Method for producing protein or peptide, method for producing 5'-capped polynucleotide, and reagent for use in said production methods

Abstract

The present invention addresses, pertaining to a technique for protein translation, the problem of promoting further translational efficiency by utilizing RNA which encodes proteins and a capped polynucleotide capable of binding to the RNA through complementary base pair formation, and addresses, pertaining to the production of a 5'-capped polynucleotide, the problem of improving the capping efficiency. The problems are solved by: configuring a 5'-capped polynucleotide and / or a single-stranded RNA to have a translational efficiency promoting structure; and inserting a base into a promoter such that the base sequence of the polynucleotide of a cap analog and the base sequence upstream from the transcription initiation point of the promoter are complementary in terms of a specific positional relationship.

Description

Method for producing proteins or peptides, method for producing 5'-capped polynucleotides, and reagents for use in these methods

[0001] The present invention relates to a method for producing a protein or peptide, a method for producing a 5'-capped polynucleotide, and reagents for use in these production methods.

[0002] In recent years, the use of mRNA vaccines has become widespread. The structure of an mRNA vaccine can be divided into the following parts from the 5' end: a cap structure, a 5' untranslated region, a protein coding region, a 3' untranslated region, and a poly(A) tail region. The cap structure is necessary for protein translation from an mRNA vaccine. However, with existing mRNA synthesis technologies, the ratio of mRNA with a cap structure to the total mRNA amount (capping efficiency) is low, and the immune response elicited by uncapped mRNA has been a problem.

[0003] In this situation, Non-Patent Document 1 reports that highly pure capped mRNA was produced by using a photoremovable hydrophobic purification tag as a cap analog, isolating and purifying only the capped mRNA by reverse-phase chromatography after the transcription reaction, and then removing the hydrophobic tag by photoreaction.

[0004] Nat Commun. 2023 May 11;14(1):2657. doi: 10.1038 / s41467-023-38244-8.

[0005] Furthermore, translation efficiency is also important for improving the performance of mRNA vaccines. In the course of their research, the present inventors have developed a protein translation technology that utilizes a protein-encoding RNA and a capped RNA that can bind to the RNA through complementary base pairing, and have focused on further enhancing the translation efficiency of this technology.

[0006] The present invention aims to further promote translation efficiency in protein or peptide translation technology by utilizing RNA encoding a protein or peptide and a capped polynucleotide that can bind to it through complementary base pairing, and to improve capping efficiency in the production of 5'-capped polynucleotides.

[0007] In light of the above problems, the present inventors have conducted extensive research and found that the structure of 5'-capped polynucleotides and / or single-stranded RNAs (RNAs encoding proteins or peptides) contributes to enhanced translation efficiency, and further found that capping efficiency can be improved by inserting bases into a promoter so that the base sequence of a cap analog polynucleotide is complementary to the base sequence upstream from the transcription start point of the promoter in a specific positional relationship. Based on these findings, the present inventors have conducted further research and completed the present invention. Specifically, the present invention encompasses the following aspects:

[0008] Item 1. A method for producing or expressing a protein or peptide, comprising carrying out a translation reaction in a reaction system containing a 5'-capped polynucleotide comprising an arbitrary base sequence A, and a base sequence B capable of binding to said base sequence A through complementary base pairing, and a single-stranded RNA comprising a protein or peptide coding sequence, wherein the 5'-capped polynucleotide and / or the single-stranded RNA has a translation efficiency-enhancing structure. Item 2. The method for producing or expressing a protein or peptide according to Item 1, wherein the 5'-capped polynucleotide is a 5'-capped RNA. Item 3. The method for producing or expressing a protein or peptide according to Item 1 or 2, wherein the 5'-capped polynucleotide is a biologically or cellularly endogenous polynucleotide. Item 4. The method for producing or expressing a protein or peptide according to Item 3, wherein the biologically or cellularly endogenous polynucleotide is an lncRNA or mRNA. Item 5. The method for producing or expressing a protein or peptide according to any one of Item 1 to 4, wherein the translation efficiency-enhancing structure is a structure that stabilizes the bond between the 5'-capped polynucleotide and the single-stranded RNA. Item 6. Item 7. The method for producing or expressing a nucleic acid according to any one of Items 1 to 5, wherein (b) the 5'-capped polynucleotide and / or the single-stranded RNA have a nuclease-binding structure and the reaction system contains the nuclease, the nuclease is RISC or Cas, and the 5'-capped polynucleotide is siRNA or miRNA, or the 5'-capped polynucleotide is lncRNA or mRNA and the single-stranded RNA comprises a tracrRNA sequence. Item 8. The method for producing or expressing a nucleic acid according to any one of Items 1 to 6, wherein at least one requirement selected from the group consisting of (a) the single-stranded RNA is a circular RNA, and (c) the 5'-capped polynucleotide and / or the single-stranded RNA have a structure that covalently bonds them. Item 9. The method for producing or expressing a nucleic acid according to Item 7, wherein requirement (a) is met and the circular RNA does not contain a stop codon, and / or requirement (c) is met and the structure is a photocrosslinkable structure.Item 9. A composition comprising a 5'-capped polynucleotide comprising an arbitrary base sequence A, and / or a base sequence B capable of binding to said base sequence A through complementary base pairing, and a single-stranded RNA comprising a protein or peptide coding sequence, wherein the 5'-capped polynucleotide and / or the single-stranded RNA has a translation efficiency-enhancing structure. Item 10. The composition according to Item 9, for use in the production or expression method according to any one of Items 1 to 8. Item 11. A method for producing or expressing a protein or peptide, comprising carrying out a translation reaction in a reaction system containing a single-stranded circular RNA of 500 bases or less, formed by 5'-capped polynucleotides linked via a linker. Item 12. A composition comprising a single-stranded circular RNA of 500 bases or less, formed by 5'-capped polynucleotides linked via a linker. Item 13. The composition according to Item 12, for use in the production or expression method according to Item 11. Item 14. Item 14. A method for producing a 5'-capped polynucleotide, comprising carrying out a transcription reaction in a reaction system containing: a double-stranded polynucleotide comprising a phage promoter having a base insertion mutant sequence adjacent to the upstream side of a transcription start point, and an antisense strand comprising a base sequence Y: (upstream side)-Y2-Y3 or (upstream side)-Y1-Y2-Y3 (in base sequence Y: Y1 and Y2 represent bases in the base insertion mutant sequence, and Y3 represents the base of the transcription start point); and a 5'-cap analog having a polynucleotide comprising the base sequence X: (cap side)-X2-X3 or (cap side)-X1-X2-X3, wherein X3 is the complementary base of Y3, and X1 is the complementary base of Y1 and / or X2 is the complementary base of Y2. Item 14. The production method according to Item 14, wherein the antisense strand of the phage promoter comprises the base sequence Y: (upstream)-Y1-Y2-Y3, where X1 is the complementary base of Y1 and X2 is the complementary base of Y2. Item 16. The production method according to Item 14 or 15, wherein the 5'-capped polynucleotide is a 5'-capped RNA. Item 17. The production method according to any of Items 14 to 16, wherein the phage promoter is a T7 promoter.Item 18. The production method according to any one of Items 14 to 17, wherein the base sequence X is (cap side) -CUG, CAG, GUG, or GAG, and Y3 is C. Item 19. A composition comprising: a double-stranded polynucleotide comprising a phage promoter having a base insertion mutant sequence adjacent to the upstream side of the transcription start site, and wherein the antisense strand comprises the base sequence Y: (upstream side)-Y2-Y3 or (upstream side)-Y1-Y2-Y3 (in base sequence Y: Y1 and Y2 represent bases in the base insertion mutant sequence, and Y3 represents the base of the transcription start site), and / or a 5' cap analog having a polynucleotide comprising the base sequence X: (cap side)-X2-X3 or (cap side)-X1-X2-X3, wherein X3 is the complementary base of Y3, and X1 is the complementary base of Y1 and / or X2 is the complementary base of Y2. Item 20. Item 21. The composition according to Item 19, for use in the production method according to any one of Items 14 to 18. Item 22. A T7 RNA polymerase having the D130W mutation.

[0009] According to the present invention, a technique for further enhancing translation efficiency in protein or peptide translation techniques by utilizing RNA encoding a protein or peptide and a capped polynucleotide capable of binding to it through complementary base pairing, and a technique for improving capping efficiency in the production of 5'-capped polynucleotides, can be provided.

[0010] The phenomenon of circular mRNA translation promotion using the RNA interference mechanism. (a) Schematic diagram of this experiment. Short double-stranded RNA (capped siRNA) bearing an m7G cap structure at the 3' end of the guide strand is incorporated into the RISC complex and binds to circular mRNA containing a mostly complementary sequence. The non-complementary region in the center of the guide strand in the resulting RNA duplex prevents mRNA cleavage. The m7G cap structure at the end of the guide strand recruits ribosomes to the start codon of the circular mRNA, promoting protein expression from the circular mRNA. (b) Schematic diagram of the circular mRNA sequence used. (c) Schematic diagram of the sequences and structures of the synthesized capped and uncapped short double-stranded RNAs (capped siRNA, uncapped siRNA). (d) The predicted duplex structure of the guide strand and mRNA formed in the RISC complex. (e) Experimental results using human-derived cultured cells (HeLa) demonstrating that capped siRNA can dose-dependently enhance the translation reaction of circular mRNA. Circular RNA and siRNA molecules were introduced into HeLa cells using a commercially available lipofection reagent. After 24 hours of culture, the cells were lysed and the amount of translation product (NanoLuc luciferase, Nluc) contained in the lysate was assessed. Circular mRNA translation promotion was demonstrated by hybridization with long non-coding RNA (lncRNA) and its stabilization by dCas13 protein. (a) (b) Experiment to evaluate the translation activity of uncapped linear mRNA encoding NanoLuc luciferase (Nluc) in HeLa cells (model experiment using linear mRNA). mRNA was introduced into HeLa cells using a commercially available lipofection reagent, and then either HULC RNA alone or HULC RNA and dRfxCas13d mRNA were introduced separately using a lipofection reagent. When HULC RNA was added alone or in combination with dRfxCas13d mRNA, protein expression from uncapped linear Nluc mRNA was improved.The sequence of the linker between the 5' cap structure and the hybridization region was varied to 5, 10, or 15 bases in length, but no significant difference was observed; a similar increase was observed in all cases. (c) In HeLa cells, interaction with HULC lncRNA promoted the translation of circular Nluc mRNA. This phenomenon was dependent on the 5' cap structure of HULC lncRNA. (d) In HeLa cells, interaction with UCA1 lncRNA promoted the translation of circular Nluc mRNA. This phenomenon was dependent on the 5' cap structure of UCA1 lncRNA. Photocrosslinkable capped RNA probes stimulate the translation of circular mRNA in a light-dependent manner. (a) Schematic diagram of the system. Capped RNA probes containing trioxalene can form covalent bonds with complementary strands through photocrosslinking. This stabilizes the binding of the capped probe to circular mRNA, enhancing its translation-stimulating effect. (b) A model experiment using a 24-base short RNA oligonucleotide as a probe target, representing a model of circular mRNA. The cross-linked complex can form a stable complex during denaturing polyacrylamide gel electrophoresis. When 15 molar equivalents of the probe were added to the mRNA model, the mRNA model formed a nearly quantitative covalent bond with the probe. (c) A capped probe was annealed to a 650-base circular mRNA encoding Nluc, followed by irradiation with 365 nm light. When transfected into HeLa cells, the translation-enhancing effect of the photocrosslinking capped probe was enhanced compared to the control group. Promotion of rolling circle translation by capped RNA. (a) Schematic diagram of this experiment. Binding capped RNA to circular RNA lacking a stop codon promotes rolling circle translation. (b) Schematic diagram of the circular mRNA sequence used. (c) Experimental results using rabbit reticulocyte lysate (RRL) demonstrating that capped oligo RNA probes promote rolling circle translation. The circular RNAs annealed with the probes were translated in RRL, and the translation products were detected by Western blot analysis using FLAG antibody.In the presence of a capped oligo-RNA probe containing a region complementary to the circular RNA sequence, multiple rotations of the ribosome on the circular RNA significantly increased the translation product. Development of a highly efficient method for synthesizing Cap-2 mRNA using TetraPureCap analogs and modified promoters. (a) Structure and sequence of the TetraPureCap-XYG series analogs and the sequence of the modified T7 RNA polymerase promoter. (b) Structure and sequence of the TetraPureCap-XYA series analogs and the sequence of the modified T7 RNA polymerase promoter. (c) Experimental results showing that the incorporation efficiency of TetraPureCap analogs was improved by transcription reactions using template DNA with modified promoters. With the natural promoter sequence, the incorporation efficiency of the cap analog was approximately 30% due to competition with GTP. However, by inserting a sequence complementary to the cap analog, thermodynamic stability was increased, improving incorporation of the cap analog to 80-90%. Development of a highly efficient method for synthesizing Cap-2 mRNA using TetraPureCap analogs and modified promoters. (d) Experimental results quantifying the total transcription amount and the amount of capped RNA synthesized by combining a TetraPureCap analog and a modified promoter using HPLC analysis. Development of a highly efficient method for synthesizing Cap-2 mRNA using a TetraPureCap analog and a modified promoter. (e) Experimental results showing that by utilizing the complementarity between the TetraPureCap analog and the promoter, the efficiency of incorporation of the analog is maintained even when the concentration of the TetraPureCap analog in the transcription reaction is lowered below the NTP concentration (2 mM). (f) Experimental results showing the effect of a mutant T7 RNA polymerase enzyme (D130W) designed and synthesized to improve the efficiency of incorporation of the TetraPureCap analog. D130W successfully improved the efficiency of incorporation of the cap analog even when template DNA with a natural promoter sequence was used. (g) Experimental data showing the translation activity of cap-2 mRNA synthesized using a TetraPureCap analog. A difference of approximately three-fold was observed in translation activity depending on the 5'-terminal base sequence. (h) PAGE analysis data confirming the purity of the mRNA used in the translation activity experiment.Experimental results demonstrating that small circular mRNAs (circRNAs; 127- and 167-base ring sizes) containing an m7G cap structure, constructed from two chemically synthesized oligonucleotide fragments, have translation activity in human cell lines. (a) Diagram showing the sequences of the starting chemically synthesized RNA oligonucleotides used to prepare the small circular mRNAs. The calculated and measured molecular weights of the chemically synthesized fragments are also shown in the figure. The two data show good agreement. The bold text in the F87 sequence represents the coding region for the peptide (translation product peptide sequence: MVSGWRLFKKISGSSGSIINFEKL (SEQ ID NO: 51)). In the figure, the nucleotide sequence of F87 is shown as SEQ ID NO: 46, the nucleotide sequences of F40c are shown as SEQ ID NOs: 47 and 48, and the nucleotide sequences of F80c are shown as SEQ ID NOs: 49 and 50. Experimental results demonstrating that small circular mRNAs (circRNAs; 127- and 167-base ring sizes) containing an m7G cap structure, constructed from two chemically synthesized oligonucleotide fragments, have translation activity in human cell lines. (b) Schematic diagram of the method for constructing circRNA by enzymatically ligating the substrate RNA oligonucleotides shown in (a). The relationship between the combination of raw material sequences and the product is shown in the figure. (c) Results of evaluation of the translation activity of the prepared circRNA in human-derived cultured cells (HeLa). RNA was introduced into HeLa cells using a commercially available transfection reagent, and the amount of HiBiT peptide encoded by the RNA was measured. Peptides were found to be produced as translation products from 127-nt and 167-nt circular RNAs. The mean values ​​(n = 4) and standard errors are shown as error bars. Assay results for Test Example 7 are shown. The upper panel shows the test scheme, the lower left panel shows the base sequences of the nucleic acids used, and the lower right panel shows a graph of the assay results. The legend in the graph indicates the shape of the chemically synthesized mRNA used. Assay results for Test Example 8 are shown. The upper left panel shows a schematic diagram of the mRNA structure used, the right panel shows the results of denaturing electrophoresis of the mRNA used, and the lower left panel shows a graph of the assay results. Hybridization with endogenous mRNA (ACTB) promotes translation of circular mRNA. (A) Translation activity of uncapped circular mRNA (circRNA) encoding NanoLuc luciferase (Nluc) in HeLa cells.The antisense sequence of human ACTB mRNA was introduced into the antisense sequence. (B) ACTB siRNA was introduced into HeLa cells using a commercially available lipofection reagent, resulting in nearly half of ACTB mRNA expression after 24 h. CircRNA was then introduced into the cells, and Nluc expression was assessed 8 h later. (C) CircRNAs containing the ACTB mRNA antisense sequence (ACTB_native, ACTB_2'OMe, ACTB_LNA) showed increased Nluc translation compared to those without (NoAntisense). Translation levels were particularly high for circRNAs with 2'OMe or LNA modifications throughout the entire antisense sequence (ACTB_2'OMe, ACTB_LNA). Furthermore, while siRNA-mediated knockdown of ACTB mRNA was unaffected by circRNAs lacking the antisense sequence, translation levels were reduced by nearly half for circRNAs containing the ACTB mRNA antisense sequence.

[0011] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0012] As used herein, polynucleotides are not particularly limited and may be DNA, RNA, or other known chemically modified, as exemplified below. To prevent degradation by hydrolases such as nucleases, the phosphate residues of each nucleotide may be substituted with chemically modified phosphate residues such as phosphorothioate (PS), methylphosphonate, or phosphorodithioate. The hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide may also be substituted with -OR (where R represents, for example, CH3(2'-O-Me), CH2CHOCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, or CH2CH2CN). Furthermore, the base moiety (pyrimidine or purine) may be chemically modified, such as by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a thiocarbonyl group for the carbonyl group at the 2-position. Further examples include those in which the phosphate moiety or hydroxyl moiety is modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, etc., but are not limited to these.

[0013] As used herein, a polynucleotide may be linked to another molecule. Examples of other molecules include fluorescent labels. Examples of fluorescent labels include fluorescein, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, phycoerythrin, 6-FAM™, Cy®3, Cy®5, and the Alexa Fluor® series.

[0014] In this specification, the bases constituting nucleic acids include not only typical bases in natural nucleic acids such as RNA and DNA (adenine (A), thymine (T), uracil (U), guanine (G), cytosine (C), etc.), but also other bases such as hypoxanthine (I) and modified bases. Modified bases include, for example, pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (e.g., 5-methylcytosine), 5-alkyluracil (e.g., 5-ethyluracil), 5-halouracil (5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidine (6-methyluracil), 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5'-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, 1-methyladenine, 1-methylhypoxanthine, etc. Examples of amino acids that can be used include uracil, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyladenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, 2-thiocytosine, purine, 2-aminopurine, isoguanine, indole, imidazole, xanthine, and cyanuric acid.

[0015] 1. Method 1 for Producing or Expressing a Protein or Peptide This section corresponds to the above sections 1 to 7 and the below-described Test Examples 1 to 4 (FIGS. 1 to 4). However, the present invention is not limited to the below-described Test Examples.

[0016] In one aspect, the present invention relates to a method for producing or expressing a protein or peptide, which comprises carrying out a translation reaction in a reaction system containing a 5'-capped polynucleotide comprising an arbitrary base sequence A, a base sequence B capable of binding to said base sequence A through complementary base pairing, and a single-stranded RNA comprising a protein or peptide coding sequence, wherein the 5'-capped polynucleotide and / or the single-stranded RNA has a translation efficiency-enhancing structure (sometimes referred to herein as the "production or expression method of the present invention"). This method is described below.

[0017] The 5'-capped polynucleotide used in the production or expression method of the present invention is not particularly limited as long as it has a 5'-cap structure and is capable of binding to single-stranded RNA through complementary base pairing. RNA is particularly preferred as the 5'-capped polynucleotide. The 5'-cap structure is not particularly limited, and examples include cap-0, cap-1, and cap-2.

[0018] The 5'-capped polynucleotide can be single-stranded or double-stranded.

[0019] The single-stranded RNA used in the production or expression method of the present invention contains a protein or peptide coding sequence. The protein or peptide coding sequence is the coding sequence for the target protein or peptide to be produced or expressed by the production or expression method of the present invention. Examples of such proteins or peptides include cancer antigens and microbial antigens. Examples of cancer antigens include common antigens (tumor-associated antigens), such as differentiation antigens (gp100, MART-1, etc.), embryonic proteins (CEA, AFP, etc.), glycoproteins, glycolipids, and carbohydrate antigens (MUC-1, CA125, etc.), and overexpressed proteins (HER2, Survivin, WT-1, etc.), and specific antigens (neoantigens), such as cancer antigens derived from driver mutations (KRAS, BRAF, EGFR, etc.) and passenger mutations (patient-specific). Examples of microbial antigens include viral antigens and bacterial antigens. The viruses from which viral antigens are derived are not particularly limited, and examples include enveloped viruses (viruses with an envelope) such as influenza virus (e.g., type A, type B, etc.), rubella virus, Ebola virus, coronavirus, measles virus, varicella-zoster virus, herpes simplex virus, mumps virus, arbovirus, respiratory syncytial virus, SARS virus, hepatitis virus (e.g., hepatitis B virus, hepatitis C virus, etc.), yellow fever virus, AIDS virus, rabies virus, hantavirus, dengue virus, Nipah virus, and lyssavirus; and non-enveloped viruses (viruses without an envelope) such as adenovirus, norovirus, rotavirus, human papillomavirus, poliovirus, enterovirus, coxsackievirus, human parvovirus, encephalomyocarditis virus, and rhinovirus. The bacteria from which bacterial antigens are derived are not particularly limited, but examples include Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae, Salmonella enterica, Helicobacter pylori, Clostridium perfringens, Clostridium botulinum, Campylobacter, Escherichia coli, Staphylococcus aureus, Streptococcus staphylococcus, Bacillus cereus, Vibrio parahaemolyticus, Propionibacterium acnes, Clostridium faecalis, Clostridium difficile, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella pneumoniae, Klebsiella pneumoniae, Koinebacterium, Streptococcus hemolyticus, Pseudomonas aeruginosa, Staphylococcus aureus, Mycoplasma, Candida, and Aspergillus.

[0020] The 5'-capped polynucleotide contains an arbitrary base sequence A, and the single-stranded RNA contains a base sequence B that can bind to base sequence A through complementary base pairing. Base sequence A and base sequence B enable the 5'-capped polynucleotide and the single-stranded RNA to bind, thereby positioning a translation complex necessary for translation of the single-stranded RNA. Note that base sequence A and base sequence B are names used to distinguish between the two, and the arbitrary base sequence B and base sequence A that can bind to base sequence B through complementary base pairing may also be used.

[0021] Complementary base pairing occurs when the base sequences are complementary to each other. "Complementary" refers not only to a perfect base complementarity (e.g., A and T or U, and G and C), but also to a degree of complementarity that allows hybridization under stringent conditions. Stringent conditions can be determined based on the melting temperature (Tm) of the nucleic acid, as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego, CA). For example, post-hybridization washing conditions typically include approximately 1x SSC, 0.1% SDS, and 37°C. Preferably, the hybridization state is maintained even after washing under such conditions. Although not particularly limited, examples of stringent hybridization conditions include washing conditions of about "0.5×SSC, 0.1% SDS, 42°C," and even stringent hybridization conditions include washing conditions of about "0.1×SSC, 0.1% SDS, 65°C." Specifically, base sequence B is a base sequence that has, for example, 85% or more identity, preferably 90% or more identity, more preferably 95% or more identity, even more preferably 98% or more identity, still more preferably 99% or more identity, and particularly preferably 100% identity to a base sequence completely complementary to base sequence A.

[0022] As used herein, the "identity" of a base sequence refers to the degree of match between the base sequences of two or more comparable base sequences. Therefore, the greater the match between two base sequences, the greater the identity or similarity between those sequences. The level of identity between base sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes," Proc Natl Acad Sci USA. 87:2264-2268 (1990); Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences," Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTX, based on the BLAST algorithm, has been developed. Specific techniques for these analysis methods are known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ).

[0023] The base length of base sequence A and base sequence B is not particularly limited, but is, for example, 8 to 1000, 10 to 500, or 25 to 200.

[0024] 5'-capped polynucleotides and single-stranded RNAs can be produced according to or in accordance with known polynucleotide production methods. 5'-capped polynucleotides or single-stranded RNAs present in living organisms or cells may also be used. 5'-capped polynucleotides can also be preferably produced by the production method of the present invention described below.

[0025] In a preferred embodiment of the present invention, the 5'-capped polynucleotide is a polynucleotide present in a living organism or a cell. Examples of such polynucleotides include lncRNA and mRNA, with lncRNA being preferred. By selecting a lncRNA or mRNA that is specifically expressed in a specific cell or tissue, a protein encoded by a single-stranded RNA exogenously introduced into a living organism or cell can be expressed in a cell- or tissue-specific manner. This can also reduce side effects and off-target effects. Furthermore, a specific lncRNA or mRNA can be detected by using a protein encoded by the single-stranded RNA as a reporter protein. Alternatively, the single-stranded RNA may be a living organism or a cell-resident RNA. Examples of such polynucleotides include circular mRNA and linear mRNA, with circular mRNA being preferred.

[0026] The production or expression method of the present invention comprises carrying out a translation reaction in a reaction system containing a 5'-capped polynucleotide and single-stranded RNA.

[0027] The reaction system is not particularly limited as long as it contains substances necessary for the translation reaction (e.g., ribosomes, amino acids, tRNA, etc.), and may be either an in vivo reaction system or an in vitro reaction system. In the case of an in vivo reaction system, the biological species is not particularly limited, and examples thereof include various mammalian animals such as humans, monkeys, mice, rats, dogs, cats, and rabbits; animal cells, etc. The type of cell is also not particularly limited, and examples thereof include blood cells, hematopoietic stem cells / progenitor cells, gametes (sperm, ova), fibroblasts, epithelial cells, vascular endothelial cells, neurons, hepatocytes, keratinocytes, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, and cancer cells. In the case of an in vitro reaction system, a reconstituted system or an extract of the above organisms or cells can also be used.

[0028] The reaction conditions for the translation reaction are not particularly limited, and appropriate conditions can be set depending on the enzymes used, etc.

[0029] When the production or expression method of the present invention is carried out in vivo, it can be carried out by introducing a 5'-capped polynucleotide and / or single-stranded RNA into a subject (organism or cell). In one embodiment, when the production or expression method of the present invention is carried out in vivo, it can be carried out by introducing a 5'-capped polynucleotide and single-stranded RNA into a subject. In one embodiment, when the production or expression method of the present invention is carried out in vivo, it can be carried out particularly preferably by introducing a 5'-capped polynucleotide or single-stranded RNA into a subject. Furthermore, when the production or expression method of the present invention is carried out in vitro, it can be carried out by adding a 5'-capped polynucleotide and / or single-stranded RNA (particularly preferably a 5'-capped polynucleotide or single-stranded RNA (i.e., either one)) to a reaction system. Therefore, in one aspect, the present invention relates to a composition (the composition of the present invention) comprising: a 5'-capped polynucleotide containing an arbitrary base sequence A; and / or a single-stranded RNA containing a base sequence B capable of binding to the base sequence A by complementary base pairing, and a protein or peptide coding sequence, wherein the 5'-capped polynucleotide and / or the single-stranded RNA has a translation efficiency-enhancing structure. In other words, when introducing or adding, the 5'-capped polynucleotide and the single-stranded RNA do not necessarily need to be bound to each other; they may be introduced (or added) separately without being bound to each other, or either the 5'-capped polynucleotide or the single-stranded RNA may be introduced and then bound to a 5'-capped polynucleotide or single-stranded RNA present in the target or reaction system.

[0030] In a particularly preferred embodiment of the present invention, the composition of the present invention preferably comprises a 5'-capped polynucleotide or a single-stranded RNA (i.e., one or the other only).

[0031] The compositions of the present invention can be pharmaceuticals or reagents. When the compositions are pharmaceuticals, they can be used, for example, as vaccines (e.g., vaccines against microbial infections) or as detectors for cellular or endogenous 5'-capped polynucleotides such as lncRNA and mRNA. The compositions may further contain other components as needed. These other components are not particularly limited as long as they are pharmaceutically acceptable, and examples include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, and chelating agents. The compositions can also be in the form of a kit, in which two or more substances are contained in two or more separate containers.

[0032] The composition of the present invention may be applied to any mammal, including, but not limited to, humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer. Examples of cells include animal cells. The types of cells are also not particularly limited, including, but not limited to, blood cells, hematopoietic stem cells and progenitor cells, gametes (sperm, eggs), fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, hepatocytes, keratinocytes, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, and cancer cells.

[0033] The compositions of the present invention may be in any dosage form, for example, oral formulations such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), and jellies; or parenteral formulations such as injectable preparations (e.g., drip injections (e.g., intravenous drip preparations), intravenous injections, intramuscular injections, subcutaneous injections, and intradermal injections), topical preparations (e.g., ointments, poultices, lotions), suppositories, inhalants, eye drops, eye ointments, nasal drops, and ear drops. The active ingredient can be administered in a complexed state with particles (e.g., lipid particles or exosomes) or encapsulated in the particles.

[0034] The route of administration of the composition of the present invention is not particularly limited as long as the desired effect can be obtained, and examples thereof include oral administration; enteral administration such as tube feeding and enema administration; and parenteral administration such as intravenous administration, intraarterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intradermal administration, intraperitoneal administration, and nasal administration.

[0035] The content of the active ingredient in the composition of the present invention depends on the mode of use, the subject to which it is applied, the condition of the subject to which it is applied, etc., and is not limited thereto, but can be, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight.

[0036] The dosage of the composition of the present invention when administered to an animal is not particularly limited as long as it is an effective amount that exhibits a medicinal effect, and is generally 0.01 to 100 mg / kg body weight, preferably 0.05 to 50 mg / kg body weight per day in terms of the weight of the active ingredient. The dosage can be increased or decreased as appropriate depending on the age, pathological condition, symptoms, etc.

[0037] The production or expression method of the present invention is characterized in that the 5'-capped polynucleotide and / or single-stranded RNA has a translation efficiency enhancing structure.

[0038] The translation efficiency-enhancing structure is preferably a structure that stabilizes the bond between the 5'-capped polynucleotide and the single-stranded RNA. The structure is not particularly limited as long as it can stabilize the bond between the 5'-capped polynucleotide and the single-stranded RNA. Examples of such a structure include requirements (b) and (c), which will be described later.

[0039] In addition, modified nucleotides constituting base sequence A of a 5'-capped polynucleotide and / or base sequence B of a single-stranded RNA that stabilize the double-stranded structure between base sequence A and base sequence B compared to standard nucleotides (phosphodiester bond, ribose / deoxyribose, basic nucleobases (A, T, G, C)) also fall under the category of the stabilizing structure. Such modified nucleotides may be known, including 2'-ome modified nucleotides in the sugar moiety and LNA. The number of modified nucleotides is preferably 30% or more, more preferably 50% or more, even more preferably 70% or more, even more preferably 80% or more, particularly preferably 90% or more, and particularly preferably 100% of the total number of nucleotides constituting base sequence A or base sequence B (100%).

[0040] The translation efficiency-enhancing structure preferably satisfies at least one requirement selected from the group consisting of: (a) the single-stranded RNA is a circular RNA; (b) the 5'-capped polynucleotide and / or the single-stranded RNA have a nuclease-binding structure, and the reaction system contains the nuclease; and (c) the 5'-capped polynucleotide and / or the single-stranded RNA have a structure that covalently bonds them.

[0041] An embodiment that satisfies requirement (a) corresponds to Test Example 4 (FIG. 4) described below. An embodiment that satisfies requirement (b) corresponds to Test Example 1 (FIG. 1) and Test Example 2 (FIG. 2) described below. An embodiment that satisfies requirement (c) corresponds to Test Example 3 (FIG. 3) described below. However, the embodiments of the present invention are not limited to the embodiments of the Test Examples described below.

[0042] When requirement (a) is satisfied, it is preferable that the circular RNA does not contain a stop codon. This allows rolling circle translation to occur, further improving translation efficiency. In this case, the production or expression method of the present invention, in which a 5'-capped polynucleotide binds to a circular RNA, was thought to inhibit translation during the second turn of the ribosome due to the presence of the 5'-capped polynucleotide, but unexpectedly improved translation efficiency.

[0043] When requirement (a) is satisfied, other preferred embodiments are as follows: From the viewpoint of translation efficiency, the base length of base sequence A is preferably 10 to 100, more preferably 12 to 50, and particularly preferably 15 to 30. From the viewpoint of translation efficiency, the base length of the base sequence between the 5'-terminal base of base sequence A and the 5'-cap is preferably 0 to 100, more preferably 5 to 50, and particularly preferably 10 to 30.

[0044] The nuclease in requirement (b) is a nucleic acid-binding nuclease, and is not particularly limited as long as it is capable of binding to a target sequence in a sequence-specific manner. Examples of such nucleases include RISC (RNA-induced silencing complex), which functions in the RNA interference mechanism, and Cas, which functions in the CRISPR / Cas system. When requirement (b) is satisfied, the nuclease is preferably RISC (corresponding to Test Example 1 (Figure 1) described below) or Cas (corresponding to Test Example 2 (Figure 2) described below). However, aspects of the present invention are not limited to the aspects of the Test Examples described below. Using a nuclease can promote the association of a 5'-capped polynucleotide with a single-stranded RNA, thereby promoting the binding of the two and improving translation efficiency.

[0045] The nuclease-binding structure varies depending on the type of nuclease, and can be appropriately selected and designed according to the type.

[0046] When the nuclease is a RISC, the nuclease-binding structure is such that the 5'-capped polynucleotide is an siRNA or miRNA.

[0047] The siRNA is preferably, for example, 18 bases or more, 19 bases or more, 20 bases or more, or 21 bases or more in length. Also, the siRNA is preferably, for example, 25 bases or less, 24 bases or less, 23 bases or less, or 22 bases or less in length. The upper and lower limits of the siRNA length described herein are assumed to be arbitrarily combined. For example, the following combinations are assumed: a lower limit of 18 bases, and an upper limit of 25 bases, 24 bases, 23 bases, or 22 bases; a lower limit of 19 bases, and an upper limit of 25 bases, 24 bases, 23 bases, or 22 bases; a lower limit of 20 bases, and an upper limit of 25 bases, 24 bases, 23 bases, or 22 bases; a lower limit of 21 bases, and an upper limit of 25 bases, 24 bases, 23 bases, or 22 bases.

[0048] The siRNA may be a small hairpin RNA (shRNA). The shRNA can be designed so that a portion thereof forms a stem-loop structure. The siRNA may have additional bases at the 5' or 3' end. The length of the additional bases is usually about 2 to 4 bases. The siRNA may have a protruding sequence (overhang) at the 3' end, specifically, one with dTdT (dT stands for deoxythymidine) added. Alternatively, the siRNA may be blunt-ended.

[0049] Unlike siRNAs, miRNAs do not cleave target mRNAs, but may inhibit their translation by pairing with the 3' untranslated region (UTR) of the target. miRNAs may be pri-miRNAs (primary miRNAs), pre-miRNAs (precursor miRNAs), or mature miRNAs. The length of a miRNA is not particularly limited; pri-miRNAs are typically several hundred to several thousand bases long, pre-miRNAs are typically 50 to 80 bases long, and mature miRNAs are typically 18 to 30 bases long.

[0050] The guide strand of the siRNA and miRNA has a 5'-cap structure and contains an arbitrary base sequence A. To suppress cleavage of the single-stranded RNA by RISC, base sequence A preferably has an identity of 95% or less, more preferably 90% or less, to a base sequence that is completely complementary to base sequence B. More specifically, in this embodiment, base sequence A contains 1 to 8, 1 to 6, or 2 to 4 mismatched bases and / or unnatural bases relative to the base sequence that is completely complementary to base sequence B.

[0051] When the nuclease is Cas, the nuclease binding structure comprises a tracrRNA sequence in addition to base sequence A or base sequence B of a 5'-capped polynucleotide and / or single-stranded RNA.

[0052] The tracrRNA sequence is not particularly limited. The tracrRNA sequence is typically an RNA consisting of a sequence of approximately 50 to 100 bases long that can form multiple (usually three) stem-loops, and the sequence varies depending on the type of Cas protein used. Furthermore, the tracrRNA sequence is positioned on the 5' or 3' side of base sequence A or base sequence B, depending on the type of Cas protein used. Various known sequences can be used as the tracrRNA sequence, depending on the type of Cas protein used.

[0053] The Cas protein is not particularly limited as long as it is used in the CRISPR / Cas system. Cas proteins derived from various organisms are known, including, for example, Cas9 protein, Cas12 protein, Cas13 protein, and Cpf1 protein (type V) derived from F. novicida. Information on the amino acid sequences and coding sequences of various Cas proteins can be easily obtained from various databases such as NCBI.

[0054] The Cas protein preferably contains amino acid mutations to suppress cleavage of single-stranded RNA. Mutations that eliminate or reduce nuclease activity are publicly known or can be easily designed based on publicly known information.

[0055] When requirement (b) is met and the nuclease is Cas, preferably, the 5'-capped polynucleotide is a lncRNA or mRNA, and the single-stranded RNA contains a tracrRNA sequence. By designing base sequence B to be complementary to a sequence (base sequence A) within the lncRNA or mRNA, the lncRNA or mRNA can be used as the 5'-capped polynucleotide. When the production or expression method of the present invention is performed in vivo, endogenous RNA can be used as the lncRNA or mRNA. By selecting a lncRNA or mRNA that is specifically expressed in a specific cell or tissue, the protein encoded by the single-stranded RNA can be expressed in a cell- or tissue-specific manner. This can also reduce side effects and off-target effects. Furthermore, a specific lncRNA or mRNA can be detected by using the protein encoded by the single-stranded RNA as a reporter protein. When Cas is used as the nuclease, a target containing a polynucleotide containing a Cas coding sequence is used, the polynucleotide is introduced into the target, or the polynucleotide is added to the reaction system.

[0056] When requirement (b) is satisfied and the nuclease is Cas, the base length of the base sequence between the 5'-terminal base of base sequence A and the 5'-cap is, from the viewpoint of translation efficiency, preferably 3 or more, more preferably 7 or more, even more preferably 12 or more, and still more preferably 20 or more. The upper limit of the base length is not particularly limited and is, for example, 100, 60, or 30.

[0057] When requirement (b) is satisfied, other preferred embodiments are as follows: From the viewpoint of translation efficiency, the base length of base sequence A is preferably 10 to 100, more preferably 12 to 50, and particularly preferably 15 to 30.

[0058] The "structure that covalently bonds the two" in requirement (c) is not particularly limited as long as it is a structure possessed by one or both of the 5'-capped polynucleotide and the single-stranded RNA, and is capable of covalently linking the 5'-capped polynucleotide and the single-stranded RNA. Such a structure is preferably a photocrosslinkable structure (a structure that can form a crosslinked structure upon light irradiation). Examples of photocrosslinkable structures include psoralens, more specifically, 8-methoxypsoralen (methoxsalen) and 4,5',8-trimethylpsoralen (trioxalen). For example, by using a 5'-capped polynucleotide or a single-stranded RNA in which some bases in base sequence A / base sequence B are replaced with the above structure, a covalent bond can be formed with the opposing base (e.g., uracil) when base sequence A and base sequence B are complementarily bonded by light irradiation (e.g., light irradiation with a wavelength of 320 to 380 nm). The formation of a covalent bond allows the 5'-capped polynucleotide to more effectively exhibit its effect of improving translation efficiency.

[0059] In one embodiment of the present invention, the protein or peptide produced by the production or expression method of the present invention can be purified from the reaction system according to or in accordance with a known method.

[0060] 1a. Protein or Peptide Production or Expression Method 2 This section corresponds to the above sections 8 to 10 and the below-described Test Examples 6 to 8 (FIGS. 8 to 11). However, the present invention is not limited to the below-described Test Examples.

[0061] In one aspect, the present invention relates to a method for producing or expressing a protein or peptide, which method comprises carrying out a translation reaction in a reaction system containing a single-stranded circular RNA in which a 5'-capped polynucleotide is linked via a linker.

[0062] For the 5'-capped polynucleotide, the description in "1. Method 1 for producing or expressing a protein or peptide" above can be used, except that the polynucleotide does not have to contain the base sequence A.

[0063] The base length of the base sequence contained in the 5'-capped polynucleotide is not particularly limited, but is, for example, 1 to 500, preferably 2 to 100, more preferably 2 to 50, even more preferably 2 to 20, still more preferably 2 to 10, and particularly preferably 3 to 6.

[0064] The single-stranded circular RNA contains a protein or peptide coding sequence. The description of "1. Protein or Peptide Production or Expression Method 1" above can be applied to the single-stranded circular RNA, except that it does not contain the base sequence B.

[0065] The base length of the single-stranded circular RNA is not particularly limited. From the viewpoint of translation efficiency, it is preferable that the base length is relatively short, for example, 500 or less, preferably 300 or less, more preferably 250 or less, and even more preferably 200 or less. In this embodiment, since an IRES is not required, the size can be kept small, thereby achieving high translation efficiency.

[0066] In the single-stranded circular RNA, all nucleotides may be linked by typical internucleotide bonds such as phosphodiester bonds or phosphorothioate bonds, or some nucleotides may be linked by linkers. In the latter case, it is preferable that a 5'-capped polynucleotide is linked to the linker.

[0067] The linker is not particularly limited as long as it has a flexibly operable structure. The linker may consist solely of a chain structure, or may contain a chain structure and a cyclic structure. The chain structure may be linear or branched. For example, in a linear structure having two ends (conveniently designated ends a and b), end a can be linked to a nucleotide (preferably the terminal nucleotide) of a chain-like single-stranded RNA, end b can be linked to a 5'-capped polynucleotide (preferably the 3' end of the 5'-capped polynucleotide), and another nucleotide of the chain-like single-stranded RNA (preferably the other terminal nucleotide other than the terminal nucleotide) can be linked to any site c of the linker structure connecting ends a and b. In this example, end a, end b, and site c can be interchanged. As another example, if the branched chain has three ends (conveniently designated as ends A to C), ends A and B can link between nucleotides of the linear single-stranded RNA (preferably between nucleotides at both ends), and end C can link to a 5'-capped polynucleotide (preferably the 3' end of the 5'-capped polynucleotide), and such a configuration is also preferable from the viewpoint of translation efficiency.

[0068] More specifically, the linker is preferably a hydrocarbon chain in which at least one carbon atom constituting the main chain may be replaced with a heteroatom and / or a linking structure. When at least one carbon atom constituting the main chain is replaced with a heteroatom and / or a linking structure, the number of replaced carbon atoms is, for example, 1 to 5.

[0069] The hydrocarbon chain is preferably an alkylene group. The alkylene group may be either linear or branched, but is preferably linear. The number of carbon atoms constituting the main chain of the alkylene group is preferably 6 or more. The number of carbon atoms is more preferably 10 or more, even more preferably 12 or more, and is preferably 100 or less, more preferably 50 or less, even more preferably 20 or less.

[0070] Examples of heteroatoms include oxygen atoms, sulfur atoms, and nitrogen atoms. When at least one carbon atom constituting the main chain of an alkylene group is replaced with an oxygen atom, specifically, -CH2- in the main chain of the alkylene group is replaced with, for example, -O-. When at least one carbon atom constituting the main chain of an alkylene group is replaced with a sulfur atom, specifically, -CH2- in the main chain of the alkylene group is replaced with, for example, -S-, -S(=O)2-, -S(=O)-. When at least one carbon atom constituting the main chain of an alkylene group is replaced with a nitrogen atom, specifically, -CH2- in the main chain of the alkylene group is replaced with, for example, -NR- (R is a hydrogen atom or a hydrocarbon group (preferably an alkyl group, more preferably an alkyl group having 1 to 8 carbon atoms)). The linking structure is not particularly limited, as long as it is a divalent group that is a bonded structure formed by the reaction of two identical or different reactive groups. Examples of reactive groups include amino, carboxy, hydroxy, ketone, ethynyl, vinyl, azide, epoxy, aldehyde, oxylamino, thiol, isocyanate, and isothiocyanate groups. Examples of reactions between reactive groups are as follows: An amino group is known to react with a carboxy group (or a group formed by esterifying a carboxy group with N-hydroxysuccinimide (NHS)) to form an amide bond. An ethynyl group is known to undergo a 1,3-dipolar cycloaddition reaction with an azide group to form a 1,2,3-triazole ring. Cycloctyne is known to undergo a 1,3-dipolar cycloaddition reaction with an azide group to form a 1,2,3-triazole ring. An amino group is known to react with a carboxy group to form an amide bond. A carboxy group is known to react with a hydroxy group to form an ester bond. A carboxy group is known to react with a thiol group to form a thioester bond. Phosphate groups are known to react with hydroxyl groups to form phosphodiester bonds. Vinyl groups react with thiol groups to form bonds. Epoxy groups react with amino and thiol groups to form bonds. Aldehyde groups react with amino groups to form Schiff bases, which can be reduced to form bonds.The oxylamino group reacts with a ketone group or an aldehyde group to form an oxime.

[0071] A single-stranded circular RNA in which 5'-capped polynucleotides are linked via a linker can be obtained by chemical synthesis.

[0072] For other aspects, the contents of "1. Protein or peptide production or expression method 1" above can be used.

[0073] 2. Method for Producing 5'-Capped Polynucleotides This section corresponds to the above-mentioned sections 11 to 18 and the below-described Test Example 5 (FIGS. 5 to 7). However, the present invention is not limited to the below-described Test Example.

[0074] In one aspect, the present invention relates to a method for producing a 5'-capped polynucleotide, the method comprising: carrying out a transcription reaction in a reaction system containing a double-stranded polynucleotide comprising a phage promoter having a base insertion mutation sequence adjacent to the upstream side of the transcription start site, and an antisense strand comprising a base sequence Y: (upstream)-Y2-Y3 or (upstream)-Y1-Y2-Y3 (in base sequence Y: Y1 and Y2 represent bases in the base insertion mutation sequence, and Y3 represents the base at the transcription start site); and a 5'-cap analog having a polynucleotide comprising a base sequence X: (cap side)-X2-X3 or (cap side)-X1-X2-X3, wherein X3 is the complementary base of Y3, and X1 is the complementary base of Y1 and / or X2 is the complementary base of Y2 (herein sometimes referred to as the "production method of the present invention"). This method is described below.

[0075] The phage promoter is not particularly limited and examples thereof include the T7 promoter, SP6 promoter, T3 promoter, etc. Among these, the T7 promoter is particularly preferred from the viewpoint of capping efficiency.

[0076] The phage promoter has a nucleotide insertion mutation sequence adjacent to the upstream side of the transcription start site. That is, the phage promoter used in the production method of the present invention is a mutant promoter obtained by nucleotide insertion mutation adjacent to the upstream side of the transcription start site of a wild-type phage promoter (e.g., T7 promoter, SP6 promoter, T3 promoter, etc.). The transcription start site of the wild-type phage promoter can be easily determined based on publicly known information.

[0077] In the double-stranded polynucleotide used in the production method of the present invention, the antisense strand comprises the nucleotide sequence Y: (upstream)-Y2-Y3 or (upstream)-Y1-Y2-Y3, where Y1 and Y2 represent nucleotides in the nucleotide insertion mutation sequence, and Y3 represents the nucleotide at the transcription start site.

[0078] The double-stranded polynucleotide contains the coding sequence of the 5'-capped polynucleotide to be produced downstream of the phage promoter, i.e., the base sequence of the 5'-capped polynucleotide is contained in the sense strand. The double-stranded polynucleotide is particularly preferably DNA.

[0079] The base length of the double-stranded polynucleotide is not particularly limited as long as it is a length that can be used as a transcription template, and is, for example, 25 to 10,000, 25 to 5,000, 25 to 3,000, 25 to 2,000, 25 to 1,000, 25 to 500, 25 to 300, or 25 to 200.

[0080] The 5' cap analog is a single-stranded polynucleotide to which a 5' cap structure has been added, and is not particularly limited as long as it can be used to produce a 5' capped polynucleotide.

[0081] The 5'-cap structure is as described above. The 5'-cap structure may include a photoremovable hydrophobic purification tag (e.g., Non-Patent Document 1). According to the production method of the present invention, it is possible to produce highly pure 5'-capped polynucleotides without using the hydrophobic purification tag, and it is also possible to produce even more pure 5'-capped polynucleotides by using the hydrophobic purification tag.

[0082] The base length of the polynucleotide having the 5' cap analog is preferably 2 to 5, more preferably 2 to 4, and particularly preferably 3.

[0083] The polynucleotide having the 5'-cap analog comprises the base sequence X: (cap side)-X2-X3 or (cap side)-X1-X2-X3. In the base sequence X, X3 is preferably at the 3' end of the polynucleotide.

[0084] In the production method of the present invention, X3 is a complementary base to Y3, and X1 is a complementary base to Y1 and / or X2 is a complementary base to Y2.

[0085] The above complementary relationship and the use of a phage promoter having a base insertion mutation sequence can improve capping efficiency. From the viewpoint of capping efficiency, it is particularly preferred that the antisense strand of the phage promoter contains the base sequence Y: (upstream)-Y1-Y2-Y3, where X1 is the complementary base of Y1 and X2 is the complementary base of Y2.

[0086] Taking the φ6.5, XY-inserted shown in Figure 5a) as an example, "XYG" in the 5'-cap analog (m7G-XYG) represents the base sequence X (X1-X2-X3), the lower strand in the duplex below it is the antisense strand, and "XYC" in the antisense strand corresponds to the base sequence Y (Y1-Y2-Y3).

[0087] The specific sequence of the base sequence X is not particularly limited, but from the viewpoints of capping efficiency, production efficiency of 5'-capped polynucleotides, etc., it is particularly preferable that the base sequence X is (cap side) -CUG, CAG, GUG, or GAG, and that Y3 is C.

[0088] From the viewpoint of translation efficiency of the 5'-capped polynucleotide to be produced, it is particularly preferred that the specific sequence of base sequence X is (cap side) UUG, UCG, UAG, CUG, CAG, AUG, or AAG, and Y3 is C, or (cap side) UUA, GUA, CUA, or AUA, and Y3 is U / T.

[0089] The double-stranded polynucleotide and 5'-cap analog can be produced according to or in accordance with known methods for producing polynucleotides and 5'-cap analogs. More specifically, the 5'-cap analog can be produced using the synthesis method described in the Examples below.

[0090] The reaction system is not particularly limited as long as it contains substances necessary for the translation reaction (for example, RNA polymerase, various nucleoside triphosphates, etc.), and may be either an in vitro reaction system or an in vivo reaction system.

[0091] In the present invention, we found that the efficiency of cap analog incorporation can be further improved by using a T7 RNA polymerase having the D130W mutation ( FIG. 7 ). Therefore, in one aspect, the present invention relates to a T7 RNA polymerase having the D130W mutation."D130W" refers to a mutation of D (aspartic acid), the 130th amino acid from the N-terminus of the amino acid sequence of T7 RNA polymerase (SEQ ID NO: 1), to W (tryptophan).The mutant T7 RNA polymerase can contain other amino acid mutations, for example, 80 or less, 50 or less, 30 or less, 20 or less, 10 or less, 5 or less, 2 or less, or 1 or less, as long as the efficiency of cap analog incorporation is not significantly impaired. The mutant T7 RNA polymerase may also have another amino acid sequence (e.g., a tag sequence, a sequence having an independent domain structure such as a fluorescent protein) added thereto, as long as the efficiency of cap analog incorporation is not significantly impaired.

[0092] The reaction conditions for the transcription reaction are not particularly limited, and appropriate conditions can be set depending on the enzymes used, etc.

[0093] The production method of the present invention can be carried out by adding a double-stranded polynucleotide and / or a 5'-cap analog to a reaction system. Therefore, in one aspect, the present invention relates to a composition comprising: a double-stranded polynucleotide comprising a phage promoter having a base insertion mutation sequence adjacent to the upstream side of a transcription start site, and an antisense strand comprising a base sequence Y: (upstream)-Y2-Y3 or (upstream)-Y1-Y2-Y3 (in base sequence Y: Y1 and Y2 represent bases in the base insertion mutation sequence, and Y3 represents the base at the transcription start site), and / or a 5'-cap analog having a polynucleotide comprising a base sequence X: (cap side)-X2-X3 or (cap side)-X1-X2-X3, wherein X3 is the complementary base of Y3, and X1 is the complementary base of Y1 and / or X2 is the complementary base of Y2.

[0094] The composition can be a medicine or a reagent. The composition may further contain other components as needed. The other components are not particularly limited as long as they are pharmaceutically acceptable, and examples include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc. The composition can also be in the form of a kit, in which two or more substances are contained in two or more separate containers.

[0095] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0096] Test Example 1. Translation reaction promotion phenomenon of circular mRNA utilizing the RNA interference mechanism A conceptual diagram of this experiment is shown in Figure 1(a).

[0097] The base sequence of the circular RNA used in this experiment is as follows: circular Nluc mRNA (620-nt) 5'__3' (SEQ ID NO: 6) (5' and 3' ends are linked).

[0098] Circular RNA was prepared as follows. First, 5'-phosphorylated linear RNA was prepared as the starting material for cyclization by transcription using the following composition: 5 ng / μL dsDNA (PCR product containing the T7 promoter), 2 mM ATP, 2 mM UTP, 2 mM CTP, 2 mM GTP, 10 mM GMP, 40 mM Tris-HCl (pH 8.0), 8 mM MgCl2, 2 mM spermidine, 5 mM DTT, 0.002 U / μL pyrophosphatase, and 4.7 ng / μL T7 RNA polymerase. The reaction mixture was incubated at 37°C for 2 hours, followed by the addition of DNase I (Takara Bio) to a final concentration of 0.1 U / μL, and then incubated at 37°C for 15 minutes. The reaction mixture was extracted with an equal volume mixture of TE-saturated phenol and chloroform to remove proteins, and the transcribed RNA was recovered by alcohol precipitation. The resulting crude transcribed RNA was purified using reverse-phase HPLC as previously reported (Nat. Commun., 14, 2657, 2023). The resulting 5'-phosphorylated transcribed RNA was cyclized by ligating the 5' and 3' ends using T4 RNA ligase 2. The composition of the ligase reaction solution is as follows: The reaction mixture contained 0.5 μM 5'-phosphorylated RNA, 1 μM template DNA oligo (5' AATATGCGCCCTAGAATTAC 3' (SEQ ID NO: 7)), 10% PEG8000, 50 mM Tris-HCl (pH 7.5), 2 mM magnesium chloride, 1 mM DTT, 400 μM ATP, and 0.1 μg / μL T4 RNA ligase. The reaction mixture was heated at 37°C for 1 hour, extracted with an equal volume mixture of TE-saturated phenol and chloroform to remove proteins, and then added with aqueous sodium acetate (pH 5.2, final concentration 0.3 M) and 2-propanol. The mixture was cooled to -30°C and centrifuged to recover the RNA precipitate. The desired cyclized RNA was isolated and purified by preparative denaturing polyacrylamide gel electrophoresis.

[0099] The sequences of the RNA oligonucleotides used in this experiment are as follows: passenger strand 5' GACGCGUGUCUACUCGAACAUA 3' (SEQ ID NO: 8); uncapped guide strand 5' p-UGUUCGAGUAGACACGCGUCAC-p 3' (SEQ ID NO: 9); capped guide strand 5' p-UGUUCGAGUAGACACGCGUCAC(3')ppp(5')m 7 G 3' (sequence number 10).

[0100] Capped RNA oligonucleotides were prepared as previously reported (ACS Chem Biol 2022, 17, 1308-1314). 5'-phosphorylated RNA oligonucleotide sequences were synthesized using commercially available phosphoramidite compounds (Chemgene) on an automated nucleic acid synthesizer, NTS T-8-A20-R8NC (Nihon Techno Service). After synthesis, the CPG solid support was added to a 1:1 mixture of 40% aqueous methylamine and 28% concentrated aqueous ammonia and heated at 65°C for 60 minutes to cleave the oligonucleotide from the support and deprotect the phosphate and base moieties. The mixture was evaporated to dryness under reduced pressure, then added to 1 M TBAF / THF solution and incubated overnight at room temperature. The oligonucleotides were desalted using a NAP-25 gel filtration column (Cytiva) and alcohol precipitated to obtain the RNA oligonucleotides. The oligonucleotides were then purified by reverse-phase HPLC. The resulting 5'-phosphorylated RNA oligonucleotides were reacted with m7GDP imidazolide (Im-m7GDP) to introduce a cap structure at the 5' end (ACS Chem Biol 2022, 17, 1308-1314). The progress of the capping reaction was monitored by gel shift of the RNA bands in denaturing PAGE and molecular weight measurement by LC-MS analysis. The passenger strand and uncapped or capped guide strand were mixed in a buffer solution (10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA (pH 8.0)) to a final concentration of 1 μM. The solution was heated at 80°C for 5 minutes and then slowly cooled to prepare a 1 μM double-stranded RNA oligonucleotide solution.

[0101] The effect of capped double-stranded RNA oligonucleotides on the translation of circular Nluc mRNA was evaluated using the human cell line HeLa (RIKEN Cell Bank, RCB0007). HeLa cells were cultured in DMEM (Wako Pure Chemical Industries, Ltd.) containing 10% FBS (Thermo Fisher Scientific) at 5% CO2 and 37°C. HeLa cells were seeded at 1.5 × 10E4 cells / well in a 96-well multiwell cell culture plate the day before transfection. Circular Nluc mRNA (30 ng / well) and double-stranded RNA oligonucleotides (0.3 pmol / well, 1.5 pmol / well, or 3 pmol / well) were added to the cells separately using Lipofectamine MessengerMAX (0.15 μL), Opti-MEM (0.15 μL), or PBS. TM Mix with 10 μL of Reduced Serum Medium (Thermo Fisher Scientific) and Opti-MEM TM These were added to wells containing HeLa cells whose growth medium had been replaced with I Reduced Serum Medium (20 μL), and RNA was introduced into the cells. After 3 hours, the supernatant was replaced with growth medium, and after a total of 6 hours of culture, the amount of Nluc protein contained in the cell lysate was assessed using the Nano-Glo® Luciferase Assay System (Promega). Chemiluminescence was measured using a TriStar5 multimode plate reader (Berthold).

[0102] The results are shown in Figure 1. It was demonstrated that translation reaction can be promoted by creating a system in which RISC can bind.

[0103] Test Example 2: Translational enhancement of circular mRNA by hybridization with long non-coding RNA (lncRNA) and its stabilization by dCas13 protein. The conceptual diagram of this experiment is shown in Figure 2(a) and (c).

[0104] The base sequence of the RNA used in this experiment is as follows:

[0105] HULC RNA (500-nt): 5'_GAUGGGGGUGGAACUCAUGAUGGAAUUGGACCCUUUACAAGGGAAUGAGACAAGAGCUCUCUUUAUUGCCACUGUGAUACAGCAAGGCCCCCAAUCUGCAAGCCAGAAGAGUCGUCACUUGAUCGUGGACAUUUUCACCUCAGAACUGUGAUCCAAAUAUUGAUCUUUGGAAGAAAACUCUGAAGUAAAGGCCGGAAUAUUCUUUGUAUAACAUUAAACAAAAAAGGCCGGAAUAUUCUUUGUUUAAAACAUUAAAAACAAAAAGCACAAAGCAUCAAGCAAGAAGUUUCCUGGCAAUAAAACUAAGCACAGUUAUUUUUAAGGACACAAAUAUAUGUGUUUCACCUGUGGCAAAUUUGUACUCCCUGAAUUAUUGUGUGUAUACAAAAAGAAAAAAAUUGGGAAGCAUGGCAAAUAUAUUCAUCACUGAAACUACUAGAAUUAAACAAAAUAUAUUGUACUCUAAA

[0106]

[0107]

[0108] Linear Nluc mRNA (705-nt) with a 5-base linker: 5'__3' (SEQ ID NO: 13) (the underlined part is the complementary sequence to HULC).

[0109] Linear Nluc mRNA (705-nt) with a 10-base linker: 5'__3' (SEQ ID NO: 14) (the underlined part is the complementary sequence to HULC).

[0110] Linear and circular Nluc mRNA (705-nt) with a 15-base linker: 5'__3' (SEQ ID NO: 15) (The underlined part is the complementary sequence to HULC; the 5' and 3' ends of the circular Nluc mRNA are linked).

[0111] Linear and circular Nluc mRNA (705-nt) with a 15-base linker: 5'__3' (SEQ ID NO: 57) (The underlined part is the complementary sequence to UCA1. The 5' and 3' ends of the circular Nluc mRNA are linked.)

[0112] Each RNA was transcribed using T7 RNA polymerase with the DNA encoding the corresponding sequence as a template. The transcription reaction mixture contained the following: 10 ng / μL dsDNA (PCR product containing the T7 promoter), 2 mM ATP, 2 mM UTP, 2 mM CTP, 2 mM GTP, 40 mM Tris-HCl (pH 8.0), 8 mM MgCl2, 2 mM spermidine, 5 mM DTT, 0.002 U / μL pyrophosphatase (New England Biolabs), and 2 U / μL T7 RNA polymerase (Takara Bio). The reaction mixture was incubated at 37°C for 2 hours. When preparing HULC RNA, dRfxCas13d mRNA, and UCA1 RNA, the hydrophobic tag-containing cap analog compound DiPure (Nat. Commun. 14, 2657, 2023) was added to a final concentration of 2 mM. Capped mRNA was isolated and purified as previously described. After the transcription reaction, DNase I (Takara Bio) was added to a final concentration of 0.1 U / μL and incubated at 37°C for 15 minutes. 7.5 M lithium chloride solution was added to the reaction mixture to a final concentration of 2.5 M. The mixture was then cooled to -30°C for 30 minutes and centrifuged (20,000 × g, 20 minutes) to recover the transcribed RNA as a precipitate. The resulting crude transcribed RNA was purified by reverse-phase HPLC as previously described (Nat. Commun. 14, 2657, 2023). Circular Nluc mRNA was prepared by ligating the 5'- and 3'-ends of 5'-phosphorylated RNA synthesized by adding GMP to the above-mentioned transcription reaction solution to a final concentration of 10 mM in the presence of template DNA oligonucleotides (30 nt, 5'AGTGGACCTGGGACCTTTTTTTTTTTTTTTT 3' (SEQ ID NO: 16)) complementary to both ends of the RNA using T4 RNA ligase 2. The composition of the ligase reaction solution is as follows:The reaction mixture contained 1 μM 5'-phosphorylated RNA, 2 μM DNA oligonucleotide, 10% PEG8000, 50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1 mM DTT, 400 μM ATP, and 0.1 μg / μL T4 RNA ligase. The reaction mixture was incubated at 37°C for 1 hour, then extracted with an equal volume mixture of TE-saturated phenol and chloroform to deproteinize. Then, sodium acetate (pH 5.2, final concentration 0.3 M) and 2-propanol were added, cooled to -30°C, and centrifuged to recover the RNA precipitate. The desired cyclized RNA was isolated and purified by preparative denaturing polyacrylamide gel electrophoresis (Sci. Rep., 5, 16435, 2015).

[0113] Experiments to evaluate the expression level of Nluc protein were performed using the human-derived cultured cell line HeLa (RIKEN Cell Bank, RCB0007). HeLa cells were cultured in DMEM (Wako Pure Chemical Industries) containing 10% FBS (Thermo Fisher Scientific) at 5% CO2 and 37°C. The day before transfection, HeLa cells were seeded at 1 x 10E4 cells per well onto a 96-well multiwell plate.

[0114] dCas RNA (20 ng / well), HULC RNA (160 ng / well), and linear Nluc RNA (20 ng / well) were mixed with 0.015 μL / well, 0.12 μL / well, and 0.015 μL / well of Lipofectamine MessengerMAX (Thermo Fisher Scientific) at concentrations of 0.015 μL / well, 0.12 μL / well, and 0.015 μL / well, respectively, and incubated for 5 minutes. TMThese were added to wells containing HeLa cells whose growth medium had been replaced with I Reduced Serum Medium (Thermo Fisher Scientific) to transfect the cells. After 4 hours, the supernatant was replaced with growth medium. After a total of 24 hours of culture, the Nluc expression level in the cells was assessed using the Nano-Glo® Luciferase Assay System (Promega). Chemiluminescence measurements were performed using a TriStar5 multimode plate reader (Berthold). In experiments using circular Nluc mRNA, 0.1 μM HULC lncRNA and 0.2 μM Nluc RNA were annealed in TE buffer containing 50 mM NaCl and transfected into HeLa cells using Lipofectamine MessengerMAX. After 4.5 hours, Nluc expression was measured using the Nano-Glo® Luciferase Assay System.

[0115] Experiments evaluating Nluc protein expression levels were performed using the human-derived cultured cell line HeLa (RIKEN Cell Bank, RCB0007). HeLa cells were cultured in DMEM (Wako Pure Chemical Industries, Ltd.) containing 10% FBS (Thermo Fisher Scientific) at 37°C with 5% CO2. The day before transfection, 1 × 10E4 HeLa cells were seeded into a 96-well multiwell plate. 0.1 μM Nluc RNA and 0.2 μM HULC RNA or UCA1 RNA were mixed in TE buffer containing 50 mM NaCl, and hybridization was performed by heating and slow cooling. 1 μL of RNA hybridization product and 0.15 μL of Lipofectamine MessengerMAX (Thermo Fisher Scientific) were prepared per well. Each was diluted with 5 μL of Opti-MEM™ I Reduced Serum Medium (Thermo Fisher Scientific), mixed, and incubated at room temperature for 5 minutes to form liposomes. The medium in the 96-well multiplate containing HeLa cells prepared the day before was replaced with fresh DMEM, and the liposomes were added. RNA was then introduced. After 4.5 hours, the expression level of Nluc in the cells was assessed using the Nano-Glo® Luciferase Assay System (Promega). Chemiluminescence was measured using a TriStar5 multimode plate reader (Berthold).

[0116] The results are shown in Figure 2. It was demonstrated that hybridization with lncRNA and the creation of a system in which Cas proteins can bind can promote translation reactions.

[0117] Test Example 3. Photocrosslinkable capped RNA probes promote the translation reaction of circular mRNA in a light-dependent manner. A conceptual diagram of this experiment is shown in Figure 3(a).

[0118] The sequences of the chemically synthesized RNAs used in this experiment are as follows: Linear 24-nt RNA 5'_AAAAGGCGCAUAUUAAGGUGACGC_3' (SEQ ID NO: 17) Probe 1 (50-nt RNA) 5'_m7G-ppp-GCGUCAXCCUUAAUAUGCGCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA_3' (SEQ ID NO: 18) Probe 2 (50-nt RNA) 5'_m7G-ppp-GCGUCACCUUAAUAXUGCGCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA_3' (SEQ ID NO: 19) Probe 3 (50-nt RNA) 5'_m2 7,2’O G-ppp-GCGUCACCUUAAUAXUGCGCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3' (SEQ ID NO: 20) Probe 4 (50-nt RNA) 5'_m 7 G-ppp-GCGUCACCUUAAUAUGCGCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA — 3' (SEQ ID NO: 21) Probes 1 and 2: X contained in the sequence represents the inserted trioxalene structure (derived from the reagent CLP-6644 manufactured by Chemgene).

[0119] The sequence of the circular RNA used in this experiment is as follows: circular Nluc mRNA (650-nt) 5'__3' (SEQ ID NO: 22) (5' and 3' ends are linked).

[0120] Unmodified and trioxalene-capped RNA oligonucleotides were prepared as previously described (ACS Chem Biol 2022, 17, 1308-1314). 5'-phosphorylated RNA oligonucleotide sequences were synthesized using commercially available phosphoramidite compounds (Chemgene) on an automated nucleic acid synthesizer, NTS T-8-A20-R8NC (Nihon Techno Service). (5'-phosphate group, CLP-1544; trioxalene structure, CLP-6644). After synthesis, the CPG solid support was added to a 1:1 mixture of 40% aqueous methylamine and 28% concentrated aqueous ammonia and heated at 65°C for 15 minutes to cleave the oligonucleotide from the support and deprotect the phosphate and base moieties. The oligonucleotides were evaporated to dryness under reduced pressure, then added to a 1 M TBAF / THF solution and incubated overnight at room temperature. The oligonucleotides were desalted using a NAP-25 gel filtration column (Cytiva) and the RNA oligonucleotides were obtained as precipitates by alcohol precipitation. The oligonucleotides were purified by reverse-phase HPLC. The resulting 5'-phosphorylated RNA oligonucleotides were reacted with m7GDP imidazolide (Im-m7GDP) to introduce a cap structure at the 5' end (ACS Chem Biol 2022, 17, 1308-1314). The progress of the capping reaction was monitored by gel shift of the RNA bands in denaturing PAGE and molecular weight measurement by LC-MS analysis.

[0121] The circular RNA used in this experiment was prepared as follows. First, 5'-phosphorylated linear RNA was prepared as the starting material for cyclization by transcription using the following composition: 5 ng / μL dsDNA (PCR product containing the T7 promoter), 2 mM ATP, 2 mM UTP, 2 mM CTP, 2 mM GTP, 10 mM GMP, 40 mM Tris-HCl (pH 8.0), 8 mM MgCl2, 2 mM spermidine, 5 mM DTT, 0.002 U / μL pyrophosphatase, and 4.7 ng / μL T7 RNA polymerase. The reaction mixture was incubated at 37°C for 2 hours, followed by the addition of DNase I (Takara Bio) to a final concentration of 0.1 U / μL and incubation at 37°C for 15 minutes. The reaction mixture was extracted with an equal volume mixture of TE-saturated phenol and chloroform to remove proteins, and the transcribed RNA was recovered by alcohol precipitation. The resulting crude transcribed RNA was purified using reverse-phase HPLC as previously reported (Nat. Commun., 14, 2657, 2023). The resulting 5'-phosphorylated transcribed RNA was cyclized by ligating the 5' and 3' ends using T4 RNA ligase 2. The composition of the ligase reaction solution is as follows: The reaction mixture contained 0.5 μM 5'-phosphorylated RNA, 1 μM template DNA oligo (5' ACCTTAATATGCGCCTTTTTTTTTTTTTTT 3' (SEQ ID NO: 23)), 10% PEG8000, 50 mM Tris-HCl (pH 7.5), 2 mM magnesium chloride, 1 mM DTT, 400 μM ATP, and 0.1 μg / μL T4 RNA ligase. The reaction mixture was heated at 37°C for 1 hour, extracted with an equal volume mixture of TE-saturated phenol and chloroform to remove proteins, and then added with aqueous sodium acetate (pH 5.2, final concentration 0.3 M) and 2-propanol. The mixture was cooled to -30°C and centrifuged to recover the RNA precipitate. The desired cyclized RNA was isolated and purified by preparative denaturing polyacrylamide gel electrophoresis.

[0122] Crosslinking experiments of probe 1 and probe 2 with linear 24-nt RNA were performed as follows. A mixed solution of RNA and probe (1 μM linear 24-nt RNA, 3, 15, or 30 μM probe 1 or probe 2, 10 mM Tris-HCl (pH 7.5), 50 mM NaCl (pH 7.5), 1 mM EDTA (pH 8.0)) was prepared and heated at 90°C for 3 minutes, then slowly cooled to room temperature. The solution was then irradiated with 365-nm light at an intensity of 4.0 mW / cm2 using a 300W xenon light source MAX-350 for 30 minutes on ice. The reaction solution was analyzed by 20% denaturing PAGE to examine the efficiency of the crosslinking reaction. The RNA bands were detected by SYBR TM After staining with Green II Nucleic Acid Gel Stain (Takara Bio), the sections were photographed and visualized using a ChemiDoc MP imager (Bio-Rad).

[0123] Circular Nluc mRNA and the photocrosslinking probe (Probe 3) and non-crosslinking probe (Probe 4) were mixed in a solution (8.8 μL: 25 ng / μL circular Nluc mRNA, 0, 0.35, or 1.75 μL Probe 3 or Probe 4, 10 mM Tris-HCl (pH 7.5), 50 mM NaCl (pH 7.5), 1 mM EDTA (pH 8.0)) and heated at 90°C for 3 minutes, then slowly cooled to room temperature. The solution was then illuminated with a 300W xenon light source (MAX-350) at 4.0 mW / cm. 2 The cells were irradiated with 365-nm light at an intensity of 1.0 × 10 for 30 minutes on ice. 4 HeLa cells were seeded individually and treated with Lipofectamine® Messenger MAX TMThe cells were transfected with 10 ng mRNA / well using the reagent, and after 24 hours of culture, the cells were lysed. The amount of Nluc protein in the cell lysate was assessed using the Nano-Glo® Luciferase Assay System (Promega). Chemiluminescence was measured using a TriStar5 multimode plate reader (Berthold).

[0124] The results are shown in Figure 3. It was demonstrated that photo-crosslinking can promote translation reactions.

[0125] The results of the above Test Examples 1 to 3 demonstrated that, as a particularly preferred embodiment of the composition, the 5'-capped polynucleotide and single-stranded RNA are tightly linked and stabilized by RISC, Cas protein, and photo-crosslinking, thereby promoting the translation reaction.

[0126] Test Example 4 Promotion of Rolling Circle Translation Reaction by Capped RNA A conceptual diagram of this experiment is shown in Figure 4(a).

[0127] The circular RNA used in this experiment had the following base sequence: Linear / Circular FLAG-EGF (264-nt) 5'_GGGAGCCACCAUGGACUACAAGGACGACGACGACAAGAUCAUCGACUAUAAAGACGACGACGAUAAAGGUGGCGACUAUAAGGACGACGACGACAAAGCCAUCAACAGCGACAGCGAGUGCCCCCUGAGCCACGACGGCUACUGCCUGCACGACGGCGUGUGCAUGUACAUCGAGGCCCUGGACAAGUACGCCUGCAACUGCGUGGUGGGCUACAUCGGCGAGAGAUGCCAGUACAGAGACCUGAAGUGGUGGGAGCUGAGACU_3' (SEQ ID NO: 2) (Circular means that the 5' and 3' ends are linked).

[0128] The base sequences of the 5' phosphorylated RNA oligonucleotides and 5' capped RNA oligonucleotides used in this experiment are as follows: 5' capped probe 1 5'_m7G_ppp_GCCACCUCUCUGUACUGGCAUCUCUC_3' (SEQ ID NO: 3), 5' capped probe 2 5'_m7G_ppp_GACAGUCUGCUCGAAGCGGCCGCUCUAGAAUCUCUGUACUGGCAUCUCUC_3' (SEQ ID NO: 4), 5' capped probe 3 5'_m7G_ppp_GACAGUCUGCUCGAAGCGGCCGCUCUAGAACCGCGUAUAAUUCCACUGCG_3' (SEQ ID NO: 5).

[0129] Circular RNA (Sci Rep 2015, 5, 16435) was prepared as follows. First, 5'-phosphorylated linear RNA was prepared as the starting material for cyclization by transcription using the following composition: 5 ng / μL dsDNA (PCR product containing the T7 promoter), 2 mM ATP, 2 mM UTP, 2 mM CTP, 2 mM GTP, 10 mM GMP, 40 mM Tris-HCl (pH 8.0), 8 mM MgCl2, 2 mM spermidine, 5 mM DTT, 0.002 U / μL pyrophosphatase, and 4.7 ng / μL T7 RNA polymerase. The reaction mixture was incubated at 37°C for 2 hours, followed by the addition of DNase I (Takara Bio) to a final concentration of 0.1 U / μL, and then incubated at 37°C for 15 minutes. A 7.5 M aqueous solution of lithium chloride was added to the reaction mixture to a final concentration of 2.5 M. After cooling at -30°C for 30 minutes, the mixture was centrifuged (20,000 × g, 20 minutes) to recover the precipitated transcribed RNA. The resulting crude transcribed RNA was purified using reverse-phase HPLC as previously described (Nat. Commun. 14, 2657, 2023). The resulting 5'-phosphorylated transcribed RNA was cyclized by ligating the 5' and 3' ends using T4 RNA ligase 2. The composition of the ligase reaction solution is as follows: The reaction mixture contained 1 μM 5'-phosphorylated RNA, 10% PEG6000, 50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1 mM DTT, 400 μM ATP, and 0.1 μg / μL T4 RNA ligase. The reaction mixture was incubated at 37°C for 1 hour, then extracted with an equal volume mixture of TE-saturated phenol and chloroform to deproteinize. Sodium acetate (pH 5.2, final concentration 0.3 M) and 2-propanol were added, cooled to -30°C, and centrifuged to recover the RNA precipitate. The desired cyclized RNA was isolated and purified by preparative denaturing polyacrylamide gel electrophoresis.

[0130] Capped RNA oligonucleotides were prepared as previously reported (ACS Chem Biol 2022, 17, 1308-1314). 5'-phosphorylated RNA oligonucleotide sequences were synthesized using commercially available phosphoramidite compounds (Chemgene) on an automated nucleic acid synthesizer, NR-2A_7MX (Nihon Techno Service). After synthesis, the CPG solid support was added to a 1:1 mixture of 40% aqueous methylamine and 28% concentrated aqueous ammonia and heated at 65°C for 30 minutes to cleave the oligonucleotide from the support and deprotect the phosphate and base moieties. The mixture was evaporated to dryness under reduced pressure, and then incubated overnight at room temperature with 1 M TBAF / THF solution to deprotect the TOM protecting group. The oligonucleotides were then desalted using a NAP-25 gel filtration column (Cytiva) and alcohol precipitation to obtain the RNA oligonucleotides. The oligonucleotides were then purified by reverse-phase HPLC. The resulting 5'-phosphorylated RNA oligonucleotides were reacted with m7GDP imidazolide (Im-m7GDP) to introduce a cap structure at the 5' end. The capping reaction mixture (20 μM phosphorylated RNA oligonucleotide, 10 mM Im-m7GDP, 1 M 1-methylimidazole, DMSO solution) was heated at 55°C for 3 hours, and the RNA was recovered by alcohol precipitation. The target RNA was then separated and purified by reverse-phase HPLC. The progress of the capping reaction was monitored by gel shift of the RNA bands in denaturing PAGE and molecular weight measurement by LC-MS analysis [5' capped probe 1; Calcd 8632.0, Found 8633.06, (+1.1): 5' capped probe 2; Calcd 16425.7, Found; 16428.8 (+3.1): 5' capped probe 3; Calcd 16511.8, Found; 16512.0 (+0.2)].

[0131] Circular RNA translation reactions using rabbit reticulocyte lysate (Promega) were performed as follows. The translation solution (2 μM circular RNA, 4 μM RNA probe, 70 (v / v)% reticulocyte lysate, nuclease-treated, 10 μM amino acid mixture minus cysteine, 10 μM amino acid mixture minus leucine, 0.8 units / μL murine RNase Inhibitor (New England Biolabs)) was incubated at 30°C for 4 hours. A 4 μL aliquot of the reaction mixture was analyzed by Western blotting. After electrophoresis on a 5-20% gradient SDS-polyacrylamide gel (ATTO), proteins and peptides were transferred to an Immobilon-P membrane (Merck) using the semi-dry method. The membrane was then reacted with anti-FLAG M2 antibody (Sigma-Aldrich) and anti-mouse IgG-HRP conjugate (Sigma-Aldrich) in sequence, and then analyzed using SuperSignal. TM Chemiluminescence generated using West Femto Maximum Sensitivity Substrate (Thermo Fisher) was imaged with a ChemiDoc MP imager (Bio-Rad).

[0132] The results are shown in Figure 4. A significant increase in translation product was confirmed in the presence of a capped oligo-RNA probe containing a region complementary to the circular RNA sequence. Test Example 5. Development of a highly efficient method for synthesizing Cap-2 mRNA using PureCap analogs and modified promoters Test Example 5-1. Synthesis of PureCap analogs The synthesized PureCap analogs are shown below.

[0133]

[0134]

[0135] Liquid-Phase Synthesis of Trinucleotides Synthesis Scheme of Trinucleotide, pAGG (33)

[0136]

[0137]

[0138]

[0139] DMTr protection, synthesis of compound (23)

[0140]

[0141] DMTrCl (11.5 g, 33.98 mmol) in pyridine (100 mL) was dissolved in N 2 The resulting mixture was added to a mixture of 22 (10.0 g, 28.3 mmol) and 2-isobutyrylguanosine. The mixture was stirred at room temperature for 15 hours. The reaction was monitored by TLC, and after completion, the mixture was quenched with water. Pyridine was removed from the reaction mixture and diluted with water. The mixture was extracted with EtOAc and dried over Na2SO4. The solvent was removed on a rotary evaporator, and the residue was purified by silica gel column chromatography using 5% MeOH (1% TEA) in DCM to give compound 23 as a magenta solid (19.0 g, quantitative). 1H-NMR (400 MHz, DMSO-d6) δ 11.57 (s, 1H), 8.08 (d, J = 2.0 Hz, 1H), 7.31 (d, J = 7.6 Hz, 2H), 7.25-7.14 (m, 7H), 6.81-6.76 (m, 4H), 5.83 (q, J = 2.2 Hz, 1H), 5.64 (s, 1H), 5.24 (d, J = 15.9 Hz, 1H), 4.50 (t, J = 4.7 Hz, 1H), 4.19-4.00 (m, 2H), 3.68 (s, 6H), 3.25-3.13 (m, 2H), 2.77-2.67 (m, 1H), 2.46 (d, J = 1.8 Hz, 1H), 1.13-1.02 (m, 6H) ppm.

[0142] Acetyl protection, synthesis of compound (24)

[0143]

[0144] To a solution of compound 23 (19.0 g, 29.0 mmol) in pyridine (200 mL) was added acetic anhydride (27.4 mL, 290 mmol). The mixture was stirred at room temperature for 15 hours. The reaction was monitored by TLC, and after completion of the reaction, it was quenched with water. Pyridine was removed from the reaction mixture and diluted with water. The mixture was extracted with EtOAc and dried over Na2SO4. The solvent was removed on a rotary evaporator, and the residue was purified by silica gel column chromatography using 5% MeOH (1% TEA) in DCM to give compound 24 as a light brown solid (23.1 g, quantitative). 1H-NMR (400 MHz, CDCl3) δ 7.77 (d, J = 5.6 Hz, 1H), 7.54-7.47 (m, 2H), 7.43-7.29 (m, 4H), 7.25-7.15 (m, 3H), 6.83-6.70 (m, 4H), 6.61-6.51 (m, 1H), 5.97-5.82 (m, 2H), 5.64 (s, 1H), 5.29 (s, 1H), 4.28-4.20 (m, 1H), 3.80-3.69 (m, 6H), 3.63 (q, J = 7.3 Hz, 1H), 3.15-3.07 (m, 1H), 2.14-2.02 (m, 6H), 1.50-1.43 (m, 1H), 0.93-0.82 (m, 3H), 0.64 (td, J = 14.9, 7.0 Hz, 3H) ppm.

[0145] Detritylation, synthesis of compound (25)

[0146]

[0147] A solution of trichloroacetic acid (25.5 g, 156 mmol) in dichloromethane (78.0 mL) was poured into a solution of compound 24 (23.1 g, 31.2 mmol) in dichloromethane (100 mL) at 0 °C. After the addition, the reaction mixture was warmed to room temperature and stirred overnight. After the reaction was completed, methanol (15.0 mL) was added to the reaction mixture to quench the reaction. The mixture was then washed with saturated aqueous NaHCO (3) and extracted with dichloromethane. The combined organic layers were concentrated and purified by silica gel column chromatography using 0-4% MeOH / DCM to give compound 25 as a white solid (3.90 g, 70.0% yield). 1H-NMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 7.23-7.11 (m, 2H), 6.19 (d, J = 6.1 Hz, 1H), 5.82 (t, J = 5.8 Hz, 1H), 5.59-5.50 (m, 1H), 4.29 (q, J = 3.1 Hz, 1H), 3.84 (ddd, J = 26.0, 12.3, 3.0 Hz, 2H), 2.77-2.70 (m, 1H), 2.15 (d, J = 8.3 Hz, 3H), 2.02 (d, J = 5.8 Hz, 3H), 1.26-1.22 (m, 6H) ppm.

[0148] Synthesis of ジヌクレオチドbody G(2'OMe)pG(2'OMe) (28)

[0149]

[0150] Compound 25 (1.00 g, 2.29 mmol), compound 26 (2.58 g, 2.97 mmol), and molecular sieves 3A (2.60 g) were dissolved in dry acetonitrile (23.0 mL). After stirring for 20 min, 1H-tetrazole (722 mg, 10.3 mmol) was added and stirred for 4 h. After complete conversion of compound 25, 5-6 M TBHP (600 μL, 2.97 mmol) in decane was added to the reaction mixture and stirred for 1 h. The reaction mixture was then diluted with dichloromethane and washed with saturated aqueous NaHCO3 and brine. The organic layer was dried over Na2SO4, concentrated, and used in the next step without purification. 27 (3.48 g, white solid) was obtained. The crude product 27 (3.48 g, 2.85 mmol) was dissolved in dichloromethane (32.0 mL) and cooled to 0 °C. A solution of trichloroacetic acid in dichloromethane (0.2 M, 71.2 mL, 14.2 mmol) was added to the mixture and stirred at room temperature for 3 hours. The reaction was monitored by TLC, and after complete conversion, the reaction mixture was quenched with saturated aqueous NaHCO3 solution, washed twice with saturated aqueous NaHCO3 solution, and extracted with dichloromethane. The solvent was removed by rotary evaporation, and the residue was purified by silica gel column chromatography using 0-5% MeOH / dichloromethane to give compound 28 as a white solid (1.10 g, 53.0% yield over two steps). 1H-NMR (400 MHz, DMSO-d6) δ 12.08-12.05 (m, 2H), 11.62 (s, 1H), 11.54 (d, J = 3.1 Hz, 1H), 8.30-8.26 (m, 1H), 8.24 (s, 1H), 6.09 (d, J = 6.7 Hz, 1H), 5.88-5.84 (m, 1H), 5.77 (t, J = 6.3 Hz, 1H), 5.47 (t, J = 2.9 Hz, 1H), 5.29 (q, J = 5.1 Hz, 1H), 5.09-5.03 (m, 1H), 4.56 (dd, J = 12.6, 7.9 Hz, 1H), 4.43-4.33 (m, 3H), 4.23-4.17 (m, 3H), 3.59-3.49 (m, 2H), 3.28 (dd, J = 10.7, 10.0 Hz, 5H), 2.91 (dt, J = 16.2, 5.4 Hz, 2H), 2.76-2.68 (m, 2H), 2.09 (d, J = 4.9 Hz, 3H), 1.98-1.95 (m, 3H), 1.09-1.06 (m, 12H) ppm.

[0151] Synthesis of trinucleotide A(2'OMe)pG(2'OMe) (31)

[0152]

[0153] Compound 28 (100 mg, 0.108 mmol), compound 29 (126 mg, 0.140 mmol), and molecular sieves 3A (125 mg) were suspended in dry acetonitrile (1.00 mL). After stirring at room temperature for 20 min, 1H-tetrazole (34.0 mg, 0.486 mmol) was added and the mixture was stirred for 4 h. After complete conversion of compound 28, 5-6 M TBHP (28.0 μL, 0.140 mmol) was added to the mixture and the mixture was stirred for 1 h.

[0154] The reaction mixture was then diluted with dichloromethane and washed with saturated aqueous NaHCO3 and brine. The organic layer was dried over Na2SO4, concentrated, and used in the next step without purification. The crude product 30 (276 mg, 0.160 mmol) was dissolved in dichloromethane (3.20 mL) and cooled to 0 °C. A solution of trichloroacetic acid in dichloromethane (0.2 M, 4.00 mL, 0.800 mmol) was added to the solution and stirred at room temperature for 3 h. The reaction was monitored by TLC. After complete conversion, the reaction mixture was quenched with saturated aqueous NaHCO3 solution, washed twice with saturated aqueous NaHCO3 solution, and extracted with dichloromethane. The solvent was removed by rotary evaporation and purified by silica gel column chromatography using 0–5% MeOH in dichloromethane to give compound 31 as a white solid (125 mg, 81% yield over two steps). 1 H-NMR (400 MHz, DMSO-d6) δ 12.09-12.06 (m, 2H), 11.55 (d, J = 6.1 Hz, 2H), 11.22 (d, J = 6.1 Hz, 1H), 8.71 (dt, J = 11.4, 2.6 Hz, 2H), 8.26-8.23 (m, 2H), 8.01 (d, J = 8.3 Hz, 2H), 7.64-7.50 (m, 3H), 6.17-6.07 (m, 2H), 5.94-5.90 (m, 1H), 5.79-5.74 (m, 1H), 5.47 (dt, J = 9.1, 3.4 Hz, 1H), 5.36 (t, J = 5.4 Hz, 1H), 5.16 (t, J = 11.6 Hz, 2H), 4.82-4.75 (m, 1H), 4.65-4.59 (m, 1H), 4.49-4.18 (m, 11H), 3.60 (t, J = 4.6 Hz, 2H), 3.35-3.26 (m, 28H), 2.95-2.88 (m, 4H), 2.74-2.63 (m, 2H), 2.28 (d, J = 2.0 Hz, 0H), 2.08 (dd, J = 6.1, 1.6 Hz, 3H), 1.98 (s, 3H), 1.09-1.07 (m, 12H) ppm.

[0155] Synthesis of the trinucleotide pA(2'OMe)pG(2'OMe) (33)

[0156]

[0157] Compound A (30 mg, 0.02 mmol, 1 eq), compound B (22.85 mg, 0.084 mmol, 4 eq), and mole sieve 3A (30 mg) were dissolved in dry ACN (1 mL). After stirring for 20 min, 1H-tetrazole 0.4 M (0.2 mL, 0.086 mmol, 4.1 eq) was added at rt and stirred for 2 h. After complete conversion of SM A, TBHP 5 M (6 μL, 0.03 mmol, 1.5 eq) was added and stirred for an additional 1 h. The reaction was then diluted with DCM and washed with NaHCO and brine. The organic portion was dried over NaSO, removed in vacuo, and used in the next step. Compound A (44 mg, 0.04 mmol, 1 eq) was dissolved in MeOH (0.5 mL), and aq. NHOH (1 mL) was added at rt and stirred for 20 h. After complete conversion of SM, the reaction mixture was diluted with MeOH and evaporated three times on a rotary evaporator. The crude material was then dissolved in MQ and purified on a DEAE column with a linear gradient of 270 min. After evaporation of the solvent, 5 mg of pure white solid compound was obtained. 1 H-NMR (392 MHz, DMSO-D6) δ 8.45 (s, 1H), 8.10-7.90 (m, 3H), 7.25 (s, 2H), 6.72 (d, J = 13.7 Hz, 4H), 5.99 (d, J = 6.7 Hz, 1H), 5.80 (d, J = 7.9 Hz, 1H), 5.64 (d, J = 6.3 Hz, 1H), 4.82-4.74 (m, 3H), 4.54-4.42 (m, 5H), 4.30-4.20 (m, 3H), 3.98-3.79 (m, 8H), 3.26-3.17 (m, 6H), 2.81 (d, J = 33.2 Hz, 3H). 31P-NMR (159 MHz, DMSO-D6) δ -0.6 (s, 1P), -1.4 (s, 2P) ppm.

[0158] Synthesis scheme of the trinucleotide pUGG (38)

[0159] Synthesis of the trinucleotide UGG (36)

[0160]

[0161] Compound 18-4 (343.3 mg, 0.37 mmol, 1 eq.) was coevaporated with toluene to remove water, and then 2'-OMe-U-CE phosphoramidite (368.96 mg, 0.48 mmol, 1.3 eq.) was added to the flask. 8 mL of acetonitrile was added to dissolve the compound, followed by the addition of 180 mg of 3Å molecular sieves. After stirring for 5 min, 1H-tetrazole (119 mg, 1.7 mmol, 4.5 eq.) was added to the mixture. The mixture was then stirred under an argon atmosphere for 4 h, after which tert-butyl hydroperoxide solution (5-6 M in decane, 0.12 mL, 0.6 mmol, 1.6 eq.) was added to oxidize the resulting compound. The mixture was then stirred for an additional 60 min, and 5 mL of dichloromethane was added to dilute the reaction mixture. The MS 3Å was filtered through filter paper and washed with 15 mL of dichloromethane, and then the mixture was washed with 40 mL of water. The organic layer was collected and trichloroacetic acid (372 mg, 3.2 mmol, 8.8 eq.) was added under ice bath. The mixture was then stirred for 30 min. After complete conversion, dichloroethane was added to the mixture and washed with NaHCO3. The organic layer was dried (Na2SO4) and concentrated. Purification was performed by column chromatography (silica gel, dichloromethane:methanol = 19:1 to 9:1). 280 mg of a white solid was obtained (yield: 58%). 1H-NMR (600 MHz, DMSO-D6) δ 12.11-12.09 (m, 2H), 11.58 (s, 2H), 11.42 (d, J = 6.0 Hz, 1H), 8.30-8.24 (m, 2H), 7.89-7.85 (m, 1H), 6.12 (t, J = 6.6 Hz, 1H), 5.95-5.91 (m, 2H), 5.80 (q, J = 6.6 Hz, 1H), 5.71 (dd, J=11.4, 8.4, 1H), 5.52-5.50 (m, 1H), 5.35 (s, 1H), 5.21-5.19 (m, 1H), 4.99-4.96 (m, 1H), 4.67-4.61 (m, 1H), 4.50-4.19 (m, 10H), 4.18-4.16 (m, 1H), 4.09 (td, J = 16.82, 5.4 Hz, 1H), 3.57 (s, 2H), 3.37-3.30 (m, 6-H), 2.97-2.91 (m,4H), 2.79-2.72 (m, 2H), 2.13 (d, J = 10.8 Hz, 3H), 2.02 (d, J = 1.2 Hz, 3H), 1.13-1.11 (m, 12H) ppm. 13 C-NMR (100MHz, ACETONITRILE-D3) δ 179.1, 168.9, 168.8, 168.5, 163.1, 154.7, 149.6, 147.6, 147.3, 139.5, 139.2, 137.8, 137.4, 122.9, 119.5, 119.3, 116.1, 116.1, 116.0, 100.6, 100.5, 85.1, 84.9, 80.4, 74.2, 71.5, 71.3, 69.2, 65.5, 62.1, 62.0, 58.8, 56.8, 56.7, 56.5, 52.1, 47.2, 47.0, 46.7, 46.5, 46.3, 46.1, 45.9, 45.7, 34.3, 17.9, 17.6, 16.8, 16.7, 16.6 ppm. 31 P-NMR (240 MHz, DMSO-D6) δ -1.6, -1.9 ppm. HRMS (ESI) Calcd. For C 49H 62 N 14 NaO 24 P + ([M+Na] + ): 1315.3429. Obsd.1315.3428.

[0162] Synthesis of the trinucleotide pUGG (38)

[0163]

[0164] Compound 18-6-1 (170 mg, 0.13 mmol, 1 eq.) was coevaporated once with toluene to remove water, and then bis(2-cyanoethyl)diisopropylphosphoramidite (144 mg, 0.52 mmol, 4 eq.), 135 mg of 3Å molecular sieves, and 5 mL of acetonitrile were added. After stirring for 9 minutes under an argon atmosphere, 1H-tetrazole (45 mg, 0.64 mmol, 5 eq.) was added. The mixture was stirred at ambient temperature for 4 hours, and TBHP (5-6 M in decane, 52 μL, 0.26 mmol, 2 eq.) was added dropwise. After 1 hour, the reaction mixture was diluted with dichloromethane and washed with water. The organic layer was dried (Na2SO4) and concentrated. The crude reaction mixture was used directly for deprotection. The resulting solid was dissolved in 1.8 mL of methanol, to which 1.7 mL of NH4OH solution (28%) was added. After stirring for 3 days, the reaction mixture was transferred to 55 °C to achieve complete conversion of the starting compound. The solvent was then removed by rotary evaporation. The resulting solid was dissolved in 5 mL of water and applied to a DEAE-Sephadex™ A-25 column (size: 70 cm). 3 The resulting mixture was loaded onto a column of 100 ml of TEAB buffer containing 10% CH3CN and purified by ion exchange chromatography (0-1.5 M TEAB buffer containing 10% CH3CN, linear gradient 15-270 min, flow rate 6 mL / min). The fraction with a retention time of 110-150 min was collected, and the solvent was removed. 50 mg of a white solid was obtained (yield: 25%, over two steps). 1H-NMR (600 MHz, D2O) δ 7.99 (d, J = 3.0 Hz, 2H), 7.95 (d, J = 8.4 Hz, 1H), 6.06 (d, J = 7.2 Hz, 1H), 5.91-5.87 (m, 3H), 4.98-4.94 (m, 1H), 4.70-4.67 (m, 1H), 4.81-4.73 (m, 1H), 4.48-4.44 (m, 2H), 4.33-4.31 (m, 2H), 4.16-4.13 (m, 4H), 4.08 (td, J =11.4, 3.6 Hz, 1H), 3.87 (dd, J = 5.4, 3.0 Hz, 1H), 3.39 (s, 3H), 3.32 (s, 3H), 3.21 (q, J = 7.2 Hz, 2H), 3.07 (q, J = 7.2 Hz, 39H, TEA), 1.21-1.18 (t, J=7.8Hz, 62H, TEA) ppm. 13 C-NMR (150 MHz, D2O) δ 165.9, 158.7, 153.8, 153.6, 151.7, 151.5, 151.4, 141.1, 138.1, 137.7, 116.7, 116.4, 102.6, 87.3, 86.3, 83.7, 83.6, 82.5, 81.5, 80.5, 73.2, 72.7, 72.1, 70.3, 65.2, 63.6, 58.9, 57.9, 57.6, 46.7, 42.3, 10.6, 8.3, 7.4 ppm. 31 P-NMR (240 MHz, D2O) δ 4.2, 0.0, -0.2 ppm. HRMS (ESI) Calcd. For C 31 H 40 N 12 O 23 P3 - ([M-H] - ): 1041.1548. Obsd.1041.1543.。

[0165] Synthetic scheme of trinucleotide pCGG(X)

[0166] Synthesis of trinucleotide CGG(41)

[0167]

[0168] Compound 18-4 (95 mg, 0.1 mmol, 1 eq.) was coevaporated with toluene to remove water, and then 2'-OMe-CE-phosphoramidite (101 mg, 0.13 mmol, 1.3 eq.) was added to the flask. 2 mL of acetonitrile was added to dissolve the compound, followed by the addition of 126 mg of 3Å molecular sieves. After stirring for 15 min, 1H-tetrazole (32 mg, 0.45 mmol, 4.5 eq.) was added to the mixture. The mixture was then stirred under an argon atmosphere for 3 h, and tert-butyl hydroperoxide solution (5-6 M in decane, 0.02 mL, 0.12 mmol, 1.3 eq.) was added to oxidize the resulting compound. The mixture was then stirred for an additional 45 min, and approximately 5 mL of dichloromethane was added to dilute the reaction mixture. The MS 3Å was filtered through filter paper and washed with 5 mL of dichloromethane, followed by rinsing the mixture with 40 mL of water. The organic layer was collected and trichloroacetic acid (220 mg, 1.3 mmol, 13 eq.) was added under ice bath. The mixture was then stirred for 30 min. After complete conversion, dichloroethane was added to the mixture and washed with NaHCO3. The organic layer was dried (Na2SO4) and concentrated. Purification was performed by column chromatography (silica gel, dichloromethane:methanol = 9:1, 1% TEA). 40 mg of a white solid was obtained (yield: 30%). 1H-NMR (400 MHz, METHANOL-D4) δ 8.47-8.39 (m, 1H), 8.14-8.12 (m, 1H), 8.08-8.03 (m, 1H), 6.20-6.16 (m, 1H), 6.03-5.87 (m, 2H), 5.67-5.62 (m, 1H), 5.28-5.11 (m, 1H), 4.98 (dt, J = 17.5, 6.1 Hz, 1H), 4.75-4.65 (m, 1H), 4.56-4.41 (m, 5H), 4.38-4.23 (m, 6H), 4.16 (t, J = 4.9 Hz, 1H), 4.06-4.02 (m, 0H), 3.74-3.70 (m, 1H), 3.55 (d, J = 3.3 Hz, 1H), 3.50-3.47 (m, 1H), 3.41 (d, J = 3.8 Hz, 1H), 3.35 (q, J = 1.5 Hz, 2H), 3.29-3.28 (m, 2H), 2.95-2.85 (m, 4H), 2.80-2.60 (m, 2H), 2.13-2.10 (m, 3H), 2.05-2.01 (m, 3H), 1.21-1.15 (m, 12H) ppm. 13C-NMR (101 MHz, METHANOL-D4) δ 180.4; 156.0, 149.1, 149.0, 148.6, 148.5, 148.4, 145.0, 144.4, 139.6, 139.1, 138.8, 138.2, 121.2, 120.9, 120.6, 117.5. 117.3, 96.9, 96.5, 88.6, 88.5, 88.4, 86.7, 86.5, 86.3, 86.1, 85.5, 82.6, 82.1, 81.2, 81.1, 73.0, 72.9, 72.7, 72.6. 72.5, 70.6, 70.3, 67.5, 67.3, 63.5, 63.4, 63.4, 60.6, 59.4, 58.2, 58.1, 57.9, 57.5, 48.5, 48.3, 48.1, 48.0, 47.9. 47.7, 47.5, 47.3, 47.0, 35.7, 23.4, 23.3, 19.2, 19.2, 19.0, 19.0, 18.9, 18.8, 18.7, 18.4, 18.1, 18.0, and 17.8 ppm. 31 P-NMR (162 MHz, METHANOL-D4) δ-2.2, -2.3, -2.5 ppm. HRMS (ESI) Calcd. For C 51 H 66 N 15 O 24 P2 + ([M+H] + ) : 1334.3875. The obsd. 1334.3874.

[0169] The structure of pCGG(43).

[0170] Compound 18-6-2 (110 mg, 0.13 mmol, 1 eq.) was coevaporated with toluene once to remove water, and then bis(2-cyanoethyl)diisopropylphosphoramidite (90 mg, 0.52 mmol, 4 eq.), 80 mg of 3Å molecular sieves, and 5 mL of acetonitrile were added. After stirring for 20 min under an argon atmosphere, 1H-tetrazole (45 mg, 0.64 mmol, 5 eq.) was added. The mixture was stirred at ambient temperature for 2 h 15 min, and TBHP (5-6 M in decane, 0.12 mL, 0.26 mmol, 2 eq.) was added dropwise. After 2 h, the reaction mixture was diluted with dichloromethane and washed with water. The organic layer was dried (Na2SO4) and concentrated. The crude reaction mixture was used directly for deprotection. The resulting solid was dissolved in 1.8 mL of methanol, to which 1.7 mL of NH4OH solution (28%) was added. After stirring for 3 days, the reaction mixture was transferred to 55 °C to achieve complete conversion of the starting compound. The solvent was then removed by rotary evaporation. The resulting solid was dissolved in 5 mL of water and applied to a DEAE-Sephadex™ A-25 column (size: 70 cm). 3 The resulting mixture was loaded onto a column of 100 ml of TEAB buffer containing 10% CH3CN and purified by ion exchange chromatography (0-1.5 M TEAB buffer containing 10% CH3CN, linear gradient 15-270 min, flow rate 6 mL / min). The fraction with a retention time of 110-150 min was collected, and the solvent was removed. 35 mg of a white solid was obtained (yield: 25%, over two steps). 1H-NMR (400 MHz, D2O) δ 7.90 (d, J = 7.6 Hz, 1H), 7.85 (s, 1H), 7.79 (s, 1H), 5.90 (d, J = 8.0 Hz, 1H), 5.85 (d, J = 4.8 Hz, 1H), 5.74 (d, J = 6.4 Hz, 2H), 4.82-4.77 (m, 1H), 4.58-4.49 (m, 2H), 4.34-4.32 (m, 1H), 4.29 (s, 1H), 4.18-4.16 (m, 2H), 4.00-4.05 (m, 3H), 3.92-3.91 (m, 2H), 3.82-3.73 (m, 2H), 3.25 (d, J = 6.8 Hz, 6H) ppm. 13 C-NMR (100 MHz, D2O) δ 166.0, 160.1, 159.6, 157.3, 154.9, 154.2, 151.5, 151.5, 141.4, 137.6, 137.5, 137.1, 116.6, 116.5, 96.7, 87.3, 87.0, 86.1, 83.5, 83.4, 82.8, 81.8, 80.5, 73.3, 72.6, 72.2, 70.2, 65.0, 62.8, 59.0, 57.9, 57.5, 46.6, 42.2, 25.6, 23.3, 18.2, 14.8, 10.5, 8.7, 8.2, 7.5 ppm. 31 P-NMR (160 MHz, D2O) δ 4.1, -0.2, -0.3 ppm. HRMS (ESI) Calcd. For C 31 H 41 N 13 O 22 P3 - ([MH] - ): 1040.1707. Obsd.1040.1710.

[0171] The automatic nucleic acid synthesizer is used for the solid-phase synthesis of いたトリヌクレオチドの

[0172]

[0173] Reaction conditions and experimental procedures Dinucleotides and trinucleotides were synthesized using a DNA / RNA synthesizer NR-2A_7MX (Nihon Techno Service, Ushiku, Ibaraki Prefecture) with primer support 5G riboG 300 (Cytiva, 15.0 μmol scale synthesis). For the coupling step, the column was circulated with 100 mM 2'-O-methyl-adenosine(n-benzoyl)-CE-phosphoramidite (ChemGene) in CH3CN, 100 mM 2'-O-methyl-adenosine(n-benzoyl)-CE-phosphoramidite (synthesized according to literature) in CH3CN, 100 mM 2'-O-methyl-guanosine(ni-Bu)-CE-phosphoramidite (ChemGenes) in CH3CN, 50–70 mM 5'-DMTr-2'-TOM-riboadenosine(n-acetyl)OP (ChemGenes) in CH3CN, 150 mM bis(2-cyanoethyl)-N,N-diisopropylphosphoramidite in CH3CN, and 0.3 M 5-(benzylthio)-1H-tetrazole in CH3CN (activator). A solution of 184 mM trichloroacetic acid in dichloromethane was used as the detritylation reagent, and 0.05 M I2 in pyridine / HO (9:1, v / v) was used for oxidation. CapA was prepared using 10% Ac2O in THF / pyridine (8:1, v / v), and CapB was prepared using 10% 1-methylimidazole in THF. Synthesis was performed using a standard RNA synthesis protocol on a 10 μmol scale. After the final cycle of synthesis, the solid support was treated with a 1:1 mixture of 28% ammonium hydroxide and 40% methylamine (1.00 mL / 15.0 μmol of solid support) at 65°C for 1–2 hours to cleave the nucleotide from the solid support and remove the protecting groups. The resulting nucleotide was concentrated to dryness and redissolved in DMSO (1.00 mL / 60.0 μmol). To this solution was added TEA-3HF (1.00 mL / 60.0 μmol), and the mixture was incubated at 65° C. for 3 to 5 hours to remove the 2′-O-silyl protecting group.The reaction mixture was neutralized with 250 mM Na2CO3 aq. (10.0 mL / 60.0 μmol). The crude product was purified by DEAE-Sephadex (φ = 4.5 cm, h = 8.4 cm, 140 cm). 3 The dinucleotides were purified by ion exchange chromatography on a 12.0 mL / min column using a linear 0-1.2 M gradient (270 min) of TEAB buffer (pH 7.9) containing 0-8% CH3CN. The product-containing fractions were pooled and concentrated to give the di- / trinucleotides. The yield was calculated using the absorbance of the product at 260 nm measured on a NanoDrop. The extinction coefficient (ε) for the dinucleotides was 260 ) = 25,000 M -1 -cm -1 and 35,100 M for trinucleotides. -1 -cm -1 was used in the calculation.

[0174] pUUG (triethylammonium salt, ε 260 = 29,700 M -1 ·cm -1 , 37.7% yield) (44): 1H NMR (600 MHz, D2O) δ 7.92 - 7.83 (m, 2H), 7.70 (dd, J= 8.2, 3.2 Hz, 1H), 5.84 - 5.73 (m, 3H), 5.70 (td, J = 6.9, 3.1 Hz, 2H), 4.62 (dt, J = 5.5, 2.7 Hz, 2H), 4.56 (dq, J = 5.5, 2.8 Hz, 1H), 4.34 - 4.29 (m, 1H), 4.26 (p, J = 2.8 Hz, 1H), 4.20 - 4.14 (m, 2H), 4.01 (dq, J = 11.4, 4.5 Hz, 3H), 3.98 - 3.94 (m, 1H), 3.92 (dq, J= 7.7, 4.6 Hz, 3H), 3.89 - 3.85 (m, 1H), 3.32 (dd, J = 6.2, 3.3 Hz, 6H), 3.14 - 2.78 (m, 28H), 1.10 (td, J = 7.1, 3.2 Hz, 36H) ppm. 13C NMR (151 MHz, D2O) δ 181.28, 165.96, 165.60, 158.69, 153.80, 151.66, 151.47, 151.30, 141.57, 140.76, 137.73, 116.43, 102.61, 102.55, 87.43, 86.66, 86.35, 83.74, 83.69, 83.07, 81.97, 81.48, 81.38, 73.40, 72.29, 71.77, 70.45, 65.31, 64.27, 63.39, 58.94, 57.93, 57.73, 55.08, 50.02, 48.63, 46.64, 42.70, 42.23, 39.88, 39.23, 34.92, 34.50, 28.78, 25.62, 23.29, 21.87, 19.20, 19.01, 18.27, 14.84, 13.42, 12.76, 12.48, 12.37, 11.88, 10.75, 10.73, 10.58, 10.54, 10.22, 8.72, 8.25, 7.48 ppm. 31P NMR (243 MHz, D2O) δ 1.90 (1P), -0.46(1P), -0.67 (1P) ppm. HR-ESI-MS calcd. for C 30 H 39 N9O 24 P3, 1002.13263 [M-H] - ; found 1002.13203.

[0175] pUCG (triethylammonium salt, ε 260 = 27,800 M -1 ·cm -1 , 20.7% yield) (45): 1H NMR (600 MHz, D2O) δ 8.04 - 7.96 (m, 3H), 6.04 (d, J = 7.7 Hz, 1H), 5.96 (d, J = 2.5 Hz, 1H), 5.90 (d, J = 3.4 Hz, 1H), 5.87 - 5.83 (m, 2H), 4.73 (t, J = 5.6 Hz, 1H), 4.70 - 4.63 (m, 2H), 4.44 (dd, J = 5.2, 3.4 Hz, 1H), 4.39 (dq, J = 4.8, 2.3 Hz, 1H), 4.33 (ddt, J = 8.5, 5.9, 2.8). Hz, 2H), 4.25 - 4.10 (m, 7H), 4.02 (dd, J = 4.9, 2.5 Hz, 1H), 3.53 (dd, J = 6.6, 4.4 Hz, 6H), 3.18 (q, J = 7.3 Hz, 13H), 1.34 - 1.23 (m, 27H); ppm. 13 C NMR (151 MHz, D2O) δ 165.93, 163.21, 158.73, 153.75, 151.68; 87.63, 86.89, 83.77, 83.71, 82.12, 81.97, 81.26, 80.93, 73.37, 71.02, 70.65, 70.33, 64.97, 63.14, 62.97, 58.62. 57.89, 57.63, 46.70, 8.27 ppm. 31 P NMR (243 MHz, D2O) δ 0.49(1P), -0.45(1P), -0.85(1P) ppm. HR-ESI-MS calcd. for C 30 H 40 N 10 O 23 P3, 1001.14861[M-H]-; found 1001.14034.

[0176] pUAG(Universal Agricultural Agency、ε 260 = 34,200 M -1 ・cm -1、28.0% yield)(46): 1 H NMR (600 MHz, D2O) δ 8.28 (s, 1H), 8.04 (s, 1H), 7.82 (s, 1H), 7.73 (d, J = 8.2 Hz, 1H), 7.24 (s, 4H), 5.98 (d, J = 4.8 Hz, 1H), 5.73 - 5.66 (m, 3H), 4.82 (dt, J = 8.0, 4.8 Hz, 1H), 4.61 - 4.55 (m, 2H), 4.36 - 4.31 (m, 3H), 4.19 (ddp, J = 6.9, 4.8, 2.3 Hz, 2H), 4.07 (q, J = 4.3 Hz, 2H), 3.98 (q, J = 2.9 Hz, 2H), 3.91 (dd, J = 4.4, 2.4 Hz, 2H), 3.86 (t, J = 5.1 Hz, 1H), 3.38 (s, 3H), 3.23 (s, 3H), 3.04 (q, J = 7.3 Hz, 18H), 1.12 (t, J = 7.4 Hz, 27H) ppm. 13 C NMR (151 MHz, D2O) δ 165.86, 158.44, 154.45, 153.64, 151.58, 151.42, 151.25, 148.56, 141.11, 139.61, 137.59, 128.47, 118.50, 116.20, 102.46, 87.54, 86.31, 85.62, 83.49, 82.49, 81.80, 81.40, 73.31, 72.30, 72.03, 70.17, 65.12, 64.62, 63.66, 58.60, 58.03, 57.58, 46.67, 8.24 ppm. 31 P NMR (243 MHz, D2O) δ 0.62 (s, 1P), -0.30 (s, 1P), -0.63 (s, 1P) ppm. ESI-TOF-MS calcd. for C 31 H 39 N 12 O 22 P3, 512.0763 [M - 2H] 2- ; found 512.0774.。

[0177] pCUG (triethylammonium salt, ε 260 = 26,300 M -1 ·cm -1 , 32.4% yield) (47): 1 H NMR (600 MHz, D2O) δ 8.06 - 8.00 (m, 2H), 7.86 (d, J = 8.2 Hz, 1H), 5.99 (d, J = 7.6 Hz, 1H), 5.94 (d, J = 2.9 Hz, 1H), 5.91 (d, J = 2.5 Hz, 1H), 5.85 (d, J = 6.1 Hz, 1H), 5.73 (d, J = 8.1 Hz, 1H), 4.72 (t, J = 5.6 Hz, 1H), 4.67 - 4.56 (m, 2H), 4.45 (dd, J = 5.3, 3.3 Hz, 1H), 4.38 - 4.30 (m, 3H), 4.24 (dt, J= 12.2, 3.3 Hz, 1H), 4.18 - 4.06 (m, 6H), 3.97 (dd, J = 4.9, 2.9 Hz, 1H), 3.58 (s, 3H), 3.50 (s, 3H), 3.17 (q, J = 7.4 Hz, 18H), 1.25 (t, J = 7.4 Hz, 27H) ppm. 13 C NMR (151 MHz, D2O) δ 165.90, 165.34, 158.87, 156.85, 153.88, 151.75, 150.90, 141.18, 139.80, 137.73, 116.44, 102.23, 96.01, 87.55, 87.37, 86.97, 83.87, 83.81, 81.76-81.03, 73.56, 70.80, 70.48, 65.09, 63.19, 62.65, 58.95, 57.85, 57.60, 46.69, 8.27 ppm. 31 P NMR (243 MHz, D2O) δ 1.12 (1P), -0.52 (1P), -0.92 (1P) ppm. HR-ESI-MS calcd. for C 30 H 40 N10 O 23 P3, 1001.14861 [M - H]-; found 1001.14034..

[0178] pCCG (Torotron, epsilon 260 = 24,800 M -1 ・cm -1 , 37.7% yield)(48): 1 H NMR (600 MHz, D2O) δ 7.90 (d, J = 7.6 Hz, 1H), 7.81 - 7.73 (m, 2H), 5.83 (d, J = 7.6 Hz, 1H), 5.81 - 5.75 (m, 2H), 5.70 (dd, J= 9.9, 3.5 Hz, 2H), 4.45 (qd, J= 11.5, 6.8 Hz, 3H), 4.27 (t, J= 4.5 Hz, 1H), 4.21 - 4.13 (m, 4H), 4.10 - 4.01 (m, 3H), 3.99 - 3.93 (m, 3H), 3.81 (d, J = 5.9 Hz, 1H), 3.49 - 3.42 (m, 6H), 3.20 - 2.84 (m, 24H), 1.10 (td, J = 7.3, 3.8 Hz, 36H) ppm. 13C NMR (151 MHz, D2O) δ 181.18, 165.59, 165.11, 158.89, 156.42, 155.99, 153.78, 151.54, 141.07, 139.28, 137.28, 116.27, 96.04, 95.72, 87.72, 87.67, 83.35, 81.96, 81.57, 81.20, 80.25, 73.72, 70.21, 70.04, 64.62, 62.58, 62.20, 58.90, 57.68, 57.52, 48.64, 46.65, 42.71, 42.24, 39.88, 34.93, 34.51, 28.78, 25.62, 24.81, 23.22, 21.87, 19.21, 19.01, 18.26, 14.84, 13.42, 12.76, 12.37, 11.88, 10.73, 10.58, 10.22, 8.71, 8.26, 7.44 ppm. 31P NMR (243 MHz, D2O) δ 1.08 (1P), -0.55 (1P), -1.05 (1P) ppm. HR-ESI-MS calcd. for C 30 H 41 N 11 O 22 P3, 1000.16460 [M - H]-; found 1000.11641 pCAG (triethylammonium salt, ε 260 = 30,600 M -1 ·cm -1 , 33.7% yield) (49): 1H NMR (600 MHz, D2O) δ 8.32 (s, 1H), 8.04 (d, J = 1.2 Hz, 1H), 7.87 (d, J = 7.6 Hz, 2H), 6.12 (d, J = 3.7 Hz, 1H), 5.89 (d, J = 7.6 Hz, 1H), 5.82 (d, J = 3.1 Hz, 1H), 5.76 (d, J = 5.4 Hz, 1H), 4.87 (dt, J = 7.9, 5.2 Hz, 1H), 4.67 (t, J = 5.4 Hz, 1H), 4.60 (q, J = 7.6 Hz, 1H), 4.42 (dt, J = 6.0, 3.7 Hz, 2H), 4.30 (dt, J = 5.9, 3.4 Hz, 3H), 4.21 - 4.10 (m, 5H), 4.09 - 4.02 (m, 2H), 3.53 (d, J= 0.8 Hz, 3H), 3.45 (s, 3H), 3.31 (q, J= 7.2 Hz, 2.5H), 3.15 (qd, J = 7.3, 0.8 Hz, 21.5H), 1.23 (td, J= 7.3, 0.9 Hz, 36H) ppm. 13 C NMR (151 MHz, D2O) δ 165.36, 158.50, 156.41, 155.14, 153.60, 152.67, 151.33, 148.30, 140.90, 138.60, 137.62, 118.45, 116.29, 95.96, 87.81, 87.40, 85.69, 83.50, 83.44, 82.15, 81.62, 73.29, 71.78, 71.22, 70.06, 64.87, 63.94, 63.02, 58.89, 58.00, 57.58, 46.68, 8.26 ppm. 31 P NMR (243 MHz, D2O) δ 0.94 (1P), -0.44 (1P), -0.71 (1P) ppm. HR-ESI-MS calcd. for C 31 H 41 N 13 O 21 P3, 1024.16583 [M - H] -; found 1024.17943..

[0179] pAUG 260 = 34,100 M -1 ・cm -1 , 35.0% yield)(50): 1 H NMR (600 MHz, D2O) δ 8.48 (s, 1H), 8.15 (s, 1H), 7.98 (s, 1H), 7.74 (d, J= 8.2 Hz, 1H), 7.37 (s, 3H), 6.15 (d, J= 4.9 Hz, 1H), 5.82 (dd, J = 9.9, 4.9 Hz, 2H), 5.66 (d, J = 8.2 Hz, 1H), 4.92 - 4.87 (m, 1H), 4.72 - 4.63 (m, 2H), 4.54 (dq, J = 5.2, 2.6 Hz, 1H), 4.49 (t, J = 4.8 Hz, 1H), 4.44 (dd, J = 5.2, 3.6 Hz, 1H), 4.35 (dq, J = 5.4, 2.6 Hz, 1H), 4.33 - 4.29 (m, 1H), 4.22 (ddd, J = 11.6, 4.6, 2.5 Hz, 1H), 4.17 - 4.09 (m, 5H), 4.02 (t, J = 4.5 Hz, 1H), 3.52 (s, 3H), 3.48 (s, 3H), 3.17 (q, J = 7.3 Hz, 17H), 1.33 - 1.23 (m, 27H) ppm. 13 C NMR (151 MHz, D2O) δ 165.42, 158.56, 154.03, 153.68, 151.49, 151.01, 150.79, 148.50, 140.27, 140.19, 137.66, 128.49, 118.51, 116.20, 102.16, 87.40, 86.91, 85.61, 83.66, 83.08, 82.15, 81.64, 73.52, 72.35, 71.39, 70.41, 65.18, 63.85, 58.58, 58.03, 57.93, 46.69, 8.26 ppm. 31P NMR (243 MHz, D2O) δ 0.71 (1P), -0.42 (1P), -0.64 (1P) ppm. HR-ESI-MS calcd. for C 31 H 40 N 12 O 22 P3, 1025.15985 [M - H]-; found 1025.15478..

[0180] pACG (Torotron, ε 260 = 31,600 M -1 ・cm -1 , 29.7% yield)(51): 1 H NMR (400 MHz, D2O) δ 8.40 (s, 1H), 8.03 (s, 1H), 7.86 (s, 1H), 7.65 (d, J = 7.6 Hz, 1H), 7.34 (s, 0.5H), 6.03 (s, 1H), 5.75 (d, J = 5.3 Hz, 1H), 5.68 - 5.58 (m, 2H), 4.70 (d, J= 7.6 Hz, 1H), 4.58 - 4.43 (m, 5H), 4.39 - 4.26 (m, 5H), 4.22 (d, J = 2.7 Hz, 0.5H), 4.17 - 4.06 (m, 5.5H), 3.93 (d, J = 5.3 Hz, 1H), 3.64 (s, 3H), 3.56 (s, 3H), 3.49 (q, J = 7.3 Hz, 3H), 3.15 (q, J= 7.3 Hz, 15H), 1.31 - 1.20 (m, 27H) ppm. 13C NMR (101 MHz, D2O) δ 162.82, 158.27, 154.47, 153.50, 153.10, 151.20, 147.69, 139.60, 137.09, 128.42, 118.57, 115.98, 94.89, 87.67, 86.17, 83.18, 81.72, 81.50, 80.40, 73.57, 70.83, 69.79, 64.51, 62.67, 58.57, 57.79, 57.72, 46.63, 42.25, 24.69, 10.55, 8.23, 7.18 ppm. 31 P NMR (162 MHz, D2O) δ 0.59 (1P), -0.53 (1P), -1.00 (1P) ppm. HR-ESI-MS calcd. for C 31 H 41 N 13 O 21 P3, 1024.17583 [M - H] - ; found 1024.17943.

[0181] pAAG (triethylammonium salt, ε 260 = 37,000 M -1 ·cm -1 , 28.3% yield) (52): 1H NMR (600 MHz, D2O) δ 8.52 (s, 1H), 8.31 (s, 1H), 8.10 (s, 1H), 7.93 (s, 1H), 7.89 (s, 1H), 6.09 (d, J = 5.0 Hz, 1H, H1’), 5.97 (d, J = 5.8 Hz, 1H, H1’), 5.81 (d, J = 5.9 Hz, 1H, H1’), 4.91 (ddd, J = 10.1, 7.6, 5.1 Hz, 2H), 4.60 (t, J = 5.6 Hz, 1H), 4.53 - 4.49 (m, 2H), 4.45 - 4.40 (m, 3H), 4.29 (dt, J= 5.5, 2.7 Hz, 1H), 4.21 (h, J= 3.4 Hz, 2H), 4.17 - 4.10 (m, 2H), 3.94 (q, J = 3.2 Hz, 2H), 3.45 (d, J= 4.8 Hz, 6H, 2’-OCH3), 2.99 (q, J = 7.3 Hz, 46H, triethylammonium), 1.16 (t, J = 7.4 Hz, 70H, triethylammonium) ppm. 13 C NMR (151 MHz, D2O) δ 165.69, 159.41, 155.19, 152.77, 152.41, 151.48, 148.70, 148.39, 140.07, 139.17, 135.56, 118.43, 118.40, 117.31, 86.56, 85.42, 84.80, 83.90, 83.30, 82.76, 82.05, 81.89, 73.69, 73.21, 72.44, 70.23, 65.11, 64.88, 63.33, 59.00, 58.17, 57.65, 46.37, 8.56 ppm. 31 P NMR (243 MHz, D2O) δ 4.31, -0.37, -0.48 ppm. ESI-TOF-MS calcd. for C 32 H 40 N 15 O 20 P3, 523.5899 [M - 2H] 2-; found 523.5926.

[0182] pGUG (triethylammonium salt, ε 260 = 31,300 M -1 ・cm -1 , 33.1% yield) (53): 1 H NMR (400 MHz, D2O) δ 8.09 (s, 1H), 7.99 (s, 1H), 7.76 (d, J = 8.2 Hz, 1H), 5.90 (d, J = 4.8 Hz, 1H), 5.87 (d, J = 4.0 Hz, 1H), 5.82 (d, J = 5.8 Hz, 1H), 5.65 (d, J = 8.1 Hz, 1H), 4.87 - 4.83 (m, 1H), 4.72 (t, J = 5.6 Hz, 1H), 4.66 (dt, J = 8.1, 5.0 Hz, 1H), 4.47 - 4.42 (m, 3H), 4.33 - 4.28 (m, 2H), 4.17 - 4.06 (m, 6H), 4.02 (t, J = 4.5 Hz, 1H), 3.47 (d, J = 8.5 Hz, 6H), 3.15 (q, J = 7.3 Hz, 18H), 1.23 (t, J = 7.3 Hz, 27H) ppm. 13 C NMR (101 MHz, D2O) δ 165.35, 158.48, 158.40, 153.75, 153.68, 151.49, 151.29, 151.08, 140.29, 137.67, 136.92, 128.42, 116.09, 115.74, 102.07, 87.51, 86.82, 85.42, 83.73, 83.64, 82.87, 81.61, 73.41, 72.18, 71.42, 70.39, 65.15, 63.87, 58.49, 57.93, 57.79, 46.63, 8.22 ppm. 31 P NMR (161 MHz, D2O) δ 0.68 (1P), -0.44 (1P), -0.65 (1P) ppm. HR-ESI-MS calcd. for C 31 H 40 N12 O 23 P3, 1041.15476 [M - H] - ; found 1041.14643.。

[0183] pGCG (Torotron, ε 260 = 28,000 M -1 ・cm -1 , 32.7% yield)(54) 1 H NMR (400 MHz, D2O) δ 8.02 (s, 1H), 7.78 (s, 1H), 7.70 (d, J = 7.6 Hz, 1H), 5.80 (s, 1H), 5.74 (d, J = 4.2 Hz, 1H), 5.71 - 5.60 (m, 1H), 5.50 (d, J = 7.8 Hz, 1H), 4.62 (s, 1H), 4.48 (d, J= 4.4 Hz, 1.5H), 4.44 - 4.31 (m, 6H), 4.26 (dt, J = 5.4, 2.5 Hz, 1.5H), 4.17 - 4.05 (m, 5H), 3.69 (s, 3H), 3.61 (s, 3H), 3.47 (q, J = 7.3 Hz, 2H), 3.15 (q, J = 7.4 Hz, 14H), 3.07 - 2.98 (m, 2H), 1.29 - 1.20 (m, 27H) ppm. 13 C NMR (100 MHz, D2O) δ 158.83, 157.90, 153.33, 153.16, 150.79, 150.53, 139.08, 136.38, 128.43, 116.07, 115.78, 94.61, 88.17, 87.66, 86.45, 82.45, 81.41, 80.80, 79.77, 73.85, 69.97, 69.43, 69.00, 63.93, 62.22, 58.60, 57.46, 57.41, 46.62, 42.24, 10.55, 8.22, 7.21 ppm. 31 P NMR (162 MHz, D2O) δ 0.62 (1P), -0.51 (1P), -1.03 (1P) ppm. HR-ESI-MS calcd. for C31 H 41 N 13 O 22 P3, 1040.17074 [M-H] - ; found 1040.16350.

[0184] pGAG (triethylammonium salt, ε 260 = 34,800 M -1 ·cm -1 , 37.7% yield) (55): 1H NMR (600 MHz, D2O, 0.2:1 rotamer mixture) δ 9.31 (s, 0.2H), 8.42 (d, J = 4.1 Hz, 0.4H), 8.29 (s, 1H), 8.09 (s, 1H), 7.99 (s, 1H), 7.90 (s, 1H), 7.78 (s, 0.2H), 7.32 (s, 0.2H), 6.35 (s, 0.2H), 6.06 (d, J = 4.7 Hz, 1H), 5.88 (d, J = 1.7 Hz, 0.2H), 5.76 (dd, J = 11.7, 5.1 Hz, 2H), 5.31 (d, J = 5.5 Hz, 0.2H), 5.08 (d, J = 8.4 Hz, 0.2H), 4.90 (dq, J = 9.6, 5.0 Hz, 1.5H), 4.87 - 4.83 (m, 1.5H), 4.76 (d, J = 1.8 Hz, 0.5H), 4.73 (d, J = 4.6 Hz, 0.2H), 4.68 (t, J = 5.3 Hz, 1H), 4.58 (dd, J = 8.1, 3.4 Hz, 0.2H), 4.54 - 4.51 (m, 0.2H), 4.44 (t, J = 4.6 Hz, 2.5H), 4.39 (q, J = 4.6 Hz, 2H), 4.35 - 4.28 (m, 2.5H), 4.24 (d, J = 9.3 Hz, 0.4H), 4.18 (q, J = 4.4 Hz, 2H), 4.14 (t, J = 3.5 Hz, 2H), 4.11 - 4.05 (m, 2H), 3.99 - 3.93 (m, 0.2H), 3.90 (s, 1H), 3.84 - 3.76 (m, 0.4H), 3.49 (s, 3H), 3.45 - 3.41 (m, 3H), 3.40 (d, J = 2.3 Hz, 3H), 3.32 (s, 0.2H), 3.14 (q, J = 7.4 Hz, 20H), 3.03 (dtd, J = 10.3, 7.3, 2.5 Hz, 2H), 2.93 (d, J = 3.2 Hz, 1H), 1.28 - 1.19 (m, 37H) ppm. 13C NMR (151 MHz, D2O, 0.2:1 rotamer mixture) δ 158.36, 158.28, 157.45, 157.09, 154.64, 153.58, 153.40, 152.60, 152.09, 151.57, 151.30, 151.14, 150.56, 148.27, 147.23, 139.13, 137.50, 136.97, 128.41, 118.33, 117.99, 116.15, 115.90, 114.60, 112.75, 87.61, 85.61, 85.33, 83.49, 83.43, 82.63, 82.41, 82.07, 81.39, 73.41, 73.28, 72.25, 70.14, 65.02, 64.46, 63.94, 58.67, 58.06, 57.63, 57.51, 46.67, 8.25 ppm. 31 P NMR (243 MHz, D2O, 0.2:1 rotamer mixture) δ 0.82 (1P), 0.66 (0.2P), 0.27 (0.2P), -0.33 (1P), -0.60 (1P), -2.26 (0.2P) ppm. HR-ESI-MS calcd. for C 32 H 41 N 15 O 21 P3, 1064.18198 [M - H] - ; found 1064.17879..

[0185] pGGG 260 = 31,700 M -1 ・cm -1 , 40.0% yield)(56): 11H NMR (400 MHz, D2O) δ 7.97 (s, 1H), 7.86 (d, J = 10.8 Hz, 2H), 5.82 - 5.69 (m, 3H), 4.87 (t, J = 4.2 Hz, 1H), 4.73 (d, J = 5.4 Hz, 1H), 4.52 (t, J = 5.3 Hz, 1H), 4.46 (t, J = 4.7 Hz, 1H), 4.34 (s, 1H), 4.31 - 4.24 (m, 3H), 4.15 (d, J = 4.9 Hz, 2H), 4.06 (s, 1.5H), 3.95 (s, 1.5H), 3.85 - 3.63 (m, 2H), 3.50 (q, J = 7.2 Hz, 2H), 3.35 (d, J = 7.9 Hz, 6H), 3.14 (q, J = 7.3 Hz, 14H), 3.02 (q, J = 7.2 Hz, 2H), 1.22 (t, J = 7.3 Hz, 27H) ppm. 13 13C NMR (101 MHz, D2O) δ 158.36, 158.24, 158.08, 153.59, 153.44, 151.38, 151.28, 151.09, 137.57, 136.70, 128.38, 116.15, 115.40, 87.65, 86.01, 85.59, 83.57, 83.48, 82.61, 81.52, 80.47, 73.26, 72.57, 72.12, 70.25, 65.17, 64.80, 63.75, 58.52, 57.82, 57.60, 46.62, 42.24, 8.22 ppm. 31 31P NMR (162 MHz, D2O) δ 0.59 (1P), -0.28 (1P), -0.52 (1P) ppm. HR-ESI-MS calcd. for C 32 H 41 N 15 O 22 P3, 1080.17689 [M - H] - ; found 1080.17185.。

[0186] pUUA (triethylammonium salt, ε 260= 34,300 M -1 ・cm -1 、33.8% yield)(57): 1 H NMR (400 MHz, D2O) δ 8.49 (s, 1H), 8.22 (s, 1H), 7.95 (d, J = 8.2 Hz, 1H), 7.84 (d, J = 8.2 Hz, 1H), 7.39 (s, 2H), 6.09 (d, J = 5.4 Hz, 1H), 5.92 (dd, J = 6.3, 4.5 Hz, 2H), 5.83 (dd, J = 8.1, 3.8 Hz, 2H), 4.77 - 4.68 (m, 3H), 4.51 (t, J = 4.5 Hz, 1H), 4.41 - 4.33 (m, 3H), 4.20 - 4.09 (m, 7H), 4.02 (t, J = 4.9 Hz, 1H), 3.50 (s, 3H), 3.39 (s, 3H), 3.18 (q, J = 7.3 Hz, 18H), 1.26 (t, J = 7.3 Hz, 27H) ppm. 13 C NMR (101 MHz, D2O) δ 165.85, 165.59, 154.80, 151.84, 151.32, 151.26, 148.96, 141.21, 140.72, 139.98, 128.50, 118.65, 102.55, 102.28, 87.31, 86.66, 86.62, 83.80, 83.70, 82.47, 81.95, 81.55-81.44, 74.26, 71.82-71.68, 70.39, 65.06, 64.21, 63.47, 58.62, 57.91, 57.81, 46.69, 8.27 ppm. 31 P NMR (162 MHz, D2O) δ 0.67 (1P), -0.47 (1P), -0.79 (1P) ppm. HR-ESI-MS calcd. for C 30 H 39 N9O 23 P3, 986.13771 [M - H] - ; found 986.13124.。

[0187] pCUA (triethylammonium salt, ε 260 = 30,900 M -1 ·cm -1 , 30.0% yield) (58): 1 H NMR (600 MHz, D2O) δ 8.51 (s, 1H), 8.22 (s, 1H), 8.03 (d, J = 7.7 Hz, 1H), 7.83 (d, J = 8.1 Hz, 1H), 7.39 (s, 2H), 6.09 (d, J = 5.5 Hz, 1H), 5.98 - 5.91 (m, 2H), 5.87 (d, J = 2.5 Hz, 1H), 5.75 (d, J = 8.1 Hz, 1H), 4.73 (dd, J = 6.8, 3.8 Hz, 2H), 4.66 (dt, J = 8.0, 5.4 Hz, 1H), 4.50 (dd, J = 5.1, 3.8 Hz, 1H), 4.36 (ddt, J = 8.3, 5.6, 2.7 Hz, 3H), 4.27 (ddd, J = 12.0, 4.8, 2.3 Hz, 1H), 4.19 - 4.06 (m, 6H), 3.97 (t, J = 4.5 Hz, 1H), 3.57 (s, 3H), 3.51 (q, J = 7.3 Hz, 2H), 3.37 (s, 3H), 3.18 (q, J = 7.3 Hz, 16H), 1.26 (t, J = 7.4 Hz, 27H) ppm. 13 C NMR (151 MHz, D2O) δ 165.43, 163.84, 154.75, 154.32, 151.78, 151.06, 148.96, 141.86, 140.20, 139.91, 128.52, 118.67, 102.37, 95.44, 87.70, 87.26, 86.65, 83.92, 83.86, 81.71, 74.36, 71.34, 70.64, 70.48, 65.01, 63.67, 62.68, 58.63, 57.86, 57.78, 46.70, 8.27 ppm. 31P NMR (243 MHz, D2O) δ 0.75 (1P), -0.46 (1P), -1.03 (1P) ppm. HR-ESI-MS calcd. for C 30 H 40 N 10 O 22 P3, 985.15370 [M - H] - ; found 985.14609..

[0188] pAUA (Torotron, ε 260 = 38,700 M -1 ・cm -1 , 27.8% yield)(59): 1 H NMR (400 MHz, D2O) δ 8.38 (s, 1H), 8.28 (s, 1H), 8.02 (s, 1H), 7.98 (s, 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.31 (s, 1H), 6.04 (d, J = 4.4 Hz, 1H), 5.99 (d, J = 5.4 Hz, 1H), 5.79 (d, J = 4.4 Hz, 1H), 5.63 (d, J = 8.2 Hz, 1H), 4.95 (dt, J = 8.7, 4.7 Hz, 1H), 4.64 (t, J = 5.3 Hz, 1H), 4.57 (dd, J = 8.0, 5.2 Hz, 1H), 4.53 - 4.41 (m, 3H), 4.35 - 4.28 (m, 2H), 4.25 - 4.04 (m, 6H), 3.88 (t, J = 4.7 Hz, 1H), 3.52 (s, 3H), 3.43 (q, J = 7.2 Hz, 2H), 3.25 (s, 3H), 3.12 (q, J = 7.3 Hz, 16H), 1.20 (t, J = 7.4 Hz, 27H) ppm. 13C NMR (101 MHz, D2O) δ 165.43, 154.63, 154.38, 151.78, 151.58, 151.00, 148.49, 148.16, 140.26, 139.73, 139.41, 128.38, 118.31, 102.18, 87.22, 86.59, 85.81, 83.68, 83.59, 81.97, 81.52, 74.19, 71.95, 71.44, 70.34, 64.91, 63.88, 63.45, 58.56, 57.99, 57.79, 46.58, 8.18 ppm. 31 P NMR (162 MHz, D2O) δ 0.89 (1P), -0.52 (1P), -0.97 (1P) ppm. HR-ESI-MS calcd. for C 31 H 40 N 12 O 21 P3, 1009.16493 [M-H] - ; found 1009.15687.

[0189] pGUA (triethylammonium salt, ε 260 = 35,900 M -1 ·cm -1 , 33.8% yield) (60): 11H NMR (600 MHz, D2O) δ 8.39 (s, 1H), 8.16 (s, 1H), 8.09 (s, 1H), 7.81 (d, J = 8.2 Hz, 1H), 7.38 (s, 1H), 6.08 (d, J = 5.3 Hz, 1H), 5.90 (dd, J = 13.3, 4.8 Hz, 2H), 5.75 (d, J = 8.2 Hz, 1H), 4.97 (dt, J = 8.2, 4.5 Hz, 1H), 4.74 (t, J = 5.3 Hz, 1H), 4.69 (dt, J = 7.8, 5.1 Hz, 1H), 4.56 - 4.48 (m, 3H), 4.38 (dp, J = 6.6, 2.8 Hz, 2H), 4.26 - 4.10 (m, 7H), 4.00 (t, J = 4.8 Hz, 1H), 3.54 (s, 3H), 3.37 (s, 3H), 3.19 (q, J = 7.4 Hz, 18H), 1.27 (t, J = 7.3 Hz, 27H). 13 13C NMR (151 MHz, D2O) δ 165.54, 158.45, 154.82, 153.63, 151.94, 151.24, 148.79, 140.59, 139.76, 一三7.07, 128.50, 118.58, 115.96, 102.37, 87.46, 86.74, 85.61, 83.74, 82.97, 82.05, 81.67, 74.29, 72.31, 71.73, 70.40, 65.07, 64.13, 63.90, 58.68, 57.99, 57.94, 46.73, 8.30 ppm. 31 31P NMR (243 MHz, D2O) δ 0.90 (1P), -0.41 (1P), -0.74 (1P) ppm. HR-ESI-MS calcd. for C 31 H 40 N 12 O 22 P3, 1025.15985 [M - H] - ; found 1025.15478.。

[0190] pUUAG (triethylammonium salt, ε260 = 43,900 M -1 ・cm -1 、% yield)(61): 1 H NMR (600 MHz, D2O) δ 8.27 (s, 1H), 8.04 (s, 1H), 7.83 (d, J = 8.2 Hz, 2H), 7.68 (d, J = 8.2 Hz, 1H), 7.24 (s, 4H), 5.97 (d, J = 4.9 Hz, 1H), 5.81 (d, J = 4.7 Hz, 1H), 5.74 (d, J = 5.0 Hz, 1H), 5.73 (s, 1H), 5.71 (s, 1H), 5.69 (d, J = 5.7 Hz, 1H), 4.85 - 4.79 (m, 1H), 4.61 (t, J= 5.4 Hz, 1H), 4.59 - 4.54 (m, 1H), 4.37 - 4.32 (m, 3H), 4.27 (dq, J = 4.9, 2.5 Hz, 1H), 4.20 - 4.17 (m, 2H), 4.08 - 3.92 (m, 10H), 3.84 (t, J= 5.1 Hz, 1H), 3.37 (s, 3H), 3.35 (s, 3H), 3.24 (s, 3H), 3.03 (t, J = 7.4 Hz, 24H), 1.12 (t, J = 7.4 Hz, 36H) ppm. 13 C NMR (151 MHz, D2O) δ 165.93, 165.60, 158.49, 154.50, 153.68, 151.56-151.21, 148.64, 141.27, 140.84, 139.78, 137.62, 128.47, 118.62, 116.24, 102.60, 102.41, 87.52, 86.55, 86.47, 85.57, 83.60, 83.54, 82.62, 82.20, 81.75, 81.44, 81.32, 73.32, 72.44, 72.09, 71.85, 70.21, 65.10, 64.81, 64.51, 63.57, 58.61, 58.02, 57.80, 57.70, 46.68, 8.26 ppm. 31P NMR (243 MHz, D2O) δ 0.62 (s, 1P), -0.30 (s, 1P), -0.64 (s, 1P), -0.70 (s, 1P) ppm. ESI-TOF-MS calcd. for C 41 H 52 N 14 O 30 P4, 672.0968 [M - 2H] 2- ; found 672.0945.

[0191] Synthesis of tetranucleotide PureCap analogues. Synthetic scheme.

[0192] Experimental protocol: A solution of trinucleotide UUG triethylammonium salt (56.5 mg, 40.1 μmol) in DMSO (802 μL) was added to N 7α-Methylguanosine diphosphate imidazolide disodium salt (92.7 mg, 120 μmol) was added, followed by zinc chloride (109 mg, 802 μmol). After incubation at 37°C for 5 days, the reaction mixture was quenched with 500 mM EDTA-NaOH aqueous solution (pH 8.0, 1.04 mmol, 2.0 mL) and diluted with water (40 mL). The crude product was purified by reverse-phase HPLC using a Shimadzu YMC-Actus Triart C8 column (250 x 20.0 mm I.D.), solvent A: 50 mM TEAA buffer (pH 6.0) containing 0.5% CH3CN, solvent B: CH3CN, linear gradient 5-80% B (25 min), flow rate: 10 mL / min, detection: 254 nm. Fractions containing the desired product were pooled, concentrated, and lyophilized to yield the tetranucleotide PureCap analog, m7GpppUUG, as its triethylammonium salt. The product was redissolved in methanol (2.0 mL) and 190 mM NaClO4 in acetone (12 mL) was added. The resulting suspension was centrifuged (4,000 rpm, 10 min). The supernatant was discarded, and the precipitate was suspended in acetone. The suspension-centrifugation process was repeated three more times. The precipitate was dried under reduced pressure to yield the desired PureCap analog, m7GpppUUG (17.7 mg, 9.96 μmol, 24.8% yield), as its sodium salt. The yield was calculated using the absorbance of the product at 260 nm measured with a NanoDrop.

[0193] Nb-7mGpppUUG (sodium salt, ε 260 = 44,800 M -1 ·cm -1 , 24.8% yield) (1): 1H NMR (600 MHz, D2O) δ7.98 (s, 1H), 7.86 (dd, J = 13.3, 8.2 Hz, 1H), 7.80 (d, J = 8.2 Hz, 1H), 7.68 (d, J = 8.2 Hz, 0.5H), 7.59 (q, J = 8.0 Hz, 1H), 7.55 - 7.49 (m, 1.5H), 7.42 (t, J= 7.7 Hz, 0.5H), 7.31 (dt, J = 8.5, 4.7 Hz, 0.5H), 6.00 (d, J= 5.1 Hz, 0.5H), 5.92 (t, J = 7.7 Hz, 1H), 5.88 (dd, J = 4.8, 2.3 Hz, 1H), 5.85 - 5.81 (m, 3H), 5.04 (s, 0.5H), 4.99 (d, J = 7.6 Hz, 0.5H), 4.93 (s, 1H), 4.86 (d, J = 7.4 Hz, 0.5H), 4.75 (d, J = 5.4 Hz, 2.5H), 4.70 (s, 1H), 4.61 (q, J = 6.4 Hz, 1H), 4.46 (td, J = 13.4, 8.0 Hz, 2H), 4.38 (s, 1H), 4.32 - 4.23 (m, 5H), 4.19 - 4.12 (m, 4H), 4.10 (s, 1.5H), 4.04 (d, J = 6.7 Hz, 6H), 3.46 - 3.41 (m, 6H), 0.66 - 0.55 (m, 9H) ppm. 13C NMR (151 MHz, D2O) δ 165.90; 140.76, 137.66, 133.74, 132.70, 132.63, 132.21, 130.03, 129.97, 128.81, 128.06, 123.80; 108.05, 102.92, 102.65, 95.10, 93.66, 87.57, 87.48, 87.40, 86.57, 86.01, 85.56, 85.11, 83.65, 83.59, 82.86, 81.83, 81.51, 81.27, 80.61, 79.41, 78.92, 73.41, 72.46, 71.93, 70.46, 68.85, 68.52, 65.33, 65.02, 64.86, 64.63. 64.36, 57.91, 57.78, 36.44, 36.05, 35.71, 30.29, 24.81, 24.74, 23.31 ppm. 31 P NMR (243 MHz, D2O) δ -0.34 (1P), -0.56 (1P), -10.84 (t, J = 16.4 Hz, 2P), -21.98 (q, J = 16.4 Hz, 1P) ppm. HR-ESI-MS calcd. for C53H69N15O37P5, 1662.26724[M-H]-; found 1662.17281.

[0194] Nb-7mGpppUCG(Nb-7mGpppUCG.ε 260 = 42,900 M -1 ・cm -1 、26.4% yield)(2) 1H NMR (600 MHz, D2O) δ 7.97 (s, 1H), 7.95 - 7.85 (m, 2H), 7.74 (d, J= 8.3 Hz, 0.5H), 7.64 (dd, J = 10.8, 7.9 Hz, 1H), 7.59 - 7.54 (m, 1.5H), 7.46 (t, J = 7.7 Hz, 0.5H), 7.37 (d, J = 5.8 Hz, 0.5H), 6.03 (d, J = 4.7 Hz, 0.5H), 6.00 - 5.94 (m, 2H), 5.92 (dd, J = 8.8, 4.4 Hz, 1H), 5.85 (dd, J = 13.6, 5.7 Hz, 1.5H), 5.79 (d, J = 4.7 Hz, 1H), 5.06 (s, 0.5H), 5.01 (d, J = 8.0 Hz, 0.5H), 4.97 (s, 0.5H), 4.94 (d, J = 7.1 Hz, 0.5H), 4.89 (d, J = 8.2 Hz, 1H), 4.86 (d, J = 6.0 Hz, 0.5H), 4.76 (s, 0.5H), 4.75 - 4.72 (m, 1H), 4.67 (q, J = 5.7 Hz, 2H), 4.61 (d, J= 3.6 Hz, 0.5H), 4.53 (t, J = 5.4 Hz, 0.5H), 4.46 (s, 1.5H), 4.39 (s, 1H), 4.35 - 4.29 (m, 4H), 4.26 (s, 0.5H), 4.21 - 4.06 (m, 10H), 4.03 (s, 1H), 3.54 - 3.52 (m, 2.5H), 3.47 - 3.45 (m, 2.5H), 3.34 (d, J = 1.2 Hz, 1H), 0.71 - 0.62 (m, 9H) ppm. 13C NMR (151 MHz, D2O) δ 165.79; 140.19, 137.94, 133.76, 132.70, 132.35; 102.68, 96.22, 95.02, 87.91, 87.69, 87.48, 86.39, 83.68, 82.56, 82.00, 81.75, 81.24, 80.60, 79.14, 78.79, 73.37,000. 71.80, 71.13, 70.37, 68.72, 68.39, 65.01, 64.47, 63.53, 57.92, 57.66, 48.93, 36.47, 36.18, 35.76, 30.29, 24.87, 24.78 ppm. 31 P NMR (243 MHz, D2O) δ -0.52 (2s, 2P), -10.86 (d, J = 16.4 Hz, 2P), -22.07 (d, J = 16.4 Hz, 1P) ppm. ESI-TOF-MS calcd. for C 53 H 69 N 16 O 36 P5, 830.1380 [M-2H] 2- ; found 830.1369.

[0195] Nb-7mGpppUAG(Nb-7mGpppUAG.ε 260 = 49,300 M -1 ・cm -1 、26.4% yield)(3) 1H NMR (600 MHz, D2O) δ 8.34 (d, J = 2.7 Hz, 1H), 8.05 (s, 1H), 7.91 (d, J = 4.2 Hz, 1H), 7.83 - 7.71 (m, 1H), 7.62 (d, J = 8.0 Hz, 0.5H), 7.59 - 7.45 (m, 2.5H), 7.38 (t, J = 7.7 Hz, 0.5H), 7.27 (dt, J = 8.3, 4.3 Hz, 0.5H), 6.11 (t, J = 4.0 Hz, 1H), 5.98 (d, J= 5.6 Hz, 0.5H), 5.90 (dd, J = 13.5, 8.2 Hz, 1H), 5.82 - 5.76 (m, 1.5H), 5.72 - 5.64 (m, 1H), 5.04 (s, 0.5H), 5.00 - 4.91 (m, 2.5H), 4.89 (s, 0.5H), 4.69 - 4.63 (m, 2H), 4.57 (t, J = 5.5 Hz, 0.5H), 4.48 - 4.41 (m, 4H), 4.35 - 4.32 (m, 3H), 4.26 - 4.10 (m, 10H), 4.06 (s, 1.5H), 4.01 (dt, J = 10.3, 5.1 Hz, 1H), 3.52 (s, 3H), 3.40 (d, J = 4.6 Hz, 3H), 3.36 (s, 0.5H), 0.62 (s, 4.5H), 0.54 (s, 4.5H) ppm. 13C NMR (151 MHz, D2O) δ 166.05; 141.00, 139.15, 137.31, 133.83, 132.76, 132.50, 132.02, 129.92, 128.73, 127.84, 123.68, 123.48, 118.58, 108.54, 102.71, 95.41, 93.78, 87.61, 87.05, 86.29, 85.47, 83.26, 82.36, 82.01, 81.30, 80.78, 80.02, 79.28, 73.57,000. 72.31, 70.05, 69.20, 68.87, 64.97, 64.55, 58.04, 57.63, 36.46, 35.98, 35.65, 30.33, 24.70, 24.65, 23.36 ppm. 31 P NMR (243 MHz, D2O) δ -0.41 (2s, 2P), -10.72 (d, J = 19.7 Hz, 2P), -21.85 (d, J = 16.4 Hz, 1P) ppm. ESI-TOF-MS calcd. for C 54 H 69 N 18 O 35 P5, 842.14359 [M-2H] 2- ; found 842.15626.

[0196] Nb-7mGpppUGG(Nb-7mGpppUGG.ε 260 = 48,920 M -1 ・cm -1 、21.0% yield)(4) 1H-NMR (400 MHz, D2O) δ 7.97 (s, 1H), 7.89-7.87 (m, 1H), 7.79-7.75 (m, 1H), 7.58-7.56 (m, 1H), 7.53-7.49 (m, 1H), 7.47-7.32 (m, 4H), 7.22-7.19 (m, 1H), 5.96-5.94 (m, 0.5H), 5.87-5.85 (m, 1.5H), 5.81-5.80 (m, 1H), 5.77-5.74 (m, 2H), 5.70-5.69 (m, 1H), 4.99-4.93 (m, 3H), 4.68-4.64 (m, 2H), 4.56-4.54 (m, 1H), 4.48-4.42 (m, 2H), 4.39-4.35 (m, 1H), 4.36-4.32 (m, 5H), 4.17-4.11 (m, 6H), 4.07-3.99 (m, 4H), 3.31-3.30 (m, 2H), 3.28-3.27 (m, 8H), 3.20-3.05 (m, 123H), 1.28-1.14 (m, 184H), 0.51 (d, J = 27.4 Hz, 9H) ppm. 13 C-NMR (101 MHz, D2O) δ 181.4, 165.9, 158.7, 153.9, 153.7, 151.7, 151.6, 151.3, 149.8, 149.6, 149.3, 148.2, 141.0, 137.5, 133.8, 132.7, 131.9, 129.9, 129.8, 128.7, 127.7, 123.6, 123.4, 116.6, 116.5, 116.3, 108.5, 102.8, 95.4, 87.2, 87.0, 86.8, 86.1, 85.9, 83.6, 83.6, 83.1, 82.7, 82.6, 81.8, 81.3, 80.6, 80.0, 79.1, 73.3, 72.9, 72.9, 72.3, 72.2, 70.4, 69.1, 68.8, 65.2, 64.9, 57.9, 57.5, 46.8, 46.6, 46.4, 36.4, 35.9, 35.9, 35.6, 24.6, 24.5, 23.3, 8.4, 8.2, 8.0 ppm.31 P-NMR (160 MHz, D2O) δ -0.3, -0.6, -11.1, -11.2, -22.3, -22.4, -22.5 ppm. HRMS (ESI) Calcd. For C 54 H 69 N 18 O 36 P5 2- ([M-2] 2- ): 850.1410. Obsd.850.1405.

[0197] Nb-7mGpppCUG (sodium salt, ε 260 = 41,400 M -1 ・cm -1 、26.0% yield)(5): 1 H NMR (600 MHz, D2O) δ 7.99 (s, 1H), 7.80 (q, J = 9.1 Hz, 2H), 7.73 (d, J = 7.3 Hz, 0.5H), 7.63 (d, J = 8.2 Hz, 1H), 7.57 (d, J = 17.2 Hz, 1.5H), 7.45 (t, J = 7.6 Hz, 0.5H), 7.36 (d, J = 7.9 Hz, 0.5H), 6.04 - 5.95 (m, 1.5H), 5.91 - 5.80 (m, 2.5H), 5.74 - 5.65 (m, 2H), 5.09 (s, 0.5H), 5.05 - 4.99 (m, 1H), 4.92 (d, J = 8.8 Hz, 1H), 4.71 - 4.69 (m, 1H), 4.66 - 4.63 (m, 1H), 4.57 (s, 1H), 4.54 - 4.47 (m, 2H), 4.45 (s, 1H), 4.41 (s, 0.5H), 4.35 - 4.26 (m, 6H), 4.17 - 4.04 (m, 10H), 3.94 (s, 1H), 3.53 (d, J = 16.0 Hz, 2.5H), 3.47 (s, 2.5H), 3.34 (d, J= 2.3 Hz, 1H), 0.71 - 0.62 (m, 9H) ppm. 13C NMR (151 MHz, D2O) δ 165.92; 137.54, 133.70, 132.82, 132.73, 132.33, 130.01, 128.84, 128.22, 123.89, 123.46, 116.37, 107.98; 96.37, 95.08, 87.85, 87.34, 86.85, 83.66, 81.71, 81.49, 80.80, 79.45, 79.10, 73.61, 71.00, 70.46, 68.55, 68.14. 65.08, 64.24, 63.34, 57.82, 57.50, 48.93, 36.40, 36.15, 35.79, 30.30, 24.92, 24.84 ppm. 31 P NMR (243 MHz, D2O) δ -0.64 (2s, 2P), -10.44 ~ -11.01 (m, 2P), -21.92 (d, J = 16.4 Hz, 1P) ppm. ESI-TOF-MS calcd. for C 53 H 69 N 16 O 36 P5, 830.1380 [M-2H] 2- ; found 830.1369.

[0198] Nb-7mGpppCCG(Nb-7mGpppCCG.ε 260 = 39,900 M -1 ・cm -1 、14.4% yield)(6) 11H NMR (600 MHz, D2O) δ7.79 - 7.58 (m, 3.5H), 7.57 - 7.41 (m, 2.5H), 7.37 - 7.20 (m, 1H), 5.88 - 5.61 (m, 5H), 5.40 (s, 0.5H), 5.02 - 4.89 (m, 1.5H), 4.81 (s, 1H), 4.46 (s, 2.5H), 4.37 - 4.26 (m, 3H), 4.18 (s, 7.5H), 4.09 - 3.93 (m, 8H), 3.90 - 3.80 (m, 3H), 3.44 (t, J = 11.0 Hz, 6H), 0.65 - 0.49 (m, 9H) ppm. 13 13C NMR (151 MHz, D2O) δ 165.79, 165.47, 158.84, 156.46, 153.66, 151.48, 149.72, 149.24, 140.36, 139.06, 133.77, 132.71, 132.26, 130.04, 128.81, 128.13, 123.87, 123.51, 116.37, 115.36, 107.96, 95.96, 95.18, 87.61, 81.96, 81.46, 80.98, 80.19, 79.60, 79.20, 73.56, 70.11, 64.58, 63.50, 62.54, 57.69, 57.42, 36.42, 36.11, 35.78, 30.29, 24.88, 24.80, 23.31 ppm. 31 31P NMR (243 MHz, D2O) δ-0.41 (1P), -0.93 (1P), -10.71 (2P), -21.78 (1P) ppm. HR-ESI-MS calcd. for C53H71N17O35P5, 1660.29920 [M - H]-; found 1660.29965.。

[0199] Nb-7mGpppCAG (sodium salt, ε 260 = 45,700 M -1 ・cm -1 , 16.6% yield) (7): 1H NMR (600 MHz, D2O) δ 8.36 (s, 1H), 8.08 (s, 1H), 7.90 (s, 2H), 7.63 (d, J= 6.5 Hz, 0.5H), 7.57 (d, J = 8.0 Hz, 1H), 7.49 (s, 1.5H), 7.37 (s, 0.5H), 7.31 (s, 0.5H), 6.12 - 6.05 (m, 2H), 6.01 (s, 0.5H), 5.83 (d, J= 4.3 Hz, 0.5H), 5.79 - 5.75 (m, 1H), 5.72 (s, 1H), 5.03 - 4.96 (m, 1H), 4.91 (s, 1H), 4.85 (d, J = 7.6 Hz, 1H), 4.71 - 4.68 (m, 2H), 4.57 (s, 1H), 4.47 - 4.42 (m, 3H), 4.36 - 4.28 (m, 6H), 4.20 - 4.04 (m, 11H), 3.48 (d, J= 2.6 Hz, 2.5H), 3.45 - 3.43 (m, 2.5H), 3.34 (d, J = 2.9 Hz, 1H), 0.61 - 0.54 (m, 9H) ppm. 13 C NMR (151 MHz, D2O) δ 163.29, 158.41, 155.35, 154.61, 153.64, 150.47, 149.74, 149.11, 148.78, 148.27, 141.81, 139.70, 132.61, 130.06, 128.22, 123.81, 123.37, 118.47, 116.13, 107.78, 107.53, 96.21, 87.92, 87.69, 86.94, 85.88, 83.42, 82.23, 81.53, 80.42, 79.01, 73.37, 72.17, 70.17, 68.10, 66.07, 65.01, 64.27, 57.98, 57.63, 48.92, 36.30, 36.14, 35.71, 30.28, 24.87, 24.79 ppm. 31P NMR (243 MHz, D2O) δ -0.48 (2s, 2P), -10.80 (2P), -21.97 (1P) ppm. ESI-TOF-MS calcd. for C 54 H 70 N 19 O 34 P5, 841.6516 [M - 2H] 2- ; found 841.6548.。

[0200] Nb-7mGpppCGG (triethylammonium salt, ε 260 = 46,330 M -1 ・cm -1 、21.0% yield)(8): 1 H-NMR (400 MHz, D2O) δ 9.90-9.89 (0H), 7.80-7.62 (m, 4H), 7.39-7.09 (m, 5H), 5.85-5.80 (m, 2H), 5.64-5.60 (m, 3H), 5.48 (s, 1H), 4.80 (s, 2H), 4.51-3.94 (m, 12H), 3.88 (d, J = 10.4 Hz, 3H), 3.28-3.15 (m, 6H), 0.32 (d, J = 12.7 Hz, 9H) ppm. 13 C-NMR (100 MHz, D2O) δ 180.6, 164.9, 158.3, 155.5, 155.4, 154.7, 153.6, 153.4, 151.3, 151.2, 149.6, 149.3, 149.0, 148.8, 137.3, 132.6, 132.1, 129.9, 129.7, 128.7, 123.7, 123.3, 116.1, 116.1, 107.5, 107.3, 96.4, 87.8, 87.5, 86.3, 81.5, 81.3, 80.7, 73.3, 72.1, 70.1, 68.3, 64.8, 64.4, 63.8, 57.8, 57.4, 46.6, 36.1, 35.7, 24.9, 24.8, 22.8, 22.6, 10.5, 8.2, 8.0, 7.3 ppm. 31P-NMR (160 MHz, D2O) δ -0.4, -0.6, -10.9, -11.1, -22.5 ppm. HRMS (ESI) Calcd. For C 54 H 72 N 19 O 35 P51701.3126. The obsd. 1701.2997.

[0201] Nb-7mGpppAUG(Nb-7mGpppAUGε 260 = 49,200 M -1 ・cm -1 、19.6% yield)(9) 1H NMR (600 MHz, D2O) δ 9.09 (s, 0.25H), 8.89 (s, 0.25H), 8.50 - 8.39 (m, 1H), 8.11 (d, J = 12.9 Hz, 1H), 7.98 (s, 1H), 7.74 (d, J = 8.2 Hz, 1H), 7.66 (d, J= 7.8 Hz, 0.5H), 7.58 (t, J = 6.7 Hz, 1H), 7.49 (d, J = 10.4 Hz, 1.5H), 7.40 (t, J = 7.2 Hz, 0.5H), 7.31 (t, J = 7.5 Hz, 0.5H), 6.05 - 5.96 (m, 1H), 5.82 (dt, J= 5.0, 2.5 Hz, 2.5H), 5.70 (d, J= 8.3 Hz, 1H), 5.64 (dd, J = 5.1, 2.3 Hz, 0.5H), 5.00 (d, J= 2.3 Hz, 0.5H), 4.95 - 4.89 (m, 2H), 4.84 (d, J = 1.9 Hz, 1H), 4.73 - 4.68 (m, 3H), 4.53 (s, 1.5H), 4.46 (t, J = 4.6 Hz, 2.5H), 4.36 (h, J = 5.0 Hz, 1H), 4.33 - 4.29 (m, 3H), 4.25 (s, 2H), 4.18 - 4.09 (m, 6H), 4.06 (d, J = 2.3 Hz, 1H), 4.01 (t, J= 4.3 Hz, 2.5H), 3.45 (dd, J = 6.2, 2.4 Hz, 5H), 3.34 (d, J = 2.5 Hz, 1H), 0.63 - 0.55 (m, 9H) ppm. 13C NMR (151 MHz, D2O) δ 165.47; 148.61, 140.40, 139.62, 133.68, 132.65, 132.30, 129.90, 128.78; 87.59, 87.35, 86.48, 85.39, 84.51, 83.52, 81.84, 81.55, 80.46, 79.34, 79.01, 73.50, 72.74, 71.75, 70.46, 68.53. 68.18, 65.32, 64.76, 64.29, 57.91, 48.92, 36.35, 36.14, 35.70, 30.28, 24.82, 24.74ppm. 31 P NMR (243 MHz, D2O) δ -0.38(2P), -10.83(2P), -22.05(1P) ppm. ESI-TOF-MS calcd. for C 54 H 69 N 18 O 35 P5, 842.1436 [M-2H] 2- ; found 842.1431.

[0202] Nb-7mGpppACG(Nb-7mGpppACG.ε 260 = 46,700 M -1 ・cm -1 、34.3% yield)(10): 1H NMR (400 MHz, D2O) δ 9.20 - 8.88 (m, 0.5H), 8.40 (s, 1H), 8.04 (d, J= 7.0 Hz, 1H), 7.90 (d, J = 6.7 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.62 - 7.18 (m, 4.5H), 5.99 (s, 1H), 5.89 - 5.64 (m, 4H), 4.89 (s, 3H), 4.61 - 4.48 (m, 4H), 4.44 - 4.10 (m, 15H), 4.07 - 3.95 (m, 4H), 3.60 - 3.46 (m, 6H), 0.58 - 0.38 (m, 9H) ppm. 13 C NMR (101 MHz, D2O) δ 164.40, 158.48, 155.23, 154.95, 154.50, 153.58, 152.22, 151.24, 149.64, 149.27, 148.86, 148.67, 148.24, 139.76, 139.28, 137.10, 133.51, 132.56, 132.22, 129.93, 128.71, 128.10, 123.74, 123.33, 118.34, 116.19, 107.48, 107.21, 95.60, 94.49, 87.65, 85.15, 83.16, 82.52, 81.77, 81.23, 80.80, 80.19, 79.34, 78.98, 73.78, 71.90, 70.68, 69.91, 68.40, 68.07, 64.62, 63.34, 57.79, 36.20, 35.61, 30.26, 24.82, 24.73 ppm. 31 P NMR (162 MHz, D2O) δ -0.53 (2P), -10.78 (2P), -21.90 (1P) ppm. ESI-TOF-MS calcd. for C 54 H 71 N 19 O 34 P5, 1684.31044 [M - H] - ; found 1684.31722.。

[0203] Nb-7mGpppAAG (sodium salt, ε 260 = 52,100 M -1 ·cm -1 , 37.4% yield) (11): 1 H NMR (600 MHz, D2O) δ 9.01 - 8.67 (2s, 1H), 8.26 (d, J = 7.6 Hz, 1H), 8.10 (d, J = 11.0 Hz, 1H), 7.98 (d, J = 7.2 Hz, 1H), 7.83 - 7.68 (m, 2H), 7.40 - 6.92 (m, 4H), 5.89 - 5.75 (m, 2H), 5.65 - 5.53 (m, 1.5H), 5.38 (s, 0.5H), 4.77 (s, 3H), 4.64 - 4.57 (m, 1.5H), 4.55 - 4.39 (m, 2H), 4.39 - 3.95 (m, 16.5H), 3.91 - 3.80 (m, 3H), 3.41 - 3.26 (m, 5.5H), 3.20 (s, 0.5H), 0.35 - 0.12 (2s, 9H) ppm. 13C NMR (151 MHz, D2O) δ 158.11; 148.68, 148.19, 148.00, 139.48, 139.11, 136.92, 133.32, 132.49, 132.13, 129.85, 129.61; 123.20, 119.15, 118.27, 117.98, 115.95, 107.46, 107.18, 93.37, 87.66, 87.45, 85.55, 84.91, 83.16, 82.77, 82.22. 81.81, 80.88, 80.03, 79.37, 78.91, 73.73, 72.38, 72.15, 70.07, 67.92, 64.97, 64.54, 58.06, 58.03, 57.75, 48.91, 36.13, 36.10, 36.03, 35.57, 24.75 ppm. 31 P NMR (243 MHz, D2O) δ -0.38 (2s, 2P), -10.76 (d, J = 19.7 Hz, 2P), -21.82 (t, J = 19.7 Hz, 1P) ppm. ESI-TOF-MS calcd. for C 55 H 70 N 21 O 33 P5, 853.65720 [M-2H] 2- ; found 853.66766.

[0204] Nb-7mGpppAGG(GpppAGG.ε 260 = 50,200 M -1 ・cm -1 、36.0% yield)(12): 1H NMR (600 MHz, D2O) δ 8.42-8.34 (m, 1H), 8.06 (d, J = 12.4 Hz, 1.5H), 7.97 (s, 1H), 7.84 (s, 1H), 7.67 (s, 0.5H), 7.57 (d, J= 7.6 Hz, 1H), 7.51-7.36 (m, 2H), 7.29 (s, 1H), 6.88 (s, 1H), 5.94 (dd, J = 11.6, 5.1 Hz, 2H), 5.88-5.80 (m, 1.5H), 5.73 (s, 1H), 5.68 (d, J= 4.3 Hz, 0.5H), 4.89 (s, 4H), 4.58 (s, 2.5H), 4.52 (s, 0.5H), 4.46 (s, 3H), 4.38 (s, 3H), 4.34-4.24 (m, 8H), 4.21-4.08 (m, 11H), 4.05 (s, 1.5H), 4.00 (s, 1.5H), 3.83-3.47 (m, 6H), 0.64-0.46 (m, 9H) ppm. 13 C NMR (151 MHz, D2O) δ 159.70-158.2 (m), 153.71-150.8 (m), 149.70-148.00 (m), 132.07, 129.36, 128.17, 123.14, 117.26, 114.91, 107.66, 87.66, 84.99, 82.62, 80.56, 79.37, 75.68, 72.44, 68.13, 65.01, 61.77, 58.27, 57.91, 56.82, 48.94, 46.69, 36.13, 35.36, 30.30, 28.25, 24.76, 20.57, 16.84, 13.30, 8.32 ppm. 31 P NMR (243 MHz, D2O) δ -0.26 (2P), -10.84 (2P), -22.09 (1P) ppm. HR-ESI-MS calcd. for C 55 H 70 N 21 O 34 P5, 861.6546 [M - 2H] 2-; found 861.6557 Nb-7mGpppGUG (sodium salt, ε 260 = 46,400 M -1 ·cm -1 , 31.5% yield) (13): 1 H NMR (400 MHz, D2O) δ 8.15 - 7.93 (m, 2H), 7.72 (s, 1H), 7.63 - 7.18 (m, 4H), 5.90 - 5.59 (m, 5H), 4.97 (d, J = 14.5 Hz, 3H), 4.73-4.63 (m, 3H), 4.58 - 3.80 (m, 20H), 3.53 - 3.30 (m, 6H), 0.72 - 0.31 (m, 9H) ppm. 13 C NMR (101 MHz, D2O) δ 165.39, 158.51, 156.35, 153.86, 153.65, 151.53, 151.16, 149.56, 149.33, 148.86, 140.34, 137.35, 136.81, 133.45, 132.56, 132.05, 129.86, 128.72, 128.09, 123.75, 123.37, 116.20, 115.72, 107.76, 107.44, 102.64, 94.32, 93.54, 87.64, 87.20, 86.18, 84.43, 83.54, 82.13, 81.64, 80.34, 79.50, 78.96, 73.37, 72.77, 72.00, 70.44, 68.12, 65.35, 64.38, 57.78, 36.18, 35.67, 30.26, 30.22, 24.76 ppm. 31 P NMR (162 MHz, D2O) δ -0.27 (2P), -10.82 (2P), -22.06 (1P) ppm. ESI-TOF-MS calcd. for C 54 H 70 N 18 O 36 P5, 1701.28937 [M - H] - ; found 1701.28013.

[0205] Nb-7mGpppGCG(Nb-7mGpppGCG.ε 260 = 43,100 M -1 ・cm -1 、11.6% yield)(14): 1 H NMR (400 MHz, D2O) δ 9.21–8.73(m,1H), 7.89(s,2H), 7.72–7.22(m,5H), 5.80(s,2H), 5.57(s,1.5H), 5.43(s,1.5H), 5.02(d,J= 16.2 Hz, 3H), 4.68 (d, J = 14.2 Hz, 3H), 4.61 - 3.90 (m, 20H), 3.61 (t, J= 13.8 Hz, 6H), 0.59 (q, J = 12.7 Hz, 9H) ppm. 13 C NMR (151 MHz, D2O) δ 164.95, 158.94, 158.48, 155.47, 155.22, 154.66, 154.54; 148.83, 148.52, 139.21, 136.70, 133.92, 132.75, 132.26, 129.96, 128.78, 128.08, 123.84; 107.62, 107.34, 95.34, 93.60, 88.29, 87.59, 85.68, 81.59, 80.68, 73.97, 70.40, 69.38, 68.82, 68.34, 65.11, 64.15. 57.53, 36.41, 36.19, 35.77, 30.28, 25.20, 24.80, 24.76 and 24.72 ppm. 31 P NMR (162 MHz, D2O) δ -0.58(2P), -10.85(2P), -22.17(1P) ppm. ESI-TOF-MS calcd. for C 54 H 71 N 19 O 35 P5, 1700.40535 [M-H] -; found 1700.36111.

[0206] Nb-7mGpppGAG (sodium salt, ε 260 = 49,900 M -1 ・cm -1 , 32.3% yield) (15): 1 H NMR (400 MHz, D2O) δ 9.08 - 8.68 (m, 0.5H), 8.18 (d, J = 8.0 Hz, 1H), 7.98 - 7.73 (m, 3H), 7.42 - 6.99 (m, 4H), 5.86 (d, J = 6.4 Hz, 1H), 5.68 - 5.38 (m, 3.5H), 4.86 (s, 2H), 4.75 (d, J = 8.1 Hz, 3.5H), 4.56 (s, 2H), 4.42 - 4.24 (m, 4H), 4.23 - 3.97 (m, 10H), 3.93 - 3.77 (m, 4.5H), 3.45 - 3.16 (m, 6H), 0.49 - 0.10 (m, 9H) ppm. 13 C NMR (101 MHz, D2O) δ 158.33, 155.12, 154.17, 153.71, 153.51, 151.46, 151.22, 148.79, 139.09, 137.08, 133.22, 129.62, 118.27, 116.04, 115.55, 107.43, 107.12, 87.99, 87.34, 85.60, 83.36, 82.84, 80.93, 73.37, 70.08, 67.82, 65.03, 58.05, 57.91, 36.14, 36.01, 35.64, 30.26, 24.81, 24.72 ppm. 31 P NMR (159 MHz, D2O) δ -0.28 (2P), -10.82 (2P), -22.11 (1P) ppm. ESI-TOF-MS calcd. for C 55 H 71 N 21 O 34 P5, 1724.31659 [M - H] - ; found 1724.31732.

[0207] Nb-7mGpppGGG(Nb-7mGpppGGGε 260 = 46,800 M -1 ・cm -1 、28.8% yield)(16): 1 H NMR (600 MHz, D2O) δ 8.20 (s, 0.5H), 7.91 (s, 3H), 7.26 (s, 4.5H), 6.12 (s, 2H), 5.96 (s, 0.5H), 5.77 (s, 1.5H), 5.12 (d, J = 1.9 Hz, 1.5H), 4.85(s,1.5H), 4.67-3.65(m,29H), 0.15(s,9H) ppm. 13 C NMR (151 MHz, D2O) δ 160.45, 159.60, 155.75–147.96, 136.37–127.56, 123.60, 116.54–113.91; 83.43, 81.62-78.68, 75.72, 71.70, 69.97, 69.59, 68.54-67.97, 67.30, 65.70; 57.92-56.88, 36.05, 35.44, 30.35, 24.78 ppm. 31 P NMR (243 MHz, D2O) δ 0.91--2.52(2P), -10.37(2P), -21.96(1P) ppm. ESI-TOF-MS calcd. for C 55 H 71 N 21 O 35 P5, 1740.31150 [M-H] - ; found 1740.31166.

[0208] Nb-7mGpppUUA(Unclean.ε 260 = 49,400 M -1 ・cm -1 7.39% yield)(17) 1H NMR (600 MHz, D2O) δ 8.44 (d, J = 4.7 Hz, 1H), 8.19 (d, J = 6.7 Hz, 1H), 7.90 - 7.84 (m, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.69 (t, J = 6.8 Hz, 0.5H), 7.62 (t, J = 6.9 Hz, 0.5H), 7.57 (d, J = 7.8 Hz, 1H), 7.53 (d, J = 5.4 Hz, 1H), 7.44 (d, J = 6.2 Hz, 0.5H), 7.31 (d, J = 6.5 Hz, 0.5H), 6.08 (t, J = 6.0 Hz, 1H), 6.00 (d, J = 5.9 Hz, 0.5H), 5.91 (dd, J = 16.9, 5.7 Hz, 2H), 5.82 (q, J = 7.1 Hz, 2.5H), 5.08 (d, J = 6.9 Hz, 1H), 5.01 (q, J = 7.1 Hz, 2H), 4.94 (d, J = 6.3 Hz, 1H), 4.63 (d, J = 17.8 Hz, 2H), 4.53 - 4.50 (m, 1H), 4.46 (s, 1H), 4.42 (s, 1H), 4.37 (s, 1H), 4.31 (s, 4H), 4.23 (s, 0.5H), 4.18 (s, 4H), 4.11 (d, J = 6.6 Hz, 1H), 4.06 (d, J = 6.5 Hz, 6.5H), 3.45 - 3.41 (m, 6H), 0.67 (d, J= 6.6 Hz, 5.5H), 0.60 (d, J = 6.5 Hz, 3.5H) ppm. 13C NMR (151 MHz, D2O) δ 165.72, 155.57, 152.91, 151.39, 149.00, 148.23, 141.19; 129.98, 127.84, 123.45, 108.43, 102.86, 102.65, 95.42, 87.34, 86.52, 86.00, 83.59, 82.28, 81.52, 81.26, 79.13, 72.51, 72.01, 70.33, 69.16, 65.10, 64.32, 57.91, 57.73, 36.53, 35.96, 30.28, 24.66 ppm. 31 P NMR (243 MHz, D2O) δ-0.53(2s,2P),-10.68--11.20(m,2P),-22.14(1P) ppm. ESI-TOF-MS calcd. for C 53 H 69 N 15 O 36 P5, 1646.2732 [M-H] - ; found 1646.44951.

[0209] Nb-7mGpppCUA(GpppCUA.ε 260 = 46,000 M -1 ・cm -1 、38.9% yield)(18): 1H NMR (600 MHz, D2O) δ 8.40 (d, J = 11.7 Hz, 1H), 8.10 (d, J = 14.2 Hz, 1H), 7.77 (s, 2.5H), 7.66 - 7.50 (m, 2.5H), 7.46 - 7.30 (m, 1H), 6.07 - 5.94 (m, 2.5H), 5.91 - 5.82 (m, 1.5H), 5.68 (s, 1H), 5.58 (s, 1H), 5.13 - 5.02 (m, 2H), 4.96 (s, 1H), 4.88 (s, 2H), 4.67 - 4.57 (m, 2H), 4.55 - 4.44 (m, 3H), 4.39 - 4.25 (m, 6H), 4.21 - 4.02 (m, 9H), 3.94 (s, 1H), 3.57 - 3.29 (m, 6H), 0.75 - 0.53 (m, 9H) ppm. 13 C NMR (151 MHz, D2O) δ 165.79, 165.31, 156.31, 156.01, 155.85, 155.32, 152.60, 150.72, 149.65, 149.44, 149.18, 148.76, 139.72, 139.37, 136.28, 133.49, 132.99, 132.73, 132.34, 130.14, 129.94, 128.77, 128.39, 123.94, 123.53, 118.46, 107.49, 102.12, 96.30, 95.03, 88.40, 87.31, 86.73, 83.47, 81.79, 81.41, 81.00, 79.53, 74.62, 70.99, 70.09, 67.90, 67.40, 64.82, 63.76, 63.21, 57.81, 57.28, 36.19, 35.85, 30.26, 24.99 ppm. 31 P NMR (243 MHz, D2O) δ -0.65 (2s, 2P), -10.73 (2P), -21.94 (1P) ppm. ESI-TOF-MS calcd. for C 53 H 70 N 16 O 35P5, 1645.28831 [M - H] - ; found 1645.28716.

[0210] Nb-7mGpppAUA (sodium salt, ε 260 = 53,800 M -1 ・cm -1 , 32.4% yield) (19): 1 H NMR (600 MHz, D2O) δ 8.55 - 8.30 (m, 2H), 8.21 - 8.02 (m, 2H), 7.76 (dd, J = 8.1, 3.4 Hz, 1H), 7.64 - 7.22 (m, 4H), 6.08 - 5.97 (m, 2H), 5.89 - 5.81 (m, 1.5H), 5.76 - 5.62 (m, 1.5H), 4.98 - 4.88 (m, 3H), 4.84 (s, 1H), 4.72 - 4.69 (m, 1H), 4.66 - 4.62 (m, 1.5H), 4.55 (d, J = 4.8 Hz, 1.5H), 4.48 (q, J = 7.4 Hz, 1.5H), 4.43 (d, J = 5.7 Hz, 1H), 4.38 - 4.32 (m, 4.5H), 4.25 (s, 2H), 4.22 - 4.17 (m, 2H), 4.17 - 4.10 (m, 3H), 4.06 - 3.97 (m, 4H), 3.43 (d, J = 4.8 Hz, 5.7H), 3.34 (d, J = 2.2 Hz, 0.3H), 0.58 - 0.46 (m, 9H) ppm. 13C NMR (151 MHz, D2O) δ 165.48; 140.00, 136.29, 133.54, 132.60, 132.27, 130.00, 129.82, 128.75, 128.16, 123.76, 123.35, 118.53, 118.13, 107.61 107.31, 102.54, 94.51, 93.49, 87.70, 87.55, 86.54, 84.85, 84.53, 83.57, 82.26, 82.14, 82.00, 81.61, 81.36, 80.26. 79.47, 79.03, 74.54, 72.90, 71.91, 70.23, 68.44, 68.13, 65.31, 65.05, 64.67, 64.33, 58.01, 57.94, 36.25, 36.13, 35.67, 30.32, 24.86, 24.78 ppm. 31 P NMR (243 MHz, D2O) δ -0.41(2P), -10.71(2P), -21.75(1P) ppm. ESI-TOF-MS calcd. for C 54 H 70 N 18 O 34 P5, 1669.29954 [M-H] - ; found 1669.29949.

[0211] Nb-7mGpppGUA(Groups.ε 260 = 51,000 M -1 ・cm -1 、24.3% yield)(20): 1H NMR (600 MHz, D2O) δ 8.24 (s, 1H), 8.00 - 7.96 (m, 1H), 7.96 - 7.88 (m, 1H), 7.63 (d, J = 8.2 Hz, 1H), 7.49 (d, J= 8.2 Hz, 0.5H), 7.42 (t, J = 9.8 Hz, 1H), 7.36 - 7.31 (m, 1.5H), 7.25 (d, J = 7.1 Hz, 0.5H), 7.16 (t, J = 7.8 Hz, 0.5H), 5.97 - 5.88 (m, 1H), 5.75 - 5.69 (m, 1.5H), 5.66 - 5.60 (m, 2H), 5.56 - 5.53 (m, 0.5H), 4.90 (s, 0.5H), 4.82 (t, J = 9.7 Hz, 2H), 4.76 (s, 1H), 4.60 - 4.56 (m, 2.5H), 4.42 (s, 0.5H), 4.40 - 4.37 (m, 2H), 4.33 (s, 1H), 4.29 (s, 0.5H), 4.25 - 4.22 (m, 1.5H), 4.18 (s, 1H), 4.13 - 4.02 (m, 7.5H), 3.94 - 3.86 (m, 6H), 3.34 - 3.29 (m, 6H), 0.47 - 0.38 (m, 9H) ppm. 13C NMR (151 MHz, D2O) δ 165.53, 158.49, 157.94, 155.43, 153.70, 152.75, 151.63, 151.26, 149.66, 149.40, 148.88, 148.76, 148.47, 140.58, 139.63, 136.95, 133.68, 132.69, 132.19, 129.93, 128.74, 127.97, 123.75, 123.43, 118.66, 115.83, 107.73, 102.58, 94.74, 93.49, 87.60, 87.38, 87.23, 86.31, 84.50, 83.54, 81.96, 81.59, 80.47, 79.76, 79.20, 74.30, 72.96, 71.98, 70.29, 68.67, 68.30, 65.50, 65.13, 64.90, 64.28, 57.89, 36.35, 36.04, 35.65, 30.29, 24.75, 24.71 ppm. 31 P NMR (243 MHz, D2O) δ -0.39 (2s, 2P), -10.73 (d, J = 19.7 Hz, 2P), -22.04 (1P) ppm. ESI-TOF-MS calcd. for C 54 H 70 N 18 O 35 P5, 1685.29445 [M-H] - ; found 1685.29855.

[0212] Synthesis of pentanucleotide PureCap analogues. Synthetic scheme.

[0213] Nb-7mGpppUUAG (sodium salt, ε 260 = 59,000 M -1 ·cm -1 , 29.2% yield) (21): 1H NMR (600 MHz, D2O) δ 8.36 (s, 1H), 8.12 (d, J = 3.1 Hz, 1H), 7.95 (s, 1H), 7.88 (dd, J = 16.5, 8.1 Hz, 1H), 7.80 (d, J = 8.2 Hz, 1H), 7.65 - 7.47 (m, 3H), 7.39 (t, J = 7.2 Hz, 0.5H), 7.29 (dt, J = 8.7, 4.1 Hz, 0.5H), 6.10 (d, J = 4.9 Hz, 1H), 6.02 (d, J = 5.4 Hz, 0.5H), 5.95 (t, J = 8.8 Hz, 1H), 5.89 - 5.81 (m, 4.5H), 5.05 (s, 0.5H), 4.98 (q, J= 7.5 Hz, 2H), 4.90 (s, 0.5H), 4.84 (d, J= 7.1 Hz, 1.5H), 4.71 (q, J = 6.8 Hz, 2.5H), 4.65 (t, J = 4.3 Hz, 0.5H), 4.60 (t, J = 5.3 Hz, 0l5H), 4.47 (tt, J = 14.9, 4.1 Hz, 5H), 4.33 (d, J = 15.4 Hz, 4.5H), 4.22 (d, J = 14.3 Hz, 5H), 4.14 (d, J = 13.3 Hz, 3H), 4.08 (d, J = 8.5 Hz, 4H), 3.99 (s, 1H), 3.51 (s, 3H), 3.44 (d, J= 4.2 Hz, 3H), 3.40 (s, 3H), 3.36 (s, 0.5H), 0.63 (s, 5H), 0.55 (s, 4H) ppm. 13C NMR (151 MHz, D2O) δ 166.08, 165.81, 161.46, 158.69, 155.43, 153.82, 152.90, 151.39, 149.88, 149.68, 149.34, 148.71, 148.32, 141.17, 140.87, 139.31, 137.40, 133.80, 132.73, 132.50, 132.03, 129.91, 128.75, 127.88, 123.69, 123.48, 118.67, 116.33, 108.60, 102.87, 102.69, 95.35, 93.75, 87.55, 87.12, 86.94, 86.48, 86.16, 85.47, 83.37, 82.78, 82.52, 82.16, 81.88, 81.37, 80.72, 79.90, 79.16, 73.59, 72.43, 72.26, 70.13, 69.15, 68.83, 65.04, 64.81, 64.66, 58.08, 57.78, 57.73, 36.46, 35.97, 35.65, 30.33, 24.73, 24.67, 23.37 ppm. 31 P NMR (241 MHz, D2O) δ -0.22 (s, 1P), -0.52 (d, J = 13.2 Hz, 2P), -10.75 (d, J = 19.7 Hz, 2P), -21.84 (q, J = 16.4 Hz, 1P) ppm. ESI-TOF-MS calcd. for C 64 H 81 N 20 O 43 P6, 667.77362 [M - 3H] 3- ; found 667.78314

[0214] Test Example 5-2. Synthesis of mRNA by co-transcription reaction using TetraPureCap analog Linear RNA was transcribed and synthesized using T7 RNA polymerase with double-stranded DNA as a template. The template double-stranded DNA was obtained by PCR amplification of pNL-TK1.1 (Promega) using KOD-Plus-Neo (TOYOBO) (1.0 ng / μL DNA vector, 1× KOD buffer, 0.2 mM dNTPs, 0.3 μM primers, 1.5 mM MgSO4, 0.02 units / μL polymerase). The primers used are as follows: Fw 1: CCCGGATCCTAATACGACTCACTATAGGCGCATATTAAGGTGACGCGT (SEQ ID NO: 24) Fw 2: CCCGGATCCTAATACGACTCACTATAAGGCGCATATTAAGGTGACGCGT (SEQ ID NO: 25) Fw 3: CCCGGATCCTAATACGACTCACTATATGGCGCATATTAAGGTGACGCGT (SEQ ID NO: 26) Fw 4: CCCGGATCCTAATACGACTCACTATACGGCGCATATTAAGGTGACGCGT (SEQ ID NO: 27) Fw 5: CCCGGATCCTAATACGACTCACTATAGGGCGCATATTAAGGTGACGCGT (SEQ ID NO: 28) Fw 6: CCCGGATCCTAATACGACTCACTATATCGGCGCATATTAAGGTGACGCGT (SEQ ID NO: 29) Fw 7: CCCGGATCCTAATACGACTCACTATATAGGCGCATATTAAGGTGACGCGT (SEQ ID NO: 30) Fw Fw 8; CCCGGATCCTAATACGACTCACTATATTGGCGCATATTAAGGTGACGCGT (SEQ ID NO: 31) Fw 9; CCCGGATCCTAATACGACTCACTATACCGGCGCATATTAAGGTGACGCGT (SEQ ID NO: 32) Fw 10; CCCGGATCCTAATACGACTCACTATACTGGCGCATATTAAGGTGACGCGT (SEQ ID NO: 33) Fw 11; CCCGGATCCTAATACGACTCACTATACAGGCGCATATTAAGGTGACGCGT (SEQ ID NO: 34) Fw 12; CCCGGATCCTAATACGACTCACTATAGTGGCGCATATTAAGGTGACGCGT (SEQ ID NO: 35) Fw 13; CCCGGATCCTAATACGACTCACTATAGCGGCGCATATTAAGGTGACGCGT (SEQ ID NO: 36) Fw 14; CCCGGATCCTAATACGACTCACTATAGAGGCGCATATTAAGGTGACGCGT (SEQ ID NO: 37) Fw 15; CCCGGATCCTAATACGACTCACTATAATGGCGCATATTAAGGTGACGCGT (SEQ ID NO: 38) Fw 16;CCCGGATCCTAATACGACTCACTATAACGGCGCATATTAAGGTGACGCGT (SEQ ID NO: 39) Fw 17; CCCGGATCCTAATACGACTCACTATAAAGGCGCATATTAAGGTGACGCGT (SEQ ID NO: 40) Fw 18; CCCGGATCCTAATACGACTCACTATTAGGCGCATATTAAGGTGACGCGT (SEQ ID NO: 41) Fw 19; CCCGGATCCTAATACGACTCACTATTTAGGCGCATATTAAGGTGACGCGT (SEQ ID NO: 42) Fw 20; CCCGGATCCTAATACGACTCACTATCTAGGCGCATATTAAGGTGACGCGT (SEQ ID NO: 43) Fw 21; CCCGGATCCTAATACGACTCACTATGTAGGCGCATATTAAGGTGACGCGT (SEQ ID NO: 44) Rev; TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTAGAATTACGCCAGAATGCG (SEQ ID NO: 45);

[0215] A transcription reaction mixture (template DNA 10 ng / μL, 1× T7 RNA Polymerase buffer (Takara) (40 mM Tris-HCl (pH 8.0), 8 mM MgCl2, 2 mM spermidine), 2.5 U / μL T7 RNA polymerase (Takara), 5 mM DTT, 2 mM ATP, 2 mM UTP, 2 mM CTP, 2 mM GTP, 0.5-2 mM TetraPureCap analog) was prepared and incubated at 37°C for 1 hour. Recombinant DNase (Takara) was added to the reaction mixture at 0.1 U / μL, and the mixture was incubated at 37°C for 30 minutes to degrade the template DNA.

[0216] After the reaction, an equal volume of 7.5 M LiCl was added to the IVT reaction mixture, and the mixture was incubated at -30°C for 30 minutes. The precipitate was then centrifuged at 15,000 rpm for 30 minutes. The precipitate was purified by reverse-phase HPLC. The purification conditions were as follows: column, YMC-TriartBioC4 (250 mm x 4.6 mm ID); solvent A, 100 mM triethylammonium acetate (pH 7.0), 5% acetonitrile; solvent B, 100 mM triethylammonium acetate (pH 7.0), 50% acetonitrile; linear gradient 10–30% solvent B (0–20 min); flow rate: 1 mL / min; detection wavelength, 260 nm; column temperature, 50°C.

[0217] The RNA solution was added to a transparent 96-well multiwell plate and illuminated with a MAX-305 light source (Asahi Spectroscopy) at 4 mW / cm 2 The sample was irradiated with 365 nm light at a light intensity of 1000 nm for 10 minutes. HPLC purification was performed using a YMC-TriartBioC4 column (250 mm x 4.6 mm ID); Solvent A: 100 mM triethylammonium acetate (pH 7.0), 5% acetonitrile; Solvent B: 100 mM triethylammonium acetate (pH 7.0), 50% acetonitrile; Linear gradient 10–30% Solvent B (0–20 min); Flow rate: 1 mL / min; Detection wavelength: 260 nm; Column temperature: 50°C.

[0218] Assessment of intracellular translation activity HeLa cells (RIKEN Cell Bank) were cultured in Dulbecco's modified Eagle's medium (DMEM; WAKO) supplemented with 10% fetal bovine serum (FBS; Invitrogen) at 37°C and 5% CO2. The day before transfection, HeLa cells (1.0 × 10) were seeded onto a 96-well plate. 4The next day, the medium was removed and replaced with 100 μL / well of Opti-MEM® (Thermo Fisher Scientific). 0.15 μL of Lipofectamine® MessengerMAX TM 5 ng of mRNA was diluted in 10 μL of Opti-MEM® and transfected into the cells. After 2 hours, the medium was replaced with Dulbecco's modified Eagle's medium (DMEM; WAKO) supplemented with 10% fetal bovine serum (FBS; Invitrogen). After 22 hours of incubation at 37°C, 20 μL / well of 1× Cell Lysis Buffer (Promega) was added to lyse the cells. Luminescence was measured using the Nano-Glo® luciferase assay® (Promega).

[0219] The results are shown in Figures 5 to 7. By inserting bases into the promoter so that the base sequence of the cap analog polynucleotide and the base sequence upstream from the transcription start point of the promoter are complementary in a specific positional relationship, we found that the ratio of the amount of capped RNA synthesized to the total amount of transcription (capping efficiency) was improved. Furthermore, we found that high translation efficiency could be achieved depending on the sequence of the cap analog polynucleotide.

[0220] Test Example 6. Translation Reaction Using Chemically Synthesized Circular mRNA Containing an m7G Cap Structure. The RNA oligonucleotides shown in Figure 8 were used as the starting material for the circular RNA. The RNA oligonucleotide sequences were synthesized using commercially available phosphoramidite compounds (Chemgene and Glen Research) using an automated nucleic acid synthesizer, NTS T8-A20R8NC (Nihon Techno Services), according to standard procedures. Branched structures were introduced using the Glen Research Asymmetric Doubler (Lev) Phosphoramidite (cat# 10-1981) reagent. After synthesis, the CPG solid support was added with a 1:1 mixture of 40% aqueous methylamine and 28% concentrated aqueous ammonia and heated at 65°C for 30 minutes to cleave the oligonucleotide from the support and deprotect the phosphate and base moieties. After drying under reduced pressure, the residue was dissolved in 1 M TBAF / THF solution and incubated at room temperature overnight to deprotect the TOM protecting group. After desalting using a NAP-25 gel filtration column (Cytiva), the RNA oligonucleotides were precipitated by alcohol precipitation. The oligonucleotides were then purified by reverse-phase HPLC. The purity and molecular weight of the target product were confirmed using a Q-TOF LC / MS system (Agilent Technologies). The 5'-terminal phosphate group of the branched chain was reacted with m7GDP imidazolide (Im-m7GDP) to introduce the m7G cap structure, as previously reported (ACS Chem Biol 2022, 17, 1308-1314). The capping reaction mixture (20 μM phosphorylated RNA oligonucleotide, 10 mM Im-m7GDP, 10 mM 2-nitroimidazole, 10 mM calcium chloride, DMSO solution) was heated at 55°C for 3 hours, after which the RNA was recovered by alcohol precipitation. The target RNA was then separated and purified by reverse-phase HPLC. The 5' ends of the oligonucleotide backbones were enzymatically phosphorylated using T4 polynucleotide kinase (Takara Bio) (8 μM oligo, 1 mM ATP, 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 5 mM DTT, 0.2 U / μL T4 PNK; incubated at 37°C for 1 hour).Two RNA oligonucleotide fragments prepared in this manner were ligated with T4 RNA ligase 2 in the presence of template DNA oligonucleotides to prepare circular RNAs. The template DNA oligonucleotides used had sequences complementary to the ends of the RNA strands to be ligated. The two types used to construct the 127-nt circRNA were 5' d(CACCATGGTGGCTCTGGACTAGGAG (SEQ ID NO: 52)) 3' and 5' d(TTTTTTTTTTTTTTTTTATCACAGTTTTTC (SEQ ID NO: 53)) 3'. The two types used to construct the 167-nt circRNA were 5' d(CACCGAGGCTCCAGCTTATCACAGTTTTTC (SEQ ID NO: 54)) 3' and 5' d(CACCATGGTGGCTCTGGACTAGGAG (SEQ ID NO: 55)) 3'. The composition of the ligase reaction solution is as follows: The reaction mixture consisted of 1 μM RNA oligonucleotides (two types), 3 μM DNA oligonucleotides (two types), 10% PEG8000, 50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1 mM DTT, 400 μM ATP, and 0.1 μg / μL T4 RNA ligase. The reaction mixture was incubated at 37°C for 2 hours, then extracted with an equal volume mixture of TE-saturated phenol and chloroform to deproteinize. Then, sodium acetate (pH 5.2, final concentration 0.3 M) and 2-propanol were added, cooled to -30°C, and centrifuged to recover the RNA precipitate. The progress of the reaction was monitored by denaturing acrylamide gel electrophoresis (PAGE), and the desired RNA ligates were isolated and purified by preparative denaturing polyacrylamide gel electrophoresis (Sci. Rep., 5, 16435, 2015). Experiments to evaluate the expression level of HiBiT peptide were performed using the human-derived cultured cell line HeLa (RIKEN Cell Bank, RCB0007). HeLa cells were cultured in DMEM (Wako Pure Chemical Industries) medium containing 10% FBS (Thermo Fisher Scientific) at 5% CO2 and 37°C. The day before transfection, HeLa cells were seeded at 1 x 10E4 cells per well onto a 96-well multiwell plate.Circular RNA was mixed with 0.3 μL of Lipofectamine MessengerMAX (Thermo Fisher Scientific) at 0.2 pmol or 2 pmol per well, incubated for 5 minutes, and then resuspended in Opti-MEM. TM These were added to wells containing HeLa cells whose growth medium had been replaced with I Reduced Serum Medium (Thermo Fisher Scientific), and RNA was transfected into the cells. After 3 hours, an equal volume (100 μL) of growth medium was added, and the cells were incubated for another 3 hours. The expression level of the HiBiT peptide in the cell lysate was assessed using the Nano Glo HiBiT Lytic Detection System (Promega). Chemiluminescence was measured using a TriStar5 multimode plate reader (Berthold).

[0221] The results are shown in Figures 8 and 9. It was found that peptides were produced with high efficiency from chemically synthesized circular mRNA containing the m7G cap structure.

[0222] Experimental Example 7: In vivo persistence of chemically synthesized circular mRNA containing m7G cap structure Experimental Section [Evaluation of in vivo expression of HiBiT peptide] Lipid nanoparticles encapsulating HiBiT-encoding mRNA (HiBiT-mRNA, sequence 59) were administered intravenously to mice, and the expression level of HiBiT peptide in the liver was measured. First, lipid nanoparticles encapsulating HiBiT-mRNA were administered intravenously to C57BL / 6N mice (5-week-old, female) at a dose of 10 μg of mRNA per mouse. Livers were harvested 6, 24, 30, or 48 hours after administration. Livers were minced with dissection scissors and homogenized by bead milling in lysis buffer (100 mM Tris-HCl, 1 mM EDTA, 0.1% Triton X-100, pH 7.8). The resulting homogenate was centrifuged (15,000 rpm, 4°C, 10 minutes), and the supernatant was collected. 30 μL of the supernatant was mixed with 60 μL of HiBiT substrate solution (Nano Glo HiBiT Lytic Detection System, Promega, N3040), and the luminescence (RLU) was measured using a luminometer. The supernatant was diluted 50-fold, and the total protein content (mg protein) was measured using the BCA protein assay. The luminescence value was corrected for the protein content (RLU / mg protein).

[0223] The results are shown in Figure 10. It was found that the chemically synthesized circular mRNA containing the m7G cap structure has a higher in vivo persistence than linear mRNA.

[0224] Test Example 8: Evaluation of translation activity of chemically synthesized circular mRNA containing an m7G cap structure The base sequence of the circular RNA used is as follows: m7G, N7-methylguanosine (cap structure linked via a 5'-5' triphosphate structure (-ppp-)); mG, 2'O-methylguanosine; Ψ, 1-methylpseudouridine; X, a branched structure derived from Asymmetric Doubler (Lev) Phosphoramidite (Glen Research).

[0225] Cap-linked circular mRNA (Cap-circ) (200-nt) 5'-GGCGCAUAUUAAGGUGACGCGUGUGGCCUCGAACACCGAGGGUA-X-AAGCCACCAUGGΨGAGCGGCΨGGCGGCΨGΨΨCAAGAAGAΨΨAGCGGGAGCΨCCGGΨAGΨAΨAAΨCAACΨΨΨΨGAAAAACΨGΨGAΨAAGCΨGGAGCCΨCGGΨGGCCAΨGCΨΨCΨΨGCCCCΨΨGGGCCΨAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3' The 5' and 3' ends are linked, forming a circular structure, and contain a branched substrand (5' m7G-ppp-mGmGAGCG-) branched from X (SEQ ID NO: 69).

[0226] HRV-B3-IRES contains circ mRNA (HRV-B3 circ / U) (816-nt) 5’GGUUAAAACAGCGGAUGGGUACCCCACCAUCCGACCCACUGGGUGUAGUACUCUGGUACUUCGUACCUUUGUACGCCUGUUCUUCCCAUUGUACCCUUCCUGAACUUCCAACCCAAGUAACGUUAGAAGCUCAACAUUUAGUACAACAGGAAGCACCACAUCCAGUGGUGUUUAGUACAAGCACUUCUGUUUCCCCGGAGCGAGGUAUAGGCUGUACCCACUGCCAAAAACCUUUAACCGUUAUCCGCCAACCAACUACGUAAAAGCUAGUAGUAUUAUGUUUUUAACUAGGCGUUCGAUCAGGUGGAUUUCCCCUCCACUAGUUUGGUCGAUGAGGCUAGGAAUUCCCCACGGGUGACCGUGUCCUAGCCUGCGUGGCGGCCAACCCAGCCCACUCACUAUUUGUUUUCGCGCCCAGUUGCAAAAAGUGUCGGGGCUGGGACGCCUUUUUAUAGACAUGGUGUGAAGACUCGCAUGUGCUUGGUUGUGAUUCCUCCGGCCCCUGAAUGCGGCUAACCUUAACCCUGGAGCCUUGUGUCACAAACCAGUGAUGAUAAGGUCGUAAUGAGCAAUUCCGGGACGGGACCGACUACUUUGGGUGUCCGUGUUUCUUAUUUUUCUUAUUAUUGUCUUAUGGUCACAGCAUAUAUAUAACAUAUACUGUGAUCAUGGUGAGCGGCUGGCGGCUGUUCAAGAAGAUUAGCGGGAGCUCCGGUAGUAUAAUCAACUUUGAAAAACUGUGAUAAGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3’. The 5’ end and the 3’ end are linked to form a circular structure (SEQ ID NO: 70).

[0227] HRV-B3-IRES Independent circ mRNA (HRV-B3 circ / m1Ψ) (816-nt) . 5'GGΨAAAACAGCGGAΨGGGΨACCCCCACCAΨCCGACCCACΨGGGΨGΨAGΨACΨCΨGGΨACΨΨCGΨACCΨΨΨGΨACGCCΨGΨΨCΨΨCCCAΨΨGΨACCCΨΨCC GAACΨCCAACCCAAGΨAACGΨGAGAGCΨCAACAΨΨΨAGΨACAACAGGAAGCACCACAΨCCAGΨGGΨGΨΨΨAGΨACAAGCACΨΨCΨGΨΨΨCCCCGGAGCG AGGCAAGGCΨGΨACCCACΨGCCAAAAACCΨΨAACCGΨΨAΨCCGCCAACCAACΨACGΨAAAAGCΨAGΨAGΨAΨΨAΨGΨΨΨΨΨAACΨAGGCGΨΨCGAΨCAGG GGAΨΨCCCCΨCCACΨAGΨΨΨGGΨCGAΨGAGGCΨAGGAAΨCCCCACGGGΨGACCGΨGΨCCΨAGCCΨGCGΨGGCGGCCAACCCAGCCCACΨCACΨAΨΨΨGΨΨ CGCGCCCAGΨΨGCAAAAAGΨGΨCGGGGCΨGGGACGCC AΨAGACAΨGGΨGΨGAAGACΨCGCAΨGΨGCΨΨGGΨΨGΨGAΨCCΨCCGGCCCCΨGAAΨG CGGCAACCΨΨAACCCΨGGAGCCΨΨGΨGΨCACAAACCAGΨGAΨGAΨAAGGΨCGΨAAΨGAGCAAΨCCGGGACGGGACCGACΨACΨΨΨGGGΨGΨCCGΨGΨΨC THE CACAGCAΨAΨAΨAΨAACAΨAΨACΨ GΨGAΨCAΨGGΨGAGCGGCΨGGCGGCΨGΨΨCAAGAAGAΨΨAGCGGGAGCΨCC GGΨAGΨAAΨCAACΨΨΨGAAAAACΨGΨGAΨAAGCΨGGAGCCΨCGGΨGGCCAΨGCΨΨCΨΨGCCCCΨΨGGGCCΨAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3' 5' walls and 3' walls are typically designed with snowflakes (St. 71).

[0228] Circular RNA was prepared as follows. First, 5'-phosphorylated linear RNA was prepared as the starting material for cyclization by transcription using the following composition: 5 ng / μL dsDNA (PCR product containing the T7 promoter), 2 mM ATP, 2 mM UTP or 1-methylpseudouridine 5'-triphosphate, 2 mM CTP, 2 mM GTP, 10 mM GMP, 40 mM Tris-HCl (pH 8.0), 8 mM MgCl2, 2 mM spermidine, 5 mM DTT, 0.002 U / μL pyrophosphatase, and 4.7 ng / μL T7 RNA polymerase. The reaction mixture was incubated at 37°C for 2 hours, followed by the addition of DNase I (Takara Bio) to a final concentration of 0.1 U / μL, and then incubated at 37°C for 15 minutes. The reaction mixture was extracted with an equal volume mixture of TE-saturated phenol and chloroform to remove proteins, and the transcribed RNA was recovered by alcohol precipitation. The resulting crude transcribed RNA was purified using reverse-phase HPLC as previously reported (Nat. Commun., 14, 2657, 2023). The resulting 5'-phosphorylated transcribed RNA was ligated using T4 RNA ligase 2 to obtain circular RNA. The composition of the ligase reaction solution is as follows: The reaction mixture contained 0.5 μM 5'-phosphorylated RNA, 1 μM template oligo DNA, 10% PEG8000, 50 mM Tris-HCl (pH 7.5), 2 mM magnesium chloride, 1 mM DTT, 400 μM ATP, and 0.1 μg / μL T4 RNA ligase. The reaction mixture was incubated at 37°C for 1 hour, then extracted with an equal volume mixture of TE-saturated phenol and chloroform to deproteinize. Sodium acetate (pH 5.2, final concentration 0.3 M) and 2-propanol were added, cooled to -30°C, and centrifuged to recover the RNA precipitate. The desired cyclized RNA was isolated and purified by preparative denaturing polyacrylamide gel electrophoresis.

[0229] Experiments evaluating HiBiT peptide expression levels were performed using the human-derived cultured cell line HeLa (RIKEN Cell Bank, RCB0007). HeLa cells were cultured in DMEM (Wako Pure Chemical Industries, Ltd.) containing 10% FBS (Thermo Fisher Scientific) at 37°C with 5% CO2. The day before transfection, HeLa cells were seeded at 1 x 10E4 cells per well in a 96-well multiwell plate. mRNA (20 ng / well) was mixed with 0.3 μL / well of Lipofectamine MessengerMAX (Thermo Fisher Scientific), incubated for 10 minutes, and then added to wells containing HeLa cells whose growth medium had been replaced with Opti-MEM™ I Reduced Serum Medium (Thermo Fisher Scientific). RNA was then introduced into the cells. After 3 hours, the supernatant was replaced with growth medium, and the cells were cultured for a total of 5, 10, 20, 30, and 45 hours. The expression level of the HiBiT peptide in the cells was then assessed using the Nano-Glo® HiBiT Lytic Detection System (Promega) and a TriStar5 multimode plate reader (Berthold).

[0230] The results are shown in Figure 11. We found that the chemically synthesized circular mRNA containing the m7G cap structure (branched cap-introduced circular mRNA) did not contain an IRES (approximately 600 nt), allowing for the minimization of its size and demonstrating high translation activity.

[0231] Test Example 9. Translation promotion of circular mRNA by hybridization with endogenous mRNA (ACTB) A conceptual diagram of this experiment is shown in FIG. 12(A).

[0232] The nucleic acid base sequences used in this experiment are as follows:

[0233] ACTB siRNA top strand: 5'_UACCUGUACACUGACUUGAGAUU_3' (sequence number 60).

[0234] ACTB siRNA bottom strand: 5'_UCUCAAGUCAGUGUACAGGUAUU_3' (sequence number 61).

[0235] NoAntisense Nluc circRNA (682-nt): 5'_GGUCCCAGGUCCACUUCAAGUAAACCCCUACCAACUGGUCGGGGUUUGAAAC<>_3' (5' and 3' ends are linked. The ACTB antisense Nluc circRNA below has the complementary sequence of ACTB mRNA inserted into the <> portion) (SEQ ID NO: 62).

[0236] ACTB antisense Nluc circRNA (705-nt): 5'__3' (The 5' and 3' ends are linked. The underlined portion indicates the complementary sequence to ACTB mRNA. All underlined nucleotides are natural for ACTB_native, 2'OMe-modified for ACTB_2'OMe, and LNA-modified for ACTB_LNA) (SEQ ID NO: 63).

[0237] NoAntisense splint (24-nt): 5'_dGAGGCCACACGCGTTTCAAACCCC_3' (SEQ ID NO: 64).

[0238] ACTB antisense splint 1 (24-nt): 5’_dCTCGCCTTTGCCGTTTCAAACCCC_3’ (SEQ ID NO: 65).

[0239] ACTB antisense splint 2 (24-nt): 5’_dGAGGCCACACGCGAGCACAGAGCC_3’ (SEQ ID NO: 66).

[0240] circRNA backbone (682 nt): 5’_phos-GCGUGUGGCCUCGAACACCGAGCGACCCUGCAGCGACCCGCUUAAAAGCUUGGCAAUCCGGUACUGUUGGUAAAGCCACCAUGGUCUUCACACUCGAAGAUUUCGUUGGGGACUGGCGACAGACAGCCGGCUACAACCUGGACCAAGUCCUUGAACAGGGAGGUGUGUCCAGUUUGUUUCAGAAUCUCGGGGUGUCCGUAACUCCGAUCCAAAGGAUUGUCCUGAGCGGUGAAAAUGGGCUGAAGAUCGACAUCCAUGUCAUCAUCCCGUAUGAAGGUCUGAGCGGCGACCAAAUGGGCCAGAUCGAAAAAAUUUUUAAGGUGGUGUACCCUGUGGAUGAUCAUCACUUUAAGGUGAUCCUGCACUAUGGCACACUGGUAAUCGACGGGGUUACGCCGAACAUGAUCGACUAUUUCGGACGGCCGUAUGAAGGCAUCGCCGUGUUCGACGGCAAAAAGAUCACUGUAACAGGGACCCUGUGGAACGGCAACAAAAUUAUCGACGAGCGCCUGAUCAACCCCGACGGCUCCCUGCUGUUCCGAGUAACCAUCAACGGAGUGACCGGCUGGCGGCUGUGCGAACGCAUUCUGGCGUAAUUCUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGUCCCAGGUCCACUUCAAGUAAACCCCUACCAACUGGUCGGGGUUUGAAAC_3’ (SEQ ID NO: 67).

[0241] ACTB antisense insert (47 nt): 5'_phos-GGGGTTTGAAACGGCAAAGGCGAGGCTCTGTGCTCGCGTGTGGCCTC_3' (The underlined portion indicates the complementary sequence to ACTB mRNA. Three types of underlined nucleotides were used: natural, 2'OMe-modified, and LNA-modified.) (SEQ ID NO: 68).

[0242] The circRNA backbone was transcribed using T7 RNA polymerase with the DNA encoding the sequence as a template. The transcription reaction mixture consisted of 10 ng / μL dsDNA (PCR product containing the T7 promoter), 2 mM ATP, 2 mM UTP, 2 mM CTP, 2 mM GTP, 10 mM GMP, 40 mM Tris-HCl (pH 8.0), 8 mM MgCl2, 2 mM spermidine, 5 mM DTT, 0.002 U / μL pyrophosphatase (New England Biolabs), and 2 U / μL T7 RNA polymerase (Takara Bio). The reaction mixture was incubated at 37°C for 2 hours. After the transcription reaction, DNase I (Takara Bio) was added to a final concentration of 0.1 U / μL and incubated at 37°C for 15 minutes. A 7.5 M aqueous solution of lithium chloride was added to the reaction mixture to a final concentration of 2.5 M. The mixture was cooled to -30°C for 30 minutes, and then centrifuged (20,000 × g, 20 minutes) to recover the transferred RNA as a precipitate. The resulting crude transferred RNA was purified by reverse-phase HPLC as previously reported (Nat. Commun. 14, 2657, 2023). For NoAntisense Nluc circRNA, 2 μM of the 5'-phosphorylated RNA prepared by the transcription reaction described above was mixed with 8 μM of NoAntisense splint. For ACTB antisense Nluc circRNA, 2 μM of the 5'-phosphorylated RNA, 8 μM of ACTB antisense splint 1, 8 μM of ACTB antisense splint 2, and 4 μM of ACTB antisense insert were mixed. Ligation was performed by treating the mixture at 37°C for 1 hour in the presence of 10% PEG8000, 50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1 mM DTT, 400 μM ATP, and 0.1 μg / μL T4 RNA ligase 2. The sample was then extracted with an equal volume mixture of TE-saturated phenol and chloroform to remove proteins, and an aqueous solution of sodium acetate (pH 5.2, final concentration 0.3 M) and 2-propanol were added. The sample was cooled to -30°C and centrifuged to recover RNA as a precipitate.The desired RNA cyclized product was isolated and purified by preparative denaturing polyacrylamide gel electrophoresis (Sci. Rep., 5, 16435, 2015).

[0243] Experiments to evaluate the expression level of Nluc protein were performed using the human-derived cultured cell line HeLa (RIKEN Cell Bank, RCB0007). HeLa cells were cultured in DMEM (Wako Pure Chemical Industries) containing 10% FBS (Thermo Fisher Scientific) at 5% CO2 and 37°C. The day before transfection, HeLa cells were seeded at 1 x 10E4 cells per well onto a 96-well multiwell plate.

[0244] For ACTB knockdown, 10 μM ACTB siRNA top strand, 10 μM ACTB siRNA bottom strand, 50 mM NaCl, and 40 mM Tris-HCl (pH 8.0) were incubated at 80°C for 5 minutes, followed by slow cooling to 15°C at -0.1°C / s. After replacing the medium in a 96-well multiwell plate containing HeLa cells prepared the day before, 1 pmol of siRNA was added to each well using Lipofectamine RNAiMAX (ThermoFisher) as a lipofection reagent and incubated at 37°C in 5% CO for 24 hours.

[0245] Twenty-four hours after siRNA addition, the medium was changed again, and then NoAntisense Nluc circRNA or ACTB antisense Nluc circRNA was added to the cells at 25 ng / well using Lipofectamine MessengerMax (ThermoFisher) as a lipofection reagent. The cells were then cultured at 37°C under 5% CO2 for 8 hours. For both Lipofectamine RNAiMAX and Lipofectamine Messenger Max, liposome formation was performed according to the manufacturer's protocol.

[0246] Eight hours after circRNA addition, each well was washed once with D-PBS (Nacalai) and then the expression level of Nluc in the cells was assessed using the Nano-Glo® Luciferase Assay System (Promega). Chemiluminescence was measured using a TriStar5 multimode plate reader (Berthold).

[0247] The results are shown in Figure 12. Hybridization with endogenous mRNA (ACTB) promoted translation of the circular mRNA.

Claims

1. A method for producing or expressing a protein or peptide, comprising carrying out a translation reaction in a reaction system containing a 5'-capped polynucleotide comprising an arbitrary base sequence A, a base sequence B capable of binding to said base sequence A through complementary base pairing, and a single-stranded RNA comprising a protein or peptide coding sequence, wherein said 5'-capped polynucleotide and / or said single-stranded RNA have a translation efficiency enhancing structure.

2. The production or expression method of claim 1, wherein the 5'-capped polynucleotide is a 5'-capped RNA.

3. The method of production or expression described in claim 1, wherein the 5'-capped polynucleotide or single-stranded RNA is an endogenous polynucleotide of a living organism or cell.

4. The production or expression method according to claim 3, wherein the biological or cellular polynucleotide is an lncRNA or an mRNA.

5. The method of production or expression described in claim 1, wherein the translation efficiency enhancing structure is a structure that stabilizes the bond between the 5'-capped polynucleotide and the single-stranded RNA.

6. The method of claim 1, wherein (b) the 5'-capped polynucleotide and / or the single-stranded RNA have a nuclease-binding structure and the reaction system contains the nuclease, the nuclease is RISC or Cas, and the 5'-capped polynucleotide is siRNA or miRNA, or the 5'-capped polynucleotide is lncRNA or mRNA and the single-stranded RNA includes a tracrRNA sequence.

7. The production or expression method according to claim 1, which satisfies at least one requirement selected from the group consisting of: (a) the single-stranded RNA is a circular RNA, and (c) the 5'-capped polynucleotide and / or the single-stranded RNA have a structure that covalently bonds the two.

8. The production or expression method according to claim 7, wherein the requirement (a) is met and the circular RNA does not contain a stop codon, and / or the requirement (c) is met and the structure is a photocrosslinkable structure.

9. A composition comprising a 5'-capped polynucleotide containing an arbitrary base sequence A, and / or a base sequence B capable of binding to the base sequence A through complementary base pairing, and a single-stranded RNA containing a protein or peptide coding sequence, wherein the 5'-capped polynucleotide and / or the single-stranded RNA has a translation efficiency-promoting structure.

10. A composition according to claim 9 for use in the production or expression method according to any one of claims 1 to 8.

11. A method for producing or expressing a protein or peptide, comprising carrying out a translation reaction in a reaction system containing a single-stranded circular RNA having a base length of 500 or less, in which a 5'-capped polynucleotide is linked via a linker.

12. A composition comprising a single-stranded circular RNA having a length of 500 or less, the single-stranded circular RNA being linked to a 5'-capped polynucleotide via a linker.

13. A composition according to claim 12 for use in the production or expression method according to claim 11.

14. A method for producing a 5'-capped polynucleotide, comprising carrying out a transcription reaction in a reaction system containing a double-stranded polynucleotide comprising a phage promoter having a base insertion mutation sequence adjacent to the upstream side of a transcription start point, and an antisense strand comprising a base sequence Y: (upstream side)-Y2-Y3 or (upstream side)-Y1-Y2-Y3 (in base sequence Y: Y1 and Y2 represent bases in the base insertion mutation sequence, and Y3 represents a base at the transcription start point), and a 5' cap analog having a polynucleotide comprising a base sequence X: (cap side)-X2-X3 or (cap side)-X1-X2-X3, wherein X3 is a complementary base of Y3, and X1 is a complementary base of Y1 and / or X2 is a complementary base of Y2.

15. The method of claim 14, wherein the antisense strand of the phage promoter comprises the base sequence Y: (upstream)-Y1-Y2-Y3, where X1 is a complementary base of Y1 and X2 is a complementary base of Y2.

16. The method of claim 14, wherein the 5'-capped polynucleotide is a 5'-capped RNA.

17. The method of claim 14, wherein the phage promoter is a T7 promoter.

18. The method according to claim 14, wherein the base sequence X is (cap side) -CUG, CAG, GUG, or GAG, and Y3 is C.

19. A composition comprising a double-stranded polynucleotide comprising a phage promoter having a base insertion mutation adjacent to the upstream side of the transcription start site, and an antisense strand comprising a base sequence Y: (upstream)-Y2-Y3 or (upstream)-Y1-Y2-Y3 (in base sequence Y: Y1 and Y2 represent bases in the base insertion mutation, and Y3 represents the base of the transcription start site), and / or a 5' cap analog having a polynucleotide comprising a base sequence X: (cap side)-X2-X3 or (cap side)-X1-X2-X3, wherein X3 is the complementary base of Y3, and X1 is the complementary base of Y1 and / or X2 is the complementary base of Y2.

20. The composition according to claim 19 for use in the manufacturing method according to any one of claims 14 to 18.

21. T7 RNA polymerase having the D130W mutation.

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