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WO2013040577A1 - Aptamères résistant à la dégradation par la nucléocapside - Google Patents

Aptamères résistant à la dégradation par la nucléocapside Download PDF

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WO2013040577A1
WO2013040577A1 PCT/US2012/055790 US2012055790W WO2013040577A1 WO 2013040577 A1 WO2013040577 A1 WO 2013040577A1 US 2012055790 W US2012055790 W US 2012055790W WO 2013040577 A1 WO2013040577 A1 WO 2013040577A1
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rna
ncp7
aptamer
degradation
virus
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Rabi Ann MUSAH
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Research Foundation of the State University of New York
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/16—Aptamers
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/532—Closed or circular
    • C—CHEMISTRY; METALLURGY
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity
    • C12N2320/51—Methods for regulating/modulating their activity modulating the chemical stability, e.g. nuclease-resistance

Definitions

  • 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.
  • HIV-1 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.
  • aptamers small non-coding nucleic acids
  • 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.
  • 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.
  • 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 ⁇ packaging element of the virus.
  • the invention relates to an
  • RNA aptamer having the nucleotide sequence of SEQ ID NO: 4 or 5.
  • 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.
  • 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 ⁇ packaging element of the virus.
  • 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.
  • the stem of the stem loop is elongated by 2 or 3 G-C pairs.
  • 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.
  • Figure 1 shows the results of an electrophoretic mobility shift assay of stem loop RNAs with WT NCp7.
  • Lane 1 control (no NCp7); lanes 2-7: 5, 10, 20, 30, 40, and 50 ⁇ 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.
  • Figure 2 shows secondary structures of RNA aptamers.
  • Figure 3 shows the results of a tryptophan fluorescence quenching assay.
  • 1 ⁇ NCp7 was titrated against increasing concentrations of nucleic acid.
  • the fluorescence signal in the absence of nucleic acids is considered as "1 " (l 0 ).
  • 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).
  • FIG. 4 Electrophoretic mobility assay of RNA aptamers incubated with NCp7.
  • (a) 4.5 ⁇ 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 ⁇ NCp7 respectively,
  • Lane 1 control (no NCp7); lanes 2-8: 5, 10, 15, 20, 25, 30, and 35 ⁇ NCp7 respectively,
  • 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 ⁇ NCp7 respectively, (d) 5.5 ⁇ 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 ⁇ 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.
  • FIG. 5 Electrophoretic mobility assay of RNA aptamers with NCp7 in the presence of SUPERase lnTM 32 P-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 ⁇ NCp7 respectively, (b) Lane 1 : control G aptamer (no NCp7); lanes 2 and 3: 0.5 and 2.5 ⁇ NCp7 respectively, (c) Lane 1 : control GR1 aptamer (no NCp7); lanes 2 and 3: 0.5 and 2.5 ⁇ NCp7 respectively; (d) Lane 1 : control GR2 aptamer (no NCp7); lanes 2 and 3: 0.5 and 2.5 ⁇ NCp7 respectively.
  • 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 ⁇ NCp7 respectively. Samples were incubated for 1 h at 37 °C and analyzed by 8% native PAGE with ethidium bromide staining.
  • 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.)
  • 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
  • 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.
  • nucleic acid 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 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.
  • 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.
  • 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-chloropur
  • 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.
  • hydrocarbon linkers e.g., an alkylene
  • a polyether linker e.g., a polyethylene glycol or PEG linker
  • 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.
  • 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.
  • the nucleic acid ligand binds to a viral protein, for example the nucleocapsid protein of human immunodeficiecy virus (HIV).
  • the retroviral nucleocapsid is the inner structure of the virus where several hundred nucleocapsid protein (NC) molecules coat the genomic RNA.
  • NC nucleocapsid protein
  • 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.
  • RNA aptamers 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.
  • 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 » lnTM. Despite the presence of the RNase inhibitor, the degradation of the RNA
  • 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.
  • 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.
  • NCp7 is responsible for the observed degradation of nucleic acids that are not of relevance to the virus.
  • oligonucleotide and not a nuclease contaminant, but this remains to be
  • 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.
  • the current hypothesis for HIV-1 is that the NCp7 domain of Gag and the cognate viral RNA ⁇ packaging signal serve as the primary determinants for the enrichment in retroviral RNA in the developing virions.
  • 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.
  • 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.
  • 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 ⁇ packaging element of HIV.
  • Nucleic acid ligands may be prepared by any method known to those of skill in the art.
  • 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.)
  • pRD2 was transformed into BL21 (DE3) pLysE.
  • the purification scheme for the recombinant NCp7 was adapted from Lee et al.
  • 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 ZnCI 2 , 5 mM dithiothreitol (DTT), and 2 mM EDTA), and stored at -80 °C.
  • lysis buffer 50 mM Tris pH 8.0, 10% v/v glycerol, 100 mM NaCI, 0.1 mM ZnCI 2 , 5 mM dithiothreitol (DTT), and 2 mM EDTA
  • the samples were thawed on ice-water, and 86 ⁇ of 10 mM phenylmethylsulfonyl fluoride (PMSF), 15 ⁇ 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 ⁇ 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 ZnCI 2 , and 10 mM ⁇ -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 ZnCI 2 , 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 ZnCI 2 , and 10 mM BME) at -80 °C.
  • the purity of NCp7 sample was confirmed by ESI-TOF and MALDI-TOF mass spectrometry.
  • SL3 and RSL3 RNA were purchased from IDT DNA Technologies
  • RNA aptamers were prepared by in vitro transcription using the
  • RNA aptamers for 32 P labeled RNA aptamers, the MAXIscript® T7 kit (Life Technologies, Grand Island, NY) was used with 32 P 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 ⁇ 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 ⁇ _ of ddH2O.
  • 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 ⁇ ZnCI 2 , 1 .5 mM MgCI 2 and 10 mM ⁇ -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.
  • a reaction buffer comprised of 50 mM Tris pH 7.5, 100 mM NaCI, 30 ⁇ ZnCI 2 , 1 .5 mM MgCI 2 and 10 mM ⁇ -
  • Nucleic acids were titrated with 1 .0 ⁇ NCp7 in a binding buffer comprised of 5 mM sodium phosphate pH 7.0, 200 mM NaCI, 0.1 mM ZnCI 2 , 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.
  • a binding buffer comprised of 5 mM sodium phosphate pH 7.0, 200 mM NaCI, 0.1 mM ZnCI 2 , 0.01 % polyethylene glycol (PEG), and 10 mM BME.
  • RNA degradation assay was performed using the RNaseAlertTM
  • 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 ⁇ signal stem loops
  • NCp7 was synthesized recombinantly using the method outlined by
  • NCp7 binds strongly to SL1 -SL3, but only weakly to SL4.
  • NCp7 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 ⁇ range, 10, 46 and NCp7 binds to SL2 and SL3 with nM affinity. 10 ' 12 ' 36"38
  • 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 ⁇ NCp7 in binding buffer comprised of 5 mM sodium phosphate pH 7.0, 200 mM NaCI, 0.1 mM ZnCI 2 , 0.01 %
  • 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).
  • 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).
  • 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
  • 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 ZnCI 2 , 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
  • RNA aptamers were unstable in the presence of ⁇ concentrations of NCp7
  • 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 others 50 to release NCp7 from bound nucleic acids.
  • 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 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.
  • RNA cleavage assay confirmed the RNA cleavage ability of NCp7
  • NCp7-mediated aptamer cleavage was obtained by conducting a fluorescence-based ribonuclease assay. Rapid RNase detection can be achieved using the RNaseAlertTM system developed by
  • 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.
  • 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.
  • NCp7 In order to differentiate between NCp7-mediated RNaseAlertTM 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.
  • DPMA 2,3-diphenylmaleic anhydride
  • Retroviral nucleocapsid domains mediate the specific recognition of genomic viral RNAs by chimeric Gag polyproteins during RNA packaging in vivo. Journal of virology 69, 6445-56.
  • NCp7 Human immunodeficiency virus Type 1 nucleocapsid protein directs specific initiation of minus-strand DNA synthesis primed by human tRNA(Lys3) in vitro: studies of viral RNA molecules mutated in regions that flank the primer binding site. Journal of virology 70, 4996-5004.
  • a HIV-1 minimal gag protein is superior to nucleocapsid at in vitro annealing and exhibits multimerization-induced inhibition of reverse transcription.
  • RNA aptamers directed to human immunodeficiency virus type 1 Gag polyprotein bind to the matrix and nucleocapsid domains and inhibit virus production. Journal of virology 85, 305-14.
  • RNA aptamers directed to discrete functional sites on a single protein structural domain. Proceedings of the National Academy of Sciences of the United States of America 104, 3742-6.
  • RNA aptamers as effective protein antagonists in a multicellular organism. Proceedings of the National Academy of Sciences of the United States of America 96, 10033-8.

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Abstract

L'invention est basée sur l'observation du fait que la protéine nucléocapside des virus ARN a la capacité de dégrader à la fois l'ARN et l'ADN non viraux. Cette dégradation par des composants viraux tels que la protéine nucléocapside de VIH-1 rend les aptamères ARN inhibiteurs généralement inefficaces in vivo. L'invention concerne des aptamères chimères qui sont recombinés par génie génétique de manière qu'ils résistent à la dégradation par des composants viraux car ils contiennent un segment du génome d'ARN viral ainsi qu'un acide nucléique anti-viral.
PCT/US2012/055790 2011-09-16 2012-09-17 Aptamères résistant à la dégradation par la nucléocapside Ceased WO2013040577A1 (fr)

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WO2015033155A1 (fr) * 2013-09-05 2015-03-12 The University Of York Thérapie antivirale
WO2015090230A1 (fr) 2013-12-19 2015-06-25 Novartis Ag Récepteurs antigéniques chimériques de la mésothéline humaine et leurs utilisations
WO2018078369A1 (fr) * 2016-10-26 2018-05-03 The University Of York Signaux d'encapsidation viraux

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WO2015033155A1 (fr) * 2013-09-05 2015-03-12 The University Of York Thérapie antivirale
WO2015090230A1 (fr) 2013-12-19 2015-06-25 Novartis Ag Récepteurs antigéniques chimériques de la mésothéline humaine et leurs utilisations
EP4026909A1 (fr) 2013-12-19 2022-07-13 Novartis AG Récepteurs antigéniques chimériques de la mésothéline humaine et leurs utilisations
WO2018078369A1 (fr) * 2016-10-26 2018-05-03 The University Of York Signaux d'encapsidation viraux

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