WO2013040577A1

WO2013040577A1 - Aptamers resistant to nucleocapsid degradation

Info

Publication number: WO2013040577A1
Application number: PCT/US2012/055790
Authority: WO
Inventors: Rabi Ann MUSAH
Original assignee: The Research Foundation Of State University Of New York
Priority date: 2011-09-16
Filing date: 2012-09-17
Publication date: 2013-03-21

Abstract

The present invention is based on the observation that the nucleocapsid protein of RNA viruses has the ability to degrade both non-viral RNA and DNA. This degradation by viral components such as HIV-1 nucleocapsid protein renders inhibitory RNA aptamers generally ineffective in vivo. Disclosed herein are chimeric aptamers that are engineered to be resistant to degradation by viral components because they contain a segment of the viral RNA genome in addition to an anti-viral nucleic acid.

Description

APTAMERS RESISTANT TO NUCLEOCAPSID DEGRADATION

Cross-Reference to Related Applications

[0001] The present application claims priority to U.S. provisional application serial number 61/535847, filed September 16, 201 1 , the contents of which are hereby incorporated by reference in their entirety.

Reference to Sequence Listing

[0002] In compliance with 37 C.F.R. §§1 .821 - 1 .825, the present application contains a sequence listing with a file name of 0794135_Sequence

Listing_ST25.txt, a creation date of September 15, 2012 and a file size of 3.3KB. The sequence listing is part of the specification and is hereby incorporated in its entirety by reference.

Technical Field of the Invention

[0003] The present invention relates generally to aptamers, and in particular to aptamers, particularly anti-viral aptamers, that are resistant to degradation by viral nucleocapsid protein.

Background of the Invention

[0004] HIV-1 , as well as other retroviruses, contains several enzymes that have obligate roles in the viral replication cycle. Because of the essential roles played by these proteins, most attempts to design anti-viral therapies have been based on the development of compounds that inhibit their functions. However, the high mutation rate of the viral RNA genome and the resultant significant changes in the protein structures targeted by current drugs have resulted in the emergence of escape mutants, which are resistant to available therapies. One technology that is perceived to hold tremendous promise in circumventing these problems is the use of small non-coding nucleic acids (aptamers) that will bind very tightly to enough of the surface area of target proteins to significantly inhibit their essential functions. Although researchers have raised numerous aptamers against diverse retroviral protein targets, the results of in vivo experiments have shown them to be ineffectual at inhibiting virus replication after only a few cycles of viral replication.

[0005] Because the small molecule therapies that are the current mainstay of the HIV drug treatment regimen ultimately encourage the development of escape variants of the virus that are highly resistant to the current repertoire of drugs, the application of gene therapy approaches holds tremendous promise as a means to circumvent the challenges imposed by virus resistance to small molecule-based therapies. However, despite significant effort and substantial financial investment on the part of biotechnology centers and academic

laboratories, the results of in vivo experiments with nucleic acid based-drugs have shown them to be ineffectual at inhibiting virus replication after only a few to several cycles of viral replication.¹ This is thought to be due in part, to their instability in in vivo systems. Demonstration of a consistently effective way to increase the in vivo stability of nucleic acid-based therapies would permit the creation of aptamers that are effective in inhibiting HIV infections, as well as infections of other retroviruses that are of agricultural and economic importance.

Summary of the Invention

[0006] The present invention is based on the observation that the nucleocapsid protein of RNA viruses has the ability to degrade both non-viral RNA and DNA. This degradation by viral components such as HIV-1 nucleocapsid protein renders inhibitory RNA aptamers generally ineffective in vivo. Disclosed herein are chimeric aptamers that are engineered to be resistant to degradation by viral components because they contain a segment of the viral RNA genome in addition to an anti-viral nucleic acid.

[0007] In one aspect, therefore, the invention relates to a non-viral nucleic acid that is resistant to degradation by retrovirus nucleocapsid protein, said non- viral nucleic acid comprising the nucleotide sequence of an aptamer susceptible to nucleocapsid degradation and the nucleotide sequence of a segment of a retroviral Ψ packaging element of the virus.

[0008] In yet another aspect of the invention, the invention relates to an

RNA aptamer having the nucleotide sequence of SEQ ID NO: 4 or 5.

[0009] In a related aspect, the invention relates to a method for producing an non-viral nucleic acid that is resistant to degradation by a nucleocapsid protein of a virus, the method comprising synthesizing a chimeric aptamer comprising the nucleotide sequence of an anti-virus aptamer in which resistance is desired and the nucleotide sequence of a segment of the genome of the virus. In one embodiment, the virus is an RNA virus, such as a retrovirus, for example, human immunodeficiency virus (HIV), the anti-virus aptamer in which resistance is desired is an aptamer directed against the nucleocapsid protein of the virus and the segment of the virus genome is a stem loop (SL) of the retroviral Ψ packaging element of the virus. In the chimeric aptamer of the invention, the 5' end of the segment of the virus genome is linked to the 3' end of the anti-virus aptamer. In some embodiments, the stem of the stem loop is elongated by 2 or 3 G-C pairs.

[0010] In yet another related aspect, the method comprises synthesizing a nucleic acid having the nucleotide sequence of SEQ ID NO: 1 and at least 14 nucleotides of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

Brief Description of the Drawings

[0011] Figure 1 shows the results of an electrophoretic mobility shift assay of stem loop RNAs with WT NCp7. In each lane of each panel, 5 μΜ of RNA was used. Lane 1 : control (no NCp7); lanes 2-7: 5, 10, 20, 30, 40, and 50 μΜ NCp7 respectively was used, (a-i) EMSA of SL1 RNA with WT NCp7; (a-ii) EMSA of SL2 RNA with WT NCp7; (a-iii) EMSA of SL3 RNA with WT NCp7; (a-iv) EMSA of SL4 RNA with WT NCp7. [0012] Figure 2 shows secondary structures of RNA aptamers. (a) G RNA;

(b) R RNA; (c) GR1 RNA; and (d) GR2 RNA. The structures were predicted using mFold.

[0013] Figure 3 shows the results of a tryptophan fluorescence quenching assay. In all cases, 1 μΜ NCp7 was titrated against increasing concentrations of nucleic acid. The fluorescence signal in the absence of nucleic acids is considered as "1 " (l₀). The signal produced upon addition of a particular amount of nucleic acids is reported as a ratio of the signal upon addition of that particular amount versus the signal in the absence of NCp7 (i.e. l/lo).

[0014] Figure 4 Electrophoretic mobility assay of RNA aptamers incubated with NCp7. (a) 4.5 μΜ R RNA was used in each lane. Lane 1 : control (no NCp7); lanes 2-10: 4.5, 13.5, 18.0, 22.5, 27.0, 31 .5, 36.0, and 45.0 μΜ NCp7 respectively, (b) 5 μΜ G RNA was used in each lane. Lane 1 : control (no NCp7); lanes 2-8: 5, 10, 15, 20, 25, 30, and 35 μΜ NCp7 respectively, (c) 6.0 μΜ GR1 RNA was used in each lane. Lane 1 : control (no NCp7); lanes 2-1 1 : 6.0, 12.0, 18.0, 24.0, 30.0, 36.0, 42.0, 48.0, 54.0, and 60.0 μΜ NCp7 respectively, (d) 5.5 μΜ GR1 RNA was used in each lane. Lane 1 : control (no NCp7); lanes 2-8: 5.5, 1 1 .0, 16.5, 22.0, 27.5, 33.0, and 38.5 μΜ NCp7 respectively. Samples were incubated for 30 min at 37 °C and run on an 8% polyacrylamide gel under native conditions, with ethidium bromide staining.

[0015] Figure 5 Electrophoretic mobility assay of RNA aptamers with NCp7 in the presence of SUPERase ln™ ³²P-labeled RNA was used in each case (not quantified), (a) Lane 1 : control R aptamer (no NCp7); lanes 2 and 3: 0.5 and 2.5 μΜ NCp7 respectively, (b) Lane 1 : control G aptamer (no NCp7); lanes 2 and 3: 0.5 and 2.5 μΜ NCp7 respectively, (c) Lane 1 : control GR1 aptamer (no NCp7); lanes 2 and 3: 0.5 and 2.5 μΜ NCp7 respectively; (d) Lane 1 : control GR2 aptamer (no NCp7); lanes 2 and 3: 0.5 and 2.5 μΜ NCp7 respectively. Samples were run on an 8% polyacrylamide gel under native conditions. [0016] Figure 6 NCp7-mediated degradation of labeled RNA monitored by fluorescence. Labeled RNA (1 μΜ) was incubated with 1 .6 μΜ NCp7 at 37 °C. The increase in fluorescence was measured as a function of time. Degradation of the labeled RNA substrate was prevented when the NCp7 was pre-treated with DPMA prior to its incubation with the RNA substrate.

[0017] Figure 7 Secondary structure of hetero-dimeric RNA (a) RSL1 RNA

(b) RSL2 RNA (c) RSL3 RNA (d) RSL4 RNA predicted using mfold.

[0018] Figure 8 Electrophoretic mobility assay of RSL3 RNA with NCp7.

Lane 1 : Control RSL3 RNA (no NCp7); lanes 2-7: 4.5, 9.0, 18.0, 27.0, 36.0 and 45.0 μΜ NCp7 respectively. Samples were incubated for 1 h at 37 °C and analyzed by 8% native PAGE with ethidium bromide staining.

Detailed Description of the Invention

[0019] All publications, patents and other references cited herein are hereby incorporated by reference in their entirety into the present disclosure.

[0020] In practicing the present invention, many conventional techniques in synthetic chemistry are used, which are within the skill of the ordinary artisan. Some techniques are described in greater detail in, for example, The Aptamer Handbook edited by S. Klussmann, Wiley-VCH Verlag GmbH and Co. KGaA, Weinheim 2006, and Oligonucleotide Synthesis, 1984 (M.L. Gait ed.) the contents of these and other references containing standard protocols, widely known to and relied upon by those of skill in the art, including manufacturers' instructions are hereby incorporated by reference as part of the present disclosure.

[0021] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0022] The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target such as a protein, for example, a viral protein. Aptamers are obtained from an in vitro evolutionary process known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment), which selects target-specific aptamer sequences from large combinatorial libraries of single stranded oligonucleotide templates comprising randomized sequences (for more information regarding the SELEX method, see U.S. patent nos. 5,567,588, 5,475,096, and 5,270,163.)

[0023] Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules. The nucleotide components of an aptamer may include modified or non-natural nucleotides, for example nucleotides that have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NH2), which may improve a desired property, e.g., resistance to ribonucleases or a longer lifetime in biological fluids, such as blood and

cerebrospinal fluid. Aptamers may also be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.

[0024] The term "nucleic acid," or "oligonucleotide," as those terms are known in the art, refers to a polymer of nucleotides. Typically, a nucleic acid comprises at least three nucleotides. The polymer may include natural

nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine,

deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) or modified nucleosides. Modifications include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil, backbone modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3' and 5' modifications such as capping. Examples of modified nucleotides include, for example, base modified nucleoside (e.g., aracytidine, inosine, isoguanosine, nebularine, pseudouridine, 2,6-diaminopurine, 2-aminopurine, 2- thiothymidine, 3-deaza-5-azacytidine, 2'-deoxyuridine, 3-nitropyrrole, 4- methylindole, 4-thiouridine, 4-thiothymidine, 2-aminoadenosine, 2-thiothymidine, 2-thiouridine, 5-bromocytidine, 5-iodouridine, inosine, 6-azauridine, 6- chloropurine, 7-deazaadenosine, 7-deazaguanosine, 8-azaadenosine, 8- azidoadenosine, benzimidazole, M1 -methyladenosine, pyrrolo-pyrimidine, 2- amino-6-chloropurine, 3-methyl adenosine, 5-propynylcytidine, 5-propynyluridine, 5-bromouridine, 5-fluorouridine, 5-methylcytidine, 7-deazaadenosine, 7- deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2- thiocytidine), chemically or biologically modified bases (e.g., methylated bases), modified sugars (e.g., 2'-fluororibose, 2'-aminoribose, 2'-azidoribose, 2'-O- methylribose, L-enantiomeric nucleosides arabinose, and hexose), modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages), and combinations thereof. Natural and modified nucleotide monomers for the chemical synthesis of nucleic acids are readily available (e.g. see the following urls: trilinkbiotech.com, appliedbiosystems.com, biogenex.com or

syngendna.com).

[0025] As used herein, a "nucleic acid ligand" is a non-naturally occurring nucleic acid that binds selectively to a target. The nucleic acid that forms the nucleic acid ligand may be composed of naturally occurring nucleosides, modified nucleosides, naturally occurring nucleosides with hydrocarbon linkers (e.g., an alkylene) or a polyether linker (e.g., a polyethylene glycol or PEG linker) inserted between one or more nucleosides, modified nucleosides with hydrocarbon or PEG linkers inserted between one or more nucleosides, or a combination of thereof. In one embodiment, nucleotides or modified nucleotides of the nucleic acid ligand can be replaced with a hydrocarbon linker or a polyether linker provided that the binding affinity and selectivity of the nucleic acid ligand is not substantially reduced by the substitution. The target molecule of a nucleic acid ligand is a three dimensional chemical structure to which the nucleic acid ligand binds. However, the nucleic acid ligand is not simply a linear complementary sequence of a nucleic acid target, but may include regions that bind via complementary Watson-Crick base pairing interrupted by other structures such as hairpin loops. In one embodiment, the nucleic acid ligand binds to a viral protein, for example the nucleocapsid protein of human immunodeficiecy virus (HIV).

[0026] The retroviral nucleocapsid is the inner structure of the virus where several hundred nucleocapsid protein (NC) molecules coat the genomic RNA. During the past twenty years, NC was found to play multiple roles in the viral life cycle, notably during the copying of the genomic RNA into the proviral DNA by viral reverse transcriptase and integrase. NC, therefore, is considered to be a prime target for anti-HIV therapy.

[0027] The present invention is based on the surprising finding that aptamers raised against HIV-1 protein targets appeared to be degraded on exposure to NCp7. In addition to painstaking efforts to ensure not only an RNase- free environment, but also that reagents and solutions were RNase free, no evidence was found by gel electrophoresis or mass spectrometry of the presence of an additional protein that might be responsible for the RNA degradation that was observed.

[0028] Whether or not the salts used in binding buffers (i.e. Zn²⁺, Mg²⁺ or

Na⁺) might either be responsible for, or contain contaminants which might have resulted in the gradually reduced structural integrity of the RNA aptamers was investigated by PAGE. However, in the absence of added recombinant NCp7, buffer solutions did not promote changes in the electrophoretic mobility of the aptamers relative to that of their respective controls. This ruled out buffer salts, in and of themselves, as being responsible for RNA cleavage. Since it was possible that an RNase contaminant might have been carried over from NCp7 samples during their recombinant synthesis using an E. coli expression system, efforts were undertaken to eliminate this species, if present, in the protein/aptamer incubation mixtures, by introducing into them the RNase inhibitor SUPERase^»ln™. Despite the presence of the RNase inhibitor, the degradation of the RNA

aptamers was still apparent, indicating either the presence of an extraneous RNase that was immune to destruction by SUPERase^»ln™, or that the

degradation was mediated by NCp7 itself. Evidence that the aptamers were likely being cleaved by a component in the recombinant NCp7 sample was obtained using an RNA cleavage assay, in which an RNA substrate harboring both a fluorescent probe and a quencher molecule was exposed to the NCp7 protein. Over a period of 1 h, the gradual increase in fluorescence intensity above background as cleavage of the substrate resulted in the separation of the quencher from the fluorescent probe, confirmed that a species with RNA cleaving ability was present. However, replacing the NCp7 in that experiment with NCp7 protein that was pre-incubated with a small molecule known to covalently modify the protein and thereby keep it from interacting with nucleic acids, showed no significant increase in fluorescence above background. The observation that an NCp7 inhibitor of protein/nucleic acid binding resulted in abrogation of aptamer cleavage supports the hypothesis that the degradation was mediated by NCp7. Lastly, when aptamers that had previously succumbed to degradation were appended to HIV-1 Ψ packaging signal stem loops 2 and 3, the aptamers were rescued from degradation.

[0029] Consideration of the results of any one of the aforementioned experiments in isolation may not provide convincing enough evidence of NCp7- mediated (as opposed to RNase-catalyzed) aptamer degradation. However, when viewed as an ensemble, the results strongly implicate the direct involvement of NCp7 in aptamer degradation. This conclusion is further supported by similar results that were observed when NCp7 was exposed to dsDNA. In earlier studies, it was demonstrated that non-lyophilized recombinant NCp7, identical to that used in the present work, facilitated the fragmentation of DNA when both mono- and divalent ions were present. Characterization of the DNA fragments formed revealed that the cleavages were non-specific, although the possibility that there may be cleavage site preferences cannot be ruled out. It was also noted that the effect was only observed at μΜ levels of NCp7, which we also observed in our experiments with the RNA aptamers used in this work. We found here that although degradation was not apparent when aptamers were exposed to nM levels of NCp7, μΜ concentrations resulted in loss of the structural integrity of the aptamers.

[0030] It is well-established that like the nucleocapsid proteins of other retroviruses, NCp7 engages in strong binding interactions with RNA of both viral and non-viral origin. Indeed, its identified activities, which include nucleic acid recognition and chaperone functions,⁵¹ all hinge on its ability to engage in purposeful interactions with nucleic acids.

[0031] That NCp7 is responsible for the observed degradation of nucleic acids that are not of relevance to the virus is supported by the following

observations. First, when NCp7 is lyophilized, the effect is abolished or reduced to such an extent that only overnight incubation of protein with a degradable nucleic acid sample would reveal the effect. Thus, experiments utilizing lyophilized NCp7, and which involve incubations of less than -12 h, will not expose the effect. In this work, non-lyophilized protein was used, which we believe is why we observed the degradation. Second, the finding that the degradative effect appears to be discriminate, and might spare nucleic acids that are of relevance to the virus but not others, implies that studies of NCp7 interactions with virus-derived RNA may not show degradation, simply because degradation does not occur in those cases. The vast majority of NCp7/nucleic acid interaction studies fall into this category. Third, in order for degradation to occur, we found that both a

monovalent cation (such as Na⁺ or K⁺) and a divalent cation (such as Mg²⁺) must be present. Examination of the literature on NCp7 interactions with RNA makes clear that by and large, the vast majority of published studies were not conducted utilizing all of the aforementioned requisite conditions. Nevertheless, there are a few reported experimental results, heretofore unexplained, that can be rationalized if the possibility that NCp7 degrades RNA is considered. A case in point is the study reported by Clever et al.⁷ These authors found that whereas a GST-fusion protein version of NCp7 binds to a 206-nucleotide HIV-1 Ψ sequence construct, "protein-dependent loss" of the antisense RNA version of the construct occurs. This reproducible NCp7 concentration -dependent loss was clearly visible by PAGE and very similar to the results reported here. The authors rationalized the observations as indicative of the presence of "highly heterodisperse

complexes or low-level RNase contamination of the protein."⁷ Since denaturation of the sample prior to PAGE analysis was not conducted, the question of whether the observed diminution in band intensity was a consequence of heterodisperse complex formation cannot be answered. Nevertheless, we hypothesize that it was the GST-NCp15 that initiated the degradation of the antisense 206-nt

oligonucleotide, and not a nuclease contaminant, but this remains to be

investigated. This would provide a logical explanation for why Clever et al.

observed binding of 206-nt oligonucleotide, but degradation of its anti-sense counterpart. Importantly, as was the case in the work reported herein, the NCp7 used in their experiment was not lyophilized.

[0032] It is also clear that the degradation bears resemblance to that catalyzed by non-specific nucleases which mediate metal-ion-dependent phosphodiester hydrolysis of DNA or RNA. Like many other non-specific nucleases, it likely exhibits sequence preferences, which would suggest that certain topological features of the nucleic acid substrate may influence the preference to react with one substrate or region of a substrate molecule over another. These reactions are ubiquitous, and play vital roles in nucleic acid synthesis, recombination, processing and degradation.⁵² Although in principle any of these could be operative, an intriguing possibility is that the nucleic acid degradative effect exhibited by NCp7 (and presumably the NCp7 domain of Gag), is instrumental in facilitating discrimination between viral and host cell nucleic acids. Gag selectively packages viral RNA by distinguishing between the retroviral genome and significant amounts of host cell derived nucleic acids.⁵³ The current hypothesis for HIV-1 is that the NCp7 domain of Gag and the cognate viral RNA Ψ packaging signal serve as the primary determinants for the enrichment in retroviral RNA in the developing virions.^{6"14, 54} Genome selection might therefore be viewed as a competitive process in which the degree of RNA enrichment is dependent on the competitive ability of Gag to bind viral versus cellular nucleic acid.⁵⁵ But given that the cellular pool of nucleic acids is probably massive compared with the amount of viral RNA present,⁶ it seems reasonable to suspect that another mechanism of viral RNA selection enhancement may be operative. One possible outcome of the nucleic acid degradative activity is that non-specific binding to non- relevant nucleic acids is managed through destruction of those nucleic acids, while the structures of nucleic acids that have structural or topological features that are immune to the cleavage effect remain intact. The nucleic acid fragments produced through the degradative effect may have less of an affinity for Gag than the original larger macromolecule from which they were derived, which would effectively keep them from binding to the polyprotein. Thus, like trying to find the proverbial "needle in a haystack", where by burning of the haystack, subsequent identification of the needle can be accomplished by exposing the ashes to a magnet, the NCp7 domain of Gag may "burn" through non-virus relevant nucleic acids by destroying them. The destruction would not occur if it comes upon a replication-relevant nucleic acid construct with which it binds tightly. Such a discrimination process, coupled with the inherently high affinity that Gag has for the Ψ recognition site within the viral genome, could provide a rationale for the high level of enrichment of gRNA found in virions. Studies are underway to explore the validity of this hypothesis.

[0033] In one embodiment, a nucleic acid ligand of the invention is rendered resistant to degradation by the HIV nucleocapsid protein by addition of a nucleotide sequence derived from the Ψ packaging element of HIV.

[0034] Nucleic acid ligands may be prepared by any method known to those of skill in the art. EXAMPLES

Expression and purification of recombinant NCp7

[0035] Expression and purification of NCp7NL4-3A: The expression vector

(pRD2) containing a gene coding for the NCp7 sequence in the HIV-1 pNL4-3 strain was obtained as a kind gift from Dr. Daniele Fabris. The pRD2 clone was designed to overexpress the 55-residue NCp7 with the primary sequence NH2- MQKGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGH- QMKDCTERQAN-COOH (SEQ ID NO: 6.) For protein expression of NCp7 in Escherichia coli, pRD2 was transformed into BL21 (DE3) pLysE. The purification scheme for the recombinant NCp7 was adapted from Lee et al.⁴⁵ Culture media were supplemented with 100 g/L ampicillin and 34 g/L chloramphenicol. A starter culture of 5 mL of lysogeny broth (LB) inoculated from a single colony was grown overnight at 37 °C. The starter culture was then transferred to 100 mL of LB and grown at 37 °C to an absorbance of 1 .0 unit at 600 nm. This was then transferred to 1 L of LB and grown at 37 °C to an absorbance of 0.5 to 0.6 units at 600 nm before induction with 1 mM isopropyl- -D-thiogalactopyranoside (IPTG). After 5 h, the cells were harvested by centrifugation, resuspended in 15 mL of lysis buffer (50 mM Tris pH 8.0, 10% v/v glycerol, 100 mM NaCI, 0.1 mM ZnCI₂, 5 mM dithiothreitol (DTT), and 2 mM EDTA), and stored at -80 °C. To lyse the cells, the samples were thawed on ice-water, and 86 μί of 10 mM phenylmethylsulfonyl fluoride (PMSF), 15 μί of 1 mg/ml Pepstatin A, and 1 .1 mL of 1 % w/v sodium deoxycholate were added. The cells were sonicated for five cycles of 1 min duration to reduce the viscosity. Typical sonication cycle parameters were: 45% amplitude, 0.3 sec pulse on and 1 .0 sec pulse off. The nucleic acids were precipitated by dropwise addition of 4% w/v polyethyleneimine (pH 7.9) to a final concentration of 0.4 % and stirred for 15 minutes before centrifugation at 25,000 rpm for 20 min at 4 °C. The supernatant was collected and filtered through a 0.22 μιτι pore size syringe filter. Ion exchange chromatography was used for further purification of the protein. A Q-Sepharose (5 mL) and a SP-Sepharose (5 mL) column (GE Healthcare) were connected in a series and were equilibrated with 50 ml_ (10 column volumes) of buffer A [50 mM Tris pH 8.0, 10% glycerol, 100 mM NaCI, 0.1 mM ZnCI₂, and 10 mM β-mercaptoethanol (BME)]. Protein samples were loaded onto the columns at 1 mL/min using a 6 ml_ loop. After washing the columns with 15 ml_ (3 column volumes) of buffer A, the Q-Sepharose column was detached, and the SP-Sepharose column was further washed with 1 .5 column volumes of buffer A. A ten column volume linear gradient from 40%-50% buffer B (50 mM Tris pH 8.0, 10% glycerol, 1 .0 M NaCI, 0.1 mM ZnCI₂, 10 mM BME) was applied to elute the NCp7. The protein eluted at approximately 43% buffer B. Protein fractions were pooled and passed through 3000 Da molecular weight cut off filters. Concentrated samples were stored in elution buffer (50 mM Tris pH 8.0, 10% glycerol, 450 mM NaCI, 0.1 mM ZnCI₂, and 10 mM BME) at -80 °C. The purity of NCp7 sample was confirmed by ESI-TOF and MALDI-TOF mass spectrometry.

RNA oligonucleotides

[0036] SL3 and RSL3 RNA were purchased from IDT DNA Technologies

(Coralville, IA, USA) and used without further purification. The RNA was heated to 95 °C for 3 min, cooled on ice, and stored on ice just prior to use.

In vitro transcription and purification of RNA aptamers

[0037] RNA aptamers were prepared by in vitro transcription using the

MEGAscript® T7 kit (Life Technologies, Grand Island, NY) according to the manufacturer's specifications. For ³²P labeled RNA aptamers, the MAXIscript® T7 kit (Life Technologies, Grand Island, NY) was used with ³²P labeled ATP. After transcription, an equal volume of 1 :1 phenol/chloroform was added. After mixing, the two layers were separated by centrifugation at 14,000 rpm for 3 min. To the aqueous layer, an equal volume of chloroform was added and after mixing, the two layers were separated by centrifugation at 14,000 rpm for 3 min. To the aqueous layer, 1 % 3 M sodium acetate, pH 5.5, three volumes of 100% ethanol and 1 μί linear acrylamide were added. The solution was allowed to stand at -80 °C for 1 h while precipitation occurred. It was then centrifuged at 4 °C for 10 min at 14,000 rpm. The supernatant was removed and the pellet was washed with 70% ethanol. Finally, the pellet was suspended in 20 μΙ_ of ddH2O.

Electrophoresis Mobility Shift Assays

[0038] For experiments with SL3 RNA and RNA aptamers, RNA (with the amounts indicated in the corresponding figures and Examples) was incubated with increasing concentrations of NCp7 (amounts indicated in corresponding figures and Examples) in a reaction buffer comprised of 50 mM Tris pH 7.5, 100 mM NaCI, 30 μΜ ZnCI₂, 1 .5 mM MgCI₂ and 10 mM β-mercaptoethanol (BME). After incubation for the indicated amount of time, samples were mixed with 1 X native gel loading dye and loaded onto an 8% polyacrylamide gel at a constant voltage of 120 V for 30 min. Gels were stained by submersion in an aqueous solution of 0.5 g/mL ethidium bromide for 15 min, and photographed using a Bio-Rad Chemidoc XRS Gel Documentation System.

Fluorescence quenching assay

[0039] Nucleic acids were titrated with 1 .0 μΜ NCp7 in a binding buffer comprised of 5 mM sodium phosphate pH 7.0, 200 mM NaCI, 0.1 mM ZnCI₂, 0.01 % polyethylene glycol (PEG), and 10 mM BME. Fluorescence measurements were made as a function of time using a Fluorolog-3 spectrofluormeter (model number FL3-221 from Horiba Jobin-Yvon Inc.) at an excitation wavelength of 290 nm and an emission wavelength of 350 nm at a 5 nm excitation and emission band-pass (slit width). The fluorescence curves were fitted to a model assuming multiple stoichiometry for the ratio of NCp7 bound to RNA.

RNA degradation assay

[0040] The RNA degradation assay was performed using the RNaseAlert™

Substrate Nuclease Detection System (IDT DNA Technologies, Coralville, IA) according to the manufacturer's specifications. Fluorescence was measured using a fluorimeter on the "fluorescein" channel, using 490 nm excitation and 520 nm emission settings. Gel retardation assays confirm recombinant NCp7 binding to individual HIV-1 Ψ signal stem loops

[0041] NCp7 was synthesized recombinantly using the method outlined by

Lee et al.⁴⁵ Gel electrophoresis analysis showed the protein had been purified to homogeneity, as indicated by the appearance of a single band by SDS PAGE using the highest amount of protein that could be accommodated by the gel. The purity of the protein was also assessed by electrospray ionization-time of flight mass spectrometry (ESI-TOF MS), which showed the presence of a single entity having a mass appropriate for NCp7 coordinated to two zinc atoms (data not shown). NCp7 is known to bind to the stem loops of the ψ packaging equence of the HIV-1 RNA genome (gRNA), and we confirmed this by electrophoretic mobility shift assays. Individual stem loop RNAs (5 μΜ) were incubated at 37 °C for 2 h with increasing concentrations of recombinant NCp7 (5-50 μΜ) in buffer comprised of 50 mM Tris pH 7.5, 100 mM NaCI, 30 μΜ ZnCI₂, 1 .5 mM MgCI₂ and 10 mM β-mercaptoethanol (BME). Subsequent electrophoretic mobility assay (EMSA) analysis (Figure 1 ) showed that in alignment with previously published findings, NCp7 binds strongly to SL1 -SL3, but only weakly to SL4. Specifically, interaction of NCp7 with SL1 showed formation of higher molecular weight complexes that in earlier studies were demonstrated to represent dimeric RNA that had been converted to a kissing dimer. Binding of NCp7 to SL2 and SL3 each featured the gradual appearance of a crisp, prominent higher molecular weight species at the expense of uncomplexed nucleic acid. On the other hand, incubation conditions and subsequent EMSA analysis did not show significant binding between NCp7 and SL4, with only a faint smear indicative of non-specific binding appearing at the highest concentrations of NCp7 used (i.e. 30 - 50 mM). These observations confirm previously reported findings. The K_d for SL4 binding of NCp7 has been estimated to be in the μΜ range,^{10, 46} and NCp7 binds to SL2 and SL3 with nM affinity.¹⁰' ¹²' ^36"38

[0042] Two representative aptamer constructs raised against NCp7 and termed "G1 " and "R1 ", were chosen from the literature for study (Figure 2a/b) of NCp7/aptamer interactions. Capitalizing on the methodology developed by Shi et al.^47"49, in which aptamers optimized for different domains on a single protein surface can be combined to provide an aptamer that has the ability to interact with more than one protein binding partner, two new aptamers were created based on combinations of the aforementioned G1 and R1 aptamers developed by Allen et al ⁴² and Berglund et al ⁴³ These are termed GR1 and GR2 respectively (Figure 2c/d). All four aptamers were prepared by in vitro

transcription.

[0043] NCp7 possesses a Trp residue within the protein's distal zinc finger at position 37. Quenching of the intrinsic Trp fluorescence occurs with binding of the protein to nucleic acids, and this phenomenon can be exploited to assess protein/macromolecular binding affinities. Thus, binding constants of the G1 , R1 , GR1 and GR2 aptamers for NCp7 were assessed using a fluorescence quenching assay. Nucleic acids were titrated with 1 μΜ NCp7 in binding buffer comprised of 5 mM sodium phosphate pH 7.0, 200 mM NaCI, 0.1 mM ZnCI₂, 0.01 %

polyethylene glycol (PEG), and 10 mM BME. Fluorescence measurements were made as a function of time using excitation and emission wavelengths of 290 nm and 350 nm respectively. The resulting fluorescence curves were fitted to a model assuming multiple stoichiometry for the ratio of NCp7 bound to RNA (Figure 3), using equation 1 , where P = protein concentration, R = RNA concentration, a and b are stoichiometric coefficients, B = RNA protein complex, P0 = initial

fluorescence intensity, P = fluorescence intensity after addition RNA, and K_d = binding constant. Origin software was used to calculate the K_d for each titration. In each case, approximately 90% of the original fluorescence signal was quenched over the concentration range used. Calculated binding constants are listed in Table 1 . Table 1

RNA Binding Constant,

SL3 RNA 0.39.23 ± (1 .5)

R aptamer 544.53 ± (8.6)

G aptamer 548.45 ± (8.7)

GR1 aptamer 439.68 ± (5.7)

GR2 aptamer 392.40 ± (7.0)

[0044] Equation 1

[P]^a [R]^b

K =

[B]

P/P₀ = 1 - a_ { P₀ ^a (P/P₀)^a [R₀- _ Po (1 - P/P₀)]^b}

P₀K

[0045] The NCp7/R1 aptamer binding constant we obtained is of the same order of magnitude as that reported by Berglund et al.⁴³ who first described the aptamer and determined the protein-nucleic acid binding affinity using a nitrocellulose filter binding assay (i.e. 544.5±8.6 nM vs. 905 nM respectively). On the other hand, the 548.5 ±8.7 nM K_d that we observed for NCp7/G1 binding was two orders of magnitude lower than that reported by Allen et al.⁴² who observed a K_d of > 10 nM). However, the experimental conditions under which they performed their filter binding assay differed significantly from ours, most notably in terms of pH and the presence of MgCI₂. Our experiments were conducted at pH 7.0 whereas theirs were done at 5.3. Their reaction buffer also contained 5 mM MgCl2, whereas ours contained no MgC^. Additionally, the aptamer we used was a truncated form of that reported by Allen et al.,⁴² and it could be that additional binding interactions that might be possible between NCp7 and the full length G1 aptamer construct, are responsible for the tighter binding reported. The GR1 and GR2 chimeric constructs exhibited binding affinities to NCp7 that were of the same order of magnitude as the R and G constructs, with GR2 showing the tightest binding (439.7 ± 5.7 nM and 393.4 ± 7.0 nM for GR1 and GR2

respectively). None of the aptamers bound to NCp7 as tightly as SL3 RNA, whose K_d for NCp7 binding was 39.2 ± 1 .5 nM. This value differs significantly from that obtained by isothermal titration calorimetry (ITC) (i.e. 170 ± 65 nM), but is closer to that observed by Shubsda et al.³⁷ (28 ± 4 nM), who determined the K_d by fluorescence quenching. The observed differences may be a consequence of the distinctions between the experimental conditions used for the ITC determination, and those which were used by us and Shubsda et al.³⁷ The K_d estimated by ITC was determined by titration of protein into RNA dissolved in a buffer comprised of 25 mM NaOAc, 25 mM NaCI and 0.1 % BME, pH 6.5. Our fluorescence quenching assay conditions were modeled after that reported by Shubsda et al.³⁷ [5 mM sodium phosphate pH 7.0, 200 mM NaCI, 0.1 mM ZnCI₂, 0.01 % polyethylene glycol (PEG)], except that we included 10 mM BME. It has been shown that changes in salt conditions can significantly influence NCp7/nucleic acid binding interactions.³⁷

EMSA analysis revealed that RNA aptamers were unstable in the presence of μΜ concentrations of NCp7

[0046] Following establishment of aptamer/NCp7 binding by fluorescence quenching, we investigated the interaction between aptamers and protein by EMSA, since this could reveal information inaccessible by fluorescence, such as higher molecular weight complex formation, nucleic acid multimerization, and/or aggregate formation. Each of the four aptamers (4.5-6.0 μΜ) was incubated at 37 0C with increasing concentrations of NCp7 at RNA:protein ratios that ranged from 1 :1 to 1 :7. Aptamer/protein samples were then analyzed by native PAGE. The results, outlined in Figure 4, show that with increasing NCp7 concentrations, the band corresponding to RNA or RNA protein complexes gradually diminished. This diminution in band intensity is reminiscent of that observed when double stranded DNA is incubated with NCp7, an observation which was subsequently shown to be a consequence of nucleic acid degradation (see accompanying manuscript). It is also in contrast to what was observed with NCp7 binding to SL3, which showed formation of a higher molecular weight RNA NCp7 complex that appeared at the expense of the RNA band (Figure 1 c). Interestingly, whereas incubation of NCp7 with the R1 , GR1 and GR2 aptamers resulted in the formation of a higher molecular weight complex whose corresponding band intensity decreased with increasing protein concentration, the G1 aptamer did not show any evidence of higher molecular weight complex formation, despite our observation of binding by tryptophan fluorescence. The diminution in band intensity could be a consequence of a number of factors, including (a) nuclease contamination; (b) formation of aggregates whose size prohibited their entry into the gel; (c) diffusion of the sample from the loading well of the gel into the electrophoresis buffer as a consequence of repulsive forces generated from increasing concentrations of the highly positively charged protein; and (d) the formation of heterodisperse complexes which were so diffuse within the gel that they were difficult to view by gel electrophoresis. Possibilities (b), (c) and (d) could be tested by rigorous denaturation of the incubated sample just prior to EMSA analysis, a technique that has been exploited by others⁵⁰ to release NCp7 from bound nucleic acids. Thus, RNA/aptamer samples that had been incubated for 30 min at 37 °C were treated with a solution comprised of 400 mM NaCI, 20 mM EDTA, 8% SDS, and 2% glycerol, followed by heating at 70 °C for 10 min. EMSA analysis of the resulting samples (data not shown) showed that in each case, the band diminution that was previously observed with increasing concentrations of protein was maintained. If the aptamers had not been degraded when incubated with the protein, but had instead been complexed within aggregates, denaturing the sample would be expected to liberate them such that upon staining, they would have appeared in the gel and exhibited an electrophoretic mobility similar to that of the RNA control. We interpreted the absence of bands representing the aptamers (following sample denaturation) to be indicative of nucleic acid degradation. This is in alignment with what has been observed when DNA has been exposed to μΜ levels of NCp7.

Exposure of aptamers to NCp7 in the presence of RNase inhibitor did not abrogate aptamer degradation

[0047] The relative instability of RNA coupled with the pervasiveness of nucleases prompted us to further examine the possibility that the apparent aptamer degradation was a result of RNase contamination. Thus, we conducted the aptamer/NCp7 incubations in the presence of the RNase inhibitor

SUPERase^»ln™, anticipating that extraneous ribonucleases that might be present in our samples would be destroyed, leading to the absence of degradation. To increase the sensitivity of our assay as well as the detection limits for our aptamers, each was labeled with ³²P. They were then incubated for 30 min at 37 °C with NCp7 (0.5 and 2.5 μΜ) in a buffer comprised of 50 mM Tris pH 7.5, 100 mM NaCI, 30 μΜ ZnCI₂, 1 .5 mM MgCI₂ and 10 mM β-mercaptoethanol (BME) and to which was added one unit of SUPERase^»ln™ . The results of EMSA analysis of these samples are shown in Figure 5. For the R1 , G1 and GR1 aptamers, the gradual reduction in band intensity on exposure to NCp7 was still maintained, even in the presence of the RNase inhibitor. The results for the GR2 aptamer show that a distribution of species with lower electrophoretic mobility than the aptamer are formed, but it is not clear whether these species are comprised of fragmented aptamers complexed to protein, or full length aptamer complexes. The results of this experiment do not completely rule out the possibility that an extraneous nuclease contaminant is responsible for what appears to be nucleic acid degradation. However, they do imply that either (a) an RNase other than one known to be destroyed by SUPERase^»ln™ was present, or (b) that the protein itself, which is not degraded by SUPERase^»ln™, was responsible for the activity.

An RNA cleavage assay confirmed the RNA cleavage ability of NCp7

[0048] Further demonstration of NCp7-mediated aptamer cleavage was obtained by conducting a fluorescence-based ribonuclease assay. Rapid RNase detection can be achieved using the RNaseAlert™ system developed by

Invitrogen (Grand Island, NY USA). The assay is optimized for the detection of RNase A, RNase T1 , RNase 1 and microccocal nuclease, but will also detect less common nucleases such as mung bean nuclease and S1 nuclease. The system is comprised of an RNase-specific five-nucleotide substrate flanked with a fluorescent reporter molecule (fluorescein, E_m = 520 nM) on the 5'-end and a dark quencher molecule on the 3'-end. The proximity of the quencher to the reporter molecule causes the entire construct to remain dark when in the absence of a nucleotide cleaving agent. However, severance of the oligonucleotide tether, such as would occur when an RNase is present, liberates the reporter, which, once separated from the quencher, emits green fluorescence which can be monitored visually or by fluorimetry. Thus, reaction solutions that contain species with RNA- cleaving activity produce a green glow in the assay when exposed to UV light, whereas solutions that do not contain an RNA cleaving species do not.

Assessment of the possible presence of a ribonuclease in our buffer solutions, and nucleic acid cleaving ability of the NCp7, was conducted by incubating the RNaseAlert™ labeled substrate (1 μΜ) with our buffer solutions and/or NCp7 (1 .6 μΜ) at 37°C according to the manufacturer's specifications. RNase A was used as a positive control, and reaction buffer made with RNase-free water was used as the negative control. The results of fluorescence monitoring as a function of time are shown in Figure 6. They show that while exposure of our reaction buffer to the labeled substrate did not appreciably increase the fluorescence, the addition of NCp7 did, indicating that the substrate had been cleaved, and confirming RNA cleavage capability on the part of a component of the sample. In order to differentiate between NCp7-mediated RNaseAlert™ substrate cleavage and that catalyzed by an extraneous nuclease, NCp7, prior to incubation with the

RNaseAlert substrate, was pre-incubated for 1 h with 7.5 mM 2,3-diphenylmaleic anhydride (DPMA), a compound that we have found inhibits NCp7 binding to nucleic acids by covalently modifying the protein. Exposure of the pre-incubated NCp7 to the labeled substrate showed that fluorescence enhancement was abrogated. This result further supported the hypothesis that it was the NCp7 itself, and not an extraneous nuclease, that was mediating nucleic acid cleavage.

Aptamers can be rescued from NCp7 '-mediated degradation if they are appended to HIV-1 Ψ packaging signal stem loops

[0049] The observation of apparent nucleic acid degradation of aptamers on their exposure to NCp7, alongside the absence of such degradation when the same NCp7 samples were exposed to HIV-1 Ψ packaging signal stem loops, indicated that degradation was discriminate, and that the SL1 -4 structures possessed features that protected them from being degraded. In order to determine the possible extent to which the presence of these stem loop structural features can confer similar protection to aptamers that might otherwise be degraded in the presence of NCp7, chimeric aptamer constructs comprised of the R1 aptamer appended to SL2 and SL3 were created. The mfold-predicted secondary structures of these constructs are shown in Figures 4 and 5.

Remarkably, incubation of each of these in binding buffer with increasing NCp7 concentrations at 37° C for 30 min, followed by EMSA analysis, showed that in every case, higher molecular weight complexes were formed, and the degradation that was previously seen when the discrete aptamers were incubated with NCp7, was no longer observed. A representative showing higher molecular weight complex formation when NCp7 is incubated with RSL3 RNA is shown in Figure 8. We interpreted this result to mean that NCp7 has the capacity to either protect or degrade RNA constructs, depending upon their structures, and that there is a logic, albeit currently unclear, that underlies whether protection versus

degradation occurs.

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Claims

Claims We claim:

1 . A non-viral nucleic acid that is resistant to degradation by a nucleocapsid protein of a virus, said non-viral nucleic acid comprising the nucleotide sequence of a segment of a retroviral Ψ packaging element of the virus.

2. The non-viral nucleic acid of claim 1 , wherein the non-viral nucleic acid has anti-viral activity.

3. An aptamer that is resistant to degradation by a nucleocapsid protein of a virus, said aptamer comprising the nucleotide sequence of a segment of a retroviral Ψ packaging element of the virus.

4. An aptamer that is resistant to degradation by a nucleocapsid protein of a virus, said aptamer comprising the nucleotide sequence of a non-viral nucleic acid susceptible to degradation by a nucleocapsid protein of a virus linked to the nucleotide sequence of a segment of a retroviral Ψ packaging element of the virus.

5. The aptamer of claim 1 , wherein said aptamer has anti-viral activity.

6. The aptamer of claim 1 , wherein the aptamer is RNA.

7. The aptamer of claim 1 , wherein the virus is a retrovirus.

8. The aptamer of claim 1 , wherein the virus is human immunodeficiency virus (HIV.)

9. The aptamer of claim 1 , wherein the segment of a retroviral Ψ packaging element of the virus is a stem loop or portion thereof.

10. The aptamer of claim 9, wherein the stem loop has the nucleotide sequence of SEQ ID NO: 2 or 3 or a portion thereof.

1 1 . The aptamer of claim 9, wherein the aptamer has the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

12. A method for producing an aptamer that is resistant to degradation by a nucleocapsid protein of a virus, the method comprising: synthesizing a chimeric aptamer comprising:

(a) the nucleotide sequence of a nucleic acid susceptible to

degradation by nucleocapsid protein of the virus; and

(b) the nucleotide sequence of a segment of a retroviral Ψ

packaging element of the virus.

13. The method of claim 12, wherein said segment of a retroviral Ψ packaging element of the virus is a stem loop or portion thereof.

14. The method of claim 12, wherein the 5' end of said nucleotide sequence of said segment is linked to the 3' end of the nucleotide sequence of said aptamer.

15. The method of claim 12, wherein the aptamer is RNA.

16. The method of claim 12, wherein the aptamer has anti-viral activity.

17. The method of claim 12, wherein the virus is a retrovirus.

18. The method of claim 12, wherein the virus is human immunodeficiency virus (HIV.)

19. The method of claim 13, wherein the stem loop is stem loop 3 or stem loop 4, or a portion thereof.