WO1993003736A1 - Cross-linking oligonucleotides for enzyme-mediated triple strand formation - Google Patents

Cross-linking oligonucleotides for enzyme-mediated triple strand formation Download PDF

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Publication number
WO1993003736A1
WO1993003736A1 PCT/US1992/007101 US9207101W WO9303736A1 WO 1993003736 A1 WO1993003736 A1 WO 1993003736A1 US 9207101 W US9207101 W US 9207101W WO 9303736 A1 WO9303736 A1 WO 9303736A1
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Prior art keywords
strand
odn
gene
crosslinking
target
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PCT/US1992/007101
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French (fr)
Inventor
Charles R. Petrie
Rich B. Meyer, Jr.
John C. Tabone
Gerald D. Hurst
Howard B. Gamper
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Microprobe Corporation
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Priority to EP92918930A priority Critical patent/EP0661979A4/en
Priority to JP5504623A priority patent/JPH06509945A/en
Publication of WO1993003736A1 publication Critical patent/WO1993003736A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6839Triple helix formation or other higher order conformations in hybridisation assays

Definitions

  • This invention relates to nucleoside crosslinking agents and to the use of these compounds in the preparation of oligonucleotides. It also relates to derivatives of pyrazolo[3,4-d]pyri_nidine which are useful as nucleic acid bases for the preparation of oligonucleotides.
  • Oligonucleotides are useful as diagnostic probes for the detection of "target" DNA or RNA
  • oligonucleotide probes has been, until the present, composed of nucleic acid containing ribose or deoxyribose or, in one case, natural ⁇ -arabinose (patent publication EP 227,459).
  • a novel class of nucleotide base the 3,4- disubstituted and 3,4, 6-trisubstituted pyrazolo[3,4-d]- pyrimidines, has now been found which offers several advantages over the prior art.
  • the de novo chemical synthesis of the pyrazolopyrimidine and the resulting nucleotide allows for the incorporation of a wide range of functional groups in a variety of different positions on the nucleotide base and for the use of different sugar moieties.
  • adenine, guanine and hypoxanthine analogs are obtained from a single nucleoside precursor. Additionally, the synthesis does not require the use of toxic heavy metals or expensive catalysts.
  • crosslinkable nucleotide probes for use in therapeutic and diagnostic applications is related to the pioneering work of B.R. Baker, "Design of Active-Site-Directed Irreversible Enzyme Inhibitors,” Wiley, New York, (1967), who used what was termed
  • active-site-directed enzyme inhibitors in chemotherapeutic applications.
  • Oligonucleotides may be used as chemotherapeutic agents to control the expression of gene sequences unique to an invading organism, such as a virus, a fungus, a parasite or a bacterium.
  • an invading organism such as a virus, a fungus, a parasite or a bacterium.
  • RNA-expression in bacteria is controlled by "antisense" RNA, which exerts its effect by forming RNA:RNA hybrids with complementary target RNAs and modulating or inactivating their biological activity.
  • antisense RNA which exerts its effect by forming RNA:RNA hybrids with complementary target RNAs and modulating or inactivating their biological activity.
  • mRNA targets in vivo (reviewed in Green, et al., Ann. Rev. Biochem. 55: 569- 597 (1986)). Additionally, a specific mRNA amongst a large number of mRNAs can be selectively inactivated for protein synthesis by hybridization with a complementary DNA restriction fragment, which binds to the mRNA and prevents its translation into protein on ribosomes
  • oligonucleotides complementary to portions of the HIV genome are capable of inhibiting protein expression and virus replication in cell culture. Inhibition of up to 95% was obtained with oligonucleotide concentrations of about 70 ⁇ M.
  • crosslinking suggests potential problems that must be circumvented.
  • the oligonucleotide containing a crosslinking. arm might covalently bond to the target sequence so readily that mismatching of sequences will occur, possibly resulting in host toxicity.
  • the crosslinking reaction must be fast enough to occur before correctly matched sequences can dissociate.
  • European Patent Application No. 86309090.8 describes the formation of chemically modified DNA probes such as 5-substituted uridinyl in which the substituent does not crosslink but contains a chemical or physical reporter group.
  • WO8707611 describes a process for labeling DNA fragments such as by chemically modifying the fragment followed by reaction with a fluorescent dye.
  • Yabusaki et al. in U.S. Patent No. 4,599,303 disclose a scheme for covalently crosslinking oligonucleotides such as by formation of furocoumarin monoadducts of thymidine which are made to covalently bond to other nucleotides upon photoexcitation.
  • EP 0259186 describes adducts of macromolecules and biotin which can be used as cross- linking nucleic acid hybridization probes.
  • WO8503075 describes crosslinking disulfonic esters useful as nucleic acid fragmentation agents.
  • DE3310337 describes the covalent crosslinking of single-stranded polynucleo- tides to such macromolecules as proteins with the
  • probe oligonucleotides consisting of sufficient base sequences to identify target sequences with high specificity, that are provided with one or more crosslinking arms which readily form covalent bonds with specific complementary bases.
  • Such oligonucleotides may be used as highly selective probes in
  • oligonucleotides may also be5used as antisensing agents of RNAs, e.g., in chemotherapy.
  • This invention is directed to crosslinking agents which accomplish crosslinking between specific sites on adjoining strands of oligonucleotides.
  • the crosslinking reaction observed is of excellent
  • the invention is also directed to oligonucleotides comprising at least one of these crosslinking agents and to the use of the resulting novel oligonucleotides for diagnostic and therapeutic purposes.
  • the crosslinking agents of this invention are derivatives of nucleotide bases with a crosslinking arm and are of the following formula (!'):
  • R 1 is hydrogen, or a sugar moiety or analog thereof optionally substituted at its 3' or its 5' position with a phosphorus derivative attached to the sugar moiety by an oxygen and including groups Q 1 ' Q 2 and Q 3 , or with a reactive precursor thereof suitable for nucleotide bond formation;
  • d is hydroxy, phosphate or diphosphate
  • Q 3 is CH 2 -R', S-R', O-R', or N-R'R";
  • each of R 1 and R" is independently hydrogen or C h alkyl
  • B is a nucleic acid base or analog thereof that ⁇ is a component of an oligonucleotide
  • Y is a functional linking group
  • each of m and q is independently 0 to 8, inclusive;
  • r is 0 or 1
  • A' is a leaving group
  • the invention also provides novel oligonucleotides comprising at least one of the above nucleotide base derivatives of formula I'.
  • Nucleotides of this invention and oligonucleotides into which the nucleotides have been incorporated may be used as probes. Since probe hybridization is reversible, albeit slow, it is desirable to ensure that each time a probe hybridizes with the correct target sequence, the probe is irreversibly attached to that sequence.
  • nucleotide bases of the present invention will permanently modify the target strand, or cause depurination.
  • the oligonucleotides of this invention are useful in the identification, isolation, localization and/or detection of complementary nucleic acid sequences of interest in cell-free and cellular systems. Therefore, the invention further provides a method for identifying target nucleic acid sequences, which method comprises utilizing an oligonucleotide probe comprising at least one of a labeled nucleotide base of the present
  • the invention further describes methods for inactivating gene function involving combination of a crosslinkable anti-gene ODN and a recombination enzyme.
  • Coating the ODN with a recombination enzyme facilitates the search for homology with in the target gene and subsequent triple strand formation.
  • Crosslinking of resultant triple strand complexes inactivates gene function.
  • a crosslinkable anti-gene nucleoprotein filament that includes (i) a nucleoside crosslinking agent covalently linked to an oligonucleotide (ODN) complementary to a target DNA sequence within a gene, and (ii) a recombination enzyme non-covalently associated with the ODN is also described.
  • This invention also provides novel substituted pyrazolo[-3,4-d]pyrimidines which are useful as a
  • nucleotide base in preparing nucleosides and nucleotides, rather than the natural purine or pyrimidine bases or the deazapurine analogs.
  • Figure 1 depicts a modified deoxyuridine residue of an oligodeoxynucleotide crosslinked via an acetamidopropyl sidearm to a deoxyguanosine residue located two sites away from the complementary base along the 5' direction.
  • Figure 2 depicts an autoradiogram of 32 P-labeled HPV target and crosslinked product following cleavage at the 3' side of the crosslinked guanosine.
  • Lane 1 :
  • Figure 3 depicts an autoradiogram of 32 P-labeled HPV target and crosslinked product showing hybrid
  • Lane 1 Control 32 P-labeled CMV target. Lane 2: 24 hour reaction at 20°C. Lane 3: 72 hour reaction at 20°C. Lane 4: 24 hour reaction at 30°C. Lane 5: 72 hour reaction at 30°C. Reaction solutions were treated with 2-aminoethanothiol, which quenches the iodoacetamido group.
  • This invention provides novel substituted nucleotide bases with a crosslinking arm which are useful in preparing nucleosides and nucleotides and are useful as crosslinking agents.
  • the substituted bases are of the following-formula (1'):
  • R 1 is hydrogen, or a sugar moiety or analog thereof optionally substituted at its 3' or its 5' position with a phosphorus derivative attached to the sugar moiety by an oxygen and including groups Q 1 , Q 2 and Q 3 , or with a reactive precursor thereof suitable for nucleotide bond formation;
  • Q 1 is hydroxy, phosphate or diphosphate
  • Q 3 is CH 2 -R' S-R', O-R', or N-R'R"; each of R' and R" is independently hydrogen or C 1-6 alkyl;
  • B is a nucleic acid base or analog thereof that is a component of an oligonucleotide
  • Y is a functional linking group
  • each of m and q is independently 0 to 8, inclusive;
  • r is 0 or 1
  • A' is a leaving group
  • the sugar moiety or analog thereof is selected from those useful as a component of a nucleotide.
  • a moiety may be selected from, for example, ribose, deoxyribose, pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, pentofuranose, xylose, lyxose, and
  • the sugar moiety is preferably ribose, deoxyribose, arabinose or 2'-O-methylribose and embraces either anomer, ⁇ or ⁇ .
  • the phosphorus derivative attached to the sugar moiety is conveniently selected from, for example, monophosphate, diphosphate, triphosphate, alkyl phosphate, alkanephosphonate, phosphorothioate, pho ⁇ phorodithioate, and the like.
  • a reactive precursor suitable for internucleotide bond formation is one which is useful during chain extension in the synthesis of an oligonucleotide.
  • Phosphoruscontaining groups suitable for internucleotide bond formation are preferably alkyl phosphorchloridites, alkyl phosphites or alkylphosphoramidites. Alternatively, activated phosphate diesters may be employed for this purpose.
  • the nucleic acid base or analog thereof (B) may be chosen from the purines, the pyrimidines, the deazapurines and the pyrazolopyrimidines. It is preferably selected from uracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl, 4-aminopyrrolo- [2,3-d]pyrimidin-5-yl, 2-amino-4-oxopyrrolo[2,3-d]pyri- midin-5-yl, 4-aminopyrazolo[3,4-d]pyrimidin-3-yl or 4- amino-6-oxopyrazolo[3,4-d]pyrimidin-3-yl, where the purines are attached to the sugar moiety of the
  • oligonucleotides via the 9-position the pyrimidines via the l-position, the pyrrolopyrimidines via the 7-position and the pyrazolopyrimidines via the l-position.
  • nucleophilic properties are capable of serving as a point of attachment of the -(CH 2 ) m -A' group. Amino groups and blocked derivatives thereof are preferred.
  • the leaving group A' may be chosen from, for example, such groups as chloro, bromo, iodo, SO 2 R'", or S + R'"R"", where each of R'" and R"" is independently
  • C 1-6 alkyl or aryl or R'" and R"" together form a C 1-6 - alkylene bridge. Chloro, bromo and iodo are preferred.
  • the leaving group will be altered by its leaving ability. Depending on the nature and reactivity of the particular leaving group, the group to be used is chosen in each case to give the desired specificity of the irreversibly binding probes.
  • 5-position of the pyrimidines lie in the major groove of the B-form duplex of double-stranded nucleic acids.
  • These positions can be substituted with side chains of considerable bulk without interfering with the hybrid- ization properties of the bases.
  • These side arms may be introduced either by derivatization of dThd or dCyd, or by straightforward total synthesis of the heterocyclic base, followed by glycosylation.
  • These modified nucleosides may be converted into the appropriate activated nucleotides for incorporation into oligonucleotides with an automated DNA synthesizer.
  • the crosslinking arm is attached at the 3-position, which is
  • the crosslinking side chain should be of sufficient length to reach across the major groove from a purine 7- or 8-position, pyrimidine 5-position, pyrrolopyrimidine 5-position or pyrazolopyrimidine 3-position and reacting with the N-7 of a purine (preferably
  • the side chain should be of at least three atoms, preferably of at least five atoms and more preferably of at least six atoms in length.
  • a generally preferred length of the side chain is from about 5 to about 9 carbon atoms.
  • the target strand base which is being attacked paired to the first or second base which is on the 3' side of the modified base in the oligonucleotide containing the crosslinking arm.
  • the target sequence for a probe containing a modified uracil should contain the complement GZA
  • oligonucleotide containing UZC preferably UCC
  • U d ⁇ rd 5-substituted with the crosslinking arm.
  • the adenine-modified AZ 1 C triplet would target GZ 1 T, where Z 1 is any base.
  • the first class is the 5-substituted-2'-deoxyuridines whose general structure is presented below:
  • the 5- (substituted) -2 ' -deoxyuridines may be prepared by the routes shown in Schemes 1 and 2.
  • acetylene-coupled product (XXII).
  • the acetylenic dUrd analog XXII is reduced, with Raney nickel for example, to give the saturated compound (XXIII), which is then used for direct conversion to a reagent for use on an automated DNA synthesizer, as described below.
  • the second class of modified nucleoside is a group of 2'-deoxy-4-aminopyrazolo[3,4-d]pyrimidine derivatives.
  • the general structure of these derivatives is presented below:
  • the above compounds are derived from a novel group of derivatives of 3,4-disubstituted and 3,4,6-trisubstituted pyrazolo[3,4-d]pyrimidines.
  • the 3,4-disubstituted and 3,4,6-trisubstituted pyrazolo[3,4-d]pyrimidines and their synthesis are disclosed in commonly owned, copending application Serial No. 250,474, the entire disclosure of which is incorporated herein by reference. They have the following formula (I):
  • R 1 is hydrogen, or a sugar moiety or analog thereof optionally substituted at its 3' or its 5' position with a phosphorus derivative attached to the sugar moiety by an oxygen and including groups Q 1 ' Q 2 and Q 3 , or with a reactive precursor thereof suitable for nucleotide bond formation; provided that when R 3 is hydrogen, then R 1 cannot be hydrogen;
  • Q 1 is hydroxy, phosphate or diphosphate
  • Q 3 is CH 2 -R', S-R', O-R', or N-R'R"; each of R' and R" is independently hydrogen or C 1-6 alkyl;
  • R 3 is hydrogen or the group -W-(X) n -A; each of W and X is independently a chemical linker arm;.
  • A is an intercalator, a metal ion chelator, an electrophilic crosslinker, a photoactivatable crosslinker, or a reporter group;
  • each of R 4 and R 6 is independently H, OR, SR,
  • R is H or C 1-6 alkyl
  • n is zero or one
  • t is zero to twelve.
  • the sugar moiety or its analog is selected from those useful as a component of a nucleotide.
  • a moiety may be selected from, for example, pentose, deoxypentose, hexose, deoxyhexose, ribose, deoxyribose, glucose, arabinose, pentofuranose, xylose, lyxose, and cyclopentyl.
  • the sugar moiety is preferably ribose, deoxyribose, arabinose or 2'-O-methylribose and embraces either anomer, ⁇ or ⁇ .
  • the phosphorus derivative attached to the sugar moiety is conveniently selected from, for example, monophosphate, diphosphate, triphosphate, alkyl phosphate, alkanephosphonate, phosphorothioate, phosphorodithioate, and the like.
  • a reactive precursor suitable for internucleotide bond formation is one which is useful during chain extension in the synthesis of an oligonucleotide.
  • Reactive groups particularly useful in the present invention are those containing phosphorus.
  • Phosphorusocontaining groups suitable for internucleotide bond formation are preferably alkyl phosphorchloridites, alkyl phosphites or alkylphosphoramidites.
  • activated phosphate diesters may be employed for this purpose.
  • Linker arms may include alkylene groups of 1 to 12 carbon atoms, alkenylene groups of 2 to 12 carbon atoms and 1 or 2 olefinic bonds, alkynylene groups of 2 to 12 carbon atoms and 1 or 2acetylenic bonds, or such groups substituted at a
  • Such functionalities including aliphatic or aromatic amines, exhibit nucleophilic properties and are capable of serving as a point of attachment of the functional group (A).
  • the linker arm moiety (W alone or together with X) is preferably of at least three atoms and more
  • the terminal nucleophilic group is preferably amino or chemically blocked derivatives thereof.
  • Intercalators are planar aromatic bi-, tri- or polycyclic molecules which can insert themselves between two adjacent base pairs in a double-stranded helix of nucleic acid. Intercalators have been used to cause frameshift mutations in DNA and RNA. It has also
  • tethered covalently bound via a linker arm
  • it increases the binding affinity of the oligonucleotide for its target sequence, resulting in strongly enhanced stability of the complementary sequence complex.
  • At least some of the tethered intercalators also protect the oligonucleotide against exonucleases, but not against endonucleases. See, Sun et al., Nucleric Acids Res., 15: 6149-6158 (1987); Le Doan etal., Nucleic Acids Res., 15:7749-7760 (1987).
  • tetherable intercalating agents are oxazolopyridocarbazole, acridine orange, proflavine, acriflavine and derivatives of proflavine and acridine such as 3-azido-6- (3-bromopropylamino) acridine, 3-amino-6-(3-bromopentyl- amino)acridine, and 3-methoxy-6-chloro-9-(5-hydroxy- pentylamino)acridine.
  • Oligonucleotides capable of crosslinking to the complementary sequence of target nucleic acids are valuable in chemotherapy because they increase the efficiency of inhibition of mRNA translation or gene expression control by covalent attachment of the oligonucleotide to the target sequence. This can be accomplished by cross linking agents being covalently attached to the oligonucleotide, which can then be chemically activated to form crosslinkages which can then induce chain breaks in the target complementary sequence, thus inducing irreversible damage in the sequence.
  • electrophilic crosslinking moieties include alpha-halocarbonyl
  • oligonucleotides comprising at least one nucleotide base moiety of the invention are utilized as a probe in nucleic acid assays
  • a label is attached to detect the presence of hybrid polynucleotides.
  • Such labels act as reporter groups and act as means for detecting duplex formation between the target nucleotides and their complementary oligonucleotide probes.
  • a reporter group as used herein is a group which has a physical or chemical characteristic which can be measured or detected. Detectability may be provided by such characteristics as color change, luminescence, fluorescence, or radioactivity; or it may be provided by the ability of the reporter group to serve as a ligand recognition site.
  • pyrazolopyrimidines of the present invention of formula. I where R 1 is hydrogen may be prepared by the procedures outlined below and as set forth by
  • malononitrile (III) is treated with acyl halide (II) in the presence of a base to yield acylmalononitrile (IV) , which is subsequently methylated with dimethyl sulfate or diazomethane, for example, to give the substituted methoxymethylenemalononitrile (V) .
  • acyl halide (II) is then reacted with hydrazine hydrate in boiling alcohol to give the 3-substituted-5-aminopyra- zole-4-carbonitrile (VI) , which is treated with cold concentrated sulfuric acid to give the 3-substituted-5- aminopyrazole-4-carboxamide (VII) .
  • the carboxamide (VII) may alternatively be prepared by treating cyanoacetamide (XII) with acid halide (II) to give the acylcyanoacetamide (XIII) , which is then methylated, and the resulting methoxy compound (XIV) is reacted with hydrazine hydrate.
  • VI and VII are obtained by treating the corresponding VI and VII with boiling formamide.
  • VI may be treated with dialkoxymethyl ester of a carboxylic acid, at room temperature or above room temperature, and then with ammonia to give VIII
  • VII may be treated with dialkoxymethyl ester of a carboxylic acid (without subsequent ammonia treatment) , at room temperature or above room temperature, to give compound X.
  • VI and VII may be treated with an alkyl xanthate salt such as potassium ethyl xanthate and with alkyl halide such as methyl iodide, at a temperature above room temperature, followed by oxidation by a peroxide such as m-chloroperbenzoic acid (MCPBA) and subsequent treatment with ammonia to give IX and XI, respectively, where R 6 is NH 2 .
  • an alkyl xanthate salt such as potassium ethyl xanthate
  • alkyl halide such as methyl iodide
  • the compounds of formula I may be recovered from the reaction mixture in which they are formed by established procedures.
  • the sugar may be either added to the 1- position of the pyrazole VI or VII prior to further treatment or added to the 1-position of the pyrazolo[3,4- d]pyrimidine VIII, IX, X or XI.
  • the pyrazole or pyrazolopyrimidine is treated with sodium hydride and then with the glycosyl halide of the blocked sugar.
  • Oligonucleotides of the present invention may comprise at least one and up to all of their nucleotides from the substituted pyrazolo[3,4-d]pyrimidines of
  • oligonucleotides To prepare oligonucleotides, protective groups are introduced onto the nucleosides of formula I or formula I' and the nucleosides are activated for use in the synthesis of oligonucleotides.
  • the conversion to protected, activated forms follows the procedures as described for 2'-deoxynucleosides in detail in several reviews. See, Sonveaux, Bioor ⁇ anic Chemistry. 14:274-325 (1986); Jones, in "Oligonucleotide Synthesis, a Practical Approach", M.J. Gait, Ed., IRL Press, p. 23-34 (1984).
  • the activated nucleotides are incorporated into oligonucleotides in a manner analogous to that for DNA0and RNA nucleotides, in that the correct nucleotides will be sequentially linked to form a chain of nucleotides which is complementary to a sequence of nucleotides in target DNA or RNA.
  • the nucleotides may be incorporated either enzymatically or via chemical synthesis.
  • the nucleotides may be converted to their 5'-O-dimethoxy- trityl-3'-(N,N-diisopropyl)phosphoramidite cyanoethyl ester derivatives, and incorporated into synthetic oligonucleotides following the procedures in
  • the activated nucleptides may be used directly on an automated DNA synthesizer according to the procedures and instructions of the particular synthesizer employed.
  • the oligonucleotides may be prepared on the synthesizer using the standard commercial phosphoramidite or H-phosphonate chemistries.
  • amino- pyrazolopyrimidine nucleotide triphosphates may be any amino- pyrazolopyrimidine nucleotide triphosphate.
  • the leaving group such as a haloacyl group
  • addition of an ⁇ -haloacetamide may be verified by a changed mobility of the modified compound on HPLC, corresponding to the removal of the positive charge of the amino group, and by subsequent readdition of a positive charge by reaction with 2-aminoethanethiol to give a derivative with reverse phase HPLC mobility similar to the original aminoalkyl- oligonucleotide.
  • each of the following electrophilic leaving groups were attached to an amino- propyl group on human papillomavirus (HPV) probes:
  • An oligonucleotide probe according to the invention includes at least one labeled substituted pyrazolo[3,4-d]pyrimidine nucleotide moiety of formula I and/or at least one labeled substituted nucleotide base of formula I'.
  • Probes may be labeled by any one of several methods typically used in the art. A common method of detection is the use of autoradiography with 3 H, 125 I, 35 S, 1 4 c, or 32 P labeled probes or the like. Other reporter groups include ligands which bind to antibodies labeled with fluorophores, chemiluminescent agents, and enzymes. Alternatively, probes can be conjugated directly with labels such as fluorophores, chemiluminescent agents, enzymes and enzyme substrates. Alternatively, the same components may be indirectly bonded through a ligand- antiligand complex, such as antibodies reactive with a ligand conjugated with label. The choice of label
  • Radioactive probes are typically made using commercially available nucleotides containing the desired radioactive isotope.
  • the radioactive nucleotides can be incorporated into probes, for example, by using DNA synthesizers, by nick-translation, by tailing of radioactive bases to the 3' end of probes with terminal transferase, by copying M13 plasmids having specific inserts with the Klenow fragment of DNA polymerase in the presence of radioactive dNTP's, or by transcribing RNA from templates using RNA polymerase in the presence of radioactive rNTP's.
  • Non-radioactive probes can be labeled directly with a signal (e.g., fluorophore, chemiluminescent agent or enzyme) or labeled indirectly by conjugation with a ligand.
  • a signal e.g., fluorophore, chemiluminescent agent or enzyme
  • a ligand molecule is covalently bound to the probe. This ligand then binds to a receptor molecule which is either inherently detectable or
  • Ligands and anti- ligands may be varied widely. Where a ligand has a natural "antiligand", namely ligands such as biotin, thyroxine, and cortisol, it can be used in conjunction with its labeled, naturally occurring antiligand.
  • any haptenic or antigenic compound can be used in combination with a suitably labeled antibody.
  • a preferred labeling method utilizes biotin-labeled analogs of oligonucleotides, as disclosed in Langer et al., Proc. Natl. Acad. Sci. USA, 78: 6633-6637 (1981), which isincorporated herein by reference.
  • Enzymes of interest as reporter groups will primarily be hydrolases, particularly phosphatases, esterases, ureases and glycosidases, or oxidoreductases, particularly peroxidases.
  • Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, rare earths, etc.
  • Chemiluminescers include luciferin, acridinium esters and 2,3-dihydrophthalazinediones, e.g., luminol.
  • hybridization conditions are not critical and will vary in accordance with the investigator's preferences and needs.
  • Various hybridization solutions may be employed, comprising from about 20% to about 60% volume, preferably about 30%, of a polar organic solvent.
  • a common hybridization solution employs about 30-60% v/v formamide, about 0.5 to 1M sodium chloride, about 0.05 to 0.1M buffers, such as sodium citrate, Tris HCl, PIPES or HEPES, about 0.05% to 0.5% detergent, such as sodium dodecylsulfate, and between 1- 10 mM EDTA, 0.01% to 5% ficoll (about 300-500 kdal), 0.1% to 5% polyvinylpyrrolidone (about 250-500 kdal), and 0.01% to 10% bovine serum albumin.
  • unlabeled carrier nucleic acids from about 0.1 to 5 mg/ml, e.g., partially fragmented calf thymus or salmon sperm, DNA, and/or partially fragmented yeast RNA and optionally from about 0.5% to 2% wt./vol. glycine.
  • Other additives may also be included, such as volume exclusion agents which include a variety of polar water-soluble or swellable agents, such as anionic polyacrylate or polymethylacrylate, and
  • charged saccharidic polymers such as dextran sulfate.
  • hybridization technique is not essential to the invention.
  • Hybridization techniques are generally described in "Nucleic Acid Hybridization, A Practical Approach", Hames and Higgins, Eds., IRL Press, 1985; Gall and Pardue, Proc. Natl. Acad. Sci., U.S.A.,
  • the amount of labeled probe which is present in the hybridization solution may vary widely. Generally, substantial excesses of probe over the stoichiometric amount of the target nucleic acid will be employed to enhance the rate of binding of the probe to the target DNA.
  • degrees of stringency of hybridization can be employed. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for the formation of a stable duplex.
  • the degree of stringency can be controlled by temperature, ionic strength, the inclusion of polar organic solvents, and the like. For example, temperatures employed will normally be in the range of about 20° to 80°C, usually 25° to 75oC. For probes of 15-50 nucleotides in 50% formamide, the optimal temperature range can vary from 22-65°C. With routine experimentation, one can define conditions which permit satisfactory hybridization at room temperature.
  • the stringency of hybridization is also conveniently varied by changing the ionic strength and polarity of the reactant solution through manipulation of the concentration of formamide within the range of about 20% to about 50%.
  • Treatment with ultrasound by immersion of the reaction vessel into commercially available sonication baths can oftentimes accelerate the hybridization rates.
  • the glass, plastic, or filter support to which the probe-target hybrid is attached is introduced into a wash solution typically containing similar
  • reagents e.g., sodium chloride, buffers, organic
  • solvents and detergent as provided in the hybridization solution.
  • These reagents may be at similar concentrations as the hybridization medium, but often they are at lower concentrations when more stringent washing conditions are desired.
  • the time period for which the supportis maintained in the wash solutions may vary from minutes to several hours or more. Either the hybridization or the wash medium can be stringent. After appropriate stringent washing, the correct hybridization complex may now be detected in accordance with the nature of the label.
  • the probe may be conjugated directly with the label.
  • the label is radioactive
  • the support surface with associated hybridization complex substrate is exposed to X-ray film.
  • the label is fluorescent
  • the sample is detected by first irradiating it with light of a particular wavelength. The sample absorbs this light and then emits light of a different wavelength which is picked up by a detector ("Physical Biochemistry", Freifelder, D., W.H. Freeman & Co., 1982, pp. 537-542).
  • the label is an enzyme
  • the signal generated may be a colored precipitate, a colored or fluorescent soluble material, or photons generated by bioluminescence or chemiluminescence.
  • alkaline phosphatase will dephosphorylate indoxyl phosphate which then will participate in a reduction reaction to convert tetrazolium salts to highly, colored and insoluble formazans.
  • a signal generating complex to a duplex of target and probe polynucleotides or nucleic acids.
  • binding occurs through ligand and antiligand interactions as between a ligand-conjugatedProbe and an antiligand conjugated with a signal.
  • the binding of the signal generation complex is also readily amenable to accelerations by exposure to ultrasonic energy.
  • the label may also allow indirect detection of the hybridization complex.
  • the label is a hapten or antigen
  • the sample can be detected by using antibodies.
  • a signal is gener ated by attaching fluorescent or enzyme molecules to the antibodies or in some cases, by attachment to a radioactive label.
  • the amount of labeled probe present in the hybridization solution may vary widely, depending upon the nature of the label, the amount of the labeled probe that can reasonably bind to the cellular target nucleic acid, and the precise stringency of the hybridization medium and/or wash medium. Generally, substantial probe excesses over the stoichiometric amount of the target will be employed to enhance the rate of binding of the probe to the target nucleic acids.
  • the invention is also directed to a method for identifying target nucleic acid sequences, which method comprises utilizing an oligonucleotide probe including at least one labeled substituted nucleotide moiety of formula I and/or formula I'.
  • the method comprises the steps of:
  • An assay for identifying target nucleic acid sequences utilizing an oligonucleotide probe including at least one labeled substituted nucleotide moiety of formula I and/or formula I' and comprising the above method is contemplated for carrying out the invention.
  • Such an assay may be provided in kit form.
  • a typical kit will include a probe reagent component comprising an oligonucleotide including at least one labeled nucleotide moiety of formula I or formula I', the oligonucleotide having a sequence complementary to that of the target nucleic acids; a denaturation reagent for converting double-stranded nucleic acid to single- stranded nucleic acid; and a hybridization reaction mixture.
  • the kit can also include a signal-generating system, such as an enzyme for example, and a substrate for the system.
  • ODNs Oligodeoxynucleotides
  • sequence specific pharmaceutical agents for the inhibition of gene expression. For instance,
  • ODNs may inhibit the expression of specific gene products through formation of duplexes upon hybridization with complementary messenger RNAs (mRNAs). More specifically, these "antisense" ODNs are believed to inhibit the processing or translation of message
  • antisense ODNs may be useful as anti-viral, antiparasitic, and anti-cancer agents. Further, antisense ODNs provide a unique opportunity for rational drug development, since a genetic target offers both extraordinar specificity and universality with respect to potential target sequences.
  • antisense technology is beset with certain fundamental disadvantages.
  • One major challenge involves development of antisense ODNs with sufficient potency to warrant in vivo testing.
  • Antisense ODN formulations that exhibit nuclease resistance, rapid cellular uptake, and efficient and stable hybridization to the target RNA sequence are desirable. Improvement of one or more of these properties without concomitant de eterious effects on other properties may be difficult.
  • phosphorothioates exhibit excellent nuclease resistance relative to unmodified ODNs, but methylphosphonate ODNs form DNA-RNA hybrids that are refractile to RNase H and phosphorothioate ODNs are poorly taken up into cells.
  • moieties to ODNs in order to potentiate their antisense activity.
  • moieties that interact directly with the RNA target upon hybridization such as pendant intercalating groups that stabilize ODN-target hybrids, ODNs with freeradical-based RNA cleaving activity or ODNs capable of covalently linking to their targets upon hybridization
  • Direct cleavage or cross- linkage of an mRNA target by an ODN renders the RNAinactive.
  • receptor-mediated cellular targeting include conjugation of a cholesteryl group that may act as an lipophilic anchor for the antisense ODN; encapsulation of ODNs into liposomes; and linkage of ODNs to soluble macromolecular complexes.
  • a cholesteryl group that may act as an lipophilic anchor for the antisense ODN
  • encapsulation of ODNs into liposomes and linkage of ODNs to soluble macromolecular complexes.
  • anti-gene A variation of the "antisense” approach to rational drug design is termed "anti-gene”.
  • antisense ODNs target single stranded mRNA
  • anti-gene ODNs hybridize with and are capable of inhibiting the function of double-stranded DNA. More specifically, anti-gene ODNs form sequence-specific triple-stranded complexes with a double stranded DNA target and thus interfere with the replication or transcription of selected target genes.
  • DNA is the repository for all genetic information, including regulatory control sequences and non-expressed genes, such as dormant proviral DNA genomes.
  • anti-gene ODNs have broader applicability and are potentially more powerful than antisense ODNs that merely inhibit mRNA processing and translation.
  • Anti-gene ODNs in the nuclei of living cells can form sequence-specific complexes with chromosomal DNA.
  • the resultant triplexes can inhibit restriction and/or transcription of the target double stranded DNA.
  • anti-gene interference with DNA functioning has longer lasting effects than the corresponding antisense inhibition of mRNA function.
  • Mammalian cell DNA does not turnover; in fact, cells possess sophisticated pathways capable of repairing lesions in DNA that may arise from environmental insults or from spontaneous rearrangements. In contrast, mRNA is transient and may exist only for minutes within a cell. The constant turnover of an mRNA species and the
  • anti-sense ODNs will provide relatively short term effects. While cellular uptake of anti-gene ODNs may need to be augmented to achieve sufficient intracellular concentrations, once within the cell the ODNs naturally concentrate in the nucleus.
  • Anti-gene therapy is based on the observation that certain DNA homopolymers can form triple-stranded complexes.
  • the third strand resides in the major groove of the Watson Crick base-paired double helix, where it hydrogen bonds to one of the two parental strands.
  • a binding code governs the recognition of base pairs by a third base (see allowed triplets below). In each case, the third strand base is presented first and is followed by the base pair;
  • Cytosine/thymidine-, guanine/adenine- and guanine/thymidine-containing ODNs can sequence- specifically bind to homopurine runs in double-stranded DNA. These recognition motifs are based on Hoogstein or reverse Hoogstein base pairing. In the C/T recognition motif, the ODN is parallel to the homopurine strand of the duplex; in the G/A recognition motif, the ODN is anti-parallel to the homopurine strand; in the G/T recognition motif, the ODN may bind parallel or antiparallel to the homopurine strand of the duplex,
  • recognition motifs may be sequence dependent.
  • the sequence specificity of anti-gene ODNs using the C/T recognition motif permits hybridization of such ODNs to homopurine runs in plasmid DNA and in yeast chromosomes. Since ODN binding is restricted to homopurine runs, it would be advantageous to identify additional heterocycles that can recognize the remaining two base pairs, i.e., C- G and T-A. While guanosine may be used in the third Strand to recognize T-A base pairs, this interaction involves only one hydrogen bond and is relatively
  • anti-gene ODNs may be modified with a variety of pendant groups designedto augment their activity.
  • intercalating groups, cleaving agents, and crosslinking moieties may be
  • substitution of 5-methyl cytosine for cytosine in the third strand ODN significantly stabilizes triplexes formed with "G"-rich homopurine runs.
  • ODNs with modified backbones, such as oligonucleoside methyl-phosphonates and phosphorothioates may form triple-stranded
  • a important disadvantage of triple strand formation as discussed above is the relatively slow kinetics of triple strand formation.
  • the claimed invention overcomes this disadvantage through enzyme catalysis of triple strand formation, with recombination enzymes particularly preferred for this purpose. More significantly, enzyme-catalyzed triple strand formation provides the immense advantage of universal sequence recognition (in contrast to the A-T and G-C recognition limitation associated with non-enzyme-mediated triple strand formation).
  • DNA molecules which plays an important role in promoting genetic diversity within a species, is catalyzed by a family of enzymes in both procaryotic andeucaryotic cells. Recombination also plays an important role in DNA repair and in the immune response.
  • Homologous recombination in E. coll serves as an illustrative example of processes that occur in both procaryotic and eucaryotic cells.
  • Studies of purifiedrecombinational enzymes from E. coli in a defined cell- free system permit division of homologous recombination into three steps.
  • the "invading" single strand is circular and the target double strand is linear.
  • the first step "presynapsis", single stranded circular DNA is coated with the multifunctional protein recA in the presence of ATP.
  • the resultant nucleoprotein filament possesses a right handed helical twist composed of 18 bases per turn and 1 monomer of recA protein per 3.6 nucleotides.
  • the single- stranded circular nucleoprotein filament conducts a two- dimensional search along a linear double-stranded DNA template for homologous sequences.
  • the search concludes with homologous alignment of the two molecules in an initial complex that has no net helical interwinding (the complex is referred to as a "paranemic joint") .
  • the DNA double helix is incorporated into the filament, and in so doing, the two DNA molecules become plectonemically coiled (i.e., helically interwound).
  • the newly incorporated third strand is homologous (in sequence and polarity) to one of the parental duplex strands, and complementary to the other parental duplex strand.
  • the three strands of DNA are believed to exist as a true hydrogen-bonded triple strand having a close association with recA.
  • the plectonemically coiled complex is stable upon
  • the plectonemically coiled synaptic complex contains 18 triads per right handed helical turn. While recA is a critical part of the complex, sequence
  • the protein-free triple stranded DNA complexes are essentially resistant to single strand-specific nucleases and exhibit very high T m 's. This is likely attributable to hydrogen bonding of the so-called "third strand" to both parental strands, as well as to the highly
  • recA may catalyze the formation of a DNA triple strand complex not otherwise attainable.
  • the two positive strands have identical sequence and
  • either positive strand is capable of forming a double stranded hybrid with the negative parental strand.
  • synaptic complexes include: (1) the length of the invading single strand; (2) the extent of shared homology between the invading single strand and the target duplex (with length of homology and absence of mismatches of particular importance); (3) the position of the shared homology region with respect to the termini of the target duplex; and (4) the superhelicity, if any, of the duplex.
  • An oligomeric 50-mer should have sufficient length to permit the formation of a stable presynaptic complex with recA.
  • Type of Joint Depending upon the positional relationship of the region of shared homology to the ends (if any) of the-targeted double-stranded DNA, the
  • Proximal joints wherein the recA-stabilized triple strand complex is located at the left hand of a linear duplex, are unstable due to recA-catalyzed strand exchange.
  • the duplex strand that is homologous to the invading third strand in the triple strand complex is displaced, resulting in a new duplex containing the invading strand and a free single strand. Since strand exchange requires a free 5' end and proceeds with a 5' to3' polarity, this process readily occurs in proximal joints.
  • distal joints and medial joints lack the appropriate ends and do not readily undergo recA-catalyzed strand exchange.
  • these joints are highly stable structures. Accordingly, within the present invention, formation of recA-stabilized triple strand complexes so as to form meidal or distal joints is preferred.
  • Superhelicity may facilitate the formation of recA-stabilized triple strands. However, high efficiency triple strand
  • the present invention combines the recA- catalyzed formation of stable triple strand complexes with synthetic anti-gene ODNs. Enzyme-catalyzed triple strand formation exhibits rapid kinetics and universal DNA sequence recognition.
  • a recA-coated anti-gene ODN serves as a "guide" that seeks homology in a target double strand DNA sequence; upon recognition and bindingof this nucleoprotein filament, recA catalyzes the formation of sequence-specific triple strand complexes.
  • an anti-gene ODN is least 30-40 nucleotides in length and has a base sequence and polarity identical to either ofthe two duplex strands in the target DNA.
  • the frequency of triplex formation and the stability of the triple strand complexes following deproteinization may be directly related to the length of the anti-gene ODN.
  • Anti-gene ODNs (of the appropriate polarity) may be usedin combination with endogenous recombinatory pathways to form sequence-specific triple strand complexes with chromosomal DNA.
  • anti-gene ODNs of a variety of lengths may be complexed with a
  • the enzyme-mediated recognition motif recognizes all four base pairs, thereby allowing targeting of any double stranded DNA sequence.
  • the recA-coated, single stranded anti-sense ODN nucleoprotein filament
  • the resultant triple strand complex is stable at physiological pH.
  • presynaptic nucleoprotein filaments such as those formed between single stranded DNA and recA, that display a weak nonspecific affinity for double stranded DNA effectively reduces the homology search from a three dimensional to a two dimensional process. Furthermore, upon homologous registry with the double strand, the nucleoprotein filament will more likely produce a triple strand complex than the corresponding interaction of double strand and a naked single strand. Because of these factors, triple strand formation between a recA-coated, single stranded ODN and an homologous double strand occurs at a reaction rate that exceeds by 1 or 2 orders of magnitude the calculated rate of spontaneous renaturation of
  • the present invention involves combination of (1) an ODN that is homologous to a portion of one target DNA duplex strand and complementary to the analogous portion of the other target DNA duplex strand; and (2) enzyme-catalyzed triple strand formation to achieve inactivation of a target DNA sequence.
  • the ODN has a crosslinking moiety covalently attached thereto.
  • cross-linkage of the third strand ODN (the anti- gene ODN) to both parental duplex strands inactivates the target DNA sequence.
  • crosslinking moiety the target DNA sequence and the environment and characteristics of the target DNA
  • inactivation of the parental duplex strands may be permanent.
  • a single administration of one or more anti-gene ODNs may abolish the expression of
  • the modified target DNA no longer supports replication or transcription. Unlike all other lesions in DNA, however, this modification is not repairable. Normally, crosslinked DNA is repaired by a combination of excision repair and homologous recombination. With crosslinked triple strand complexes, however, there will be no undamaged copies of the targeted gene to
  • the eucaryotic cell may attempt to use a misrepair (or SOS) pathway wherein the crosslink will be removed, but at the expense of mutagenesis. In such case, gene function would be irreversibly silenced by the resultant mutations.
  • misrepair or SOS
  • anti-gene ODN is combined with a recombination enzyme (for instance, in a nucleoprotein complex).
  • suitable target DNA sequences include structural genes and both up-stream and down-stream regulatory control sequences. These regulatory sequences may be involved in either
  • the anti-gene ODN will be determined and designed according to the target DNA sequence chosen for alteration of function, and will have a sequence complementary to one of the two strands of the chosen target DNA.
  • Crosslinkers suitable for use within the invention include photochemical agents, such as
  • psoralens and chemical crosslinking agents.
  • Preferred chemical crosslinking agents include those described in Section A., above; electrophilic moieties attached to the 3' and/or 5' termini of the ODN; and masked electrophilic moieties attached to the ODN.
  • Photo-crosslinking agents may be useful for topical or extracorporeal applications, for targets accessible to light exposure, as well as for in vitro use. Chemical crosslinkers may be used without limitation and are particularly preferred for use within the claimed invention.
  • Preferred recombination enzymes include
  • procaryotic and eucaryotic recombination enzymes such as recA, human recombinase and Drosophila recombinase, with human recombinase particularly preferred.
  • an anti-gene ODN is administered to a cell or a host, and upon entry to a target cell nucleus, the anti-gene ODN combines with recombination enzymes present within the nucleus.
  • the anti-gene ODN and recombination enzyme are combined ex vivo and then administered to a cell or a host as a nucleoprotein filament. In this embodiment, it may be advantageous to administer the nucleoprotein filament in a liposome.
  • RT means room temperature
  • Thin layer chromatography was performed on silica gel 60 F 254 plates (Analtech) using the followingsolvent mixtures: A- 90% methylene chloride: 10% methanol; B- 50% ethyl acetate:50% hexanes; C- 70% ethyl acetate: 10% methanol: 10% water:10% acetone; D- 50% ether:50% hexanes. Flash chromatography was performed using 60 F 254 silica (Merck). Oligonucleotides were synthesized onan Applied Biosystems Model 380B Synthesizer. Oligonucleotides were isotopically labeled using T4
  • 6-Aminocaproic acid (26 g, 0.2 mole) was dissolved in dichloromethane (200 mL) by the addition of triethylamine (100 mL). Trityl chloride (120 g, 0.45 mole) was added and the solution stirred for 36 hr. The resulting solution was extracted with 1NHCl and the organic layer evaporated to dryness. The residue was suspended in 2-propanol/1N NaOH (300 mL/100 mL) and refluxed for 3 hr. The solution was evaporated to a thick syrup and added to dichloromethane (500 mL). Water was added and acidified. The phases were separated, and the organic layer dried over sodium sulfate and evaporated to dryness. The residue was suspended in hot 2- propanol, cooled, and filtered to give 43.5 g (58%) of 6- (tritylamino) caproic acid, useful as an intermediate compound.
  • EXAMPLE 2 6-Aminocaproic acid
  • the dichloromethane solution was washed with ice cold 2N HCl (300 mL) and the biphasic mixture was filtered to remove product that precipitated (13.2 g). The phases were separated and the organic layer dried and evaporated to a thick syrup. The syrup was covered with dichloromethane and on standing deposited fine crystals of product. The crystals were filtered and dried to give 6.3 g for a total yield of 19.5 g (87%) of the product, which is useful as an intermediate.
  • Example 4 (3.5 g, 8 mmole) was treated with sodium hydride and stirred for 30 min at 0-4°C. 1-Chloro-1,2- dideoxy-3,5-di-O-toluoylribofuranose was added and the solution stirred for 1 hr at 0-4°C. The solution was poured into a saturated solution of sodium bicarbonate and extracted with dichloromethane. The organic layer was dried over sodium sulfate and evaporated to dryness. The residue was flash chromatographed on silica gel using toluene/ethyl acetate (5/1) as eluent. Two major
  • N-1 and N-2 isomers were isolated and identified as the N-1 and N-2 isomers in 57% (3.6 g) and 20% (1.2 g) N-1 and N-2 yields, respectively. Approximately 1 g of a mixture of N-1 and N-2 isomers was also collected. Overall yield of glycosylated material was 5.8 g (92%).
  • the triphosphate of Example 9 was incorporated into pHPV-16 using the nick tanslation protocol of Langer et al. (supra).
  • the probe prepared with the triphosphate of Example 9 was compared with probe prepared using commercially available bio-ll-dUTP (Sigma Chemical Co). No significant differences could be observed in both a filter hybridization and in in situ smears.
  • DNA polymerase 1 (U.S. Biochemicals) - 8
  • pHPV - 16 - 2.16 mg/mL which is a
  • Nucleic acid was isolated by ethanol precipitation and hybridized to pHPV-16 slotted onto nitrocellulose.
  • the hybridized biotinylated probe was visualized by a streptavidin-alkaline phosphatase conjugate with BCIP/NBT substrate.
  • Probe prepared using either biotinylated nucleotide gave identical signals.
  • the probes were also tested in an in situ format on cervical smears and showed no qualitative differences in signal and background.
  • 5-Iodo-2'-deoxyuridine (354 mg, 1 mmol) was dissolved in 10 mL of dimethylformamide. Cuprous iodide (76 mg, 0.4 mmol), tetrakis(triphenylphosphine)palladium(0) (230 mg, 0.2 mmol), and triethylamine (200 mg, 2.0 mmol) were added. 4-Phthalimidobut-1-yne (300 mg, 1.5 mmol) was added all at once and the reaction kept at 60°C for three hours. The clear yellow reaction was then evaporated and methylene chloride was added. Scratching of the flask induced crystallization of nearly all of the product which was filtered and recrystallized from 95% ethanol to give 335 mg (78%) of title compound as fine, feathery needles.
  • Nucleosides were 5'-dimethoxytritylated, following known procedures, to give around 85% yield, and the 3'-phosphoramidite was made using diisopropylamino ⁇ - cyanoethylchlorophosphite (as described in
  • Oligonucleotides were removed from the DNA synthesizer in tritylated form and deblocked using 30% ammonia at 55°C for 6 hours. Ten ⁇ L of 0.5M sodium bicarbonate was added to prevent acidification duringconcentration. The oligonucleotide was evaporated to dryness under vacuum and redissolved in 1.0 mL water.
  • oligonucleotides were purified by HPLC using 15-55% acetonitrile in 0.1N triethylammonium acetate over 20 minutes. Unsubstituted oligonucleotides came off at 10 minutes; amino derivatives took 11-12 minutes. The desired oligonucleotide was collected and evaporated to dryness, then it was redissolved in 80% aqueous acetic acid for 90 minutes to remove the trityl group.
  • nucleoside 5-(3-trifluoroacetamidoprop-1-yl)-2'-deoxyuridine was converted to the 5'-O-dimethoxytrityl-3'-(N,N- diisopropyl) phosphoramidite cyanoethyl ester derivative. This was added to a DNA synthesizer and the following 14- mer oligonucleotide sequence was prepared:
  • a corresponding 14-mer oligonucleotide was also prepared where U 1 is the unmodified deoxyuridine.
  • B (Dupont) eluted with a 20 minute gradient of 60% to 80% B composed of: A (20% acetonitrile: 80% 0.02 N NaH 2 PO 4 ) and B (1.2 N NaCl in 20% acetonitrile: 80% 0.02 N NaH 2 PO 4 ).
  • Oligo A and oligo B, as well as the above 14- mer where U 1 is the unmodified deoxyuridine were resolved in the Zorbax column, all of identical sequence, with the following retention times: unmodified 14-mer, 9.31 min; aminopropyl 14-mer (oligo A), 7.36 min; and iodoacetamidopropyl 14-mer (oligo B), 10.09 min.
  • aminobutyl 14-mer (oligo C, Example 23) was reacted with either N-hydroxysuccinimide ⁇ -iodo- acetate or N-hydroxysuccinimide 4-bromobutyrate to give the 14-mer where U 1 is 5-(4-iodoacetamidobut-1-yl)-2'- deoxyuridine or 5-(4-(4-bromobutyramido)but-1-yl)-2'- deoxyuridine, respectively.
  • the reaction of crosslinking a DNA probe to a target nucleic acid sequence contained 1 ⁇ g of haloacyl- amidoalkyl probe and 10 ng of 32 P-labeled cordycepintailed target in 200 ⁇ L of 0.1 M Tris, pH 8.0, and 0.9 M NaCl incubated at 20° or 30°C. Aliquots were removed at 24- or 72-hour intervals and diluted in 20 ⁇ L of 10 mM cysteamine to quench the haloacylamido group. These0solutions were stored at RT, and 1 ⁇ L was used for analysis by denaturing polyacrylamide gel electrophoresis (PAGE).
  • PAGE denaturing polyacrylamide gel electrophoresis
  • the target for HPV is a 30-mer, and for CMV it is a 24-mer.
  • the crosslinking probes were a 14-mer for HPV and two 15-mers for CMV. Each probe contained a single modified deoxyuridine designated as U in the sequences above.
  • Example 25 the crosslinked HPV hybrid of Example 25 (where U is 5-(3-iodoacetamidoprop-1-yl)-2'-deoxyuridine) was subjected to a 10% piperidine solution at 90°C for 60 minutes. As shown by Maxam et al. (Proc. Natl. Acad.
  • Psoralen-modified, photo-crosslinkable ODNs were prepared using a photochemical procedure (REF??). Briefly, ODN was hybridized to a complementary DNA sequence and irradiated at 360 nm in the presence of 4'-hydroxymethy-4,5',8-trimethylpsoralen (HMT), thereby forming a sequence-specific, interstrand crosslink at a unique 5'-TpA-3' sequence.
  • HMT 4'-hydroxymethy-4,5',8-trimethylpsoralen
  • the HMT furan-side monoadducted ODN was isolated by denaturing PAGE.
  • a psoralen derivative to an ODN may be accomplished by chemical conjugation.
  • These synthetic schemes may be used to obtain ODNs that are coupled to a 4,5',8-trimethyl- psoralen (TMP) via a linker arm that spans the 5' terminus of the ODN and the 4' position of the psoralen.
  • TMP 4,5',8-trimethyl- psoralen
  • a modified TMP is reacted with a suitably 5'-activated, deblocked ODN (B.L. Lee et al., Biochem. 27: 3197-3202, 1988).
  • a TMP phosphoramidite linker compound is added in the last cycle of solid phase synthesis (U. Pieles and U.
  • TMP is modified at its 4' position with linker which is attached to the C8 position of deoxyadenosine.
  • a prototype, psoralenated ODN that can efficiently crosslink to both Watson and Crick strands when complexed to homologous DNA in a recA-stabilized triple strand is employed.
  • the ODN 5'-modified with a psoralenated tail is synthesized so as to optimize photo- crosslinkage within a 5'-TpA-3' sequence flanking the recA-stabilized triplex.
  • a psoralenated ODN in the presence of a recombination enzyme is used to
  • the ODN which Hoogstein base pairs to the DNA target, is modified with a 5-hydroxypsoralen moiety or a 4,5',8-trimethylpsoralen moiety at its 5' terminus.
  • the furocourmain is able to readily intercalate into a double stranded 5'-TpA-3' sequence immediately adjacent to the triple strand. Both the furan and pyrone rings are properly positioned to photoreact with thymidines.
  • psoralenated tail may be altered to obtain optimal intercalation of the psoralen into the duplex-triple strand junction.

Abstract

The invention disclosed is directed to cross-linking between specific sites on adjoining oligonucleotides or oligodeoxynucleotides wherein the nucleoside monomers used to effect the cross-linking are (alkylating substituent substituted) pyrazolol[3,4-d]pyrimidine ribosides or 2'-deoxyribosides. This cross-linking is assisted by the presence in mammalian host of a recombination enzyme such as RecA. The cross-linking of either double or triple stranded nucleic acids is expected to have utility in the inhibiting the expression of the targeted nucleic acids in vivo and as a diagnostic tool as well.

Description

CROSS-LINKING OLIGONUCLEOTIDES FOR
ENZYME-MEDIATED TRIPLE STRAND FORMATION This application is a continuation-in-part of application Serial No. 07/353,857, filed on May 18, 1989, which in turn is a continuation-in-part of application Serial No. 07/250,474, filed on September 28, 1988 (now abandoned).
BACKGROUND OF THE INVENTION
This invention relates to nucleoside crosslinking agents and to the use of these compounds in the preparation of oligonucleotides. It also relates to derivatives of pyrazolo[3,4-d]pyri_nidine which are useful as nucleic acid bases for the preparation of oligonucleotides.
Oligonucleotides are useful as diagnostic probes for the detection of "target" DNA or RNA
sequences. In the past, such probes were made up of sequences of nucleic acid containing purine, pyrimidine or 7-deazapurine nucleotide bases (U.S. Patent 4,711,955; Robins et al., J. Can. J. Chem., 60:554 (1982); Robins et aI., J. Org. Chem., 48:1854 (1983)). The method for attaching chemical moieties to these bases has been via an acetoxy-mercuration reaction, which introduces
covalently bound mercury atoms into the 5-position of the pyrimidine ring, the C-8 position of the purine ring or the C-7 position of a 7-deazapurine ring (Dale et al., Proc. Natl. Acad. Sci. USA, 70:2238 (1973); Dale et al., Biochemistry, 14:2447 (1975)), or by the reaction of organomercurial compounds with olefinic compounds in the presence of palladium catalysts (Ruth et al., J. Org.
Chem., 43:2870 (1978); Bergstrom et al., J. Am. Chem.
Soc., 100:8106 (1978); Bigge et al., J. Am. Chem. Soc., 102:2033 (1980)). The sugar component of oligonucleotide probes has been, until the present, composed of nucleic acid containing ribose or deoxyribose or, in one case, natural β-arabinose (patent publication EP 227,459).
A novel class of nucleotide base, the 3,4- disubstituted and 3,4, 6-trisubstituted pyrazolo[3,4-d]- pyrimidines, has now been found which offers several advantages over the prior art. The de novo chemical synthesis of the pyrazolopyrimidine and the resulting nucleotide allows for the incorporation of a wide range of functional groups in a variety of different positions on the nucleotide base and for the use of different sugar moieties. Also, adenine, guanine and hypoxanthine analogs are obtained from a single nucleoside precursor. Additionally, the synthesis does not require the use of toxic heavy metals or expensive catalysts. Similar pyrazolo[3,4-d]pyrimidines are known (Kobayashi, Chem. Pharm. Bull., 21:941 (1973)); however, the substituents on the group are different from those of the present invention and their only use is as xanthine oxidase inhibitors.
The concept of crosslinkable nucleotide probes for use in therapeutic and diagnostic applications is related to the pioneering work of B.R. Baker, "Design of Active-Site-Directed Irreversible Enzyme Inhibitors," Wiley, New York, (1967), who used what was termed
"active-site-directed enzyme inhibitors" in chemotherapeutic applications.
In recent years, the concept of incorporating a crosslink in an oligonucleotide has been sporadically discussed in efforts to develop superior sequence probes. Knorre and Vlassov, Prog. Nucl. Acid Res. Mol. Biol.,
32: 291 (1985), have discussed sequence-directed cross- linking ("complementary addressed modification") using an W-(2-chloroethyl)-N''methylaniline group attached to either the 3'- or 5'-terminus of oligonucleotides.
Summerton and Bartlett, J. Mol. Biol., 122:145 (1978) have shown that an 8-atom chain, attached to a cytosine residue at its C-4 position and terminating in the highly reactive bromomethyl ketone group, can crosslink to the N-7 of guanosine.
Webb and Matteucci, Nucleic Acids Res., 14:7661 (1986), have prepared oligonucleotides containing a 5- methyl-N,N-ethanocytosine base which is capable of slow crosslinking with a complementary strand. In a conceptually related alkylation via a linker arm within a DNA hybrid, Iverson and Dervan, Proc. Natl. Acad. Sci. USA. 85:4615 (1988), have shown opposite strand methylation, triggered by BrCN activation of a methylthio ether, predominately on a guanine base located two pairs from the base bearing the linker.
Oligonucleotides may be used as chemotherapeutic agents to control the expression of gene sequences unique to an invading organism, such as a virus, a fungus, a parasite or a bacterium. In nature, some RNA- expression in bacteria is controlled by "antisense" RNA, which exerts its effect by forming RNA:RNA hybrids with complementary target RNAs and modulating or inactivating their biological activity. A variety of recent studies using plasmid vectors for the introduction of antisense RNAs into eukaryotic cells have shown that they
effectively inhibit expression of mRNA targets in vivo (reviewed in Green, et al., Ann. Rev. Biochem. 55: 569- 597 (1986)). Additionally, a specific mRNA amongst a large number of mRNAs can be selectively inactivated for protein synthesis by hybridization with a complementary DNA restriction fragment, which binds to the mRNA and prevents its translation into protein on ribosomes
(Paterson, et al., Proc. Natl. Acad. Sci 74: 4370-4374 (1977); Hastie et al., Proc. Natl. Acad. Sci. 75: 1217- 1221 (1978)).
In the first demonstration of the concept of using sequence-specific, antisense oligonucleotides as regulators of gene expression and as chemotherapeutic agents, Zamecnik and Stephenson, Proc. Natl. Acad. Sci. USA. 75:280 (1978), showed that a small antisense oligodeoxynucleotide probe can inhibit replication of Rous Sarcoma Virus in cell culture, and that RSV viral RNA translation is inhibited under these conditions
(Stephenson et al., Proc. Natl. Acad. Sci. USA 75:285 (1978)). Zamecnik et al., Proc. Natl. Acad. Sci. USA, 83:4143 (1986), have also shown that oligonucleotides complementary to portions of the HIV genome are capable of inhibiting protein expression and virus replication in cell culture. Inhibition of up to 95% was obtained with oligonucleotide concentrations of about 70 μM.
Importantly, they showed with labeled phosphate studies that the oligonucleotides enter cells intact and are reasonably stable to metabolism.
Uncharged methylphosphonate oligodeoxynucleo- tides with a sequence complementary to the initiation codon regions of rabbit globin mRNA inhibited the
translation of the mRNA in both cell-free systems and in rabbit reticulocytes (Blake et al., Biochemistry 24:6139 (1985)). Another uncharged methylphosphonate
oligonucleotide analog, an 8-nucleotide sequence
complementary to the acceptor splice junction of a mRNA of Herpes simplex virus, Type 1, can inhibit virusreplication in intact Vero cells. However, fairly high concentrations (>25 mM) of this nonionic probe were required for this inhibition.
Although the impact of crosslinking oligonucleotides in the chemotherapeutic field might be of great significance, their impact in DNA probe-based diagnostics is of equally great importance. The ability to covalently crosslink probe-target hybrids has the potential to dramatically improve background and sensitivity limits in diagnostic assays as well as permit novel assay formats. Specific innovations (discussed previously by Gamper et al., Nucl. Acids Res., 14, 9943 (1988)) include:
(a) incorporation of a denaturing wash step to remove background;
(b) use of the crosslink as an additional tier of discrimination;
(c) crosslinking occurring at or near the melting temperature of the expected hybrid to insure exquisite specificity and to substantially reduce secondary structure in the target, thereby increasing the efficiency of hybrid formation; and
(d) novel solution hybridization formats as
exemplified by the Reverse Southern
protocol.
The concept of crosslinking, however, suggests potential problems that must be circumvented. For instance, the oligonucleotide containing a crosslinking. arm might covalently bond to the target sequence so readily that mismatching of sequences will occur, possibly resulting in host toxicity. On the other hand, the crosslinking reaction must be fast enough to occur before correctly matched sequences can dissociate.
This issue can be addressed by constructing an oligonucleotide that, upon hybridization, results in a duplex whose Tm is just above the physiological
temperature of 37°C. Thus, even a single mismatched base will prevent hybrid formation and therefore crosslinkage. The optimization can be accomplished by judicious choice of oligonucleotide length and base composition, as well as position of the modified base within the probe. The probe must be long enough, however, to insure specific targeting of a unique site.
European Patent Application No. 86309090.8 describes the formation of chemically modified DNA probes such as 5-substituted uridinyl in which the substituent does not crosslink but contains a chemical or physical reporter group. WO8707611 describes a process for labeling DNA fragments such as by chemically modifying the fragment followed by reaction with a fluorescent dye. Yabusaki et al. in U.S. Patent No. 4,599,303 disclose a scheme for covalently crosslinking oligonucleotides such as by formation of furocoumarin monoadducts of thymidine which are made to covalently bond to other nucleotides upon photoexcitation. EP 0259186 describes adducts of macromolecules and biotin which can be used as cross- linking nucleic acid hybridization probes. WO8503075 describes crosslinking disulfonic esters useful as nucleic acid fragmentation agents. DE3310337 describes the covalent crosslinking of single-stranded polynucleo- tides to such macromolecules as proteins with the
resulting complex subsequently used as a marker in hybridization experiments in the search for complementary sequences in foreign polynucleotides.
A need exists for probe oligonucleotides, consisting of sufficient base sequences to identify target sequences with high specificity, that are provided with one or more crosslinking arms which readily form covalent bonds with specific complementary bases. Such oligonucleotides may be used as highly selective probes in
hybridization assays. The oligonucleotides may also be5used as antisensing agents of RNAs, e.g., in chemotherapy.
SUMMARY OF THE INVENTION
This invention is directed to crosslinking agents which accomplish crosslinking between specific sites on adjoining strands of oligonucleotides. The crosslinking reaction observed is of excellent
specificity. The invention is also directed to oligonucleotides comprising at least one of these crosslinking agents and to the use of the resulting novel oligonucleotides for diagnostic and therapeutic purposes. More particularly, the crosslinking agents of this invention are derivatives of nucleotide bases with a crosslinking arm and are of the following formula (!'):
R1 - B - (CH2)q- (Y)r - (CH2)m - A' (I') wherein.
R1 is hydrogen, or a sugar moiety or analog thereof optionally substituted at its 3' or its 5' position with a phosphorus derivative attached to the sugar moiety by an oxygen and including groups Q1 ' Q2 and Q3, or with a reactive precursor thereof suitable for nucleotide bond formation;
d is hydroxy, phosphate or diphosphate;
Q2 is =O or =S;
Q3 is CH2-R', S-R', O-R', or N-R'R";
each of R1 and R" is independently hydrogen or Chalkyl;
B is a nucleic acid base or analog thereof thatθis a component of an oligonucleotide;
Y is a functional linking group;
each of m and q is independently 0 to 8, inclusive;
r is 0 or 1; and
A' is a leaving group.
The invention also provides novel oligonucleotides comprising at least one of the above nucleotide base derivatives of formula I'.
Nucleotides of this invention and oligonucleotides into which the nucleotides have been incorporated may be used as probes. Since probe hybridization is reversible, albeit slow, it is desirable to ensure that each time a probe hybridizes with the correct target sequence, the probe is irreversibly attached to that sequence. The covalent crosslinking arm of the
nucleotide bases of the present invention will permanently modify the target strand, or cause depurination. As such, the oligonucleotides of this invention are useful in the identification, isolation, localization and/or detection of complementary nucleic acid sequences of interest in cell-free and cellular systems. Therefore, the invention further provides a method for identifying target nucleic acid sequences, which method comprises utilizing an oligonucleotide probe comprising at least one of a labeled nucleotide base of the present
invention.
The invention further describes methods for inactivating gene function involving combination of a crosslinkable anti-gene ODN and a recombination enzyme. Coating the ODN with a recombination enzyme facilitates the search for homology with in the target gene and subsequent triple strand formation. Crosslinking of resultant triple strand complexes inactivates gene function. A crosslinkable anti-gene nucleoprotein filament that includes (i) a nucleoside crosslinking agent covalently linked to an oligonucleotide (ODN) complementary to a target DNA sequence within a gene, and (ii) a recombination enzyme non-covalently associated with the ODN is also described.
This invention also provides novel substituted pyrazolo[-3,4-d]pyrimidines which are useful as a
nucleotide base in preparing nucleosides and nucleotides, rather than the natural purine or pyrimidine bases or the deazapurine analogs.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts a modified deoxyuridine residue of an oligodeoxynucleotide crosslinked via an acetamidopropyl sidearm to a deoxyguanosine residue located two sites away from the complementary base along the 5' direction.
Figure 2 depicts an autoradiogram of 32P-labeled HPV target and crosslinked product following cleavage at the 3' side of the crosslinked guanosine. Lane 1:
32P-labeled 15-mer size marker. Lane 2: 24 hour reaction at 20°C. Lane 3: 72 hour reaction at 20°C. Lane 4: 24 hour reaction at 30ºC. Lane 5: 72 hour reaction at 30ºC. Reactions were quenched with 2-aminoethanothiol and treated with piperidine solution to effect cleavage.
Figure 3 depicts an autoradiogram of 32P-labeled HPV target and crosslinked product showing hybrid
separation by denaturing polyacrylamide gel electrophor- esis. Lane 1: Control 32P-labeled CMV target. Lane 2: 24 hour reaction at 20°C. Lane 3: 72 hour reaction at 20°C. Lane 4: 24 hour reaction at 30°C. Lane 5: 72 hour reaction at 30°C. Reaction solutions were treated with 2-aminoethanothiol, which quenches the iodoacetamido group.
DETAILED DESCRIPTION OF THE INVENTION
A. Crosslinking oligonucleotides
This invention provides novel substituted nucleotide bases with a crosslinking arm which are useful in preparing nucleosides and nucleotides and are useful as crosslinking agents. The substituted bases are of the following-formula (1'):
R1 - B - (CH2)q - (Y)r - (CH2) m - A' (I') wherein,
R1 is hydrogen, or a sugar moiety or analog thereof optionally substituted at its 3' or its 5' position with a phosphorus derivative attached to the sugar moiety by an oxygen and including groups Q1, Q2 and Q3, or with a reactive precursor thereof suitable for nucleotide bond formation;
Q1 is hydroxy, phosphate or diphosphate;
Q2 is =O or =S;
Q3 is CH2-R' S-R', O-R', or N-R'R"; each of R' and R" is independently hydrogen or C1-6alkyl;
B is a nucleic acid base or analog thereof that is a component of an oligonucleotide;
Y is a functional linking group;
each of m and q is independently 0 to 8, inclusive;
r is 0 or 1; and
A' is a leaving group.
In the practice of the present invention, the sugar moiety or analog thereof is selected from those useful as a component of a nucleotide. Such a moiety may be selected from, for example, ribose, deoxyribose, pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, pentofuranose, xylose, lyxose, and
cyclopentyl. The sugar moiety is preferably ribose, deoxyribose, arabinose or 2'-O-methylribose and embraces either anomer, α or β .
The phosphorus derivative attached to the sugar moiety is conveniently selected from, for example, monophosphate, diphosphate, triphosphate, alkyl phosphate, alkanephosphonate, phosphorothioate, phoεphorodithioate, and the like.
A reactive precursor suitable for internucleotide bond formation is one which is useful during chain extension in the synthesis of an oligonucleotide.
Reactive groups particularly useful in the present
invention are those containing phosphorus. Phosphoruscontaining groups suitable for internucleotide bond formation are preferably alkyl phosphorchloridites, alkyl phosphites or alkylphosphoramidites. Alternatively, activated phosphate diesters may be employed for this purpose.
The nucleic acid base or analog thereof (B) may be chosen from the purines, the pyrimidines, the deazapurines and the pyrazolopyrimidines. It is preferably selected from uracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl, 4-aminopyrrolo- [2,3-d]pyrimidin-5-yl, 2-amino-4-oxopyrrolo[2,3-d]pyri- midin-5-yl, 4-aminopyrazolo[3,4-d]pyrimidin-3-yl or 4- amino-6-oxopyrazolo[3,4-d]pyrimidin-3-yl, where the purines are attached to the sugar moiety of the
oligonucleotides via the 9-position, the pyrimidines via the l-position, the pyrrolopyrimidines via the 7-position and the pyrazolopyrimidines via the l-position.
The functional linking group Y may be chosen from nucleophilic groups such as oxy, thio, amino or chemically blocked derivatives thereof, for example trifluoroacetamido, phthalimido, CONR' , NR'CO, and SO2NR', where R1 = H or C1-6alkyl. Such functionalities,
including aliphatic or aromatic amines, exhibit
nucleophilic properties and are capable of serving as a point of attachment of the -(CH2)m-A' group. Amino groups and blocked derivatives thereof are preferred.
The leaving group A' may be chosen from, for example, such groups as chloro, bromo, iodo, SO2R'", or S+R'"R"", where each of R'" and R"" is independently
C1-6alkyl or aryl or R'" and R"" together form a C1-6- alkylene bridge. Chloro, bromo and iodo are preferred. The leaving group will be altered by its leaving ability. Depending on the nature and reactivity of the particular leaving group, the group to be used is chosen in each case to give the desired specificity of the irreversibly binding probes.
Examination of double-stranded DNA by ball- and-stick models and high resolution computer graphics indicates that the 7-position of the purines and the
5-position of the pyrimidines lie in the major groove of the B-form duplex of double-stranded nucleic acids.
These positions can be substituted with side chains of considerable bulk without interfering with the hybrid- ization properties of the bases. These side arms may be introduced either by derivatization of dThd or dCyd, or by straightforward total synthesis of the heterocyclic base, followed by glycosylation. These modified nucleosides may be converted into the appropriate activated nucleotides for incorporation into oligonucleotides with an automated DNA synthesizer. With the pyrazolo[3,4-d]- pyrimidines, which are analogs of adenine, the crosslinking arm is attached at the 3-position, which is
equivalent to the 7-position of purine.
The crosslinking side chain should be of sufficient length to reach across the major groove from a purine 7- or 8-position, pyrimidine 5-position, pyrrolopyrimidine 5-position or pyrazolopyrimidine 3-position and reacting with the N-7 of a purine (preferably
guanine) located above (on the oligomer 3'-side) the base pair containing the modified analog. Thus, the side chain should be of at least three atoms, preferably of at least five atoms and more preferably of at least six atoms in length. A generally preferred length of the side chain is from about 5 to about 9 carbon atoms.
To optimize strand crosslinking, it would be desirable to have the target strand base which is being attacked paired to the first or second base which is on the 3' side of the modified base in the oligonucleotide containing the crosslinking arm. For example, in the case where the target strand base under attack is a guanine, the target sequence for a probe containing a modified uracil should contain the complement GZA
(preferably GGA), where Z is any base, with the probe oligonucleotide containing UZC (preferably UCC), where U is dϋrd 5-substituted with the crosslinking arm. In oligonucleotides containing crosslinking adenine derivatives, for example, the adenine-modified AZ1C triplet would target GZ1T, where Z1 is any base.
It has been found that when the modified base containing the crosslinking arm is a uracil and thetarget sequence is GGA, alkylation of the second guanine on the target's 5' side of the crosslinker-modified base pair is the exclusive action observed (as shown in Figure 1). The crosslinking reaction seems to be very specific for the "best fit" of electrophile to nucleophile, i.e., two or more guanine residues may need to neighbor the complement of the modified base to discover the site of alkylation.
Two classes of modified 2'-deoxynucleosides have demonstrated particular usefulness in the present invention for incorporation into oligonucleotides as sequence-directed crosslinking agents. The first class is the 5-substituted-2'-deoxyuridines whose general structure is presented below:
Figure imgf000015_0001
The 5- (substituted) -2 ' -deoxyuridines may be prepared by the routes shown in Schemes 1 and 2.
Scheme 1 :
Figure imgf000016_0001
For example, the general procedure of Robins etal. (J. Can. J. Chem., 60:554 (1982); J. Org. Chem.,
48:1854 (1983)) may be adapted, as shown in Scheme 1, to the palladium-mediated coupling of a substituted 1-alkyne (XXI) to 5-iodo-2'-deoxyuridine (XX) to give the
acetylene-coupled product (XXII). The acetylenic dUrd analog XXII is reduced, with Raney nickel for example, to give the saturated compound (XXIII), which is then used for direct conversion to a reagent for use on an automated DNA synthesizer, as described below.
Scheme 2:
Figure imgf000017_0001
When 5-chloromercurio-2'-deoxyuridine (XXIV) is used as a starting compound, it cannot be directly coupled to an olefin group to give the olefinic compound (XXVII) by palladium-catalyzed coupling with functional ized olefins. Instead, as shown in Scheme 2, a substituted alkene (XXV) and 5-chloromercurio-2'-deoxyuridine (XXIV) are reacted together with methanol to give the alpha-methoxy adduct (XXVI), which is converted to the olefinic compound XXVII by trifluoroacetic acid and trifluoroacetic anhydride. Reduction gives the saturated compound (XXIII), to be converted to the DNA synthesizerready reagent as described below.
B. Pyrazolo[3,4-d,]pyrimidines
The second class of modified nucleoside is a group of 2'-deoxy-4-aminopyrazolo[3,4-d]pyrimidine derivatives. The general structure of these derivatives is presented below:
Figure imgf000018_0001
The above compounds are derived from a novel group of derivatives of 3,4-disubstituted and 3,4,6-trisubstituted pyrazolo[3,4-d]pyrimidines. The 3,4-disubstituted and 3,4,6-trisubstituted pyrazolo[3,4-d]pyrimidines and their synthesis are disclosed in commonly owned, copending application Serial No. 250,474, the entire disclosure of which is incorporated herein by reference. They have the following formula (I):
Figure imgf000018_0002
wherein,
R1 is hydrogen, or a sugar moiety or analog thereof optionally substituted at its 3' or its 5' position with a phosphorus derivative attached to the sugar moiety by an oxygen and including groups Q1 ' Q2 and Q3, or with a reactive precursor thereof suitable for nucleotide bond formation; provided that when R3 is hydrogen, then R1 cannot be hydrogen;
Q1 is hydroxy, phosphate or diphosphate;
Q2 is =O or =S;
Q3 is CH2-R', S-R', O-R', or N-R'R"; each of R' and R" is independently hydrogen or C1-6alkyl;
R3 is hydrogen or the group -W-(X)n-A; each of W and X is independently a chemical linker arm;.
A is an intercalator, a metal ion chelator, an electrophilic crosslinker, a photoactivatable crosslinker, or a reporter group;
each of R4 and R6 is independently H, OR, SR,
NHOR, NH2, or NH(CH2)tNH2;
R is H or C1-6alkyl;
n is zero or one; and
t is zero to twelve.
The synthesis of 3,4-disubstituted and 3,4,6- trisubstituted pyrazolo[3,4-d]pyrimidine nucleosides and their use as reagents for incorporation into nucleic acids either enzymatically or via chemical synthesis offers several advantages over current procedures. The de novo chemical synthesis of the nucleotide allows for the incorporation of a wide range of functional groups (e.g., NH2, SH, OH, halogen, COOH, CN, CONH2) and the use of different sugar moieties. Also, adenine, guanine, and hypoxanthine analogs are obtained from a single nucleoside precursor. And, the synthesis does not require the use of toxic heavy metals or expensive catalysts. In the practice of the present invention, the sugar moiety or its analog is selected from those useful as a component of a nucleotide. Such a moiety may be selected from, for example, pentose, deoxypentose, hexose, deoxyhexose, ribose, deoxyribose, glucose, arabinose, pentofuranose, xylose, lyxose, and cyclopentyl. The sugar moiety is preferably ribose, deoxyribose, arabinose or 2'-O-methylribose and embraces either anomer, α or β .
The phosphorus derivative attached to the sugar moiety is conveniently selected from, for example, monophosphate, diphosphate, triphosphate, alkyl phosphate, alkanephosphonate, phosphorothioate, phosphorodithioate, and the like.
A reactive precursor suitable for internucleotide bond formation is one which is useful during chain extension in the synthesis of an oligonucleotide.
Reactive groups particularly useful in the present invention are those containing phosphorus. Phosphorusocontaining groups suitable for internucleotide bond formation are preferably alkyl phosphorchloridites, alkyl phosphites or alkylphosphoramidites. Alternatively, activated phosphate diesters may be employed for this purpose..
In the above formula I, a chemical linker arm
(W alone or together with X) serves to make the
functional group (A) more able to readily interact with antibodies, detector proteins, or chemical reagents, for example. The linkage holds the functional group away from the base when the base is paired with another within the double-stranded complex. Linker arms may include alkylene groups of 1 to 12 carbon atoms, alkenylene groups of 2 to 12 carbon atoms and 1 or 2 olefinic bonds, alkynylene groups of 2 to 12 carbon atoms and 1 or 2acetylenic bonds, or such groups substituted at a
terminal point with nucleophilic groups such as oxy, thio, amino or chemically blocked derivatives thereof (e.g., trifluoroacetamido, phthalimido, CONR', NR'CO, and SO2NR', where R1 = H or C1-6alkyl). Such functionalities, including aliphatic or aromatic amines, exhibit nucleophilic properties and are capable of serving as a point of attachment of the functional group (A).
The linker arm moiety (W alone or together with X) is preferably of at least three atoms and more
preferably of at least five atoms. The terminal nucleophilic group is preferably amino or chemically blocked derivatives thereof.
Intercalators are planar aromatic bi-, tri- or polycyclic molecules which can insert themselves between two adjacent base pairs in a double-stranded helix of nucleic acid. Intercalators have been used to cause frameshift mutations in DNA and RNA. It has also
recently been shown that when an intercalator is
covalently bound via a linker arm ("tethered") to the end of a deoxyoligonucleotide, it increases the binding affinity of the oligonucleotide for its target sequence, resulting in strongly enhanced stability of the complementary sequence complex. At least some of the tethered intercalators also protect the oligonucleotide against exonucleases, but not against endonucleases. See, Sun et al., Nucleric Acids Res., 15: 6149-6158 (1987); Le Doan etal., Nucleic Acids Res., 15:7749-7760 (1987). Examples of tetherable intercalating agents are oxazolopyridocarbazole, acridine orange, proflavine, acriflavine and derivatives of proflavine and acridine such as 3-azido-6- (3-bromopropylamino) acridine, 3-amino-6-(3-bromopentyl- amino)acridine, and 3-methoxy-6-chloro-9-(5-hydroxy- pentylamino)acridine.
Oligonucleotides capable of crosslinking to the complementary sequence of target nucleic acids are valuable in chemotherapy because they increase the efficiency of inhibition of mRNA translation or gene expression control by covalent attachment of the oligonucleotide to the target sequence. This can be accomplished by cross linking agents being covalently attached to the oligonucleotide, which can then be chemically activated to form crosslinkages which can then induce chain breaks in the target complementary sequence, thus inducing irreversible damage in the sequence. Examples of electrophilic crosslinking moieties include alpha-halocarbonyl
compounds, 2-chloroethylamines and epoxides.
When oligonucleotides comprising at least one nucleotide base moiety of the invention are utilized as a probe in nucleic acid assays, a label is attached to detect the presence of hybrid polynucleotides. Such labels act as reporter groups and act as means for detecting duplex formation between the target nucleotides and their complementary oligonucleotide probes.
A reporter group as used herein is a group which has a physical or chemical characteristic which can be measured or detected. Detectability may be provided by such characteristics as color change, luminescence, fluorescence, or radioactivity; or it may be provided by the ability of the reporter group to serve as a ligand recognition site.
The pyrazolopyrimidines of the present invention of formula. I where R1 is hydrogen may be prepared by the procedures outlined below and as set forth by
Kobayashi in Chem. Pharm. Bull., 21: 941-951 (1973), the disclosure of which is incorporated herein by reference.
Figure imgf000022_0001
In general, malononitrile (III) is treated with acyl halide (II) in the presence of a base to yield acylmalononitrile (IV) , which is subsequently methylated with dimethyl sulfate or diazomethane, for example, to give the substituted methoxymethylenemalononitrile (V) . This, compound is then reacted with hydrazine hydrate in boiling alcohol to give the 3-substituted-5-aminopyra- zole-4-carbonitrile (VI) , which is treated with cold concentrated sulfuric acid to give the 3-substituted-5- aminopyrazole-4-carboxamide (VII) .
The carboxamide (VII) may alternatively be prepared by treating cyanoacetamide (XII) with acid halide (II) to give the acylcyanoacetamide (XIII) , which is then methylated, and the resulting methoxy compound (XIV) is reacted with hydrazine hydrate.
Figure imgf000023_0001
Syntheses of pyrazolo[3 , 4-d]pyrimidines are accomplished from the two pyrazole intermediates, VI, and
VII . Thus, 3 , 4-disubstituted pyrazolo[3 , 4-d]pyrimidines
(VIII and X) are obtained by treating the corresponding VI and VII with boiling formamide. Alternatively, VI may be treated with dialkoxymethyl ester of a carboxylic acid, at room temperature or above room temperature, and then with ammonia to give VIII, and VII may be treated with dialkoxymethyl ester of a carboxylic acid (without subsequent ammonia treatment) , at room temperature or above room temperature, to give compound X. 3 , 4 , 6-Tri- substituted pyrazolo [3 , 4-d]pyrimidines (IX and XI) are obtained by fusing the corresponding VI and VTI with urea and thiourea (H2N)2C=R6 (where R6 is O or S). Alternatively, VI and VII may be treated with an alkyl xanthate salt such as potassium ethyl xanthate and with alkyl halide such as methyl iodide, at a temperature above room temperature, followed by oxidation by a peroxide such as m-chloroperbenzoic acid (MCPBA) and subsequent treatment with ammonia to give IX and XI, respectively, where R6 is NH2.
Figure imgf000024_0001
The compounds of formula I may be recovered from the reaction mixture in which they are formed by established procedures.
In the compounds of formula I where R1 is a sugar moiety, the sugar may be either added to the 1- position of the pyrazole VI or VII prior to further treatment or added to the 1-position of the pyrazolo[3,4- d]pyrimidine VIII, IX, X or XI. To add the sugar, the pyrazole or pyrazolopyrimidine is treated with sodium hydride and then with the glycosyl halide of the blocked sugar.
Oligonucleotides of the present invention may comprise at least one and up to all of their nucleotides from the substituted pyrazolo[3,4-d]pyrimidines of
formula I and/or at least one and up to all of their nucleotides from the substituted nucleotide bases offormula I'.
To prepare oligonucleotides, protective groups are introduced onto the nucleosides of formula I or formula I' and the nucleosides are activated for use in the synthesis of oligonucleotides. The conversion to protected, activated forms follows the procedures as described for 2'-deoxynucleosides in detail in several reviews. See, Sonveaux, Bioorσanic Chemistry. 14:274-325 (1986); Jones, in "Oligonucleotide Synthesis, a Practical Approach", M.J. Gait, Ed., IRL Press, p. 23-34 (1984).
The activated nucleotides are incorporated into oligonucleotides in a manner analogous to that for DNA0and RNA nucleotides, in that the correct nucleotides will be sequentially linked to form a chain of nucleotides which is complementary to a sequence of nucleotides in target DNA or RNA. The nucleotides may be incorporated either enzymatically or via chemical synthesis. The nucleotides may be converted to their 5'-O-dimethoxy- trityl-3'-(N,N-diisopropyl)phosphoramidite cyanoethyl ester derivatives, and incorporated into synthetic oligonucleotides following the procedures in
"Oligonucleotide Synthesis: A Practical Approach", supra . The N-protecting groups are then removed, along with the other oligonucleotide blocking groups, by post-synthesis aminolysis, by procedures generally known in the aft.
In a preferred embodiment, the activated nucleptides may be used directly on an automated DNA synthesizer according to the procedures and instructions of the particular synthesizer employed. The oligonucleotides may be prepared on the synthesizer using the standard commercial phosphoramidite or H-phosphonate chemistries.
In another preferred embodiment, the amino- pyrazolopyrimidine nucleotide triphosphates may
substitute for an adenine using the nick translation procedure, as described by Langer et al., Proc. Natl.
Acad. Sci. USA, 78:6633-6637 (1981), the disclosure of which is incorporated herein by reference.
The leaving group, such as a haloacyl group, may be added to the aminoalkyl tails (-CH2)q-Y) following incorporation into oligonucleotides and removal of any blocking groups. For example, addition of an α-haloacetamide may be verified by a changed mobility of the modified compound on HPLC, corresponding to the removal of the positive charge of the amino group, and by subsequent readdition of a positive charge by reaction with 2-aminoethanethiol to give a derivative with reverse phase HPLC mobility similar to the original aminoalkyl- oligonucleotide.
In specific embodiments, each of the following electrophilic leaving groups were attached to an amino- propyl group on human papillomavirus (HPV) probes:
bromoacetyl, iodoacetyl and the less reactive but cσnformationally more flexible 4-bromobutyryl. Bromoacetyl and iodoacetyl were found to be of equal reactivity in crosslinking.
An oligonucleotide probe according to the invention includes at least one labeled substituted pyrazolo[3,4-d]pyrimidine nucleotide moiety of formula I and/or at least one labeled substituted nucleotide base of formula I'.
Probes may be labeled by any one of several methods typically used in the art. A common method of detection is the use of autoradiography with 3H, 125I, 35S, 14c, or 32P labeled probes or the like. Other reporter groups include ligands which bind to antibodies labeled with fluorophores, chemiluminescent agents, and enzymes. Alternatively, probes can be conjugated directly with labels such as fluorophores, chemiluminescent agents, enzymes and enzyme substrates. Alternatively, the same components may be indirectly bonded through a ligand- antiligand complex, such as antibodies reactive with a ligand conjugated with label. The choice of label
depends on sensitivity required, ease of conjugation with 5the probe, stability requirements, and available
instrumentation. The choice of label dictates the manner in which the label is incorporated into the probe. Radioactive probes are typically made using commercially available nucleotides containing the desired radioactive isotope. The radioactive nucleotides can be incorporated into probes, for example, by using DNA synthesizers, by nick-translation, by tailing of radioactive bases to the 3' end of probes with terminal transferase, by copying M13 plasmids having specific inserts with the Klenow fragment of DNA polymerase in the presence of radioactive dNTP's, or by transcribing RNA from templates using RNA polymerase in the presence of radioactive rNTP's.
Non-radioactive probes can be labeled directly with a signal (e.g., fluorophore, chemiluminescent agent or enzyme) or labeled indirectly by conjugation with a ligand. For example, a ligand molecule is covalently bound to the probe. This ligand then binds to a receptor molecule which is either inherently detectable or
covalently bound to a detectable signal, such as an enzyme or photoreactive compound. Ligands and anti- ligands may be varied widely. Where a ligand has a natural "antiligand", namely ligands such as biotin, thyroxine, and cortisol, it can be used in conjunction with its labeled, naturally occurring antiligand.
Alternatively, any haptenic or antigenic compound can be used in combination with a suitably labeled antibody. A preferred labeling method utilizes biotin-labeled analogs of oligonucleotides, as disclosed in Langer et al., Proc. Natl. Acad. Sci. USA, 78: 6633-6637 (1981), which isincorporated herein by reference.
Enzymes of interest as reporter groups will primarily be hydrolases, particularly phosphatases, esterases, ureases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, rare earths, etc. Chemiluminescers include luciferin, acridinium esters and 2,3-dihydrophthalazinediones, e.g., luminol.
The specific hybridization conditions are not critical and will vary in accordance with the investigator's preferences and needs. Various hybridization solutions may be employed, comprising from about 20% to about 60% volume, preferably about 30%, of a polar organic solvent. A common hybridization solution employs about 30-60% v/v formamide, about 0.5 to 1M sodium chloride, about 0.05 to 0.1M buffers, such as sodium citrate, Tris HCl, PIPES or HEPES, about 0.05% to 0.5% detergent, such as sodium dodecylsulfate, and between 1- 10 mM EDTA, 0.01% to 5% ficoll (about 300-500 kdal), 0.1% to 5% polyvinylpyrrolidone (about 250-500 kdal), and 0.01% to 10% bovine serum albumin. Also included in the typical hybridization solution will be unlabeled carrier nucleic acids from about 0.1 to 5 mg/ml, e.g., partially fragmented calf thymus or salmon sperm, DNA, and/or partially fragmented yeast RNA and optionally from about 0.5% to 2% wt./vol. glycine. Other additives may also be included, such as volume exclusion agents which include a variety of polar water-soluble or swellable agents, such as anionic polyacrylate or polymethylacrylate, and
charged saccharidic polymers, such as dextran sulfate.
The particular hybridization technique is not essential to the invention. Hybridization techniques are generally described in "Nucleic Acid Hybridization, A Practical Approach", Hames and Higgins, Eds., IRL Press, 1985; Gall and Pardue, Proc. Natl. Acad. Sci., U.S.A.,
63: 378-383 (1969); and John et al., Nature, 223:582-587
(1969). As improvements are made in hybridization
techniques, they can readily be applied.
The amount of labeled probe which is present in the hybridization solution may vary widely. Generally, substantial excesses of probe over the stoichiometric amount of the target nucleic acid will be employed to enhance the rate of binding of the probe to the target DNA.
Various degrees of stringency of hybridization can be employed. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for the formation of a stable duplex. The degree of stringency can be controlled by temperature, ionic strength, the inclusion of polar organic solvents, and the like. For example, temperatures employed will normally be in the range of about 20° to 80°C, usually 25° to 75ºC. For probes of 15-50 nucleotides in 50% formamide, the optimal temperature range can vary from 22-65°C. With routine experimentation, one can define conditions which permit satisfactory hybridization at room temperature. The stringency of hybridization is also conveniently varied by changing the ionic strength and polarity of the reactant solution through manipulation of the concentration of formamide within the range of about 20% to about 50%.
Treatment with ultrasound by immersion of the reaction vessel into commercially available sonication baths can oftentimes accelerate the hybridization rates.
After hybridization at a temperature and timeperiod appropriate for the particular hybridization solution used, the glass, plastic, or filter support to which the probe-target hybrid is attached is introduced into a wash solution typically containing similar
reagents (e.g., sodium chloride, buffers, organic
solvents and detergent), as provided in the hybridization solution. These reagents may be at similar concentrations as the hybridization medium, but often they are at lower concentrations when more stringent washing conditions are desired. The time period for which the supportis maintained in the wash solutions may vary from minutes to several hours or more. Either the hybridization or the wash medium can be stringent. After appropriate stringent washing, the correct hybridization complex may now be detected in accordance with the nature of the label.
The probe may be conjugated directly with the label. For example, where the label is radioactive, the support surface with associated hybridization complex substrate is exposed to X-ray film. Where the label is fluorescent, the sample is detected by first irradiating it with light of a particular wavelength. The sample absorbs this light and then emits light of a different wavelength which is picked up by a detector ("Physical Biochemistry", Freifelder, D., W.H. Freeman & Co., 1982, pp. 537-542). Where the label is an enzyme, the sample is detected by incubation with an appropriate substrate for the enzyme. The signal generated may be a colored precipitate, a colored or fluorescent soluble material, or photons generated by bioluminescence or chemiluminescence. The preferred label for dipstick assays
generates a colored precipitate to indicate a positive reading. For example, alkaline phosphatase will dephosphorylate indoxyl phosphate which then will participate in a reduction reaction to convert tetrazolium salts to highly, colored and insoluble formazans.
Detection of a hybridization complex may
require the binding of a signal generating complex to a duplex of target and probe polynucleotides or nucleic acids. Typically, such binding occurs through ligand and antiligand interactions as between a ligand-conjugatedProbe and an antiligand conjugated with a signal. The binding of the signal generation complex is also readily amenable to accelerations by exposure to ultrasonic energy.
The label may also allow indirect detection of the hybridization complex. For example, where the label is a hapten or antigen, the sample can be detected by using antibodies. In these systems, a signal is gener ated by attaching fluorescent or enzyme molecules to the antibodies or in some cases, by attachment to a radioactive label. (Tijssen, P., "Practice and Theory of Enzyme Immunoassays, Laboratory Techniques in Biochemistry and Molecular Biology", Burdon, R.H., van Knippenberg, P.H., Eds., Elsevier, 1985, pp. 9-20).
The amount of labeled probe present in the hybridization solution may vary widely, depending upon the nature of the label, the amount of the labeled probe that can reasonably bind to the cellular target nucleic acid, and the precise stringency of the hybridization medium and/or wash medium. Generally, substantial probe excesses over the stoichiometric amount of the target will be employed to enhance the rate of binding of the probe to the target nucleic acids.
The invention is also directed to a method for identifying target nucleic acid sequences, which method comprises utilizing an oligonucleotide probe including at least one labeled substituted nucleotide moiety of formula I and/or formula I'.
In one embodiment, the method comprises the steps of:
(a) denaturing nucleic acids in the sample to be-testedτ
(b) hybridizing to the target nucleic acids an oligonucleotide probe including at least one labeled substituted nucleotide moiety of formula I or formula I', wherein the probe comprises a sequence complementary to that of the target nucleic acids;
(c) washing the sample to remove unbound probe;
(d) incubating the sample with detection agents; and
(e) inspecting the sample.
The above method may be conducted following procedures well known in the art. An assay for identifying target nucleic acid sequences utilizing an oligonucleotide probe including at least one labeled substituted nucleotide moiety of formula I and/or formula I' and comprising the above method is contemplated for carrying out the invention. Such an assay may be provided in kit form. For example, a typical kit will include a probe reagent component comprising an oligonucleotide including at least one labeled nucleotide moiety of formula I or formula I', the oligonucleotide having a sequence complementary to that of the target nucleic acids; a denaturation reagent for converting double-stranded nucleic acid to single- stranded nucleic acid; and a hybridization reaction mixture. The kit can also include a signal-generating system, such as an enzyme for example, and a substrate for the system.
C. Enzyme-Mediated Triple Strand Formation
Antisense ODNs
Oligodeoxynucleotides (ODNs) have great
potential as sequence specific pharmaceutical agents for the inhibition of gene expression. For instance,
chemically synthesized ODNs may inhibit the expression of specific gene products through formation of duplexes upon hybridization with complementary messenger RNAs (mRNAs). More specifically, these "antisense" ODNs are believed to inhibit the processing or translation of message
primarily through an RNase H-mediated cleavage of the target mRNA sequence. Because of this inhibitory effect, antisense ODNs may be useful as anti-viral, antiparasitic, and anti-cancer agents. Further, antisense ODNs provide a unique opportunity for rational drug development, since a genetic target offers both exquisite specificity and universality with respect to potential target sequences.
However, "antisense" technology is beset with certain fundamental disadvantages. One major challenge involves development of antisense ODNs with sufficient potency to warrant in vivo testing. Antisense ODN formulations that exhibit nuclease resistance, rapid cellular uptake, and efficient and stable hybridization to the target RNA sequence are desirable. Improvement of one or more of these properties without concomitant de eterious effects on other properties may be difficult. For instance, antisense ODNs with modified backbones, such as oligonucleoside methylphosphonates and
phosphorothioates, exhibit excellent nuclease resistance relative to unmodified ODNs, but methylphosphonate ODNs form DNA-RNA hybrids that are refractile to RNase H and phosphorothioate ODNs are poorly taken up into cells.
Another approach to improving effectiveness of antisense ODNs involves attaching moieties to ODNs in order to potentiate their antisense activity. For instance, moieties that interact directly with the RNA target upon hybridization (such as pendant intercalating groups that stabilize ODN-target hybrids, ODNs with freeradical-based RNA cleaving activity or ODNs capable of covalently linking to their targets upon hybridization) may be useful in this regard. Direct cleavage or cross- linkage of an mRNA target by an ODN renders the RNAinactive.
Cellular uptake of antisense ODNs is another area of concern. While charged ODNs may be actively taken up into mammalian cells through an energy dependent pathway, augmentation of this pathway to achieve high intracellular concentrations of antisense ODN is
desirable. "Endocytic approaches", which enhance
receptor-mediated cellular targeting, include conjugation of a cholesteryl group that may act as an lipophilic anchor for the antisense ODN; encapsulation of ODNs into liposomes; and linkage of ODNs to soluble macromolecular complexes. Application of the above-noted approaches, alone or in combination, may provide an increase in antisense ODN potency in the range of 1000-fold and may significantly reduce the cost of ODN synthesis.
Anti-gene ODNs
A variation of the "antisense" approach to rational drug design is termed "anti-gene". Whereas antisense ODNs target single stranded mRNA, anti-gene ODNs hybridize with and are capable of inhibiting the function of double-stranded DNA. More specifically, anti-gene ODNs form sequence-specific triple-stranded complexes with a double stranded DNA target and thus interfere with the replication or transcription of selected target genes. Except for certain RNA viruses and nucleic acid-free viroids, DNA is the repository for all genetic information, including regulatory control sequences and non-expressed genes, such as dormant proviral DNA genomes. In contrast, the target for antisense ODNs, mRNA, represents a very small subset of the information encoded in DNA. Thus, anti-gene ODNs have broader applicability and are potentially more powerful than antisense ODNs that merely inhibit mRNA processing and translation.
Anti-gene ODNs in the nuclei of living cells can form sequence-specific complexes with chromosomal DNA. The resultant triplexes can inhibit restriction and/or transcription of the target double stranded DNA. Based on the known stabilities of the two target nucleic acid species (i.e., DNA and RNA), anti-gene interference with DNA functioning has longer lasting effects than the corresponding antisense inhibition of mRNA function.
Mammalian cell DNA does not turnover; in fact, cells possess sophisticated pathways capable of repairing lesions in DNA that may arise from environmental insults or from spontaneous rearrangements. In contrast, mRNA is transient and may exist only for minutes within a cell. The constant turnover of an mRNA species and the
potentially high copy number of such mRNA species suggest that anti-sense ODNs will provide relatively short term effects. While cellular uptake of anti-gene ODNs may need to be augmented to achieve sufficient intracellular concentrations, once within the cell the ODNs naturally concentrate in the nucleus.
Anti-gene therapy is based on the observation that certain DNA homopolymers can form triple-stranded complexes. In these triple-standed complexes, the third strand resides in the major groove of the Watson Crick base-paired double helix, where it hydrogen bonds to one of the two parental strands. A binding code governs the recognition of base pairs by a third base (see allowed triplets below). In each case, the third strand base is presented first and is followed by the base pair;
hydrogen bonding between the first two bases maintains the third base interaction. A-A-T G-G-C
T-A-T C-G-C
Certain limitations of this base pair recognition code are apparent from the allowed triplets. First, there isno capability for the recognition of T-A and C-G base pairs; hence, triple strand formation is restricted to runs of homσpurine bases on one strand and homopyrimidine bases on the other strand of the duplex. Second, if cytosine is in the third strand ("C"), it must be
protonated to be able to hydrogen bond to the guanine of a G-C base pair. The pKa for protonation of cytosine is 4.6, suggesting that at physiological pH the stability of C-G-C triads is likely to be impaired. The substitution of 5-methyl cytosine or the use of polyvalent cations (such as spermine or spermidine) may stabilize the C-G-C triplets at pH 7.0. Third, in all cases triads are maintained by two hydrogen bonds between the third strand base and the purine residue of the base pair. Hence, triple-stranded complexes are generally less stable than the parental double-stranded DNA, which is maintained by a combination of two (A-T) or three (G-C) hydrogen bonds between purine and pyrimidine pairs.
Cytosine/thymidine-, guanine/adenine- and guanine/thymidine-containing ODNs can sequence- specifically bind to homopurine runs in double-stranded DNA. These recognition motifs are based on Hoogstein or reverse Hoogstein base pairing. In the C/T recognition motif, the ODN is parallel to the homopurine strand of the duplex; in the G/A recognition motif, the ODN is anti-parallel to the homopurine strand; in the G/T recognition motif, the ODN may bind parallel or antiparallel to the homopurine strand of the duplex,
depending on the G content of the third strand. These recognition motifs may be sequence dependent. The sequence specificity of anti-gene ODNs using the C/T recognition motif permits hybridization of such ODNs to homopurine runs in plasmid DNA and in yeast chromosomes. Since ODN binding is restricted to homopurine runs, it would be advantageous to identify additional heterocycles that can recognize the remaining two base pairs, i.e., C- G and T-A. While guanosine may be used in the third Strand to recognize T-A base pairs, this interaction involves only one hydrogen bond and is relatively
unstable.
Analogous to anti-sense ODNs, anti-gene ODNs may be modified with a variety of pendant groups designedto augment their activity. Hence, intercalating groups, cleaving agents, and crosslinking moieties may be
appended to the termini of anti-gene ODNs. Upon triplex formation, these groups interact with the adjacent duplex and intercalate, cleave or crosslink, respectively.
Further, in the C/T recognition motif, substitution of 5-methyl cytosine for cytosine in the third strand ODN significantly stabilizes triplexes formed with "G"-rich homopurine runs. In addition, ODNs with modified backbones, such as oligonucleoside methyl-phosphonates and phosphorothioates, may form triple-stranded
complexes.
Enzyme-Mediated Triple Strand Formation
A important disadvantage of triple strand formation as discussed above is the relatively slow kinetics of triple strand formation. The claimed invention overcomes this disadvantage through enzyme catalysis of triple strand formation, with recombination enzymes particularly preferred for this purpose. More significantly, enzyme-catalyzed triple strand formation provides the immense advantage of universal sequence recognition (in contrast to the A-T and G-C recognition limitation associated with non-enzyme-mediated triple strand formation).
Briefly, the genetic material of all living organisms is occasionally subject to homologous
rearrangement. Recombination between like (or
homologous) DNA molecules, which plays an important role in promoting genetic diversity within a species, is catalyzed by a family of enzymes in both procaryotic andeucaryotic cells. Recombination also plays an important role in DNA repair and in the immune response.
Homologous recombination in E. coll serves as an illustrative example of processes that occur in both procaryotic and eucaryotic cells. Studies of purifiedrecombinational enzymes from E. coli in a defined cell- free system permit division of homologous recombination into three steps. For purposes of this illustration, the "invading" single strand is circular and the target double strand is linear.
In the first step, "presynapsis", single stranded circular DNA is coated with the multifunctional protein recA in the presence of ATP. The resultant nucleoprotein filament possesses a right handed helical twist composed of 18 bases per turn and 1 monomer of recA protein per 3.6 nucleotides.
In the second step, "synapsis", the single- stranded circular nucleoprotein filament conducts a two- dimensional search along a linear double-stranded DNA template for homologous sequences. The search concludes with homologous alignment of the two molecules in an initial complex that has no net helical interwinding (the complex is referred to as a "paranemic joint") .
Paranemic joints are highly unstable and upon
deproteinization readily dissociate. Very rapidly, the DNA double helix is incorporated into the filament, and in so doing, the two DNA molecules become plectonemically coiled (i.e., helically interwound). In this triple strand form, the newly incorporated third strand is homologous (in sequence and polarity) to one of the parental duplex strands, and complementary to the other parental duplex strand. In the resultant synaptic complex, the three strands of DNA are believed to exist as a true hydrogen-bonded triple strand having a close association with recA. In contrast to paramenic joints, the plectonemically coiled complex is stable upon
deproteinization (removal of recA).
The plectonemically coiled synaptic complex contains 18 triads per right handed helical turn. While recA is a critical part of the complex, sequence
specificity resides entirely in the hydrogen bonding between bases. In a plectonemically coiled synaptic complex, two alternative conformations exist. In one,the negative linear strand of parental DNA is Watson
Crick base-paired to the positive linear strand; in the other, the negative linear strand of parental DNA is
Watson Crick base-paired to the positive circular single strand. In both conformations, the bases of the third strand are presumed to be hydrogen-bonded in a Hoogstein or reverse Hoogstein fashion to the purines of the other two strands (according to the binding code described above). The single stranded circular DNA within the triple stranded nucleoprotein filament subsequently displaces the homologous linear strand ("strand
exchange"). This displacement features a recA-catalyzed, energy-dependent release of the linear positive strand, coupled with hybridization of the circular single strand to the complementary linear strand. During both synapsis and strand exchange, recA remains associated with the complex. Finally, recA dissociates from the newly formed double strand and recombination is complete.
Upon deproteinization of synaptic complexes, the protein-free triple stranded DNA complexes are essentially resistant to single strand-specific nucleases and exhibit very high Tm's. This is likely attributable to hydrogen bonding of the so-called "third strand" to both parental strands, as well as to the highly
underwound state of the complex. The highly underwound, deproteinized synaptic complexes therefore are analogous to highly underwound recA-containing complexes. Thus, recA may catalyze the formation of a DNA triple strand complex not otherwise attainable. In this complex, the two positive strands have identical sequence and
polarity, and either positive strand is capable of forming a double stranded hybrid with the negative parental strand.
Recent evidence indicates that recA alters the conformation of the triplex, such that strand switching is allowed. That is, the bases of the third strand can hydrogen bond to the appropriate purine base regardless of which duplex strand contains the purine base.
Several factors may affect the stability of synaptic complexes, including: (1) the length of the invading single strand; (2) the extent of shared homology between the invading single strand and the target duplex (with length of homology and absence of mismatches of particular importance); (3) the position of the shared homology region with respect to the termini of the target duplex; and (4) the superhelicity, if any, of the duplex.
The following factors may be important when selecting and designing an optimal system for stable triple strand formation with an anti-gene:
Length: An oligomeric 50-mer should have sufficient length to permit the formation of a stable presynaptic complex with recA.
Shared Homology: A minimal shared homology of 13 bases appears to be necessary for formation of
recA-stabilized synaptic filaments. Photo-fixation of a recA-stabilized triple strand by an HMT furan side monoadducted ODN may add further contraints to a minimal or preferred level of shared homology. With photo- fixation, a conformational change within the triple strand complex allows the invading third strand to interchange position with the homologous duplex strand. This results in the third strand bases being Watson Crick base-paired to the complementary strand. Thus, in a preferred embodiment, the ODN and target share 50 bases of homology.
Type of Joint: Depending upon the positional relationship of the region of shared homology to the ends (if any) of the-targeted double-stranded DNA, the
resultant synaptic complex is classified as a proximal joint, a medial joint, or a distal joint. Proximal joints, wherein the recA-stabilized triple strand complex is located at the left hand of a linear duplex, are unstable due to recA-catalyzed strand exchange. Duringstrand exhange, the duplex strand that is homologous to the invading third strand in the triple strand complex is displaced, resulting in a new duplex containing the invading strand and a free single strand. Since strand exchange requires a free 5' end and proceeds with a 5' to3' polarity, this process readily occurs in proximal joints. By contrast, distal joints and medial joints lack the appropriate ends and do not readily undergo recA-catalyzed strand exchange. As a result, these joints are highly stable structures. Accordingly, within the present invention, formation of recA-stabilized triple strand complexes so as to form meidal or distal joints is preferred.
Superhelicity: Superhelical targets may facilitate the formation of recA-stabilized triple strands. However, high efficiency triple strand
formation (even using a DNA target and ODN with a very small region of shared homology) may be obtained using linear targets. Selection of a synthetic, oligomeric double stranded target DNA versus a supercoiled target in the model system will vary with the characteristics of the target duplex DNA.
The present invention combines the recA- catalyzed formation of stable triple strand complexes with synthetic anti-gene ODNs. Enzyme-catalyzed triple strand formation exhibits rapid kinetics and universal DNA sequence recognition. A recA-coated anti-gene ODN serves as a "guide" that seeks homology in a target double strand DNA sequence; upon recognition and bindingof this nucleoprotein filament, recA catalyzes the formation of sequence-specific triple strand complexes. Within the present invention, it is preferred that an anti-gene ODN is least 30-40 nucleotides in length and has a base sequence and polarity identical to either ofthe two duplex strands in the target DNA. The frequency of triplex formation and the stability of the triple strand complexes following deproteinization may be directly related to the length of the anti-gene ODN.
Anti-gene ODNs (of the appropriate polarity) may be usedin combination with endogenous recombinatory pathways to form sequence-specific triple strand complexes with chromosomal DNA. Alternatively, anti-gene ODNs of a variety of lengths may be complexed with a
recombinational enzyme prior to combination with target double strand DNA.
Using the recombinational machinery of cells to catalyze the formation of hydrogen bonded triple-stranded complexes overcomes several disadvantages associated with current anti-gene approaches. First, the enzyme-mediated recognition motif recognizes all four base pairs, thereby allowing targeting of any double stranded DNA sequence. second, the recA-coated, single stranded anti-sense ODN (nucleoprotein filament) searches for target double strand DNA homology much more efficiently than does a small naked anti-gene ODN, thus decreasing the
concentration of anti-gene ODN required for efficient triple strand complex formation. Third, due to the hydrogen bonding patterns and the novel helical twist involved in enzyme-mediated recognition, the resultant triple strand complex is stable at physiological pH.
Fourth, since the cellular recombinational pathway is being harnessed, the DNA in higher order chromatin structures will be accessible for targeting. And fifth, the resultant triple strand complexes display increased stability with increased anti-gene ODN length.
The ability to conduct an efficient homology search is a significant advantage. Preliminary data indicate that ODNs are very inefficient at scanning double stranded DNA for complementary homopurine
sequences. For instance, non-enzyme-catalyzed triple strand formation in vitro has been analyzed after 60 min at 37°C (F.M. Orson et al., Nucl. Acids Res. 19:3435-41, 1991). In contrast, a classical hybridization between two complementary single strands would occur within seconds, rather than hours. Since the human genome contains over 3 X 109 base pairs, the homology search timemay be inordinately long, especially if anti-gene ODNs are used at relatively low concentration. The use of presynaptic nucleoprotein filaments, such as those formed between single stranded DNA and recA, that display a weak nonspecific affinity for double stranded DNA effectively reduces the homology search from a three dimensional to a two dimensional process. Furthermore, upon homologous registry with the double strand, the nucleoprotein filament will more likely produce a triple strand complex than the corresponding interaction of double strand and a naked single strand. Because of these factors, triple strand formation between a recA-coated, single stranded ODN and an homologous double strand occurs at a reaction rate that exceeds by 1 or 2 orders of magnitude the calculated rate of spontaneous renaturation of
complementary single strands under standard hybridization conditions.
The present invention involves combination of (1) an ODN that is homologous to a portion of one target DNA duplex strand and complementary to the analogous portion of the other target DNA duplex strand; and (2) enzyme-catalyzed triple strand formation to achieve inactivation of a target DNA sequence. In a preferred embodiment, the ODN has a crosslinking moiety covalently attached thereto. Upon hybridization to the target DNA sequence cross-linkage of the third strand ODN (the anti- gene ODN) to both parental duplex strands inactivates the target DNA sequence. Depending on the ODN, the
crosslinking moiety, the target DNA sequence and the environment and characteristics of the target DNA
sequence, inactivation of the parental duplex strands may be permanent. Thus, a single administration of one or more anti-gene ODNs may abolish the expression of
integrated retroviral genomes, of episomal herpesvirus genomes, or of mutant oncogenes.
Following triple strand formation and covalentcrosslinkage, the modified target DNA no longer supports replication or transcription. Unlike all other lesions in DNA, however, this modification is not repairable. Normally, crosslinked DNA is repaired by a combination of excision repair and homologous recombination. With crosslinked triple strand complexes, however, there will be no undamaged copies of the targeted gene to
participate in recombination. By analogy with
procaryotic models, the eucaryotic cell may attempt to use a misrepair (or SOS) pathway wherein the crosslink will be removed, but at the expense of mutagenesis. In such case, gene function would be irreversibly silenced by the resultant mutations.
The use of recombination enzymes in combination- with anti-gene ODNs significantly enhances the efficiency with which the single strand ODN "finds" its
complementary target DNA sequence. Accordingly, the efficiency of triple strand formation is greatly
increased when the anti-gene ODN is combined with a recombination enzyme (for instance, in a nucleoprotein complex).
Within the present invention, suitable target DNA sequences include structural genes and both up-stream and down-stream regulatory control sequences. These regulatory sequences may be involved in either
transcription or replication. The anti-gene ODN will be determined and designed according to the target DNA sequence chosen for alteration of function, and will have a sequence complementary to one of the two strands of the chosen target DNA.
Crosslinkers suitable for use within the invention include photochemical agents, such as
psoralens, and chemical crosslinking agents. Preferred chemical crosslinking agents include those described in Section A., above; electrophilic moieties attached to the 3' and/or 5' termini of the ODN; and masked electrophilic moieties attached to the ODN. Photo-crosslinking agentsmay be useful for topical or extracorporeal applications, for targets accessible to light exposure, as well as for in vitro use. Chemical crosslinkers may be used without limitation and are particularly preferred for use within the claimed invention.
Preferred recombination enzymes include
procaryotic and eucaryotic recombination enzymes, such as recA, human recombinase and Drosophila recombinase, with human recombinase particularly preferred.
In a particularly preferred embodiment, an anti-gene ODN is administered to a cell or a host, and upon entry to a target cell nucleus, the anti-gene ODN combines with recombination enzymes present within the nucleus. In an alternative embodiment, the anti-gene ODN and recombination enzyme are combined ex vivo and then administered to a cell or a host as a nucleoprotein filament. In this embodiment, it may be advantageous to administer the nucleoprotein filament in a liposome.
The following examples are provided to illustrate the present invention without limiting same. "RT" means room temperature.
General
Thin layer chromatography was performed on silica gel 60 F 254 plates (Analtech) using the followingsolvent mixtures: A- 90% methylene chloride: 10% methanol; B- 50% ethyl acetate:50% hexanes; C- 70% ethyl acetate: 10% methanol: 10% water:10% acetone; D- 50% ether:50% hexanes. Flash chromatography was performed using 60 F 254 silica (Merck). Oligonucleotides were synthesized onan Applied Biosystems Model 380B Synthesizer. Oligonucleotides were isotopically labeled using T4
Polynucleotide kinase (BRL) and τ-32P-ATP (New England Nuclear). EXAMPLE 1:
6-(Tritylamino)caproic Acid.
6-Aminocaproic acid (26 g, 0.2 mole) was dissolved in dichloromethane (200 mL) by the addition of triethylamine (100 mL). Trityl chloride (120 g, 0.45 mole) was added and the solution stirred for 36 hr. The resulting solution was extracted with 1NHCl and the organic layer evaporated to dryness. The residue was suspended in 2-propanol/1N NaOH (300 mL/100 mL) and refluxed for 3 hr. The solution was evaporated to a thick syrup and added to dichloromethane (500 mL). Water was added and acidified. The phases were separated, and the organic layer dried over sodium sulfate and evaporated to dryness. The residue was suspended in hot 2- propanol, cooled, and filtered to give 43.5 g (58%) of 6- (tritylamino) caproic acid, useful as an intermediate compound. EXAMPLE 2:
5-(Tritylamino)pentylhydroxymethylenemalononitrile.
To a dichloromethane solution of 6-(tritylamino)- caproic acid (20.0 g, 53 mmole) and triethylamine (20 mL) in an ice bath was added dropwise over 30 min isobutyl- chloroformate (8.3 mL, 64 mmole). After the mixture was stirred for 2 hr in an ice bath, freshly distilled malononitrile (4.2 g, 64 mmole) was added all at once. The solution was stirred for 2 hr in an ice bath and for2 hr at RT. The dichloromethane solution was washed with ice cold 2N HCl (300 mL) and the biphasic mixture was filtered to remove product that precipitated (13.2 g). The phases were separated and the organic layer dried and evaporated to a thick syrup. The syrup was covered with dichloromethane and on standing deposited fine crystals of product. The crystals were filtered and dried to give 6.3 g for a total yield of 19.5 g (87%) of the product, which is useful as an intermediate.
EXAMPLE 3 :
5-(Tritylamino)pentylmethoxymethylenemalononitrile.
A suspension of the malononitrile of Example 2 (13 g, 31 mmole) in ether/dichloromethane (900 mL/100 mL), cooled in an ice bath, was treated with a freshly prepared ethereal solution of diazomethane (from 50 mmole of Diazald® (Aldrich Chemical Company)). The solution was stirred for 6 hr and then neutralized with acetic acid (10 mL). The solution was evaporated to dryness and the residue chromatographed on silica gel using dichloromethane/acetone (4/1) as the eluent. Fractions
containing product were pooled and evaporated to a syrup. The syrup was triturated with dichloromethane to induce crystallization. The crystals were filtered and dried to give 8.3 g (61%) of chromatographically pure product, useful as an intermediate compound.
EXAMPLE 4:
5-Amino-3-[(5-tritylamino)pentyl]pyrazole-4-carbonitrile.
To a methanol solution (100 mL) of the product of Example 3 (7.0 g, 16 mmole) in an ice bath was added hydrazine monohydrate (7.8 mL, 160 mmole) dropwise over is min. After stirring for 30 min in an ice bath, the solution was evaporated to dryness. The residue was suspended in cold methanol and filtered to give 7.1 g (100%) of 5-amino-3-[(5-tritylamino)pentyl]pyrazole-4- carbonitrile, useful as an intermediate, after drying.An analytical sample was prepared by recrystallization from water. EXAMPLE 5 :
5-Amino-1-(2-deoxy-3,5-di-o-toluoyl-β-D-erythropentofuranosyl)-3-[(5-tritylamino)pentyl]pyrazole-4-carbonitrile. An ice cold solution of the carbonitrile from
Example 4 (3.5 g, 8 mmole) was treated with sodium hydride and stirred for 30 min at 0-4°C. 1-Chloro-1,2- dideoxy-3,5-di-O-toluoylribofuranose was added and the solution stirred for 1 hr at 0-4°C. The solution was poured into a saturated solution of sodium bicarbonate and extracted with dichloromethane. The organic layer was dried over sodium sulfate and evaporated to dryness. The residue was flash chromatographed on silica gel using toluene/ethyl acetate (5/1) as eluent. Two major
products were isolated and identified as the N-1 and N-2 isomers in 57% (3.6 g) and 20% (1.2 g) N-1 and N-2 yields, respectively. Approximately 1 g of a mixture of N-1 and N-2 isomers was also collected. Overall yield of glycosylated material was 5.8 g (92%). The N-1 isomer, 5-amino-1-(2-deoxy-3,5-di-O-toluoyl-β-D-erythropentofuranosyl)-3-[(5-tritylamino)pentyl]pyrazole-4-carbonitrile, was used without further purification in Example 6.
EXAMPLE 6:
1-(2-Deoxy-β-D-erythropentofuranosyl)-3-[5-(tritylamino)- pentyl]pyrazolo[3,4-d]pyrimidin-4-amine.
To a toluene (100 mL) solution of the pyrazole- 4-carbonitrile of Example 5 (3.5 g, 4.4 mmole) was added diethoxymethyl acetate (1.1 mL, 6.7 mmole). The solution was kept at 80-90°C for 5 hr and then evaporated to a syrup. The syrup was dissolved in dichloromethane (10 mL) and added to ice cold methanolic ammonia (100 mL) in a glass pressure bottle. After two days at RT the
contents of the bottle were evaporated to dryness. The residue was dissolved in methanol and adjusted to pH 8 with freshly prepared sodium methoxide to complete the deprotection. After stirring overnight the solution was treated with Dowex®-50 H+ resin, filtered, and evaporated to dryness. The residue was chromatographed on silica gel using acetone/hexane (3/2) as eluent to give 2.0 g (77%) of analytically pure product.
EXAMPLE 7:
1-(2-Deoxy-β-D-erythropentofuranosyl)-3-[5-(tritylamino)- pentyl]pyrazolo[3,4-d]pyrimidin-4-amine 5'-monophosphate.
To an ice cold solution of the pyrazolo- pyrimidin-4-amine of Example 6 (250 mg, 0.43 mmole) in trimethyl phosphate (5 mL) was added phosphoryl chloride (50 μL) and the solution was kept at 0-4°C. The reaction was monitored by reversed phase HPLC using a linear gradient from 0 to 100% acetonitrile in water over 25 min. After stirring for 5 hr, an additional aliquot of phosphoryl chloride (25 μL) was added and the solution was stirred another 30 min. The solution was poured into 0.1M ammonium bicarbonate and kept in the cold overnight. The solution was then extracted with ether and the aqueous layer evaporated to dryness. The residue was dissolved in water (5 mL) and purified by reversed phase HPLC using a 22mm X 50cm C18 column. The column was equilibrated in water and eluted with a gradient of 0 to 100% acetonitrile over 20 min. Fractions containing the desired material were pooled and lyophilized to give 160 mg (56%) of chromatographically pure nucleotide. EXAMPLE 8 :
1-(2-Deoxy-β-D-erythropentofuranosyl)-3-{5-[(6-biotin- amido)hexanamido]pentyl}pyrazolo[3,4-d]pyrimidin-4-amine 5'-monophosphate. An ethanol solution (10 mL) of the nucleotide of Example 7, palladium hydroxide on carbon (50 mg), and cyclohexadiene (1 mL) was refluxed for 3 days, filtered. and evaporated to dryness. The residue was washed with dichloromethane, dissolved in DMF (1.5 mL) containing triethylamine (100 mL), and treated with N-hydroxysuccinimidyl biotinylaminocaproate (50 mg). After stirring overnight an additional amount of N-hydroxysuccinimidyl 6-biotinamidocaproate (50 mg) was added and the solution was stirred for 18 hr. The reaction mixture was evaporated to dryness and chromatographed following the procedure in Example 7. Fractions were pooled and lyophilized to give 80 mg of chromatographically pure biotinamido-substituted nucleotide.
EXAMPLE 9:
1-(2-Deoxy-β-D-erythropentofuranosyl)-3-[5-(6-biotin- amido)hexanamidopentyl]pyrazolo[3,4-d]pyrimidin-4-amine 5'-triphosphate.
The monophosphate of Example 8 (80 mg, ca. 0.1 mmole) was dissolved in DMF with the addition of
triethylamine (14 μL). Carbonyldiimidazole (81 mg, 0.5 mmole) was added and the. solution stirred at RT for 18 hr. The solution was treated with methanol (40 μL) , and after stirring for 30 min tributylammonium pyrophosphate (0.5 g in 0.5 mL-DMF) was added. After stirring for 24hr another aliquot of tributylammonium pyrophosphate was added and the solution was stirred overnight. The reaction mixture was evaporated to dryness and chromatographed following the procedure in Example 8. Two products were collected and were each separately treatedwith cone, ammonium hydroxide (1 mL) for 18 hr at 55°C. UV and HPLC analysis indicated that both products were identical after ammonia treatment and were pooled and lyophilized to give 35.2 mg of nucleoside triphosphate. EXAMPLE 10:
NICK-TRANSLATION REACTION
The triphosphate of Example 9 was incorporated into pHPV-16 using the nick tanslation protocol of Langer et al. (supra). The probe prepared with the triphosphate of Example 9 was compared with probe prepared using commercially available bio-ll-dUTP (Sigma Chemical Co). No significant differences could be observed in both a filter hybridization and in in situ smears.
More specifically, the procedure involved the following materials and steps:
Materials:
DNase (ICN Biomedicals) - 4μg/mL
DNA polymerase 1 (U.S. Biochemicals) - 8
U/mL
pHPV - 16 - 2.16 mg/mL which is a
plasmid containing the genomic sequence of human papillomavirus type 16.
10X-DP - 1M Tris,pH7.5(20mL); 0.5M
DTT(80 mL); 1M MgCl2(2.8 mL);
H2O(17mL)
Nucleotides - Mix A - 2mM each dGTP,
dCTP, TTP (Pharmacia)
Mix U - 2mM each dGTP, dCTP, dATP
Bio-ll-dUTP - 1.0 mg/mL (BRL) Bio-12-dAPPTP - 1.0 mg/mL
Steps:
To an ice cold mixture of 10X-DP (4 mL), pHPV-16 (2 mL), nucleotide mix A (6 mL), Bio-12-dAPPTP (2 mL), and H2O (20 mL) was added DNase (1 mL) and DNA polymerase 1 (2.4 mL). The reaction mixture was
incubated at 16°C for 1 hr. The procedure was repeated using Bio-11-dUTP and nucleotide mix U in place of Bio- 12-dAPPTP (comprising the triphosphate of Example 9) and nucleotide mix A.
Nucleic acid was isolated by ethanol precipitation and hybridized to pHPV-16 slotted onto nitrocellulose. The hybridized biotinylated probe was visualized by a streptavidin-alkaline phosphatase conjugate with BCIP/NBT substrate. Probe prepared using either biotinylated nucleotide gave identical signals. The probes were also tested in an in situ format on cervical smears and showed no qualitative differences in signal and background.
EXAMPLE 11:
5-Amino-3-[(5-tritylamino)pentyl]pyrazole-4-carboxamide.
Following the procedure of Example 2, except that cyanoacetamide is used instead of malononitrile, 5- (tritylamino)pentylhydroxymethylenecyanoacetamide is0prepared from 6- (tritylamino) caproic acid. This is then treated with diazomethane to give the methoxy derivative, following the procedures of Example 3, which is then reacted with hydrazine monohydrate, as in Example 4, to give 5-amino-3 [(5-tritylamino)pentyl]pyrazole-4- carboxamide.
EXAMPLE 12:
4-Hydroxy-6-methylthio-3-[(5-tritylamino)pentyl]pyrazolo-
[3,4-d]pyrimidine.
The carboxamide from Example 11 is reacted with potassium ethyl xanthate and ethanol at an elevated temperature to give the potassium salt of 4-hydroxypyrazolo[3,4-d]pyrimidine-6-thiol. This salt is then reacted with iodomethane to give 4-hydroxy-6-methylthio-3-[(5- tritylamino)pentyl]pyrazolo[3,4-d]pyrimidine. EXAMPLE 13 :
1-(2-Deoxy-β-D-erythropentofuranosyl)-4-hydroxy-3-[5-
(tritylamino)pentyl]pyrazolo[3,4-d]pyrimidin-6-amine. Following the procedure of Example 5, the pyrazolopyrimidine of Example 12 is treated with sodium hydride and reacted with l-chloro-l,2-dideoxy-3,5-di-O- toluoylribofuranose. The resulting compound is reacted with MCPBA and with methanolic ammonia, and the toluoyl protecting groups are removed to give the product.
EXAMPLE 14:
1-(2-Deoxy-β-D-erythropentofuranosyl)-4-hydroxy-3-[5-(6- biotinamido) hexanamidopentyl]pyrazolo[3,4-d]pyrimidin-6- aX52ne 5'-monophosphate.
Following the procedure of Example 1 , the pyrazolopyrimidine of Example 13 is reacted with
phosphoryl chloride to give the corresponding 5'-mono- phosphate.
Following the procedure of Example 8, the above 5'-monophosphate is reacted with palladium/carbon and cyclohexadiene, and the residue is reacted with N- hydrpxysuccinimidyl biotinylaminocaproate to give 1-(2- deoxy-β-D-erythropentofuranosyl)-4-hydroxy-3-[5-(6- biotinamido) hexanamidopentyl]pyrazolo[3,4-d]pyrimidin-6- amine 5'-monophosphate.
EXAMPLE 15:
1-(2-Deoxy-β-D-erythropentofuranosyl)-4-hydroxy-3-[5-(6- biotinamido)hexanamidopentyl]pyrazolo[3,4-d]pyrimidin-6- amine 5'-triphosphate.
Following the procedure of Example 9, the 5'-monophosphate of Example 14 is treated with carbonyldiimidazole and then reacted with tributylammonium pyrophosphate to give the corresponding 5'-t-iphosphate. EXAMPLE 16 :
1-(2-Deoxy-β-D-erythropentofuranosyl)-3-[5-(tritylamino)- pentyl]pyrazolo[3,4-d]pyrimidine-4-benzoylamine. 1-(2-Deoxy-β-D-erythropentofuranosyl)-3-[5-
(tritylamino)penty-]pyrazolo[3,4-d]pyrimidine-4-amine from Example 6 is reacted with benzoyl chloride and pyridine to give 1-(2-deoxy-3,5-di-O-benzoyl-β-D-erythropentofuranosyl)-3-[5-(tritylamino)pentyl]pyrazolo[3,4-d]- pyrimidine-4-dibenzoylamine. This is treated with aqueous sodium hydroxide to partially deprotect the compound, giving 1-(2-deoxy-β-D-erythropentofuranosyl)-3- [5-(tritylamino)pentyl]pyrazolo[3,4-d]pyrimidine-4- benzoylamine.
EXAMPLE 17:
1-(2-Deoxy-β-D-erythropentofuranosyl)-3-[5-(trifluoro- acetamido)pentyl]pyrazolo[3,4-d]pyrimidine-4-benzoyl- amine.
Following the procedure of Example 8, the benzoylamine of Example 16 is treated with palladium hydroxide on carbon and then with trifluoroacetic
anhydride to give 1-(2-deoxy-β-D-erythropentofuranosyl)- 3-[5-(trifluoroacetamido)pentyl]pyrazolo[3,4-d]pyrimi- dine-4-benzoylamine.
EXAMPLE 18:
1-(2-Deoxy-5-O-dimethoxytrityl-β-D-erythropentofurano- syl)-3-[5-(trifluoroacetamido)pentyl]pyrazolo[3,4-d]- pyrimidine-4-benzoylamine 3'-O-(N,N-diisoproρyl)phosphoramidite cyanoethyl ester.
The compound of Example 17 is reacted withdimethoxytrityl chloride and pyridine to give the
corresponding 5'-dimethoxytrityl compound. This compound is then reacted with cyanoethyl chloro-N,N-diisopropyl- phosphoramidite (according to the method of Sinha et al., Nucleic Acids Res., 12 : 4539 (1984)) to give the 3'-O- activated nucleoside. EXAMPLE 19:
5-(4-Phthalimidobut-1-yn-1-yl)-2'-deoxyuridine.
5-Iodo-2'-deoxyuridine (354 mg, 1 mmol) was dissolved in 10 mL of dimethylformamide. Cuprous iodide (76 mg, 0.4 mmol), tetrakis(triphenylphosphine)palladium(0) (230 mg, 0.2 mmol), and triethylamine (200 mg, 2.0 mmol) were added. 4-Phthalimidobut-1-yne (300 mg, 1.5 mmol) was added all at once and the reaction kept at 60°C for three hours. The clear yellow reaction was then evaporated and methylene chloride was added. Scratching of the flask induced crystallization of nearly all of the product which was filtered and recrystallized from 95% ethanol to give 335 mg (78%) of title compound as fine, feathery needles.
EXAMPLE 20:
5-(4-Phthalimidobut-1-yl)-2'-deoxyuridine. 1.00 Gram of deoxyridine from Example 19 was dissolved in 95% EtOH and about 3 g of neutral Raney nickel was added. After 48 hours, the catalyst was removed by cautious filtration and the filtrate was evaporated to a solid which was recrystallized from methanol-water to give 960 mg (97%) of the title
compound.
EXAMPLE 21:
5-(3-Iodoacetamidopropyl)-2'-deoxyuridine. 5-(3-Trifluoroacetamidoprop-1-yl)-2'-deoxyuridine (0.3 mmol) is treated with ammonia and then with N-hydroxysuccinimidyl α-iodoacetate (0.5 mmol). The reaction mixture is evaporated to dryness and purified by chromatography to give 5-(3-iodoacetamidopropyl)-2'- deoxyuridine. EXAMPLE 22:
5-(4-(4-Bromobutyramido)butyl)-2'-deoxyuridine.
Following the procedure of Example 21, 5-(4- phthalimidobut-1-yl)-2'-deoxyuridine, from Example 20, is treated with ammonia and then with N-hydroxysuccinimidyl 4-bromobutyrate to give 5-(4-(4-bromobutyramido)butyl)- 2'-deoxyuridine.
Preparation of Synthetic Oligonucleotides
EXAMPLE 23:
Phosphoramidite Preparation and DNA Synthesis.
Nucleosides were 5'-dimethoxytritylated, following known procedures, to give around 85% yield, and the 3'-phosphoramidite was made using diisopropylamino β- cyanoethylchlorophosphite (as described in
"Oligonucleotide Synthesis: A Practical Approach", supra) with diisopropylethylamine in methylene chloride. The phosphoramidite was made into a 0.2N solution in
acetonitrile and placed on the automated DNA synthesizer. Incorporation of these new and modified phosphoramidites gave incorporation similar to ordinary phosphoramidites (97-99% as judged by assay of the trityl color releasedby UV.)
Oligonucleotides were removed from the DNA synthesizer in tritylated form and deblocked using 30% ammonia at 55°C for 6 hours. Ten μL of 0.5M sodium bicarbonate was added to prevent acidification duringconcentration. The oligonucleotide was evaporated to dryness under vacuum and redissolved in 1.0 mL water.
The oligonucleotides were purified by HPLC using 15-55% acetonitrile in 0.1N triethylammonium acetate over 20 minutes. Unsubstituted oligonucleotides came off at 10 minutes; amino derivatives took 11-12 minutes. The desired oligonucleotide was collected and evaporated to dryness, then it was redissolved in 80% aqueous acetic acid for 90 minutes to remove the trityl group.
Desalting was accomplished with a G25 Sephadex column and appropriate fractions were taken. The fractions were concentrated, brought to a specific volume, dilution Oreading taken to ascertain overall yield and an
analytical HPLC done to assure purity. Oligonucleotides were frozen at -20°C until use.
Following the above procedures, the nucleoside 5-(3-trifluoroacetamidoprop-1-yl)-2'-deoxyuridine was converted to the 5'-O-dimethoxytrityl-3'-(N,N- diisopropyl) phosphoramidite cyanoethyl ester derivative. This was added to a DNA synthesizer and the following 14- mer oligonucleotide sequence was prepared:
3' -CT TCC U1TG TAG GTC-5 '
0where U1 is 5-(3-aminoprop-1-yl)-2'-deoxyuridine (oligo A).
In the same manner, 5-(4-phthalimidobut-1-yl)- 2 ' -deoxyuridine was converted to the 5'-O-dimethoxytrityl-3'-(N,N=diisopropyl)phosphoramidite cyanoethyl ester derivative and added to a DNA synthesizer to prepare the above 14-mer oligonucleotide sequence where U1 is 5-(4-aminobut-1-yl)-2'-deoxyuridine (oligo C).
A corresponding 14-mer oligonucleotide was also prepared where U1 is the unmodified deoxyuridine.
EXAMPLE 24:
Derivatization of Oligonucleotides.
In general, to add the crosslinking arm to an aminoalkyloligonucleotide, a solution of 10 μg of the aminoalkyloligonucleotide and a 100X molar excess of n- hydroxysuccinimide haloacylate such as α-haloacetate or 4-halobutyrate in 10 μL of 0.1 M borate buffer, pH 8.5, was incubated at ambient temperature for 30 min. in the dark. The entire reaction was passed over a NAP-10 column equilibrated with and eluted with distilled water. Appropriate fractions based on UV absorbance were
combined and the concentration was determined spectrophotometrically.
Introduction of the haloacyl moiety was
examined by HPLC. A Zorbax® oligonucleotide column
(Dupont) eluted with a 20 minute gradient of 60% to 80% B composed of: A (20% acetonitrile: 80% 0.02 N NaH2PO4) and B (1.2 N NaCl in 20% acetonitrile: 80% 0.02 N NaH2PO4).
The presence of a reactive α-haloacyl moiety was indicated by return of the retention time of the α-haloacylamidoalkyl oligonucleotide to the corresponding amino- alkyl oligonucleotide after exposure to IN cysteamine. Introduction of cysteamine created equivalent charge patterns between the aminoalkyl oligonucleotide and the α-haloacylamido oligonucleotide.
Following the above procedure, the 14-mer oligonucleotide:
3'-CT TCC U1TG TAG GTC-5'
where U1 is 5-(3-aminoprop-1-yl)-2'-deoxyuridine (oligo A, Example 23), was reacted with n-hydroxysuccinimideα-iodoacetate to give the above 14-mer oligonucleotide where U1 is 5-(3-iodoacetamidoprop-1-yl)-2'-deoxyuridine (oligo B).
Oligo A and oligo B, as well as the above 14- mer where U1 is the unmodified deoxyuridine were resolved in the Zorbax column, all of identical sequence, with the following retention times: unmodified 14-mer, 9.31 min; aminopropyl 14-mer (oligo A), 7.36 min; and iodoacetamidopropyl 14-mer (oligo B), 10.09 min.
In the same manner, the aminopropyl 14-mer
(oligo A) was reacted with N-hydroxysuccinimide 4-bromobutyrate to give the 14-mer where U1 is 5-(3-(4-bromobutyramido)prop-1-yl)-2'-deoxyuridine. Also, the aminobutyl 14-mer (oligo C, Example 23) was reacted with either N-hydroxysuccinimide α-iodo- acetate or N-hydroxysuccinimide 4-bromobutyrate to give the 14-mer where U1 is 5-(4-iodoacetamidobut-1-yl)-2'- deoxyuridine or 5-(4-(4-bromobutyramido)but-1-yl)-2'- deoxyuridine, respectively.
Assays
EXAMPLE 25:
Assay of Crosslinking Reaction.
The reaction of crosslinking a DNA probe to a target nucleic acid sequence contained 1 μg of haloacyl- amidoalkyl probe and 10 ng of 32P-labeled cordycepintailed target in 200 μL of 0.1 M Tris, pH 8.0, and 0.9 M NaCl incubated at 20° or 30°C. Aliquots were removed at 24- or 72-hour intervals and diluted in 20 μL of 10 mM cysteamine to quench the haloacylamido group. These0solutions were stored at RT, and 1 μL was used for analysis by denaturing polyacrylamide gel electrophoresis (PAGE).
Following the above procedure, two model oligonucleotide sequences were utilized to evaluate the crosslinkage petential of the modified probe to its complement. The sequences, derived from human papilloma- virus (HPV) or human cytomegalovirus (CMV), are shown below:
HPV System:
Figure imgf000060_0001
Target : 5 ' -AGA CAG CAC AGA ATT CGA AGG AAC ATC CAG-3 '
Probe : 3 ' -CT TCC UTG TAG GTC-5 ' CMV System:
Figure imgf000061_0001
Target: 5'-ACC GTC CTT GAC ACG ATG GAC TCC-3'
Probe: 3'-GAA CTG TGC UAC CTC-5' U = 5-[3-(α-iodoacetamido)- or 3-(4-bromo- butyramido)-propyl]-2'-deoxyuridine, or U = 5-[3-(α-iodoacetamido)- or 4-(4- bromobutyramido)-butyl]-2'-deoxyuridine.
The target for HPV is a 30-mer, and for CMV it is a 24-mer. The crosslinking probes were a 14-mer for HPV and two 15-mers for CMV. Each probe contained a single modified deoxyuridine designated as U in the sequences above.
Results of the reaction of HPV target with a limiting amount of crosslinking probe containing a
5-(3-iodoacetamidopropyl) sidearm are shown in Figure 2. Analysis of the cleavage pattern on a denaturing PAGE gel showed the loss of the crosslinked hybrid with the concomitant appearance of a discrete low molecular weight (band. The intensity of this band was dependent upon the extent of crosslinkage in the initial reaction. The localization of signal into two discrete bands on the gel strongly argues that no non-seguence-directed alkylation of either target or probe strands had occurred (including intramolecular probe alkylation).
Comparison to an authentic 15-mer run in an adjacent lane suggested that the major cleaved fragment is a 9-mer. Upon close examination of the original auto- radiogram, a slower moving band of very weak intensity was visible. This pattern would be consistent with major alkylation at G-21 and minor alkylation at G-20. An examination of a Dreiding model of the crosslinkable HPV hybrid shows that the 5-(3-iodoacetamidopropyl) sidearm can contact the G-21 residue of the target strand with only minor distortion of the helix. If alkylation occurs predominately at a guano- sine on the target strand located two units on the 5' side of the modified-deoxyuridine base pair, the CMV sequence should not react. This result was in fact observed. The absence of reaction with CMV further supports the specificity of crosslinking scheme of the invention.
EXAMPLE 26:
Time and Temperature Dependence.
Time and temperature dependence studies were carried out with the HPV system of Example 25 where U is 5-(3-iodoacetamidoprop-1-yl)-2'-deoxyuridine. The target was 32P-labeled by cordycepin tailing with terminal transferase (Maniatis et al., "Molecular Cloning - A Laboratory Manual", Cold Spring Harbor Laboratory, 1982, p. 239) and incubated with excess probe in a pH 8.0 Tris buffer at either 20° or 30°C. Aliquots were removed after 0, 24, or 72 hours incubation, quenched with an equivalent volume of 10 mM mercaptoethylamine (which reacts with the iodoacetamide), and stored at RT for subsequent analysis by denaturing or non-denaturing PAGE.
Crosslinkage of the hybrid, which was monitored by denaturing PAGE, was evident for the 24 and 72 hour time points at both temperatures (see Figure 3). The amount of crosslinked hybrid increased with both temperature and time. Approximately 20% of the hybrid was crosslinked after 72 hours incubation at 30°C.
Separate experiments at a range of temperatures indicated that the half-life for crosslinking at 37°C is approximately 2 days, and that the reaction is complete after 24 hours at 58°C. This time-dependent reaction implies that the iodoacetamido moiety does not hydrolyze or react with the buffer. The increased reaction rate at higher temperature indicates that the hybrid is maintained, and subsequently the rate of alkylation shows the expected increase with temperature.
EXAMPLE 27:
Site Specificity of Alkylation.
To elucidate the site specificity of alkylation, the crosslinked HPV hybrid of Example 25 (where U is 5-(3-iodoacetamidoprop-1-yl)-2'-deoxyuridine) was subjected to a 10% piperidine solution at 90°C for 60 minutes. As shown by Maxam et al. (Proc. Natl. Acad.
Sci. USA. 74:560 (1977), this treatment quantitatively cleaves the target strand 3'- to the site of alkylation. The resulting data indicated that the alkylation of the second guanine above the crosslinker-modified base pair (i.e., the guanine above the target base) was the
exclusive action observed, indicating that the crosslinking reaction in the HPV model system is remarkably specific.
EXAMPLE 28 :
Chemically-Modified Crosslinkable ODNs.
Psoralen-modified, photo-crosslinkable ODNs were prepared using a photochemical procedure (REF??). Briefly, ODN was hybridized to a complementary DNA sequence and irradiated at 360 nm in the presence of 4'-hydroxymethy-4,5',8-trimethylpsoralen (HMT), thereby forming a sequence-specific, interstrand crosslink at a unique 5'-TpA-3' sequence. Following partial
photoreversal of the crosslink by brief exposure to 260 nm light, the HMT furan-side monoadducted ODN was isolated by denaturing PAGE.
Alternatively, the attachment of a psoralen derivative to an ODN may be accomplished by chemical conjugation. These synthetic schemes may be used to obtain ODNs that are coupled to a 4,5',8-trimethyl- psoralen (TMP) via a linker arm that spans the 5' terminus of the ODN and the 4' position of the psoralen. In one approach, a modified TMP is reacted with a suitably 5'-activated, deblocked ODN (B.L. Lee et al., Biochem. 27: 3197-3202, 1988). In a second approach, a TMP phosphoramidite linker compound is added in the last cycle of solid phase synthesis (U. Pieles and U.
Englisch, Nucl. Acids Res. 17:285-99, 1989). Similar adducts may be prepared using a thiolated derivative of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) (J. Teare and P. Wollenzien, Nucl. Acids Res. 17: 3359-72, 1989). In a further alternative, a TMP-modified deoxyadenosine phosphoramidite may be inserted at any position in an ODN during solid phase synthesis (U, Pieles et al., Nucl.
Acids Res. 17:8967-78, 1989). In this synthon, TMP is modified at its 4' position with linker which is attached to the C8 position of deoxyadenosine.
{Analogous w/ chemical XLers of app'n) EXAMPLE 29 :
Use of Chemically Modified ODNs to Target Double Stranded
DNA. A prototype, psoralenated ODN that can efficiently crosslink to both Watson and Crick strands when complexed to homologous DNA in a recA-stabilized triple strand is employed. The ODN 5'-modified with a psoralenated tail is synthesized so as to optimize photo- crosslinkage within a 5'-TpA-3' sequence flanking the recA-stabilized triplex. Briefly, a psoralenated ODN in the presence of a recombination enzyme is used to
introduce a sequence-specific crosslink the complementary site in a double stranded DNA target. The ODN, which Hoogstein base pairs to the DNA target, is modified with a 5-hydroxypsoralen moiety or a 4,5',8-trimethylpsoralen moiety at its 5' terminus. By linking the psoralen through the 5' position, the furocourmain is able to readily intercalate into a double stranded 5'-TpA-3' sequence immediately adjacent to the triple strand. Both the furan and pyrone rings are properly positioned to photoreact with thymidines. The length of the
psoralenated tail may be altered to obtain optimal intercalation of the psoralen into the duplex-triple strand junction.

Claims

WHAT IS CLAIMED IS:
1. A method for altering gene function, comprising:
attaching a crosslinking agent to an oligonucleotide (ODN) that is homologous to one strand and complementary to the other strand of target double stranded DNA, wherein the target double stranded DNA corresponds to a structural gene, a regulatory sequence that affects gene function, or a portion thereof, thereby forming a crosslinking ODN;
contacting the crosslinking ODN and a recombination enzyme, thereby forming a
nucleoprotein filament;
combining the nucleoprotein filament and the target double stranded DNA for a time period
sufficient for formation of a triple strand complex; and
crosslinking the triple strand complex, thereby altering gene function.
2. The method of Claim 1 wherein the crosslinking agent is a nucleoside crosslinking agent.
3. The method of Claim 2 wherein the nucleoside crosslinking agent is represented by the following formula:
R1 - B - (CH2)q- (Y)r - (CH2)m- A' (I') wherein,
R1 is hydrogen, or a sugar moiety or analog thereof optionally substituted at its 3' or its 5' position with a phosphorus derivative attached to the sugar moiety by an oxygen and including groups Q1, Q2 and Q3, or with a reactive precursor thereof suitable for nucleotide bond formation;
Q1 is hydroxy, phosphate or diphosphate;
Q2 is =O or =S;
Q3 is CH2-R', S-R', O-R', or N-R'R"; each of R' and R" is independently hydrogen or C1-6alkyl;
B is a nucleic acid base or analog thereof that is a component of an oligonucleotide;
Y is a functional linking group;
each of m and q is independently 0 to 8, inclusive;
r is 0 or 1; and
A' is a leaving group.
4. A chemical crosslinking anti-gene nucleoprotein filament comprising:
a chemical crosslinking agent covalently linked to an oligonucleotide (ODN) that is homologous to one strand and complementary to the other strand of target double stranded DNA that corresponds to a structural gene, a regulatory sequence that affects gene function, or a portion thereof; and
a recombination enzyme non-covalently associated with the ODN.
5. The chemical crosslinking anti-gene
nucleoprotein filament of Claim 4 wherein the chemical crosslinking agent is a nucleoside crosslinking agent.
6. The chemical crosslinking anti-gene nucleoprotein filament of Claim 5 wherein the nucleoside crosslinking agent is represented by the following formula:
R1 - B - (CH2)q- (Y)r - (CH2)m - A' (I') wherein,
R1 is hydrogen, or a sugar moiety or analog thereof optionally substituted at its 3' or its 5' position with a phosphorus derivative attached to the sugar moiety by an oxygen and including groups Q1, Q2 and Q3, or with a reactive precursor thereof suitable for nucleotide bond formation;
Q1 is hydroxy, phosphate or diphosphate;
Q2 is =O or =S;
Q3 is CH2-R', S-R', O-R', or N-R'R"; each of R' and R" is independently hydrogen or C1-6alkyl;
B is a nucleic acid base or analog thereof that is a component of an oligonucleotide;
Y is a functional linking group;
each of m and q is independently 0 to 8, inclusive;
r is 0 or 1; and
A' is a leaving group.
PCT/US1992/007101 1991-08-19 1992-08-19 Cross-linking oligonucleotides for enzyme-mediated triple strand formation WO1993003736A1 (en)

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EP92918930A EP0661979A4 (en) 1991-08-21 1992-08-19 Cross-linking oligonucleotides for enzyme-mediated triple strand formation.
JP5504623A JPH06509945A (en) 1991-08-19 1992-08-19 Crosslinkable oligonucleotides for enzyme-mediated triplex formation

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US748,138 1991-08-19
US74813891A 1991-08-21 1991-08-21

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WO1994017092A1 (en) * 1993-01-26 1994-08-04 Microprobe Corporation Bifunctional crosslinking oligonucleotides adapted for linking to a desired gene sequence of invading organism or cell
WO1994024144A2 (en) * 1993-04-19 1994-10-27 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified purines
EP0635023A1 (en) * 1992-03-05 1995-01-25 Isis Pharmaceuticals, Inc. Covalently cross-linked oligonucleotides
WO1996040711A1 (en) * 1995-06-07 1996-12-19 Microprobe Corporation Cross-linking oligonucleotides
US5594121A (en) * 1991-11-07 1997-01-14 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified purines
US5659022A (en) * 1996-01-05 1997-08-19 Epoch Pharmaceuticals, Inc. Oligonucleotide-cyclopropapyrroloindole conjugates as sequence specific hybridization and crosslinking agents for nucleic acids
US5763240A (en) * 1992-04-24 1998-06-09 Sri International In vivo homologous sequence targeting in eukaryotic cells
US5824796A (en) * 1988-09-28 1998-10-20 Epoch Pharmaceuticals, Inc. Cross-linking oligonucleotides
US5912340A (en) * 1995-10-04 1999-06-15 Epoch Pharmaceuticals, Inc. Selective binding complementary oligonucleotides
US5948653A (en) * 1997-03-21 1999-09-07 Pati; Sushma Sequence alterations using homologous recombination
US5955590A (en) * 1996-07-15 1999-09-21 Worcester Foundation For Biomedical Research Conjugates of minor groove DNA binders with antisense oligonucleotides
US6136601A (en) * 1991-08-21 2000-10-24 Epoch Pharmaceuticals, Inc. Targeted mutagenesis in living cells using modified oligonucleotides
US6312894B1 (en) 1995-04-03 2001-11-06 Epoch Pharmaceuticals, Inc. Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
US6312925B1 (en) 1997-05-08 2001-11-06 Epoch Pharmaceuticals, Inc. Methods and compositions to facilitate D-loop formation by oligonucleotides
USRE38416E1 (en) 1988-09-28 2004-02-03 Epoch Biosciences, Inc. Cross-linking oligonucleotides
US7205105B2 (en) 1999-12-08 2007-04-17 Epoch Biosciences, Inc. Real-time linear detection probes: sensitive 5′-minor groove binder-containing probes for PCR analysis
US7348146B2 (en) 2003-10-02 2008-03-25 Epoch Biosciences, Inc. Single nucleotide polymorphism analysis of highly polymorphic target sequences
US7759126B2 (en) 2003-10-28 2010-07-20 Elitech Holding B.V. Real-time linear detection probes: sensitive 5′-minor groove binder-containing probes for amplification (or PCR) analysis
US7794945B2 (en) 1995-04-03 2010-09-14 Elitech Holding B.V. Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders

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USRE38416E1 (en) 1988-09-28 2004-02-03 Epoch Biosciences, Inc. Cross-linking oligonucleotides
US5824796A (en) * 1988-09-28 1998-10-20 Epoch Pharmaceuticals, Inc. Cross-linking oligonucleotides
US6136601A (en) * 1991-08-21 2000-10-24 Epoch Pharmaceuticals, Inc. Targeted mutagenesis in living cells using modified oligonucleotides
US5594121A (en) * 1991-11-07 1997-01-14 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified purines
EP0635023A1 (en) * 1992-03-05 1995-01-25 Isis Pharmaceuticals, Inc. Covalently cross-linked oligonucleotides
EP0635023A4 (en) * 1992-03-05 1996-03-20 Isis Pharmaceuticals Inc Covalently cross-linked oligonucleotides.
US5763240A (en) * 1992-04-24 1998-06-09 Sri International In vivo homologous sequence targeting in eukaryotic cells
WO1994017092A1 (en) * 1993-01-26 1994-08-04 Microprobe Corporation Bifunctional crosslinking oligonucleotides adapted for linking to a desired gene sequence of invading organism or cell
WO1994024144A3 (en) * 1993-04-19 1995-03-16 Gilead Sciences Inc Enhanced triple-helix and double-helix formation with oligomers containing modified purines
WO1994024144A2 (en) * 1993-04-19 1994-10-27 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified purines
US6492346B1 (en) 1995-04-03 2002-12-10 Epoch Pharmaceuticals, Inc. Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
US7794945B2 (en) 1995-04-03 2010-09-14 Elitech Holding B.V. Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
US7556923B1 (en) 1995-04-03 2009-07-07 Epoch Biosciences, Inc. Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
US6884584B2 (en) 1995-04-03 2005-04-26 Epoch Biosciences, Inc. Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
US6312894B1 (en) 1995-04-03 2001-11-06 Epoch Pharmaceuticals, Inc. Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
WO1996040711A1 (en) * 1995-06-07 1996-12-19 Microprobe Corporation Cross-linking oligonucleotides
US5935830A (en) * 1995-06-07 1999-08-10 Epoch Pharmaceuticals, Inc. Targeted mutagenesis in living cells using modified oligonucleotides
AU709924B2 (en) * 1995-06-07 1999-09-09 Epoch Pharmaceuticals, Inc. Cross-linking oligonucleotides
US5912340A (en) * 1995-10-04 1999-06-15 Epoch Pharmaceuticals, Inc. Selective binding complementary oligonucleotides
US5659022A (en) * 1996-01-05 1997-08-19 Epoch Pharmaceuticals, Inc. Oligonucleotide-cyclopropapyrroloindole conjugates as sequence specific hybridization and crosslinking agents for nucleic acids
US5955590A (en) * 1996-07-15 1999-09-21 Worcester Foundation For Biomedical Research Conjugates of minor groove DNA binders with antisense oligonucleotides
US6200812B1 (en) 1997-03-21 2001-03-13 Sri International Sequence alterations using homologous recombination
US6074853A (en) * 1997-03-21 2000-06-13 Sri Sequence alterations using homologous recombination
US5948653A (en) * 1997-03-21 1999-09-07 Pati; Sushma Sequence alterations using homologous recombination
US6312925B1 (en) 1997-05-08 2001-11-06 Epoch Pharmaceuticals, Inc. Methods and compositions to facilitate D-loop formation by oligonucleotides
US7205105B2 (en) 1999-12-08 2007-04-17 Epoch Biosciences, Inc. Real-time linear detection probes: sensitive 5′-minor groove binder-containing probes for PCR analysis
US7485442B2 (en) 1999-12-08 2009-02-03 Epoch Biosciences, Inc. Real-time linear detection probes: sensitive 5'-minor groove binder-containing probes for PCR analysis
US7348146B2 (en) 2003-10-02 2008-03-25 Epoch Biosciences, Inc. Single nucleotide polymorphism analysis of highly polymorphic target sequences
US7718374B2 (en) 2003-10-02 2010-05-18 Elitech Holding B.V. Single nucleotide polymorphism analysis of highly polymorphic target sequences
US7759126B2 (en) 2003-10-28 2010-07-20 Elitech Holding B.V. Real-time linear detection probes: sensitive 5′-minor groove binder-containing probes for amplification (or PCR) analysis

Also Published As

Publication number Publication date
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JPH06509945A (en) 1994-11-10
EP0661979A4 (en) 1995-09-13

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