Method for producing nucleoside triphosphate
The method utilizing polyphosphate kinase (MAN) from Mangrovibacterium marinum efficiently converts NMP or NDP into NTP, addressing the challenges of high costs and import reliance in mRNA vaccine production.
Patent Information
- Application Number
- JP2023182220
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2023-10-24
- Publication Date
- 2025-05-09
AI Technical Summary
The production of nucleoside triphosphates (NTP) from nucleoside monophosphates (NMP) or nucleoside diphosphates (NDP) is hindered by the high cost and reliance on imported raw materials, particularly for mRNA vaccine production.
A method involving the use of polyphosphate kinase (MAN) derived from Mangrovibacterium marinum, which converts NMP or NDP into NTP in the presence of polyphosphate and divalent metal ions, with the reaction conducted at temperatures between 10 to 60°C.
This method enables the efficient and cost-effective production of NTP from NMP or NDP, reducing dependence on expensive imports and facilitating the production of mRNA vaccines.
Smart Images
Figure 2025071848000001 
Figure 2025071848000002 
Figure 2025071848000003
Abstract
Description
[Technical field]
[0001] The present invention relates to a method for producing nucleoside triphosphates (nucleoside-5'-triphosphates, NTPs). [Background technology]
[0002] Polyphosphate kinase is an enzyme whose presence was first identified in Escherichia coli, and it has now been revealed that homologues of polyphosphate kinase genes exist in the genomes of various microorganisms.
[0003] There are two types of polyphosphate kinase: polyphosphate kinase 1 (PPK1) and polyphosphate kinase 2 (PPK2). PPK1 is an enzyme that transfers the terminal phosphate group of adenosine-5'-triphosphate (ATP) to short-chain polyphosphate to generate long-chain polyphosphate. PPK1 also catalyzes the reverse reaction, but is known to preferentially catalyze the generation of polyphosphate.
[0004] In contrast to PPK1, PPK2 is known to preferentially catalyze the phosphorylation of nucleic acids. PPK2 is classified into three subfamilies. Enzymes belonging to PPK2 class I are known to catalyze the polyphosphate-dependent phosphorylation of nucleoside-5'-diphosphate (NDP) to nucleoside-5'-triphosphate (NTP). Enzymes belonging to PPK2 class II are known to catalyze the polyphosphate-dependent phosphorylation of nucleoside-5'-monophosphate (NMP) to nucleoside-5'-diphosphate (NDP). Enzymes belonging to PPK2 class III are known to catalyze the phosphorylation of both NMP and NDP (see, for example, Non-Patent Document 1). [Prior art documents] [Non-patent literature]
[0005] [Non-Patent Document 1] Motomura K., et al., A New Subfamily of Polyphosphate Kinase 2 (Class III PPK2) Catalyzes both Nucleoside Monophosphate Phosphorylation and Nucleoside Diphosphate Phosphorylation, Applied and Environmental Microbiology, 80 (8), 2602-2608, 2014. Summary of the Invention [Problem to be solved by the invention]
[0006] While NMP (adenosine-5'-monophosphate (AMP), guanosine-5'-monophosphate (GMP), cytidine-5'-monophosphate (CMP), uridine-5'-monophosphate (UMP)) is relatively inexpensive, NTPs (adenosine-5'-triphosphate (ATP), guanosine-5'-triphosphate (GTP), cytidine-5'-triphosphate (CTP), uridine-5'-triphosphate (UTP)), which are used as raw materials for mRNA vaccines and the like, are expensive and are currently dependent on imports from overseas. An object of the present invention is to provide a technique for producing NTPs from NMP or NDP. [Means for solving the problem]
[0007] The present invention includes the following aspects. [1] A method for producing NTP, comprising the step of incubating a reaction solution containing a nucleoside monophosphate (nucleoside-5'-monophosphate, NMP) or a nucleoside diphosphate (nucleoside-5'-diphosphate, NDP), polyphosphate, and a polyphosphate kinase, thereby converting the NMP or the NDP into a nucleoside triphosphate (nucleoside-5'-triphosphate, NTP), wherein the polyphosphate kinase is polyphosphate kinase (MAN) derived from Mangrovibacterium marinum. [2] The method according to [1], wherein the reaction solution further contains a divalent metal ion. [3] The method according to [1] or [2], wherein the incubation step is carried out at 10 to 60°C. [4] A method for producing RNA, comprising the step of incubating a reaction solution containing NMP or NDP, polyphosphate, polyphosphate kinase, template DNA, and RNA polymerase, wherein the polyphosphate kinase is MAN, and as a result, the NMP or the NDP is converted into NTP, and further, the RNA polymerase transcribes the template DNA using the NTP as a substrate to produce RNA. [5] The method according to [4], wherein the reaction solution further contains a divalent metal ion. [6] The method according to [4] or [5], wherein the incubation step is carried out at 10 to 60°C. Effect of the Invention
[0008] According to the present invention, a technique for producing NTP from NMP or NDP can be provided. [Brief description of the drawings]
[0009] [Figure 1] FIG. 1 shows chromatograms of HPLC measurement results when AMP was reacted with polyphosphate kinase (MAN) and polyphosphate kinase (CHU) in Experimental Example 2. [Diagram 2] FIG. 2 is a graph summarizing the results of measuring the enzyme activities of MAN and CHU in Experimental Example 2. [Diagram 3] FIG. 3 is a graph summarizing the results of measuring the enzyme activity of MAN in Experimental Example 3. [Figure 4] FIG. 4 is a graph showing the area ratio of each nucleotide (AMP, ADP, ATP, AP4, APn) calculated from the HPLC chromatogram measured in Experimental Example 4. [Diagram 5] FIG. 5 is a graph showing the area ratios of each nucleotide (AMP, ADP, ATP, AP4, APn) calculated from the HPLC chromatogram measured in Experimental Example 5. [Figure 6]FIG. 6 is a graph showing the area ratios of each nucleotide (AMP, ADP, ATP, AP4) calculated from the HPLC chromatogram measured in Experimental Example 6. [Figure 7] FIG. 7 is a graph showing the area ratios of each nucleotide (AMP, ADP, ATP, AP4) calculated from the HPLC chromatogram measured in Experimental Example 7. [Figure 8] FIG. 8 is a graph showing the change over time in fluorescence at a wavelength of 516 nm measured in Experimental Example 7. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0010] [NTP manufacturing method] In one embodiment, the present invention provides a method for producing NTP, comprising the step of incubating a reaction solution containing NMP or NDP, polyphosphate, and polyphosphate kinase, thereby converting the NMP or the NDP into NTP, and the polyphosphate kinase is polyphosphate kinase (MAN) derived from Mangrovibacterium marinum.
[0011] As described later in the Examples, the inventors have revealed that, among various microorganism-derived polyphosphate kinases, Mangrovibacterium marinum-derived polyphosphate kinase (MAN), an enzyme belonging to polyphosphate kinase 2 (PPK2) class III, has broad substrate specificity and can efficiently produce adenosine-5'-triphosphate (ATP), guanosine-5'-triphosphate (GTP), cytidine-5'-triphosphate (CTP) and uridine-5'-triphosphate (UTP) using all of adenosine-5'-monophosphate (AMP), adenosine-5'-diphosphate (ADP), guanosine-5'-monophosphate (GMP), guanosine-5'-diphosphate (GDP), cytidine-5'-monophosphate (CMP), cytidine-5'-diphosphate (CDP), uridine-5'-monophosphate (UMP) and uridine-5'-diphosphate (UDP) as substrates. As used herein, NMP means AMP, GMP, CMP, and UMP, NDP means ADP, GDP, CDP, and UDP, and NTP means ATP, GTP, CTP, and UTP.
[0012] Therefore, according to the production method of this embodiment, NTP can be produced from NMP or NDP. In addition, as described later in the Examples, NMP can be used instead of NTP to convert NMP to NTP and transcribe template DNA to RNA by RNA polymerase in the same vessel consecutively. The base sequence of MAN cDNA is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.
[0013] The polyphosphate is not particularly limited as long as it is a substrate for the reaction in which polyphosphate kinase converts NMP or NDP into NTP. Polyphosphate may be a salt such as a sodium salt or a potassium salt. Polyphosphates are available in various chain lengths. The chain length of the polyphosphate used in the production method of the present embodiment may be, for example, about 2 to 10,000. The lower limit of the chain length of the polyphosphate may be 2, 10, 15, 50, 60, 100, 300, 500, 700, or 1,000. The upper limit of the chain length of the polyphosphate may be 10,000, 5,000, 1,000, 800, 500, 300, 100, 70, 50, 30, 20, or 15. These upper and lower limits can be combined arbitrarily. The chain length of the polyphosphoric acid may be, for example, 60-70.
[0014] In the production method of this embodiment, the reaction solution containing NMP or NDP, polyphosphate, and polyphosphate kinase preferably further contains a divalent metal ion.
[0015] As described later in the Examples, by including a divalent metal ion in the reaction solution, the activity of MAN to convert NMP or NDP to NTP is significantly improved. 2+ Ion, Mg 2+ Ion, Ni 2+ Ion, Ca 2+ Ion, Co 2+ Among them, Mn ions are preferred because of their high activity in converting NMP or NDP to NTP. 2+ Ion, Mg 2+ Ion, Ni 2+ Ions are preferred.
[0016] In the production method of this embodiment, the step of incubating the reaction solution containing NMP or NDP, polyphosphate, and polyphosphate kinase is preferably carried out at 10 to 60° C. The lower limit of the above temperature range may be 25° C., 30° C., or 35° C. The upper limit of the above temperature range may be 55° C., 50° C., or 45° C. These upper and lower limits can be combined in any combination.
[0017] As described later in the Examples, within the above temperature range, the activity of MAN to convert NMP or NDP to NTP tends to be high.
[0018] [RNA manufacturing method] In one embodiment, the present invention provides a method for producing RNA, comprising the step of incubating a reaction solution containing NMP or NDP, polyphosphate, polyphosphate kinase, template DNA, and RNA polymerase, wherein the polyphosphate kinase is MAN, such that the NMP or the NDP is converted into an NTP, and further, the RNA polymerase transcribes the template DNA using the NTP as a substrate to produce RNA.
[0019] As described later in the Examples, according to the production method of this embodiment, inexpensive NMP is used instead of expensive NTPs, and the conversion of NMP to NTPs by PPK2 (MAN) and the transcription of template DNA to RNA by RNA polymerase can be carried out continuously in the same container.
[0020] In the production method of this embodiment, the reaction solution containing NMP or NDP, polyphosphate, polyphosphate kinase, template DNA, and RNA polymerase preferably further contains a divalent metal ion. The divalent metal ion is the same as that described above.
[0021] In the production method of this embodiment, the step of incubating a reaction solution containing NMP or NDP, polyphosphate, polyphosphate kinase, template DNA, and RNA polymerase is preferably carried out at 10 to 60°C.
[0022] Moreover, from the viewpoint that it is preferable to perform incubation at a temperature at which the activity of RNA polymerase is expressed, the lower limit of the above temperature range may be 30° C. or 35° C. Moreover, the upper limit of the above temperature range may be 55° C. or 50° C. These upper and lower limits may be combined in any manner. EXAMPLES
[0023] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.
[0024] [Experimental Example 1] (Preparation of MAN and CHU proteins) Among various microbial polyphosphate kinases, we focused on polyphosphate kinase 2 (PPK2) class III. Furthermore, as a result of phylogenetic analysis of PPK2 class III, we focused on polyphosphate kinase (MAN) derived from Mangrovibacterium marinum.
[0025] To analyze the enzymatic activity of MAN, we prepared MAN protein and, for comparison, CHU protein, a polyphosphate kinase 2 class III enzyme from Cytophaga hutchinsonii.
[0026] The nucleotide sequence of MAN cDNA is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. The nucleotide sequence of CHU cDNA is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4.
[0027] The DNA fragments encoding MAN and CHU were prepared by chemical synthesis. Each DNA fragment was introduced into an expression vector and expressed in E. coli. MAN was expressed as a fusion protein with a 6x histidine tag and a maltose binding protein linked in that order to its N-terminus. CHU was also expressed as a fusion protein with a 6x histidine tag and a maltose binding protein linked in that order to its N-terminus, similar to MAN.
[0028] MAN and CHU expressed in E. coli were purified on a Ni-nitrilotriacetic acid (NTA) affinity column and used in the following experiments, in which MAN and CHU were used in the form of fusion proteins with a 6x histidine tag and maltose binding protein.
[0029] [Experimental Example 2] (Evaluation of MAN and CHU enzyme activities) The activity of MAN and CHU to add phosphate groups to NMP and NDP was measured. PolyP-60 (BioEnex) was used as polyphosphate. PolyP-60 is a polymer with 60 to 70 phosphate groups. AMP, ADP, GMP, GDP, CMP, CDP, UMP, and UDP were used as NMP and NDP.
[0030] A reaction solution was prepared by adding 65 mM polyP-60, 4 mM AMP, ADP, GMP, GDP, CMP, CDP, UMP or UDP, 18.0 to 18.9 μM polyphosphate kinase 2 (PPK2, MAN or CHU), and 10 mM MnCl2 to a 50 mM MOPS-NaOH (pH 7.0) buffer solution. The polyphosphate concentration was expressed in terms of the molar concentration of phosphate monomers.
[0031] After reacting at 37°C for 1 hour, the same volume of ultrapure water and 2x volume of 5% trichloroacetic acid solution were added to stop the reaction. For comparison, a reaction solution without PPK2 was also prepared and reacted at 37°C for 1 hour (negative control). Then, the mixture was centrifuged at 15,000 rpm, 4°C, for 10 minutes, the supernatant was transferred to another tube, and 2x volume of 50 mM triethylamine acetate (pH 7.0)-2 mM EDTA solution was added. Then, the mixture was centrifuged at 15,000 rpm, 4°C, for 10 minutes, the supernatant was transferred to an HPLC autosampler vial, and HPLC measurement was performed.
[0032] FIG. 1 shows, as an example, chromatograms of HPLC measurement results when AMP was reacted with MAN and CHU. In FIG. 1, "AP4" indicates a compound in which one phosphate group is further bound to ATP, and "APn" indicates a compound in which another phosphate group is further bound to AP4. In FIG. 1, the leftmost column shows the results of HPLC measurement of a mixture of AMP, ADP and ATP preparations, the second column from the left shows the results of a negative control in which no PPK2 was added to the reaction solution, the third column from the left shows the results of using CHU as PPK2, and the rightmost column shows the results of using MAN as PPK2. Similar measurements were also performed for ADP, GMP, GDP, CMP, CDP, UMP and UDP.
[0033] Figure 2 is a graph summarizing the results of measuring the enzyme activity of MAN and CHU. In Figure 2, the values on the graph indicate the percentage of the substrate (AMP, ADP, GMP, GDP, CMP, CDP, UMP, or UDP) added to the reaction solution that was converted to ATP, GTP, CTP, or UTP.
[0034] As a result, it was revealed that MAN has the activity to efficiently convert AMP, ADP, GMP, GDP, CMP, CDP, UMP and UDP into ATP, GTP, CTP and UTP.
[0035] [Experimental Example 3] (Polyphosphate study) The activity of MAN was evaluated in the same manner as in Experimental Example 2, except that polyphosphates with different degrees of polymerization were used as substrates. PolyP-10, PolyP-60, and PolyP-700 (all manufactured by Bioenex) were used as polyphosphates. PolyP-10 is a polymer having 10 to 15 phosphate groups. PolyP-60 is a polymer having 60 to 70 phosphate groups. PolyP-700 is a polymer having 700 to 800 phosphate groups. Polyphosphate was reacted at a concentration of 65 mM calculated as the molar concentration of phosphate monomer.
[0036] Figure 3 is a graph summarizing the results of measuring the enzyme activity. In Figure 3, the values on the graph indicate the percentage of the substrate (AMP, ADP, GMP, GDP, CMP, CDP, UMP, or UDP) added to the reaction solution that was converted to ATP, GTP, CTP, or UTP.
[0037] As a result, it was revealed that MAN has high activity in converting AMP, ADP, GMP, GDP, CMP, CDP, UMP and UDP to ATP, GTP, CTP and UTP when PolyP-60 is used as polyphosphate.
[0038] [Experimental Example 4] (Evaluation of temperature dependence of MAN) The effect of temperature on MAN activity was examined. MAN activity was evaluated in the same manner as in Experimental Example 2, except that AMP was used as the substrate and the reaction temperatures were 20°C, 30°C, 37°C, 45°C, and 55°C. PolyP-60 (BioEnex) was used as polyphosphate. Polyphosphate was reacted at a concentration of 65 mM, calculated as the molar concentration of phosphate monomer. As a negative control, a reaction solution was also prepared in which PPK2 (MAN) was not added and the reaction was carried out at 37°C.
[0039] Figure 4 is a graph showing the area ratio of each nucleotide calculated from the HPLC chromatogram. In Figure 4, "AP4" represents a compound in which one phosphate group is further bound to ATP, and "APn" represents a compound in which another phosphate group is further bound to AP4.
[0040] As a result, it was revealed that MAN converts AMP to ATP at reaction temperatures between 10 and 50°C. In particular, it was revealed that the activity of converting AMP to ATP was high at temperatures between 37°C and 45°C.
[0041] [Experimental Example 5] (Effect of divalent metal ions on MAN activity) The effect of divalent metal ions on the activity of MAN was examined. The activity of MAN was evaluated in the same manner as in Experimental Example 2, except that AMP was used as the substrate, and 10 mM of MnCl2, MgCl2, CaCl2, CoCl2, and NiCl2 were used as the divalent metal ions. PolyP-60 (manufactured by Bioenex) was used as polyphosphate. Polyphosphate was reacted at a concentration of 65 mM, calculated as the molar concentration of phosphate monomer. For comparison, a reaction solution not containing PPK2 (MAN) and a reaction solution not containing metal ions were also prepared.
[0042] Figure 5 is a graph showing the area ratio of each nucleotide calculated from the HPLC chromatogram. In Figure 5, "AP4" indicates a compound in which one phosphate group is further bound to ATP, and "APn" indicates a compound in which another phosphate group is further bound to AP4. "Negative" indicates the result of a reaction solution that does not contain PPK2 (MAN), and "No Metal" indicates the result of a reaction solution that does not contain metal ions.
[0043] As a result, it was revealed that the activity of MAN requires the presence of metal ions. Moreover, AMP could be converted to ATP regardless of the metal ion used. In particular, Mn 2+ Ion, Mg 2+ Ion, Ni 2+ It was revealed that the activity of converting AMP to ATP was high when ions were added.
[0044] [Experimental Example 6] (Consideration of MAN concentration) The effect of MAN concentration on activity was examined. MAN activity was evaluated in the same manner as in Experimental Example 2, except that AMP was used as the substrate and the MAN concentrations were 2.4 μM, 4.7 μM, 9.5 μM, and 18.9 μM. PolyP-60 (manufactured by Bioenex) was used as polyphosphate. Polyphosphate was reacted at a concentration of 65 mM, calculated as the molar concentration of phosphate monomer. For comparison, a reaction solution not containing PPK2 (MAN) was also prepared.
[0045] Figure 6 is a graph showing the area ratio of each nucleotide calculated from the HPLC chromatogram. In Figure 6, "AP4" indicates a compound in which one phosphate group is further bound to ATP. In addition, "Negative" indicates the result of a reaction solution that does not contain PPK2 (MAN).
[0046] As a result, it was confirmed that the higher the concentration of MAN, the higher the activity of converting AMP to ATP.
[0047] [Experimental Example 7] (Mg 2+ (Consideration of ion concentration) Mg as divalent metal ion 2+ Using ions, Mg 2+ The effect of ion concentration on the activity of MAN was examined. AMP was used as the substrate, and the activity of MAN was evaluated in the same manner as in Experimental Example 2, except that 1 mM, 3 mM, 5 mM, and 10 mM MgCl2 were used. PolyP-60 (manufactured by Bioenex) was used as polyphosphate. Polyphosphate was reacted at a concentration of 65 mM, calculated as the molar concentration of phosphate monomer. For comparison, a reaction solution not containing AMP and a reaction solution containing 10 mM MnCl2 instead of MgCl2 were also prepared.
[0048] Figure 7 is a graph showing the area ratio of each nucleotide calculated from the HPLC chromatogram. In Figure 7, "AP4" indicates a compound in which one phosphate group is further bound to ATP. "AMP Neg" indicates the result of a reaction solution that does not contain AMP.
[0049] The results showed that divalent metal ions are required for MAN activity, Mg 2+ In the case of ions, Mg 2+ It was revealed that the higher the concentration of ions, the higher the activity of converting AMP to ATP.
[0050] [Experimental Example 8] (In vitro transcription) In an in vitro transcription reaction system, we used NMP instead of NTP to examine whether the conversion of NMP to NTP by PPK2 (MAN) and the transcription of template DNA to RNA by RNA polymerase could be performed consecutively in the same vessel.
[0051] A reaction solution containing NMP, PolyP-60, PPK2 (MAN), template DNA and RNA polymerase was prepared without using NTP contained in a commercially available in vitro transcription kit (ScriptMAX Thermo T7 Transcription Kit, cat. No. TSK-101).
[0052] As the template DNA, a single-stranded DNA fragment was used in which DNA encoding a fluorescent aptamer RNA (Pepper) was linked downstream of a T7 promoter. The base sequence of the template DNA is shown in SEQ ID NO: 5, and the base sequence of Pepper RNA is shown in SEQ ID NO: 6. When Pepper binds to the fluorescent dye ligand HBC530 (CAS number: 156840-13-0), it emits fluorescence of 495 to 595 nm when irradiated with an excitation wavelength of 488 nm.
[0053] More specifically, a reaction solution containing the buffer provided in the in vitro transcription kit, T7 RNA polymerase, 5.6 mM NMPs, 420 ng of template DNA, 1 μM HBC530, 6.5 mM PolyP-60, and 9.4 μM PPK2 (MAN) (hereinafter sometimes referred to as "NTP / 6.5 mM PolyP60") was prepared.
[0054] For comparison, a reaction solution containing the buffer provided in the in vitro transcription kit, T7 RNA polymerase, 5.6 mM NMPs, 420 ng of template DNA, and 1 μM HBC530, but not PolyP-60 or PPK2 (MAN) (hereinafter sometimes referred to as "NMP / Neg"), was also prepared.
[0055] As a positive control, a reaction solution was also prepared by mixing the buffer provided with the in vitro transcription kit, NTPs, T7 RNA polymerase, 420 ng of template DNA, and 1 μM HBC530 (hereinafter sometimes referred to as "NTP / Posi").
[0056] For comparison, a reaction solution containing the buffer provided in the in vitro transcription kit, T7 RNA polymerase, and 1 μM HBC530, but no template DNA (hereinafter sometimes referred to as "NTP / Neg") was also prepared.
[0057] Next, each reaction solution was placed in a real-time PCR device (model "Mx3005P", Agilent Technologies) and incubated at 37°C for 3 hours. During this incubation, light with an excitation wavelength of 492 nm was irradiated every minute, and fluorescence with a wavelength of 516 nm was detected. In this experimental system, fluorescence corresponding to the amount of RNA synthesis was detected.
[0058] Figure 8 is a graph showing the change over time in fluorescence at a wavelength of 516 nm. The bottom of Figure 8 is an enlarged graph of the range of values on the vertical axis of the graph in the top of Figure 8, from 0 to 45. In Figure 8, "NTP / Neg", "NTP / Posi", "NMP / Neg", and "NMP / 6.5mM PolyP60" respectively indicate the results of the reaction solutions described above.
[0059] As a result, it was revealed that by using NMP instead of NTP, the conversion of NMP to NTP by PPK2(MAN) and the transcription of template DNA to RNA by RNA polymerase could be carried out continuously in the same vessel. [Industrial Applicability]
[0060] According to the present invention, a technique for producing NTP from NMP or NDP can be provided.
Claims
1. incubating a reaction solution containing nucleoside monophosphate (NMP) or nucleoside diphosphate (NDP), polyphosphate and polyphosphate kinase, so that the NMP or NDP is converted to a nucleoside triphosphate (NTP); A method for producing NTP, wherein the polyphosphate kinase is polyphosphate kinase (MAN) derived from Mangrovibacterium marinum.
2. The method according to claim 1 , wherein the reaction solution further contains a divalent metal ion.
3. The method according to claim 1 or 2, wherein the incubating step is carried out at 10 to 60°C.
4. A method for producing RNA, comprising the steps of incubating a reaction solution containing NMP or NDP, polyphosphate, polyphosphate kinase, template DNA and RNA polymerase, wherein the polyphosphate kinase is MAN, and as a result, the NMP or the NDP is converted into an NTP, and further, the RNA polymerase transcribes the template DNA using the NTP as a substrate to produce RNA.
5. The method according to claim 4 , wherein the reaction solution further contains a divalent metal ion.
6. The method according to claim 4 or 5, wherein the incubating step is carried out at 10 to 60°C.