Method for synthesizing RNA

Abstract

To provide a method for synthesizing RNA that enables efficient and low-cost RNA synthesis by providing a readily and economically producible substrate for RNA synthesis, and to provide an RNA synthesis kit usable in the synthesis method.SOLUTION: This invention has been completed based on the finding that: that a nucleoside polyphosphate, obtained by adding polyphosphate to an RNA degradation product, can serve as a substrate for DNA-dependent RNA polymerase.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for synthesizing RNA and a kit for synthesizing RNA. [Background technology]

[0002] With the spread of new virus infections, the demand for RNA synthesis is rapidly increasing, as mRNA vaccines are being developed and used as infectious disease vaccines, and research into mRNA as a cancer treatment vaccine is also progressing.

[0003] Currently, RNA synthesis is carried out by in vitro transcription using RNA polymerase, NTPs, etc., with DNA into which a target sequence has been inserted as a template.

[0004] It is known that the substrate NTP is mainly obtained by enzymatic synthesis, but due to the need for multiple types of enzymes and the substrate specificity of the enzymes, ATP, CTP, GTP, and UTP must each be synthesized in a separate system, which is known to be costly and inefficient (Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 5800218 [Patent Document 2] Patent No. 4256341 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides a substrate for RNA synthesis that can be produced easily and at low cost, thereby providing a method for synthesizing RNA that can synthesize RNA efficiently and at low cost, and provides an RNA synthesis kit that can be used in the synthesis method. [Means for solving the problem]

[0007] In the present invention, it was found that nucleoside polyphosphates obtained by adding polyphosphate to RNA degradation products can be used as substrates for DNA-dependent RNA polymerase, and the present invention was completed based on this finding.

[0008] That is, the present invention relates to the following aspects [1] to [6]. [1] A method for synthesizing RNA, comprising carrying out in vitro transcription using a reaction solution containing a DNA-dependent RNA polymerase, a template DNA, a nucleoside polyphosphate (NPP), a magnesium salt, and a buffer solution, wherein the NPP is a nucleoside monophosphate obtained by enzymatically decomposing RNA, to which polyphosphate has been added. [2] The method for synthesizing RNA according to [1], wherein the NPP is an NPP to which polyphosphate has been added by chemical synthesis using cyclic polyphosphate. [3] The method for synthesizing RNA according to [1] or [2], wherein the cyclic polyphosphate is trimetaphosphate and NPP is nucleoside tetraphosphate. [4] A kit for RNA synthesis, comprising a DNA-dependent RNA polymerase, a nucleoside polyphosphate (NPP), a magnesium salt, and a buffer solution, wherein the NPP is a nucleoside monophosphate obtained by enzymatically decomposing RNA, to which polyphosphate has been added. [5] The kit for synthesizing RNA according to [4], characterized in that the NPP has polyphosphate added thereto by chemical synthesis using cyclic polyphosphate. [6] The kit for RNA synthesis according to [4] or [5], wherein the cyclic polyphosphate is trimetaphosphate and NPP is a nucleoside tetraphosphate. Effect of the Invention

[0009] INDUSTRIAL APPLICABILITY The present invention provides a substrate for RNA synthesis that can be produced easily and at low cost, and thereby makes it possible to provide a method for synthesizing RNA and a kit for RNA synthesis that can synthesize RNA efficiently and at low cost. [Brief description of the drawings]

[0010] [Figure 1] RNA synthesis reactions were carried out for a total of three samples: a positive control (PC), a negative control (NC), and Test 4 using the ATaP composition (Preparation 1). The results of electrophoresis using TapeStation for each reaction solution are shown. A1: Marker, B1: PC, C1: NC, and D1: Test 4. [Diagram 2] RNA synthesis reactions were carried out for a total of three samples: a positive control (PC), a negative control (NC), and Test 5 using the NTeP composition (Preparation 3). The results of electrophoresis using TapeStation for each reaction solution are shown. A1: Marker, B1: PC, C1: NC, and D1: Test 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The RNA synthesis method of the present invention is a method capable of synthesizing RNA by in vitro transcription using a reaction solution containing a DNA-dependent RNA polymerase, a template DNA, a nucleoside polyphosphate (NPP), a magnesium salt, and a buffer solution, and the NPP is a nucleoside monophosphate obtained by enzymatic degradation of RNA to which polyphosphate has been added.

[0012] The DNA-dependent RNA polymerase is not particularly limited as long as it is an enzyme that catalyzes a reaction to synthesize single-stranded RNA using DNA as a template, and is preferably an enzyme consisting of a single subunit, examples of which include T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase.

[0013] The RNA synthesis method of the present invention can synthesize RNA by in vitro transcription using a reaction solution containing DNA-dependent RNA polymerase, template DNA, NPP, magnesium salt and a buffer solution, and the reaction solution can further contain an RNase inhibitor such as DEPC, a reducing agent such as DTT, a stabilizer such as spermidine, etc., as appropriate. The NPP used as the substrate can be adenosine polyphosphate, guanosine polyphosphate, cytidine polyphosphate and uridine polyphosphate, and is not particularly limited as long as it is an NPP in which three or more linear polyphosphates are added to the phosphate group of a nucleoside monophosphate, but nucleoside tetraphosphates such as adenosine tetraphosphate, guanosine tetraphosphate, cytidine tetraphosphate and uridine tetraphosphate are preferred, and the NPP is an NPP in which polyphosphate is added to a nucleoside monophosphate obtained by enzymatically decomposing RNA.

[0014] The enzymatic degradation of RNA may be, for example, by treating a commercially available RNA raw material with a hydrolase that cleaves phosphodiester bonds between nucleotides, and is not particularly limited as long as it is treated with an enzyme that degrades RNA and generates nucleoside monophosphate, and a general RNase may be used. After the enzymatic degradation of RNA, the RNase is inactivated and / or removed by heat treatment, preferably by pressurized heat treatment, and / or by removal of RNase, RNA raw material, etc. using an ultrafiltration membrane, and more preferably by purification, to recover the nucleoside monophosphate composition.

[0015] The NPP described in the present invention can be synthesized by a general method, and can be synthesized chemically or enzymatically. For example, polyphosphate can be chemically added to nucleoside monophosphate, and linear polyphosphate or cyclic polyphosphate can be used. Examples of cyclic polyphosphate include trimetaphosphate and tetrametaphosphate, and cyclic polyphosphate salts can be used, with sodium trimetaphosphate being preferred. For example, nucleoside tetraphosphate can be synthesized according to Samy Mohamady and Scott D. Taylor, "Synthesis of Nucleoside Tetraphosphates and Dinucleoside Pentaphosphaes via Activation of Cyclic Trimetaphosphate", Organic Letters, May 13, 2013, Vol. 15, No. 11, p. 2612-2615 and the Supporting Information of the paper.

[0016] The template DNA may be any DNA in which a gene sequence of interest is inserted downstream of a promoter sequence, and the promoter sequence is not particularly limited as long as it is a promoter that can be recognized by the RNA polymerase used, but a DNA-dependent promoter is preferred, and a sequence obtained by partially modifying a known promoter sequence may be used, and a natural or modified T7 promoter may also be used. In addition, it is preferable to include a terminator sequence downstream of the gene sequence of interest, and the terminator sequence is not particularly limited as long as it is a terminator that can be recognized by the RNA polymerase used.

[0017] The concentration of the buffer in the reaction solution is not particularly limited, but the upper limit is preferably 200 mM or less, more preferably 100 mM or less, and even more preferably 50 mM or less. The lower limit is preferably 1 mM or more, more preferably 2 mM or more, and even more preferably 5 mM or more.

[0018] In the present invention, in vitro transcription may be performed under conditions suitable for DNA-dependent RNA polymerase. The reaction temperature is not particularly limited as long as it is within a range in which the enzyme is not inactivated; however, in vitro transcription is preferably performed at 15°C or higher, more preferably 20°C or higher, even more preferably 25°C or higher, and particularly preferably 30°C or higher; in vitro transcription is preferably performed at 45°C or lower, more preferably 42°C or lower, and even more preferably 40°C or lower.

[0019] In the present invention, an RNA synthesis kit can be produced using NPP as a substrate for DNA-dependent RNA polymerase, and an RNA synthesis kit containing DNA-dependent RNA polymerase, NPP, magnesium salt, and buffer solution can be produced. Furthermore, the kit can appropriately contain RNase inhibitors such as DEPC, reducing agents such as DTT, stabilizers such as spermidine, and the like. The NPP used as the substrate can be adenosine polyphosphate, guanosine polyphosphate, cytidine polyphosphate, or uridine polyphosphate, and is not particularly limited as long as it is an NPP in which three or more linear polyphosphates are added to the phosphate group of a nucleoside monophosphate, but nucleoside tetraphosphates such as adenosine tetraphosphate, guanosine tetraphosphate, cytidine tetraphosphate, and uridine tetraphosphate are preferred, and the NPP is an NPP in which polyphosphate is added to a nucleoside monophosphate obtained by enzymatically decomposing RNA. EXAMPLES

[0020] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples. In the present invention, all % is by weight unless otherwise specified.

[0021] [Example 1] 1. Preparation of Adenosine Tetraphosphate (ATeP) Adenosine tetraphosphate was prepared by adding phosphate to AMP according to Samy Mohamady and Scott D. Taylor, “Synthesis of Nucleoside Tetraphosphates and Dinucleoside Pentaphosphaes via Activation of Cyclic Trimetaphosphate”, Organic Letters, May 13, 2013, Vol. 15, No. 11, p. 2612-2615 and the Supporting Information of the paper. In detail, 10 mM AMP solution and 10 mM trimetaphosphate solution were each subjected to a strong cation exchange column, and 40% tetrabutylammonium hydroxide solution was added to the permeate so that the pH was 7. Thereafter, the solvent was distilled off with an evaporator, and the residue was recovered with N,N-dimethylformamide (DMF) to obtain an AMP tetrabutylammonium salt (AMP-TBA) solution and a trimetaphosphate tetrabutylammonium salt (TriMP-TBA) solution. In the case of 1 equivalent of trimetaphosphoric acid, 3.2 equivalents of 1-methylimidazole and 0.86 equivalents of 2-mesitylenesulfonyl chloride were added to the TriMP-TBA solution and reacted at room temperature for 25 minutes. Then, 0.64 equivalents of AMP-TBA were added and reacted at room temperature for 3 hours. After the reaction, the same volume of 100 mM triethylammonium acetate as the reaction solution was added in ice to stop the reaction, thereby preparing an adenosine tetraphosphate (ATeP) composition (Preparation 1).

[0022] (2-1. Preparation of Nucleoside Monophosphate (NMP)) 3 g of RNA raw material (LS ribonucleic acid, RNA: 70%) was suspended in 97 g of distilled water, adjusted to pH 5 with sodium hydroxide, sterilized, and then 0.1 g of nuclease (Amano G) was added and subjected to enzyme treatment at 72°C for 2 hours. The enzyme was then inactivated by pressurized heating (121°C, 40 minutes), and the mixture was passed through a UF membrane (5 kDa) and the membrane permeate was collected to prepare an RNA degradation product (Preparation 2).

[0023] (2-2. Preparation of Nucleoside Tetraphosphate (NTeP)) A nucleoside tetraphosphate (NTeP) composition (Preparation 3) was prepared according to the preparation of ATiP above, except that Preparation 2, an RNA degradant, was used instead of the AMP used in the preparation of ATiP above.

[0024] (3.RNA synthesis 1) (3-1. Use of ATaP Composition) The RNA synthesis reaction was confirmed by fluorescence intensity using the ProFoldin T7 RNA Polymerase Assay Kit (ProFoldin) and T7 RNA polymerase. Fluorescence intensities were measured for three samples according to the kit: a positive control (PC) using T7 RNA polymerase and a reagent included in the kit; a negative control (NC) not adding the NTP included in the kit, which is a substrate for T7 RNA polymerase; and test 1 using the reagents CTP, GTP, and UTP instead of the NTP included in the kit, which is a substrate for T7 RNA polymerase, and the ATaP composition, which is preparation 1. The results are shown in Table 1. The values ​​obtained by subtracting the fluorescence intensity value of NC from each fluorescence intensity are shown in Table 1 as Δfluorescence intensity, and the Δfluorescence intensity ratio to PC was also calculated and shown in Table 1.

[0025] [Table 1]

[0026] Even when the ATaP composition in which phosphate was added to AMP instead of ATP was used as a substrate for T7 RNA polymerase, the same fluorescence intensity was confirmed as when ATP was used as the substrate, confirming that the ATaP composition functions as a substrate for T7 RNA polymerase.

[0027] (3-2. Use of RNA decomposition products) The sample in Test 2 was prepared using RNA degradation product (Preparation 2) instead of NTP included in the kit, which is a substrate for T7 RNA polymerase. The fluorescence intensity was measured for a total of three samples, including PC and NC as in 3-1, according to the kit. The results are shown in Table 2. As in 3-1, the Δ fluorescence intensity and the Δ fluorescence intensity ratio to PC were also calculated and shown in Table 2.

[0028] [Table 2]

[0029] When RNA degradation products were used instead of NTPs as substrates for T7 RNA polymerase, the fluorescence intensity was extremely low compared to when NTPs were used as the substrate, confirming that RNA degradation products do not function as substrates for T7 RNA polymerase.

[0030] (3-3. Use of NTeP Composition) The sample in which the NTeP composition, preparation 3, was used instead of the NTP included in the kit, which is a substrate for T7 RNA polymerase, was designated as Test 3, and the fluorescence intensity was measured according to the kit for a total of three samples, including PC and NC as in 3-1. The results are shown in Table 3. As in 3-1, the Δ fluorescence intensity and the Δ fluorescence intensity ratio to PC were also calculated and shown in Table 3.

[0031] [Table 3]

[0032] Even when the NTeP composition in which phosphate is added to an RNA degradant instead of NTPs was used as a substrate for T7 RNA polymerase, high fluorescence intensity was confirmed, confirming that the NTeP composition in which phosphate is added to an RNA degradant functions as a substrate for T7 RNA polymerase.

[0033] (4.RNA synthesis 2) (4-1. Use of ATaP Composition) RNA synthesis reaction was carried out using the CUGA7 in vitro Transcription Kit (Nippon Genetech Co., Ltd.), followed by electrophoresis. A total of three samples, a positive control (PC) using the reagents included in the kit, a negative control (NC) without the addition of the substrate ATP included in the kit, and Test 4 using Preparation 1, the ATaP composition, instead of the ATP included in the kit, were reacted according to the kit, and the electrophoretic patterns of each reaction solution were confirmed using TapeStation (Agilent). The electrophoretic results are shown in Figure 1. The DNA template used was the Control DNA (100 bp) included in the kit.

[0034] In both PC and Test 4, bands were confirmed around 100 bp, indicating that even when the ATaP composition in which phosphate was added to AMP instead of ATP was used, the RNA synthesis reaction could be carried out in the same way as when ATP was used as the substrate, and that the ATaP composition functions as a substrate for T7 RNA polymerase.

[0035] Furthermore, for PC and Test 4 in which RNA synthesis was confirmed, the RNA concentrations were measured and are shown in Table 4.

[0036] [Table 4]

[0037] In Test 4, which used the ATaP composition in which phosphate was added to AMP instead of ATP, the concentration of synthesized RNA was approximately four times higher than that of PC, suggesting that the use of the ATaP composition may enable RNA synthesis more efficiently than RNA synthesis using ATP.

[0038] (4-2. Use of NTeP Composition) The sample in which the NTeP composition, Preparation 3, was used instead of the mixture of ATP, CTP, GTP and UTP included in the kit, which are substrates for T7 RNA polymerase, was used as Test 5. A total of three samples, including PC and NC similar to those in 4-1, were reacted according to the kit, and the electrophoretic pattern of each reaction solution was confirmed using TapeStation (Agilent). The electrophoretic results are shown in FIG. 2. The DNA template used was 400 bp DNA. The electrophoretic pattern of each solution after reaction was confirmed using TapeStation (Agilent). The electrophoretic results are shown in FIG. 2. The RNA concentration of the test section using PC and Preparation 3 obtained from the electrophoretic results is shown in Table 4. Since a band was confirmed near 400 bp in the test section using PC and Preparation 3, it was confirmed that the RNA synthesis reaction proceeds even if Preparation 3 is used instead of NTP, that is, RNA synthesis using Preparation 3 is possible.

[0039] In both PC and Test 5, a band was confirmed around 400 bp, which indicates that even when the NTeP composition in which phosphate is added to an RNA degradation product is used instead of the reagent mixture of ATP, CTP, GTP, and UTP (NTP), the RNA synthesis reaction can be carried out in the same manner as when the reagent NTP is used as the substrate, and that the NTeP composition functions as a substrate for T7 RNA polymerase.

Claims

1. A method for synthesizing RNA, comprising carrying out in vitro transcription using a reaction solution containing a DNA-dependent RNA polymerase, a template DNA, a nucleoside polyphosphate (NPP), a magnesium salt, and a buffer solution, wherein the NPP is an NPP in which polyphosphate has been added to a nucleoside monophosphate obtained by enzymatically decomposing RNA.

2. 2. The method for synthesizing RNA according to claim 1, wherein the NPP has polyphosphate added thereto by chemical synthesis using cyclic polyphosphate.

3. 3. The method for synthesizing RNA according to claim 1, wherein the cyclic polyphosphate is trimetaphosphate and the NPP is a nucleoside tetraphosphate.

4. A kit for RNA synthesis comprising a DNA-dependent RNA polymerase, a nucleoside polyphosphate (NPP), a magnesium salt and a buffer solution, wherein the NPP is an NPP in which polyphosphate is added to a nucleoside monophosphate obtained by enzymatically decomposing RNA.

5. 5. The kit for RNA synthesis according to claim 4, wherein the NPP has polyphosphate added thereto by chemical synthesis using cyclic polyphosphate.

6. 6. The kit for RNA synthesis according to claim 4 or 5, wherein the cyclic polyphosphate is trimetaphosphate and the NPP is a nucleoside tetraphosphate.

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