Method for producing capped RNA

Abstract

A method for producing a capped RNA that is a 5' end cap-modified RNA, said method being characterized by comprising reacting an activated cap compound represented by formula (1) with a monophosphate RNA that is monophosphorylated at the 5' end. (In the formula, L represents a leaving group.)　Preferably, the activated cap compound is a compound represented by formula (2).

Description

[Technical field]

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

[0002] In eukaryotic mRNA, a 5'-cap structure is known in which 7-methylguanylic acid is linked to the 5'-5' end via a triphosphate bond. The cap structure is known to promote the translation of mRNA, and there is a demand for efficient introduction of the cap structure into mRNA in order to efficiently synthesize target proteins in protein expression systems.

[0003] There are two types of mRNA synthesis: enzymatic transcription and chemical synthesis using the amidite method. The latter method has a great advantage in that it allows the free introduction of chemical modifications (which contribute to improving mRNA stability and translation ability) that are essential for mRNA medicines. On the other hand, there is no method that allows easy introduction of a cap structure to chemically synthesized mRNA. The only known method is to enzymatically introduce a cap after diphosphorylating the 5' end in chemical synthesis (for example, Non-Patent Document 1). Figure 16 is a conceptual diagram showing this conventional cap introduction method. In this method, RNA that is diphosphorylated by transcription synthesis or chemical synthesis using a CAP enzyme is prepared, and a cap structure is further introduced to the 5' end using a CAP enzyme.

[0004] Another known method for CAP addition is to use an enzyme to methylate guanine using solid-phase synthesis and activation of monophosphate (see, for example, Non-Patent Document 2). According to the method described in this document, an imidazole group is introduced into the monophosphate group at the 5' end of RNA, and a diphosphate group such as GDP (guanine diphosphate) is reacted with this to introduce a CAP structure into the 5' end of RNA.

[0005] Furthermore, another method is also known (see, for example, 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 then a monophosphate group such as GMP (guanine monophosphate) is reacted with this to introduce a CAP structure into the 5' end of RNA. [Prior art documents] [Patent documents]

[0006] [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 [Non-Patent Document 2] “Practical Synthesis of Cap-4 RNA, Josef Leiter, et.al, ChemBioChem 2020 [Non-Patent Document 3] “Chemical Synthesis of U1 snRNA Derivatives”, Akihiro Ohkubo et.al, Organic Letter2013 Summary of the Invention [Problem to be solved by the invention]

[0007] The above conventional methods are not suitable for industrial application because the efficiency of diphosphorylation is unstable and quantitative synthesis is difficult because of the use of capping enzymes. For this reason, a method for chemically introducing a cap to the 5' end of RNA with a relatively simple procedure has been desired.

[0008] An object of the present invention is to provide a method for producing capped RNA, which enables chemically introducing a cap structure into RNA through a simple procedure. [Means for solving the problem]

[0009] The present inventors have conducted extensive research to solve the above problems, and as a result have found that a cap structure can be chemically introduced at the 5' end of RNA by using an activated cap compound in which a diphosphate of the cap structure is activated with imidazole and reacting this with monophosphate RNA under specific conditions, thereby completing the present invention.

[0010] That is, the present invention is a method for producing capped RNA, which is RNA whose 5' end has been cap-modified, characterized by reacting an activated cap compound represented by the following formula (1) with monophosphate RNA whose 5' end has been monophosphorylated. [ka] (wherein L represents a leaving group.)

[0011] In this case, the activating cap compound is preferably a compound represented by the following formula (2): [ka]

[0012] It is also preferred that the activated cap compound and the monophosphate RNA are reacted in the presence of a heteroaromatic compound, a metal salt, and a solvent.

[0013] In this case, the metal salt is preferably a calcium salt.

[0014] In this case, the reaction temperature is preferably within the range of 30 to 60°C.

[0015] Furthermore, in this case, the reaction time is preferably within the range of 1 to 25 hours.

[0016] Furthermore, in this case, it is preferable that the solvent is an organic solvent containing water in the range of 0 to 20% by weight.

[0017] Furthermore, in this case, the concentration of the activated cap compound is preferably within the range of 5 to 30 mM.

[0018] Furthermore, in this case, the heteroaromatic compound is preferably 2-nitroimidazole and / or 1-methylimidazole. Effect of the Invention

[0019] According to the present invention, it is possible to chemically introduce a cap structure into RNA by a simple procedure. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 is a diagram showing an outline of a method for producing capped RNA of the present invention. [Diagram 2] FIG. 1 is a diagram showing the results of Experimental Example 1 in the Examples. [Diagram 3] FIG. 1 is a diagram showing the results of Experimental Example 2 in the Examples. [Figure 4] FIG. 1 is a diagram showing the results of Experimental Example 3 in the Examples. [Diagram 5] FIG. 1 is a diagram showing the results of Experimental Example 4 in the Examples. [Figure 6] FIG. 1 shows the results of Experimental Example 5 in the Examples. [Figure 7] FIG. 13 is a diagram showing the results of Experimental Example 6 in the Examples. [Figure 8]FIG. 13 is a diagram showing the results of Experimental Example 7 in the Examples. [Figure 9] FIG. 13 is a diagram showing the results of Experimental Example 8 in the Examples. [Figure 10] FIG. 13 is a diagram showing the results of Experimental Example 9 in the Examples. [Figure 11] FIG. 1 shows the results of Experimental Example 10 in the Examples. [Figure 12] FIG. 1 shows the results of Experimental Example 11 in the Examples. [Figure 13] FIG. 13 is a diagram showing the results of Experimental Example 12 in the Examples. [Figure 14] FIG. 13 is a diagram showing the results of Experimental Example 13 in the Examples. [Figure 15] FIG. 13 is a diagram showing the results of Experimental Example 14 in the Examples. [Figure 16] FIG. 1 is a diagram showing an outline of a conventional enzymatic method for producing capped RNA. [Figure 17] FIG. 1 shows the results of Experimental Example 15 in the Examples. [Figure 18] FIG. 1 shows the results of Experimental Example 16 in the Examples. [Figure 19] FIG. 13 is a diagram showing the results of Experimental Example 17 in the Examples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The method for producing the capped RNA of the present invention will be described below. The method for producing the capped RNA of the present invention is a method for introducing a cap into the 5'-end of an RNA molecule such as mRNA, that is, a method for producing a capped RNA, which is an RNA whose 5'-end is cap-modified. Figure 1 shows an outline of the method for producing the capped RNA of the present invention.

[0022] In the method of the present invention, first, an activated cap compound represented by the following formula (1) and monophosphate RNA whose 5' end is monophosphorylated are prepared. [ka] (wherein L represents a leaving group.)

[0023] Here, 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 in which a diphosphate of a cap structure is activated with imidazole as a leaving group L, and is a compound in which imidazole is bonded to a diphosphate of 7-methylguanylic acid. [ka]

[0024] Examples of the leaving group L in formula (1) include the above-mentioned imidazole group, as well as heteroaromatic ring compounds such as pyrazoles, oxazoles, thiazoles, pyridines, pyrimidines, pyrazines and triazines.

[0025] The activated cap compound of formula (2) can be synthesized by diphosphorylation of guanosine, followed by dehydration condensation with imidazole. Specifically, it can be synthesized by the scheme described in the examples below. The outline is that the 5' position of ribose of guanosine is first phosphorylated to synthesize guanosine monophosphate (guanosine-5'-phosphate), and then imidazole is reacted to bind the imidazole to the phosphate group. Next, triethanolamine phosphate or the like is reacted to synthesize guanosine diphosphate, and further iodomethane or the like is reacted to methylate the 7th position of the base. Finally, imidazole is reacted to bind the imidazole to the phosphate group.

[0026] Monophosphate RNA with monophosphorylated 5' end is a target compound to which the above-mentioned activated cap compound binds. 5' monophosphate RNA can be synthesized by a method of removing pyrophosphate from 5' triphosphate RNA using RNA 5' pyrophosphohydrolase (RppH) or by chemical solid-phase synthesis. Examples of counter salts of monophosphate RNA include tetraalkylammonium salts, trialkylacetates, and sodium acetate. In particular, reactivity can be improved by using an organic salt as the counter cation of phosphate.

[0027] The activated cap compound and monophosphate RNA are then reacted with a heteroaromatic compound and at least one metal salt selected from the group consisting of calcium salts, zinc salts, magnesium salts, nickel salts and copper salts in the presence of a solvent.

[0028] As the heteroaromatic compound, an imidazole compound having an imidazole group is preferred. As the imidazole compound, an N-alkylimidazole in which an alkyl group is bonded to the nitrogen of imidazole can be mentioned, and in particular, a compound in which the alkyl group has 1 to 5 carbon atoms can be mentioned. As the N-alkylimidazole, 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 mentioned. Among these N-alkylimidazoles, 1-methylimidazole is preferred from the viewpoint of high cap introduction activity. In addition, as the imidazole compound, imidazoles other than N-alkylimidazole can be mentioned, such as 1-(2-hydroxyethyl)imidazole and 2-nitroimidazole. Among these imidazole compounds, 2-nitroimidazole is particularly preferred from the viewpoint of high capping activity.

[0029] The metal salt is selected from the group consisting of calcium salts, zinc salts, magnesium salts, nickel salts, and copper salts, and may be a mixture thereof (e.g., calcium salt and zinc salt). Calcium salts include calcium chloride (CaCl2), calcium hydroxide (Ca(OH)2), etc. Zinc salts include zinc chloride (ZnCl2), etc. Among these, CaCl2 is particularly preferred in terms of its high capping activity.

[0030] Examples of the solvent include water and organic solvents. Examples of the organic solvent include dimethyl sulfoxide (DMSO), acetone, acetonitrile, tetrahydrofuran (THF), dioxane, methyl ethyl ketone, N,N-dimethylformamide (DMF), 1-methyl-2-pyrrolidone (NMP), dimethylformamide (DMF), methanol, and ethanol. Among these, dimethyl sulfoxide (DMSO) is preferred from the viewpoint of high solubility of organic salts and the like. By using an organic solvent as the main solvent, hydrolysis of the capping reagent, which is a side reaction, can be suppressed, and a highly efficient capping reaction can be achieved. From the viewpoint of high cap introduction activity, the solvent is preferably an organic solvent containing water in the range of 0 to 20% by weight, and more preferably an organic solvent containing water in the range of 1 to 10% by weight.

[0031] The concentration of the activated cap compound in the reaction solution is preferably within the range of 5 to 30 mM. The concentration of the heteroaromatic compound in the reaction solution is preferably within 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 within the range of 0.5 to 10 mM. The reaction conditions can be appropriately set, but for example, the reaction temperature is within the range of 30 to 60°C, preferably within the range of 35 to 40°C, and particularly preferably 37°C. In addition, the reaction time is within the range of 1 to 25 hours, preferably within the range of 5 to 15 hours, and particularly preferably 9 hours. Within these conditions, the cap introduction activity is high, and the cap structure can be efficiently introduced into mRNA. EXAMPLES

[0032] The present invention will be described in detail below with reference to examples, but the object of the present invention is not limited thereto. In the following examples, "%" is based on mass (mass percent) unless otherwise specified.

[0033] The activated cap compound (compound 13) was synthesized according to the following scheme, where the percentages in the figure indicate yields. [ka]

[0034] (1) Synthesis of Compound 9 Guanosine (748 mg, 2.64 mmol, 1.0 eq.) was suspended in trimethyl phosphate (7.0 mL). Phosphonyl 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. 1M TBAE buffer was added to quench the reaction. The solution was concentrated and then purified by DEAE-sephadax. The fractions were collected and concentrated to obtain a white solid (TEA salt).

[0035] (2) Synthesis of compound 10 Compound 9 (1.63 g, 4.48 mmol, 1.0 eq. TEA salt), imidazole (3.67 g, 53.9 mmol, 12 eq.), 2,2'-dithiodipyridine (3.30 g, 14.9 mmol, 3.3 eq.) were dissolved in dehydrated DMSO (10 mL), and then 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 allowed to stand at 4°C. The precipitated solid was filtered by suction and washed with dry, cold acetone. The obtained solid was dried under vacuum to obtain a white powder (Na salt).

[0036] (3) Synthesis of compound 11 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 with stirring at room temperature, and the mixture was stirred at room temperature for 16 hours. The reaction was quenched by adding 1M TBAE buffer. The resulting precipitate was removed by suction filtration, and the solution was concentrated. It was purified by DEAE-sephadax. The fraction was collected and concentrated to obtain a white solid (TEA salt).

[0037] (4) Synthesis of compound 12 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. The reaction was quenched by adding 1M TBAE buffer. The mixture was separated with diethyl ether, and the aqueous layer was collected and concentrated. The mixture was purified by DEAE-sephadax. The fractions were collected and concentrated to obtain a white solid (TEA salt).

[0038] (5) Synthesis of compound 13 Compound 12 (20 mg, 43.7 μmol, 1.0 eq. TEA salt), imidazole (46.6 mg, 0.685 mmol, 15 eq.), 2,2'-dithiodipyridine (61.3 mg, 0.685 mmol, 6.0 eq.) were dissolved in dehydrated DMSO (400 μL), and then 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 allowed to stand at 4°C. The precipitated solid was filtered by suction and washed with dry, cold acetone. The obtained solid was dried under vacuum to obtain a white powder (Na salt).

[0039] The production conditions are as follows: Column: DEAE Sephadax Eluent: A) MQ B) 1.5M TEAB Buffer + 10% ACN Gradient: 0-10min B conc. 0% 10-210min B conc. 0-100% 210-240min B conc. 100% 240min- B conc. 0% Flow rate: 5ml / min Detection: 260nm

[0040] 1. Experimental Example 1 (Example 1): Temperature Condition Investigation The studies were carried out at room temperature, 37°C (Example 1-1), and 55°C (Example 1-2). (1) Experimental items The required amounts of RNA, activated capping compound, and CaCl2 were mixed. The solution was dried using a centrifugal evaporator. DMSO was added and incubated at room temperature (rt), 37°C, or 55°C overnight. The reaction was quenched by desalting using an Amicon (3K). The reaction efficiency was analyzed by gel electrophoresis. The results are shown in Figure 2. (a) in this figure shows the concentration of each component in the reaction solution, (b) shows the results of electrophoresis, and (c) shows the yield. (2) Results No reaction was observed at rt or 37° C. At 55° C., the reaction proceeded, although at a low efficiency. In the case of the activated monophosphate, it was found that the reaction efficiency improved by about 20% at 55° C.

[0041] 2. Experimental Example 2 (Example 2): Examination of MCl2 As Lewis acids, six metal salts were examined: 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). (1) Experimental items The required amounts of RNA, activated capping compound, and MCl2 were mixed. The solution was dried using a centrifugal evaporator. DMSO was added and incubated at 55°C for 17 hours. The reaction was quenched by desalting using an Amicon (3K). The reaction efficiency was analyzed by gel electrophoresis. The results are shown in Figure 3. (a) in this figure shows the concentration of each component in the reaction solution, (b) shows the results of electrophoresis, and (c) shows the yield. (2) Results It was found that the reaction efficiency was improved by using CaCl2 (Example 2-1) and MgCl2 (Example 2-2).

[0042] 3. Experimental Example 3 (Example 3): Addition of 1-methylimidazole and reaction time examination The reaction of ZnCl2 and CaCl2 was examined by adding 1-methylimidazole. (1) Experimental items The required amounts of RNA, activated capping compound, and MCl2 were mixed. The solution was dried using a centrifugal evaporator. 1-Methylimidazole, anh. DMSO were added and incubated at 55°C for 17 hours. The reaction was quenched by desalting using an Amicon (3K). The reaction efficiency was analyzed by gel electrophoresis. The results are shown in Figure 4. (a) in this figure shows the concentration of each component in the reaction solution, (b) the results of electrophoresis, (c) the yield, and (d) a graph of the yield. (2) Results It was found that the addition of 1-methylimidazole improved the overall yield regardless of the type of salt.

[0043] 4. Experimental Example 4 (Example 4): Examination of 1-methylimidazole concentration The reaction efficiency was improved by the addition of 1-methylimidazole, so the concentration conditions were examined. (1) Experimental items The required amounts of RNA, activated capping compound, and CaCl2 were mixed. The solution was dried using a centrifugal evaporator. The required amounts of 1-methylimidazole and anh.DMSO (anhydrous dimethyl sulfoxide) were added and incubated at 55°C for 3 hours. The reaction was quenched by desalting using an Amicon (3K). The reaction efficiency was analyzed by gel electrophoresis. The results are shown in Figure 5. (a) in this figure shows the concentration of each component in the reaction solution, (b) shows the results of electrophoresis, and (c) shows the yield. (2) Results It was found that changes in concentration did not affect the reaction efficiency.

[0044] 5. Experimental Example 5 (Example 5): Nucleic Acid Concentration Study We investigated whether the nucleic acid concentration affected the reaction efficiency. (1) Experimental items The required amounts of RNA, activated capping compound, and CaCl2 were mixed. The solution was dried using a centrifugal evaporator. The required amounts of 1-methylimidazole and anh.DMSO were added and incubated at 55°C for 3 hours. The reaction was quenched by desalting using an Amicon (3K). The reaction efficiency was analyzed by gel electrophoresis. The results are shown in Figure 6. (a) in this figure shows the concentration of each component in the reaction solution, (b) shows the results of electrophoresis, and (c) shows the yield. (2) Results It was found that the nucleic acid concentration did not significantly affect the reaction efficiency.

[0045] 6. Experimental Example 6 (Example 6): Study in the presence of water The reaction efficiency in the presence of water was investigated. (1) Experimental Section (Lane 1 (Example 6-1), Lane 2 (Example 6-2)) The required amounts of RNA, activated capping compound, and CaCl2 were mixed. The solution was dried using a centrifugal evaporator. The required amounts of 1-methylimidazole and each solvent (anh.DMSO / MQ (ultrapure water) = 1 / 1 or anh.DMSO) were added and incubated at 55°C for 3 hours. The reaction was desalted using an Amicon (3K) and quenched. The reaction efficiency was analyzed by gel electrophoresis. (2) Experimental section (Lane 3 (Example 6-3)) The required amounts of activated capping compound and CaCl2 were mixed. The solution was dried using a centrifugal evaporator. The required amounts of 1-methylimidazole, anh.DMSO, and RNA were added and incubated at 55°C for 3 hours. The reaction was quenched by desalting using an Amicon (3K). The reaction efficiency was analyzed by gel electrophoresis. The results are shown in Figure 7. (a) in this figure shows the concentration of each component in the reaction solution, (b) shows the results of electrophoresis, and (c) shows the yield. (3) Results Comparing lane 2, which was used as the positive control, with the other lanes, it was found that the reaction efficiency was significantly reduced under the 50% MQ condition (lane 1), but was slightly improved under the approximately 10% MQ condition (lane 3).

[0046] 7. Experimental Example 7 (Example 7): Examination of CaCl2 concentration We investigated whether the salt concentration affected the reaction efficiency. (1) Experimental items The required amounts of activated capping compound and CaCl2 were mixed. The solution was dried using a centrifugal evaporator. The required amounts of 1-methylimidazole, anh.DMSO, and RNA were added and incubated at 55°C for 3 hours. The reaction was quenched by desalting using an Amicon (3K). The reaction efficiency was analyzed by gel electrophoresis. The results are shown in Figure 8. (a) in this figure shows the concentration of each component in the reaction solution, (b) shows the results of electrophoresis, and (c) shows the yield. (2) Results A certain salt concentration is necessary for the reaction to proceed efficiently, but it was found that efficiency decreases at high concentrations.

[0047] 8. Experimental Example 8 (Example 8): Examination of the concentration of the activated capping compound We investigated whether the concentration of the activated capping compound affected the reaction efficiency. (1) Experimental items The required amounts of activated capping compound and CaCl2 were mixed. The solution was dried using a centrifugal evaporator. The required amounts of 1-methylimidazole, anh.DMSO, and RNA were added and incubated at 55°C for 3 hours. The reaction was quenched by desalting using an Amicon (3K). The reaction efficiency was analyzed by gel electrophoresis. The results are shown in Figure 9. (a) in this figure shows the concentration of each component in the reaction solution, (b) shows the results of electrophoresis, and (c) shows the yield. (2) Results As the concentration increased, the reaction efficiency improved, with the best yield (20 mM) being 84%.

[0048] 9. Experimental Example 9 (Example 9): Consideration of the last reagent added Under the conditions of "6. Experimental Example 6 (Example 6): Study in the presence of water", the reaction efficiency was examined depending on the reagent added last. Lane 1: 5'-PO RNA, lane 2: activated cap compound, lane 3: CaCl2 were added last. (1) Experimental items (Lane 1) Activated capping compound, CaCl2, (Lane 2) RNA, CaCl2, (Lane 3) RNA, and activated capping compound were mixed in the required amounts. This solution was dried using a centrifugal evaporator. 1-Methylimidazole, anh. DMSO, (Lane 1) RNA, (Lane 2) Activated capping compound, and (Lane 3) CaCl2 were added in the required amounts, and incubated at 55°C for 3 hours. The reaction was quenched by desalting using an Amicon (3K). The reaction efficiency was analyzed by gel electrophoresis. The results are shown in Figure 10. In this figure, (a) shows the concentration of each component in the reaction solution, (b) shows the results of electrophoresis, and (c) shows the yield. (2) Results The last addition of any reagent had no significant effect on reaction efficiency.

[0049] 10. Experimental Example 10 (Example 10): Study of reaction time The reaction mixture was sampled at 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, and 20 hours from the start of the reaction to examine the change in yield due to reaction time. The reaction was incubated at 55°C. In the case of 1-methylimidazole (+), the reaction had almost completely proceeded after 1 hour of reaction, so the reaction mixture was sampled at shorter time intervals (10, 20, 30, 40, 50, 60, 90, and 120 minutes) to examine the difference in reaction efficiency. (1) Experimental items The required amounts of RNA and CaCl2 were mixed. The solution was dried using a centrifugal evaporator. The required amounts of 1-methylimidazole (+ only), anh.DMSO, and activated cap compound were added and incubated at 55°C. The reaction was quenched by desalting using an Amicon (3K). The reaction efficiency was analyzed by gel electrophoresis. The results are shown in Figure 11. (a) in this figure shows the concentration of each component in the reaction solution, (b) shows the results of electrophoresis, and (c) shows the yield. (2) Results The reaction was completed in 2 to 3 hours, and the yield was slightly improved by reacting overnight. In the case of 1-methylimidazole (-), a large difference in reaction rate was observed.

[0050] 11. Experimental Example 11 (Example 11): Study of reaction in aqueous solution 3. Experimental Example 3 (Example 3): Addition of 1-methylimidazole and reaction time examination In the study of "CaCl2 and MgCl2, which gave high yields without the need for the addition of an activator, we investigated the reaction in aqueous solution. MQ and 10 mM HEPES Buffer (pH = 7.4) were used as the solvent. The reaction was incubated at 55°C overnight. (1) Experimental items RNA, activated capping compound, MCl2, and solvent were mixed so that the final concentrations were as shown in the table in Figure 12(a). The mixture was incubated at 55°C overnight. The reaction was quenched by desalting using Amicon (3K). The reaction efficiency was analyzed by gel electrophoresis. The results are shown in Figure 12. In this figure, (a) shows the concentration of each component in the reaction solution, (b) shows the results of electrophoresis, and (c) shows the yield. (2) Results When MQ was used as the solvent, the reaction proceeded efficiently, but the band intensity was low, suggesting that RNA hydrolysis may have also proceeded.

[0051] 12. Experimental Example 12 (Example 12): Examination of salt concentration (Mg) The effect of MgCl2 concentration in MQ solvent was examined. Four concentrations were examined: 5, 10, 20, and 30 mM. The reaction was incubated at 55°C overnight. (1) Experimental items RNA, activated capping compound, CaCl2, and MQ were mixed in the required amounts so that the final concentrations were as shown in the table in Figure 13(a). The mixture was incubated at 55°C overnight. The reaction was desalted using Amicon (3K) and quenched. The reaction efficiency was analyzed by gel electrophoresis. The results are shown in Figure 13. In this figure, (a) shows the concentration of each component in the reaction solution, (b) shows the results of electrophoresis, (c) shows the yield, and (d) shows a graph of the yield. (2) Results The MgCl2 concentration did not significantly affect the reaction efficiency.

[0052] 13. Experimental Example 13 (Example 13): Examination of salt concentration (Ca) The effect of CaCl2 concentration in MQ solvent was examined. Seven concentrations were examined: 5, 10, 20, 30, 45, 60, and 90 mM. The reaction was incubated at 55°C overnight. (1) Experimental items RNA, activated capping compound, CaCl2, and MQ were mixed in the required amounts so that the final concentrations were as shown in the table in Figure 14(a). The mixture was incubated at 55°C overnight. The reaction was desalted using Amicon (3K) and quenched. The reaction efficiency was analyzed by gel electrophoresis. The results are shown in Figure 14. In this figure, (a) shows the concentration of each component in the reaction solution, (b) shows the results of electrophoresis, (c) shows the yield, and (d) shows a graph of the yield. (2) Results The CaCl2 concentration had a significant effect on the reaction efficiency. The yield increased with increasing salt concentration, reaching a maximum at 30 mM. Excessive salt concentration promoted hydrolysis, leading to a decrease in the reaction efficiency.

[0053] 14. Experimental Example 14 (Example 14): Study of reaction time The reaction time was examined under 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 reaction time was examined. The reaction was incubated at 55°C. (1) Experimental items RNA, activated capping compound, CaCl2, and MQ were mixed in the required amounts so that the final concentrations were as shown in the table in Figure 15(a). The mixture was incubated at 55°C. The reaction was desalted using Amicon (3K) and quenched. The reaction efficiency was analyzed by gel electrophoresis. The results are shown in Figure 15. In this figure, (a) shows the concentration of each component in the reaction solution, (b) shows the results of electrophoresis, and (c) shows a graph of the yield. (2) Results Although the reaction efficiency improved over time, hydrolysis was also significantly promoted, leading to a decrease in the amount of RNA.

[0054] 15. Experimental Example 15 (Example 14): Screening of additives (heteroaromatic compounds) The heteroaromatic compounds used as additives were subjected to CAP reactions using various imidazole derivatives. The imidazole derivatives evaluated are as follows: [ka]

[0055] (1) Experimental items The outline of the experiment is shown in FIG. 17(a). [Ion exchange] The RNA solution obtained by isopropanol precipitation was loaded onto a cation exchange resin column (Dowex 50Wx8) that had been equilibrated with 20% (v / w) tetraethylammonium chloride solution and washed with MQ, and eluted with MQ. The fractions with A260 absorbance were collected to obtain an RNA solution that had been salt-exchanged with tetraethylammonium chloride. [Capping reaction] The ion-exchanged RNA solution (and CaCl2 solution) was added to an Eppendorf tube, and the water was removed using a freeze dryer. 7 Gpp-Im (activated capping compound) and a DMSO solution of an imidazole derivative were added, and DMSO was added to adjust the concentration. The final concentrations were RNA 10 μM, CaCl2 10 mM, m 7 Gpp-Im was 10 mM, and the imidazole derivative was 50 mM. 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, 7.5M urea). The results of the electrophoresis are shown in Figure 17(c). (2) Results From these results, 2-nitroimidazole (compound F) had the highest yield (yield = 98%) in the presence of CaCl2 (+). Therefore, it was found that 2-nitroimidazole is the most suitable additive.

[0056] 16. Experimental Example 16 (Example 15): Screening of additive concentration, temperature, and reaction time Using 2-nitroimidazole as an additive, the CAP reaction was carried out at various concentrations, reaction temperatures, and reaction times under the reaction conditions shown in Figure 18(b). (1) Experimental items The outline of the experiment is shown in FIG. 18(a). [Capping reaction] The ion-exchanged RNA solution and CaCl2 solution were added to an Eppendorf tube, and the water was removed using a freeze dryer. m7Gpp-Im, a DMSO solution of an imidazole derivative, and DMSO for concentration adjustment were added. The final concentrations were 10 μM RNA, 10 mM CaCl2, 7 Gpp-Im was 10 mM, and the imidazole derivative was 0 to 50 mM. 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, 7.5M urea). The results of the electrophoresis are shown in Figure 18(c). (2) Results From these results, it was found that the conditions of lane 5 (concentration 10 mM, reaction temperature 37°C, reaction time 9 hours) were optimal.

[0057] 17. Experimental Example 17 (Example 16): Evaluation of the effect of counter salt on RNA As counter salts for RNA, CAP reactions were performed using various salts. The salts evaluated are shown in Figure 19(b) as K440'A, etc. (1) Experimental items The outline of the experiment is shown in Figure 19(a). Tetraethylammonium salt (NEt4Cl) and sodium acetate salt (NaOAc) were used as counter salts. [Capping reaction] The RNA solution and CaCl2 solution were added to an Eppendorf tube, and the water was removed using a freeze dryer. 7 DMSO solutions of Gpp-Im and imidazole derivatives, and DMSO for adjusting the concentrations were added. The final concentrations were 10 μM RNA, 10 mM CaCl2, 7 Gpp-Im was 10 mM, and 2-nitroimidazole was 10 mM. The reaction solution was incubated at 37° C. for 9 hours, and a portion of the reaction solution was mixed with 2× loading buffer and analyzed by gel electrophoresis (10% acrylamide, 7.5 M urea). The results of the electrophoresis are shown in FIG. 19(c). (2) Results These results show that the reactivity is the same whether tetraethylammonium salt or sodium acetate salt is used.

Claims

1. A method for producing capped RNA, which is RNA whose 5' end is cap-modified, comprising the steps of: A method for producing capped RNA, comprising reacting an activated cap compound represented by the following formula (2) with monophosphate RNA having a monophosphorylated 5' end in the presence of an imidazole compound, a metal salt, and a solvent: 【Chemistry 1】

2. The method for producing capped RNA according to claim 1, characterized in that the metal salt is a calcium salt.

3. The method for producing capped RNA according to claim 1 or 2, characterized in that the reaction temperature is within the range of 30 to 60°C.

4. The method for producing capped RNA according to claim 1 or 2, characterized in that the reaction time is within the range of 1 to 25 hours.

5. 3. The method for producing capped RNA according to claim 1, wherein the solvent is an organic solvent containing water in the range of 0 to 20% by weight.

6. The method for producing capped RNA according to claim 1 or 2, characterized in that the concentration of the activated cap compound is within the range of 5 to 30 mM.

7. The method for producing a capped RNA according to claim 1 or 2, characterized in that the imidazole compound is 1-methylimidazole.

8. The method for producing capped RNA described in claim 1 or 2, characterized in that the imidazole compound is 2-nitroimidazole.

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