Method for producing protein or peptide, method for producing 5 '-cap polynucleotide, and reagents used in said production methods

Abstract

The purpose of the present invention is to further improve the efficiency of translation by means of a technique for translating a protein using RNA that encodes a protein and a capped polynucleotide that can bind thereto by complementary base pairing, and to improve the efficiency of capping for producing a 5 '-capped polynucleotide. This problem is solved by causing a 5 '-capped polynucleotide and / or the single-stranded RNA to have a translation efficiency promoting structure, and inserting a base into the promoter such that the base sequence of the polynucleotide of the cap analogue and the base sequence upstream from the transcription start point of the promoter complement each other in a specific positional relationship.

Description

Technical Field

[0001] This invention relates to methods for manufacturing proteins or peptides, methods for manufacturing 5′ capped polynucleotides, and reagents used in these manufacturing methods. Background Technology

[0002] In recent years, the use of mRNA vaccines has become increasingly widespread. The structure of an mRNA vaccine, starting from the 5′ end, consists of a cap region, a 5′ untranslated region, a protein-coding region, a 3′ untranslated region, and a PolyA tail region. A cap region is required for protein translation from an mRNA vaccine. However, in current mRNA synthesis technologies, the proportion of mRNA with a cap region relative to the total amount of mRNA (capping efficiency) is low, making the immune response triggered by non-capped mRNA a problem.

[0003] In this context, Non-Patent Literature 1 reports the use of a photosensitive purification tag as a cap analogue, in which the capped mRNA is isolated and purified by reversed-phase chromatography after transcription, and then the hydrophobic tag is removed by photoreaction, thereby achieving the production of capped mRNA with high purity.

[0004] Existing technical documents Non-patent literature Non-patent literature 1: Nat Commun. 2023 May 11;14(1):2657. doi: 10.1038 / s41467-023-38244-8. Summary of the Invention

[0005] The problem that the invention aims to solve Furthermore, translation efficiency is also important for improving the performance of mRNA vaccines. In their research, the inventors of this invention developed a technique for protein translation using protein-encoding RNA and capped RNA that can bind to it via complementary base pairing, with a focus on further improving the translation efficiency of this technique.

[0006] The objective of this invention is to further improve the translation efficiency of technologies for translating proteins or peptides using RNA encoding proteins or peptides and capped polynucleotides that can bind to them through complementary base pairing, and to improve the capping efficiency of manufacturing 5′ capped polynucleotides.

[0007] Technical solutions for solving the problem The inventors of this invention conducted in-depth research on the aforementioned issues and discovered that the structure of 5′ capped polynucleotides and / or single-stranded RNA (RNA encoding proteins or peptides) helps to improve translation efficiency. Furthermore, they found that by inserting bases into the promoter in a specific positional relationship where the base sequence of the capped polynucleotide is complementary to the base sequence upstream of the transcription start site of the promoter, capping efficiency is improved. Based on these findings, the inventors of this invention conducted further research, resulting in this invention. That is, this invention includes the following embodiments.

[0008] Item 1. A method for the manufacture or expression of a protein or peptide, comprising the step of carrying out a translation reaction in a reaction system, The reaction system contains: 5′ capped polynucleotides containing any base sequence A; and It contains a single-stranded RNA consisting of a base sequence B that can bind to the aforementioned base sequence A through complementary base pairing, and a protein or peptide coding sequence. The above-mentioned 5′ capped polynucleotides and / or the above-mentioned single-stranded RNAs have translation efficiency-enhancing structures.

[0009] Item 2. The manufacturing or expression method as described in Item 1, wherein the 5′ capped polynucleotide is a 5′ capped RNA.

[0010] Item 3. The manufacturing or expression method as described in Item 1 or 2, wherein the 5′ capped polynucleotide is an endogenous polynucleotide of an organism or cell.

[0011] Item 4. The manufacturing or expression method as described in Item 3, wherein the endogenous polynucleotide of the organism or cell is lncRNA or mRNA.

[0012] Item 5. The manufacturing or expression method as described in any one of items 1 to 4, wherein the translation efficiency promoting structure is a stabilizing structure that stabilizes the binding of the 5′ capped polynucleotide to the single-stranded RNA.

[0013] Item 6. The manufacturing or expression method as described in any one of items 1 to 5, wherein (b) the above-mentioned 5′ capped polynucleotide and / or the above-mentioned single-stranded RNA has a nuclease-binding structure, and the above-mentioned reaction system contains the above-mentioned nuclease, The above-mentioned nucleases are RISC or Cas, and The aforementioned 5′ capped polynucleotide is siRNA or miRNA, or The 5′ capped polynucleotides mentioned above are lncRNAs or mRNAs, and the single-stranded RNAs mentioned above contain tracrRNA sequences.

[0014] Item 7. A manufacturing or expression method as described in any one of items 1 to 6, which satisfies at least one condition selected from (a) and (c). (a) The above single-stranded RNA is a circular RNA. (c) The above-mentioned 5′ capped polynucleotide and / or the above-mentioned single-stranded RNA have a structure that allows the two to be covalently linked.

[0015] Item 8. The manufacturing or expression method as described in Item 7, which satisfies condition (a) above, and the circular RNA does not contain a stop codon, and / or The above condition (c) is met, and the above structure is a photocrosslinking structure.

[0016] Item 9. A composition comprising: 5′ capped polynucleotides containing any base sequence A; and / or It contains a single-stranded RNA consisting of a base sequence B that can bind to the aforementioned base sequence A through complementary base pairing, and a protein or peptide coding sequence. 5′ capped polynucleotides and / or the above-mentioned single-stranded RNAs have translation efficiency-enhancing structures.

[0017] Item 10. The composition as described in Item 9, used in any of the manufacturing or expression methods described in Items 1 to 8.

[0018] Item 11. A method for the manufacture or expression of a protein or peptide, comprising the step of carrying out a translation reaction in a reaction system, The above reaction system contains single-stranded circular RNA with a base length of less than 500, formed by linking 5′ capped polynucleotides via a linker.

[0019] Item 12. A composition containing a single-stranded circular RNA of less than 500 bases, formed by linking 5′ capped polynucleotides via a linker.

[0020] Item 13. The composition as described in Item 12, used in the manufacturing or expression method described in Item 11.

[0021] Item 14. A method for producing a 5′ capped polynucleotide, comprising the step of carrying out a translation reaction in a reaction system thereof, The above reaction system contains: A double-stranded polynucleotide containing a phage promoter adjacent to the upstream side of the transcription start site and having a base insertion mutation sequence, and whose antisense strand contains the base sequence Y: (upstream side)-Y2-Y3 or (upstream side)-Y1-Y2-Y3 (where Y1 and Y2 represent bases within the base insertion mutation sequence, and Y3 represents the base at the transcription start site); and 5′ cap analogs containing a polynucleotide containing the base sequence X: (cap side)-X2-X3 or (cap side)-X1-X2-X3 X3 is the complementary base of Y3, and X1 is a complementary base of Y1 and / or X2 is a complementary base of Y2.

[0022] Item 15. The manufacturing method as described in Item 14, wherein the antisense strand of the phage promoter comprises the base sequence Y: (upstream side) -Y1-Y2-Y3, X1 being the complementary base of Y1, and X2 being the complementary base of Y2.

[0023] Item 16. The manufacturing method as described in Item 14 or 15, wherein the 5′ capped polynucleotide is a 5′ capped RNA.

[0024] Item 17. The manufacturing method as described in any one of items 14 to 16, wherein the phage promoter is the T7 promoter.

[0025] Item 18. The manufacturing method as described in any one of items 14 to 17, wherein the base sequence X is (cap-side)-CUG, CAG, GUG or GAG, and Y3 is C.

[0026] Item 19. A composition comprising: The phage promoter containing a base insertion mutation sequence adjacent to the upstream side of the transcription start site, and the antisense strand containing the base sequence Y: (upstream side)-Y2-Y3 or (upstream side)-Y1-Y2-Y3 (where Y1 and Y2 represent bases within the base insertion mutation sequence, and Y3 represents the base at the transcription start site), and / or a double-stranded polynucleotide. 5′ cap analogs containing a polynucleotide containing the base sequence X: (cap side)-X2-X3 or (cap side)-X1-X2-X3 X3 is the complementary base of Y3, and X1 is a complementary base of Y1 and / or X2 is a complementary base of Y2.

[0027] Item 20. The composition as described in Item 19, used in any one of Items 14 to 18 in the manufacturing method.

[0028] Item 21. A T7 RNA polymerase with the D130W mutation.

[0029] Invention Effects According to the present invention, techniques are provided for further improving the translation efficiency of protein or peptide translation by utilizing RNA encoding proteins or peptides and capped polynucleotides that can bind to them via complementary base pairing, and techniques are provided for improving the capping efficiency of 5′ capped polynucleotides. Attached Figure Description

[0030] Figure 1 This is a phenomenon that promotes the translation of circular mRNA using RNA interference mechanisms. (a) Conceptual diagram of this experiment. A short double-stranded RNA (capped siRNA) with an m7G cap structure at the 3′ end of the guide strand is incorporated into the RISC complex, where it largely binds to the circular mRNA containing the complementary sequence. In the resulting RNA double-stranded structure, there is a non-complementary region in the central portion of the guide strand, thus not triggering the mRNA cleavage response. The m7G cap structure at the end of the guide strand recruits ribosomes to the vicinity of the start codon of the circular mRNA, thereby 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) Predicted double-stranded structure of the guide strand and mRNA to form in the RISC complex. (e) shows the experimental results demonstrating that capped siRNA from human cultured cells (HeLa) can promote the translation of circular mRNA in a dose-dependent manner. Circular RNA and siRNA molecules were introduced into HeLa cells using a commercially available lipid transfection reagent. After culturing for 24 hours, the cells were lysed, and the amount of translational product (NanoLuc luciferase, Nluc) in the lysate was evaluated, representing the experimental results.

[0031] Figure 2This study utilizes the translation-enhancing effect of hybridization with long non-coding RNA (lncRNA) and the stabilization of its dCas13 protein by circular mRNA. (a) and (b) show the translational activity evaluation experiments in HeLa cells using non-capped linear mRNA encoding NanoLuc luciferase (Nluc) (using a linear mRNA model experiment). The mRNA was introduced into HeLa cells using a commercially available lipid transfection reagent, or separately mixed with the lipid transfection reagent, either HULC RNA alone or HULC RNA and dRfxCas13d mRNA separately mixed with the lipid transfection reagent and then introduced. Protein expression from non-capped linear Nluc mRNA was increased with the addition of HULC RNA alone or in combination with dRfxCas13d mRNA. Changing the sequence corresponding to the linker portion between the 5′ cap structure and the hybridization region to 5, 10, and 15 bases in length did not show significant differences; the same increasing tendency was observed in all cases. (c) In HeLa cells, interaction with HULC lncRNA promotes the translation of circular Nluc mRNA. This phenomenon is 5′ cap structure dependent on HULC lncRNA. (d) In HeLa cells, interaction with UCA1 lncRNA promotes the translation of circular Nluc mRNA. This phenomenon is 5′ cap structure dependent on UCA1 lncRNA.

[0032] Figure 3 This describes the phenomenon that photocrosslinked capped RNA probes promote the translation of circular mRNAs in a photodependent manner. (a) is a conceptual diagram. Capped RNA probes containing trioxsalen can form covalent bonds with complementary strands through photocrosslinking. This stabilizes the binding of the capped probe to circular mRNA, enhancing its translation-promoting effect. (b) A model experiment using short-chain RNA oligonucleotides consisting of 24 bases as probe targets was conducted as a model of circular mRNA. The crosslinked reaction formed a stable complex in denaturing polyacrylamide gel electrophoresis. It was found that when 15 molar equivalents of the probe were added to the mRNA model, the mRNA model formed covalent bonds with the probe in a essentially quantitative manner. (c) After annealing a 650-base circular mRNA encoding Nluc with the capped probe, it was irradiated with 365 nm light. It was found that when introduced into HeLa cells, the translation-promoting effect of the photocrosslinked capped probe was improved compared to the control group.

[0033] Figure 4This demonstrates the use of capped RNA to promote rolling circle translation. (a) Conceptual diagram of this experiment. Rolling circle translation is promoted by binding capped RNA to circular RNA that does not have a stop codon. (b) Schematic diagram of the circular mRNA sequence used. (c) Experimental results showing the use of capped oligoRNA probes in rabbit reticulocyte lysate (RRL) to promote rolling circle translation. Circular RNA annealed to the probe was translated in RRL, and the translation products were detected by Western blotting using FLAG antibodies. A significant increase in translation products was confirmed by rolling ribosomes multiple times on the circular RNA in the presence of capped oligoRNA probes containing complementary regions of the circular RNA sequence.

[0034] Figure 5 This describes the development of a highly efficient method for synthesizing Cap-2 mRNA using TetraPureCap analogs and mutant promoters. (a) Structure and sequence of the TetraPureCap-XYG series analogs and the sequence of the mutant promoter of T7 RNA polymerase. (b) Structure and sequence of the TetraPureCap-XYA series analogs and the sequence of the mutant promoter of T7 RNA polymerase. (c) Experimental results showing the increased incorporation efficiency of TetraPureCap analogs through transcription using template DNA with a mutant promoter. With the native promoter sequence, the incorporation efficiency of the cap analogs is approximately 30% due to competition with GTP; however, by inserting a sequence complementary to the cap analog, thermodynamic stability is improved, and the incorporation efficiency of the cap analogs is increased to 80–90%.

[0035] Figure 6 This involves the development of a highly efficient method for synthesizing Cap-2 mRNA using a TetraPureCap analog and a mutant promoter. (d) Quantitative results of total transcription and capped RNA synthesis using the combination of the TetraPureCap analog and the mutant promoter, obtained by HPLC analysis.

[0036] Figure 7This section describes the development of a highly efficient method for synthesizing Cap-2 mRNA using TetraPureCap analogs and mutant promoters. (e) shows the experimental results demonstrating that, by utilizing the complementarity of TetraPureCap analogs and promoters, the incorporation efficiency of the analog can be maintained during transcription even at concentrations below NTPs (2 mM). (f) shows the experimental results of a mutant enzyme of T7 RNA polymerase (D130W) designed and synthesized to improve the incorporation efficiency of TetraPureCap analogs. Even using template DNA with a native promoter sequence, D130W successfully improved the incorporation efficiency of the cap analog. (g) shows the experimental data on the translational activity of cap-2 mRNA synthesized using TetraPureCap analogs. A difference of approximately 3-fold in translational activity was observed through the 5′ end base sequence. (h) PAGE analysis data confirming the purity of the mRNA used in the translational activity experiments.

[0037] Figure 8 This diagram shows the experimental results demonstrating the translational activity of a small circular mRNA (circRNA; with a ring size of 127 and 167 bases) constructed from two chemically synthesized oligonucleotide fragments in human cell lines. (a) is a diagram showing the chemically synthesized RNA oligonucleotide sequences used to prepare the small circular mRNA, with calculated and measured molecular weights of the chemically synthesized fragments. Both show high agreement. Bold text in the F87 sequence indicates the coding region of the peptide (translation product peptide sequence, MVSGWRLFKKISGSSGSIINFEKL (Sequence No. 51)). In the diagram, the base sequence of F87 is indicated by sequence No. 46, the base sequence of F40c by sequences Nos. 47 and 48, and the base sequence of F80c by sequences Nos. 49 and 50.

[0038] Figure 9(a) Experimental results showing the translational activity of small circular mRNAs (circRNAs; rings of 127 and 167 bases) constructed from two chemically synthesized oligonucleotide fragments containing an m7G cap in human cell lines. (b) Conceptual diagram of the method for constructing circRNAs by enzymatic ligation of the substrate RNA oligonucleotides shown in (a). The diagram illustrates the relationship between the combination of raw material sequences and the products. (c) Results of the evaluation of the translational activity of the prepared circRNAs in cultured human cells (HeLa). RNA was introduced into HeLa cells using commercially available transfection reagents, and the amount of HiBiT peptide encoded by the RNA was measured. It was found that the 127-nt and 167-nt circular RNAs produced peptides as translational products. Mean values ​​(n=4) and standard errors are presented as error bars.

[0039] Figure 10 This section shows the test results for Example 7. The top section describes the experimental protocol, the left side of the bottom section shows the base sequence of the nucleic acid used, and the right side of the bottom section shows a graph of the test results. In the graph, the legend indicates the shape of the chemically synthesized mRNA used.

[0040] Figure 11 This section shows the test results for Example 8. The upper left section shows a schematic diagram of the mRNA used, the right section shows the denaturing electrophoresis results of the mRNA used, and the lower left section shows a graph of the test results.

[0041] Figure 12This study investigated the translation-enhancing effect of circular mRNAs obtained through hybridization with endogenous mRNA (ACTB). (a) Evaluation of translational activity of uncapped circular mRNAs (circRNAs) encoding NanoLuc luciferase (Nluc) in HeLa cells. Antisense sequences of human ACTB mRNA were introduced into the antisense sequence region. (b) Introduction of ACTB siRNA into HeLa cells using a commercially available lipid transfection reagent reduced ACTB mRNA levels to nearly half after 24 h. Nluc expression was evaluated 8 h after circRNA introduction. (c) Compared to cells without ACTB mRNA (NoAntisense), circRNAs containing antisense sequences of ACTB mRNA (ACTB_native, ACTB_2′OMe, ACTB_LNA) showed increased translation levels, particularly those circRNAs modified with 2′OMe or LNA in the fully antisense sequence region (ACTB_2′OMe, ACTB_LNA), which showed a significant increase in translation. Furthermore, when ACTB mRNA was knocked down using siRNA, no effect was observed in circRNAs without antisense sequences, but the translation level was reduced to nearly half in circRNAs containing antisense sequences of ACTB mRNA. Detailed Implementation

[0042] In this specification, expressions of "containing" and "including" include the concepts of "containing", "comprising", "substantially constituted by", and "consisting solely of".

[0043] In this specification, there are no particular limitations on polynucleotides. Besides DNA and RNA, known chemical modifications can be applied as illustrated below. To prevent degradation by hydrolytic enzymes such as nucleases, the phosphate residues (phosphate esters) of each nucleotide can be replaced with chemically modified phosphate residues such as thiophosphate (PS), methylphosphonate, or dithiophosphate. Additionally, the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide can be replaced with -OR (R, for example, represents CH3(2′-O-Me), CH2CH2OCH3(2′-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, CH2CH2CN, etc.). Furthermore, chemical modifications can be applied to the base moiety (pyrimidine, purine). For example, methyl or cationic functional groups can be introduced at the 5-position of the pyrimidine base, or the carbonyl group at the 2-position can be replaced with a thiocarbonyl group. In addition, examples such as polynucleotides modified with biotin, amino, lower alkylamine, acetyl, etc., can be listed, but are not limited to these.

[0044] In this specification, polynucleotides may be linked to other molecules. Examples of other molecules include fluorescent markers. Examples of fluorescent markers include fluorescein, rhodamine, Texas red, tetramethylrhodamine, carboxyrhodamine, phycoerythrin, 6-FAM (trademark), Cy (registered trademark) 3, Cy (registered trademark) 5, and the Alexa Fluor (registered trademark) series.

[0045] In this specification, the bases constituting nucleic acids include not only typical bases found in natural nucleic acids such as RNA and DNA (adenine (A), thymine (T), uracil (U), guanine (G), cytosine (C), etc.), but also bases other than these, such as hypoxanthine (I) and modified bases. Examples of 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, and 1-methylhypoxanthine. Purines, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyladenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-methoxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, 2-thiocytosine, purines, 2-aminopurines, isoguanine, indole, imidazole, xanthine, cyanuric acid, etc.

[0046] 1. Methods for the manufacture or expression of proteins or peptides. This item corresponds to items 1 to 7 above and test examples 1 to 4 described below. Figures 1-4 However, the present invention is not limited to the experimental examples described later.

[0047] In one aspect, this invention relates to a method for manufacturing or expressing a protein or peptide (in this specification, it is sometimes referred to as "the method for manufacturing or expressing the present invention"), comprising the step of performing a translation reaction in a reaction system containing: a 5′-capped polynucleotide comprising any base sequence A; and a single-stranded RNA comprising a base sequence B capable of binding to the base sequence A via complementary base pairing, and a protein or peptide coding sequence, wherein the 5′-capped polynucleotide and / or the single-stranded RNA have translation efficiency-enhancing structures. This will be described below.

[0048] The 5′ capped polynucleotide used in the manufacturing or expression method of the present invention is not particularly limited as long as it has a 5′ cap structure and can bind to single-stranded RNA through complementary base pairing. RNA is particularly preferred as the 5′ capped polynucleotide. There are no particular limitations on the 5′ cap structure; for example, cap-0, cap-1, cap-2, etc., can be listed.

[0049] 5′ capped polynucleotides can be single-stranded or double-stranded.

[0050] The single-stranded RNA used in the manufacturing or expression method of the present invention contains a protein or peptide coding sequence. This protein or peptide coding sequence is the coding sequence of the target protein or peptide manufactured or expressed by the manufacturing or expression method of the present invention. Examples of such proteins or peptides include cancer antigens or microbial antigens. Examples of cancer antigens include, for example, common antigens (tumor-associated antigens), differentiation antigens (gp100, MART-1, etc.), fetal proteins (CEA, AFP, etc.), glycoprotein / glycolipid / glycan antigens (MUC-1, CA125, etc.), overexpressed proteins (HER2, Survivin, WT-1, etc.); examples of intrinsic antigens (neoantigens) include cancer antigens originating from driver mutations (KRAS, BRAF, EGFR, etc.) and cancer antigens originating from transient mutations (patient-specific). Examples of microbial antigens include viral antigens and bacterial antigens. There are no particular restrictions on the viruses that can be used as sources of viral antigens. Examples include influenza viruses (such as type A and type B), rubella virus, Ebola virus, coronavirus, measles virus, varicella-zoster virus, herpes simplex virus, mumps virus, arbovirus, RS virus, SARS virus, hepatitis viruses (such as hepatitis B virus and hepatitis C virus), yellow fever virus, HIV, rabies virus, Hantavirus, dengue virus, Nipah virus, Lyssa virus, etc., enveloped viruses; and non-enveloped viruses such as adenovirus, norovirus, rotavirus, human papillomavirus, poliovirus, enterovirus, Coxsackievirus, human parvovirus, encephalomyelitis virus, and rhinovirus. There are no particular restrictions on the types of bacteria that can be used as sources of bacterial antigens. Examples include Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae, Salmonella enterica, Helicobacter pylori, Clostridium perfringens, Clostridium botulinum, Campylobacter, Escherichia coli, Staphylococcus aureus, Streptococcus, Bacillus cereus, Vibrio parahaemolyticus, Propionibacterium acnes, Enterococcus faecalis, Clostridium difficile, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Klebsiella pneumoniae, Corynebacterium, hemolytic streptococci, Pseudomonas aeruginosa, Staphylococcus, Mycoplasma, Candida albicans, Aspergillus, etc.

[0051] The 5′ capped polynucleotide contains any base sequence A, and the single-stranded RNA contains a base sequence B that can bind to base sequence A through complementary base pairing. Through base sequences A and B, the 5′ capped polynucleotide can bind to the single-stranded RNA, thereby configuring the translation complex required for the translation of the single-stranded RNA. Furthermore, base sequences A and B are names used to distinguish the two; either B can be any base sequence and A can bind to base sequence B through complementary base pairing.

[0052] Complementary base pairing occurs through the complementarity of base sequences. "Complementary" includes not only complete complementarity (e.g., A with T or U, and G with C), but also the degree of complementarity required to hybridize under stringent conditions. These stringent conditions, as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA), can be determined based on the melting temperature (Tm) of the nucleic acid. For example, wash conditions after hybridization typically include "1×SSC, 0.1% SDS, 37°C". It is preferable to maintain hybridization even under such conditions. Without particular limitation, more stringent hybridization conditions include "0.5×SSC, 0.1% SDS, 42°C", and even more stringent wash conditions include "0.1×SSC, 0.1% SDS, 65°C". Specifically, the base sequence B is a base sequence that has, for example, 85% or more identity with a base sequence that is completely complementary to the base sequence A, preferably 90% or more identity, more preferably 95% or more identity, further preferably 98% or more identity, even more preferably 99% or more identity, and particularly preferably 100% identity with respect to the base sequence A.

[0053] In this specification, the “identity” of a base sequence refers to the degree of similarity between two or more comparable base sequences relative to each other. Therefore, the higher the similarity between two base sequences, the higher the identity or similarity of these sequences. The level of identity of a base sequence can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm of 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 has been developed based on such a BLAST algorithm. The specific methods used in these analysis are well-known and can be found on the website of the National Center for Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov / ).

[0054] There are no particular restrictions on the base lengths of base sequences A and B, for example, 8–1000, 10–500, or 25–200.

[0055] 5′-capped polynucleotides and single-stranded RNAs can be manufactured according to or based on known methods for manufacturing polynucleotides. 5′-capped polynucleotides or single-stranded RNAs can utilize sequences present in organisms and cells. Additionally, 5′-capped polynucleotides can be suitably manufactured using the manufacturing methods of the present invention described later.

[0056] In a preferred embodiment of the present invention, the 5′ capped polynucleotide is an endogenous polynucleotide derived from the organism or cell. Examples of such polynucleotides include lncRNA and mRNA, with lncRNA being preferred. By selecting RNA specifically expressed in a particular cell or tissue as the lncRNA or mRNA, proteins encoded by single-stranded RNA introduced from the organism or cell can be expressed specifically in the cell or tissue. This also enables the suppression of side effects or off-target effects. Furthermore, by using the protein encoded by the single-stranded RNA as a reporter protein, specific lncRNAs or mRNAs can be detected. Alternatively, the single-stranded RNA can be an endogenous RNA derived from the organism or cell. Examples of such polynucleotides include circular mRNA and linear mRNA, with circular mRNA being preferred.

[0057] The manufacturing or expression method of the present invention includes a step of performing a translation reaction in a reaction system containing 5′ capped polynucleotides and single-stranded RNA.

[0058] The reaction system only needs to contain the substances required for the translation reaction (such as ribosomes, amino acids, tRNA, etc.), and there are no particular restrictions. It can be any system, whether in vivo or in vitro. In the case of an in vivo reaction system, there are no particular restrictions on the type of organism; examples include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits; and animal cells. Furthermore, there are no particular restrictions on the type of cell; examples include blood cells, hematopoietic stem cells, progenitor cells, gametes (sperm, egg), 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. In the case of an in vitro reaction system, a reconstitution system or extracts from the above-mentioned organisms and cells can be used.

[0059] There are no particular restrictions on the reaction conditions for translation reactions; appropriate conditions can be set according to the enzymes used.

[0060] When performing the manufacturing or expression method of the present invention in vivo, it can be carried out by introducing 5′-capped polynucleotides and / or single-stranded RNA into the target (organism or cell). In one embodiment, when performing the manufacturing or expression method of the present invention in vivo, it can be carried out by introducing 5′-capped polynucleotides and single-stranded RNA into the target. In one embodiment, when performing the manufacturing or expression method of the present invention in vivo, it is particularly preferred to introduce 5′-capped polynucleotides or single-stranded RNA into the target.

[0061] Furthermore, when performing the manufacturing or expression method of the present invention in vitro, it can be done by adding 5′-capped polynucleotides and / or single-stranded RNA (particularly preferably 5′-capped polynucleotides or single-stranded RNA (i.e., either one)) to the 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 any base sequence A; and / or a single-stranded RNA containing a base sequence B capable of binding to the aforementioned base sequence A through complementary base pairing, and a protein or peptide coding sequence, wherein the 5′-capped polynucleotide and / or the aforementioned single-stranded RNA have a translation efficiency-enhancing structure. That is, during introduction or addition, the 5′-capped polynucleotide and single-stranded RNA do not necessarily need to bind; they can be introduced (or added) separately without binding, or either the 5′-capped polynucleotide or single-stranded RNA can be introduced and then bound to the 5′-capped polynucleotide or single-stranded RNA inherent in the object or reaction system.

[0062] In a particularly preferred embodiment of the invention, the composition of the invention preferably contains 5′ capped polynucleotide or single-stranded RNA (i.e., either one).

[0063] The compositions of the present invention can be pharmaceuticals or reagents. When the composition is a pharmaceutical, it can be used, for example, as a detection reagent for vaccines (e.g., vaccines for microbial infections), cellular or biological endogenous 5′-cap polynucleotides such as lncRNA and mRNA. The composition may also contain other ingredients as needed. These other ingredients are not particularly limited as long as they are pharmaceutically acceptable; examples include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents, etc. The composition can also be in the form of a kit containing two or more substances in two or more separate containers.

[0064] The composition of this invention is not particularly applicable to any particular species. Among mammals, examples include humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer. Furthermore, as cells, animal cells can be included. The types of cells are also not particularly limited; examples include blood cells, hematopoietic stem cells, progenitor cells, gametes (sperm, egg), 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.

[0065] The compositions of the present invention can be in any dosage form, such as oral formulations (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, gel drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including decoctions, suspensions, syrups), gels, etc., or non-oral formulations (e.g., intravenous drip injections, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), topical formulations (e.g., ointments, patches, lotions), suppositories, inhalers, eye drops, eye ointments, nasal drops, ear drops, etc.). Furthermore, the active ingredient can be administered in a state of being complexed with particles (e.g., lipid particles or exosomes) or encapsulated within such particles.

[0066] As for the route of administration of the composition of the present invention, there are no particular limitations as long as the desired effect can be obtained. Examples include: oral administration; enteral administration such as tube feeding or enema; non-oral administration such as intravenous administration, arterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intradermal administration, intraperitoneal administration, and nasal administration.

[0067] The content of the active ingredient in the composition of the present invention is affected by the method of use, the target of application, the state of the target of application, etc., and is not limited. For example, it can be set to 0.0001 to 100% by weight, preferably 0.001 to 50% by weight.

[0068] When administering the composition of the present invention to animals, the dosage is not particularly limited as long as it is an effective amount that demonstrates the therapeutic effect. Generally, it is based on the weight of the active ingredient, typically 0.01 to 100 mg / kg body weight per day, preferably 0.05 to 50 mg / kg body weight. The above dosage can also be appropriately increased or decreased according to age, disease condition, symptoms, etc.

[0069] The manufacturing or expression method of the present invention is characterized in that the 5′ capped polynucleotide and / or single-stranded RNA has a translation efficiency promoting structure.

[0070] The preferred translation efficiency-enhancing structure is a stabilizing structure that stabilizes the binding of a 5′-capped polynucleotide to single-stranded RNA. There are no particular restrictions on this structure; any structure capable of stabilizing the binding of a 5′-capped polynucleotide to single-stranded RNA is acceptable. Examples of such structures include conditions (b) and (c), which are described later.

[0071] In addition to these, the nucleotides constituting the 5′ capped polynucleotide base sequence A and / or the base sequence B of the single-stranded RNA are also considered to be modified nucleotides that can more stably form the double strands of base sequence A and base sequence B compared to the case of the reference nucleotides (phosphodiester bonds, ribose / deoxyribose, basic nucleic acid bases (A, T, G, C)). Known nucleotides can be used as such modified nucleotides, for example, 2′Ome modified nucleotides of the sugar moiety, LNA, etc. The amount of this modified nucleotide is preferably 30% or more, more preferably 50% or more, further preferably 70% or more, even more preferably 80% or more, particularly preferably 90% or more, and especially preferably 100%, relative to 100% of the constitutive nucleotides of base sequence A or base sequence B.

[0072] As a translation efficiency-enhancing structure, it is preferable to satisfy at least one of the conditions selected from (a), (b), and (c): (a) The above single-stranded RNA is a circular RNA. (b) The above-mentioned 5′ capped polynucleotide and / or the above-mentioned single-stranded RNA have a nuclease-binding structure, and the above-mentioned reaction system contains the above-mentioned nuclease, and (c) The above-mentioned 5′ capped polynucleotide and / or the above-mentioned single-stranded RNA have a structure that allows the two to be covalently linked.

[0073] The way that satisfies condition (a) corresponds to test example 4 described later. Figure 4 The way that condition (b) is met corresponds to Experimental Example 1 described later. Figure 1 ) and Experimental Example 2 ( Figure 2 The way that condition (c) is met corresponds to Experimental Example 3 described later. Figure 3 However, the present invention is not limited to the experimental examples described later.

[0074] Under condition (a), the circular RNA preferably does not contain a stop codon. This allows rolling circular translation to occur, further improving translation efficiency. In this case, in the method of manufacturing or expressing the circular RNA of the present invention, in which the ribosome was originally thought to inhibit translation in the second round due to the presence of the 5′-capped polynucleotide, the translation efficiency was surprisingly improved.

[0075] Other preferred methods are as follows, provided that condition (a) is met. 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 and the 5′ cap of base sequence A is preferably 0 to 100, more preferably 5 to 50, and particularly preferably 10 to 30.

[0076] The nuclease in condition (b) is a nucleic acid-binding nuclease; any nuclease capable of sequence-specific binding to the target sequence is acceptable, with no particular restrictions. Examples of such nucleases include RISC (RNA-induced silencing complex), which functions in RNA interference mechanisms, and Cas, which functions in CRISPR / Cas systems. Under condition (b) met, RISC is preferred (corresponding to Experimental Example 1 described later). Figure 1 )) or Cas (corresponding to test example 2 described later) Figure 2 However, the present invention is not limited to the experimental methods described later. By utilizing nucleases, the association between 5′ capped polynucleotides and single-stranded RNA can be promoted, thereby enhancing the binding of the two and improving translation efficiency.

[0077] The binding structure of a nuclease varies depending on the type of nuclease, and can be appropriately selected and designed according to its type.

[0078] When the nuclease is RISC, the nuclease-binding structure is a 5′ capped polynucleotide siRNA or miRNA.

[0079] The siRNA is preferably 18 or more bases, 19 or more bases, 20 or more bases, or 21 or more bases in length. Alternatively, the siRNA is preferably 25 or less bases, 24 or less bases, 23 or less bases, or 22 or less bases in length. The upper and lower limits of the siRNA length described herein can be arbitrarily combined. For example, combinations of the following are possible: a lower limit of 18 bases and an upper limit of 25, 24, 23, or 22 bases; a lower limit of 19 bases and an upper limit of 25, 24, 23, or 22 bases; a lower limit of 20 bases and an upper limit of 25, 24, 23, or 22 bases; and a lower limit of 21 bases and an upper limit of 25, 24, 23, or 22 bases.

[0080] siRNA can be shRNA (small hairpin RNA). shRNA can be engineered to form a stem-loop structure as part of its structure. siRNA can have an added base at the 5′ or 3′ end. This added base is typically 2–4 bases long. siRNA can have an overhang sequence (dangling sequence) at the 3′ end, specifically dTdT (dT represents deoxythymidine). Alternatively, it can have a smooth end (blunt end) without an added end.

[0081] Unlike siRNAs, which cleave target mRNAs, miRNAs can inhibit translation by pairing with the target's 3′ untranslated region (UTR). miRNAs can be pri-miRNAs (primary miRNAs), pre-miRNAs (precursor miRNAs), or mature miRNAs. There are no particular restrictions on miRNA length; pri-miRNAs are typically hundreds to thousands of bases long, pre-miRNAs are typically 50 to 80 bases long, and mature miRNAs are typically 18 to 30 bases long.

[0082] The guide strand of siRNA and miRNA has a 5′ cap structure and contains any base sequence A. To suppress RISC-induced cleavage of single-stranded RNA, base sequence A preferably has less than 95% identity with a base sequence that is completely complementary to base sequence B, more preferably less than 90%. In this manner, more specifically, base sequence A contains 1 to 8, 1 to 6, or 2 to 4 mismatched bases and / or non-natural bases relative to a base sequence that is completely complementary to base sequence B.

[0083] When the nuclease is Cas, the nuclease-binding structure contains a tracrRNA sequence in addition to the base sequence A or base sequence B of the 5′ capped polynucleotide and / or single-stranded RNA.

[0084] There are no particular restrictions on the tracrRNA sequence. A typical tracrRNA sequence is an RNA of 50–100 bases in length capable of forming multiple (usually three) stem-loops, and its sequence varies depending on the type of Cas protein used. Furthermore, depending on the type of Cas protein used, the tracrRNA sequence is positioned on the 5′ or 3′ side of base sequence A or base sequence B. Various known sequences can be used as tracrRNA sequences, depending on the type of Cas protein used.

[0085] Cas proteins are any proteins used in the CRISPR / Cas system; there are no particular restrictions. Known Cas proteins come from various organisms, such as Cas9, Cas12, Cas13, and the Cpf1 protein (type V) from *F. novicida*. The amino acid sequences and coding sequences of various Cas proteins are readily available in databases such as NCBI.

[0086] To inhibit single-stranded RNA cleavage, Cas proteins preferably contain amino acid variations. Variations that eliminate or reduce nuclease activity are well-known and can be easily determined based on publicly available information.

[0087] When condition (b) is met and the nuclease is Cas, the 5′ capped polynucleotide is preferably lncRNA or mRNA, and the single-stranded RNA contains a tracrRNA sequence. By designing base sequence B to be complementary to the sequence (base sequence A) within the lncRNA or mRNA, the lncRNA or mRNA can be used as a 5′ capped polynucleotide. When the manufacturing or expression method of the present invention is performed in vivo, endogenous RNA can be used as the lncRNA or mRNA. By selecting RNA specifically expressed in specific cells or tissues as the lncRNA or mRNA, the protein encoded by the single-stranded RNA can be specifically expressed in cells or tissues. This also enables the suppression of side effects and off-target effects. In addition, by using the protein encoded by the single-stranded RNA as a reporter protein, specific lncRNAs or mRNAs can be detected. When Cas is used as the nuclease, a target containing a polynucleotide containing a Cas coding sequence is used, and the polynucleotide is introduced into the target or added to the reaction system.

[0088] When condition (b) is met and the nuclease is Cas, from the viewpoint of translation efficiency, the base length of the sequence between the 5′ end and the 5′ cap of base sequence A is preferably 3 or more, more preferably 7 or more, further preferably 12 or more, and even more preferably 20 or more. There is no particular upper limit to this base length, for example, it can be 100, 60 or 30.

[0089] Other preferred methods are as follows, provided that condition (b) is met. From the viewpoint of translation efficiency, the base length of the base sequence A is preferably 10 to 100, more preferably 12 to 50, and particularly preferably 15 to 30.

[0090] The "structure that allows the two to be covalently bonded" in condition (c) is any structure possessed by one or both of the 5′-capped polynucleotide and single-stranded RNA, and is capable of covalently bonding the 5′-capped polynucleotide and single-stranded RNA; there are no particular limitations. As such a structure, a photocrosslinking structure (a structure capable of forming a crosslinking structure through light irradiation) is preferred. Examples of photocrosslinking structures include psoralen, and more specifically, 8-methoxypsoralen (methoxsalen) and 4,5′,8-trimethylpsoralen (trimethasone). For example, by replacing a portion of the bases in base sequence A / base sequence B with the sequence described above, the complementary bases of base sequence A and base sequence B (e.g., uracil) can form a covalent bond through light irradiation (e.g., light irradiation at a wavelength of 320–380 nm). By forming a covalent bond, the improved translation efficiency brought about by the 5′-capped polynucleotide can be more effectively utilized.

[0091] In one aspect of the invention, the protein or peptide generated by the manufacturing or expression method of the invention can be purified by the reaction system according to or based on known methods.

[0092] 1a. Methods for the manufacture or expression of proteins or peptides 2 This item corresponds to items 8 to 10 above and test examples 6 to 8 described later. Figures 8-11 However, the present invention is not limited to the experimental examples described later.

[0093] In one aspect, the present invention also relates to a method for the manufacture or expression of a protein or peptide, comprising a step of performing a translation reaction in a reaction system containing a single-stranded circular RNA composed of 5′ capped polynucleotides linked via a linker.

[0094] Regarding 5′ capped polynucleotides, except for the absence of a limitation regarding the inclusion of base sequence A, the description in “1. Method for the manufacture or expression of proteins or peptides 1” above can be cited.

[0095] The base length of the base sequence contained in the 5′ capped polynucleotide is not particularly limited, for example, it is 1 to 500, preferably 2 to 100, more preferably 2 to 50, further preferably 2 to 20, even more preferably 2 to 10, and particularly preferably 3 to 6.

[0096] Single-stranded circular RNA contains protein or peptide coding sequences. Regarding single-stranded circular RNA, except for the limitation that it does not contain the base sequence B, the description in "1. Methods for the manufacture or expression of proteins or peptides" above can be cited.

[0097] There is no particular limitation on the base length of the single-stranded circular RNA. From the viewpoint of translation efficiency, a shorter length is preferred, for example, 500 or less, more preferably 300 or less, more preferably 250 or less, and even more preferably 200 or less. In this embodiment, since IRES is not required, the size can be kept low, thereby achieving high translation efficiency.

[0098] All nucleotides in a single-stranded circular RNA can be linked by typical internucleotide bonds such as phosphodiester bonds or thiophosphate bonds, or some nucleotides can be linked by a linker. In the latter case, it is preferable that the linker is attached to a 5′ capped polynucleotide.

[0099] The linker can function flexibly and is not particularly limited. It can consist solely of a chain structure, or it can include both chain and loop structures. Furthermore, the chain structure can be straight or branched. For example, if a straight chain with two ends (referred to as ends a to b for convenience) is used, end a can be linked to a nucleotide (preferably a terminal nucleotide) of the single-stranded RNA, end b can be linked to a 5'-capped polynucleotide (preferably the 3' end of the 5'-capped polynucleotide), and other nucleotides of the single-stranded RNA (preferably another terminal nucleotide different from the aforementioned terminal nucleotide) can be linked at any point c in the linker structure connecting ends a and b. In this example, ends a, b, and point c can be interchanged. In addition, as another example, if it is a branched structure with three ends (represented as ends A to C for convenience), then ends A and B can be connected between nucleotides of the single-stranded RNA (preferably between the two end nucleotides), and end C can be connected to a 5′ capped polynucleotide (preferably the 3′ end of the 5′ capped polynucleotide). These methods are also preferred from the point of view of translation efficiency.

[0100] More specifically, the linker preferably has at least one carbon atom constituting the main chain that can be replaced by a heteroatom and / or a linking structure in the hydrocarbon chain. When at least one carbon atom constituting the main chain is replaced by a heteroatom and / or a linking structure, the number of replaced carbon atoms is, for example, 1 to 5.

[0101] Alkylene chains are preferred as examples of hydrocarbon chains. Alkylene chains can be either straight-chain or branched, but are preferably straight-chain. The main chain of the alkylene chain preferably has 6 or more carbon atoms. More preferably, it has 10 or more carbon atoms, even more preferably 12 or more carbon atoms, and even more preferably 100 or less, more preferably 50 or less, and even more preferably 20 or less carbon atoms.

[0102] Examples of heteroatoms include oxygen atoms, sulfur atoms, and nitrogen atoms. When at least one carbon atom in the main chain of the alkylene group is replaced by an oxygen atom, specifically, the -CH2- group in the main chain of the alkylene group is replaced by, for example, -O-. When at least one carbon atom in the main chain of the alkylene group is replaced by a sulfur atom, specifically, the -CH2- group in the main chain of the alkylene group is replaced by, for example, -S-, -S(=O)2-, -S(=O)-. When at least one carbon atom in the main chain of the alkylene group is replaced by a nitrogen atom, specifically, the -CH2- group in the main chain of the alkylene group is replaced by, for example, -NR- (R represents a hydrogen atom or a hydrocarbon group (preferably an alkyl group, more preferably an alkyl group with 1 to 8 carbon atoms)). The linking structure can be any divalent group formed by the reaction of two identical or different reactive groups; there are no particular limitations. Examples of reactive groups include amino, carboxyl, hydroxyl, ketone, ethynyl, vinyl, azide, epoxy, aldehyde, oxyamino, thiol, isocyanate, and isothiocyanate groups. Examples of reactions between reactive groups are as follows: It is known that amino groups react with carboxyl groups (or groups formed by esterifying carboxyl groups with N-hydroxysuccinimide (NHS)) to form amide bonds. It is known that ethynyl groups form 1,2,3-triazole rings through a 1,3-dipolar cycloaddition with an azide group. It is known that cyclooctyne groups form 1,2,3-triazole rings through a 1,3-dipolar cycloaddition with an azide group. Amino groups react with carboxyl groups to form amide bonds. It is known that carboxyl groups form ester bonds with hydroxyl groups. It is known that carboxyl groups react with thiol groups to form thioester bonds. It is known that phosphate groups react with hydroxyl groups to form phosphodiester bonds. Vinyl groups react with thiol groups to form bonds. Epoxy groups react with amino or thiol groups to form bonds. Aldehydes react with amino groups to form Schiff bases, which, if reduced, form bonds. Oxyamino groups react with ketone or aldehyde groups to form oximes.

[0103] Single-stranded circular RNAs formed by linking 5′ capped polynucleotides via adapters can be obtained through chemical synthesis.

[0104] In addition to the above, the contents of "1. Methods for the manufacture or expression of proteins or peptides" above may be cited.

[0105] 2. Method for manufacturing 5′ capped polynucleotides This item corresponds to items 11 to 18 above and test example 5 described later. Figures 5-7 However, the present invention is not limited to the experimental examples described later.

[0106] In one embodiment, the present invention relates to a method for manufacturing a 5′ capped polynucleotide (in this specification, it is sometimes referred to as "the manufacturing method of the present invention"), comprising a step of performing a transcription reaction in a reaction system containing: a phage promoter comprising an insertion mutation sequence adjacent to the upstream side of the transcription start site, and the antisense strand comprising the base sequence Y: (upstream side)-Y2-Y3 or (upstream side)-Y1-Y2-Y3 (in the base sequence Y: Y1 and Y2 represent bases within the insertion mutation sequence, and Y3 represents a base at the transcription start site); and a 5′ capped analog having a polynucleotide comprising the base sequence X: (cap side)-X2-X3 or (cap side)-X1-X2-X3, where X3 is a complementary base of Y3, and X1 is a complementary base of Y1 and / or X2 is a complementary base of Y2. This will be described below.

[0107] There are no particular restrictions on the type of phage promoter; examples include the T7 promoter, SP6 promoter, and T3 promoter. Among these, the T7 promoter is particularly preferred from the perspective of capping efficiency.

[0108] The phage promoter is adjacent to the upstream side of the transcription start site and contains a base insertion mutation sequence. That is, the phage promoter used in the manufacturing method of this invention is a mutant promoter obtained by inserting a base 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 known information.

[0109] The antisense strand of the double-stranded polynucleotide used in the manufacturing method of the present invention contains the base sequence Y: (upstream side) - Y2 - Y3 or (upstream side) - Y1 - Y2 - Y3. In the base sequence Y: Y1 and Y2 represent bases inserted into the mutation sequence, and Y3 represents the base at the transcription start site.

[0110] The double-stranded polynucleotide contains the coding sequence of the 5′ capped polynucleotide of the target material downstream of the phage promoter, i.e., the base sequence of the 5′ capped polynucleotide is contained in the positive strand. This double-stranded polynucleotide is particularly preferably DNA.

[0111] The base length of the double-stranded polynucleotide can be any length that can be used as a transcription template, without any particular restrictions, such as 25–10000, 25–5000, 25–3000, 25–2000, 25–1000, 25–500, 25–300, or 25–200.

[0112] 5′ cap analogs are single-chain polynucleotides with a 5′ cap structure added. There are no particular restrictions as long as they can be used in the manufacture of 5′ capped polynucleotides.

[0113] The 5′ cap structure is as described above. The 5′ cap structure may include a hydrophobic purification tag that can be removed by light (e.g., Non-Patent Document 1). Using the manufacturing method of the present invention, high-purity 5′-capped polynucleotides can be manufactured even without using the hydrophobic purification tag, and even higher-purity 5′-capped polynucleotides can be manufactured by using the hydrophobic purification tag.

[0114] The polynucleotides of the 5′ cap analogues are preferably 2 to 5, more preferably 2 to 4, and particularly preferably 3.

[0115] The 5′ cap analogue has a polynucleotide containing the base sequence X: (cap side)-X2-X3 or (cap side)-X1-X2-X3. In the base sequence X, X3 is preferably the 3′ end of the polynucleotide.

[0116] In the manufacturing method of the present invention X3 is the complementary base of Y3, and X1 is a complementary base of Y1 and / or X2 is a complementary base of Y2.

[0117] By using a phage promoter with the aforementioned complementary relationship and base insertion mutation sequence, the capping efficiency can be improved. From the viewpoint of capping efficiency, it is particularly preferred that the antisense strand of the phage promoter contains the base sequence Y: (upstream side) -Y1-Y2-Y3, where X1 is the complementary base of Y1 and X2 is the complementary base of Y2.

[0118] by Figure 5 In the case of φ6.5, XY-inserted in a), "XYG" in the 5′ cap analog (m7G-XYG) is the base sequence X (X1-X2-X3), and the lower strand of the double strand below it is the antisense strand. "XYC" in the antisense strand is equivalent to the base sequence Y (Y1-Y2-Y3).

[0119] There are no particular restrictions on the specific sequence of the base sequence X. However, from the viewpoint of capping efficiency and the production efficiency of 5′ capped polynucleotides, it is particularly preferred that (cap side) -CUG, CAG, GUG or GAG, and Y3 is C.

[0120] Furthermore, from the viewpoint of translation efficiency of the produced 5′ capped polynucleotide, the specific sequence of the base sequence X is particularly preferably (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.

[0121] Double-stranded polynucleotides and 5′-capped analogs can be manufactured according to or based on known methods for manufacturing polynucleotides and 5′-capped analogs. More specifically, for 5′-capped analogs, synthetic methods, such as those described later, can be referenced.

[0122] The reaction system only needs to contain the substances necessary for the translation reaction (such as RNA polymerase, various nucleoside triphosphates, etc.), and there are no special restrictions. It can be any type of in vitro or in vivo reaction system.

[0123] In this invention, by using a T7 RNA polymerase with the D130W mutation, the incorporation efficiency of cap analogs can be further improved. Figure 7Therefore, in one aspect of the present invention, therein relates to a T7 RNA polymerase having the D130W mutation. “D130W” refers to the mutation of amino acid D (aspartic acid) at position 130 from the N-terminus of the amino acid sequence of the T7 RNA polymerase (Sequence No. 1) to W (tryptophan).The mutant T7 RNA polymerase can contain other amino acid mutations, provided that it does not significantly impair the cap analog incorporation efficiency. These mutations can be in quantities of, for example, less than 80, 50, 30, 20, 10, 5, 2, or 1. The mutant T7 RNA polymerase can also contain other amino acid sequences (e.g., tag sequences, fluorescent protein sequences with independent domain structures), provided that it does not significantly impair the cap analog incorporation efficiency.

[0124] There are no particular restrictions on the reaction conditions for transcription; appropriate conditions can be set according to the enzymes used.

[0125] In the manufacturing method of the present invention, it can be carried out by adding a double-stranded polynucleotide and / or a 5′ cap analog to the reaction system. Therefore, in one aspect, the present invention relates to a composition 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 the base sequence Y: (upstream side)-Y2-Y3 or (upstream side)-Y1-Y2-Y3 (where in the base sequence Y: Y1 and Y2 represent bases within the base insertion mutation sequence, and Y3 represents a base at 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, where X3 is a complementary base to Y3, and X1 is a complementary base to Y1 and / or X2 is a complementary base to Y2.

[0126] This composition can be a pharmaceutical product or a reagent. It may also contain other ingredients as needed. These other ingredients are pharmaceutically acceptable and are not particularly limited, but can include, for example, base agents, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents, etc. This composition can also be in the form of a kit containing two or more substances in two or more separate containers.

[0127] Example The present invention will now be described in detail based on embodiments, but the present invention is not limited to these embodiments.

[0128] Experimental Example 1. Promotion of Translation Response by Circular mRNA Using RNA Interference Mechanism The concept diagram of this experiment is shown in... Figure 1 In (a).

[0129] The base sequence of the circular RNA used in this experiment is shown below.

[0130] Circular Nluc mRNA (620-nt) 5′_GGCGCAUAUUAAGGUGACGCGUGUGGCCUCGAACACCGAGCGACCCUGCAGCGACCCGCUUAAAAGCUUGGCAAUCCGGUACUGUUGGUAAAGCCACCAUGGUCUUCACACUCGAAGAUUUCGUUGGGGACUGGCGACAGACAGCCGGCUACAACCUGGACCAAGUCCUUGAACAGGGAGGUGUGUCCAGUUUGUUUCAGAAUCUCGGGGUGUCCGUAACUCCGAUCCAAAGGAUUGUCCUGAGCGGUGAAAAUGGGCUGAAGAUCGACAUCCAUGUCAUCAUCCCGUAUGAAGGUCUGAGCGGCGACCAAAUGGGCCAGAUCGAAAAAAUUUUUAAGGUGGUGUACCCUGUGGAUGAUCAUCACUUUAAGGUGAUCCUGCACUAUGGCACACUGGUAAUCGACGGGGUUACGCCGAACAUGAUCGACUAUUUCGGACGGCCGUAUGAAGGCAUCGCCGUGUUCGACGGCAAAAAGAUCACUGUAACAGGGACCCUGUGGAACGGCAACAAAAUUAUCGACGAGCGCCUGAUCAACCCCGACGGCUCCCUGCUGUUCCGAGUAACCAUCAACGGAGUGACCGGCUGGCGGCUGUGCGAACGCAUUCUGGCGUAAUUCUAG_3′ (SEQ ID NO: 6) (5′, 3′ end binding).

[0131] Circular RNA was prepared as follows. First, 5′ phosphorylated linear RNA was prepared as the raw material for circularization using a transcription reaction with 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 solution was heated at 37°C for 2 hours, followed by the addition of DNase I (TakaraBio) to a final concentration of 0.1 U / μL, and incubation at 37°C for 15 minutes. The reaction solution was extracted with an equal volume mixture of TE saturated phenol and chloroform to remove proteins, and the transcribed RNA was precipitated with ethanol and recovered. The crude transcribed RNA obtained was purified by reverse-phase HPLC according to existing reports (Nat. Commun., 14, 2657, 2023). The 5′ phosphorylated transcribed RNA was circularized by ligating the 5′ and 3′ ends using T4 RNA ligase 2. The composition of the ligase reaction solution is shown below: 0.5 μM 5′ phosphorylated RNA, 1 μM template DNA oligomer (5′ AATATGCGCCCTAGAATTAC 3′ (Sequence 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 2. After heating the reaction solution at 37°C for 1 hour, it was extracted with an equal volume mixture of TE saturated phenol-chloroform to remove proteins. Sodium acetate aqueous solution (pH 5.2, final concentration 0.3 M) and 2-propanol were added, and the mixture was centrifuged after cooling at -30°C. The RNA was recovered as the precipitate. The target RNA circularized bodies were purified by preparative denaturing polyacrylamide gel electrophoresis.

[0132] The sequences of the RNA oligonucleotides used in this experiment are shown below.

[0133] Passerby Chain 5′ GACGCGUGUCUACUCGAACAUA 3′ (Serial Number 8) Uncapped guide chain 5′ p-UGUUCGAGUAGACACGCGUCAC-p 3′ (Serial No. 9) Capped Guide Chain 5′ p-UGUUCGAGUAGACACGCGUCAC(3′)ppp(5′)m 7 G 3′ (Serial No. 10).

[0134] Capped RNA oligonucleotides were prepared according to existing reports (ACS Chem Biol 2022, 17, 1308-1314). The 5′ phosphorylated RNA oligonucleotide sequences were synthesized using a commercially available phosphoramide compound (ChemGenes) and an automated nucleic acid synthesizer, NTS T-8-A20-R8NC (Techno Service, Japan). After synthesis, a 1:1 mixture of 40% methylamine aqueous solution and 28% concentrated ammonia was added to the CPG solid-phase support, and the mixture was heated at 65°C for 60 minutes to cleave the oligonucleotides from the support, deprotecting the phosphate and base portions. The oligonucleotides were dried under reduced pressure, incubated overnight at room temperature with 1 M TBAF / THF solution, and desalted using a NAP-25 gel filtration column (Cytiva). The precipitate was obtained by ethanol precipitation. The oligonucleotides were then purified by reversed-phase HPLC. The obtained 5′ phosphorylated RNA oligonucleotides were reacted with m7GDP imidazole (Im-m7GDP) to introduce a cap structure at the 5′ end (ACS Chem Biol 2022, 17, 1308-1314). The capping reaction was confirmed by detecting the gel migration of RNA bands using denaturing PAGE and determining the molecular weight using LC-MS. The guest strand and the uncapped or capped guide strand were mixed at a final concentration of 1 μM in buffer (10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA (pH 8.0)). The solution was heated at 80 °C for 5 minutes and then slowly cooled to prepare a 1 μM double-stranded RNA oligonucleotide solution.

[0135] The effect of capped double-stranded RNA oligonucleotides on the translation response of cyclic Nluc mRNA was evaluated using the human cell line HeLa (RIKEN Cellbank, RCB0007). HeLa cells were cultured in DMEM (Wako Pure Chemical Industries Co., Ltd.) containing 10% FBS (Thermo Fisher Scientific) at 5% CO2 concentration and 37°C. For HeLa cells seeded at 1.5 × 10⁴ cells / well in 96-well multi-well plates one day prior to transfection, cyclic Nluc mRNA (30 ng / well), double-stranded RNA oligonucleotides (0.3 pmol / well, 1.5 pmol / well, 3 pmol / well), and Opti-MEM were respectively added to Lipofectamine MessengerMAX (0.15 μL) and Opti-MEM. TM Mix 10 μL of low serum medium (Thermo Fisher) and add it to Opti-MEM. TMI. Low serum medium (20 μL) was used to replace the growth medium in wells containing HeLa cells, 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 in the cell lysate was evaluated using a Nano-Glo (registered trademark) Luciferase Assay System (Promega). Chemiluminescence assays were performed using a TriStar5 multi-functional plate reader (Berthold).

[0136] The results are shown in Figure 1 This indicates that forming a RISC-compatible system can promote the translation response.

[0137] Experimental Example 2. Utilizing hybridization with long non-coding RNA (lncRNA) and stabilization of its dCas13 protein. Translation-promoting phenomena of circular mRNAs The concept diagram of this experiment is shown in... Figure 2 (a) and (c).

[0138] The base sequence of the RNA used in this experiment is shown below.

[0139] HULC RNA (500-nt): 5′_GAUGGGGGUGGAACUCAUGAUGGAAUUGGAGCCUUUACAAGGGAAUGAAGAGACAAGAGCUCUCUUUAUGCCACGUGAGGAUACAGCAAGGCCCCAAUCUGCAAGCCAGGAAGAGUCGUCACG AGAACCAGACCAUGCAGGAACUCUGAUCGUGGACAUUUCAACCUCCAGAACUGUGAUCCAAAAUGCAUGUAUCUUUGGAAGAAACUCUGAAGUAAAGGCCGGAAUAUUCUUUGUUUAAAACAUUA AAAACAAAACAGACCAAAGCAUCAAGCAAGAAGUUUCCUGGCAAUAAACUAAGCACAGCAUUAUUUUUAAGGAACACAAAUUAAGUGUUCAACCUGUGGCAAAUUUGUACUUUCUCCCUGAAUUAU GUUGUUAUCAAAGAAAAAAAUUGGGAAGCAUGGCAAAAUAUCAUCAAAACUGAAACUAGAAUUAAACAAAACUAAAUUAAAAUGAAAUAAAAUGAUGUCCAUUCUUAAAAAAAAAAAAAAAAAA_3′ (Serial number 11).

[0140] UCA1 RNA(1678 nt):

[0141] dRfxCas13d mRNA(3184-nt):

[0142] Straight-chain Nluc mRNA (705-nt) with a 5-base linker: 5′_GGUCCCAGGUCCACUUCAAGUAAACCCCUACCAACUGGUCGGGGGUUUGAAAC CCAAUUCCAUCAU GAGUUCCACC _3′ (Sequence number 13) (The underlined part is the sequence complementary to HULC).

[0143] Straight-chain Nluc mRNA (705-nt) with a 10-base-length linker: 5′_GGUCCCAGGUCCACUUCAAGUAAACCCCUACCAACUGGUCGGGGGUUUGAAAC AGGCUCCAAUUCC AUCAUGAGUU_3′ (Sequence No. 14) (The underlined part is the sequence complementary to HULC).

[0144] Straight and circular Nluc mRNAs with 15-base-length linkers (705-nt): 5′_GGUCCCAGGUCCACUUCAAGUAAACCCCUACCAACUGGUCGGGGUUUGAAAC UGUAAAGGCUCCAAUUCCAUCAU_3′ (Sequence number 15) (The underlined part is the sequence complementary to HULC, which binds to the 5′ and 3′ ends of the circular Nluc mRNA).

[0145] Straight-chain and circular Nluc mRNAs (705-nt) with 15-base-length linkers: 5′_GGUCCCAGGUCCACUUCAAGUAAACCCCUACCAACUGGUCGGGGGUUUGAAAC UGGAGAGAUGAUG GGACUCAUUGGCGUGUGGCCUCGAACACCGAGCGACCCUGCAGCGACCCGCUUAAAAGCUUGGCAAUCCGGUACUGUUGGUAAAGCCACCAUGGUCUUCACACUCGAAGAUUUCGUUGGGGACUGGCGACAGACAGCCGGCUACAACCUGGACCAAGUCCUUGAACAGGGAGGUGUGUCCAGUUUGUUUCAGAAUCUCGGGGUGUCCGUAACUCCGAUCCAAAGGAUUGUCCUGAGCGGUGAAAAUGGGCUGAAGAUCGACAUCCAUGUCAUCAUCCCGUAUGAAGGUCUGAGCGGCGACCAAAUGGGCCAGAUCGAAAAAAUUUUUAAGGUGGUGUACCCUGUGGAUGAUCAUCACUUUAAGGUGAUCCUGCACUAUGGCACACUGGUAAUCGACGGGGUUACGCCGAACAUGAUCGACUAUUUCGGACGGCCGUAUGAAGGCAUCGCCGUGUUCGACGGCAAAAAGAUCACUGUAACAGGGACCCUGUGGAACGGCAACAAAAUUAUCGACGAGCGCCUGAUCAACCCCGACGGCUCCCUGCUGUUCCGAGUAACCAUCAACGGAGUGACCGGCUGGCGGCUGUGCGAACGCAUUCUGGCGUAAUUCUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA_3′ (SEQ ID NO: 57) (The underlined part is the sequence complementary to UCA1, binding to the 5′ and 3′ ends in the circular Nluc mRNA) Each RNA was synthesized using T7 RNA polymerase as a template with the DNA encoding that sequence. The composition of the transcription reaction is shown below: 10 ng / μL dsDNA (PCR product containing the T7 promoter), 2 mM ATP, 2 mM UTP, 2 mM CTP, 2 mM MGITP, 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 solution was heated at 37°C for 2 hours. During the preparation of HULC RNA, dRfxCas13d mRNA, and UCA1 RNA, a capped analog compound DiPure (Nat. Commun. 14, 2657, 2023) with a hydrophobic tag was added to a final concentration of 2 mM. The capped mRNA was then isolated and purified according to existing reports. After transcription, DNase I (Takara Bio) was added to a final concentration of 0.1 U / μL, and the mixture was heated at 37°C for 15 minutes. 7.5 M lithium chloride aqueous solution was added to the reaction mixture to a final concentration of 2.5 M, and the mixture was cooled at -30°C for 30 minutes before centrifugation (20,000 × g, 20 minutes). The transcribed RNA was recovered as a precipitate. The crude transcribed RNA was purified by reverse-phase HPLC according to existing reports (Nat. Commun. 14, 2657, 2023). The 5′ phosphorylated RNA synthesized by adding GMP to the above transcription reaction mixture at a final concentration of 10 mM was ligated to the 5′ and 3′ ends using T4 RNA ligase 2 in the presence of template DNA oligonucleotides complementary to the RNA ends (30-nt, 5′AGTGGACCTGGGACCTTTTTTTTTTTTTTTTT 3′ (Sequence No. 16)) to prepare circular Nluc mRNA. The composition of the ligase reaction solution is shown below: 1 μM 5′ phosphorylated RNA, 2 μM DNA oligonucleotides, 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. After incubating the reaction solution at 37°C for 1 hour, extraction was performed with an equal volume mixture of TE saturated phenol-chloroform to remove proteins. Sodium acetate aqueous solution (pH 5.2, final concentration 0.3 M) and 2-propanol were added, and the mixture was centrifuged after cooling at -30°C. The RNA was recovered as the precipitate. The target RNA circularized body was purified by preparative denaturing polyacrylamide gel electrophoresis (Sci. Rep., 5, 16435, 2015).

[0146] The expression level evaluation assay for Nluc protein was performed using the human cultured cell line HeLa (Riken Cellbank, RCB0007). HeLa cells were cultured in DMEM (Wako Pure Chemical Industries Co., Ltd.) containing 10% FBS (Thermo Fisher Scientific) at a concentration of 5% CO2 at 37°C. One day before transfection, HeLa cells were seeded at 1×10E4 cells per well in a 96-well multi-well plate.

[0147] 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), respectively. After incubation for 5 minutes, they were added to Opti-MEM. TM I. Low-serum medium (Thermo Fisher) was used to replace the growth medium in wells containing HeLa cells, and RNA was introduced into the cells. After 4 hours, the supernatant was replaced with growth medium, and after a total of 24 hours of culture, Nluc expression in the cells was evaluated using a Nano-Glo (trademarked) luciferase assay system (Promega). Chemiluminescence assays were performed using a TriStar5 multi-functional 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 similarly introduced into HeLa cells using a Lipofectamine MessengerMAX. After 4.5 hours, Nluc expression was measured using a Nano-Glo (trademarked) Luciferase Assay System.

[0148] In addition, Nluc protein expression level evaluation experiments were performed using the human cultured cell line HeLa (Riken Cellbank, RCB0007). HeLa cells were cultured in DMEM (Wako Pure Chemical Industries Co., Ltd.) containing 10% FBS (Thermo Fisher) at 37°C with 5% CO2. One day before transfection, HeLa cells were seeded at 1×10E4 cells per well in a 96-well multi-well 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 hybridized by slow cooling from heating. 1 μL of RNA hybridization product and 0.15 μL of Lipofectamine MessengerMAX (Thermo Fisher) were prepared in each well, diluted with 5 μL of Opti-MEM (trademark) I low-serum medium (Thermo Fisher), and mixed to form liposomes by incubation at room temperature for 5 minutes. The culture medium in the 96-well multi-well plates containing HeLa cells prepared the previous day was replaced with fresh DMEM, and the aforementioned liposomes were added to introduce RNA. 4.5 hours later, Nluc expression levels in the cells were evaluated using a Nano-Glo (registered trademark) luciferase assay system (Promega). Chemiluminescence assays were performed using a TriStar5 multi-functional plate reader (Berthold).

[0149] The results are shown in Figure 2 This indicates that through Forming a hybridization with lncRNA and being able to The Cas protein-binding system can promote the translation response.

[0150] Experimental Example 3. The phenomenon that photocrosslinked capped RNA probes promote the translation of circular mRNAs in a photodependent manner. elephant The concept diagram of this experiment is shown in... Figure 3 (a)

[0151] The sequence of the chemically synthesized RNA used in this experiment is shown below: Straight-chain 24-nt RNA 5′_AAAAGGCGCAUAUUAAGGUGACGC_3′ (Serial Number 17) Probe 1 (50-nt RNA) 5′_m7G-ppp-GCGUCA X CCUUAAUAUGCGCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA_3′ (Serial Number 18) Probe 2 (50-nt RNA) 5′_m7G-ppp-GCGUCACCUUAAUAX UGCGCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA_3′ (Serial Number 19) Probe 3 (50-nt RNA) 5′_m2 7,2′O G-ppp-GCGUCACCUUAAUA X UGCGCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA_3′ (Serial Number 20) Probe 4 (50-nt RNA) 5′_m 7 G-ppp-GCGUCACCUUAAUAUGCGCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA_3′ (Serial Number 21) The sequences contained in probes 1 and 2 X This indicates the inserted trimethasone structure (from ChemGenes reagent CLP-6644).

[0152] The sequence of the circular RNA used in this experiment is shown below: Circular Nluc mRNA (650-nt) 5′_GGCGCAUAUUAAGGUGACGCGUGUGGCCUCGAACACCGAGCGACCCUGCAGCGACCCGCUUAAAAGCUUGGCAAUCCGGUACUGUUGGUAAAGCCACCAUGGUCUUCACACUCGAAGAUUUCGUUGGGGACUGGCGACAGACAGCCGGCUACAACCUGGACCAAGUCCUUGAACAGGGAGGUGUGUCCAGUUUGUUUCAGAAUCUCGGGGUGUCCGUAACUCCGAUCCAAAGGAUUGUCCUGAGCGGUGAAAAUGGGCUGAAGAUCGACAUCCAUGUCAUCAUCCCGUAUGAAGGUCUGAGCGGCGACCAAAUGGGCCAGAUCGAAAAAAUUUUUAAGGUGGUGUACCCUGUGGAUGAUCAUCACUUUAAGGUGAUCCUGCACUAUGGCACACUGGUAAUCGACGGGGUUACGCCGAACAUGAUCGACUAUUUCGGACGGCCGUAUGAAGGCAUCGCCGUGUUCGACGGCAAAAAGAUCACUGUAACAGGGACCCUGUGGAACGGCAACAAAAUUAUCGACGAGCGCCUGAUCAACCCCGACGGCUCCCUGCUGUUCCGAGUAACCAUCAACGGAGUGACCGGCUGGCGGCUGUGCGAACGCAUUCUGGCGUAAUUCUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA_3′ (SEQ ID NO: 22) (5′, 3′ end binding).

[0153] Unmodified RNA oligonucleotides and trimethylsalicyline-capped RNA oligonucleotides were prepared according to existing reports (ACSChem Biol 2022, 17, 1308-1314). The 5′ phosphorylated RNA oligonucleotide sequences were synthesized using a commercially available phosphoramide compound (manufactured by ChemGenes) via an automated nucleic acid synthesizer NTS T-8-A20-R8NC (manufactured by TechnoService, Japan). (5′ phosphate group CLP-1544; trimethylsalicyline structure, CLP-6644). After synthesis, a 1:1 mixture of 40% aqueous methylamine and 28% concentrated ammonia was added to the CPG solid-phase support, and the mixture was heated at 65°C for 15 minutes, thereby cleaving the oligonucleotides from the support and deprotecting the phosphate and base portions. The RNA oligonucleotides were dried and cured under reduced pressure, incubated overnight at room temperature with 1 M TBAF / THF solution, desalted using a NAP-25 gel filtration column (Cytiva), and precipitated with ethanol to obtain the precipitate. The oligonucleotides were then purified by reversed-phase HPLC. The obtained 5′ phosphorylated RNA oligonucleotides were reacted with m7GDP imidazole (Im-m7GDP) to introduce a cap structure at the 5′ end (ACS Chem Biol 2022, 17, 1308-1314). The capping reaction was confirmed by detecting gel migration of the RNA bands using denaturing PAGE and determining the molecular weight using LC-MS.

[0154] The circular RNA used in this experiment was prepared as follows. First, 5′ phosphorylated linear RNA, used as raw material before circularization, was prepared by transcription with 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 solution was heated at 37°C for 2 hours, followed by the addition of DNase I (Takara Bio) at a final concentration of 0.1 U / μL, and incubation at 37°C for 15 minutes. The reaction solution was extracted with an equal volume mixture of TE saturated phenol and chloroform to remove proteins, then precipitated with ethanol and the transcribed RNA was recovered. The crude transcribed RNA obtained was purified by reverse-phase HPLC according to existing reports (Nat. Commun., 14, 2657, 2023). The 5′ and 3′ ends of the obtained 5′ phosphorylated transcribed RNA were ligated and circularized using T4 RNA ligase 2. The composition of the ligase reaction solution is shown below: 0.5 μM 5′ phosphorylated RNA, 1 μM template DNA oligomer (5′ACCTTAATATGCGCCTTTTTTTTTTTTTTTTT 3′ (Sequence 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 2. The reaction solution was heated at 37°C for 1 hour, then extracted with an equal volume of TE saturated phenol-chloroform mixture to remove proteins. Sodium acetate aqueous solution (pH 5.2, final concentration 0.3 M) and 2-propanol were added, and the mixture was cooled at -30°C and centrifuged. The RNA was recovered as the precipitate. The target RNA circularized bodies were purified by preparative denaturing polyacrylamide gel electrophoresis.

[0155] The cross-linking experiments of probe 1 and probe 2 with linear 24-nt RNA were performed as follows. A mixed solution of RNA and probes (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), and 1 mM EDTA (pH 8.0)) was prepared, heated at 90°C for 3 minutes, and 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 MAX-350 light source under ice-cold conditions for 30 minutes. The reaction solution was analyzed using 20% ​​denaturing PAGE to study the efficiency of the cross-linking reaction. RNA bands were analyzed using SYBR Green.TM After staining with Green II Nucleic Acid Gel Stain (Takara Bio), the images were visualized using a ChemiDoc MP imager (Bio-Rad).

[0156] Circular Nluc mRNA was mixed with a photocrosslinked probe (probe 3) and a non-crosslinked probe (probe 4) in a solution (8.8 μL: containing 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), and 1 mM EDTA (pH 8.0)). The solution was then heated at 90°C for 3 minutes and slowly cooled to room temperature. The solution was then subjected to a 300W xenon light source (MAX-350) at 4.0 mW / cm². 2 Irradiated with 365nm light for 30 minutes under cold conditions. Lipofectamine (registered trademark) was used in Messenger MAX. TM Reagent introduced it at 10 ng mRNA / well the day before into a 96-well multi-well plate at 1.0 × 10⁻⁶ mRNA / well. 4 After a total of 24 hours of culture, the seeded HeLa cells were lysed. The amount of Nluc protein in the cell lysates was evaluated using a Nano-Glo (registered trademark) luciferase assay system (Promega). Chemiluminescence assays were performed using a TriStar5 multi-functional plate reader (Berthold).

[0157] The results are shown in Figure 3 This indicates that photocrosslinking can promote the translation response.

[0158] Based on the results of the above experimental examples 1 to 3, it is shown that, as a particularly preferred method of composition, 5′ capped polynucleotides and single-stranded RNA are firmly linked and stabilized by RISC, Cas proteins and photocrosslinking, thereby promoting the translation reaction.

[0159] Experimental Example 4. Promotion of Rolling Circle Translation Reaction by Capped RNA The concept diagram of this experiment is shown in... Figure 4 (a).

[0160] The base sequence of the circular RNA used in this experiment is shown below.

[0161] Linear / Circular FLAG-EGF (264-nt) 5′_GGGAGCCACCAUGGACUACAAGGACGACGACGACAAGAUCAUCGACUAUAAAGACGACGACGAUAAAGGUGGCGACUAUAAGGACGACGACGACAAAGCCAUCAACAGCGACAGCGAGUGCCCCCUGAGCCACGACGGCUACUGCCUGCACGACGGCGUGUGCAUGUACAUCGAGGCCCUGGACAAGUACGCCUGCAACUGCGUGGUGGGCUACAUCGGCGAGAGAUGCCAGUACAGAGACCUGAAGUGGUGGGAGCUGAGACU_3′ (Sequence number 2) (in the ring, 5′ and 3′ ends are bound).

[0162] The base sequences of the 5′ phosphorylated RNA oligonucleotides and 5′ capped RNA oligonucleotides used in this experiment are shown below.

[0163] 5' Capped Probe 1 5′_m7G_ppp_GCCACCUCUCUGUACUGGCAUCUCUC_3′ (Serial Number 3) 5' Capped Probe 2 5′_m7G_ppp_GACAGUCUGCUCGAAGCGGCCGCUCUAGAAUCUCUGUACUGGCAUCUCUC_3′ (Serial Number 4) 5' Capped Probe 3 5′_m7G_ppp_GACAGUCUGCUCGAAGCGGCCGCUCUAGAACCGCGUAUAAUUCCACUGCG_3′ (serial number 5).

[0164] Circular RNA (Sci Rep 2015, 5, 16435) was prepared as follows. First, 5′ phosphorylated linear RNA, used as the raw material before circularization, was prepared by a transcription reaction consisting of the following components: 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 solution was heated 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 heated at 37°C for 15 minutes. 7.5 M lithium chloride aqueous solution was added to the reaction solution 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 transcribed RNA as a precipitate. The crude transcribed RNA product was purified by reverse-phase HPLC according to existing reports (Nat. Commun. 14, 2657, 2023). The 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 shown below: 1 μM 5′ phosphorylated RNA, 10% PEG6000, 50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1 mM DTT, 400 μM ATP, 0.1 μg / μL T4 RNA ligase 2. The reaction solution was heated at 37°C for 1 hour, then extracted with an equal volume of TE saturated phenol-chloroform mixture to remove proteins. Sodium acetate aqueous solution (pH 5.2, final concentration 0.3 M) and 2-propanol were added, and the mixture was cooled at -30°C and centrifuged. The RNA was recovered as the precipitate. The target RNA circularized bodies were purified by preparative denaturing polyacrylamide gel electrophoresis.

[0165] Capped RNA oligonucleotides were prepared according to existing reports (ACS Chem Biol 2022, 17, 1308-1314). The 5′ phosphorylated RNA oligonucleotide sequences were synthesized using a commercially available phosphoramide compound (ChemGenes) via an automated nucleic acid synthesizer NR-2A_7MX (Techno Service, Japan). After synthesis, a 1:1 mixture of 40% methylamine aqueous solution and 28% concentrated ammonia was added to the CPG solid-phase support, and the mixture was heated at 65°C for 30 minutes to cleave the oligonucleotides from the support and deprotect the phosphate and base portions. The oligonucleotides were dried under reduced pressure, incubated overnight at room temperature with 1 M TBAF / THF solution to deprotect the TOM protecting group, desalted using a NAP-25 gel filtration column (Cytiva), and precipitated with ethanol to obtain the RNA oligonucleotides as the precipitate. The oligonucleotides were then purified by reversed-phase HPLC. The obtained 5′ phosphorylated RNA oligonucleotides were reacted with m7GDP imidazole (Im-m7GDP) to introduce a cap structure at the 5′ end. The capping reaction solution (20 μM phosphorylated RNA oligonucleotides, 10 mM Im-m7GDP, 1 M 1-methylimidazole, DMSO solution) was heated at 55 °C for 3 hours, and the RNA was recovered by ethanol precipitation. The target RNA was then separated and purified by reversed-phase HPLC. The capping reaction was confirmed by detecting gel migration of RNA bands using denaturing PAGE and by determining the molecular weight using LC-MS [5′ capping probe 1: Calcd 8632.0, Found 8633.06, (+1.1); 5′ capping probe 2: Calcd 16425.7, Found; 16428.8 (+3.1); 5′ capping probe 3: Calcd 16511.8, Found; 16512.0 (+0.2)].

[0166] The translation reaction using circular RNA from rabbit reticulocyte lysate (Promega) was performed as follows. The translation buffer (2 μM circular RNA, 4 μM RNA probe, 70 (v / v)% nuclease-treated reticulocyte lysate, 10 μM cysteine-free amino acid mixture, 10 μM leucine-free amino acid mixture, and 0.8 units / μL murine RNase inhibitor (New England Biolabs)) was incubated at 30°C for 4 hours. A portion (4 μL) of the reaction solution was aliquoted and analyzed by Western blotting. After electrophoresis with a 5–20% gradient SDS-polyacrylamide (Atto), proteins and peptides were semi-dry transferred onto an Immobilon-P membrane (Merck). The membrane was then reacted sequentially with anti-FLAG M2 antibody (Sigma-Aldrich) and anti-mouse IgG-HRP complex (Sigma-Aldrich) using ChemiDoc. MP Imager (Bio-Rad) captured images using SuperSignal TM Chemiluminescence produced by the West FemtoMaximum Sensitivity Substrate (Thermo Fisher).

[0167] The results are shown in Figure 4 A significant increase in translational products was confirmed in the presence of capped oligoRNA probes containing regions complementary to the circular RNA sequence.

[0168] Experimental Example 5. A Highly Efficient Synthesis of Cap-2 Type mRNA Using PureCap Analogs and Mutant Promoters development Experimental Example 5-1. Synthesis of PureCap Analogs The synthesized PureCap analogues are represented as follows. Liquid-phase synthesis of trinucleotides Trinucleotides Synthesis Scheme of Trinucleotide, pAGG(33) DMTr protection, synthesis of compound (23) DMTrCl (11.5 g, 33.98 mmol) from pyridine (100 mL) was added to N in pyridine (200 mL). 2 The mixture of -isobutyrylguanosine (22) (10.0 g, 28.3 mmol) was stirred at room temperature for 15 hours. The reaction was monitored by TLC, and quenched with water after completion. 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 by rotary evaporator, and the residue was purified by silica gel column chromatography in DCM with 5% MeOH (1% TEA) to give compound 23 (19.0 g, quantified) as a fuchsin solid. 1 H-NMR (400 MHz, DMSO-d6) δ11.57 (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.

[0169] Acetyl protection, synthesis of compound (24) Anhydrous acetic acid (27.4 mL, 290 mmol) was added to a solution of compound 23 (19.0 g, 29.0 mmol) in pyridine (200 mL). The mixture was stirred at room temperature for 15 hours. The reaction was monitored by TLC, and quenched with water after completion. 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 by rotary evaporation, and the residue was purified by silica gel column chromatography with 5% MeOH (1% TEA) in DCM to give compound 24 (23.1 g, quantified) as a pale brown solid. 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, 7.0Hz, 3H) ppm.

[0170] Detriphenylmethylation and synthesis of compound (25) A solution of trichloroacetic acid (25.5 g, 156 mmol) in dichloromethane (78.0 mL) was injected at 0 °C into a solution of compound 24 (23.1 g, 31.2 mmol) in dichloromethane (100 mL). After addition, the reaction mixture was warmed to room temperature and stirred overnight. After the reaction was complete, methanol (15.0 mL) was added to the reaction mixture to quench the reaction. The mixture was then washed with a saturated aqueous solution of NaHCO3 (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 (3.90 g, 70.0% yield) as a white solid. 1 H-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.

[0171] Synthesis of the dinucleotide nucleotide G(2'OMe)pG(2'OMe) (28) Compound 25 (1.00 g, 2.29 mmol), compound 26 (2.58 g, 2.97 mmol), and molecular sieve 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 the mixture was stirred for 4 h. After complete conversion of compound 25, 5-6 M TBHP (600 μL, 2.97 mmol) from decane was added to the reaction mixture, and the mixture was 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 and concentrated on Na2SO4 without purification for use in the next step, yielding (3.48 g, white solid). 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 the mixture was stirred at room temperature for 3 hours. 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 the mixture was purified by silica gel column chromatography using 0–5% MeOH / dichloromethane to give compound 28 as a white solid (1.10 g, yield 53.0%, after two stages). 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 = 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..

[0172] Synthesis of the trinucleotide A(2'OMe)pG(2'OMe) (31) Compound 28 (100 mg, 0.108 mmol), compound 29 (126 mg, 0.140 mmol), and molecular sieve 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.

[0173] The reaction mixture was then diluted with dichloromethane and washed with saturated aqueous NaHCO3 and brine. The organic layer was dried and concentrated on Na2SO4 without purification and used in the next step. 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 the mixture was stirred at room temperature for 3 hours. 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 the mixture was purified by silica gel column chromatography using 0–5% MeOH / dichloromethane to give compound 31 (125 mg, 81% yield, in two stages) as a white solid. 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, 2.6Hz, 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 = 2.0Hz, 0H), 2.08 (dd, J = 6.1, 1.6 Hz, 3H), 1.98 (s, 3H), 1.09-1.07 (m, 12H)ppm.

[0174] Synthesis of the trinucleotide polymorphism pA(2'OMe)pG(2'OMe) (33) Compound A (30 mg, 0.02 mmol, 1 eq), compound B (22.85 mg, 0.084 mmol, 4 eq), and molecular sieve 3A (30 mg) were dissolved in dry ACN (1 mL). After stirring for 20 min, 0.4 M 1H-tetrazole (0.2 mL, 0.086 mmol, 4.1 eq) was added at room temperature, and the mixture was stirred for 2 h. After complete conversion of SM A, 5 M TBHP (6 μL, 0.03 mmol, 1.5 eq) was added, and the mixture was stirred for another 1 h. The reaction mixture was then diluted with DCM and washed with NaHCO3 and brine. The organic fraction was dried on Na2SO4 and removed under vacuum for use in the next step. Compound A (44 mg, 0.04 mmol, 1 eqw) was dissolved in MeOH (0.5 mL), and 1 mL of aqueous NH4OH solution was added at room temperature, and the mixture was stirred for 20 h. After complete conversion of SM, the reaction mixture was diluted with MeOH and evaporated three times by rotary evaporation. The crude compound was then dissolved in MQ and purified using a DEAE column with a linear gradient of 270 min. After evaporation of the solvent, 5 mg of the compound as a pure white solid 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 = 13.7Hz, 4H), 5.99 (d, J = 6.7 Hz, 1H), 5.80 (d, J = 7.9 Hz, 1H), 5.64 (d, J = 6.3Hz, 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.

[0175] Synthetic scheme of the trinucleotide pUGG (38) Synthesis of the trinucleotide UGG (36) Compound 18-4 (343.3 mg, 0.37 mmol, 1 eq.) was co-evaporated with toluene to remove water. Then, 2'-OMe-U-CE phosphorous amide (368.96 mg, 0.48 mmol, 1.3 eq.) was added to a flask. 8 mL of acetonitrile was added to dissolve the compound, followed by the addition of 180 mg of molecular sieve (3 Å). After stirring for 5 minutes, 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 hours, followed by the addition of tert-butyl hydroperoxide solution (in decane, 5–6 M, 0.12 mL, 0.6 mmol, 1.6 eq.) to oxidize the resulting compound. The mixture was then stirred further for 60 minutes, and 5 mL of dichloromethane was added to dilute the reaction mixture. The mixture was filtered through MS 3 Å filter paper, washed with 15 mL of dichloromethane, and then washed with 40 mL of water. The organic layer was collected, and trichloroacetic acid (372 mg, 3.2 mmol, 8.8 eq.) was added in an ice bath. The mixture was then stirred for 30 minutes. After complete conversion, dichloroethane was added to the mixture, and the mixture was washed with NaHCO3. The organic layer was dried (Na2SO4) and concentrated. Purification was performed using column chromatography (silica gel, dichloromethane:methanol = 19:1 to 9:1). 280 mg of a white solid was given (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, 16.6ppm. 31 P-NMR (240 MHz, DMSO-D6) δ -1.6, -1.9 ppm. HRMS (ESI) Calcd. ForC 49 H62 N 14 NaO 24 P + ([M+Na]) + ): 1315.3429. Obsd.1315.3428.

[0176] Synthesis of the trinucleotide pUGG (38) Compound 18-6-1 (170 mg, 0.13 mmol, 1 eq.) was co-evaporated once with toluene to remove water. Bis(2-cyanoethyl)diisopropylphosphonamide (144 mg, 0.52 mmol, 4 eq.), 3 Å molecular sieve (135 mg), and acetonitrile (5 mL) were added. After stirring under argon atmosphere for 9 minutes, 1H-tetrazole (45 mg, 0.64 mmol, 5 eq.) was added. The mixture was stirred at ambient temperature for 4 hours, and TBHP (in decane, 5–6 M, 52 μL, 0.26 mmol, 2 eq.) was added dropwise. The reaction mixture was diluted with dichloromethane after 1 hour and washed with water. The organic layer was dried (Na₂SO₄) and concentrated. The crude product was used directly for deprotection. The resulting solid was dissolved in 1.8 mL of methanol, and 1.7 mL of NH₄OH solution (28%) was added. After stirring for several days, the reaction mixture was transferred to 55 °C to achieve complete conversion of the starting compound. The solution was then removed by rotary evaporation. The resulting solid was dissolved in 5 mL of water and loaded onto a DEAE-Sephadex™ A-25 column (70 cm⁻¹). 3 The fraction was purified by ion exchange chromatography (0–1.5 M TEAB buffer containing 10% CH3CN, linear gradient of 15–270 min, flow rate 6 mL / min). Fractions with retention times of 110–150 min were collected, and the solvent was removed. 50 mg of a white solid was obtained (yield: 25%, after two stages). 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. ForC 31 H 40 N 12 O 23 P3 - ([M-H] - ): 1041.1548. Obsd.1041.1543.。

[0177] Synthetic scheme of trinucleotide pCGG(X) Synthesis of the trinucleotide CGG (41) Compound 18-4 (95 mg, 0.1 mmol, 1 eq.) was co-evaporated with toluene to remove water. Then, 2'-OMe-CE-phosphamide (101 mg, 0.13 mmol, 1.3 eq.) was added to a flask. 2 mL of acetonitrile was added to dissolve the compound, followed by the addition of 126 mg of molecular sieve (3 Å). After stirring for 15 minutes, 1H-tetrazole (32 mg, 0.45 mmol, 4.5 eq.) was added to the mixture. The mixture was then stirred for 3 hours under an argon atmosphere, and tert-butyl hydroperoxide solution (in decane, 5–6 M, 0.02 mL, 0.12 mmol, 1.3 eq.) was added to oxidize the resulting compound. The mixture was then stirred further for 45 minutes, and approximately 5 mL of dichloromethane was added to dilute the reaction mixture. The mixture was filtered through MS 3 Å filter paper, washed with 5 mL of dichloromethane, and then washed with 40 mL of water. The organic layer was collected, and trichloroacetic acid (220 mg, 1.3 mmol, 13 eq.) was added in an ice bath. The mixture was then stirred for 30 minutes. After complete conversion, dichloroethane was added to the mixture, and the mixture was washed with NaHCO3. The organic layer was dried (Na2SO4) and concentrated. Purification was performed using column chromatography (silica gel, dichloromethane:methanol = 9:1, 1% TEA). 40 mg of a white solid was given (yield: 30%). 1 H-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.13 C-NMR (101 MHz, METHANOL-D4) δ 180.4, 180.3, 171.8,171.7, 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, 20.6, 17.5, 17.3. 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, 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, 47.7, 47.7, 47.9, 47.9. 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, 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 THEIR 24 P2 + ([M+H] + ): 1334.3875. Obsd. 1334.3874。

[0178] Synthesis of the trinucleotide pCGG (43) Compound 18-6-2 (110 mg, 0.13 mmol, 1 eq.) was co-evaporated once with toluene to remove water. Bis(2-cyanoethyl)diisopropylphosphonamide (90 mg, 0.52 mmol, 4 eq.), 3 Å molecular sieve (80 mg), and acetonitrile (5 mL) were added. After stirring under argon atmosphere for 20 min, 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 (in decane, 5–6 M, 0.12 mL, 0.26 mmol, 2 eq.) was added dropwise. The reaction mixture was diluted with dichloromethane after 2 hours and washed with water. The organic layer was dried (Na₂SO₄) and concentrated. The crude product was used directly for deprotection. The resulting solid was dissolved in 1.8 mL of methanol, and 1.7 mL of NH₄OH solution (28%) was added. After stirring for several days, the reaction mixture was transferred to 55 °C to achieve complete conversion of the starting compound. The solution was then removed by rotary evaporation. The resulting solid was dissolved in 5 mL of water and loaded onto a DEAE-Sephadex™ A-25 column (70 cm⁻¹). 3 The fraction was purified by ion exchange chromatography (0–1.5 M TEAB buffer containing 10% CH3CN, linear gradient of 15–270 min, flow rate 6 mL / min). Fractions with retention times between 110 and 150 min were collected, and the solvent was removed. 35 mg of a white solid was obtained (yield: 25%, after two stages). 1 H-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. 13C-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..

[0179] Solid-phase synthesis of trinucleotides using an automated nucleic acid synthesizer Synthesis scheme Reaction conditions and experimental procedures Dinucleotides and trinucleotides were synthesized using a DNA / RNA synthesizer NR-2A_7MX (TechnoService, Ushiku City, Ibaraki Prefecture, Japan) with a Primer Support 5G riboG 300 (Cytiva, 15.0 μmol scale synthesis). In the coupling process, 100 mM 2'-O-methyl-adenosine (n-benzoyl)-CE-phosphoramide (ChemGenes) from CH3CN, 100 mM 2'-O-methyl-adenosine (n-benzoyl)-CE-phosphoramide (synthesized according to literature) from CH3CN, 100 mM 2'-O-methyl-guanosine (ni-Bu)-CE-phosphoramide (ChemGenes) from CH3CN, 50-70 mM 5'-DMTr-2'-TOM-riboadenosine (n-acetyl)OP (ChemGenes) from CH3CN, 150 mM bis(2-cyanoethyl)-N,N-diisopropylphosphoramide from CH3CN, and 0.3 M 5-(benzylthio)-1H-tetrazole (activator) from CH3CN are circulated in the column. A 184 mM trichloroacetic acid / dichloromethane solution was used as the detriphenylmethylation reagent. Oxidation was performed using a 0.05 mM pyridine solution / H₂O (9:1, v / v). For Cap A, a 10% Ac₂O THF / pyridine solution (8:1, v / v) was used, and for Cap B, a 10% 1-methylimidazole THF solution was used. Synthesis was performed using standard RNA synthesis procedures at a 10 μmol scale. After the final cycle of synthesis, the solid support was treated with a 1:1 mixture of 28% ammonium hydroxide / 40% methylamine (1.00 mL / 15.0 μmol of solid support) at 65 °C for 1–2 h to cleave the nucleotides from the solid support and remove the protecting groups. The obtained nucleotides were concentrated until dry and then dissolved in DMSO (1.00 mL / 60.0 μmol). TEA-3HF (1.00 mL / 60.0 μmol) was added to the solution, and the mixture was incubated at 65 °C for 3–5 hours to remove the 2'-O-methylsilyl protecting group. The reaction mixture was then neutralized by adding 250 mM Na₂CO₃ aqueous solution (10.0 mL / 60.0 μmol). The solution was then analyzed using DEAE-Sephadex (φ = 4.5 cm, h = 8.4 cm, 140 cm⁻¹). 3 Ion-exchange chromatography was performed at 12.0 mL / min, and the crude product was purified using a linear 0–1.2 M gradient (270 min) with TEAB buffer (pH 7.9) containing 0–8% CH3CN. The fraction containing the product was collected and concentrated to obtain di / trinucleotides. Yields were calculated using the absorbance of the product at 260 nm as measured by NanoDrop. Extinction coefficients (ε) were used in the calculations for dinucleotides.260 ) = 25,000 M -1 -cm -1 Regarding the use of 35,100 M trinucleotides in calculations -1 -cm -1 .

[0180] pUUG (triethylammonium salt, ε) 260 = 29,700 M -1 •cm -1 (37.7% yield) (44): 1 H 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, 3.3Hz, 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] - ; found1002.13203.

[0181] pUCG (triethylammonium salt, ε) 260 = 27,800 M -1 •cm -1 (20.7% yield) (45): 11H 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 13C NMR (151 MHz, D2O) δ 165.93, 163.21, 158.73, 153.75, 151.68, 151.22, 141.26, 140.86, 137.92, 116.40, 102.12, 95.72, 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 31P 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.

[0182] pUAG (triethylammonium salt, ε 260 = 34,200 M -1 •cm -1 −1, 28.0% yield) (46): 11H 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 13C 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 31P 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.

[0183] 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 N 10 O 23 P3, 1001.14861 [M - H]-; found 1001.14034.

[0184] pCCG (triethylammonium salt, ε) 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. 13 C NMR (151 MHz, D2O) δ181.18, 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 C30 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): 1 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 ¹³C 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. 31P 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.

[0185] pAUG (triethylammonium salt, ε 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. 13CNMR (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. 31 P 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.

[0186] pACG (triethylammonium salt, 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), 4.22 (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. forC 31 H 41 N 13 O 21 P3, 1024.17583 [M - H] - ; found 1024.17943.

[0187] pAAG (triethylammonium salt, ε) 260 = 37,000 M -1 •cm -1 (28.3% yield) (52): 11H 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 13C 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 31P 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.

[0188] 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 = 4.8Hz, 1H), 5.87 (d, J = 4.0 Hz, 1H), 5.82 (d, J = 5.8 Hz, 1H), 5.65 (d, J =8.1Hz, 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, 192.07, 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 N 12 O 23 P3, 1041.15476 [M - H] -; found 1041.14643.

[0189] pGCG (triethylammonium salt, 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 C 31 H 41 N 13 O 22 P3,1040.17074 [M - H] -; found 1040.16350.

[0190] pGAG (triethylammonium salt, ε) 260 = 34,800 M -1 •cm -1 (37.7% yield) (55): 11H NMR (600 MHz, D2O, 0.2:1 mixture of rotamers) 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, \(1.5\)H), 4.87 - 4.83 (m, \(1.5\)H), 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 mixture of rotational isomers) 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 (243MHz, D2O, 0.2:1 rotational isomer 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.

[0191] pGGG (triethylammonium salt, ε) 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.

[0192] pUUA (triethylammonium salt, ε 260 = 34,300 M -1 •cm-1 , (57) in a yield of 33.8%: 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.

[0193] pCUA (triethylammonium salt, ε 260 = 30,900 M -1 •cm-1 (58): Yield: 30.0% 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. 31 P 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 22P3, 985.15370 [M -H] - ; found 985.14609.

[0194] pAUA (triethylammonium salt, 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. 13 C 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. 31P 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] - ; found1009.15687.

[0195] pGUA (triethylammonium salt, ε) 260 = 35,900 M -1 •cm -1 33.8% yield (60): 1 H 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). 13C NMR (151MHz, D2O) δ 165.54, 158.45, 154.82, 153.63, 151.94, 151.24, 148.79, 140.59,139.76, 137.07, 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 P 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] - ; found1025.15478.

[0196] pUUAG (triethylammonium salt, ε) 260 = 43,900 M -1 •cm -1 (61) % yield 1H 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 (151MHz, 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. 31 P 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 C41 H 52 N 14 O 30 P4,672.0968 [M - 2H] 2- ; found 672.0945.

[0197] Synthesis of tetranucleotide PureCap analogues Synthesis scheme Experimental steps N was added to a DMSO (802 μL) solution of trinucleotide UUG triethylammonium salt (56.5 mg, 40.1 μmol). 7 Sodium methylguanosine diphosphate imidazole (92.7 mg, 120 μmol) was added to 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 purified product was purified by reversed-phase HPLC: Shimadzu preparative column YMC-Actus Triart C8 (preparative, 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. The fraction containing the target analyte was collected, concentrated, freeze-dried, and the tetranucleotide PureCap analogue m7GpppUUG was obtained as triethylammonium salt. The product was redissolved in methanol (2.0 mL), and a solution of 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 resuspended in acetone. This suspension-centrifugation process was repeated three more times. The precipitate was dried under reduced pressure to obtain the target PureCap analog, m7GpppUUG (17.7 mg, 9.96 μmol, yield 24.8%), as its sodium salt. The yield was calculated using the absorbance of the product at 260 nm as measured by NanoDrop.

[0198] 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) δ165.90; 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, 82.00. 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, 64.36, 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.

[0199] Nb-7mGpppUCG(X.ε 260 = 42,900 M -1 •cm -1 、26.4% share)(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, 165.48, 158.79,156.68, 153.70, 151.64, 151.22, 149.82, 149.67, 149.24, 148.65, 141.12,140.19, 137.94, 133.76, 132.70, 132.35, 130.17, 130.02, 128.86, 128.20,123.88, 123.38, 116.51, 108.04, 107.72, 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, 73.37,71.80, 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.

[0200] Nb-7mGpppUAG (sodium salt, 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, 161.52,158.66, 155.40, 153.79, 152.79, 151.33, 149.86, 149.63, 149.30, 148.58,148.27, 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, 116.32, 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, 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-MScalcd. for C 54 H 69 N 18 O 35 P5, 842.14359 [M - 2H] 2- ; found 842.15626.

[0201] Nb-7mGpppUGG (triethylammonium salt, 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) δ181.4, 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. 31P-NMR (160 MHz, D2O) δ -0.3, -0.6, -11.1, -11.2, -22.3, -22.4, -22.5 ppm.HRMS (ESI) Calcd. ForC 54 H 69 N 18 O 36 P5 2- ([M-2] 2- ): 850.1410. Obsd. 850.1405.

[0202] 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 165.92,165.33, 158.79, 156.70, 153.83, 151.67, 150.88, 149.76, 149.23, 148.73,140.56, 139.83, 137.54, 133.70, 132.82, 132.73, 132.33, 130.01, 128.84,128.22, 123.89, 123.46, 116.37, 107.98, 107.75, 102.26, 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. forC 53 H 69 N 16 O 36 P5, 830.1380 [M - 2H] 2- ; found 830.1369.

[0203] Nb-7mGpppCCG (sodium salt, ε) 260 = 39,900 M -1 •cm -1 (6): 14.4% yield 11H NMR (600 MHz, D2O) δ 7.79 - 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.�4, 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.

[0204] 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 = 7.6Hz, 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-MScalcd. for C 54 H 70 N 19 O 34 P5, 841.6516 [M - 2H] 2- ; found 841.6548.

[0205] Nb-7mGpppCGG (triethylammonium salt, ε 260 = 46,330 M -1 •cm -1 (21.0% yield) (8): 1 H-NMR (400MHz, 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. ForC 54 H 72 N 19 O 35 P51701.3126. Obsd. 1701.2997.

[0206] Nb-7mGpppAUG (sodium salt, ε) 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, 2.4Hz, 5H), 3.34 (d, J = 2.5 Hz, 1H), 0.63 - 0.55 (m, 9H) ppm. 13C NMR (151 MHz,D2O) δ 165.47, 158.54, 155.38, 155.15, 154.94, 154.34, 153.65, 152.38,151.14, 149.70, 149.29, 148.84, 148.61, 140.40, 139.62, 133.68, 132.65,132.30, 129.90, 128.78, 128.12, 123.81, 123.34, 107.23, 102.50, 94.71, 93.48,87.59, 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.

[0207] Nb-7mGpppACG (sodium salt, 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 CNMR (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. forC 54 H 71 N 19 O 34 P5, 1684.31044 [M - H] - ; found 1684.31722.。

[0208] 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. 13 C NMR(151 MHz, D2O) δ 158.11, 155.27, 155.07, 154.25, 154.13, 153.90, 153.50, 151.46, 151.06, 149.52, 149.08, 148.81, 148.68, 148.19, 148.00, 139.48, 139.11, 136.92, 133.32, 132.49, 132.13, 129.85, 129.61, 128.58, 128.06, 123.62, 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. 31P 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-MScalcd. for C 55 H 70 N 21 O 33 P5, 853.65720 [M - 2H] 2- ; found 853.66766.。

[0209] Nb-7mGpppAGG (sodium salt, ε 260 = 50,200 M -1 •cm -1 、36.0% yield) (12): 1 H 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. 13CNMR (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 PNMR (243 MHz, D2O) δ -0.26 (2P), -10.84 (2P), -22.09 (1P) ppm. HR-ESI-MScalcd. 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. 1313C 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 31P 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.

[0210] Nb-7mGpppGCG (sodium salt, ε 260 = 43,100 M -1 •cm -1 −1, 11.6% yield) (14): 1 1H 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. 13C NMR (151 MHz, D2O) δ 164.95, 158.94, 158.48, 155.47, 155.22,154.66, 154.54, 154.13, 153.51, 150.97, 149.55, 149.35, 148.83, 148.52,139.21, 136.70, 133.92, 132.75, 132.26, 129.96, 128.78, 128.08, 123.84,123.43, 116.45, 115.80, 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, 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.

[0211] Nb-7mGpppGAG (sodium salt, ε) 260 = 49,900 M -1 •cm -1 (32.3% yield) (15): 11H 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 13C 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 31P 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.

[0212] Nb-7mGpppGGG (sodium salt, ε 260 = 46,800 M -1 •cm -1 、28.8% yield) (16): 11H 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 13C NMR (151 MHz, D2O) δ 160.45, 159.60, 155.75 - 147.96, 136.37 - 127.56, 123.60, 116.54 - 113.91, 107.66, 94.16, 88.36 - 85.23, 83.43, 81.62 - 78.68, 75.72, 71.70, 69.97, 69.59, 68.54 - 67.97, 67.30, 65.70, 64.85 - 63.23, 61.99 - 61.52, 60.01, 57.92 - 56.88, 36.05, 35.44, 30.35, 24.78 ppm. 31 31P 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.

[0213] Nb-7mGpppUUA (sodium salt, 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 = 6.9Hz, 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 = 6.3Hz, 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) δ165.72, 155.57, 152.91, 151.39, 149.00, 148.23, 141.19, 140.82, 139.68,133.90, 132.59, 132.12, 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, 74.27, 72.51,72.01, 70.33, 69.16, 65.10, 64.32, 57.91, 57.73, 36.53, 35.96, 30.28, 24.66ppm. 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] - ; found1646.44951.

[0214] Nb-7mGpppCUA (sodium salt, 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. forC 53 H 70 N 16 O 35P5, 1645.28831 [M - H] - ; found 1645.28716.

[0215] Nb-7mGpppAUA (sodium salt, ε[[ID=六]] 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. 13 It should be noted that there seems to be an error in the original text where "六" is used instead of "6" in the relevant part. This has been corrected in the translation for better understanding.C NMR (151 MHz, D2O) δ165.48, 155.36, 155.16, 154.41, 154.30, 154.14, 151.19, 149.70, 149.23,148.84, 148.63, 140.58, 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] - ; found1669.29949.

[0216] Nb-7mGpppGUA (sodium salt, 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) δ165.53, 158.49, 157.94, 155.43, 153.70, 152.75, 151.63, 151.26; 148.47, 140.58, 139.63, 136.95, 133.68, 132.69,132.19, 129.93, 128.74, 127.97; 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] - ; found1685.29855.

[0217] Synthesis of PureCap pentanucleotide analogs Synthesis scheme Nb-7mGpppUUAG(High.ε 260 = 59,000 M -1 •cm -1 、29.2% likes)(21): 1H NMR (600MHz, 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 = 8.5Hz, 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) δ166.08, 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 Experimental Example 5-2. Synthesis of mRNA via co-transcriptional reaction using a TetraPureCap analog. Straight-stranded RNA was synthesized by transcription using double-stranded DNA as a template via T7 RNA polymerase. 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 shown below. Fw 1; CCCGGATCCTAATACGACTCACTATAGGCGCATATTAAGGTGACGCGT (Serial Number 24) Fw 2; CCCGGATCCTAATACGACTCACTATAAGGCGCATATTAAGGTGACGCGT (Serial Number 25) Fw 3; CCCGGATCCTAATACGACTCACTATATGGCGCATATTAAGGTGACGCGT (Serial Number 26) Fw 4; CCCGGATCCTAATACGACTCACTATACGGCGCATATTAAGGTGACGCGT (Serial Number 27) Fw 5; CCCGGATCCTAATACGACTCACTATAGGGCGCATATTAAGGTGACGCGT (Serial Number 28) Fw 6; CCCGGATCCTAATACGACTCACTATATCGGCGCATATTAAGGTGACGCGT (Serial Number 29) 7; CCCGGATCCTAATACGACTCACTATATAGGCGCATATTAAGGTGACGCGT (Serial No. 30) Fw 8; CCCGGATCCTAATACGACTCACTATATTGGCGCATATTAAGGTGACGCGT (Serial No. 31) Fw 9; CCCGGATCCTAATACGACTCACTATACCGGCGCATATTAAGGTGACGCGT (Serial No. 32) Fw 10; CCCGGATCCTAATACGACTCACTATACTGGCGCATATTAAGGTGACGCGT (Serial No. 33) Fw 11; CCCGGATCCTAATACGACTCACTATACAGGCGCATATTAAGGTGACGCGT (Serial No. 34) Fw 12; CCCGGATCCTAATACGACTCACTATAGTGGCGCATATTAAGGTGACGCGT (Serial No. 35) Fw 13; CCCGGATCCTAATACGACTCACTATAGCGGCGCATATTAAGGTGACGCGT (Serial Number 36) Fw 14;Serial Number 37 (Fw 15); Serial Number 38 (Fw 16); Serial Number 39 (Fw 17); Serial Number 40 (Fw 18); Serial Number 41 (Fw 19); Serial Number 42 (Fw 20); CCCGGATCCTAATACGACTCACTATCTAGGCGCATATTAAGGTGACGCGT (Serial No. 43) Fw 21; CCCGGATCCTAATACGACTCACTATGTAGGCGCATATTAAGGTGACGCGT (Serial No. 44) Rev; TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTAGAATTACGCCAGAATGCG (Serial No. 45); The transcription reaction solution was prepared (10 ng / μL template DNA, 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 MTT, 2 mM ATP, 2 mM UTP, 2 mM CTP, 2 mM GTP, 0.5–2 mM TetraPureCap analog), and incubated at 37°C for 1 hour. Recombinant DNase (Takara) was added to the reaction solution to a concentration of 0.1 U / μL, and the solution was incubated at 37°C for 30 minutes to degrade the template DNA.

[0218] After the reaction, an equal volume of 7.5 M LiCl was added to the IVT reaction solution, and the mixture was incubated at -30°C for 30 minutes. The precipitate was then obtained by centrifugation at 15,000 rpm for 30 minutes. The precipitate was purified by reversed-phase HPLC. The purification conditions were as follows: column: YMC-TriartBioC4 (250 mm × 4.6 mm ID); solution 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.

[0219] Deprotection through light irradiation RNA solution was added to a clear 96-well multi-well plate and filtered through a MAX-305 light source (Asahita spectroscopy) at 4 mW / cm². 2 Irradiate with 365 nm light for 10 minutes. Perform HPLC purification. Chromatographic column: YMC-TriartBioC4 (250 mm × 4.6 mm I.D.); 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 Evaluation of intracellular translational activity HeLa cells (RIKEN Cell Bank) were cultured in Dulbecco modified Eagle medium (DMEM; WAKO) supplemented with 10% fetal bovine serum (FBS; Invitrogen) (37°C, 5% CO2). One day prior to transfection, HeLa cells (1.0 × 10⁶ cells / well) were seeded into 96-well plates. 4 Cells / well). The next day, remove the culture medium and replace it with 100 μL / well Opti-MEM (registered trademark) (Thermo Fisher Scientific). Add 0.15 μL Lipofectamine (registered trademark) MessengerMAX. TM5 ng mRNA was diluted with 10 μL of Opti-MEM (trademarked) and introduced into cells. After 2 hours, the medium was replaced with Dulbecco modified Eagle medium (DMEM; WAKO) supplemented with 10% fetal bovine serum (FBS; Invitrogen). After incubation at 37°C for 22 hours, cells were lysed by adding 20 μL / well 1× cell lysis buffer (Promega). Luminescence assay was performed using the Nano-Glo (trademarked) luciferase assay (Promega).

[0220] The results are shown in Figures 5-7 It has been shown that by inserting bases into the promoter in a manner complementary to the base sequence of the cap-like polynucleotide at a specific positional relationship with the upstream base sequence from the transcription start site of the promoter, the proportion of capped RNA synthesized relative to the total transcription (capping efficiency) is increased. Furthermore, it has been shown that high translation efficiency can be obtained by using the sequence of the cap-like polynucleotide.

[0221] Example 6. Translation reaction using chemically synthesized circular mRNA containing an m7G cap structure. As a raw material for circular RNA Figure 8RNA oligonucleotides representing the sequence were synthesized using commercially available phosphoramidite compounds (manufactured by ChemGenes and Glen Research) via a standard nucleic acid automated synthesizer, NTS T8-A20R8NC (manufactured by Techno Service, Japan). Branching structures were introduced using Glen Research's Asymmetric Doubler (Lev) Phosphoramidite (cat# 10-1981). After synthesis, a 1:1 mixture of 40% methylamine aqueous solution and 28% concentrated ammonia was added to the CPG solid-phase support, and the mixture was heated at 65°C for 30 minutes to cleave the oligonucleotides from the support, thereby deprotecting the phosphate and base portions. After drying and curing under reduced pressure, the residue was dissolved in 1 M TBAF / THF solution and incubated overnight at room temperature to deprotect the TOM protecting group. After desalting using a NAP-25 gel filtration column (Cytiva), the RNA oligonucleotides were precipitated with ethanol to obtain the precipitate. The oligonucleotides were then purified by reversed-phase HPLC. The purity and molecular weight of the target analytes were confirmed using a Q-TOF LC / MS system (Agilent Technologies). The 5′ phosphate group of the branched chain was reacted with m7GDP imidazole (Im-m7GDP) according to existing reports (ACS ChemBiol 2022, 17, 1308-1314) to introduce an m7G cap structure. The capping reaction solution (20 μM phosphorylated RNA oligonucleotides, 10 mM Im-m7GDP, 10 mM 2-nitroimidazole, 10 mM calcium chloride, DMSO solution) was heated at 55°C for 3 hours, followed by ethanol precipitation to recover the RNA. The target RNA was then separated and purified by reversed-phase HPLC. The 5′ end of the oligonucleotide backbone was enzymatically phosphorylated (8 μM) using T4 polynucleotide kinase (Takara Bio). uM oligo, 1 mM ATP, 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 5 mM DTT, 0.2 U / uL T4PNK; incubated at 37°C for 1 hour). Each pair of RNA oligonucleotide fragments prepared in this way was ligated with T4 RNA ligase 2 in the presence of template DNA oligonucleotides to prepare circular RNA. The template DNA oligonucleotides used had sequences complementary to the terminal portions of the ligated RNA strands. The two types used in the construction of the 127-nt circRNA were 5′ d (CACCATGGTGGCTCTGGACTAGGAG (SEQ ID NO. 52)) 3′ and 5′ d (TTTTTTTTTTTTTTTTTATCACAGTTTTTC (SEQ ID NO. 53)) 3′.Two 5′ d (CACCGAGGCTCCAGCTTATCACAGTTTTTC (Sequence No. 54)) 3′ and 5′ d (CACCATGGTGGCTCTGGACTAGGAG (Sequence No. 55)) 3′ ligases were used for the construction of 167-nt circRNAs. The composition of the ligase reaction solution is shown below: 1 μM RNA oligonucleotides (2 types), 3 μM DNA oligonucleotides (2 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 2. After heating the reaction solution at 37°C for 2 hours, the RNA was extracted with an equal volume mixture of TE saturated phenol-chloroform and deproteinized. Sodium acetate aqueous solution (pH 5.2, final concentration 0.3 M) and 2-propanol were added, and the mixture was centrifuged after cooling at -30°C. The RNA was recovered as the precipitate. The reaction was confirmed by denaturing acrylamide gel electrophoresis (PAGE), and the target RNA linkers were isolated and purified by preparative denaturing polyacrylamide gel electrophoresis (Sci. Rep., 5, 16435, 2015). The expression level of HiBiT peptide was evaluated using the human cell line HeLa (Riken Cellbank, RCB0007). HeLa cells were cultured in DMEM (Wako Pure Chemical Industries Co., Ltd.) medium containing 10% FBS (Thermo Fisher) at 5% CO2 concentration and 37°C. One day before transfection, HeLa cells were seeded at 1×10E4 cells / well in 96-well multi-well plates. Circular RNA was mixed with 0.2 pmol or 2 pmol per well with 0.3 μL of Lipofectamine MessengerMAX (Thermo Fisher), incubated for 5 minutes, and then added to Opti-MEM. TM I. The HeLa cells were replaced with low-serum medium (Thermo Fisher) and RNA was introduced into the cells. After 3 hours, an equal volume (100 μL) of growth medium was added, and the cells were cultured for another 3 hours. The expression level of HiBiT peptide in the cell lysate was evaluated using the Nano Glo HiBiTLytic Detection System (Promega). Chemiluminescence assays were performed using a TriStar5 multi-functional plate reader (Berthold).

[0222] The results are shown in Figure 8 and 9 It can be seen that chemically synthesized circular mRNA containing an m7G cap structure can efficiently produce peptides.

[0223] Example 7. In vivo persistence of chemically synthesized circular mRNA containing an m7G cap structure. Experimental item [Evaluation of HiBiT peptide expression in vivo] Lipid nanoparticles encapsulating HiBiT mRNA (HiBiT-mRNA, sequence 59) were administered intravenously to the tail vein of 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 weeks old, female) at a dose of 10 μg of mRNA per mouse. Liver samples were collected at 6, 24, 30, or 48 hours after administration. The liver was minced with scissors and homogenized using magnetic beads 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 min), and the supernatant was collected. After mixing 30 μL of supernatant with 60 μL of HiBiT substrate solution (NanoGlo HiBiT Lytic Detection System, Promega, N3040), the luminescence value (RLU) was measured using a photometer. The supernatant was diluted 50-fold, and the total protein mass (mg protein) was determined by the BCA protein assay. The luminescence value (RLU / mg protein) was then corrected using the protein mass.

[0224] The results are shown in Figure 10 It can be seen that chemically synthesized circular mRNAs containing the m7G cap structure have higher persistence in vivo compared to stranded mRNAs.

[0225] Example 8. Evaluation of the translational activity of chemically synthesized circular mRNAs containing an m7G cap structure. The base sequence of the circular RNA used is shown below.

[0226] in, m7G represents N7-methylguanosine (a cap structure linked by a 5′-5′ triphosphate (-ppp-) structure). mG represents 2′O-methylguanosine. Ψ represents 1-methylpseuuridine X represents the branch structure derived from Asymmetric Doubler (Lev) phosphorous amide (Glen Research).

[0227] Cap-bound circular mRNA (Cap-circ) (200-nt) 5′-GGCGCAUAUUAAGGUGACGCGUGUGGCCUCGAACACCGAGGGUA-X-AAGCCACCAUGGΨGAGCGGCΨGGCGGCΨGΨΨCAAGAAGAΨΨAGCGGGAGCΨCCGGΨAGΨAΨAAΨCAACΨΨΨGAAAAACΨGΨGAΨAAGCΨGGAGCCΨCGGΨGGCCAΨGCΨΨCΨΨGCCCCΨΨGGGCCΨAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3′ The 5′ end and the 3′ end are connected to form a circular structure Contains a branch side chain (5′ m7G-ppp-mGmGAGCG-) branched from X (SEQ ID NO: 69).

[0228] circ mRNA containing HRV-B3-IRES (HRV-B3 circ / U) (816-nt) 5′ GGUUAAAACAGCGGAUGGGUACCCCACCAUCCGACCCACUGGGUGUAGUACUCUGGUACUUCGUACCUUUGUACGCCUGUUCUUCCCAUUGUACCCUUCCUGAACUUCCAACCCAAGUAACGUUAGAAGCUCAACAUUUAGUACAACAGGAAGCACCACAUCCAGUGGUGUUUAGUACAAGCACUUCUGUUUCCCCGGAGCGAGGUAUAGGCUGUACCCACUGCCAAAAACCUUUAACCGUUAUCCGCCAACCAACUACGUAAAAGCUAGUAGUAUUAUGUUUUUAACUAGGCGUUCGAUCAGGUGGAUUUCCCCUCCACUAGUUUGGUCGAUGAGGCUAGGAAUUCCCCACGGGUGACCGUGUCCUAGCCUGCGUGGCGGCCAACCCAGCCCACUCACUAUUUGUUUUCGCGCCCAGUUGCAAAAAGUGUCGGGGCUGGGACGCCUUUUUAUAGACAUGGUGUGAAGACUCGCAUGUGCUUGGUUGUGAUUCCUCCGGCCCCUGAAUGCGGCUAACCUUAACCCUGGAGCCUUGUGUCACAAACCAGUGAUGAUAAGGUCGUAAUGAGCAAUUCCGGGACGGGACCGACUACUUUGGGUGUCCGUGUUUCUUAUUUUUCUUAUUAUUGUCUUAUGGUCACAGCAUAUAUAUAACAUAUACUGUGAUCAUGGUGAGCGGCUGGCGGCUGUUCAAGAAGAUUAGCGGGAGCUCCGGUAGUAUAAUCAACUUUGAAAAACUGUGAUAAGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3′ The 5′ end and 3′ end are connected to form a circular structure (SEQ ID NO: 70).

[0229] Circular mRNA containing HRV-B3-IRES (HRV-B3 circ / m1Ψ) (816-nt) 5′ GGΨΨAAAACAGCGGAΨGGGΨACCCCACCAΨCCGACCCACΨGGGΨGΨAGΨACΨCΨGGΨACΨΨCGΨACCΨΨΨGΨACGCCΨGΨΨCΨΨCCCAΨΨGΨACCCΨΨCCΨGAACΨΨCCAACCCAAGΨAACGΨΨAGAAGCΨCAACAΨΨΨAGΨACAACAGGAAGCACCACAΨCCAGΨGGΨGΨΨΨAGΨACAAGCACΨΨCΨGΨΨΨCCCCGGAGCGAGGΨAΨAGGCΨGΨACCCACΨGCCAAAAACCΨΨΨAACCGΨΨAΨCCGCCAACCAACΨACGΨAAAAGCΨAGΨAGΨAΨΨAΨGΨΨΨΨΨAACΨAGGCGΨΨCGAΨCAGGΨGGAΨΨΨCCCCΨCCACΨAGΨΨΨGGΨCGAΨGAGGCΨAGGAAΨΨCCCCACGGGΨGACCGΨGΨCCΨAGCCΨGCGΨGGCGGCCAACCCAGCCCACΨCACΨAΨΨΨGΨΨΨΨCGCGCCCAGΨΨGCAAAAAGΨGΨCGGGGCΨGGGACGCCΨΨΨΨΨAΨAGACAΨGGΨGΨGAAGACΨCGCAΨGΨGCΨΨGGΨΨGΨGAΨΨCCΨCCGGCCCCΨGAAΨGCGGCΨAACCΨΨAACCCΨGGAGCCΨΨGΨGΨCACAAACCAGΨGAΨGAΨAAGGΨCGΨAAΨGAGCAAΨΨCCGGGACGGGACCGACΨACΨΨΨGGGΨGΨCCGΨGΨΨΨCΨΨAΨΨΨΨΨCΨΨAΨΨAΨΨGΨCΨΨAΨGGΨCACAGCAΨAΨAΨAΨAACAΨAΨACΨGΨGAΨCAΨGGΨGAGCGGCΨGGCGGCΨGΨΨCAAGAAGAΨΨAGCGGGAGCΨCCGGΨAGΨAΨAAΨCAACΨΨΨGAAAAACΨGΨGAΨAAGCΨGGAGCCΨCGGΨGGCCAΨGCΨΨCΨΨGCCCCΨΨGGGCCΨAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3′ The 5′ end and the 3′ end are connected to form a circular structure (Sequence No. 71).

[0230] Circular RNA was prepared as follows. First, 5′ phosphorylated linear RNA, serving as the raw material for circularization, was prepared using a transcription reaction with 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 solution 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 solution was extracted with an equal volume mixture of TE saturated phenol and chloroform to remove proteins, and the transcribed RNA was recovered by ethanol precipitation. The crude transcribed RNA obtained was purified by reverse-phase HPLC according to existing reports (Nat. Commun., 14, 2657, 2023). The 5′ phosphorylated transcribed RNA was ligated using T4 RNA ligase 2 to obtain circular RNA. The composition of the ligase reaction solution is shown below: 0.5 μM 5′ phosphorylated RNA, 1 μM template oligoDNA, 10% PEG8000, 50 mM Tris-HCl (pH 7.5), 2 mM magnesium chloride, 1 mM DTT, 400 μM ATP, 0.1 μg / μL T4 RNA ligase 2. After incubating the reaction solution at 37°C for 1 hour, extraction was performed with an equal volume mixture of TE saturated phenol-chloroform to remove proteins. Sodium acetate aqueous solution (pH 5.2, final concentration 0.3 M) and 2-propanol were added, and the mixture was centrifuged after cooling at -30°C. The RNA was recovered as a precipitate. The target RNA circular body was purified by preparative denaturing polyacrylamide gel electrophoresis.

[0231] The expression level evaluation of HiBiT peptide was performed using the human cultured cell line HeLa (Riken Cellbank, RCB0007). HeLa cells were cultured in DMEM (Wako Pure Chemical Industries Co., Ltd.) containing 10% FBS (Thermo Fisher) at 5% CO2 concentration and 37°C. One day before transfection, 1×10E4 HeLa cells / well were seeded in 96-well multi-well plates. mRNA (20 ng / well) was mixed with 0.3 μL / well of Lipofectamine MessengerMAX (Thermo Fisher), and after incubation for 10 minutes, it was added to the wells containing HeLa cells after the growth medium had been replaced with Opti-MEM (trademark) I low serum medium (Thermo Fisher), thereby introducing RNA into the cells. After 3 hours, the supernatant was replaced with growth medium. After a total of 5, 10, 20, 30 and 45 hours of culture, the expression level of HiBiT peptide in cells was evaluated using Nano-Glo (registered trademark) HiBiT Lytic Detection System (Promega) and TriStar5 multi-functional plate reader (Berthold).

[0232] The results are shown in Figure 11 It is known that chemically synthesized circular mRNAs containing the m7G cap structure (branched cap-introduced circular mRNAs) can minimize their size and exhibit high translational activity because they do not contain IRES (approximately 600-nt).

[0233] Experimental Example 9. Translation-promoting phenomenon of circular mRNA hybridizing with endogenous mRNA (ACTB). The concept diagram of this experiment is shown in... Figure 12 (A)

[0234] The nucleic acid base sequences used in this experiment are shown below.

[0235] ACTB siRNA top strand: 5′_UACCUGUACACUGACUUGAGAUU_3′ (Serial number 60).

[0236] ACTB siRNA guide strand (bottom strand): 5′_UCUCAAGUCAGUGUACAGGUAUU_3′ (Serial No. 61).

[0237] NoAntisense Nluc circRNA (682-nt): 5′_GGUCCCAGGUCCACUUCAAGUAAACCCCUACCAACUGGUCGGGGUUUGAAAC<>GCGUGUGGCCUCGAACACCGAGCGACCCUGCAGCGACCCGCUUAAAAGCUUGGCAAUCCGGUACUGUUGGUAAAGCCACCAUGGUCUUCACACUCGAAGAUUUCGUUGGGGACUGGCGACAGACAGCCGGCUACAACCUGGACCAAGUCCUUGAACAGGGAGGUGUGUCCAGUUUGUUUCAGAAUCUCGGGGUGUCCGUAACUCCGAUCCAAAGGAUUGUCCUGAGCGGUGAAAAUGGGCUGAAGAUCGACAUCCAUGUCAUCAUCCCGUAUGAAGGUCUGAGCGGCGACCAAAUGGGCCAGAUCGAAAAAAUUUUUAAGGUGGUGUACCCUGUGGAUGAUCAUCACUUUAAGGUGAUCCUGCACUAUGGCACACUGGUAAUCGACGGGGUUACGCCGAACAUGAUCGACUAUUUCGGACGGCCGUAUGAAGGCAUCGCCGUGUUCGACGGCAAAAAGAUCACUGUAACAGGGACCCUGUGGAACGGCAACAAAAUUAUCGACGAGCGCCUGAUCAACCCCGACGGCUCCCUGCUGUUCCGAGUAACCAUCAACGGAGUGACCGGCUGGCGGCUGUGCGAACGCAUUCUGGCGUAAUUCUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA_3′ (5′, 3′ end binding. The sequence into which the complementary sequence of ACTB mRNA was introduced in the <> portion is the following ACTB antisense (antisense) Nluc circRNA) (SEQ ID NO: 62).

[0238] ACTB antisense Nluc circRNA (705-nt): 5′_GGUCCCAGGUCCACUUCAAGUAAACCCCUACCAACUGGUCGGGGUUUGAAAC GGCAAAGGCGAGG CTCTGTGCTC_3′ (5′, 3′ end binding. The underlined portion indicates the sequence complementary to ACTB mRNA. All nucleotides in the underlined portion are ACTB_native (natural type), ACTB_2′OMe has 2′OMe modification, and ACTB_LNA has LNA modification.) (Sequence number 63).

[0239] NoAntisense splint (24-nt): 5′_dGAGGCCACACGCGTTTCAAACCCC_3′ (Serial number 64).

[0240] ACTB antisense splint 1 (24-nt): 5′_dCTCGCCTTTGCCGTTTCAAACCCC_3′ (Serial number 65).

[0241] ACTB antisense splint 2 (24-nt): 5′_dGAGGCCACACGCGAGCACAGAGCC_3′ (SEQ ID NO: 66).

[0242] circRNA backbone (682 nt): 5′phos-GCGUGUGGCCUCGAACACCGAGCGACCCUGCAGCGACCCGCUUAAAAGCUUGGCAAUCCGGUACUGUUGGUAAAGCCACCAUGGUCUUCACACUCGAAGAUUUCGUUGGGGACUGGCGACAGACAGCCGGCUACAACCUGGACCAAGUCCUUGAACAGGGAGGUGUGUCCAGUUUGUUUCAGAAUCUCGGGGUGUCCGUAACUCCGAUCCAAAGGAUUGUCCUGAGCGGUGAAAAUGGGCUGAAGAUCGACAUCCAUGUCAUCAUCCCGUAUGAAGGUCUGAGCGGCGACCAAAUGGGCCAGAUCGAAAAAAUUUUUAAGGUGGUGUACCCUGUGGAUGAUCAUCACUUUAAGGUGAUCCUGCACUAUGGCACACUGGUAAUCGACGGGGUUACGCCGAACAUGAUCGACUAUUUCGGACGGCCGUAUGAAGGCAUCGCCGUGUUCGACGGCAAAAAGAUCACUGUAACAGGGACCCUGUGGAACGGCAACAAAAUUAUCGACGAGCGCCUGAUCAACCCCGACGGCUCCCUGCUGUUCCGAGUAACCAUCAACGGAGUGACCGGCUGGCGGCUGUGCGAACGCAUUCUGGCGUAAUUCUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGUCCCAGGUCCACUUCAAGUAAACCCCUACCAACUGGUCGGGGUUUGAAAC_3′ (SEQ ID NO: 67).

[0243] ACTB antisense insert (47 nt): 5′_phos-GGGGTTTGAAAC GGCAAAGGCGAGGCTCTGTGCTC GCGTGTGGCCTC_3′ (The underlined portion indicates the sequence complementary to ACTB mRNA. All nucleotides in the underlined portion are of three types: natural, 2′OMe modified, and LNA modified.) (Sequence number 68).

[0244] The circRNA backbone is synthesized using T7 RNA polymerase transcribed from DNA encoding the sequence. The transcription reaction composition is as follows: 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 solution 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 the mixture was incubated at 37°C for 15 minutes. A 7.5 M lithium chloride aqueous solution was added to the reaction solution to a final concentration of 2.5 M. After cooling at -30 °C for 30 min, the mixture was centrifuged (20,000 × g, 20 min), and the transcribed RNA was recovered as a precipitate. The crude transcribed RNA product was purified by reversed-phase HPLC according to existing reports (Nat. Commun. 14, 2657, 2023). For NoAntisense Nluc circRNA, 2 μM of 5′ phosphorylated RNA prepared by the above transcription reaction was mixed with 8 μM of NoAntisense splint. For ACTB antisense Nluc circRNA, 2 μM of 5′ phosphorylated RNA, 8 μM ACTB antisense splint 1, 8 μM ACTB antisense splint 2, and 4 μM ACTB antisense insert were mixed and treated 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. Ligation was then performed by extraction with an equal volume of TE saturated phenol-chloroform mixture and protein removal. Sodium acetate aqueous solution (pH 5.2, final concentration 0.3 M) and 2-propanol were added, and the mixture was centrifuged after cooling at -30°C. The RNA was recovered as a precipitate. The target RNA circularized body was separated and purified by preparative denaturing polyacrylamide gel electrophoresis (Sci. Rep., 5, 16435, 2015).

[0245] The expression level of Nluc protein was evaluated using the human cell line HeLa (Riken Cellbank, RCB0007). HeLa cells were cultured in DMEM (Wako Pure Chemical Industries Co., Ltd.) containing 10% FBS (Thermo Fisher Scientific) at 37°C with 5% CO2. One day before transfection, 1×10⁴ HeLa cells were seeded into each of 96-well multi-well plates.

[0246] To knock down ACTB, 10 μM ACTB siRNA top strand, 10 μM ACTB siRNA bottom strand, 50 mM NaCl, and 40 mM Tris-HCl (pH 8.0) were treated at 80°C for 5 minutes, followed by slow cooling to 15°C at -0.1°C / second to prepare siRNA. The culture medium for HeLa cells in the prepared 96-well multi-well plate was changed the day before, and siRNA was added at 1 pmol / well using Lipofectamine RNAiMAX (ThermoFisher) as the lipid transfection reagent. The plate was then incubated at 37°C for 24 hours with 5% CO2.

[0247] 24 hours after adding siRNA, the culture medium was changed again, and Lipofectamine MessengerMax (ThermoFisher) was used as the lipid transfection reagent. NoAntisense Nluc circRNA or ACTB antisense Nluc circRNA was added at a rate of 25 ng / well, and the mixture was incubated at 37°C with 5% CO2 for 8 hours. Liposome formation of LipefectamineRNAiMAX and Lipofectamine Messenger Max was performed according to the manufacturer's experimental instructions.

[0248] Eight hours after adding circRNA, each well was washed once with D-PBS (nacalai), and the expression level of Nlucose in cells was evaluated using a Nano-Glo (registered trademark) luciferase assay system (Promega). Chemiluminescence assays were performed using a TriStar5 multi-functional plate reader (Berthold).

[0249] The results are shown in Figure 12 It promotes the translation of circular mRNA by hybridizing with endogenous mRNA (ACTB).

Claims

1. A method for manufacturing or expressing a protein or peptide, characterized in that: The steps include translation reactions carried out in the following reaction systems. The reaction system contains: 5′ capped polynucleotides containing any base sequence A; and A single-stranded RNA comprising a base sequence B capable of binding to the base sequence A via complementary base pairing, and a protein or peptide coding sequence. The 5′ capped polynucleotide and / or the single-stranded RNA have translation efficiency-enhancing structures.

2. The manufacturing or expression method as described in claim 1, characterized in that: The 5′ capped polynucleotide is a 5′ capped RNA.

3. The manufacturing or expression method as described in claim 1, characterized in that: The 5′ capped polynucleotide or single-stranded RNA is an endogenous polynucleotide of an organism or cell.

4. The manufacturing or expression method as described in claim 3, characterized in that: The endogenous polynucleotides of the organism or cell are lncRNA or mRNA.

5. The manufacturing or expression method as described in claim 1, characterized in that: The translation efficiency enhancement structure is a stabilization structure that stabilizes the binding of the 5′ capped polynucleotide to the single-stranded RNA.

6. The manufacturing or expression method as described in claim 1, characterized in that: Satisfying (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 contains a tracrRNA sequence.

7. The manufacturing or expression method as described in claim 1, characterized in that: Satisfy at least one of the conditions selected from (a) and (c) (a) The single-stranded RNA is a circular RNA. (c) The 5′ capped polynucleotide and / or the single-stranded RNA have a structure that allows them to be covalently linked.

8. The manufacturing or expression method as described in claim 7, characterized in that: The condition (a) is met, and the circular RNA does not contain a stop codon, and / or The condition (c) is met, and the structure is a photocrosslinking structure.

9. A composition, characterized in that, contain: 5′ capped polynucleotides containing any base sequence A; and / or A single-stranded RNA comprising a base sequence B capable of binding to the base sequence A via complementary base pairing, and a protein or peptide coding sequence. The 5′ capped polynucleotide and / or the single-stranded RNA have translation efficiency-enhancing structures.

10. The composition according to claim 9, characterized in that: The manufacturing or expression method according to any one of claims 1 to 8.

11. A method for manufacturing or expressing a protein or peptide, characterized in that: The steps include translation reactions carried out in the following reaction systems. The reaction system contains single-stranded circular RNA with a base length of less than 500, formed by linking 5′ capped polynucleotides via a linker.

12. A composition, characterized in that: A single-stranded circular RNA with a base length of less than 500, containing 5′ capped polynucleotides linked by a linker.

13. The composition according to claim 12, characterized in that: Used in the manufacturing or expression method as described in claim 11.

14. A method for manufacturing a 5′ capped polynucleotide, characterized in that: The steps include translation reactions carried out in the following reaction systems. The reaction system contains: The phage promoter contains a base insertion mutation sequence adjacent to the upstream side of the transcription start site, and the antisense strand contains a double-stranded polynucleotide with the base sequence Y: (upstream side)-Y2-Y3 or (upstream side)-Y1-Y2-Y3, wherein Y1 and Y2 represent bases within the base insertion mutation sequence, and Y3 represents a base at the transcription start site; and 5′ cap analogs containing a polynucleotide containing the base sequence X: (cap side)-X2-X3 or (cap side)-X1-X2-X3 X3 is the complementary base of Y3, and X1 is a complementary base of Y1 and / or X2 is a complementary base of Y2.

15. The manufacturing method as described in claim 14, characterized in that: The antisense strand of the phage promoter contains the base sequence Y: (upstream side) -Y1-Y2-Y3, where X1 is the complementary base of Y1 and X2 is the complementary base of Y2.

16. The manufacturing method as described in claim 14, characterized in that: The 5′ capped polynucleotide is a 5′ capped RNA.

17. The manufacturing method as described in claim 14, characterized in that: The phage promoter is the T7 promoter.

18. The manufacturing method as described in claim 14, characterized in that: The base sequence X is (cap-side) -CUG, CAG, GUG or GAG, and Y3 is C.

19. A composition, characterized in that, contain: The phage promoter comprises a base insertion mutation sequence adjacent to the upstream side of the transcription start site, and the antisense strand comprises a double-stranded polynucleotide containing the base sequence Y: (upstream side)-Y2-Y3 or (upstream side)-Y1-Y2-Y3, wherein Y1 and Y2 represent bases within the base insertion mutation sequence, and Y3 represents a base at the transcription start site; and / or 5′ cap analogs containing a polynucleotide containing the base sequence X: (cap side)-X2-X3 or (cap side)-X1-X2-X3 X3 is the complementary base of Y3, and X1 is a complementary base of Y1 and / or X2 is a complementary base of Y2.

20. The composition of claim 19, characterized in that: The manufacturing method according to any one of claims 14 to 18.

21. A T7 RNA polymerase, characterized in that: It has the D130W mutation.

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