polynucleotide

Abstract

To provide a technique that enables compatibility between capping efficiency and yield of 5'-capped polynucleotide.SOLUTION: A polynucleotide comprising a nucleotide sequence a consisting of a T7 promoter sequence-insert sequence q-G (transcription initiation site)-GGN0-5 (sequence p), and / or a nucleotide sequence b complementary to the nucleotide sequence a, are provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to polynucleotides that can be used as templates for transcription of 5'-capped polynucleotides. [Background technology]

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

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

[0004] [Non-Patent Document 1] Nat Commun. 2023 May 11;14(1):2657. doi: 10.1038 / s41467-023-38244-8. Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors have been conducting research to improve the capping efficiency in the production of 5'-capped polynucleotides. They discovered that the ratio of the amount of capped polynucleotide synthesized to the total amount of transcription (capping efficiency) can be improved by inserting bases between the promoter and the transcription start site (G) so that the base sequence of the cap analog polynucleotide and the base sequence upstream from the transcription start site of the promoter are complementary in a specific positional relationship. However, although this technique improved capping efficiency, it also resulted in a decrease in the yield of 5'-capped polynucleotides.

[0006] An objective of the present invention is to provide a technique that uses this technique to achieve both high capping efficiency and high yield of 5'-capped polynucleotides. [Means for solving the problem]

[0007] In view of the above problems, the present inventors have conducted extensive research and have found that the T7 promoter sequence - insertion sequence qG (transcription start site) - GGN 0-5 It has been found that the above-mentioned problems can be solved by using a polynucleotide comprising a base sequence a consisting of (sequence p) and / or a base sequence b that is complementary to the base sequence a. Based on this finding, the present inventors have conducted further research and have completed the present invention. That is, the present invention encompasses the following aspects.

[0008] Section 1. T7 promoter sequence - insertion sequence qG (transcription start site) - GGN 0-5 A polynucleotide comprising a base sequence a consisting of (sequence p) and / or a base sequence b that is complementary to the base sequence a.

[0009] Item 2. The T7 promoter sequence The base sequence c (TAATACGACTCACTATA) shown in SEQ ID NO: 1, or a base sequence d having promoter activity, wherein one or more bases are mutated relative to the base sequence c; Item 2. The polynucleotide according to Item 1,

[0010] Item 3. The polynucleotide according to Item 1 or 2, wherein the insertion sequence q has a base length of 1 to 3.

[0011] Item 4. The polynucleotide according to any one of Items 1 to 3, comprising a coding sequence for a protein or peptide downstream of the base sequence a and / or the base sequence b.

[0012] Item 5. The polynucleotide according to any one of Items 1 to 4, which is a double-stranded polynucleotide comprising the base sequence a and the base sequence b.

[0013] Item 6. The insertion sequence q is N 0-2 A polynucleotide according to any one of Items 1 to 5, wherein A is A.

[0014] Item 7. The array p is GGAN 0-4 Item 7. The polynucleotide according to any one of Items 1 to 6,

[0015] Item 8. The polynucleotide according to any one of Items 1 to 7, which is DNA.

[0016] Item 9. A 5'-capped polynucleotide transcription template comprising the polynucleotide according to any one of Items 1 to 8.

[0017] Item 10. A method for producing a 5'-capped polynucleotide, comprising: A polynucleotide according to any one of Items 1 to 8, wherein the base sequence b comprises a base sequence Y: (upstream)-Y2-Y3 or (upstream)-Y1-Y2-Y3 (in the base sequence Y: Y1 and Y2 represent bases in the complementary sequence of the insertion sequence q, and Y3 represents a base at the transcription start point), and is a double-stranded polynucleotide; 5' cap analog having a polynucleotide containing the base sequence X: (cap side)-X2-X3 or (cap side)-X1-X2-X3 carrying out a transcription reaction in a reaction system containing X3 is the complementary base of Y3, and X1 is the complementary base of Y1 and / or X2 is the complementary base of Y2; Manufacturing method. [Effects of the Invention]

[0018] According to the present invention, a technique can be provided that achieves both high capping efficiency and high yield of 5'-capped polynucleotides. [Brief explanation of the drawings]

[0019] [Figure 1] The figures show the measurement results of capping efficiency (left side of the figure: Capping efficiency) and 5'-capped mRNA yield (right side of the figure: Capped mRNA yield) in Test Example 4. The upper row shows the case where the base sequence of the cap analog is CAG, and the lower row shows the case where the base sequence of the cap analog is GAG. The horizontal axis shows the 5'-capped mRNA transcription template used (see Table 1) and the base sequence of the cap analog. [Figure 2] This figure shows the structure and sequence of the TetraPureCap-XYG series analogs and the sequence of the modified promoter for T7 RNA polymerase in the development of a highly efficient mRNA synthesis method using TetraPureCap analogs and modified promoters. DETAILED DESCRIPTION OF THE INVENTION

[0020] 1.Definition In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0021] In the present specification, the polynucleotide is not particularly limited and may be DNA, RNA, or the like, and may be chemically modified as exemplified below. To prevent degradation by hydrolases such as nucleases, the phosphate residue of each nucleotide may be substituted with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithioate. Furthermore, the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide may be substituted with -OR (where R represents, for example, CH3(2'-O-Me), CH2CHOCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, or CH2CH2CN). Furthermore, the base moiety (pyrimidine or purine) may be chemically modified, for example, by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a thiocarbonyl group for the carbonyl group at the 2-position. Further examples include those in which the phosphate moiety or hydroxyl moiety is modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, etc., but are not limited to these.

[0022] As used herein, a polynucleotide may be linked to another molecule. Examples of other molecules include fluorescent labels. Examples of fluorescent labels include fluorescein, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, phycoerythrin, 6-FAM™, Cy®3, Cy®5, and the Alexa Fluor® series.

[0023] In this specification, the bases constituting nucleic acids include not only typical bases in natural nucleic acids such as RNA and DNA (adenine (A), thymine (T), uracil (U), guanine (G), cytosine (C), etc.), but also other bases such as hypoxanthine (I) and modified bases. Modified bases include, for example, pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (e.g., 5-methylcytosine), 5-alkyluracil (e.g., 5-ethyluracil), 5-halouracil (5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidine (6-methyluracil), 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5'-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, 1-methyladenine, 1-methylhypoxanthine, etc. Examples of suitable amino acids include uracil, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyladenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, 2-thiocytosine, purine, 2-aminopurine, isoguanine, indole, imidazole, xanthine, and cyanuric acid.

[0024] As used herein, the term "upstream" refers to the direction toward the promoter when the promoter and transcription initiation site are located there.

[0025] In this specification, "N" in a base sequence represents any nucleic acid base. N is, for example, A, T, G, or C. The number displayed to the right of "N" indicates the number of consecutive Ns. For example, "N 0-5 " indicates that N is 0 (no base), N, NN, NNN, NNNN, or NNNNN.

[0026] Complementary base pairing occurs when the base sequences are complementary to each other. "Complementary" refers not only to a perfect base complementarity (e.g., A and T or U, and G and C), but also to a degree of complementarity that allows hybridization under stringent conditions. Stringent conditions can be determined based on the melting temperature (Tm) of the nucleic acid, as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego, CA). For example, washing conditions after hybridization typically include approximately "1x SSC, 0.1% SDS, 37°C." It is preferable that the hybridized state is maintained even after washing under such conditions. Although not particularly limited, examples of stringent hybridization conditions include washing conditions of about "0.5×SSC, 0.1% SDS, 42°C," and even stringent hybridization conditions include washing conditions of about "0.1×SSC, 0.1% SDS, 65°C." Specifically, base sequence B is a base sequence that has, for example, 85% or more identity, preferably 90% or more identity, more preferably 95% or more identity, even more preferably 98% or more identity, still more preferably 99% or more identity, and particularly preferably 100% identity to a base sequence completely complementary to base sequence A.

[0027] As used herein, the "identity" of a base sequence refers to the degree of match between the base sequences of two or more comparable base sequences. Therefore, the greater the match between two base sequences, the greater the identity or similarity between those sequences. The level of identity between base sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes," Proc. Natl. Acad. Sci. USA. 87:2264-2268 (1990); Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences," Proc. Natl. Acad. Sci. USA. 90:5873-7 (1993)). A program called BLASTX, based on the BLAST algorithm, has been developed. Specific techniques for these analysis methods are known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ).

[0028] In the present specification, when a range consisting of an upper limit and a lower limit is indicated, a range consisting of only the upper limit, a range consisting of only the lower limit, a range formed by combining any upper limit and any lower limit, etc. are also unambiguously disclosed in the present specification.

[0029] 2. Polynucleotides In one aspect, the present invention provides a T7 promoter sequence-insertion sequence qG (transcription start site)-GGN 0-5The present invention relates to a polynucleotide (sometimes referred to herein as the "polynucleotide of the present invention") comprising a base sequence a consisting of (sequence p) and / or a base sequence b that is complementary to the base sequence a. This will be explained below.

[0030] In the base sequence a, "-" indicates that the sequences on both sides of it are directly linked without any intervening sequences.

[0031] The T7 promoter sequence is known to those skilled in the art and is a sequence that does not include the transcription start site but is located upstream of the transcription start site (usually adjacent to the upstream of the transcription start site). The T7 promoter sequence may be mutated (by base substitution, deletion, insertion, etc.) as long as it has promoter activity.

[0032] The T7 promoter sequence is preferably the base sequence c (TAATACGACTCACTATA) shown in SEQ ID NO: 1, or the base sequence d, which is a base sequence in which one or more (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, and even more preferably 1) bases are mutated (by base substitution, deletion, insertion, etc.) with respect to the base sequence c and which has promoter activity.

[0033] The base length of the base sequence d is, for example, 15 to 18, preferably 16 to 17, and particularly preferably 17.

[0034] The promoter activity of a certain base sequence can be determined by whether or not a transcription product is produced as a result of an in vitro transcription reaction (e.g., a transcription reaction under conditions according to or similar to the conditions of Test Example 3) using wild-type T7 polymerase and a linear double-stranded DNA containing the base sequence, a transcription start point (G) located adjacent to the base sequence downstream, and a template sequence located further downstream of the transcription start point as an RNA transcription template.

[0035] In base sequence a, an "insertion sequence q" is located adjacent to the downstream side of the T7 promoter sequence. By adding this insertion sequence q, the base sequence of the cap analog polynucleotide and the base sequence upstream from the transcription start site are designed to be complementary in a specific positional relationship (see below, e.g., Figure 2), thereby improving capping efficiency.

[0036] The insertion sequence q is not particularly limited as long as it is designed to satisfy the above conditions. The base length of the insertion sequence q is, for example, 1 to 5, preferably 1 to 3, and more preferably 1 to 2.

[0037] The insert sequence q is preferably N 0-2 A, more preferably N 0-1 A, more preferably A.

[0038] In the base sequence a, G is positioned adjacent to the downstream side of the insertion sequence q, and this G functions as a transcription initiation site for the T7 promoter sequence.

[0039] In base sequence a, there is a GGN sequence adjacent to the downstream side of the transcription start site. 0-5 (sequence p) is placed downstream of the transcription initiation site. Typically, any sequence is placed downstream of the transcription initiation site and is designed to serve as a template depending on the sequence of the mRNA to be synthesized. In the present invention, it has been found that by using a sequence containing GG as the sequence, a higher yield of 5'-capped polynucleotide can be achieved while maintaining a high level of capping efficiency.

[0040] The sequence p is preferably GGAN 0-4 This can further improve the yield of 5'-capped polynucleotides.

[0041] Specific examples of sequence p include GGAAATA, GGATCCT, GGTTCCC, etc. In addition to these, examples include base sequences in which one or more (for example, 1 to 3, preferably 1 to 2, more preferably 1) bases in the portion other than GG of these sequences have been mutated (by substitution, deletion, insertion, etc. of the base).

[0042] The base sequence b is a sequence complementary to the base sequence a.

[0043] In one embodiment, the polynucleotide of the present invention is a double-stranded polynucleotide comprising base sequence a and base sequence b. In this case, base sequence a and base sequence b form a complementary base pair. The double-stranded polynucleotide can be used as a transcription template for a 5'-capped polynucleotide.

[0044] In one embodiment, the polynucleotide of the present invention is a single-stranded polynucleotide comprising base sequence a or base sequence b. The single-stranded polynucleotide can be used as a primer for preparing a transcription template for a 5'-capped polynucleotide by PCR. In this PCR, for example, a polynucleotide containing a coding sequence for a protein / peptide to be expressed by translation from the 5'-capped polynucleotide is prepared, and a PCR reaction is carried out using this polynucleotide as a template and the above-mentioned primers.

[0045] In the polynucleotide of the present invention, base sequence a / base sequence b are preferably DNA, and it is particularly preferred that the entire polynucleotide of the present invention is DNA.

[0046] The polynucleotide of the present invention preferably contains a coding sequence for a protein or peptide downstream of base sequence a and / or base sequence b.

[0047] The protein or peptide coding sequence is a coding sequence for a protein or peptide to be expressed from a 5'-capped polynucleotide obtained using the polynucleotide of the present invention. Examples of such proteins or peptides include microbial antigens, such as viral antigens and fungal antigens. The viruses from which viral antigens are derived are not particularly limited, and examples include enveloped viruses (viruses with an envelope) such as influenza virus (e.g., type A, type B, etc.), rubella virus, Ebola virus, coronavirus, measles virus, varicella-zoster virus, herpes simplex virus, mumps virus, arbovirus, respiratory syncytial virus, SARS virus, hepatitis virus (e.g., hepatitis B virus, hepatitis C virus, etc.), yellow fever virus, AIDS virus, rabies virus, hantavirus, dengue virus, Nipah virus, and lyssavirus; and non-enveloped viruses (viruses without an envelope) such as adenovirus, norovirus, rotavirus, human papillomavirus, poliovirus, enterovirus, coxsackievirus, human parvovirus, encephalomyocarditis virus, and rhinovirus. The bacteria from which bacterial antigens are derived are not particularly limited, but examples include Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae, Salmonella enterica, Helicobacter pylori, Clostridium perfringens, Clostridium botulinum, Campylobacter, Escherichia coli, Staphylococcus aureus, Streptococcus staphylococcus, Bacillus cereus, Vibrio parahaemolyticus, Propionibacterium acnes, Clostridium faecalis, Clostridium difficile, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella pneumoniae, Klebsiella pneumoniae, Koinebacterium, Streptococcus hemolyticus, Pseudomonas aeruginosa, Staphylococcus aureus, Mycoplasma, Candida, and Aspergillus.

[0048] The base length of the polynucleotide of the present invention is not particularly limited, but can be, for example, 20 to 10,000. When used as a 5'-capped polynucleotide transcription template, the base length is not particularly limited as long as it is a length that can be used as a transcription template, and is, for example, 25 to 10,000, 25 to 5,000, 25 to 3,000, 25 to 2,000, 25 to 1,000, 25 to 500, 25 to 300, or 25 to 200. When used as a primer, the base length can be, for example, 20 to 100, 25 to 80, 30 to 70, or 40 to 60.

[0049] As described above, the polynucleotides of the present invention can be used as 5'-capped polynucleotide transcription templates, particularly as 5'-capped polynucleotide transcription templates for use in the production methods of the present invention described below. Thus, in one aspect, the present invention relates to 5'-capped polynucleotide transcription templates comprising the polynucleotides of the present invention, and 5'-capped polynucleotide transcription compositions (e.g., reagents, reaction solutions, etc.) containing the polynucleotides of the present invention.

[0050] The composition may further contain other components as needed. The other components are not particularly limited as long as they are pharmaceutically acceptable, and examples thereof include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, flavorings, chelating agents, etc. The composition may also be in the form of a kit in which two or more substances are contained in two or more separate containers.

[0051] 3. Method for producing 5'-capped polynucleotides In one aspect, the present invention comprises: 1. A method for producing a 5'-capped polynucleotide, comprising: The polynucleotide of the present invention, wherein the base sequence b comprises a base sequence Y: (upstream)-Y2-Y3 or (upstream)-Y1-Y2-Y3 (in the base sequence Y: Y1 and Y2 represent bases in the complementary sequence of the insertion sequence q, and Y3 represents the base of the transcription start site), and is a double-stranded polynucleotide; and 5' cap analog having a polynucleotide containing the base sequence X: (cap side)-X2-X3 or (cap side)-X1-X2-X3 carrying out a transcription reaction in a reaction system containing X3 is the complementary base of Y3, and X1 is the complementary base of Y1 and / or X2 is the complementary base of Y2; The present invention relates to a method for producing the same (also referred to as the "production method of the present invention" in this specification), which will be described below.

[0052] The 5' cap analog is a single-stranded polynucleotide to which a 5' cap structure has been added, and is not particularly limited as long as it can be used to produce a 5' capped polynucleotide.

[0053] The 5'-cap structure is not particularly limited and examples include cap-0, cap-1, and cap-2. The 5'-cap structure may include a hydrophobic purification tag that can be removed by light (e.g., Non-Patent Document 1). According to the production method of the present invention, it is possible to produce highly pure 5'-capped polynucleotides without using the hydrophobic purification tag, and it is also possible to produce even more pure 5'-capped polynucleotides by using the hydrophobic purification tag.

[0054] The base length of the polynucleotide of the 5' cap analog is preferably 2 to 5, more preferably 2 to 4, and particularly preferably 3.

[0055] The polynucleotide having the 5'-cap analogue comprises the base sequence X: (cap side)-X2-X3 or (cap side)-X1-X2-X3. In the base sequence X, X3 is preferably at the 3' end of the polynucleotide.

[0056] In the production method of the present invention, X3 is the complementary base of Y3, and X1 is the complementary base of Y1 and / or X2 is the complementary base of Y2.

[0057] By using the polynucleotide of the present invention and satisfying the above-mentioned complementary relationship, it is possible to achieve both capping efficiency and a high yield of 5'-capped polynucleotide. From the viewpoint of capping efficiency, it is particularly preferred that the antisense strand of the phage promoter contains the base sequence Y:(upstream)-Y1-Y2-Y3, where X1 is the complementary base of Y1 and X2 is the complementary base of Y2.

[0058] Taking the φ6.5, XY-inserted in Figure 2 as an example, "XYG" in the 5'-cap analog (m7G-XYG) represents the base sequence X (X1-X2-X3), the lower strand in the duplex below it is the antisense strand, and "XYC" in the antisense strand corresponds to the base sequence Y (Y1-Y2-Y3).

[0059] The specific sequence of the base sequence X is not particularly limited, but from the viewpoint of capping efficiency, production efficiency of 5'-capped polynucleotides, etc., it is particularly preferable that the base sequence X be (cap side) -CUG, CAG, GUG, or GAG.

[0060] The double-stranded polynucleotide and 5'-cap analog can be produced according to or in accordance with known methods for producing polynucleotides and 5'-cap analogs. More specifically, the 5'-cap analog can be produced using the synthesis method described in the Examples below.

[0061] The reaction system is not particularly limited as long as it contains substances necessary for the translation reaction (for example, T7 RNA polymerase, various nucleoside triphosphates, etc.), and may be either an in vitro reaction system or an in vivo reaction system.

[0062] The reaction conditions for the transcription reaction are not particularly limited, and appropriate conditions can be set depending on the enzymes used, etc.

[0063] The 5'-capped polynucleotide is not particularly limited as long as it has a 5'-cap structure and can bind to a single-stranded RNA through complementary base pairing. The 5'-capped polynucleotide is particularly preferably RNA. [Example]

[0064] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0065] Test Example 1. Synthesis of TetraPureCap analogue

[0066] [ka]

[0067] The trinucleotide CAG triethylammonium salt and the trinucleotide GAG ​​triethylammonium salt were each synthesized in solid phase using an automated nucleic acid synthesizer.

[0068] A solution of trinucleotide CAG triethylammonium salt or trinucleotide GAG ​​triethylammonium salt (56.5 mg, 40.1 μmol) in DMSO (802 μL) was added to N 71-Methylguanosine diphosphate imidazolide disodium salt (92.7 mg, 120 μmol) was added, followed by zinc chloride (109 mg, 802 μmol). After incubation at 37°C for 5 days, the reaction mixture was quenched with 500 mM EDTA-NaOH aqueous solution (pH 8.0, 1.04 mmol, 2.0 mL) and diluted with water (40 mL). The crude product was purified by reverse-phase HPLC using a Shimadzu YMC-Actus Triart C8 column (250 x 20.0 mm I.D.), solvent A: 50 mM TEAA buffer (pH 6.0) containing 0.5% CH3CN, solvent B: CH3CN, linear gradient 5-80% B (25 min), flow rate: 10 mL / min, detection: 254 nm. Fractions containing the desired product were pooled, concentrated, and lyophilized to yield the tetranucleotide PureCap analog, m7Gppp CAG / GAG, as its triethylammonium salt. The product was redissolved in methanol (2.0 mL) and 190 mM NaClO4 in acetone (12 mL) was added. The resulting suspension was centrifuged (4,000 rpm, 10 min). The supernatant was discarded, and the precipitate was suspended in acetone. The suspension-centrifugation process was repeated three more times. The precipitate was dried under reduced pressure to yield the desired PureCap analogs, m7Gppp CAG (12.0 mg, 6.66 μmol, 16.6% yield) and m7Gppp GAG (23.9 mg, 13.0 μmol, 32.3% yield), as their sodium salts. Yields were calculated using the absorbance of the products at 260 nm measured with a NanoDrop.

[0069] Nb-7mGpppCAG (sodium salt, ε 260 = 45,700 M -1 ·cm -1 , 16.6% yield)(7): 1H NMR (600 MHz, D2O) δ 8.36 (s, 1H), 8.08 (s, 1H), 7.90 (s, 2H), 7.63 (d, J= 6.5 Hz, 0.5H), 7.57 (d, J = 8.0 Hz, 1H), 7.49 (s, 1.5H), 7.37 (s, 0.5H), 7.31 (s, 0.5H), 6.12 - 6.05 (m, 2H), 6.01 (s, 0.5H), 5.83 (d, J= 4.3 Hz, 0.5H), 5.79 - 5.75 (m, 1H), 5.72 (s, 1H), 5.03 - 4.96 (m, 1H), 4.91 (s, 1H), 4.85 (d, J = 7.6 Hz, 1H), 4.71 - 4.68 (m, 2H), 4.57 (s, 1H), 4.47 - 4.42 (m, 3H), 4.36 - 4.28 (m, 6H), 4.20 - 4.04 (m, 11H), 3.48 (d, J= 2.6 Hz, 2.5H), 3.45 - 3.43 (m, 2.5H), 3.34 (d, J = 2.9 Hz, 1H), 0.61 - 0.54 (m, 9H) ppm. 13 C NMR (151 MHz, D2O) δ 163.29, 158.41, 155.35, 154.61, 153.64, 150.47, 149.74, 149.11, 148.78, 148.27, 141.81, 139.70, 132.61, 130.06, 128.22, 123.81, 123.37, 118.47, 116.13, 107.78, 107.53, 96.21, 87.92, 87.69, 86.94, 85.88, 83.42, 82.23, 81.53, 80.42, 79.01, 73.37, 72.17, 70.17, 68.10, 66.07, 65.01, 64.27, 57.98, 57.63, 48.92, 36.30, 36.14, 35.71, 30.28, 24.87, 24.79 ppm. 31P NMR (243 MHz, D2O) δ -0.48 (2s, 2P), -10.80 (2P), -21.97 (1P) ppm. ESI-TOF-MS calcd. for C 54 H 70 N 19 O 34 P5, 841.6516 [M - 2H] 2- ; found 841.6548.。

[0070] Nb-7mGpppGAG (sodium salt, ε 260 = 49,900 M -1 ·cm -1 、32.3% yield)(15): 1 H NMR (400 MHz, D2O) δ 9.08 - 8.68 (m, 0.5H), 8.18 (d, J = 8.0 Hz,  1H), 7.98 - 7.73 (m, 3H), 7.42 - 6.99 (m, 4H), 5.86 (d, J = 6.4 Hz, 1H), 5.68 - 5.38 (m, 3.5H), 4.86 (s, 2H), 4.75 (d, J = 8.1 Hz, 3.5H), 4.56 (s, 2H), 4.42 - 4.24 (m, 4H), 4.23 - 3.97 (m, 10H), 3.93 - 3.77 (m, 4.5H), 3.45 - 3.16 (m, 6H), 0.49 - 0.10 (m, 9H) ppm. 13 C NMR (101 MHz, D2O) δ 158.33, 155.12, 154.17, 153.71, 153.51, 151.46, 151.22, 148.79, 139.09, 137.08, 133.22, 129.62, 118 .27, 116.04, 115.55, 107.43, 107.12, 87.99, 87.34, 85.60, 83.36, 82.84, 80.93, 73.37, 70.08, 67.82, 65.03, 58.05, 57.91, 36.14, 36.01, 35.64, 30.26, 24 .81, 24.72 ppm. 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.

[0071] Test Example 2. Preparation of 5'-capped mRNA transcription template As a template for 5´-capped mRNA transcription, Starting from the 5' side, T7 promoter sequence (SEQ ID NO: 1: TAATACGACTCACTATA), Insertion sequence q(A) or no insertion sequence q, ·Transcription start site (G), Sequence p:GCGCATA, GGAAATA, GGATCCT, or GGTTCCC A base sequence a' consisting of the following linked sequences: Contains a base sequence b' which is a complementary strand of the base sequence a, Linear double-stranded DNA, It was produced by PCR using a vector containing the NanoLuc coding sequence (pNL1.1.TK[Nluc / TK] Vector) as a template.

[0072] Primers (SEQ ID NOS: 2 to 6) containing the nucleotide sequence a' and a complementary sequence at the 3' end to the upstream region of the NanoLuc (registered trademark) coding sequence in the vector were designed as forward primers for PCR. The designed primer sequences, as well as the names of the primers and 5'-capped mRNA transcription templates prepared using them, are shown in Table 1. In the sequences in Table 1, the underlined sequence represents the sequence p, and the capital letter G adjacent to the 5' side represents the transcription start point (+1). In the names in Table 1, "A-inserted" indicates that an A has been inserted between the transcription start point (G) and the T7 promoter.

[0073] [Table 1]

[0074] Double-stranded DNA containing the NanoLuc coding sequence was amplified by PCR using the above forward primer and the reverse primer (5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTAGAATTACGCCAGAATGCG-3' (SEQ ID NO: 7)) and the pNL1.1.TK[Nluc / TK] vector as a template. The PCR reaction mixture consisted of 1x NEB standard buffer (NEB), 0.2 mM dNTPs, 0.2 μM forward primer, 0.2 μM reverse primer, 0.5 ng / μL pNL1.1.TK, and 1.25 units / 50 μL Taq DNA polymerase (NEB). After a hot start at 95°C for 30 seconds, the PCR was repeated for 30 cycles, consisting of 95°C for 15 seconds, 51°C for 15 seconds, and 68°C for 45 seconds. The PCR product was purified using the QIAquick PCR Purification Kit (QIAGEN) according to the protocol. The concentration of the purified DNA was measured using a DeNovix DS-11 (SCRUM) spectrophotometer. The purified DNA was also subjected to agarose gel electrophoresis to confirm that DNA of the desired length had been obtained.

[0075] Test Example 3. Production of 5'-capped mRNA 5'-capped mRNA transcription templates (Test Example 2) with or without an A inserted between the transcription start site (G) and the T7 promoter and with various sequences (sequence p) in the 3' flanking sequence of the transcription start site were mixed with cap analogs (Test Example 1) and subjected to co-transcriptional capping using T7 RNA polymerase. The mixture was prepared to contain 10 ng / mL template DNA, 2 mM NTP, 40 mM Hepes (pH 7.6), 8 mM Mg(OAc)2, 2 mM SPD, 2 mM DTT, 1 μM T7 RNA polymerase, 0.1 ng / μL PPiase, and 2 mM cap analog. The co-transcriptional capping reaction was performed by incubating at 37°C for 60 min. The resulting transcripts were purified using Lithium Chloride Precipitation Solution (Invitrogen). In this test example, the specified protocol was partially modified, and the sample was washed with 500 μL of 80% ethanol and eluted with 20 μL of nuclease-free water.

[0076] Test Example 4: Measurement of capping efficiency and yield of 5'-capped mRNA The purified sample obtained in Experiment 3 was analyzed by high-performance liquid chromatography (HPLC) using a YMC-Triart Bio C4 analytical column (particle size 5 μm, pore size 30 nm, column size 4.6 × 250 mm), and the peak areas of 5'-capped and uncapped mRNA were calculated. The mobile phase consisted of solvent A (100 mM triethylammonium acetate (TEAA), 5% acetonitrile (ACN)) and solvent B (100 mM TEAA, 50% ACN). Equilibration was performed with 100% solvent A, and the concentration of solvent B was increased from 10 to 30% over a period of 0.1 to 20 min to separate the transcripts. The column and line were then washed with 100% solvent B from 20.1 to 25 min, and equilibration was continued with 100% solvent A from 25.1 to 40 min.

[0077] The capping efficiency (the ratio of 5'-capped mRNA to the total of 5'-capped and non-capped mRNA) and capped mRNA yield were calculated from the peak areas. Co-transcriptional capping reactions were performed four times independently, and the capping efficiency and capped mRNA yield were calculated by HPLC.

[0078] The results are shown in Figure 1.

[0079] Comparison of No-inserted_000 and A-inserted_000 revealed that inserting a base between the promoter and the transcription start site (G) so that the base sequence of the cap analog polynucleotide and the base sequence upstream from the promoter transcription start site are complementary in a specific positional relationship improves the ratio of capped RNA synthesis to total transcription (capping efficiency) (Figure 1: Capping efficiency). However, although the capping efficiency was improved, the yield of 5'-capped mRNA decreased (Figure 1: Capped mRNA yield).

[0080] Comparison of A-inserted_000 with A-inserted_001, A-inserted_474, and A-inserted_949 revealed that when GG was placed adjacent to the downstream side of the transcription start site (G), the yield of 5'-capped mRNA increased while maintaining a high level of capping efficiency (Figure 1). When GGA was placed adjacent to the downstream side of the transcription start site (G), the yield of 5'-capped mRNA was particularly high.

[0081] The above tendency was also confirmed when the inserted sequence between the promoter and the transcription start site (G), the base sequence of the cap analog, the sequence 3' of the transcription start site (G), etc. were changed.

[0082] In addition, for A-inserted_001, A-inserted_474, and A-inserted_949 in Table 1, when the A between the transcription start site (G) and the T7 promoter was removed (No-inserted_001, No-inserted_474, and No-inserted_949), the transcription amount when transcribed using a cap analog (Test Example 1) was equivalent to that of No-inserted_000. This suggests that the increase in 5'-capped mRNA yield when GG is located adjacent to the downstream side of the transcription start site (G) is a decrease specific to the presence of an inserted sequence between the transcription start site (G) and the T7 promoter.

Claims

1. T7 promoter sequence - insertion sequence q - G (transcription start site) - GGN 0－5 A polynucleotide comprising a base sequence a consisting of (sequence p) and / or a base sequence b that is complementary to the base sequence a.

2. The T7 promoter sequence The base sequence c (TAATACGACTCACTATA) shown in SEQ ID NO: 1, or a base sequence d having promoter activity, wherein one or more bases are mutated relative to the base sequence c; The polynucleotide of claim 1,

3. The polynucleotide of claim 1, wherein the insertion sequence q has a base length of 1 to 3.

4. The polynucleotide according to claim 1, comprising a coding sequence for a protein or peptide downstream of said base sequence a and / or said base sequence b.

5. The polynucleotide according to claim 1, which is a double-stranded polynucleotide comprising the base sequence a and the base sequence b.

6. The insertion sequence q is N 0－2 The polynucleotide of claim 3, wherein the polynucleotide is A.

7. The array p is GGAN 0－4 The polynucleotide of claim 1,

8. The polynucleotide of claim 1 which is DNA.

9. A 5'-capped polynucleotide transcription template comprising the polynucleotide of any one of claims 1 to 8.

10. 1. A method for producing a 5'-capped polynucleotide, comprising: The polynucleotide according to any one of claims 1 to 8, wherein the base sequence b comprises a base sequence Y: (upstream)-Y2-Y3 or (upstream)-Y1-Y2-Y3 (in the base sequence Y: Y1 and Y2 represent bases in the complementary sequence of the insertion sequence q, and Y3 represents a base at the transcription start point), and is a double-stranded polynucleotide; 5' cap analog having a polynucleotide containing the base sequence X: (cap side)-X2-X3 or (cap side)-X1-X2-X3 carrying out a transcription reaction in a reaction system containing X3 is the complementary base of Y3, and X1 is the complementary base of Y1 and / or X2 is the complementary base of Y2; Manufacturing method.

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