WO2017104796A1 - Artificial rna restriction enzyme - Google Patents

Artificial rna restriction enzyme Download PDF

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WO2017104796A1
WO2017104796A1 PCT/JP2016/087538 JP2016087538W WO2017104796A1 WO 2017104796 A1 WO2017104796 A1 WO 2017104796A1 JP 2016087538 W JP2016087538 W JP 2016087538W WO 2017104796 A1 WO2017104796 A1 WO 2017104796A1
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rna
restriction enzyme
peptide linker
artificial
hpuf
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PCT/JP2016/087538
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French (fr)
Japanese (ja)
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貴史 世良
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国立大学法人 岡山大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • the present invention relates to an artificial RNA restriction enzyme.
  • the present inventor as a nucleic acid cleaving agent for cleaving DNA, comprises a zinc finger protein that specifically binds to a base sequence located upstream of the target cleavage site, and a nucleic acid cleaving unit that binds to the zinc finger protein.
  • a nucleic acid cleaving agent was proposed (for example, see Patent Document 1).
  • RNA viruses such as influenza virus.
  • the artificial RNA restriction enzyme of the present invention is an artificial RNA restriction enzyme obtained by linking an RNA binding protein that binds to RNA and an enzyme that cleaves a predetermined site of RNA via a peptide linker.
  • the length of the peptide linker is 5 amino acids
  • the enzyme linked via the peptide linker is PIN
  • the peptide linker is characterized in that the length is 5 amino acids or 10 amino acids.
  • the RNA binding protein that binds to RNA is preferably human Pumilio and FBF homology (hPUF) protein or a variant thereof.
  • the peptide linker is preferably a peptide composed of GlyGlyGlyGlySer or a peptide composed of GlyGlyGlyGlySer GlyGlyGlyGlySer.
  • a recombinant expression vector further comprising a promoter, DNA encoding an RNA binding protein that binds RNA, DNA encoding a peptide linker, and DNA encoding an enzyme that cleaves a predetermined site of RNA in this order.
  • RNA restriction enzyme that can effectively cleave RNA viruses such as influenza viruses and render them harmless.
  • FIG. 1 is an electrophoresis of a reaction product generated by cleavage of a substrate RNA with an artificial RNA restriction enzyme.
  • FIG. 2 is an electrophoresis of peptide linker length optimization experiments.
  • FIG. 3 is an electrophoresis of peptide linker length optimization experiments.
  • FIG. 4 shows an outline of the function evaluation (non-replication system) of the artificial RNA restriction enzyme in the cell.
  • FIG. 5 shows the cloning of pCMV (-H) -hPUF-L10-SNase-FLAG.
  • FIG. 6 shows the cloning of pCMV (-H) -hPUF-L10-hPIN-FLAG.
  • FIG. 7 shows the cloning of pCMV (-H) -SNase-FLAG.
  • FIG. 8 shows the cloning of pCMV (-H) -hPIN-FLAG.
  • FIG. 9 shows the cloning of pCMV (-H) -hPUF-L10-SNase-FLAG (-NLS).
  • FIG. 10 shows the cloning of pCMV (-H) -hPUF-L10-hPIN-FLAG (-NLS).
  • FIG. 11 shows the cloning of pCMV (-H) -SNase-FLAG (-NLS).
  • FIG. 12 shows the cloning of pCMV (-H) -hPIN-FLAG (-NLS).
  • FIG. 13 shows the cloning of pCAGGS-hPUF-L10-SNase-FLAG (-NLS).
  • FIG. 14 shows the cloning of pCAGGS-hPUF-L10-hPIN-FLAG (-NLS).
  • FIG. 15 shows the cloning of pCAGGS-SNase-FLAG (-NLS).
  • FIG. 16 shows the cloning of pCAGGS-hPIN-FLAG (-NLS).
  • FIG. 17 shows the cloning of pcDNA-NRE-Luc.
  • FIG. 18 shows the cloning of pcDNA-NRE3X-Luc.
  • FIG. 19 shows the cloning of pcDNA-NRE6X-Luc.
  • FIG. 20 shows the cloning of pcDNA-MT-Luc.
  • FIG. 21 shows the cloning of pcDNA-MT3X-Luc.
  • FIG. 22 shows the cloning of pcDNA-MT6X-Luc.
  • FIG. 23 shows the results of functional evaluation (non-replicating system) of an artificial RNA restriction enzyme in cells.
  • FIG. 24 shows an outline of the function evaluation (replication system) of an artificial RNA restriction enzyme in a cell.
  • FIG. 25 shows the cloning of pCMV (-H) -hPUF-L5-SNase-FLAG.
  • FIG. 26 shows the cloning of pCMV (-H) -hPUF-L5-hPIN-FLAG.
  • FIG. 27 shows the cloning of pCAGGS-hPUF-L5-SNase-FLAG.
  • FIG. 28 shows the cloning of pCAGGS-hPUF-L5-hPIN-FLAG.
  • FIG. 29 shows the cloning of pUC-vRNA (MCS_Luc) (NCR_NS).
  • FIG. 30 shows the cloning of pUC-vRNA (Luc_MCS) (NCR_NS).
  • FIG. 31 shows the cloning of pUC-vRNA (TGTATATA_Luc) (NCR_NS).
  • FIG. 32 shows the cloning of pUC-vRNA [(TGTATATA) 3 _Luc] (NCR_NS).
  • FIG. 33 shows the cloning of pUC-vRNA (Luc_TGTATATA) (NCR_NS).
  • FIG. 34 shows the cloning of pUC-vRNA [Luc_ (TGTATATA) 3 ] (NCR_NS).
  • FIG. 35 shows an RNA expression vector used when targeting vRNA (corresponding to FIG. 36).
  • FIG. 36 shows the results of functional evaluation (replication system) of artificial RNA restriction enzymes in cells.
  • FIG. 37 shows the RNA expression vector used when cRNA was targeted (corresponding to FIG. 38).
  • FIG. 38 shows the results of functional evaluation (replication system) of artificial RNA restriction enzymes in cells.
  • FIG. 39 shows an outline of the function evaluation (replication system) of an artificial RNA restriction enzyme in a cell.
  • FIG. 40 shows the cloning of pUC-vRNA (Luc_Kuma_NP) (NCR_NS).
  • FIG. 41 shows the cloning of pUC-vRNA (Luc_Aki_PA) (NCR_NS).
  • FIG. 42 shows the RNA expression vector used when targeting Kuma_NP (corresponding to FIG. 43).
  • FIG. 43 shows the results of functional evaluation (replication system) of an artificial RNA restriction enzyme in cells.
  • FIG. 44 shows an RNA expression vector used when targeting Aki_PA (corresponding to FIG. 45).
  • FIG. 45 shows the results of functional evaluation (replication system) of artificial RNA restriction enzymes in cells.
  • FIG. 46 shows an outline of functional evaluation (replication system) of an artificial RNA restriction enzyme in cells.
  • FIG. 47 shows the RNA expression vector used when targeting Kuma_NP (corresponding to FIG. 49).
  • FIG. 48 shows the cloning of pUC-vRNA (Kuma_NP) (NCR_NS).
  • FIG. 49 shows the results of functional evaluation (replication system) of an artificial RNA restriction enzyme in cells.
  • FIG. 50 shows the RNA expression vector used in the cytotoxicity evaluation (corresponding to FIG. 51).
  • FIG. 51 shows the results of cytotoxicity evaluation (replication system).
  • the artificial RNA restriction enzyme of the present invention is an artificial RNA restriction enzyme obtained by linking an RNA binding protein that binds to RNA and an enzyme that cleaves a predetermined site of RNA via a peptide linker.
  • an enzyme that cleaves a predetermined site of RNA via a peptide linker is an enzyme that cleaves a predetermined site of RNA via a peptide linker.
  • RNA binding protein that binds to RNA in the present invention is not particularly limited as long as it is a protein that can bind to RNA.
  • RNA binding protein that binds to RNA human Pumilio and FBF homology (hPUF) proteins or their variants are preferably used, but other RNA binding proteins may also be used.
  • hPUF human Pumilio and FBF homology
  • examples of other RNA binding proteins include U1 snRNP, U2 snRNP, U6 snRNP contained in the spliceosome complex, poly A binding protein, RNA modifying enzyme, RISC complex, and the like.
  • the hPUF protein is composed of eight repeat motifs R1, R2, R3, R4, R5, R6, R7, R8, an N-terminal R1 'domain, and a C-terminal R8' domain. That is, the natural (wild type) hPUF protein has a configuration of R1′-R1-R2-R3-R4-R5-R6-R7-R8 -R8 ′.
  • Examples of the modified hPUF protein include modified products in which a part of the motifs R1, R2, R3, R4, R5, R6, R7, and R8 described above are deleted, substituted, or added.
  • An example is a modified form in which R7 is replaced with R5.In this case, it has a configuration of R1′-R1-R2- R3-R4-R5-R6-R5-R8 -R8 ′. become. As long as it has the ability to bind to RNA, it is also possible to use a variant of the hPUF protein as described above.
  • RNA cleaving enzyme such as an RNA cleaving enzyme
  • a nucleic acid cleavage domain that is the entire RNA cleaving enzyme or a part of the full length of the enzyme can be used.
  • nucleic acid cleaving enzymes modified by genetic engineering techniques or other techniques may be used. Nucleic acid cleavage domains in the nucleic acid cleavage enzyme can be easily identified by those skilled in the art by conventional methods, and can be easily obtained by genetic engineering techniques.
  • nucleic acid cleavage enzyme examples include SNase having a cleavage activity for DNA and RNA, or a nucleic acid cleavage domain thereof, or one or more of SNase, preferably several amino acid residues substituted, inserted, or deleted. Mutant SNase and the like can be used.
  • the type of peptide linker used in the present invention is not particularly limited as long as the effects of the present invention are not impaired.
  • a peptide linker usually consisting of 1 to 100 amino acids, preferably 3 to 50 amino acids, more preferably 5 to 30 amino acids, and even more preferably 5 to 15 amino acids can be used.
  • Gly-Ser, Gly-Gly-Ser, Ser-Gly-Gly, Gly-Gly-Gly-Ser, Ser-Gly-Gly-Gly, Gly-Gly-Gly-Gly-Ser, Ser-Gly-Gly-Gly- Gly, Gly-Gly-Gly-Gly-Ser, Ser-Gly-Gly-Gly-Gly-Gly, Gly-Gly-Gly-Gly-Gly-Ser, Ser-Gly-Gly-Gly-Gly-Gly-Gly, (Gly-Gly-Gly-Gly-Ser ) n, (Ser-Gly-Gly-Gly-Gly) n and the like can be used (wherein, n is an integer of 2 to 20, preferably 2 to 10 and more preferably 2). It is an integer of ⁇ 5, particularly preferably 2 or 3, and is not particularly limited.
  • the peptide linker is preferably a peptide composed of GlyGlyGlyGlySer or a peptide composed of GlyGlyGlyGlySer GlyGlyGlyGlySer.
  • the recombinant expression vector of the present invention is a vector for expressing the above-described artificial RNA restriction enzyme of the present invention.
  • the recombinant expression vector of the present invention has in this order a promoter, DNA encoding an RNA binding protein that binds to RNA, DNA encoding a peptide linker, and DNA encoding an enzyme that cleaves a predetermined site of RNA.
  • a promoter DNA encoding an RNA binding protein that binds to RNA
  • DNA encoding a peptide linker DNA encoding an enzyme that cleaves a predetermined site of RNA.
  • T7 promoter, T3 promoter, SP6 promoter etc. can be used.
  • a non-viral vector for example, either a non-viral vector or a viral vector may be used.
  • a viral vector a retrovirus vector (oncoretrovirus vector, lentivirus vector, pseudotype vector, etc.), adenovirus vector, adeno-associated virus vector, simian virus vector, vaccinia virus vector or Sendai virus vector, Epstein-Barr virus vector Virus vectors such as HSV vectors can be used, and various pCMV vectors can be used.
  • RNA restriction enzyme a nucleic acid cleaving enzyme amplified with a primer added with a sequence encoding a peptide linker is introduced into an expression vector for an RNA binding protein, An expression vector in E. coli was constructed.
  • RNA binding domain a human Pumilio and FBF homology (hPUF) protein derivative, which is frequently used in experiments, was used.
  • the hPUF protein is composed of eight repeat motifs R1, R2, R3, R4, R5, R6, R7, R8 that differ in length from the amino acid sequence, an N-terminal R1 'domain, and a C-terminal R8' domain
  • the amino acid sequence of each domain is as follows.
  • R1 HIMEFSQDQHGSRFIQLKLERATPAERQLVFNEILQ (SEQ ID NO: 1)
  • R2 AAYQLMVDVFGCYVIQKFFEFGSLEQKLALAERIRG (SEQ ID NO: 11)
  • R3 HVLSLALQMYGCRVIQKALEFIPSDQQNEMVRELDG (SEQ ID NO: 3)
  • R4 HVLKCVKDQNGNHVVQKCIECVQPQSLQFIIDAFKG (SEQ ID NO: 4)
  • R6 HTEQLVQDQYGNYVIQHVLEHGRPEDKSKIVAEIRG (SEQ ID NO: 6)
  • R7 NVLVLSQHK
  • SNase staphylococcal nuclease
  • a peptide linker a peptide linker in which three flexible GlyGlyGlyGlySers, which are frequently used in experiments, are connected in tandem was used.
  • IPTG was added when the OD600 was ⁇ 0.65, and the mixture was cultured at 37 ° C. for 3 hours to express the artificial RNA restriction enzyme.
  • Escherichia coli expressing the artificial RNA restriction enzyme was collected and disrupted by ultrasonic waves.
  • the suspension obtained by grinding was analyzed by SDS-PAGE to confirm that the artificial RNA restriction enzyme was soluble.
  • the suspension obtained by pulverization was centrifuged to obtain a supernatant. By purifying the supernatant using an affinity column, an artificial RNA restriction enzyme could be obtained with high purity.
  • RNA having target sequence A DNA encoding 190-base RNA RNA derived from the influenza genome containing the target sequence was synthesized and introduced downstream of the T7 promoter of the pET21a vector.
  • RNA region of this vector was cleaved with a restriction enzyme, and then transcribed in a test tube using T7 RNA polymerase. However, since the amount of RNA produced was not sufficient, various reaction conditions and kits were tested. Finally, by using MEGAscript Kit (manufactured by Invitrogen), substrate RNA having a sufficient amount of the target sequence was simultaneously obtained. I was able to get it.
  • RNA cleavage activity of the artificial RNA restriction enzyme purified by the method described above was verified in a test tube.
  • the reaction conditions were a reaction buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% Glycerol, 1 ⁇ g / ⁇ l) containing 0.6 to 1 ⁇ g of substrate RNA having the target sequence and a final concentration of artificial RNA restriction enzyme of 5 nM.
  • the reaction was started by adding a final concentration of 1 mM Ca 2+ to tRNA).
  • FIG. 1 shows the result of electrophoresis. Whether the cleavage product seen after the reaction was cleaved by the artificial RNA restriction enzyme correctly binding to the substrate RNA sequence having the target sequence was evaluated by comparison with the migration position of an appropriate length size marker.
  • the target base sequence in the substrate RNA is mutated so that the RNA-binding protein of the artificial RNA restriction enzyme cannot bind, and the same
  • the band considered to be the target cleavage product was reduced. That is, it was confirmed that cleavage with the substrate RNA was caused by binding to the target sequence.
  • the peptide linker is a 15 amino acid peptide linker obtained by repeating three peptides consisting of 5 amino acids of GlyGlyGlyGlySer.
  • the artificial RNA restriction enzyme is referred to as L15 type.
  • the artificial RNA restriction enzyme having the 10 amino acid peptide linker (GlyGlyGlyGlySer GlyGlyGlyGlySer) by repeating the peptide linker consisting of two peptides consisting of 5 amino acids of GlyGlyGlyGlySer in the same manner as the artificial RNA restriction enzyme production method described above and the L10 type
  • the artificial RNA restriction enzyme consisting of GlyGlyGlyGlySer's 5-amino acid peptide is called L5 type, and there is no peptide linker, and the artificial RNA restriction enzyme that directly binds RNA-binding protein and nucleic acid cleaving enzyme is L0. Let's call it a type.
  • L10 type, L5 type and L0 type artificial RNA restriction enzymes were used for protein expression in Escherichia coli, and the prepared vectors were introduced into BL21 (DE3). The solubility was confirmed.
  • the L10 type, L5 type and L0 type artificial RNA restriction enzymes were not sufficiently soluble at 37 ° C. for 3 hours as the L15 type artificial RNA restriction enzymes. Thus, when culturing was performed under various conditions, the amount of expression was high and the amount of solubility was high when cultured at 30 ° C. for 3 hours at a final inducer concentration of 0.1 ⁇ mM. Therefore, L10 type, L5 type and L0 type artificial RNA restriction enzymes were cultured under these conditions.
  • FIG. 2 shows the results of verifying the cleavage activity of artificial RNA restriction enzymes of L15 type, L10 type, L5 type and L0 type having different peptide linker lengths under the same conditions. From FIG. 2, L5 type artificial RNA restriction enzyme had the highest reactivity.
  • RNA is cleaved instead of the SNase domain. It was decided to use a known PIN domain.
  • an expression vector for a PIN-type artificial RNA restriction enzyme was constructed by the same production method as the above-described SNase-type artificial RNA restriction enzyme.
  • four types of PIN-type artificial RNA restriction enzymes of L15 type, L10 type, L5 type and L0 type having different peptide linker lengths were prepared.
  • each of the prepared vectors was introduced into BL21 (DE3) to confirm the solubility.
  • the solubility was not sufficient when cultured at 37 ° C for 3 hours, the final inducer concentration was 0.1 ⁇ mM at 25 ° C for 7 hours.
  • the expression level was high and the soluble level was high. Therefore, PIN type artificial RNA restriction enzymes of L15 type, L10 type, L5 type and L0 type were cultured under these conditions.
  • FIG. 3 shows the results of verifying the cleavage activity of PIN type artificial RNA restriction enzymes of L15 type, L10 type, L5 type and L0 type having different peptide linker lengths under the same conditions. From FIG. 3, the artificial RNA restriction enzyme of L5 type or L10 type had the highest reactivity.
  • Example 1 Functional evaluation of artificial RNA restriction enzyme in cells (non-replicating system) (Fig. 4) (1) Construction of plasmid (FIGS. 5 to 22) DNA sequence encoding hPUF between EcoRI site and HindIII site of artificial RNA restriction enzyme expression vectors for animal cells pCMV (-H) -hPUF_MT-L10-SNase-FLAG and pCMV (-H) -hPUF_MT-L10-hPIN-FLAG was cloned to construct pCMV (-H) -hPUF-L10-SNase-FLAG and CMV (-H) -hPUF-L10-hPIN-FLAG.
  • PCR was performed using these four types of plasmids as templates, and the amplified products were cloned between the XhoI sites of the pCAGGS plasmid, respectively, and pCAGGS-hPUF-L10-SNase-FLAG (-NLS), pCAGGS-hPUF-L10 -hPIN-FLAG (-NLS), pCAGGS-SNase-FLAG (-NLS), and pCAGGS-hPIN-FLAG (-NLS) were constructed.
  • a DNA sequence encoding the hPUF binding site (TGTATATA) downstream of it and a DNA sequence encoding the luciferase gene are commissioned and cloned between the NdeI site and EcoRI site of pcDNA3.1 (+)
  • TGTATATA hPUF binding site
  • DNA sequences encoding 3 and 6 copies of the hPUF binding site were prepared by annealing and ligation of synthetic oligonucleotides, which were cloned between the SacI site and the BamHI site of pcDNA-NRE-Luc and pcDNA-NRE3X- Luc and pcDNA-NRE6X-Luc were constructed.
  • a DNA sequence (TCATATTA) that encodes 1, 3 and 6 copy sites of mutation sites with mutations introduced into 5 sites of the hPUF binding site was prepared in the same manner, and these were designated as pcDNA-NRE-Luc SacI sites. It was cloned between BamHI sites to construct pcDNA-MT-Luc, pcDNA-MT3X-Luc, and pcDNA-MT6X-Luc.
  • Example 2 Functional evaluation of artificial RNA restriction enzyme in cells (replication system, using luciferase gene) (FIG. 24) (1) Construction of plasmid (FIGS. 25 to 34) Artificial RNA restriction enzyme expression vector for animal cells pCMV (-H) -hPUF_MT-L5-SNase-FLAG and pCMV (-H) -hPUF_MT-L5-hPIN-FLAG DNA sequence encoding hPUF between EcoRI site and HindIII site was cloned to construct pCMV (-H) -hPUF-L5-SNase-FLAG and pCMV (-H) -hPUF-L5-hPIN-FLAG.
  • PCR was performed using these plasmids as templates, and the amplified products were cloned between the XhoI sites of the pCAGGS plasmid to construct pCAGGS-hPUF-L5-SNase-FLAG and pCAGGS-hPUF-L5-hPIN-FLAG. .
  • 5 'untranslated region derived from the segment encoding NS of influenza A virus (A / PR / 8/34 (H1N1)) downstream of human RNA polymerase I promoter, minus strand of luciferase gene , (A / PR / 8/34 (H1N1)) NS segment-derived 3 'untranslated region (3'NCR) and mouse-derived RNA polymerase I terminator are placed in two types of DNA sequences Synthesized.
  • DNA sequences encoding 1 and 3 copies of the hPUF binding site were generated by annealing synthetic oligonucleotides, which were cloned between the AgeI and BglII sites of pUC-vRNA (MCS_Luc) (NCR_NS) vRNA (TGTATATA_Luc) (NCR_NS) and pUC-vRNA [(TGTATATA) 3 _Luc] (NCR_NS) were constructed.
  • DNA sequences encoding 1 and 3 copies of the hPUF binding site were prepared by annealing synthetic oligonucleotides, and these were transcribed into the negative strand between the AgeI and BglII sites of pUC-vRNA (Luc_MCS) (NCR_NS).
  • PUC-vRNA (Luc_TGTATATA) (NCR_NS) and pUC-vRNA [Luc_ (TGTATATA) 3 ] (NCR_NS) were constructed by cloning in the direction.
  • cell lysate was prepared with 100 ml of 1 ⁇ Passive Lysis Buffer, and luciferase activity was measured using Luciferase Assay System.
  • ⁇ -galactosidase activity was measured using Luminescent b-galactosidase Detection Kit II to standardize transfection efficiency between wells. After normalizing the luciferase activity value with the ⁇ -galactosidase activity value, the relative luciferase activity was evaluated based on the condition where the empty vector pcDNA3.1 (+) was transfected.
  • TGTATATA_Luc When pUC-vRNA (TGTATATA_Luc) (NCR_NS) and pUC-vRNA [(TGTATATA) 3 _Luc] (NCR_NS) are used for the reporter plasmid, there is an hPUF binding site in the vRNA (minus strand) and pUC-vRNA (Luc_TGTATATA ) When (NCR_NS) and pUC-vRNA [Luc_ (TGTATATA) 3 ] (NCR_NS) are used, there are hPUF binding sites in cRNA (plus strand) and mRNA.
  • Example 3 Functional evaluation of artificial RNA restriction enzyme in cells (replication system) (FIG. 39)
  • Construction of plasmid (FIGS. 40 to 41) A DNA sequence encoding NP derived from influenza A virus (A / chicken / kumamoto / 1-7 / 2014 (H5N8)) is commissioned and synthesized using this as a template, and the amplified product is pUC-vRNA (Luc_MCS PUC-vRNA (Luc_Kuma_NP) (NCR_NS) was constructed by cloning in the direction in which the minus strand was transcribed between the AgeI and BglII sites of (NCR_NS).
  • cell lysate was prepared with 100 ml of 1 ⁇ Passive Lysis Buffer, and luciferase activity was measured using Luciferase Assay System.
  • ⁇ -galactosidase activity was measured using Luminescent b-galactosidase Detection Kit II to standardize transfection efficiency between wells. After normalizing the luciferase activity value with the ⁇ -galactosidase activity value, the relative luciferase activity was evaluated based on the condition where the empty vector pcDNA3.1 (+) was transfected.
  • NCR_NS When pUC-vRNA (Luc_Kuma_NP) (NCR_NS) is used as the reporter plasmid, there is an hPUF binding site in vRNA (minus strand), and when pUC-vRNA (Luc_Aki_PA) (NCR_NS) is used, cRNA (plus strand) And there is an hPUF binding site in the mRNA.
  • Luciferase activity was reduced by the artificial RNA restriction enzyme of the present invention.
  • Example 4 Functional evaluation of artificial RNA restriction enzyme in cells (replication system, using viral genome) Analysis at RNA level)
  • Figure 46 (1) Construction of plasmid (FIGS. 47 to 48) 5 'untranslated region (5'NCR) derived from NS coding segment of influenza A virus (A / PR / 8/34 (H1N1)) downstream of human RNA polymerase I promoter, influenza A virus ( A / chicken / kumamoto / 1-7 / 2014 (H5N8))-derived NP gene minus strand, (A / PR / 8/34 (H1N1)) NS-derived segment 3 'untranslated region (3 'NCR) and a DNA sequence in which a mouse RNA polymerase I terminator was placed were commissioned and synthesized. This was cloned between the XbaI site and Acc65I site of pUC19 to construct a viral RNA expression plasmid pUC-vRNA (Kuma_NP) (NCR_NS
  • RNA Extract the total RNA using TRIzol and RNeasy Mini kit after incubation for 48 hours in a 37 ° C, 5% CO 2 culture tank. After DNaseI treatment, perform reverse transcription using viral RNA-specific primers and RNaseH treatment. In order to quantify viral RNA replicated in the cells, real-time PCR analysis was performed using TaqMan Fast Advanced Master Mix. In addition, in order to standardize transfection efficiency between dishes, reverse transcription reaction was performed with random primers using total RNA extracted, and real-time PCR analysis was performed to quantify ⁇ -galactosidase mRNA expression after RNaseH treatment. went. After normalizing the amount of viral RNA in the cells with the expression level of ⁇ -galactosidase mRNA, the relative amount of viral RNA replication was evaluated based on the condition of transfection with the empty vector pcDNA3.1 (+).
  • the measurement results are shown in FIG.
  • the relative amount of viral RNA replication was reduced by the artificial RNA restriction enzyme of the present invention.
  • Example 5 Functional evaluation of artificial RNA restriction enzyme in cells (replication system, using viral genome, cytotoxicity evaluation) 2 ⁇ 10 4 293T cells are seeded in poly-D-lysine 96-well plate, cultured for 24 hours in 10% inactivated FBS-containing DMEM medium at 37 ° C in CO 2 5% culture tank, then artificial RNA restriction enzyme expression 96 ng of plasmid or 96 ng of empty vector pcDNA3.1 (+) 4 kinds of influenza virus-derived protein expression plasmids pCAGGS-PA, pCAGGS-PB1, pCAGGS-PB2, pCAGGS-NP each 1 ng, viral RNA expression plasmid pUC- 293T cells were co-transfected using Lipofectamine 3000 with 0.2 ng of vRNA (Kuma_NP) (NCR_NS) and 0.2 ng of ⁇ -galactosidase expression plasmid pCMV-b.
  • Lipofectamine 3000 with
  • the measurement results are shown in FIG. It was confirmed that the artificial RNA restriction enzyme of the present invention does not show cytotoxicity.

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Abstract

The present invention addresses the problem of providing an artificial RNA restriction enzyme with which it is possible to disable an RNA virus such as an influenza virus by cleavage. The invention is an artificial RNA restriction enzyme formed by linking an RNA-binding protein that binds with RNA and an enzyme that cleaves a predetermined site of the RNA, via a peptide linker. The invention is also characterized in that the length of the peptide linker is five amino acids when the enzyme linked via the peptide linker is an SNase and the length of the peptide linker is five amino acids or ten amino acids when the enzyme linked via the peptide linker is a PIN.

Description

人工ＲＮＡ制限酵素Artificial RNA restriction enzyme
　本発明は、人工ＲＮＡ制限酵素に関するものである。 The present invention relates to an artificial RNA restriction enzyme.
　本発明者は、ＤＮＡを切断する核酸切断剤として、標的切断部位の上流に位置する塩基配列に対して特異的に結合するジンクフィンガータンパク質と、このジンクフィンガータンパク質に結合した核酸切断部とで構成した核酸切断剤を提案した（例えば、特許文献１参照。）。 The present inventor, as a nucleic acid cleaving agent for cleaving DNA, comprises a zinc finger protein that specifically binds to a base sequence located upstream of the target cleavage site, and a nucleic acid cleaving unit that binds to the zinc finger protein. A nucleic acid cleaving agent was proposed (for example, see Patent Document 1).
特表２００９－５２８８１６号公報Special table 2009-528816
　核酸切断剤の考え方をＲＮＡに展開すれば、インフルエンザウイルス等のＲＮＡウイルスに効果的な薬剤となり得ると考えた。 We thought that if the concept of nucleic acid cleaving agent was developed in RNA, it could be an effective drug for RNA viruses such as influenza virus.
　本発明の人工ＲＮＡ制限酵素は、ＲＮＡと結合するＲＮＡ結合タンパク質と、ＲＮＡの所定の部位を切断する酵素とを、ペプチドリンカーを介して連結して成る人工ＲＮＡ制限酵素である。 The artificial RNA restriction enzyme of the present invention is an artificial RNA restriction enzyme obtained by linking an RNA binding protein that binds to RNA and an enzyme that cleaves a predetermined site of RNA via a peptide linker.
　さらに、本発明の人工ＲＮＡ制限酵素では、ペプチドリンカーを介して連結する酵素がSNaseの場合には、ペプチドリンカーの長さを５アミノ酸とし、ペプチドリンカーを介して連結する酵素がPINの場合には、ペプチドリンカーの長さを５アミノ酸または１０アミノ酸とすることにも特徴を有するものである。 Furthermore, in the artificial RNA restriction enzyme of the present invention, when the enzyme linked via the peptide linker is SNase, the length of the peptide linker is 5 amino acids, and when the enzyme linked via the peptide linker is PIN Further, the peptide linker is characterized in that the length is 5 amino acids or 10 amino acids.
　本発明においては、好ましくは、ＲＮＡと結合するＲＮＡ結合タンパク質は、ヒトPumilio及びFBFホモロジー（hPUF)タンパク質又はその改変体である。
　本発明において、好ましくは、ペプチドリンカーは、GlyGlyGlyGlySerからなるペプチド、又はGlyGlyGlyGlySer GlyGlyGlyGlySerからなるペプチドである。 In the present invention, the RNA binding protein that binds to RNA is preferably human Pumilio and FBF homology (hPUF) protein or a variant thereof.
In the present invention, the peptide linker is preferably a peptide composed of GlyGlyGlyGlySer or a peptide composed of GlyGlyGlyGlySer GlyGlyGlyGlySer.
　本発明によればさらに、プロモーター、ＲＮＡと結合するＲＮＡ結合タンパク質をコードするＤＮＡ、ペプチドリンカーをコードするＤＮＡ、及びＲＮＡの所定の部位を切断する酵素をコードするＤＮＡとをこの順に有する組み換え発現ベクターが提供される。 According to the present invention, a recombinant expression vector further comprising a promoter, DNA encoding an RNA binding protein that binds RNA, DNA encoding a peptide linker, and DNA encoding an enzyme that cleaves a predetermined site of RNA in this order. Is provided.
　本発明によれば、インフルエンザウイルス等のＲＮＡウイルスを効果的に切断して無害化可能とする人工ＲＮＡ制限酵素を提供できる。 According to the present invention, it is possible to provide an artificial RNA restriction enzyme that can effectively cleave RNA viruses such as influenza viruses and render them harmless.
図１は、人工ＲＮＡ制限酵素による基質ＲＮＡの切断によって生じる反応生成物の電気泳動である。FIG. 1 is an electrophoresis of a reaction product generated by cleavage of a substrate RNA with an artificial RNA restriction enzyme. 図２は、ペプチドリンカーの長さの最適化実験の電気泳動である。FIG. 2 is an electrophoresis of peptide linker length optimization experiments. 図３は、ペプチドリンカーの長さの最適化実験の電気泳動である。FIG. 3 is an electrophoresis of peptide linker length optimization experiments. 図４は、細胞内での人工RNA制限酵素の機能評価（非複製系）の概要を示す。FIG. 4 shows an outline of the function evaluation (non-replication system) of the artificial RNA restriction enzyme in the cell. 図５は、pCMV(-H)-hPUF-L10-SNase-FLAGのクローニングを示す。FIG. 5 shows the cloning of pCMV (-H) -hPUF-L10-SNase-FLAG. 図６は、pCMV(-H)-hPUF-L10-hPIN-FLAGのクローニングを示す。FIG. 6 shows the cloning of pCMV (-H) -hPUF-L10-hPIN-FLAG. 図７は、pCMV(-H)-SNase-FLAGのクローニングを示す。FIG. 7 shows the cloning of pCMV (-H) -SNase-FLAG. 図８は、pCMV(-H)-hPIN-FLAGのクローニングを示す。FIG. 8 shows the cloning of pCMV (-H) -hPIN-FLAG. 図９は、pCMV(-H)-hPUF-L10-SNase-FLAG(-NLS)のクローニングを示す。FIG. 9 shows the cloning of pCMV (-H) -hPUF-L10-SNase-FLAG (-NLS). 図１０は、pCMV(-H)-hPUF-L10-hPIN-FLAG(-NLS)のクローニングを示す。FIG. 10 shows the cloning of pCMV (-H) -hPUF-L10-hPIN-FLAG (-NLS). 図１１は、pCMV(-H)-SNase-FLAG(-NLS)のクローニングを示す。FIG. 11 shows the cloning of pCMV (-H) -SNase-FLAG (-NLS). 図１２は、pCMV(-H)-hPIN-FLAG(-NLS)のクローニングを示す。FIG. 12 shows the cloning of pCMV (-H) -hPIN-FLAG (-NLS). 図１３は、pCAGGS-hPUF-L10-SNase-FLAG(-NLS)のクローニングを示す。FIG. 13 shows the cloning of pCAGGS-hPUF-L10-SNase-FLAG (-NLS). 図１４は、pCAGGS-hPUF-L10-hPIN-FLAG(-NLS)のクローニングを示す。FIG. 14 shows the cloning of pCAGGS-hPUF-L10-hPIN-FLAG (-NLS). 図１５は、pCAGGS-SNase-FLAG（-NLS）のクローニングを示す。FIG. 15 shows the cloning of pCAGGS-SNase-FLAG (-NLS). 図１６は、pCAGGS-hPIN-FLAG（-NLS）のクローニングを示す。FIG. 16 shows the cloning of pCAGGS-hPIN-FLAG (-NLS). 図１７は、pcDNA-NRE-Lucのクローニングを示す。FIG. 17 shows the cloning of pcDNA-NRE-Luc. 図１８は、pcDNA-NRE3X-Lucのクローニングを示す。FIG. 18 shows the cloning of pcDNA-NRE3X-Luc. 図１９は、pcDNA-NRE6X-Lucのクローニングを示す。FIG. 19 shows the cloning of pcDNA-NRE6X-Luc. 図２０は、pcDNA-MT-Lucのクローニングを示す。FIG. 20 shows the cloning of pcDNA-MT-Luc. 図２１は、pcDNA-MT3X-Lucのクローニングを示す。FIG. 21 shows the cloning of pcDNA-MT3X-Luc. 図２２は、pcDNA-MT6X-Lucのクローニングを示す。FIG. 22 shows the cloning of pcDNA-MT6X-Luc. 図２３は、細胞内での人工RNA制限酵素の機能評価（非複製系）の結果を示す。FIG. 23 shows the results of functional evaluation (non-replicating system) of an artificial RNA restriction enzyme in cells. 図２４は、細胞内での人工RNA制限酵素の機能評価（複製系）の概要を示す。FIG. 24 shows an outline of the function evaluation (replication system) of an artificial RNA restriction enzyme in a cell. 図２５は、pCMV(-H)-hPUF-L5-SNase-FLAGのクローニングを示す。FIG. 25 shows the cloning of pCMV (-H) -hPUF-L5-SNase-FLAG. 図２６は、pCMV(-H)-hPUF-L5-hPIN-FLAGのクローニングを示す。FIG. 26 shows the cloning of pCMV (-H) -hPUF-L5-hPIN-FLAG. 図２７は、pCAGGS-hPUF-L5-SNase-FLAGのクローニングを示す。FIG. 27 shows the cloning of pCAGGS-hPUF-L5-SNase-FLAG. 図２８は、pCAGGS-hPUF-L5-hPIN-FLAGのクローニングを示す。FIG. 28 shows the cloning of pCAGGS-hPUF-L5-hPIN-FLAG. 図２９は、pUC-vRNA(MCS_Luc)(NCR_NS)のクローニングを示す。FIG. 29 shows the cloning of pUC-vRNA (MCS_Luc) (NCR_NS). 図３０は、pUC-vRNA(Luc_MCS)(NCR_NS)のクローニングを示す。FIG. 30 shows the cloning of pUC-vRNA (Luc_MCS) (NCR_NS). 図３１は、pUC-vRNA(TGTATATA_Luc)(NCR_NS)のクローニングを示す。FIG. 31 shows the cloning of pUC-vRNA (TGTATATA_Luc) (NCR_NS). 図３２は、pUC-vRNA[(TGTATATA)3_Luc] (NCR_NS)のクローニングを示す。FIG. 32 shows the cloning of pUC-vRNA [(TGTATATA) 3 _Luc] (NCR_NS). 図３３は、pUC-vRNA(Luc_TGTATATA)(NCR_NS)のクローニングを示す。FIG. 33 shows the cloning of pUC-vRNA (Luc_TGTATATA) (NCR_NS). 図３４は、pUC-vRNA[Luc_(TGTATATA)3](NCR_NS) のクローニングを示す。FIG. 34 shows the cloning of pUC-vRNA [Luc_ (TGTATATA) 3 ] (NCR_NS). 図３５は、vRNAを標的とした際に使用したRNA発現ベクターを示す（図３６に対応）。FIG. 35 shows an RNA expression vector used when targeting vRNA (corresponding to FIG. 36). 図３６は、細胞内での人工RNA制限酵素の機能評価（複製系）の結果を示す。FIG. 36 shows the results of functional evaluation (replication system) of artificial RNA restriction enzymes in cells. 図３７は、cRNAを標的とした際に使用したRNA発現ベクターを示す（図３８に対応）。FIG. 37 shows the RNA expression vector used when cRNA was targeted (corresponding to FIG. 38). 図３８は、細胞内での人工RNA制限酵素の機能評価（複製系）の結果を示す。FIG. 38 shows the results of functional evaluation (replication system) of artificial RNA restriction enzymes in cells. 図３９は、細胞内での人工RNA制限酵素の機能評価（複製系）の概要を示す。FIG. 39 shows an outline of the function evaluation (replication system) of an artificial RNA restriction enzyme in a cell. 図４０は、pUC-vRNA(Luc_Kuma_NP)(NCR_NS)のクローニングを示す。FIG. 40 shows the cloning of pUC-vRNA (Luc_Kuma_NP) (NCR_NS). 図４１は、pUC-vRNA(Luc_Aki_PA)(NCR_NS)のクローニングを示す。FIG. 41 shows the cloning of pUC-vRNA (Luc_Aki_PA) (NCR_NS). 図４２は、Kuma_NPを標的とした際に使用したRNA発現ベクターを示す（図４３に対応）。FIG. 42 shows the RNA expression vector used when targeting Kuma_NP (corresponding to FIG. 43). 図４３は、細胞内での人工RNA制限酵素の機能評価（複製系）の結果を示す。FIG. 43 shows the results of functional evaluation (replication system) of an artificial RNA restriction enzyme in cells. 図４４は、Aki_PAを標的とした際に使用したRNA発現ベクターを示す（図４５に対応）FIG. 44 shows an RNA expression vector used when targeting Aki_PA (corresponding to FIG. 45). 図４５は、細胞内での人工RNA制限酵素の機能評価（複製系）の結果を示す。FIG. 45 shows the results of functional evaluation (replication system) of artificial RNA restriction enzymes in cells. 図４６は、細胞内での人工RNA制限酵素の機能評価（複製系）の概要を示す。FIG. 46 shows an outline of functional evaluation (replication system) of an artificial RNA restriction enzyme in cells. 図４７は、Kuma_NPを標的とした際に使用したRNA発現ベクターを示す（図４９に対応）。FIG. 47 shows the RNA expression vector used when targeting Kuma_NP (corresponding to FIG. 49). 図４８は、pUC-vRNA(Kuma_NP)(NCR_NS)のクローニングを示す。FIG. 48 shows the cloning of pUC-vRNA (Kuma_NP) (NCR_NS). 図４９は、細胞内での人工RNA制限酵素の機能評価（複製系）の結果を示す。FIG. 49 shows the results of functional evaluation (replication system) of an artificial RNA restriction enzyme in cells. 図５０は、細胞毒性評価の際に使用したRNA発現ベクターを示す（図５１に対応）。FIG. 50 shows the RNA expression vector used in the cytotoxicity evaluation (corresponding to FIG. 51). 図５１は、細胞毒性評価（複製系）の結果を示す。FIG. 51 shows the results of cytotoxicity evaluation (replication system).
　本発明の人工ＲＮＡ制限酵素は、ＲＮＡと結合するＲＮＡ結合タンパク質と、ＲＮＡの所定の部位を切断する酵素とを、ペプチドリンカーを介して連結して成る人工ＲＮＡ制限酵素である。以下において、本発明の実施形態を具体的に説明する。 The artificial RNA restriction enzyme of the present invention is an artificial RNA restriction enzyme obtained by linking an RNA binding protein that binds to RNA and an enzyme that cleaves a predetermined site of RNA via a peptide linker. In the following, embodiments of the present invention will be specifically described.
　本発明における「ＲＮＡと結合するＲＮＡ結合タンパク質」は、ＲＮＡと結合することができるタンパク質であればよく、その種類は特に限定されない。 The “RNA binding protein that binds to RNA” in the present invention is not particularly limited as long as it is a protein that can bind to RNA.
　ＲＮＡと結合するＲＮＡ結合タンパク質としては、ヒトPumilio及びFBFホモロジー（hPUF)タンパク質又はその改変体を使用することが好ましいが、その他のＲＮＡ結合タンパク質を使用してもよい。その他のＲＮＡ結合タンパク質としては、例えば、スプライソソーム複合体に含まれるＵ１ｓｎＲＮＰ、Ｕ２ｓｎＲＮＰ、Ｕ６ｓｎＲＮＰ、あるいはポリＡ結合タンパク質、ＲＮＡ修飾酵素、ＲＩＳＣ複合体等が挙げられる。 As the RNA binding protein that binds to RNA, human Pumilio and FBF homology (hPUF) proteins or their variants are preferably used, but other RNA binding proteins may also be used. Examples of other RNA binding proteins include U1 snRNP, U2 snRNP, U6 snRNP contained in the spliceosome complex, poly A binding protein, RNA modifying enzyme, RISC complex, and the like.
　hPUFタンパク質は、後記する８つのリピートモチーフR1,R2,R3,R4,R5,R6,R7,R8と、N末端のR1’ドメインと、C末端のをR8’ドメインとで構成されている。即ち、天然(野生型) hPUFタンパク質は、R1’-R1-R2-R3-R4-R5-R6-R7-R8 -R8’という構成を有している。hPUFタンパク質の改変体としては、上記したR1,R2,R3,R4,R5,R6,R7,R8というモチーフの一部を欠失、置換、又は付加させた改変体を挙げることができる。一例としては、R7をR5に置き換えた改変体等を挙げることができ、この場合には、R1’-R1-R2- R3-R4-R5-R6-R5-R8 -R8’という構成を有することになる。ＲＮＡへの結合能を有する限り、上記したようなhPUFタンパク質の改変体を使用することも可能である。 The hPUF protein is composed of eight repeat motifs R1, R2, R3, R4, R5, R6, R7, R8, an N-terminal R1 'domain, and a C-terminal R8' domain. That is, the natural (wild type) hPUF protein has a configuration of R1′-R1-R2-R3-R4-R5-R6-R7-R8 -R8 ′. Examples of the modified hPUF protein include modified products in which a part of the motifs R1, R2, R3, R4, R5, R6, R7, and R8 described above are deleted, substituted, or added. An example is a modified form in which R7 is replaced with R5.In this case, it has a configuration of R1′-R1-R2- R3-R4-R5-R6-R5-R8 -R8 ′. become. As long as it has the ability to bind to RNA, it is also possible to use a variant of the hPUF protein as described above.
　ＲＮＡの所定の部位を切断する酵素としては、ＲＮＡ切断酵素等の核酸切断酵素を使用することができる。例えば、ＲＮＡ切断酵素の全体又は該酵素の全長の一部である核酸切断ドメインを用いることができる。天然型の核酸切断酵素、例えばスタフィロコッカルヌクレアーゼ(ＳＮａｓｅ)などのほか、遺伝子工学的手法や他の手法により改変された核酸切断酵素を用いてもよい。核酸切断酵素中の核酸切断ドメインは常法により当業者が容易に特定することができ、遺伝子工学的な手法により容易に入手することが可能である。核酸切断酵素としては、例えば、ＤＮＡ及びＲＮＡに対して切断活性を有するＳＮａｓｅ又はその核酸切断ドメイン、あるいはＳＮａｓｅの１又は２以上、好ましくは数個の構成アミノ酸残基を置換、挿入、欠失した変異体ＳＮａｓｅなどを用いることができる。 As the enzyme that cleaves a predetermined site of RNA, a nucleic acid cleaving enzyme such as an RNA cleaving enzyme can be used. For example, a nucleic acid cleavage domain that is the entire RNA cleaving enzyme or a part of the full length of the enzyme can be used. In addition to natural nucleic acid cleaving enzymes such as staphylococcal nuclease (SNase), nucleic acid cleaving enzymes modified by genetic engineering techniques or other techniques may be used. Nucleic acid cleavage domains in the nucleic acid cleavage enzyme can be easily identified by those skilled in the art by conventional methods, and can be easily obtained by genetic engineering techniques. Examples of the nucleic acid cleavage enzyme include SNase having a cleavage activity for DNA and RNA, or a nucleic acid cleavage domain thereof, or one or more of SNase, preferably several amino acid residues substituted, inserted, or deleted. Mutant SNase and the like can be used.
　本発明で用いるペプチドリンカーの種類は、本発明の効果が損なわれない限り特に限定されない。例えば、通常、1から100アミノ酸、好ましくは3から50アミノ酸、より好ましくは5から30アミノ酸、さらに好ましくは5から15アミノ酸からなるペプチドリンカーを使用することができる。 The type of peptide linker used in the present invention is not particularly limited as long as the effects of the present invention are not impaired. For example, a peptide linker usually consisting of 1 to 100 amino acids, preferably 3 to 50 amino acids, more preferably 5 to 30 amino acids, and even more preferably 5 to 15 amino acids can be used.
　ペプチドリンカーとしては、
Gly-Ser、Gly-Gly-Ser、Ser-Gly-Gly、Gly-Gly-Gly-Ser、Ser-Gly-Gly-Gly、Gly-Gly-Gly-Gly-Ser、Ser-Gly-Gly-Gly-Gly、Gly-Gly-Gly-Gly-Gly-Ser、
Ser-Gly-Gly-Gly-Gly-Gly、Gly-Gly-Gly-Gly-Gly-Gly-Ser、Ser-Gly-Gly-Gly-Gly-Gly-Gly、 (Gly-Gly-Gly-Gly-Ser)n、(Ser-Gly-Gly-Gly-Gly)n等を使用することができるが（式中、ｎは２～２０の整数であり、好ましくは２～１０整数であり、より好ましくは２～５の整数であり、特に好ましくは２又は３である）、特に限定されない。 As a peptide linker,
Gly-Ser, Gly-Gly-Ser, Ser-Gly-Gly, Gly-Gly-Gly-Ser, Ser-Gly-Gly-Gly, Gly-Gly-Gly-Gly-Ser, Ser-Gly-Gly-Gly- Gly, Gly-Gly-Gly-Gly-Gly-Ser,
Ser-Gly-Gly-Gly-Gly-Gly, Gly-Gly-Gly-Gly-Gly-Gly-Ser, Ser-Gly-Gly-Gly-Gly-Gly-Gly, (Gly-Gly-Gly-Gly-Ser ) n, (Ser-Gly-Gly-Gly-Gly) n and the like can be used (wherein, n is an integer of 2 to 20, preferably 2 to 10 and more preferably 2). It is an integer of ˜5, particularly preferably 2 or 3, and is not particularly limited.
　本発明において、好ましくは、ペプチドリンカーは、GlyGlyGlyGlySerからなるペプチド、又はGlyGlyGlyGlySer GlyGlyGlyGlySerからなるペプチドである。 In the present invention, the peptide linker is preferably a peptide composed of GlyGlyGlyGlySer or a peptide composed of GlyGlyGlyGlySer GlyGlyGlyGlySer.
　本発明の組み換え発現ベクターは、上記した本発明の人工ＲＮＡ制限酵素を発現するためのベクターである。本発明の組み換え発現ベクターは、プロモーター、ＲＮＡと結合するＲＮＡ結合タンパク質をコードするＤＮＡ、ペプチドリンカーをコードするＤＮＡ、及びＲＮＡの所定の部位を切断する酵素をコードするＤＮＡとをこの順に有する。本発明で用いるプロモーターの種類は特に限定されないが、Ｔ７プロモーター、Ｔ３プロモーター、ＳＰ６プロモーターなどを使用することができる。 The recombinant expression vector of the present invention is a vector for expressing the above-described artificial RNA restriction enzyme of the present invention. The recombinant expression vector of the present invention has in this order a promoter, DNA encoding an RNA binding protein that binds to RNA, DNA encoding a peptide linker, and DNA encoding an enzyme that cleaves a predetermined site of RNA. Although the kind of promoter used by this invention is not specifically limited, T7 promoter, T3 promoter, SP6 promoter etc. can be used.
　ベクターとしては、例えば、非ウイルスベクター又はウイルスベクターの何れでもよい。ウイルスベクターとしては、レトロウイルスベクター（オンコレトロウイルスベクター、レンチウイルスベクター、シュードタイプベクター等）、アデノウイルスベクター、アデノ随伴ウイルスベクター、シミアンウイルスベクター、ワクシニアウイルスベクター又はセンダイウイルスベクター、エプスタイン－バーウイルスベクター、ＨＳＶベクターなどのウイルスベクターが使用できるまた、ｐＣＭＶ系の各種ベクターを使用することができる。 As the vector, for example, either a non-viral vector or a viral vector may be used. As a viral vector, a retrovirus vector (oncoretrovirus vector, lentivirus vector, pseudotype vector, etc.), adenovirus vector, adeno-associated virus vector, simian virus vector, vaccinia virus vector or Sendai virus vector, Epstein-Barr virus vector Virus vectors such as HSV vectors can be used, and various pCMV vectors can be used.
　以下に本発明の具体例をさらに説明する。 Specific examples of the present invention will be further described below.
＜人工ＲＮＡ制限酵素の作製＞
　まず、本発明の人工ＲＮＡ制限酵素を大腸菌を用いて作製可能とするために、ＲＮＡ結合タンパク質の発現ベクターに、ペプチドリンカーをコードする配列を付加したプライマーで増幅した核酸切断酵素を導入して、大腸菌での発現ベクターを構築した。 <Production of artificial RNA restriction enzyme>
First, in order to be able to produce the artificial RNA restriction enzyme of the present invention using Escherichia coli, a nucleic acid cleaving enzyme amplified with a primer added with a sequence encoding a peptide linker is introduced into an expression vector for an RNA binding protein, An expression vector in E. coli was constructed.
　ここで、ＲＮＡ結合ドメインとしては、実験上、よく利用している、human Pumilio and FBF homology （hPUF)タンパク質誘導体を用いた。hPUFタンパク質は、アミノ酸配列と長さが異なる８つのリピートモチーフR1,R2,R3,R4,R5,R6,R7,R8と、N末端のR1’ドメインと、C末端のをR8’ドメインとで構成しており、各ドメインのアミノ酸配列は、以下のようになっている。
　　R1'：GRSRLLEDFRNNRYPNLQLREIAG （配列番号９）
　　R1 ：HIMEFSQDQHGSRFIQLKLERATPAERQLVFNEILQ（配列番号１）
　　R2 ：AAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRG（配列番号２）
　　R3 ：HVLSLALQMYGCRVIQKALEFIPSDQQNEMVRELDG（配列番号３）
　　R4 ：HVLKCVKDQNGNHVVQKCIECVQPQSLQFIIDAFKG（配列番号４）
　　R5 ：QVFALSTHPYGCRVIQRILEHCLPDQTLPILEELHQ（配列番号５）
　　R6 ：HTEQLVQDQYGNYVIQHVLEHGRPEDKSKIVAEIRG（配列番号６）
　　R7 ：NVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHS（配列番号７）
　　R8 ：ALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRP（配列番号８）
　　R8'：HIATLRKYTYGKHILAKLEKYYMKNGVDLG　（配列番号１０） Here, as an RNA binding domain, a human Pumilio and FBF homology (hPUF) protein derivative, which is frequently used in experiments, was used. The hPUF protein is composed of eight repeat motifs R1, R2, R3, R4, R5, R6, R7, R8 that differ in length from the amino acid sequence, an N-terminal R1 'domain, and a C-terminal R8' domain The amino acid sequence of each domain is as follows.
R1 ': GRSRLLEDFRNNRYPNLQLREIAG (SEQ ID NO: 9)
R1: HIMEFSQDQHGSRFIQLKLERATPAERQLVFNEILQ (SEQ ID NO: 1)
R2: AAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRG (SEQ ID NO: 2)
R3: HVLSLALQMYGCRVIQKALEFIPSDQQNEMVRELDG (SEQ ID NO: 3)
R4: HVLKCVKDQNGNHVVQKCIECVQPQSLQFIIDAFKG (SEQ ID NO: 4)
R5: QVFALSTHPYGCRVIQRILEHCLPDQTLPILEELHQ (SEQ ID NO: 5)
R6: HTEQLVQDQYGNYVIQHVLEHGRPEDKSKIVAEIRG (SEQ ID NO: 6)
R7: NVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHS (SEQ ID NO: 7)
R8: ALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRP (SEQ ID NO: 8)
R8 ': HIATLRKYTYGKHILAKLEKYYMKNGVDLG (SEQ ID NO: 10)
　また、変異型であるhPUF_MTは、８つのリピートモチーフR1,R2,R3,R4,R5,R6,R7,R8が以下の配列を有している。
　　R1 ：HIMEFSQDQHGSRFIQLKLERATPAERQLVFNEILQ（配列番号１）
　　R2 ：AAYQLMVDVFGCYVIQKFFEFGSLEQKLALAERIRG（配列番号１１）
　　R3 ：HVLSLALQMYGCRVIQKALEFIPSDQQNEMVRELDG（配列番号３）
　　R4 ：HVLKCVKDQNGNHVVQKCIECVQPQSLQFIIDAFKG（配列番号４）
　　R5 ：QVFALSTHPYGCRVIQRILEHCLPDQTLPILEELHQ（配列番号５）
　　R6 ：HTEQLVQDQYGNYVIQHVLEHGRPEDKSKIVAEIRG（配列番号６）
　　R7 ：NVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHS（配列番号７）
　　R8 ：ALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRP（配列番号８） Further, in the hPUF_MT which is a mutant type, eight repeat motifs R1, R2, R3, R4, R5, R6, R7, and R8 have the following sequences.
R1: HIMEFSQDQHGSRFIQLKLERATPAERQLVFNEILQ (SEQ ID NO: 1)
R2: AAYQLMVDVFGCYVIQKFFEFGSLEQKLALAERIRG (SEQ ID NO: 11)
R3: HVLSLALQMYGCRVIQKALEFIPSDQQNEMVRELDG (SEQ ID NO: 3)
R4: HVLKCVKDQNGNHVVQKCIECVQPQSLQFIIDAFKG (SEQ ID NO: 4)
R5: QVFALSTHPYGCRVIQRILEHCLPDQTLPILEELHQ (SEQ ID NO: 5)
R6: HTEQLVQDQYGNYVIQHVLEHGRPEDKSKIVAEIRG (SEQ ID NO: 6)
R7: NVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHS (SEQ ID NO: 7)
R8: ALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRP (SEQ ID NO: 8)
　また、核酸切断酵素としては、実験上、よく利用している核酸切断酵素であるstaphylococcal nuclease（SNase）を用いた。さらに、ペプチドリンカーとしては、実験上、よく利用しているフレキシブルなGlyGlyGlyGlySerを３つタンデムにつなげたペプチドリンカーを用いた。 Further, as the nucleic acid cleaving enzyme, staphylococcal nuclease (SNase), which is a nucleic acid cleaving enzyme that is frequently used in experiments, was used. Further, as a peptide linker, a peptide linker in which three flexible GlyGlyGlyGlySers, which are frequently used in experiments, are connected in tandem was used.
　上記の発現ベクターを発現用大腸菌株BL21(DE3)に導入した後、OD600が～0.65になった時にIPTGを添加して、37℃で３時間培養して、人工ＲＮＡ制限酵素を発現させた。 After introducing the above expression vector into the expression Escherichia coli strain BL21 (DE3), IPTG was added when the OD600 was ˜0.65, and the mixture was cultured at 37 ° C. for 3 hours to express the artificial RNA restriction enzyme.
　人工ＲＮＡ制限酵素を発現した大腸菌を回収して、超音波により破砕した。粉砕して得られた懸濁液をSDS-PAGEにより解析することで人工ＲＮＡ制限酵素が可溶性であることを確認した。粉砕して得られた懸濁液を遠心分離して上清を得た。この上清をアフィニティーカラムを用いて精製することで人工ＲＮＡ制限酵素を高純度で得ることができた。 Escherichia coli expressing the artificial RNA restriction enzyme was collected and disrupted by ultrasonic waves. The suspension obtained by grinding was analyzed by SDS-PAGE to confirm that the artificial RNA restriction enzyme was soluble. The suspension obtained by pulverization was centrifuged to obtain a supernatant. By purifying the supernatant using an affinity column, an artificial RNA restriction enzyme could be obtained with high purity.
＜標的配列を有する基質ＲＮＡの作製＞
　標的配列を含むインフルエンザゲノム由来の190塩基からなるＲＮＡをコードするＤＮＡを合成し、これをpET21aベクターのT７プロモーターの下流に導入した。 <Preparation of substrate RNA having target sequence>
A DNA encoding 190-base RNA RNA derived from the influenza genome containing the target sequence was synthesized and introduced downstream of the T7 promoter of the pET21a vector.
　このベクターのＲＮＡ領域の末端を制限酵素で切断した後、試験管内でT7 RNA polymeraseを用いて転写させた。しかし、RNA生成量が十分ではなかったため、様々な反応条件やキットを試したところ、最終的に、MEGAscript Kit（Invitrogen社製）を用いることにより、十分量の標的配列を有する基質ＲＮＡを一度に得ることができた。 The end of the RNA region of this vector was cleaved with a restriction enzyme, and then transcribed in a test tube using T7 RNA polymerase. However, since the amount of RNA produced was not sufficient, various reaction conditions and kits were tested. Finally, by using MEGAscript Kit (manufactured by Invitrogen), substrate RNA having a sufficient amount of the target sequence was simultaneously obtained. I was able to get it.
＜標的配列を有する基質ＲＮＡの切断試験＞
　上述した方法で得られた基質ＲＮＡを用い、上述した方法で精製した人工ＲＮＡ制限酵素のＲＮＡ切断活性を試験管内で検証した。 <Cleavage test of substrate RNA having target sequence>
Using the substrate RNA obtained by the method described above, the RNA cleavage activity of the artificial RNA restriction enzyme purified by the method described above was verified in a test tube.
　反応条件は、標的配列を有する基質ＲＮＡを0.6～１μｇと、人工ＲＮＡ制限酵素を終濃度５ｎＭで含む反応緩衝液（20 mM Tris-HCl、pH 8.0、150 mM NaCl、10% Glycerol、1μg/μl tRNA）に、終濃度１mMのCa2+を添加することで反応を開始させた。 The reaction conditions were a reaction buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% Glycerol, 1 μg / μl) containing 0.6 to 1 μg of substrate RNA having the target sequence and a final concentration of artificial RNA restriction enzyme of 5 nM. The reaction was started by adding a final concentration of 1 mM Ca 2+ to tRNA).
　37℃で所定時間（２分または１０分）反応させた後、EDTAの添加により反応を停止させ、分解しやすいRNAの取り扱いに注意しながら反応生成物を回収した。 After reacting at 37 ° C. for a predetermined time (2 minutes or 10 minutes), the reaction was stopped by adding EDTA, and the reaction product was recovered while paying attention to handling of RNA that is easily degraded.
　回収した反応生成物を10%変性ゲルでの電気泳動により解析した。図１に電気泳動の結果を示す。反応後にみられる切断生成物が、人工ＲＮＡ制限酵素が標的配列を有する基質ＲＮＡ配列に正しく結合して切断したものかどうかは、適切な長さのサイズマーカーの泳動位置との比較により評価した。 The collected reaction product was analyzed by electrophoresis on a 10% denaturing gel. FIG. 1 shows the result of electrophoresis. Whether the cleavage product seen after the reaction was cleaved by the artificial RNA restriction enzyme correctly binding to the substrate RNA sequence having the target sequence was evaluated by comparison with the migration position of an appropriate length size marker.
　さらに、得られた２つの主切断生成物が目的の生成物かどうかを確認するために、基質ＲＮＡ内の標的塩基配列を、人工ＲＮＡ制限酵素のＲＮＡ結合タンパク質が結合できないように変異させ、同様の切断実験を行ったところ、目的の切断生成物と思われるバンドの減少が確認された。すなわち、基質ＲＮＡでの切断が、標的配列への結合により生じていることを確認した。 Furthermore, in order to confirm whether the obtained two main cleavage products are the target products, the target base sequence in the substrate RNA is mutated so that the RNA-binding protein of the artificial RNA restriction enzyme cannot bind, and the same As a result of the cutting experiment, it was confirmed that the band considered to be the target cleavage product was reduced. That is, it was confirmed that cleavage with the substrate RNA was caused by binding to the target sequence.
＜ペプチドリンカー長の最適化＞
　上述した人工ＲＮＡ制限酵素では、ペプチドリンカーを、GlyGlyGlyGlySerの５アミノ酸からなるペプチドを３つ繰り返して、１５アミノ酸のペプチドリンカーとしており、この場合の人工ＲＮＡ制限酵素をL15型と呼ぶこととする。 <Optimization of peptide linker length>
In the artificial RNA restriction enzyme described above, the peptide linker is a 15 amino acid peptide linker obtained by repeating three peptides consisting of 5 amino acids of GlyGlyGlyGlySer. In this case, the artificial RNA restriction enzyme is referred to as L15 type.
　上述した人工ＲＮＡ制限酵素の作製方法と同じ方法で、ペプチドリンカーを、GlyGlyGlyGlySerの５アミノ酸からなるペプチドを２つ繰り返して、１０アミノ酸のペプチドリンカー（GlyGlyGlyGlySer GlyGlyGlyGlySer）を有する人工ＲＮＡ制限酵素をL10型と呼び、ペプチドリンカーを、GlyGlyGlyGlySerの５アミノ酸からなるペプチドで構成した人工ＲＮＡ制限酵素をL5型と呼び、ペプチドリンカーがなく、ＲＮＡ結合タンパク質と核酸切断酵素とを直接結合させた人工ＲＮＡ制限酵素をL0型と呼ぶこととする。 The artificial RNA restriction enzyme having the 10 amino acid peptide linker (GlyGlyGlyGlySer GlyGlyGlyGlySer) by repeating the peptide linker consisting of two peptides consisting of 5 amino acids of GlyGlyGlyGlySer in the same manner as the artificial RNA restriction enzyme production method described above and the L10 type The artificial RNA restriction enzyme consisting of GlyGlyGlyGlySer's 5-amino acid peptide is called L5 type, and there is no peptide linker, and the artificial RNA restriction enzyme that directly binds RNA-binding protein and nucleic acid cleaving enzyme is L0. Let's call it a type.
　上述したL15型の人工ＲＮＡ制限酵素と同様に、L10型、L5型及びL0型の人工ＲＮＡ制限酵素において、大腸菌でのタンパク質発現を行い、それぞれの作製したベクターをBL21(DE3)に導入して、可溶性を確認した。 Like the L15 type artificial RNA restriction enzyme described above, L10 type, L5 type and L0 type artificial RNA restriction enzymes were used for protein expression in Escherichia coli, and the prepared vectors were introduced into BL21 (DE3). The solubility was confirmed.
　L10型、L5型及びL0型の人工ＲＮＡ制限酵素では、L15型の人工ＲＮＡ制限酵素と同じ37℃で３時間培養では可溶性が十分ではなかった。そこで、各種条件で培養を行ったところ、誘導剤終濃度：0.1 mMで、30℃・３時間培養で行った場合が、発現量が高く、かつ可溶量が多かった。そこで、この条件でL10型、L5型及びL0型の人工ＲＮＡ制限酵素を培養した。 The L10 type, L5 type and L0 type artificial RNA restriction enzymes were not sufficiently soluble at 37 ° C. for 3 hours as the L15 type artificial RNA restriction enzymes. Thus, when culturing was performed under various conditions, the amount of expression was high and the amount of solubility was high when cultured at 30 ° C. for 3 hours at a final inducer concentration of 0.1 μmM. Therefore, L10 type, L5 type and L0 type artificial RNA restriction enzymes were cultured under these conditions.
　図２は、ペプチドリンカー長の異なるL15型、L10型、L5型及びL0型の人工ＲＮＡ制限酵素の切断活性を同一条件下で検証した結果である。図２より、L5型の人工ＲＮＡ制限酵素が最も反応性が高かった。 FIG. 2 shows the results of verifying the cleavage activity of artificial RNA restriction enzymes of L15 type, L10 type, L5 type and L0 type having different peptide linker lengths under the same conditions. From FIG. 2, L5 type artificial RNA restriction enzyme had the highest reactivity.
＜他の実施形態について＞
　上述した実施形態では、核酸切断酵素としてSNaseを用いたが、他の核酸切断酵素を用いた場合でも同様の切断活性を有していることを確認するため、SNaseドメインの代わりにＲＮＡを切断することが知られているPINドメインを用いることとした。 <About other embodiments>
In the above-described embodiment, SNase is used as the nucleic acid cleaving enzyme. However, in order to confirm that the same cleaving activity is obtained even when another nucleic acid cleaving enzyme is used, RNA is cleaved instead of the SNase domain. It was decided to use a known PIN domain.
　すなわち、上述したSNase型の人工ＲＮＡ制限酵素と同じ作製方法により、PIN型の人工ＲＮＡ制限酵素の発現ベクターを構築した。ここで、ペプチドリンカーの長さの異なるL15型、L10型、L5型及びL0型の４種類のPIN型の人工ＲＮＡ制限酵素を作製した。 That is, an expression vector for a PIN-type artificial RNA restriction enzyme was constructed by the same production method as the above-described SNase-type artificial RNA restriction enzyme. Here, four types of PIN-type artificial RNA restriction enzymes of L15 type, L10 type, L5 type and L0 type having different peptide linker lengths were prepared.
　なお、作製したベクターをそれぞれBL21(DE3)に導入して可溶性を確認したが、37℃で３時間培養では可溶性が十分ではなかったため、誘導剤終濃度：0.1 mMで25℃・７時間培養で行った場合が、発現量が高く、かつ可溶量が多かった。そこで、この条件でL15型、L10型、L5型及びL0型のPIN型の人工ＲＮＡ制限酵素を培養した。 In addition, each of the prepared vectors was introduced into BL21 (DE3) to confirm the solubility. However, since the solubility was not sufficient when cultured at 37 ° C for 3 hours, the final inducer concentration was 0.1 µmM at 25 ° C for 7 hours. When performed, the expression level was high and the soluble level was high. Therefore, PIN type artificial RNA restriction enzymes of L15 type, L10 type, L5 type and L0 type were cultured under these conditions.
　図３は、ペプチドリンカー長の異なるL15型、L10型、L5型及びL0型のPIN型の人工ＲＮＡ制限酵素の切断活性を同一条件下で検証した結果である。図３より、L5型またはL10型の人工ＲＮＡ制限酵素が最も反応性が高かった。 FIG. 3 shows the results of verifying the cleavage activity of PIN type artificial RNA restriction enzymes of L15 type, L10 type, L5 type and L0 type having different peptide linker lengths under the same conditions. From FIG. 3, the artificial RNA restriction enzyme of L5 type or L10 type had the highest reactivity.
実施例１：細胞内での人工RNA制限酵素の機能評価（非複製系）(図４)
（１）プラスミドの構築（図５～図２２）
　動物細胞用人工RNA制限酵素発現ベクターpCMV(-H)-hPUF_MT-L10-SNase-FLAGとpCMV(-H)-hPUF_MT-L10-hPIN-FLAGのEcoRIサイトとHindIIIサイト間にhPUFをコードするDNA配列をクローニングしてpCMV(-H)-hPUF-L10-SNase-FLAGとCMV(-H)-hPUF-L10-hPIN-FLAGを構築した。次に、pCMV(-H)-hPUF-L5-SNase-FLAGとpCMV(-H)-hPUF-L5-hPIN-FLAGからhPUFをコードするDNA配列を除去するためにEcoRIとHindIIIで切断・平滑化・ライゲーションを行い、pCMV(-H)-SNase-FLAGとpCMV(-H)-hPIN-FLAGを構築した。これらの4種類のプラスミドから核局在化シグナルをコードするDNA配列を除去するために、合成オリゴヌクレオチドのアニーリングにより作製したT7タグをコードするDNA配列をNheIサイトとBamHIサイトの間にクローニングしてpCMV(-H)-hPUF-L10-SNase-FLAG(-NLS)、pCMV(-H)-hPUF-L10-hPIN-FLAG(-NLS)、pCMV(-H)-SNase-FALG(-NLS)、およびpCMV(-H)-hPIN-FLAG(-NLS)を構築した。そして、これらの4種類のプラスミドを鋳型にしてPCRを行い、その増幅産物をそれぞれpCAGGSプラスミドのXhoIサイト間にクローニングしてpCAGGS-hPUF-L10-SNase-FLAG(-NLS)、pCAGGS-hPUF-L10-hPIN-FLAG(-NLS)、pCAGGS-SNase-FLAG(-NLS)、およびpCAGGS-hPIN-FLAG(-NLS)を構築した。 Example 1: Functional evaluation of artificial RNA restriction enzyme in cells (non-replicating system) (Fig. 4)
(1) Construction of plasmid (FIGS. 5 to 22)
DNA sequence encoding hPUF between EcoRI site and HindIII site of artificial RNA restriction enzyme expression vectors for animal cells pCMV (-H) -hPUF_MT-L10-SNase-FLAG and pCMV (-H) -hPUF_MT-L10-hPIN-FLAG Was cloned to construct pCMV (-H) -hPUF-L10-SNase-FLAG and CMV (-H) -hPUF-L10-hPIN-FLAG. Next, cut and smooth with EcoRI and HindIII to remove the DNA sequence encoding hPUF from pCMV (-H) -hPUF-L5-SNase-FLAG and pCMV (-H) -hPUF-L5-hPIN-FLAG -Ligation was performed to construct pCMV (-H) -SNase-FLAG and pCMV (-H) -hPIN-FLAG. To remove the DNA sequence encoding the nuclear localization signal from these four plasmids, the DNA sequence encoding the T7 tag generated by annealing the synthetic oligonucleotide was cloned between the NheI site and the BamHI site. pCMV (-H) -hPUF-L10-SNase-FLAG (-NLS), pCMV (-H) -hPUF-L10-hPIN-FLAG (-NLS), pCMV (-H) -SNase-FALG (-NLS), And pCMV (-H) -hPIN-FLAG (-NLS) was constructed. Then, PCR was performed using these four types of plasmids as templates, and the amplified products were cloned between the XhoI sites of the pCAGGS plasmid, respectively, and pCAGGS-hPUF-L10-SNase-FLAG (-NLS), pCAGGS-hPUF-L10 -hPIN-FLAG (-NLS), pCAGGS-SNase-FLAG (-NLS), and pCAGGS-hPIN-FLAG (-NLS) were constructed.
　CMVプロモーターの一部、その下流にhPUF結合サイトをコードするDNA配列（TGTATATA）とルシフェラーゼ遺伝子をコードするDNA配列を委託合成し、これをpcDNA3.1(+)のNdeIサイトとEcoRIサイト間にクローニングしてレポータープラスミドpcDNA-NRE-Lucを構築した。次に、hPUF結合サイトを3、および6コピーコードするDNA配列を合成オリゴヌクレオチドのアニーリング・ライゲーションにより作製し、これらをpcDNA-NRE-LucのSacIサイトとBamHIサイト間にクローニングしてpcDNA-NRE3X-Luc、およびpcDNA-NRE6X-Lucを構築した。また、同様の方法によりhPUF結合サイトの5か所に変異を導入した変異サイトを1、3、および6コピーコードするDNA配列（TCATATTA）を作製し、これらをpcDNA-NRE-LucのSacIサイトとBamHIサイト間にクローニングしてpcDNA-MT-Luc、pcDNA-MT3X-Luc、およびpcDNA-MT6X-Lucを構築した。 A part of the CMV promoter, a DNA sequence encoding the hPUF binding site (TGTATATA) downstream of it and a DNA sequence encoding the luciferase gene are commissioned and cloned between the NdeI site and EcoRI site of pcDNA3.1 (+) Thus, a reporter plasmid pcDNA-NRE-Luc was constructed. Next, DNA sequences encoding 3 and 6 copies of the hPUF binding site were prepared by annealing and ligation of synthetic oligonucleotides, which were cloned between the SacI site and the BamHI site of pcDNA-NRE-Luc and pcDNA-NRE3X- Luc and pcDNA-NRE6X-Luc were constructed. In addition, a DNA sequence (TCATATTA) that encodes 1, 3 and 6 copy sites of mutation sites with mutations introduced into 5 sites of the hPUF binding site was prepared in the same manner, and these were designated as pcDNA-NRE-Luc SacI sites. It was cloned between BamHI sites to construct pcDNA-MT-Luc, pcDNA-MT3X-Luc, and pcDNA-MT6X-Luc.
（２）レポーターアッセイ（非複製系）(図２３)
　poly-D-lysine 96ウェルプレートに2×104個の293T細胞を播種し、10%非働化FBS含有DMEM培地で37℃・CO2 5%培養槽において24時間培養後、人工RNA制限酵素発現プラスミド81 ng、RNA分解ドメイン発現プラスミド81 ng、もしくは空ベクターpcDNA3.1(+) 81 ngをレポータープラスミド9 ng、およびβ-ガラクトシダーゼ発現プラスミドpCMV-b 10 ngとともにLipofectamine 3000を用いて293T細胞へコトランスフェクションした。37℃・CO2 5%培養槽で48時間培養後に1×Passive Lysis Buffer 100 mlで細胞ライセートを調製し、Luciferase Assay Systemを用いてルシフェラーゼ活性を測定した。また、ウェル間のトランスフェクション効率を標準化するためにLuminescent b-galactosidase Detection Kit IIを用いてβ-ガラクトシダーゼ活性を測定した。ルシフェラーゼ活性値をβ-ガラクトシダーゼ活性値で標準化した後、空ベクターpcDNA3.1(+)をトランスフェクションした条件を基準にして相対的なルシフェラーゼ活性を評価した。測定結果を図２３に示す。標的配列のコピー数に依存してルシフェラーゼ活性が減少した。 (2) Reporter assay (non-replicating system) (FIG. 23)
2 × 10 4 293T cells are seeded in poly-D-lysine 96-well plate, cultured for 24 hours in 10% inactivated FBS-containing DMEM medium at 37 ° C in CO 2 5% culture tank, then artificial RNA restriction enzyme expression 81 ng of plasmid, 81 ng of RNA degradation domain expression plasmid, or 81 ng of empty vector pcDNA3.1 (+) was transferred to 293T cells using Lipofectamine 3000 with 9 ng of reporter plasmid and 10 ng of β-galactosidase expression plasmid pCMV-b. Transfected. After culturing in a 37 ° C./CO 2 5% culture tank for 48 hours, cell lysate was prepared with 100 ml of 1 × Passive Lysis Buffer, and luciferase activity was measured using Luciferase Assay System. In addition, β-galactosidase activity was measured using Luminescent b-galactosidase Detection Kit II to standardize transfection efficiency between wells. After normalizing the luciferase activity value with the β-galactosidase activity value, the relative luciferase activity was evaluated based on the condition where the empty vector pcDNA3.1 (+) was transfected. The measurement results are shown in FIG. Luciferase activity decreased depending on the copy number of the target sequence.
実施例２：細胞内での人工RNA制限酵素の機能評価（複製系、ルシフェラーゼ遺伝子使用）(図２４)
（１）プラスミドの構築（図２５～図３４）
　動物細胞用人工RNA制限酵素発現ベクターpCMV(-H)-hPUF_MT-L5-SNase-FLAGとpCMV(-H)-hPUF_MT-L5-hPIN-FLAGのEcoRIサイトとHindIIIサイト間にhPUFをコードするDNA配列をクローニングしてpCMV(-H)-hPUF-L5-SNase-FLAGとpCMV(-H)-hPUF-L5-hPIN-FLAGを構築した。そして、これらのプラスミドを鋳型にしてPCRを行い、その増幅産物をそれぞれpCAGGSプラスミドのXhoIサイト間にクローニングしてpCAGGS-hPUF-L5-SNase-FLAGとpCAGGS-hPUF-L5-hPIN-FLAGを構築した。 Example 2: Functional evaluation of artificial RNA restriction enzyme in cells (replication system, using luciferase gene) (FIG. 24)
(1) Construction of plasmid (FIGS. 25 to 34)
Artificial RNA restriction enzyme expression vector for animal cells pCMV (-H) -hPUF_MT-L5-SNase-FLAG and pCMV (-H) -hPUF_MT-L5-hPIN-FLAG DNA sequence encoding hPUF between EcoRI site and HindIII site Was cloned to construct pCMV (-H) -hPUF-L5-SNase-FLAG and pCMV (-H) -hPUF-L5-hPIN-FLAG. PCR was performed using these plasmids as templates, and the amplified products were cloned between the XhoI sites of the pCAGGS plasmid to construct pCAGGS-hPUF-L5-SNase-FLAG and pCAGGS-hPUF-L5-hPIN-FLAG. .
　ヒト由来のRNAポリメラーゼIプロモーターの下流にA型インフルエンザウイルス（A/PR/8/34（H1N1））のNSをコードするセグメント由来の5’非翻訳領域（5’NCR）、ルシフェラーゼ遺伝子のマイナス鎖、（A/PR/8/34（H1N1））のNSをコードするセグメント由来の3’非翻訳領域（3’NCR）、およびマウス由来のRNAポリメラーゼIターミネーターを配置した2種類のDNA配列を委託合成した。一つは5’NCRとルシフェラーゼ遺伝子の間にクローニングサイトAgeIとBglIIが配置されていて、もう一つはルシフェラーゼ遺伝子と3’NCRの間にクローニングサイトAgeIとBglIIが配置されている。これらをpUC19のXbaIサイトとAcc65Iサイト間にクローニングして2種類のレポータープラスミド前駆体pUC-vRNA(MCS_Luc)(NCR_NS)とpUC-vRNA(Luc_MCS)(NCR_NS)を構築した。次に、hPUF結合サイトを1、および3コピーコードするDNA配列を合成オリゴヌクレオチドのアニーリングにより作製し、これらをpUC-vRNA(MCS_Luc)(NCR_NS)のAgeIサイトとBglIIサイト間にクローニングしてpUC-vRNA(TGTATATA_Luc)(NCR_NS)とpUC-vRNA[(TGTATATA)3_Luc](NCR_NS)を構築した。また、hPUF結合サイトを1、および3コピーコードするDNA配列を合成オリゴヌクレオチドのアニーリングにより作製し、これらをpUC-vRNA(Luc_MCS)(NCR_NS)のAgeIサイトとBglIIサイト間にマイナス鎖が転写される向きにクローニングしてpUC-vRNA(Luc_TGTATATA)(NCR_NS)とpUC-vRNA[Luc_(TGTATATA)3](NCR_NS)を構築した。 5 'untranslated region (5'NCR) derived from the segment encoding NS of influenza A virus (A / PR / 8/34 (H1N1)) downstream of human RNA polymerase I promoter, minus strand of luciferase gene , (A / PR / 8/34 (H1N1)) NS segment-derived 3 'untranslated region (3'NCR) and mouse-derived RNA polymerase I terminator are placed in two types of DNA sequences Synthesized. One has a cloning site AgeI and BglII located between the 5′NCR and the luciferase gene, and the other has a cloning site AgeI and BglII located between the luciferase gene and the 3′NCR. These were cloned between the XbaI and Acc65I sites of pUC19 to construct two reporter plasmid precursors pUC-vRNA (MCS_Luc) (NCR_NS) and pUC-vRNA (Luc_MCS) (NCR_NS). Next, DNA sequences encoding 1 and 3 copies of the hPUF binding site were generated by annealing synthetic oligonucleotides, which were cloned between the AgeI and BglII sites of pUC-vRNA (MCS_Luc) (NCR_NS) vRNA (TGTATATA_Luc) (NCR_NS) and pUC-vRNA [(TGTATATA) 3 _Luc] (NCR_NS) were constructed. In addition, DNA sequences encoding 1 and 3 copies of the hPUF binding site were prepared by annealing synthetic oligonucleotides, and these were transcribed into the negative strand between the AgeI and BglII sites of pUC-vRNA (Luc_MCS) (NCR_NS). PUC-vRNA (Luc_TGTATATA) (NCR_NS) and pUC-vRNA [Luc_ (TGTATATA) 3 ] (NCR_NS) were constructed by cloning in the direction.
（２）レポーターアッセイ（複製系）(図３５～図３８)
　poly-D-lysine 96ウェルプレートに2×104個の293T細胞を播種し、10%非働化FBS含有DMEM培地で37℃・CO2 5%培養槽において24時間培養後、人工RNA制限酵素発現プラスミド86 ng、もしくは空ベクターpcDNA3.1(+) 86 ngを4種類のインフルエンザウイルス由来タンパク質発現プラスミドpCAGGS-PA、pCAGGS-PB1、pCAGGS-PB2、pCAGGS-NP各1 ng、レポータープラスミド0.1 ng、およびβ-ガラクトシダーゼ発現プラスミドpCMV-b 10 ngとともにLipofectamine 3000を用いて293T細胞へコトランスフェクションした。37℃、CO2 5%培養槽で48時間培養後に1×Passive Lysis Buffer 100 mlで細胞ライセートを調製し、Luciferase Assay Systemを用いてルシフェラーゼ活性を測定した。また、ウェル間のトランスフェクション効率を標準化するためにLuminescent b-galactosidase Detection Kit IIを用いてβ-ガラクトシダーゼ活性を測定した。ルシフェラーゼ活性値をβ-ガラクトシダーゼ活性値で標準化した後、空ベクターpcDNA3.1(+)をトランスフェクションした条件を基準にして相対的なルシフェラーゼ活性を評価した。レポータープラスミドにpUC-vRNA(TGTATATA_Luc)(NCR_NS)とpUC-vRNA[(TGTATATA)3_Luc](NCR_NS)を使用した時はvRNA（マイナス鎖）内にhPUF結合サイトが存在し、pUC-vRNA(Luc_TGTATATA)(NCR_NS)とpUC-vRNA[Luc_(TGTATATA)3](NCR_NS)を使用した時はcRNA（プラス鎖）およびmRNA内にhPUF結合サイトが存在する。 (2) Reporter assay (replication system) (FIGS. 35 to 38)
2 × 10 4 293T cells are seeded in poly-D-lysine 96-well plate, cultured for 24 hours in 10% inactivated FBS-containing DMEM medium at 37 ° C in CO 2 5% culture tank, then artificial RNA restriction enzyme expression 86 ng of plasmid, or 86 ng of empty vector pcDNA3.1 (+), 4 types of influenza virus-derived protein expression plasmids pCAGGS-PA, pCAGGS-PB1, pCAGGS-PB2, pCAGGS-NP 1 ng each, reporter plasmid 0.1 ng, and Co-transfected into 293T cells using Lipofectamine 3000 together with 10 ng of β-galactosidase expression plasmid pCMV-b. After culturing at 37 ° C. in a CO 2 5% culture tank for 48 hours, cell lysate was prepared with 100 ml of 1 × Passive Lysis Buffer, and luciferase activity was measured using Luciferase Assay System. In addition, β-galactosidase activity was measured using Luminescent b-galactosidase Detection Kit II to standardize transfection efficiency between wells. After normalizing the luciferase activity value with the β-galactosidase activity value, the relative luciferase activity was evaluated based on the condition where the empty vector pcDNA3.1 (+) was transfected. When pUC-vRNA (TGTATATA_Luc) (NCR_NS) and pUC-vRNA [(TGTATATA) 3 _Luc] (NCR_NS) are used for the reporter plasmid, there is an hPUF binding site in the vRNA (minus strand) and pUC-vRNA (Luc_TGTATATA ) When (NCR_NS) and pUC-vRNA [Luc_ (TGTATATA) 3 ] (NCR_NS) are used, there are hPUF binding sites in cRNA (plus strand) and mRNA.
　測定結果を図３６及び図３８に示す。標的配列のコピー数に依存してルシフェラーゼ活性が減少した。また、図３６及び図３８の対比から、本発明の人工RNA制限酵素は、ｖＲＮＡよりもｃＲＮＡに用いる方が有利であることも示唆される。 The measurement results are shown in FIGS. Luciferase activity decreased depending on the copy number of the target sequence. 36 and 38 also suggest that the artificial RNA restriction enzyme of the present invention is more advantageous for cRNA than vRNA.
実施例３：細胞内での人工RNA制限酵素の機能評価（複製系）(図３９)
（１）プラスミドの構築（図４０～図４１）
　A型インフルエンザウイルス（A/chicken/kumamoto/1-7/2014(H5N8)）由来NPをコードするDNA配列を委託合成し、これを鋳型にしてPCRを行い、その増幅産物をpUC-vRNA(Luc_MCS)(NCR_NS)のAgeIサイトとBglIIサイト間にマイナス鎖が転写される向きにクローニングにしてpUC-vRNA(Luc_Kuma_NP)(NCR_NS)を構築した。同様に、A型インフルエンザウイルス（A/whooper swan/Akita/1/2008(H5N1)）由来PAをコードするDNA配列を委託合成し、これを鋳型にしてPCRを行い、その増幅産物をpUC-vRNA(Luc_MCS)(NCR_NS)のAgeIサイトとBglIIサイト間にマイナス鎖が転写される向きにクローニングしてpUC-vRNA(Luc_Aki_PA)(NCR_NS)を構築した。 Example 3: Functional evaluation of artificial RNA restriction enzyme in cells (replication system) (FIG. 39)
(1) Construction of plasmid (FIGS. 40 to 41)
A DNA sequence encoding NP derived from influenza A virus (A / chicken / kumamoto / 1-7 / 2014 (H5N8)) is commissioned and synthesized using this as a template, and the amplified product is pUC-vRNA (Luc_MCS PUC-vRNA (Luc_Kuma_NP) (NCR_NS) was constructed by cloning in the direction in which the minus strand was transcribed between the AgeI and BglII sites of (NCR_NS). Similarly, consigned synthesis of a DNA sequence encoding PA derived from influenza A virus (A / whooper swan / Akita / 1/2008 (H5N1)), and using this as a template, PCR was performed, and the amplified product was pUC-vRNA PUC-vRNA (Luc_Aki_PA) (NCR_NS) was constructed by cloning in such a direction that the minus strand was transcribed between the AgeI and BglII sites of (Luc_MCS) (NCR_NS).
（２）レポーターアッセイ（複製系、ウイルスゲノムにルシフェラーゼ遺伝子を連結）（図４２～図４５）
　poly-D-lysine 96ウェルプレートに2×104個の293T細胞を播種し、10%非働化FBS含有DMEM培地で37℃・CO25%培養槽において24時間培養後、人工RNA制限酵素発現プラスミド85 ng、もしくは空ベクターpcDNA3.1(+) 85 ngを4種類のインフルエンザウイルス由来タンパク質発現プラスミドpCAGGS-PA、pCAGGS-PB1、pCAGGS-PB2、pCAGGS-NP各1 ng、レポータープラスミド1 ng、およびβ-ガラクトシダーゼ発現プラスミドpCMV-b 10 ngとともにLipofectamine 3000を用いて293T細胞へコトランスフェクションした。37℃、CO2 5%培養槽で48時間培養後に1×Passive Lysis Buffer 100 mlで細胞ライセートを調製し、Luciferase Assay Systemを用いてルシフェラーゼ活性を測定した。また、ウェル間のトランスフェクション効率を標準化するためにLuminescent b-galactosidase Detection Kit IIを用いてβ-ガラクトシダーゼ活性を測定した。ルシフェラーゼ活性値をβ-ガラクトシダーゼ活性値で標準化した後、空ベクターpcDNA3.1(+)をトランスフェクションした条件を基準にして相対的なルシフェラーゼ活性を評価した。レポータープラスミドにpUC-vRNA(Luc_Kuma_NP)(NCR_NS)を使用した時はvRNA（マイナス鎖）内にhPUF結合サイトが存在し、pUC-vRNA(Luc_Aki_PA)(NCR_NS)を使用した時はcRNA（プラス鎖）およびmRNA内にhPUF結合サイトが存在する。 (2) Reporter assay (replication system, luciferase gene linked to viral genome) (FIGS. 42 to 45)
2 × 10 4 293T cells are seeded in poly-D-lysine 96-well plate, cultured for 24 hours in 10% inactivated FBS-containing DMEM medium at 37 ° C in CO 2 5% culture tank, then artificial RNA restriction enzyme expression 85 ng of plasmid, or 85 ng of empty vector pcDNA3.1 (+), 4 ng of influenza virus-derived protein expression plasmids pCAGGS-PA, pCAGGS-PB1, pCAGGS-PB2, pCAGGS-NP 1 ng each, reporter plasmid 1 ng, and Co-transfected into 293T cells using Lipofectamine 3000 together with 10 ng of β-galactosidase expression plasmid pCMV-b. After culturing at 37 ° C. in a CO 2 5% culture tank for 48 hours, cell lysate was prepared with 100 ml of 1 × Passive Lysis Buffer, and luciferase activity was measured using Luciferase Assay System. In addition, β-galactosidase activity was measured using Luminescent b-galactosidase Detection Kit II to standardize transfection efficiency between wells. After normalizing the luciferase activity value with the β-galactosidase activity value, the relative luciferase activity was evaluated based on the condition where the empty vector pcDNA3.1 (+) was transfected. When pUC-vRNA (Luc_Kuma_NP) (NCR_NS) is used as the reporter plasmid, there is an hPUF binding site in vRNA (minus strand), and when pUC-vRNA (Luc_Aki_PA) (NCR_NS) is used, cRNA (plus strand) And there is an hPUF binding site in the mRNA.
　測定結果を図４３及び図４５に示す。本発明の人工ＲＮＡ制限酵素によりルシフェラーゼ活性が減少した。 The measurement results are shown in FIGS. Luciferase activity was reduced by the artificial RNA restriction enzyme of the present invention.
実施例４：細胞内での人工RNA制限酵素の機能評価（複製系、ウイルスゲノムを使用した
RNAレベルでの解析）（図４６）
（１）プラスミドの構築（図４７～図４８）
　ヒト由来のRNAポリメラーゼIプロモーターの下流にA型インフルエンザウイルス（A/PR/8/34（H1N1））のNSをコードするセグメント由来の5’非翻訳領域（5’NCR）、A型インフルエンザウイルス（A/chicken/kumamoto/1-7/2014(H5N8)）由来のNP遺伝子のマイナス鎖、（A/PR/8/34（H1N1））のNSをコードするセグメント由来の3’非翻訳領域（3’NCR）、およびマウス由来のRNAポリメラーゼIターミネーターを配置したDNA配列を委託合成した。これをpUC19のXbaIサイトとAcc65Iサイト間にクローニングしてウイルスRNA発現プラスミドpUC-vRNA(Kuma_NP)(NCR_NS)を構築した。 Example 4: Functional evaluation of artificial RNA restriction enzyme in cells (replication system, using viral genome)
Analysis at RNA level) (Figure 46)
(1) Construction of plasmid (FIGS. 47 to 48)
5 'untranslated region (5'NCR) derived from NS coding segment of influenza A virus (A / PR / 8/34 (H1N1)) downstream of human RNA polymerase I promoter, influenza A virus ( A / chicken / kumamoto / 1-7 / 2014 (H5N8))-derived NP gene minus strand, (A / PR / 8/34 (H1N1)) NS-derived segment 3 'untranslated region (3 'NCR) and a DNA sequence in which a mouse RNA polymerase I terminator was placed were commissioned and synthesized. This was cloned between the XbaI site and Acc65I site of pUC19 to construct a viral RNA expression plasmid pUC-vRNA (Kuma_NP) (NCR_NS).
（２）定量リアルタイムPCRでのRNAレベルでの解析（複製系、ウイルスゲノムを使用したRNAレベルでの解析）
 poly-D-lysine 35 mmディッシュに4×105個の293T細胞を播種し、10%非働化FBS含有DMEM培地で37℃・CO25%培養槽において24時間培養後、人工RNA制限酵素発現プラスミド2400 ng、もしくは空ベクターpcDNA3.1(+) 2400 ngを4種類のインフルエンザウイルス由来タンパク質発現プラスミドpCAGGS-PA、pCAGGS-PB1、pCAGGS-PB2、pCAGGS-NP各25 ng、ウイルスRNA発現プラスミドpUC-vRNA(Kuma_NP)(NCR_NS)2.5 ng、およびβ-ガラクトシダーゼ発現プラスミドpCMV-b 2.5 ngとともにLipofectamine 3000を用いて293T細胞へコトランスフェクションした。37℃、CO2 5%培養槽で48時間培養後にTRIzolとRNeasy Mini kitを用いてトータルRNAを抽出し、DNaseI処理後にウイルスRNA特異的プライマーを用いて逆転写反応をし、RNaseH処理をしてから細胞内で複製したウイルスRNAを定量するためにTaqMan Fast Advanced Master Mixを用いてリアルタイムPCR解析を行った。また、ディッシュ間のトランスフェクション効率を標準化するために抽出したトータルRNAを用いてランダムプライマーで逆転写反応をし、RNaseH処理をしてからβ-ガラクトシダーゼmRNA発現量を定量するためにリアルタイムPCR解析を行った。細胞内のウイルスRNA量をβ-ガラクトシダーゼmRNA発現量で標準化した後、空ベクターpcDNA3.1(+)をトランスフェクションした条件を基準にして相対的なウイルスRNA複製量を評価した。 (2) Analysis at the RNA level in quantitative real-time PCR (analysis at the RNA level using a replication system and viral genome)
Artificial RNA restriction enzyme expression after seeding 4 × 10 5 293T cells in poly-D-lysine 35 mm dish and culturing in DMEM medium containing 10% inactivated FBS for 24 hours in a 37 ° C / CO 2 5% culture tank Plasmid 2400 ng, or empty vector pcDNA3.1 (+) 2400 ng, 4 types of influenza virus-derived protein expression plasmids pCAGGS-PA, pCAGGS-PB1, pCAGGS-PB2, pCAGGS-NP 25 ng each, viral RNA expression plasmid pUC- 293T cells were co-transfected using Lipofectamine 3000 with 2.5 ng of vRNA (Kuma_NP) (NCR_NS) and 2.5 ng of β-galactosidase expression plasmid pCMV-b. Extract the total RNA using TRIzol and RNeasy Mini kit after incubation for 48 hours in a 37 ° C, 5% CO 2 culture tank. After DNaseI treatment, perform reverse transcription using viral RNA-specific primers and RNaseH treatment. In order to quantify viral RNA replicated in the cells, real-time PCR analysis was performed using TaqMan Fast Advanced Master Mix. In addition, in order to standardize transfection efficiency between dishes, reverse transcription reaction was performed with random primers using total RNA extracted, and real-time PCR analysis was performed to quantify β-galactosidase mRNA expression after RNaseH treatment. went. After normalizing the amount of viral RNA in the cells with the expression level of β-galactosidase mRNA, the relative amount of viral RNA replication was evaluated based on the condition of transfection with the empty vector pcDNA3.1 (+).
　測定結果を図４９に示す。本発明の人工ＲＮＡ制限酵素により相対的なウイルスRNA複製量が減少した。 The measurement results are shown in FIG. The relative amount of viral RNA replication was reduced by the artificial RNA restriction enzyme of the present invention.
実施例５：細胞内での人工RNA制限酵素の機能評価（複製系、ウイルスゲノムを使用、細胞毒性評価）
　poly-D-lysine 96ウェルプレートに2×104個の293T細胞を播種し、10%非働化FBS含有DMEM培地で37℃・CO2 5%培養槽において24時間培養後、人工RNA制限酵素発現プラスミド96 ng、もしくは空ベクターpcDNA3.1(+) 96 ngを4種類のインフルエンザウイルス由来タンパク質発現プラスミドpCAGGS-PA、pCAGGS-PB1、pCAGGS-PB2、pCAGGS-NP各1 ng、ウイルスRNA発現プラスミドpUC-vRNA(Kuma_NP)(NCR_NS)0.2 ng、およびβ-ガラクトシダーゼ発現プラスミドpCMV-b 0.2 ngとともにLipofectamine 3000を用いて293T細胞へコトランスフェクションした。37℃、CO2 5%培養槽で48時間培養後にCell Counting Kit-8のWST-8を10 ml添加し、37℃、CO25%培養槽で培養してからマルチプレートリーダーにて450 nmの吸光度を測定した。空ベクターpcDNA3.1(+)をトランスフェクションした条件を基準にして細胞生存率を評価した。 Example 5: Functional evaluation of artificial RNA restriction enzyme in cells (replication system, using viral genome, cytotoxicity evaluation)
2 × 10 4 293T cells are seeded in poly-D-lysine 96-well plate, cultured for 24 hours in 10% inactivated FBS-containing DMEM medium at 37 ° C in CO 2 5% culture tank, then artificial RNA restriction enzyme expression 96 ng of plasmid or 96 ng of empty vector pcDNA3.1 (+) 4 kinds of influenza virus-derived protein expression plasmids pCAGGS-PA, pCAGGS-PB1, pCAGGS-PB2, pCAGGS-NP each 1 ng, viral RNA expression plasmid pUC- 293T cells were co-transfected using Lipofectamine 3000 with 0.2 ng of vRNA (Kuma_NP) (NCR_NS) and 0.2 ng of β-galactosidase expression plasmid pCMV-b. After culturing in a 37 ° C, 5% CO 2 culture tank for 48 hours, add 10 ml of Cell Counting Kit-8 WST-8, and incubate in a 37 ° C, 5% CO 2 culture tank, and then use a multiplate reader for 450 nm The absorbance was measured. Cell viability was evaluated based on the conditions transfected with empty vector pcDNA3.1 (+).
　測定結果を図５１に示す。本発明の人工ＲＮＡ制限酵素は細胞毒性を示さないことが確認された。 The measurement results are shown in FIG. It was confirmed that the artificial RNA restriction enzyme of the present invention does not show cytotoxicity.

Claims (6)

  1. 　ＲＮＡと結合するＲＮＡ結合タンパク質と、ＲＮＡの所定の部位を切断する酵素とを、ペプチドリンカーを介して連結して成る人工ＲＮＡ制限酵素。 An artificial RNA restriction enzyme comprising an RNA-binding protein that binds to RNA and an enzyme that cleaves a predetermined site of RNA via a peptide linker.
  2. 　前記酵素がSNaseの場合には、ペプチドリンカーの長さを５アミノ酸とする請求項１に記載の人工ＲＮＡ制限酵素。 The artificial RNA restriction enzyme according to claim 1, wherein when the enzyme is SNase, the length of the peptide linker is 5 amino acids.
  3. 　前記酵素がPINの場合には、ペプチドリンカーの長さを５アミノ酸または１０アミノ酸とする請求項１に記載の人工ＲＮＡ制限酵素。 The artificial RNA restriction enzyme according to claim 1, wherein when the enzyme is PIN, the length of the peptide linker is 5 amino acids or 10 amino acids.
  4. ＲＮＡと結合するＲＮＡ結合タンパク質が、ヒトPumilio及びFBFホモロジー（hPUF)タンパク質又はその改変体である、請求項１から３の何れか一項に記載の人工ＲＮＡ制限酵素。 The artificial RNA restriction enzyme according to any one of claims 1 to 3, wherein the RNA-binding protein that binds to RNA is human Pumilio and FBF homology (hPUF) protein or a variant thereof.
  5. ペプチドリンカーが、GlyGlyGlyGlySerからなるペプチド、又はGlyGlyGlyGlySer GlyGlyGlyGlySerからなるペプチドである、請求項１から４の何れか一項に記載の人工ＲＮＡ制限酵素。 The artificial RNA restriction enzyme according to any one of claims 1 to 4, wherein the peptide linker is a peptide composed of GlyGlyGlyGlySer or a peptide composed of GlyGlyGlyGlyGlySer GlyGlyGlyGlySer.
  6. プロモーター、ＲＮＡと結合するＲＮＡ結合タンパク質をコードするＤＮＡ、ペプチドリンカーをコードするＤＮＡ、及びＲＮＡの所定の部位を切断する酵素をコードするＤＮＡとをこの順に有する組み換え発現ベクター。 A recombinant expression vector comprising, in this order, a promoter, DNA encoding an RNA binding protein that binds to RNA, DNA encoding a peptide linker, and DNA encoding an enzyme that cleaves a predetermined site of RNA.
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