAAGGCCGAA AGGACAU 603 CCUUUGU CUGAUGAGGCCGAAAGGCCGAA ACGUUUC 604 GCCUUUG CUGAUGAGGCCGAAAGGCCGAA AACGUUU 613 GGGUAGU CUGAUGAGGCCGAAAGGCCGAA AGCCUCU 614 UGGGCAG CUGAUGAGGCCGAAAGGCCGAA AAGCCUU 617 CCUUGGG CUGAUGAGGCCGAAAGGCCGAA AGUAAGC 629 UGUUGGC CUGAUGAGGCCGAAAGGCCGAA AUGUCCU 640 UUCAUAG CUGAUGAGGCCGAAAGGCCGAA AGCUGUU 641 CUUCAUA CUGAUGAGGCCGAAAGGCCGAA AAGCUGU 643 CACUUCA CUGAUGAGGCCGAAAGGCCGAA AGAAGCU 652 UUUUUCA CUGAUGAGGCCGAAAGGCCGAA ACACUUC 653 GUUUUUC CUGAUGAGGCCGAAAGGCCGAA AACACUU 663 AAGUGGG CUGAUGAGGCCGAAAGGCCGAA AUGUUUU 670 AUCUAUA CUGAUGAGGCCGAAAGGCCGAA AGUGGGG 671 CAUCUAU CUGAUGAGGCCGAAAGGCCGAA AAGUGGG 672 ACAUCUA CUGAUGAGGCCGAAAGGCCGAA AAAGUGG 674 AAACAUC CUGAUGAGGCCGAAAGGCCGAA AUAAAGU 680 GAACAAA CUGAUGAGGCCGAAAGGCCGAA ACAUCUA 681 UGAACAA CUGAUGAGGCCGAAAGGCCGAA AAGAUCU 682 AUGAACA CUGAUGAGGCCGAAAGGCCGAA AAACAUC 683 AAUGAAC CUGAUGAGGCCGAAAGGCCGAA AAAACAU 686 CAAAAUG CUGAUGAGGCCGAAAGGCCGAA ACAAAAA 687 CCAAAAU CUGAUGAGGCCGAAAGGCCGAA AACAAAA 690 AUACCAA CUGAUGAGGCCGAAAGGCCGAA AUGAACA 691 UAUACCA CUGAUGAGGCCGAAAGGCCGAA AAUGAAC 692 CUAUACC CUGAUGAGGCCGAAAGGCCGAA AAAUGAA 696 UGUGCUA CUGAUGAGGCCGAAAGGCCGAA ACCAAAA 698 AUUGCGC CUGAUGAGGCCGAAAGGCCGAA AUACCAA 706 GGUAGAA CCGACGAGGCCGAAAGGCCGAA ACCGCGC 708 CUGGUAG CUGAUGAGGCCGAAAGGCCGAA AGAUUGU 709 UCUGGUA CUGAUGAGGCCGAAAGGCCGAA AAGAUCG 711 CCUCUGG CUGAUGAGGCCGAAAGGCCGAA AGAAGAU 726 UCAACUC CUGAUGAGGCCGAAAGGCCGAA ACUGCCA 731 UCCCUUC CUGAUGAGGCCGAAAGGCCGAA ACUCUAC 740 CUGCAAA CUGAUGAGGCCGAAAGGCCGAA AUCCCUU

741 CCUGCAA CUGAUGAGGCCGAAAGGCCGAA AAUCCCU

742 UCCUGCA CUGAUGAGGCCGAAAGGCCGAA AAAUCCC

743 AUCCUGC CUGAUGAGGCCGAAAGGCCGAA AAAAUCC 751 CAUAAAC CUGAUGAGGCCGAAAGGCCGAA AUCCUGC

754 AUUCAUA CUGAUGAGGCCGAAAGGCCGAA ACAAUCC

755 CAUUCAU CCGAUGAGGCCGAAAGGCCGAA AACAAUC

756 GCAUUCA CUGAUGAGGCCGAAAGGCCGAA AAACAAU 766 UGCACCA CUGAUGAGGCCGAAAGGCCGAA AGGCAUU

787 CCACCGU CUGAUGAGGCCGAAAGGCCGAA ACAUCAC

788 CCCACCG CUGAUGAGGCCGAAAGGCCGAA AACAUCA 800 UUGCUAA CUGAUGAGGCCGAAAGGCCGAA ACUCCCC

802 UUCCGCU CUGAUGAGGCCGAAAGGCCGAA AGACUCC

803 AUUCUGC CUGAUGAGGCCGAAAGGCCGAA AAGACUC 811 UUUAACU CUGAUGAGGCCGAAAGGCCGAA AUUUUGC

815 UAUUUUU CUGAUGAGGCCGAAAGGCCGAA ACUGAUU

816 AUAUUUU CUGAUGAGGCCGAAAGGCCGAA AACUGAU 822 AACAUAA CUGAUGAGGCCGAAAGGCCGAA AUUUUUA

824 CUAACAU CCGAUGAGGCCGAAAGGCCGAA AUAUUUU

825 CCUAACA CUGAUGAGGCCGAAAGGCCGAA AAUAUUU

829 AUGUCCU CUGAUGAGGCCGAAAGGCCGAA ACAUAAU

830 CAUGUCC CUGAUGAGGCCGAAAGGCCGAA AACAUAA 840 UGCACAC CUGAUGAGGCCGAAAGGCCGAA AGCAUGU 866 CCUCAAC CCGAUGAGGCCGAAAGGCCGAA ACUUGUU 869 AAACCUC CCGAUGAGGCCGAAAGGCCGAA ACAACUU

875 AUUCAUA CUGAUGAGGCCGAAAGGCCGAA ACCUCAA

876 UAUUCAU CUGAUGAGGCCGAAAGGCCGAA AACCUCA

877 AUAUUCA CUGAUGAGGCCGAAAGGCCGAA AAACCUC 883 UUGGGCA CUGAUGAGGCCGAAAGGCCGAA AUUCAUA 895 ACCACCC CUGAUGAGGCCGAAAGGCCGAA AUUUUUG

913 AUGGUAG CUGAUGAGGCCGAAAGGCCGAA AUCCUGC

914 UAUGGUA CUGAUGAGGCCGAAAGGCCGAA AAUCCUG 916 UAUAUGG CUGAUGAGGCCGAAAGGCCGAA AGAAUCC 921 UUCAAUA CUGAUGAGGCCGAAAGGCCGAA AUGGUAG 923 UGUUCAA CUGAUGAGGCCGAAAGGCCGAA AUAUGGU 925 GUUGUUC CUGAUGAGGCCGAAAGGCCGAA AUAUAUG 943 UAAUAAU CUGAUGAGGCCGAAAGGCCGAA AUGCUUU

946 AGAUAAU CUGAUGAGGCCGAAAGGCCGAA AUGAUGC

947 AAGAUAA CUGAUGAGGCCGAAAGGCCGAA AAUGAUG

949 CAAAGAU CUGAUGAGGCCGAAAGGCCGAA AUAAUGA

950 UCAAAGA CUGAUGAGGCCGAAAGGCCGAA AAUAAUG 952 AGUCAAA CUGAUGAGGCCGAAAGGCCGAA AUAAUAA

954 UGAGUCA CUGAUGAGGCCGAAAGGCCGAA AGAUAAU

955 UUGAGUC CUGAUGAGGCCGAAAGGCCGAA AAGAUAA 960 GGAAAUU CUGAUGAGGCCGAAAGGCCGAA AGUCAAA

964 GUGAGGA CUGAUGAGGCCGAAAGGCCGAA AUUGAGU

965 AGUGAGG CUGAUGAGGCCGAAAGGCCGAA AAUUGAG

966 AAGUGAG CUGAUGAGGCCGAAAGGCCGAA AAAUUGA 969 GAGAAGU CUGAUGAGGCCGAAAGGCCGAA AGGAAAU 973 ACUGGAG CUGAUGAGGCCGAAAGGCCGAA AGUGAGG

974 CACUGGA CUGAUGAGGCCGAAAGGCCGAA AAGUGAG 976 UACACUG CUGAUGAGGCCGAAAGGCCGAA AGAAGUG 983 CUAAUAC CUGAUGAGGCCGAAAGGCCGAA AGACUGG 986 UGCCUAA CUGAUGAGGCCGAAAGGCCGAA ACUACAC

988 AUUGCCU CUGAUGAGGCCGAAAGGCCGAA AUACUAC

989 CAUUGCC CUGAUGAGGCCGAAAGGCCGAA AAUACUA 1007 UUAUGCC CUGAUGAGGCCGAAAGGCCGAA AGGCCUG 1013 CUCCCAU CUGAUGAGGCCGAAAGGCCGAA AUGCCUA 1024 ACCUCUG CUGAUGAGGCCGAAAGGCCGAA ACUCUCC 1032 CUCGGUG CUGAUGAGGCCGAAAGGCCGAA ACCUCUG 1044 AGAUCUU CUGAUGAGGCCGAAAGGCCGAA AUUCCUC 1050 UCAUAUA CUGAUGAGGCCGAAAGGCCGAA AUCUUGA 1052 CAUCAUA CUGAUGAGGCCGAAAGGCCGAA AGAUCCU 1054 UGCAUCA CUGAUGAGGCCGAAAGGCCGAA AUAGAUC 1072 UUCAGCA CUGAUGAGGCCGAAAGGCCGAA AUGCCUU 1085 UUUCUUU CUGAUGAGGCCGAAAGGCCGAA AGUUGUU

1103 UGUAGUU CUGAUGAGGCCGAAAGGCCGAA AUCACAC

1104 CUGUAGU CUGAUGAGGCCGAAAGGCCGAA AAUCACA 1108 UACACUG CUGAUGAGGCCGAAAGGCCGAA AGUUAAU 1115 AGUCUAG CUGAUGAGGCCGAAAGGCCGAA ACACUGU 1118 UCAAGUC CUGAUGAGGCCGAAAGGCCGAA AGUACAC 1123 UGCUGUC CUGAUGAGGCCGAAAGGCCGAA AGUCUAG 1139 UAGCCUC CUGAUGAGGCCGAAAGGCCGAA AGUUCUU 1146 UGUUUGA CUGAUGAGGCCGAAAGGCCGAA AGCCUCU 1148 GAUGUUU CUGAUGAGGCCGAAAGGCCGAA AUAGCCU 1155 UUAAGCU CUGAUGAGGCCGAAAGGCCGAA AUGUUUG

1160 UUGGAUU CUGAUGAGGCCGAAAGGCCGAA AGCUGAU

1161 UUUGGAU CUGAUGAGGCCGAAAGGCCGAA AAGCUGA 1154 UCUUUUG CUGAUGAGGCCGAAAGGCCGAA AUUAAGC 1173 ACAUCAU CUGAUGAGGCCGAAAGGCCGAA AUCUUUU 1181 AAAGCUC CUGAUGAGGCCGAAAGGCCGAA ACAUCAU

1187 UAACUCA CUGAUGAGGCCGAAAGGCCGAA AGCCCUA

1188 CUAACUC CUGAUGAGGCCGAAAGGCCGAA AAGCUCU

1193 UUUUAUU CUGAUGAGGCCGAAAGGCCGAA ACUCAAA

1194 UUUUUAU CUGAUGAGGCCGAAAGGCCGAA AACUCAA

Table 39: Large-Scale Synthesis

Sequence Activator Amidite Time* % Full

[Added/Final] [Added/Final] Length

(min) (min) Product

A₉T T [0.50/0.33] [0.1/0.02] 15 m 85 A₉T S [0.25/0.17] [0.1/0.02] 15 m 89

(GGU)₃GGT T [0.50/0.33] [0.1/0.02] 15 m 78 (GGU)₃GGT S [0.25/0.17] [0.1/0.02] 15 m 81

C₉T T [0.50/0.33] [0.1/0.02] 15 m 90 C₉T S [0.25/0.17] [0.1/0.02] 15 m 97

U₉T T [0.50/0.33] [0.1/0.02] 15 m 80 U₉T S [0.25/0.17] [0.1/0.02] 15 m 85

A (36-mer) T [0.50/0.33] [0.1/0.02] 15/15m 21 A (36-mer) S [0.25/0.17] [0.1/0.02] 15/15 m 25 A (36-mer) S [0.50/0.24] [0.1/0.03] 15/15 m 25 A (36-mer) S [0.50/0.18] [0.1/0.05] 15/15 m 38 A (36-mer) S [0.50/0.18] [0.1/0.05] 10/5 m 42

*Where two coupling times are indicated the first refers to RNA coupling and the second to 2'-O-methyl coupling. S = 5-S-Ethyltetrazole, T = tetrazole activator. A is 5' -ucu ccA UCU GAU GAG GCC GAA AGG CCG AAA Auc ccu -3' where lowerecase represents 2'-O-methylnucleotides. Table 40: Base Deprotection

Sequence Deprotection Time T °C % Full

Reagent (min) Length

Product iBu(GGU)₄ NH₄OH/EtOH 16h 55 62.5

MA 10m 65 62.7

AMA 10m 65 74.8

MA 10m 55 75.0

AMA 10m 55 77.2 iPrP(GGU)₄ NH₄OH/EtOH 4h 65 44.8

MA 10m 65 65.9

AMA 10 m 65 59.8

MA 10 m 55 61.3

AMA 10m 55 60.1

C₉U NH₄OH/EtOH 4h 65 75.2

MA 10m 65 79.1

AMA 10m 65 77.1

MA 10 m 55 79.8

AMA 10m 55 75.5 A (36-mer) NH₄OH/EtOH 4h 65 22.7

MA 10m 65 28.9

Table 41 : 2'-O-AIkylsiIyI Deprotection

Sequence Deprotection Time T °C % Full

Reagent (min) Length

Product

AgT TBAF 24 h 20 84.5

1.4 MHF 0.5 h 65 81.0

(GGU)₄ TBAF 24 h 20 60.9

1.4 MHF 0.5 h 65 67.8

C₁₀ TBAF 24 h 20 86.2

1.4 MHF 0.5 h 65 86.1

U₁₀ TBAF 24 h 20 84.8

1.4 MHF 0.5 h 65 84.5

B (36-mer) TBAF 24 h 20 25.2

1.4 MHF 1.5 h 65 30.6

A (36-mer) TBAF 24 h 20 29.7

1.4 M HF 1.5 h 65 30.4

B is 5'- UCU CCA UCU GAU GAG GCC GAA AGG CCG AAA AUC CCU

-3'.

Table 44. Kinetics of Self-Processing In Vitro

* k represents the unimolecular rate constant for ribozyme self-cleavage determined from a non-linear, least-squares fit (KaleidaGraph, Synergy Software, Reeding, PA) to the equation:

The equation describes the extent of ribozyme processing in the presense of ongoing transcription (Long & Uhlenbeck, 1994 Proc. Natl. Acad. Sci. USA 91 , 6977) as a function of time (t) and the unimolecular rate constant for cleavage (k). Each value of k represents the average (± range) of values determined from two experiments.

Table 45

Entry Modification t_1/2 (m ) t_1/2 (m) β = t_S/t_A

Activity Stability x 10 (tA) (ts) 1 U4 & U7 = U 1 0.1 1 2 U4 & U7 = 2'-O-Me-U 4 260 650 3 U4 = 2'=CH₂-U 6.5 120 180 4 U7 = 2'=CH₂-U 8 280 350 5 U4 & U7 = 2'=CH₂-U 9.5 120 13⁰ 6 U4 = 2'=CF₂-U 5 320 640 7 U7 = 2'=CF₂-U 4 220 550 8 U4 & U7 = 2'=CF₂-U 20 320 160 9 U4 = 2'-F-U 4 320 800 10 U7 = 2'-F-U 8 400 500 11 U4 & U7 = 2'-F-U 4 300 750 12 U4 = 2'-C-Allyl-U 3 >500 >1700 13 U7 = 2'-C-Allyl-U 3 220 730 14 U4 & U7 = 2'-C-Allyl-U 3 120 400 15 U4 = 2'-araF-U 5 >500 >1000 16 U7 = 2'-araF-U 4 350 875

17 U4 & U7 = 2'-araF-U 15 500 330

18 U4 = 2'-NH₂-U 10 500 500

19 U7 = 2'-NH₂-U 5 500 1000 20 U4 & U7 = 2'-NH₂-U 2 300 1500 21 U4 = dU 6 100 170

22 U4 & U7 = dU 4 240 600

Claims

CLAIMS What is claimed is:

1. An enzymatic nucleic acid molecule which cleaves ICAM-1 mRNA, IL- 5 mRNA, rel A mRNA, TNF-α mRNA sites shown in Table 23, 25, 27, or 28, CML associated mRNA selected from those identified as

SEQ. ID NOS 1-25, or RSV mRNA or RSV genomic RNA in a region selected from the group consisting of 1C, 1B and N.

2. The enzymatic nucleic acid molecule of claim 1, the binding arms of which contain sequences complementary to any one of the sequences defined in any of those in Tables 2, 3, 6-9, 11, 13, 15- 23, 27, 28, 31 , 33, 34, 36, and 37.

3. The enzymatic nucleic acid molecule of claim 1 or 2, wherein said nucleic acid molecule is in a hammerhead motif.

4. The enzymatic nucleic acid molecule of claim 1 or 2, wherein said RNA molecule is in a hairpin, hepatitis delta virus, group 1 intron,

Neurospora VS RNA or RNaseP RNA motif.

5. The enzymatic nucleic acid molecule of claim 1 or 2, comprising between 12 and 100 bases complementary to said mRNA or genomic RNA.

6. The enzymatic nucleic acid molecule of claim 5 comprising between

14 and 24 bases complementary to said mRNA or genomic RNA.

7. The enzymatic nucleic acid molecule of claim 1 or 2, comprising between 5 and 23 bases complementary to said mRNA or genomic RNA.

8. The enzymatic nucleic acid molecule of claim 7 comprising between

10 and 18 bases complementary to said mRNA or genomic RNA.

9. An enzymatic nucleic acid molecule consisting essentially of a sequence selected from the group of those shown in Tables 4-8, 10, 12, 14-16, 19-22, 24, 26-28, 30, 32, 34 and 36-38.

10. A mammalian cell including an enzymatic nucleic acid molecule of claims 1 or 2.

11. The ceil of claim 10, wherein said cell is a human cell.

12. An expression vector including nucleic acid encoding an enzymatic nucleic acid molecule or multiple enzymatic molecules of claims 1 or 2 in a manner which allows expression of that enzymatic RNA molecule(s) within a mammalian cell.

13. A mammalian cell including an expression vector of claim 12.

14. The cell of claim 13, wherein said cell is a human cell.

15. A method for treatment of a pathological condition related to the mRNA level of ICAM-1 , IL-5, rel A, TNF-α, or RSV by administering to a patient an enzymatic nucleic acid molecule of claim 1 or 2.

16. A method for treatment of a pathological condition related to the mRNA level of ICAM-1 , IL-5, rel A, TNF-α, or RSV by administering to a patient an expression vector of claim 12.

17. The method of claims 15 or 16, wherein said patient is a human.

18. The method of claim 17 wherein said condition is selected from the group consisting of atherosclerosis, myocardial infraction, stroke, restenosis, heart diseases, cancer, rheumatoid arthritis, asthma, reperfusion injury, inflammatory or autoimmune disorders, transplant rejection, myocardial ischemia, stroke, psoriasis, Kawasaki disease, HIV and AIDS, and septic shock.

19. A nucleoside selected from the group consisting of 5'-C- alkylnucleoside, 2'-deoxy-2'-alkylnucleoside, nucleoside 5'-deoxy- 5'-dihalo-methylphosphonate, nucleoside 5'-deoxy-5'-difluoromethylphosphonate, nucleoside 3'-deoxy-3'-dihalomethylphosphonate, and 5',3'-dideoxy-5',3'-bis(dihalo)- methylphosphonate.

20. A nucleotide selected from the group consisting of 5'-C- alkylnucleotide, 2'-deoxy-2'-alkylnucleotide, 5'-deoxy-5'-dihalomethylnucleotide, 5'-deoxy-5'-difluoro-methylnucleotide, 3'-deoxy3'-dihalo-methylnucleotide, and 5',3^,-dideoxy-5',3'-bis(dihalo)- methylphosphonate.

21. A nucleotide triphosphate comprising a nucleotide selected from the group consisting of 5'-C-alkylnucleotide, 2'-deoxy-2'- alkylnucleotide, 5'-deoxy-5'-dihalo-methylnucleotide, 5'-deoxy-5'- difluoro-methylnucleotide, 3'-deoxy-3'-dihalo-methylnucleotide, and 5',3'-dideoxy-5',3'-bis(dihalo)-methylphosphonate.

22. The 5'-C-alkylnucleoside of claim 19, wherein the sugar portion is in a talo configuration.

23. The 5'-C-alkylnucleoside of claim 19, wherein the sugar portion is in an allo configuration.

24. An oligonucleotide comprising a nucleotide selected from the group consisting of 5'-C-alkylnucleotide, 2'-deoxy-2'-alkylnucleotide, 5'- deoxy-5'-dihalo-methylnucleotide, 5'-deoxy-5'-difluoromethylnucleotide, 3'-deoxy-3'-dihalo-methylnucleotide, and 5',3'- dideoxy-5',3'-bis(dihalo)-methylphosphonate.

25. An oligonucleotide comprising a moiety having the formula: wherein B is a nucleotide base or hydrogen; R1 , R2 and R3 independently is selected from the group consisting of hydrogen, an alkyl group containing between 2 and 10 carbon atoms inclusive, an amine, an amino acid, and a peptide containing between 2 and 5 amino acids inclusive; and the zigzag lines are independently hydrogen or a bond.

26. An oligonucleotide comprising a 3'-amido or peptido group.

27. An oligonucleotide comprising a 5'-amido or peptido group.

28. The oligonucleotide of claim 24, 25, 26, or 27 having enzymatic activity.

29. Method for producing an enzymatic nucleic acid molecule having activity to cleave an RNA or single-stranded DNA molecule, comprising the step of forming said enzymatic molecule with at least one nucleotide having an alkyl group at its 5'-position or 2'- position.

30. Method for conversion of a protected allo sugar to a protected talo sugar, comprising the step of contacting said protected allo sugar with triphenyl phosphine, diethylazodicarboxylate, p-nitrobenzoic acid under inversion causing conditions to provide said protected talo sugar.

31. Method for the synthesis of a nucleoside 5' or a 3'-dihalomethylphosphonate comprising the step of condensing a difluoromethylphosphonate-containing sugar with a pyrimidine or purine under conditions suitable for forming a nucleoside 5'- or 3'- difluoromethylphosphonate.

32. The oligonucleotide of claim 3, wherein the normal hammerhead U4 and/or U7 positions are substituted with 2'-NH-amino acid.

33. A method for the synthesis of RNA comprising the step of providing 5-S-alkyitetrazole at a delivered 0.1-1.0 M concentration for the activation of a RNA amidite during a coupling step for less than or equal to 10 minutes.

34. A method for the synthesis of RNA comprising the step of providing 5-S-alkyltetrazole at 0.15-0.35 M effective, or final, concentration for the activation of a RNA amidite during a coupling step for less than or equal to 10 minutes.

35. A method for the deprotection of RNA comprising the step of providing alkylamine (MA) or NH₄OH/alkylamine (AMA) at between 60°C - 70°C for 5 to 15 minutes to remove any exocyclic amino protecting groups from protected RNA; wherein said alkyl is selected from the group consisting of methyl, ethyl, propyl and butyl.

36. A method for the deprotection of RNA alkylsilyl protecting groups comprising, contacting said groups with anhydrous triethylamine-hydrogen fluoride (aHF•TEA) trimethylamine or disopropylethylamine at between 60 °C-70 °C for 0.25-24 h.

37. A method for the purification of an RNA molecule by passing said enzymatic RNA molecule over an HPLC column, wherein said HPCC column is an anion exchange chromatography column.

38. Method for one pot deprotection of RNA comprising, contacting a protected base with anhydrous methyl amine at between 60 °C-70 °C for at least 5 min, cooling the resulting mixture and contacting said mixture with TEA-3HF reagents under conditions which remove a protecting group of the 2'-hydroxyl position.

39. Method for synthesizing RNA containing a phosphorothioate linkage comprising the step of contacting 6-10 equivalents of 3H-1 ,2- benzodithiole-3-one 1 ,1 -dioxide (Beaucage reagent) with the growing RNA chain for 5 seconds with a reaction time of at least 300 seconds.

40. Method of synthesizing RNA containing a phosphorothioate linkage comprising the step of achieving coupling with 5-S-ethyltetrazole or 5-S-methyltetrazole prior to sulfurization.

41. Method of claims 38, 39 or 40 wherein said RNA is enzymatically active.

42. Method for synthesizing 2'-deoxy-2'-amino-nucleoside phosphoramidite, comprising the step of protecting the 2'-amino group with a N-phtaloyl group.

43. The method of claim 42 wherein the said nucleoside lacks a base.

44. Method for synthesis of RNA comprising the step of: protecting the

2'-position of a nucleotide during said synthesis with a (trimethylsilyl)ethoxymethyl (SEM) group.

45. Method for covalently linking a SEM group to the 2'-position of a nucleotide, comprising the step of: contacting a nucleoside with an SEM-containing molecule under SEM bonding conditions.

46. The method of claim 45, wherein said conditions comprise dibutyltin oxide and tetrabutylammonium fluoride and SEM-Cl.

47. Method for removal of an SEM group from a nucleoside molecule or an oligonucleotide, comprising the step of: contacting said molecule or oligonucleotide with boron trifluoride etherate

(BF₃•OEt₂) under SEM removing conditions.

48. The method of claim 57 wherein said (BF₃•OEt₂) is provided in acetonitrile.

49. One or more vectors comprising a first nucleic acid sequence encoding a first ribozyme having intramolecular or intermolecular cleaving activity, said first ribozyme being selected from the group consisting of a hammerhead, hairpin, hepatitis delta virus, Neurospora VS RNA,

Group I, and RNaseP motif; and a second nucleic acid sequence encoding a second ribozyme having intermolecular cleaving activity, said Second ribozyme being selected from the group consisting of a hammerhead, hairpin, hepatitis delta virus, Neurospora VS RNA, Group I, and RNaseP motif and said second nucleic acid being flanked by other nucleic acid sequences encoding RNA which is cleaved by said first ribozyme to release said second ribozyme from RNA encoded by said vector; wherein said first and second nucleic acid sequences may be on the same or separate nucleic acid molecules, and said vector encodes mRNA or comprises RNA which lacks secondary structure which reduces release of said second ribozyme by more than 20%.

50. Cell comprising the vector of claim 49.

51. A transcribed non-naturally occurring RNA molecule, comprising a desired therapeutic RNA portion, wherein said molecule comprises an intramolecular stem formed by base-pairing interactions between a 3' region and 5' complementary nucleotides in said

RNA, wherein said stem comprises at least 8 base pairs.

52. The RNA molecule of claim 51, wherein said molecule is transcribed by a RNA polymerase III based promoter system.

53. The RNA molecule of claim 51, wherein said molecule is transcribed by a type 2 pol III promoter system.

54. The RNA molecule of claim 51 , wherein said molecule is a chimeric tRNA.

55. The RNA molecule of claim 53, said RNA having A and B boxes of a type 2 pol III promoter separated by between 0 and 300 bases.

56. The RNA molecule of claim 53, wherein said desired RNA molecule is at the 3' end of said B box.

57. The RNA molecule of claim 53, wherein said desired RNA molecule is in between the said A and the B box.

58. The RNA molecule of claim 53, wherein said desired RNA molecule includes said B box.

59. The RNA molecule of claim 51 , wherein said desired RNA molecule is selected from the group consisting of antisense RNA, decoy RNA, therapeutic editing RNA, enzymatic RNA, agonist RNA and antagonist RNA.

60. The RNA molecule of claim 51 , wherein said 5' terminus is able to base-pair with at least 12 bases of said 3' region.

61. The RNA molecule of claim 51 , wherein said 5' terminus is able to base-pair with at least 15 bases of said 3' region.

62. DNA vector encoding the RNA molecule of claim 51

63. The vector of claim 62, wherein said vector is derived from an AAV or adeno virus.

64. RNA vector encoding the RNA molecule of claim 51.

65. The vector of claim 64, wherein said vector is derived from an alpha virus or retro virus.

66. The vector of claim 62 wherein the portions of the vector encoding said RNA function as a RNA pol III promoter.

67. Cell comprising the vector of claim 62.

68. Cell comprising the vector of claim 53.

69. Cell comprising the RNA of claim 51 .

70. Method to provide a desired RNA molecule in a cell, comprising introducing said molecule into said cell a RNA comprising a desired RNA molecule, having a 5' terminus able to base pair with at least 8 bases of a 3' region of said RNA molecule.

71. The method of claim 70, wherein said introducing comprises providing a vector encoding said RNA molecule.

72. Hammerhead ribozyme having 2 or 3 base pairs in stem II with an interconnecting loop of 4 or more bases between said base pairs.

73. Hairpin ribozyme lacking a substrate moiety, comprising at least six bases in helix 2 and able to base-pair with a separate substrate

RNA, wherein the said ribozyme comprises one or more bases 3' of helix 3 able to base-pair with the said substrate RNA to form a helix 5 and wherein the said ribozyme can cleave and/or ligate said separate RNA(s) in trans.

74. The ribozyme of claim 73, wherein said ribozyme comprises six bases in helix 2.

75. The ribozyme of claim 73, having the structure of Fig. 3, wherein each N and N' is independently any base and each dash may represent a hydrogen bond, r is 1-20, q is 2-20, o is 0 - 20, n is 1 - 4, and m is 1 - 20.

76. Method for increasing the activity of a hairpin ribozyme by providing one or more bases 3' of helix 3 able to base-pair with a substrate RNA to form a helix 5.

77. Trans-cleaving Hairpin ribozyme comprising at least 6 base pairs in helix 2 lacking a substrate RNA moiety.

78. Trans-ligating Hairpin ribozyme comprising at least 6 base pairs in helix 2 lacking a substrate RNA moiety.

79. The ribozyme of claim 73 having the structure of Fig. 73.

80. The ribozyme of claim 73 having the structure of Fig. 74.

81. A cell including the ribozyme of any of claims 73-80.

82. An expression vector comprising nucleic acid encoding the ribozyme of any of claims 73-80, in a manner which allows expression of that ribozyme within a cell.

83. A cell including an expression vector of claim 82.

84. Method for altering in vivo the nucleotide base sequence of a naturally occurring mutant nucleic acid molecule, comprising the steps of: contacting said nucleic acid molecule in vivo with an oligonucleotide or peptide nucleic acid able to form a duplex or triplex molecule with said nucleic acid molecule, wherein formation of said duplex or triplex molecule directly, or after nucleic acid repair in vivo, causes at least one base in said nucleic acid molecule to be chemically modified to functionally alter the nucleotide base sequence of said nucleic acid sequence.

85. The method of claim 84, wherein said oligonucleotide is of a length sufficient to activate dsRNA deaminase in vivo to cause conversion of an adenine base to inosine in an RNA molecule.

86. The method of claim 84, wherein said oligonucleotide comprises an enzymatic nucleic acid molecule which is active to chemically modify a base.

87. The method claim 84, wherein said nucleic acid molecule is DNA or

RNA.

88. The method of claim 84, wherein said oligonucleotide comprises a chemical mutagen.

89. The method of claim 88, wherein said mutagen is nitrous acid.

90. The method of claim 84 wherein said oligonucleotide causes deamination of 5-methylcytosine to thymidine, cytosine to uracil, or adenine to inosine, or methylation of cytosine to 5-methylcytosine.

91. The method of claim 84, wherein an endogenous mammalian editing system is co-opted to cause said chemical modification.

92. Method for introduction of enzymatic nucleic acid into a cell or tissue, comprising the steps of; providing a complex of a first nucleic acid molecule encoding said enzymatic nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired structure under physiological conditions with said first nucleic acid molecule; wherein said R-loop is formed in a region of said first nucleic acid molecule at a location which promotes expression of RNA from said first nucleic acid under said conditions; and contacting said complex with said cell or tissue under conditions in which said enzymatic nucleic acid molecule is produced in said cell or tissue.

93. Method for introduction of a desired nucleic acid into a cell or tissue, comprising the steps of; providing a complex of a first nucleic acid molecule encoding said desired nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired structure under physiological conditions with said first nucleic acid molecule; wherein said first nucleic acid molecule lacks a promoter region and said R-loop is formed in a region of said first nucleic acid molecule at a location which promotes expression of RNA from said first nucleic acid under said conditions; and contacting said complex with said cell or tissue under conditions in which said desired acid molecule is produced in said cell or tissue.

94 Method for introduction of a desired nucleic acid into a cell or tissue, comprising the steps of; providing a complex of a first nucleic acid molecule encoding said enzymatic nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired structure under physiological conditions with said first nucleic acid molecule; wherein said R-loop is formed in a region of said first nucleic acid molecule at a location which promotes expression of RNA from said first nucleic acid under said conditions; and wherein said second nucleic acid further comprises a localization factor; and contacting said complex with said cell or tissue under conditions in which said desired nucleic acid molecule is produced in said cell or tissue.

95. Complex of a first nucleic acid molecule encoding an enzymatic nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired structure under physiological conditions with said first nucleic acid molecule; wherein said R-loop is formed in a region of said first nucleic acid molecule at a location which promotes expression of RNA from said first nucleic acid under said conditions.

96. Complex of a first nucleic acid molecule encoding a desired nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired structure under physiological conditions with said first nucleic acid molecule; wherein said first nucleic acid molecule lacks a promoter region and said R-loop is formed in a region of said first nucleic acid molecule at a location which promotes expression of RNA from said first nucleic acid under said conditions.

97. Complex of a first nucleic acid molecule encoding an enzymatic nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired structure under physiological conditions with said first nucleic acid molecule; wherein said R-loop is formed in a region of said first nucleic acid molecule at a location which promotes expression of RNA from said first nucleic acid under said conditions, and wherein said second nucleic acid further comprises a localization factor.