Methods for producing capped RNA

Abstract

The present invention provides a method for producing a capped RNA, wherein the capped RNA is an RNA with a cap modified at the 5' end, characterized in that an activated cap compound represented by the following formula (1) is reacted with a monophosphate RNA with a monophosphorylated 5' end (wherein L represents a leaving group). The activated cap compound is preferably a compound represented by the following formula (2).

Description

Technical Field

[0001] The present invention relates to a method for preparing a capped RNA having a cap structure at the 5' end. Background Art

[0002] It is known that in eukaryotic mRNA, etc., there is a 5' cap structure that is 5'-5' bound to 7-methylguanylate via a triphosphate bond at the 5' end. The cap structure is known to promote the translation of mRNA, and in order to efficiently synthesize the target protein in a protein expression system, etc., it is necessary to efficiently introduce the cap structure into mRNA.

[0003] Regarding the synthesis of mRNA, there are transcription methods using enzymes and chemical synthesis methods based on the amide method, but the latter has great advantages in that chemical modifications necessary for mRNA medicines can be freely introduced (which helps to improve the stability and translation ability of mRNA). On the other hand, there is no method for easily introducing a cap structure into chemically synthesized mRNA. The only known method is a method in which the 5' end is diphosphorylated in chemical synthesis and then the cap is introduced using an enzyme (e.g., Non-Patent Document 1). Fig.16 : is a conceptual diagram showing this conventional cap introduction method. In this method, diphosphorylated RNA is prepared by transcription synthesis or chemical synthesis using CAP decapping enzyme (capping enzyme), and a cap structure is further introduced into the 5' end using CAP decapping enzyme.

[0004] In addition, as another method of CAP, a method of methylating guanine by enzyme through solid phase synthesis and activation of monophosphate is known (for example, refer to non-patent document 2). According to the method of this document, an imidazole group is introduced into the monophosphate group at the 5' end of RNA, and it is reacted with a diphosphate group such as GDP (guanosine diphosphate), thereby introducing a CAP structure (cap structure) into the 5' end of RNA.

[0005] In addition, another method is known (for example, see Non-Patent Document 3). In this method, an imidazole group is introduced into the triphosphate group at the 5' end of RNA by solid phase synthesis, and reacted with a monophosphate group such as GMP (guanosine monophosphate), thereby introducing a CAP structure at the 5' end of RNA.

[0006] Prior art literature

[0007] Patent Literature

[0008] Non-patent document 1: "Synthesis and biological activity of artificial mRNA prepared with novel phosphorylating reagents" Seigo Nagata, Tomohiro Hamasaki, Koichi Uetake, Hirofumi Masuda, Kazuchika Takagaki, Natsuhisa Oka, Takeshi Wada, Tadaaki Ohgi, Junichi Yano, Nucleic Acids Research, 2010, 38, 7845

[0009] Non-patent literature 2: “Practical Synthesis of Cap-4RNA, Josef Leiter, et al., ChemBioChem 2020

[0010] Non-patent literature 3: "Chemical Synthesis of U1 snRNA Derivatives", Akihiro Ohkubo et al., Organic Letter 2013 Summary of the invention

[0011] Technical problem to be solved by the invention

[0012] The above-mentioned conventional methods are not suitable for industrial application because the efficiency of diphosphorylation is unstable and quantitative synthesis is difficult due to the use of a capping enzyme. Therefore, a method for chemically introducing a cap at the 5' end of RNA by a relatively simple operation is needed.

[0013] An object of the present invention is to provide a method for producing capped RNA, which is capable of chemically introducing a cap structure into RNA by a simple operation.

[0014] Solutions to technical problems

[0015] The inventors have repeatedly conducted in-depth studies to solve the above technical problems and found that the cap structure can be chemically introduced into the 5' end of RNA by using an activated cap compound obtained by activating the diphosphate of the cap structure with imidazole and reacting it with monophosphate RNA under specified conditions, thereby completing the present invention.

[0016] That is, the present invention is a method for producing capped RNA, which is RNA with a cap modification at the 5' end, characterized in that an activated cap compound represented by the following formula (1) is reacted with a monophosphate RNA with a monophosphorylated 5' end.

[0017]

[0018] (Wherein, L represents a leaving group.)

[0019] In this case, the activation cap compound is preferably a compound represented by the following formula (2).

[0020]

[0021] In addition, it is preferred that the activated cap compound is reacted with the monophosphate RNA in the presence of a heteroaromatic compound, a metal salt, and a solvent.

[0022] Also in this case, it is preferred that the metal salt is a calcium salt.

[0023] Also in this case, it is preferred that the reaction temperature be in the range of 30 to 60°C.

[0024] Also in this case, it is preferred that the reaction time is within the range of 1 to 25 hours.

[0025] Also in this case, it is preferred that the solvent is an organic solvent containing water in the range of 0 to 20% by weight.

[0026] Also in this case, it is preferred that the concentration of the activated cap compound is in the range of 5 to 30 mM.

[0027] Also in this case, it is preferred that the heteroaromatic compound is 2-nitroimidazole and / or 1-methylimidazole.

[0028] Effects of the Invention

[0029] According to the present invention, a cap structure can be chemically introduced into RNA through a simple operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a diagram schematically showing the method for producing capped RNA of the present invention.

[0031] Figure 2 It is a figure which shows the result of Experimental Example 1 in an Example.

[0032] Figure 3 It is a figure which shows the result of Experimental Example 2 in an Example.

[0033] Figure 4 It is a figure which shows the result of Experimental Example 3 in an Example.

[0034] Figure 5 It is a figure which shows the result of Experimental Example 4 in an Example.

[0035] Figure 6 It is a figure which shows the result of Experimental Example 5 in an Example.

[0036] Figure 7 It is a figure which shows the result of Experimental Example 6 in an Example.

[0037] Figure 8 It is a figure which shows the result of Experimental Example 7 in an Example.

[0038] Fig. 9 It is a figure which shows the result of Experimental Example 8 in an Example.

[0039] Fig.10 It is a figure which shows the result of Experimental Example 9 in an Example.

[0040] Fig.11 It is a figure which shows the result of Experimental Example 10 in an Example.

[0041] Fig.12 It is a figure which shows the result of Experimental Example 11 in an Example.

[0042] Fig.13 It is a figure which shows the result of Experimental Example 12 in an Example.

[0043] Fig.14 It is a figure which shows the result of Experimental Example 13 in an Example.

[0044] Fig.15 It is a figure which shows the result of Experimental Example 14 in an Example.

[0045] Fig.16 It is a probability diagram showing the method of producing capped RNA using existing enzymes.

[0046] Fig.17 It is a figure which shows the result of Experimental Example 15 in an Example.

[0047] Fig.18 It is a figure which shows the result of Experimental Example 16 in an Example.

[0048] Fig.19 It is a figure which shows the result of Experimental Example 17 in an Example. DETAILED DESCRIPTION

[0049] The method for producing capped RNA of the present invention is described below. The method for producing capped RNA of the present invention is a method for introducing a cap at the 5' end of an RNA molecule such as mRNA, that is, a method for producing capped RNA which is an RNA with a cap modified at the 5' end. Figure 1 The method for producing the capped RNA of the present invention is shown.

[0050] In the method of the present invention, first, an activated cap compound represented by the following formula (1) and a monophosphorylated 5'-end monophosphate RNA are prepared.

[0051]

[0052] (Wherein, L represents a leaving group.)

[0053] Among them, the activated cap compound represented by the above formula (1) is preferably a compound represented by the following formula (2). The activated cap compound of the following formula (2) is a compound that activates the diphosphate of the cap structure by the leaving group L, which is a compound in which the diphosphate of 7-methylguanylate is bound to imidazole.

[0054]

[0055] Examples of the leaving group L of the formula (1) include, in addition to the above-mentioned imidazole group, heteroaromatic ring compounds such as pyrazoles, oxazoles, thiazoles, pyridines, pyrimidines, pyrazines, and triazines.

[0056] The activated cap compound of formula (2) can be synthesized by diphosphorylating guanosine and then dehydrating and condensing it with imidazole. Specifically, it can be synthesized by the scheme described in the examples described below. The outline is that the 5' position of the ribose of guanosine is first phosphorylated to synthesize guanosine monophosphate (guanosine-5'-phosphate), and then reacted with imidazole to bind the imidazole to the phosphate group. Next, it is reacted with triethanolamine phosphate to synthesize guanosine diphosphate, and further reacted with methyl iodide to methylate the 7-position of the base. Finally, it is reacted with imidazole to bind the imidazole to the phosphate group.

[0057] The monophosphate RNA monophosphorylated at the 5' end is a target compound combined with the above-mentioned activation cap compound. 5' monophosphate RNA can be synthesized by using RNA 5' pyrophosphohydrolase (RppH) to remove pyrophosphate from 5' triphosphate RNA, chemical solid phase synthesis, etc. As countersalts of monophosphate RNA, tetraalkylammonium salts, trialkylacetates, sodium acetate, etc. can be listed. In particular, by making the counter cation of phosphate an organic salt, the reactivity can be improved.

[0058] Next, the activated cap compound is reacted with the monophosphate RNA in the presence of a heteroaromatic compound, at least one metal salt selected from the group consisting of calcium salt and zinc salt, magnesium salt, nickel salt and copper salt, and a solvent.

[0059] As the heteroaromatic compound, an imidazole compound having an imidazole group is preferred. As the imidazole compound, N-alkylimidazoles in which the nitrogen of imidazole is bonded to an alkyl group can be cited, and in particular, compounds having 1 to 5 carbon atoms can be cited as the alkyl group. As N-alkylimidazoles, for example, 1-methylimidazole, 1-ethylimidazole, 1-propylimidazole, 4-methylimidazole, 1-methyl-1H-imidazole-2-carboxylate, 1-methylimidazole-4-carboxylate, 5-chloro-1-methyl-4-nitroimidazole, 2-hydroxymethyl-1-methylimidazole, etc. can be cited. Among these N-alkylimidazoles, 1-methylimidazole is preferred based on the high degree of cap introduction activity. In addition, as the imidazole compound, imidazoles other than N-alkylimidazole can be cited, and 1-(2-hydroxyethyl)imidazole, 2-nitroimidazole, etc. can also be cited. Among these imidazole compounds, 2-nitroimidazole is particularly preferred based on the high degree of cap introduction activity.

[0060] As the metal salt, it can be selected from the group consisting of calcium salts, zinc salts, magnesium salts, nickel salts, and copper salts, or a mixture thereof (e.g., calcium salts and zinc salts). Examples of calcium salts include calcium chloride (CaCl2), calcium hydroxide (Ca(OH)2), and the like. Examples of zinc salts include zinc chloride (ZnCl2), and the like. Among them, CaCl2 is particularly preferred from the perspective of the high activity of cap introduction.

[0061] As the solvent, water and organic solvents can be cited. As the organic solvent, dimethyl sulfoxide (DMSO), acetone, acetonitrile, tetrahydrofuran (THF), dioxane, methyl ethyl ketone, N,N-dimethylformamide (DMF), 1-methyl-2-pyrrolidone (NMP), dimethylformamide (DMF), methanol, ethanol, etc. can be cited. Among them, dimethyl sulfoxide (DMSO) is preferred based on the high solubility of organic salts, etc. By selecting an organic solvent as the main solvent, the hydrolysis of the capping reagent as a side reaction can be suppressed, and a highly efficient capping reaction can be obtained. As a solvent, based on the high viewpoint of the cap introduction activity, an organic solvent containing water in the range of 0 to 20 weight % is preferred, and an organic solvent containing water in the range of 1 to 10 weight % is more preferred.

[0062] The concentration of the activated cap compound in the reaction solution is preferably in the range of 5 to 30 mM. The concentration of the heteroaromatic compound in the reaction solution is preferably in the range of 0.5 to 20 mM, more preferably 5 to 15 mM, and particularly preferably 10 mM. The concentration of the metal salt in the reaction solution is preferably in the range of 0.5 to 10 mM. The reaction conditions can be appropriately set, for example, the reaction temperature is in the range of 30 to 60° C., preferably in the range of 35 to 40° C., and particularly preferably 37° C. In addition, the reaction time is in the range of 1 to 25 hours, preferably in the range of 5 to 15 hours, and particularly preferably 9 hours. Within the range of these conditions, the cap introduction activity is increased, and the cap structure can be efficiently introduced into mRNA.

[0063] Example

[0064] Hereinafter, the present invention will be specifically described based on examples, but the purpose of the present invention is not limited thereto. In the following examples, "%" is a mass basis (mass percentage) unless otherwise indicated.

[0065] The activated cap compound (Compound 13) was synthesized based on the following scheme. The percentages in the figure are the yields.

[0066]

[0067] (1) Synthesis of Compound 9

[0068] Guanosine (748 mg, 2.64 mmol, 1.0 eq.) was suspended in trimethyl phosphate (7.0 mL). Phosphoryl chloride (743 μL, 7.92 mmol, 3.0 eq.) was added dropwise while stirring at -10°C, and the mixture was stirred at -10°C for 20 hours. 1 M TBAE buffer was added to quench the reaction. After concentrating the solution, it was purified by DEAE-dextran. The fractions were recovered and concentrated to obtain a white solid (TEA salt: triethylamine salt).

[0069] (2) Synthesis of Compound 10

[0070] Compound 9 (1.63 g, 4.48 mmol, 1.0 eq. TEA salt), imidazole (3.67 g, 53.9 mmol, 12 eq.), and 2,2'-disulfide bipyridine (3.30 g, 14.9 mmol, 3.3 eq.) were dissolved in dehydrated DMSO (10 mL), and TEA (1.90 mL, 13.5 mmol, 3.0 eq.) and Ph3P (3.74 g, 14.2 mmol, 3.2 eq.) were added, and stirred at room temperature for 18 hours. Sodium perchlorate (2.04 g, 16.6 mmol, 3.7 eq.) dissolved in dry acetone (80 mL) was added, and the mixture was allowed to stand at 4°C. The precipitated solid was filtered out by suction and washed with dry and cold acetone. The obtained solid was dried and solidified under vacuum to obtain a white powder (Na salt).

[0071] (3) Synthesis of Compound 11

[0072] Compound 10 (500 mg, 1.21 mmol, 1.0 eq.) was suspended in dehydrated DMSO (8.0 mL). Zinc chloride (849 mg, 6.23 mmol, 5.1 eq.) and triethylamine phosphate (1.0 g, 5.02 mmol, 4.1 eq., dehydrated DMSO aq.) were added while stirring at room temperature, and stirred at room temperature for 16 hours. 1 M TBAE buffer was added to quench the reaction. The generated precipitate was removed by suction filtration, and the solution was concentrated. Purification was performed by DEAE-dextran. The fractions were recovered and concentrated to obtain a white solid (TEA salt).

[0073] (4) Synthesis of Compound 12

[0074] Compound 11 (100 mg, 0.226 mmol, 1.0 eq. TEA salt) was dissolved in dehydrated DMSO (3.0 mL). Methyl iodide (90 μL, 1.42 mmol, 6.3 eq.) was added at room temperature and stirred for 18 hours. 1 M TBAE buffer was added to quench the reaction. The mixture was separated from diethyl ether, recovered in the water tank, and concentrated. Purification was performed by DEAE-dextran. The fractions were recovered and concentrated to obtain a white solid (TEA salt).

[0075] (5) Synthesis of Compound 13

[0076] Compound 12 (20 mg, 43.7 μmol, 1.0 eq. TEA salt), imidazole (46.6 mg, 0.685 mmol, 15 eq.), and 2,2'-disulfide bipyridine (61.3 mg, 0.685 mmol, 6.0 eq.) were dissolved in dehydrated DMSO (400 μL), and TEA (18.0 μL, 0.129 mmol, 3.0 eq) and Ph3P (57.7 mg, 0.220 mmol, 5.0 eq) were added, and stirred at room temperature for 4.0 hours. Sodium perchlorate (68.2 mg, 0.606 mmol, 14 eq.) dissolved in dry acetone (3.6 mL) was added, and the mixture was allowed to stand at 4°C. The precipitated solid was filtered by suction and washed with dry, cold acetone. The obtained solid was dried and solidified under vacuum to obtain a white powder (Na salt).

[0077] The production conditions are as follows.

[0078] Column:

[0079] DEAE dextran

[0080] Eluent:

[0081] A)MQ(Ultra-pure water)

[0082] B) 1.5M TEAB buffer (triethylamine-carbonic acid buffer) + 10% ACN (acetonitrile)

[0083] ·gradient:

[0084] 0~10min B conc.0%

[0085] 10~210min B conc.0-100%

[0086] 210~240min B conc.100%

[0087] 240min-B conc.0%

[0088] Flow rate:

[0089] 5ml / min

[0090] Detection:

[0091] 260nm

[0092] 1. Experimental Example 1 (Example 1): Discussion on Temperature Conditions

[0093] Discussions were conducted at room temperature, 37°C (Example 1-1), and 55°C (Example 1-2).

[0094] (1) Experimental items

[0095] The required amount of RNA, activated capping compound, and CaCl2 were mixed separately. The solution was dried and solidified by centrifugal evaporator. Anh.DMSO (anhydrous dimethyl sulfoxide) was added and incubated at room temperature (rt), 37°C or 55°C overnight. The reaction was quenched by desalting with Amicon (3K). The reaction efficiency was analyzed by gel electrophoresis. The results were Figure 2 Shown. Figure 2 (a) shows the concentration of each component in the reaction solution, (b) shows the result of electrophoresis, and (c) shows the yield.

[0096] (2) Results

[0097] The reaction did not proceed at rt and 37° C. At 55° C., the reaction proceeded, although the efficiency was low. In the case of the monophosphate activated form, the reaction efficiency was improved by about 20% at 55° C.

[0098] 2. Experimental Example 2 (Example 2): Discussion of MCl2

[0099] As Lewis acids, a total of 6 metal salts were discussed, including NiCl2 (Reference Example 2-1), ZnCl2 (Reference Example 2-2), CaCl2 (Example 2-1), MgCl2 (Example 2-2), CuCl2 (Reference Example 2-3), and FeCl2 (Reference Example 2-4).

[0100] (1) Experimental items

[0101] The required amount of RNA, activated cap compound, and MCl2 were mixed separately. The solution was dried and solidified by centrifugal evaporator. Anh.DMSO was added and incubated at 55°C for 17 hours. The reaction was quenched by desalting with Amicon (3K). The reaction efficiency was analyzed by gel electrophoresis. The results were Figure 3 Shown. Figure 3 (a) shows the concentration of each component in the reaction solution, (b) shows the result of electrophoresis, and (c) shows the yield.

[0102] (2) Results

[0103] It can be seen that the reaction efficiency is improved in CaCl2 (Example 2-1) and MgCl2 (Example 2-2).

[0104] 3. Experimental Example 3 (Example 3): Discussion on the addition of 1-methylimidazole and reaction time

[0105] For ZnCl2 and CaCl2, discuss the reaction of adding 1-methylimidazole.

[0106] (1) Experimental items

[0107] The required amount of RNA, activated cap compound, and MCl2 were mixed separately. The solution was dried and solidified by centrifugal evaporator. 1-Methylimidazole and anh.DMSO were added and incubated at 55°C for 17 hours. The reaction was rapidly cooled by Amicon (3K) desalting. The reaction efficiency was analyzed by gel electrophoresis. The results were Figure 4 Shown in. Figure 4 (a) shows the concentration of each component in the reaction solution, (b) shows the result of electrophoresis, (c) shows the yield, and (d) shows a histogram of the yield.

[0108] (2) Results

[0109] It can be seen that, regardless of the type of salt, the addition of 1-methylimidazole increases the overall yield.

[0110] 4. Experimental Example 4 (Example 4): Discussion on the Concentration of 1-Methylimidazole

[0111] Since the reaction efficiency was improved by the addition of 1-methylimidazole, its concentration conditions were discussed.

[0112] (1) Experimental items

[0113] The required amount of RNA, activated cap compound, and CaCl2 were mixed separately. The solution was dried and solidified by centrifugal evaporator. The required amount of 1-methylimidazole and anh.DMSO (anhydrous dimethyl sulfoxide) were added separately, and incubated at 55°C for 3 hours. The reaction was quenched by desalting with Amicon (3K). The reaction efficiency was analyzed by gel electrophoresis. The results are shown in Figure 5 Shown. Figure 5 (a) shows the concentration of each component in the reaction solution, (b) shows the result of electrophoresis, and (c) shows the yield.

[0114] (2) Results

[0115] The concentration change did not affect the reaction efficiency.

[0116] 5. Experimental Example 5 (Example 5): Discussion on Nucleic Acid Concentration

[0117] Investigate whether nucleic acid concentration affects reaction efficiency.

[0118] (1) Experimental items

[0119] The required amount of RNA, activated cap compound, and CaCl2 were mixed separately. The solution was dried and solidified by centrifugal evaporator. The required amount of 1-methylimidazole and anh.DMSO were added separately and incubated at 55°C for 3 hours. The reaction was rapidly cooled by Amicon (3K) desalting. The reaction efficiency was analyzed by gel electrophoresis. The results are shown in Figure 6 Shown. Figure 6 (a) shows the concentration of each component in the reaction solution, (b) shows the result of electrophoresis, and (c) shows the yield.

[0120] (2) Results

[0121] The nucleic acid concentration has little effect on the reaction efficiency.

[0122] 6. Experimental Example 6 (Example 6): Discussion in the Presence of Water

[0123] The reaction efficiency in the presence of water was investigated.

[0124] (1) Experimental items (lane 1 (Example 6-1), lane 2 (Example 6-2))

[0125] The required amount of RNA, activated cap compound, and CaCl2 were mixed separately. The solution was dried and solidified by centrifugal evaporator. The required amount of 1-methylimidazole and each solvent (anh.DMSO / MQ (ultrapure water) = 1 / 1 or anh.DMSO) were added separately, and incubated at 55°C for 3 hours. The reaction was rapidly cooled by Amicon (3K) desalting. The reaction efficiency was analyzed by gel electrophoresis.

[0126] (2) Experimental item (lane 3 (Example 6-3))

[0127] The required amount of activated cap compound and CaCl2 were mixed. The solution was dried and solidified by centrifugal evaporator. The required amount of 1-methylimidazole, anh.DMSO, and RNA were added and incubated at 55°C for 3 hours. The reaction was quenched by desalting with Amicon (3K). The reaction efficiency was analyzed by gel electrophoresis. The results were Figure 7 Shown. Figure 7 (a) shows the concentration of each component in the reaction solution, (b) shows the result of electrophoresis, and (c) shows the yield.

[0128] (3) Results

[0129] Lane 2 was selected as a positive control. Compared with other lanes, the reaction efficiency was greatly reduced under the condition of 50% MQ (lane 1), but was slightly increased under the condition of about 10% MQ (lane 3).

[0130] 7. Experimental Example 7 (Example 7): Discussion on the Concentration of CaCl2

[0131] Investigate whether salt concentration affects reaction efficiency.

[0132] (1) Experimental items

[0133] The required amount of activated cap compound and CaCl2 were mixed. The solution was dried and solidified by centrifugal evaporator. The required amount of 1-methylimidazole, anh.DMSO, and RNA were added and incubated at 55°C for 3 hours. The reaction was quenched by desalting with Amicon (3K). The reaction efficiency was analyzed by gel electrophoresis. The results were Figure 8 Shown. Figure 8 (a) shows the concentration of each component in the reaction solution, (b) shows the result of electrophoresis, and (c) shows the yield.

[0134] (2) Results

[0135] Although a certain salt concentration is required to efficiently promote the reaction, the efficiency decreases at high concentrations.

[0136] 8. Experimental Example 8 (Example 8): Discussion on the concentration of the activated cap compound

[0137] It was investigated whether the concentration of the activated cap compound affects the reaction efficiency.

[0138] (1) Experimental items

[0139] The required amount of activated cap compound and CaCl2 were mixed. The solution was dried and solidified by centrifugal evaporator. The required amount of 1-methylimidazole, anh.DMSO, and RNA were added and incubated at 55°C for 3 hours. The reaction was quenched by desalting with Amicon (3K). The reaction efficiency was analyzed by gel electrophoresis. The results were Fig. 9 Shown. Fig. 9 (a) shows the concentration of each component in the reaction solution, (b) shows the result of electrophoresis, and (c) shows the yield.

[0140] (2) Results

[0141] The reaction efficiency increased with increasing concentration, and the best (20 mM) obtained a yield of 84%.

[0142] 9. Experimental Example 9 (Example 9): Discussion of the Last Reagent Added

[0143] Under the conditions of "6. Experimental Example 6 (Example 6): Discussion in the presence of water", the reaction efficiency obtained by the difference in the reagent added last was discussed. 5'-PO RNA was added last in lane 1, the activated cap compound was added last in lane 2, and CaCl2 was added last in lane 3.

[0144] (1) Experimental items

[0145] (lane1): Mix the required amount of activated cap compound and CaCl2, (lane2): Mix the required amount of RNA and CaCl2, (lane3): Mix the required amount of RNA and activated cap compound. Dry and solidify the solution using a centrifugal evaporator. (lane1): Add the required amount of 1-methylimidazole, anh.DMSO and RNA, (lane2): Add the required amount of 1-methylimidazole, anh.DMSO and activated cap compound, (lane3): Add the required amount of 1-methylimidazole, anh.DMSO and CaCl2, and incubate at 55°C for 3 hours. Desalt using Amicon (3K) to quench the reaction. The reaction efficiency is analyzed by gel electrophoresis. The results are shown in Fig.10 Shown. Fig.10 (a) shows the concentration of each component in the reaction solution, (b) shows the result of electrophoresis, and (c) shows the yield.

[0146] (2) Results

[0147] Which reagent is added last has no significant effect on the reaction efficiency.

[0148] 10. Experimental Example 10 (Example 10): Discussion of Reaction Time

[0149] The reaction solution was sampled every 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, and 20 hours from the start of the reaction to investigate changes in yield due to reaction time. The reaction conditions were cultured at 55°C. In the case of 1-methylimidazole (+), the reaction basically did not proceed when the reaction time was 1 hour, so the reaction solution was sampled at shorter time intervals (10, 20, 30, 40, 50, 60, 90, and 120 minutes) to investigate differences in reaction efficiency.

[0150] (1) Experimental items

[0151] The required amount of RNA and CaCl2 were mixed separately. The solution was dried and solidified by centrifugal evaporator. The required amount of 1-methylimidazole (only +), anh.DMSO, and activated cap compound were added separately and incubated at 55°C. The reaction was quenched by desalting with Amicon (3K). The reaction efficiency was analyzed by gel electrophoresis. The results are shown in Fig.11 Shown. Fig.11 (a) shows the concentration of each component in the reaction solution, (b) shows the result of electrophoresis, and (c) shows the yield.

[0152] (2) Results

[0153] The reaction was basically completed in 2 to 3 hours, and the yield was slightly increased when the reaction was carried out overnight. In the case of 1-methylimidazole (-), the reaction rate was greatly different.

[0154] 11. Experimental Example 11 (Example 11): Discussion of Reaction in Aqueous Solution

[0155] In the discussion of "3. Experimental Example 3 (Example 3): Addition of 1-methylimidazole, discussion of reaction time", the reaction in aqueous solution was discussed for obtaining high yields of CaCl2 and MgCl2 without adding an activator. MQ and 10mM HEPES buffer (pH=7.4) were used as solvents. The reaction conditions were incubation at 55°C overnight.

[0156] (1) Experimental items

[0157] Mix RNA, activated capping compound, MCl2, and solvent to make the final concentration Fig.12 (a) is shown in the table. The cells were cultured overnight at 55°C. The reaction was rapidly cooled by Amicon (3K) desalting. The reaction efficiency was analyzed by gel electrophoresis. The results are shown in Fig.12 Shown in. Fig.12 (a) shows the concentration of each component in the reaction solution, (b) shows the result of electrophoresis, and (c) shows the yield.

[0158] (2) Results

[0159] When MQ was used as the solvent, it was confirmed that the reaction proceeded efficiently, but since the band intensity was reduced, there was a possibility that RNA hydrolysis also proceeded in the same manner.

[0160] 12. Experimental Example 12 (Example 12): Discussion of Salt Concentration (Mg)

[0161] The effect of MgCl2 concentration in MQ solvent was discussed. The concentration was 5, 10, 20, and 30 mM. The reaction was incubated at 55°C overnight.

[0162] (1) Experimental items

[0163] Mix the required amount of RNA, activated capping compound, CaCl2, and MQ to give a final concentration of Fig.13 (a) is shown in the table. The cells were cultured overnight at 55°C. The reaction was rapidly cooled by Amicon (3K) desalting. The reaction efficiency was analyzed by gel electrophoresis. The results are shown in Fig.13 Shown. Fig.13 (a) shows the concentration of each component in the reaction solution, (b) shows the result of electrophoresis, (c) shows the yield, and (d) shows a histogram of the yield.

[0164] (2) Results

[0165] The MgCl2 concentration had no significant effect on the reaction efficiency.

[0166] 13. Experimental Example 13 (Example 13): Discussion of Salt Concentration (Ca)

[0167] The effect of CaCl2 concentration in MQ solvent was discussed. The concentrations were 5, 10, 20, 30, 45, 60, and 90 mM. The reaction was cultured at 55°C overnight.

[0168] (1) Experimental items

[0169] Mix the required amount of RNA, activated capping compound, CaCl2, and MQ to give a final concentration of Fig.14 (a) is shown in the table. The cells were cultured overnight at 55°C. The reaction was rapidly cooled by Amicon (3K) desalting. The reaction efficiency was analyzed by gel electrophoresis. The results are shown in Fig.14 Shown. Fig.14 (a) shows the concentration of each component in the reaction solution, (b) shows the result of electrophoresis, (c) shows the yield, and (d) shows a histogram of the yield.

[0170] (2) Results

[0171] The CaCl2 concentration has a great influence on the reaction efficiency. The yield increases as the salt concentration increases, reaching the maximum at 30 mM. Excessive salt concentration promotes hydrolysis, which is associated with a decrease in reaction efficiency.

[0172] 14. Experimental Example 14 (Example 14): Discussion of Reaction Time

[0173] The time dependence of the reaction conditions was discussed in MQ solvent conditions. The reaction solution was sampled at 1.0, 2.0, 3.0, 15, 40, and 70 hours from the start of the reaction, and the change in yield due to the reaction time was discussed. The reaction conditions were cultured at 55°C.

[0174] (1) Experimental items

[0175] Mix the required amount of RNA, activated capping compound, CaCl2, and MQ to make the final concentration Fig.15 (a) is shown in the table. The culture was carried out at 55°C. The reaction was rapidly cooled by Amicon (3K) desalting. The reaction efficiency was analyzed by gel electrophoresis. The results are shown in Fig.15 Shown. Fig.15 (a) shows the concentration of each component in the reaction solution, (b) shows the result of electrophoresis, and (c) shows a histogram of the yield.

[0176] (2) Results

[0177] Although the reaction efficiency increased with the passage of time, hydrolysis was also greatly promoted, which was associated with a decrease in the amount of RNA.

[0178] 15. Experimental Example 15 (Example 14): Screening of additives (heteroaromatic compounds)

[0179] For the heteroaromatic compound as an additive, CAP reaction (capping reaction) was carried out using various imidazole derivatives. The imidazole derivatives evaluated are as follows.

[0180]

[0181] (1) Experimental items

[0182] The outline of the experiment is as follows Fig.17 (a) shown.

[0183] [Ion Exchange]

[0184] The RNA solution obtained by isopropanol precipitation was loaded onto a cation exchange resin column (Dowex 50Wx8) equilibrated with a 20% (v / w) tetraethylammonium chloride solution and washed with MQ, and eluted with MQ. The fraction having an absorbance of A260 was recovered and salt-exchanged with tetraethylammonium chloride to obtain an RNA solution.

[0185] [Capping reaction]

[0186] In a microcentrifuge tube, add the ion-exchanged RNA solution (and CaCl2 solution) and remove the water using a freeze dryer. 7 Gpp-Im (activated cap compound), DMSO solution of imidazole derivatives, and DMSO for concentration adjustment. The final concentrations are: RNA 10μM, CaCl2 10mM, m7Gpp-Im 10mM, imidazole derivative 50mM.

[0187] The reaction solution was incubated at 55°C for 3 hours, and a portion of the reaction solution was mixed with 2x loading buffer (80% formamide, 10 mM EDTA in MQ) and analyzed by gel electrophoresis (10% acrylamide, 7.5 M urea). Fig.17 (c) is shown.

[0188] (2) Results

[0189] From the results, it can be seen that the yield of 2-nitroimidazole (Compound F) is the highest when CaCl2(+) is present (yield = 98%). Therefore, it can be seen that 2-nitroimidazole is the most suitable additive.

[0190] 16. Experimental Example 16 (Example 15): Screening of Additive Concentration, Temperature, and Reaction Time

[0191] 2-nitroimidazole was used as an additive to carry out CAP reaction at various concentrations, reaction temperatures and reaction times. Fig.18 (b) as shown.

[0192] (1) Experimental items

[0193] The outline of the experiment is as follows Fig.18 (a) shown.

[0194] [Capping reaction]

[0195] In a microcentrifuge tube, add the ion-exchanged RNA solution and CaCl2 solution, and remove the water using a freeze dryer. 7 Gpp-Im, DMSO solution of imidazole derivatives and DMSO for adjusting concentration.

[0196] The final concentrations are: RNA 10 μM, CaCl2 10 mM, m 7 Gpp-Im 10mM, imidazole derivatives 0~50mM.

[0197] The reaction solution was incubated at various temperatures (37 or 55°C) for 3 to 23 hours, and a portion of the reaction solution was mixed with 2x loading buffer and analyzed by gel electrophoresis (10% acrylamide, 7.5 M urea). Fig.18 (c) is shown.

[0198] (2) Results

[0199] From this result, it can be seen that the conditions in lane 5 (concentration 10 mM, reaction temperature 37° C., reaction time 9 hours) are the most suitable.

[0200] 17. Experimental Example 17 (Example 16): Evaluation of the effect of RNA counter salt

[0201] For RNA counter salts, various salts were used for CAP reaction. Fig.19 (b) shows K440'A etc.

[0202] (1) Experimental items

[0203] The outline of the experiment is as follows Fig.19 As the counter salt, tetraethylammonium salt (NEt4Cl) and sodium acetate salt (NaOAc) were used.

[0204] [Capping reaction]

[0205] Add RNA solution and CaCl2 solution to a microcentrifuge tube and remove water using a freeze dryer. 7 DMSO solutions of Gpp-Im and imidazole derivatives and DMSO for adjusting concentrations.

[0206] The final concentrations are: RNA 10 μM, CaCl2 10 mM, m 7 Gpp-Im 10mM, 2-nitroimidazole 10mM.

[0207] The reaction solution was incubated at 37°C for 9 hours, and a portion of the reaction solution was mixed with 2x loading buffer and analyzed by gel electrophoresis (10% acrylamide, 7.5 M urea). Fig.19 (c) is shown.

[0208] (2) Results

[0209] From this result, it is understood that the reactivity of either the tetraethylammonium salt or the sodium acetate salt does not change.

Claims

1. A method for producing a capped RNA, wherein the capped RNA is an RNA whose 5' end is capped and modified, characterized in that: The activated cap compound represented by the following formula (2) is reacted with the monophosphate RNA whose 5' end is monophosphorylated: The reaction is carried out in the presence of an imidazole compound having an imidazole group, at least one metal salt selected from the group consisting of calcium salts, zinc salts, magnesium salts, nickel salts, and copper salts, and a solvent. The imidazole compound having an imidazole group is at least one selected from the group consisting of 1-methylimidazole and compounds represented by the following formulas C, D, E and F: 。 2. The method for producing capped RNA according to claim 1, wherein The metal salt is a calcium salt.

3. The method for producing capped RNA according to claim 1, wherein The reaction temperature is in the range of 30 to 60°C.

4. The method for producing capped RNA according to claim 1, wherein The reaction time is in the range of 1 to 25 hours.

5. The method for producing capped RNA according to claim 1, wherein The solvent contains water in a range of 0 to 20% by weight.

6. The method for producing capped RNA according to claim 1, wherein The concentration of the activated cap compound is in the range of 5 to 30 mM.

7. The method for producing capped RNA according to claim 1, wherein The imidazole compound is 1-methylimidazole.

8. The method for producing capped RNA according to claim 2, wherein: The imidazole compound is a compound represented by the formula F.

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