Modified RNA polymerase

Abstract

The objective is to provide an RNA polymerase with improved performance. [Solution] The above problem is solved by a specific modified RNA polymerase.

Description

Technical Field

[0004] , ,

[0001] The present invention relates to a modified RNA polymerase. More specifically, the present invention relates to a modified RNA polymerase capable of reducing 3'-extended RNA by a high-temperature reaction and reducing dsRNA generated after a transcription reaction, a method for producing the RNA polymerase, and uses thereof.

Background Art

[0002] DNA-dependent RNA polymerase is an enzyme that recognizes a promoter sequence in DNA and synthesizes RNA using DNA as a template, and has been the subject of many studies because it is responsible for the transcription reaction that synthesizes mRNA from genomic DNA in vivo. Among them, phage-derived RNA polymerases have a relatively simple structure and function with a single subunit, and have thus been the subject of biochemical and structural biological studies for a long time. These RNA polymerases are also widely used in applications for synthesizing RNA in vitro (in vitro), and the RNA synthesized thereby is used as an RNA probe for molecular biological techniques, as various functional RNAs, or as a template RNA for protein expression in cells or for use in a cell-free protein synthesis system.

[0003] In addition, isothermal amplification reactions such as the NASBA method and the TMA method have been developed using the property of RNA polymerase to synthesize a large number of RNAs from template DNA, and these methods are applied to clinical diagnosis.

[0004] In recent years, RNA synthesized by RNA polymerase has been applied to mRNA drugs, including mRNA vaccines, and RNA polymerase is widely used in the field of pharmaceutical manufacturing. One of the impurities that poses a problem in the manufacturing process of mRNA drugs is dsRNA. Since dsRNA causes inflammatory responses in the body, it is desirable to remove it as much as possible from mRNA drugs. Non-patent document 1 describes a modified T7 RNA polymerase in which the dsRNA content during the transcription reaction is reduced. Another impurity is 3' elongated RNA, which is produced when the 3' end of RNA is further elongated after the transcription reaction. Non-patent document 2 describes that 3' elongated RNA can be reduced by high-temperature reactions, but it also states that dsRNA may not be reduced depending on the template DNA used. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Dousis, A., Ravichandran, K., Hobert, EM, Moore, MJ, & Rabideau, AE (2023). An engineered T7 RNA polymerase that produces mRNA free of immunostimulatory byproducts. Nature biotechnology, 41(4), 560-568. [Non-Patent Document 2] WU, MZ, ASAHARA, H., TZERTZINIS, G., & ROY, B. (2020). SYNTHESIS OF LOW IMMUNOGENICITY RNA WITH HIGH-TEMPERATURE IN VITRO TRANSCRIPTION. RNA, 26(3), 345-360. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] As mentioned above, with the expanding applications of RNA polymerase, further improvements in its performance are desired. [Means for solving the problem]

[0007] As a result of diligent research, the inventors discovered that a specific modified RNA polymerase can reduce 3' elongated RNA produced by transcription reactions at high temperatures, thereby reducing the amount of dsRNA generated after the transcription reaction, and thus completed the present invention.

[0008] In other words, the representative aspects of the present invention are as follows: [Section 1] It has more than 90% identical amino acid sequence to Sequence ID No. 1, One or more amino acids at positions 430, 633, 849, and 880 in Sequence ID No. 1 have been modified, and RNA polymerase in which one or more amino acids at positions corresponding to the 61st, 156th, 189th, 165th, and 661st positions in SEQ ID NO: 1 have been modified. [Section 2] RNA polymerases described in item 1 that have 90% or more identity with the amino acid sequence of Sequence ID No. 1 and satisfy one or more of the following conditions (i) to (iv) and one or more of the following conditions (v) to (ix): (i) The amino acid at position 430 in Sequence ID No. 1 is proline, alanine, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (ii) The amino acid at position 633 in Sequence ID No. 1 is proline, alanine, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (iii) The amino acid at position 849 in Sequence ID No. 1 is isoleucine, proline, alanine, glycine, leucine, methionine, valine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (iv) The amino acid at position 880 in Sequence ID No. 1 is tyrosine, isoleucine, proline, alanine, glycine, leucine, methionine, valine, tryptophan, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (v) The amino acid at position 61 in Sequence ID No. 1 is proline, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (vi) The amino acid at position 156 in Sequence ID No. 1 is asparagine, glutamine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, serine, threonine, arginine, histidine, lysine, or glutamic acid; (vii) The amino acid at position 189 in Sequence ID No. 1 is asparagine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, glutamine, serine, threonine, arginine, histidine, lysine, or glutamic acid; (viii) The amino acid at position 165 in Sequence ID No. 1 is serine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (ix) The amino acid at position 661 in Sequence ID No. 1 is asparagine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid. [Section 3] RNA polymerases described in item 1 or 2 that satisfy all of the following conditions (i) to (v): (i) The amino acid at position 430 in Sequence ID No. 1 is proline; (ii) The amino acid at position 633 in Sequence ID No. 1 is proline; (iii) The amino acid at position 849 in Sequence ID No. 1 is isoleucine; The amino acid at position 880 in (iv) Sequence ID No. 1 is tyrosine; The amino acid at the position corresponding to the 61st position in (v) Sequence ID No. 1 is proline. [Section 4] An RNA polymerase capable of synthesizing RNA at temperatures above 48°C, wherein the proportion of dsRNA in the RNA synthesized at temperatures above 48°C is 70% or less compared to the proportion of dsRNA in the RNA synthesized at 37°C using the corresponding wild-type RNA polymerase. [Section 5] RNA polymerases described in item 4 that satisfy one or more of the following (i) to (iv), and optionally one or more of the following (v) to (ix): (i) The amino acid at position 430 in Sequence ID No. 1 is proline, alanine, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (ii) The amino acid at position 633 in Sequence ID No. 1 is proline, alanine, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (iii) The amino acid at position 849 in Sequence ID No. 1 is isoleucine, proline, alanine, glycine, leucine, methionine, valine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (iv) The amino acid at position 880 in Sequence ID No. 1 is tyrosine, isoleucine, proline, alanine, glycine, leucine, methionine, valine, tryptophan, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (v) The amino acid at position 61 in Sequence ID No. 1 is proline, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (vi) The amino acid at position 156 in Sequence ID No. 1 is asparagine, glutamine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, serine, threonine, arginine, histidine, lysine, or glutamic acid; (vii) The amino acid at position 189 in Sequence ID No. 1 is asparagine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, glutamine, serine, threonine, arginine, histidine, lysine, or glutamic acid; (viii) The amino acid at position 165 in Sequence ID No. 1 is serine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (ix) The amino acid at position 661 in Sequence ID No. 1 is asparagine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid. [Section 6] RNA polymerases described in item 4 or 5 that satisfy all of the following conditions (i) to (iv), or all of the following conditions (i) to (v): (i) The amino acid at position 430 in Sequence ID No. 1 is proline; (ii) The amino acid at position 633 in Sequence ID No. 1 is proline; (iii) The amino acid at position 849 in Sequence ID No. 1 is isoleucine; (iv) The amino acid at position 880 in Sequence ID No. 1 is tyrosine; (v) The amino acid at position 61 in sequence number 1 is proline. [Section 7] A method for producing RNA polymerase using a polynucleotide encoding an RNA polymerase as described in any of items 1 to 6, a vector containing the polynucleotide, or recombinant host cells transformed using the vector. [Section 8] A reagent comprising an RNA polymerase described in any of items 1 to 6, a polynucleotide encoding the RNA polymerase, a vector containing the polynucleotide, or a recombinant host cell transformed using the vector. [Section 9] A method for performing an in vitro transcription reaction using an RNA polymerase described in any of sections 1 to 6. [Item 10] The method according to item 9, wherein the ratio of dsRNA in the RNA after the in vitro transcription reaction is 90% or less as compared with the case where the in vitro transcription reaction is carried out using the corresponding wild-type RNA polymerase. [Item 11] A method for synthesizing mRNA using the RNA polymerase according to any one of items 1 to 6. [Item 12] The method according to item 11, wherein the ratio of dsRNA in the mRNA is 90% or less as compared with the case where mRNA synthesis is carried out using the corresponding wild-type RNA polymerase. [Item 13] A method for synthesizing non-coding RNA using the RNA polymerase according to any one of items 1 to 6. [Item 14] A method for synthesizing an RNA drug using the RNA polymerase according to any one of items 1 to 6. [Effect of the Invention]

[0009] By using the modified RNA polymerase provided by the present invention, it is possible to reduce the 3'-extended RNA by the transcription reaction at high temperature and the dsRNA after the transcription reaction. [Modes for Carrying Out the Invention]

[0010] Hereinafter, the present invention will be described in more detail while showing embodiments of the present invention, but the present invention is not limited thereto. All non-patent documents and patent documents described in this specification are incorporated herein by reference in their entirety. In addition, "~" in this specification means "above and below", for example, if "X~Y" is described in the specification, it means "X or more and Y or less". "And / or" in this specification means either one or both. "Including" in this specification includes the concepts of "consisting essentially of" and "consisting only of".

[0011] The present invention provides a modified RNA polymerase. A modified RNA polymerase (hereinafter also referred to as "modified RNA polymerase") is one in which at least one amino acid residue in the amino acid sequence of wild-type RNA polymerase has been mutated, i.e., substituted, deleted, or added to another amino acid.

[0012] In this specification, "wild-type RNA polymerase" (hereinafter also referred to as "wild-type" or "WT") means RNA polymerase that has not been artificially mutated. Examples of wild-type RNA polymerases include T7 RNA polymerase, T3 RNA polymerase, K11 RNA polymerase, SP6 RNA polymerase, Syn5 RNA polymerase, KP34 RNA polymerase, and VSW-3 RNA polymerase. A preferred RNA polymerase is, for example, wild-type T7 RNA polymerase consisting of the amino acid sequence shown in Sequence ID No. 1. T7 RNA polymerase means T7 phage-derived RNA polymerase, as is well known to those skilled in the art.

[0013] The RNA polymerase of the present invention may have improved specific activity compared to the corresponding wild-type RNA polymerase. Here, "specific activity" refers to the enzyme activity per unit protein weight when the enzyme activity is measured at a temperature of 20°C to 100°C, for example. Furthermore, "corresponding wild-type RNA polymerase" means the wild-type RNA polymerase of the same type as a given modified RNA polymerase. For example, the "corresponding wild-type RNA polymerase" for a modified T7 RNA polymerase is the wild-type T7 RNA polymerase.

[0014] The RNA polymerase of the present invention has a different amino acid sequence from conventionally known wild-type RNA polymerase. Therefore, in this specification, the RNA polymerase of the present invention may be referred to as mutant RNA polymerase or modified RNA polymerase. In this specification, the terms "mutant" and "modified" are used interchangeably and mean that the RNA polymerase has a different amino acid sequence from conventionally known RNA polymerase, and do not distinguish whether the mutation is due to artificial mutation or natural mutation. Therefore, the mutant RNA polymerase of the present invention is an RNA polymerase in which one or more amino acids in the amino acid sequence described in Sequence ID No. 1, which shows the wild-type T7 RNA polymerase sequence, have been modified, and have a different amino acid sequence from Sequence ID No. 1, regardless of whether the mutant RNA polymerase is an RNA polymerase obtained by artificial mutation or an RNA polymerase obtained by natural mutation.

[0015] In this specification, nucleotide sequences, amino acid sequences, and their individual components may be represented using simplified alphabetical symbols, following the conventions of molecular biology and genetic engineering. Furthermore, in this specification, to concisely indicate amino acid sequence mutations, notations such as "A61E" are used. "A61E" indicates the substitution of alanine (A) at position 61 with glutamic acid (E). That is, it indicates the type of amino acid residue before substitution, its location, and the type of amino acid residue after substitution. Unless otherwise specified, the sequence number corresponds to the sequence number listed in the sequence listing. In the case of multiple mutants, the above notations can be connected with a " / " (for example, A61E / D156N / N165S).

[0016] The mutant RNA polymerase of the present invention has 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1. The mutant RNA polymerase is not particularly limited as long as RNA polymerase activity is not lost, but it is preferable that it is composed of an amino acid sequence that has 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the amino acid sequence described in SEQ ID NO: 1. Here, the identity of the amino acid sequence can be evaluated by any means known in the art. For example, it can be calculated using commercially available analysis tools or tools available via telecommunication lines (Internet), and as an example, it is possible to calculate the identity of the amino acid sequence by using the default parameters of the homology algorithm BLAST (Basic local alignment search tool) of the National Center for Biotechnology Information (NCBI) http: / / www.ncbi.nlm.nih.gov / BLAST / . Furthermore, the amino acid sequence of the mutant RNA polymerase may be an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and / or added to the amino acid sequence described in SEQ ID NO: 1. Here, "one or several" is not particularly limited as long as RNA polymerase activity is not lost, but for example, it is 1 to 30, more preferably 1 to 20, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. Such an amino acid sequence may be artificially created by, for example, genetic engineering techniques, or it may be an amino acid sequence of a naturally occurring protein.

[0017] In one embodiment, if the mutant RNA polymerase has substitutions in the amino acid sequence described in Sequence ID No. 1, it is preferable that the substitutions are between structurally and / or chemically similar amino acids (so-called conserved substitutions). Examples of conserved substitutions include, but are not limited to, substitutions between basic amino acids (H, K, R), substitutions between acidic amino acids (D, E), substitutions between neutral nonpolar amino acids (A, G, V, L, I, P, F, M, W), substitutions between neutral polar amino acids (N, Q, S, T, Y, C), substitutions between aromatic amino acids (W, F, H, Y), substitutions between nitrogen-containing amino acids (K, R, N, Q, P), substitutions between sulfur-containing amino acids (C, M), substitutions between oxygen-containing amino acids (S, T), substitutions between β-branched-chain amino acids (V, L, I), and substitutions between amino acids (A, G) having linear alkyl or hydrogen side chains.

[0018] In certain embodiments, the RNA polymerase of the present invention may contain a peptide other than RNA polymerase at either the N-terminus, the C-terminus, or both. Examples of such peptides include, but are not limited to, extracellular secretory signals, protein purification tags (His tags, Flag tags, Strep tags, GST tags, MBP tags, etc.), proteins with other functions, and linkers.

[0019] The RNA polymerase of the present invention may be composed of an amino acid sequence containing the above-described amino acid residues. Those skilled in the art can produce an RNA polymerase protein of the desired amino acid sequence by, for example, appropriately designing a base sequence encoding the desired amino acid sequence using any genetic engineering method known in the art, incorporating it into an expression vector or the like, and transforming and expressing it in host cells.

[0020] The RNA polymerase of the present invention has one or more amino acids modified at positions corresponding to the 430th, 633rd, 849th, and 880th positions in Sequence ID No. 1. In Sequence ID No. 1, the amino acid at position 430 is serine, and examples of amino acids that substitute the serine residue at position 430 include proline, alanine, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid. In a more preferred embodiment, the RNA polymerase of the present invention is an RNA polymerase in which the amino acid at position 430 is substituted with a neutral nonpolar amino acid. Examples of neutral nonpolar amino acids include proline, alanine, glycine, valine, leucine, isoleucine, phenylalanine, methionine, and tryptophan, with proline, alanine, glycine, valine, leucine, and isoleucine being preferred, and proline being even more preferred. While not intending to be bound by any particular theory, the amino acid mutation at position 430 in Sequence ID No. 1 is thought to contribute to improved heat resistance of the modified RNA polymerase. This is thought to suppress the reduction in specific activity and contribute to an increase in RNA yield, even when using the modified RNA polymerase for RNA synthesis via transcription reactions under high-temperature conditions, such as 48°C or higher. Furthermore, while not intending to be bound by any particular theory, the amino acid mutation at position 430 in Sequence ID No. 1 is also thought to contribute to enhanced transcriptional activity of the modified RNA polymerase.

[0021] In this specification, "position corresponding to position 430 in SEQ ID NO: 1" means position 430 in the amino acid sequence of wild-type T7 RNA polymerase (SEQ ID NO: 1), or the position in the amino acid sequence of mutant RNA polymerase corresponding to position 430 in the amino acid sequence of wild-type T7 RNA polymerase (SEQ ID NO: 1). The "position corresponding to position 430 in SEQ ID NO: 1" in the amino acid sequence of mutant RNA polymerase can be easily identified, for example, by comparing and aligning the amino acid sequence of wild-type T7 RNA polymerase (SEQ ID NO: 1) with the amino acid sequence of mutant RNA polymerase using any known means. For example, the comparison and alignment of amino acid sequences can be performed using commercially available analysis tools or tools available via telecommunication lines (the Internet), but as an example, it is possible to perform amino acid sequence alignment using the default parameters of the multi-alignment program Clustal Omega (https: / / www.ebi.ac.uk / jdispatcher / msa / clustalo) provided by the European Bioinformatics Laboratory (EMBL). The same applies in this specification when a position corresponding to any amino acid other than the 430th position in Sequence ID No. 1 is described.

[0022] In Sequence ID No. 1, the amino acid at position 633 is serine, and examples of amino acids that substitute the serine residue at position 633 include proline, alanine, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid. In a more preferred embodiment, the RNA polymerase of the present invention is an RNA polymerase in which the amino acid at position 633 is substituted with a neutral nonpolar amino acid. Examples of neutral nonpolar amino acids include alanine, glycine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan, with alanine, glycine, valine, leucine, isoleucine, and proline being preferred, and proline being even more preferred. While not intended to be bound by any particular theory, the amino acid mutation at position 633 in Sequence ID No. 1 is thought to contribute to improved heat resistance of the modified RNA polymerase. This is thought to suppress the reduction in specific activity and contribute to an increase in RNA yield, even when using the modified RNA polymerase for RNA synthesis via transcription reactions under high-temperature conditions, such as 48°C or higher. Furthermore, while not intended to be bound by any particular theory, the amino acid mutation at position 633 in Sequence ID No. 1 is also thought to contribute to enhanced transcriptional activity of the modified RNA polymerase.

[0023] In Sequence ID No. 1, the amino acid at position 849 is phenylalanine, and examples of amino acids that substitute the phenylalanine residue at position 849 include isoleucine, proline, alanine, glycine, leucine, methionine, valine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid. In a more preferred embodiment, the RNA polymerase of the present invention is an RNA polymerase in which the amino acid at position 849 is substituted with a β-branched chain amino acid. Examples of β-branched chain amino acids include valine, leucine, and isoleucine, with isoleucine being more preferred. While not intended to be bound by any particular theory, the amino acid mutation at position 849 in Sequence ID No. 1 is thought to contribute to improved heat resistance of the modified RNA polymerase. This is thought to suppress the reduction in specific activity and contribute to an increase in RNA yield, even when using the modified RNA polymerase for RNA synthesis via transcription reactions under high-temperature conditions, such as 48°C or higher. Furthermore, while not intended to be bound by any particular theory, the amino acid mutation at position 849 in Sequence ID No. 1 is also thought to contribute to enhanced transcriptional activity of the modified RNA polymerase.

[0024] In Sequence ID No. 1, the amino acid at position 880 is phenylalanine, and examples of amino acids that substitute the phenylalanine residue at position 880 include tyrosine, isoleucine, proline, alanine, glycine, leucine, methionine, valine, tryptophan, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid. In a more preferred embodiment, the RNA polymerase of the present invention is an RNA polymerase in which the amino acid at position 880 is substituted with a neutral polar amino acid. Examples of neutral polar amino acids include asparagine, glutamine, serine, threonine, tyrosine, and cysteine, with tyrosine being more preferred. While not intending to be bound by any particular theory, the amino acid mutation at position 880 in Sequence ID No. 1 is thought to contribute to improved heat resistance of the modified RNA polymerase. This is thought to suppress the reduction in specific activity and contribute to an increase in RNA yield, even when using the modified RNA polymerase for RNA synthesis via transcription reactions under high-temperature conditions, such as 48°C or higher. Furthermore, while not intending to be bound by any particular theory, the amino acid mutation at position 880 in Sequence ID No. 1 is also thought to contribute to enhanced transcriptional activity of the modified RNA polymerase.

[0025] The mutant RNA polymerase of the present invention has one or more amino acids modified at positions corresponding to the 61st, 156th, 189th, 165th, and 661st positions in Sequence ID No. 1. The amino acid at position 61 in Sequence ID No. 1 is alanine, and the amino acids that can substitute the alanine residue at position 61 include proline, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid. Proline, glutamic acid, aspartic acid, serine, threonine, and glycine are preferred, and proline, glutamic acid, aspartic acid, serine, and glycine are more preferred. Although not intended to be bound by any particular theory, it is thought that the amino acid mutation at position 61 in Sequence ID No. 1 contributes to reducing the proportion of dsRNA in RNA produced by transcription reactions using modified RNA polymerase.

[0026] In Sequence ID No. 1, the amino acid at position 156 is aspartic acid, and examples of amino acids that substitute the aspartic acid residue at position 156 include, but are not limited to, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, and glutamic acid. In a more preferred embodiment, the RNA polymerase of the present invention is an RNA polymerase in which the amino acid at position 156 is modified to be a basic amino acid or a neutral amino acid. Examples of basic or neutral amino acids include asparagine, glycine, alanine, proline, isoleucine, leucine, valine, phenylalanine, tryptophan, tyrosine, glutamine, cysteine, methionine, serine, threonine, arginine, histidine, and lysine, and are more preferably asparagine or glutamine, and even more preferably asparagine. While not intending to be bound by any particular theory, the amino acid mutation at position 156 in Sequence ID No. 1 is thought to contribute to reducing the proportion of dsRNA in RNA produced by transcription reactions using modified RNA polymerase.

[0027] In Sequence ID No. 1, the amino acid at position 165 is asparagine. Amino acids that can substitute for the asparagine residue at position 165 include glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, and glutamic acid, with serine being preferred. While not intended to be bound by any particular theory, it is thought that the amino acid mutation at position 165 in Sequence ID No. 1 contributes to reducing the proportion of dsRNA in RNA produced by transcription reactions using modified RNA polymerase.

[0028] In Sequence ID No. 1, the amino acid at position 189 is aspartic acid, and examples of amino acids that substitute the aspartic acid residue at position 189 include, but are not limited to, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, and glutamic acid. In a more preferred embodiment, the RNA polymerase of the present invention preferably has the amino acid at position 189 substituted with a basic amino acid or a neutral amino acid, and examples of basic or neutral amino acids include glycine, alanine, proline, isoleucine, leucine, valine, phenylalanine, tryptophan, tyrosine, asparagine, glutamine, cysteine, methionine, serine, threonine, arginine, histidine, and lysine, with substitution with asparagine or glutamine being more preferred, and substitution with asparagine being particularly preferred. While not intending to be bound by any particular theory, the amino acid mutation at position 189 in Sequence ID No. 1 is thought to contribute to reducing the proportion of dsRNA in RNA produced by transcription reactions using modified RNA polymerase.

[0029] In Sequence ID No. 1, the amino acid at position 661 is serine. Amino acids that can substitute serine at position 661 include glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, and glutamic acid. Substitution with asparagine or glutamine is more preferable, and substitution with asparagine is particularly preferable. While not intended to be bound by any particular theory, it is thought that the amino acid mutation at position 661 in Sequence ID No. 1 contributes to reducing the proportion of dsRNA in RNA produced by transcription reactions using modified RNA polymerase.

[0030] In a preferred embodiment, the RNA polymerase of the present invention has 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more identity with the amino acid sequence of SEQ ID NO: 1, with one or more amino acids at positions corresponding to the 430th, 633rd, 849th, and 880th positions in SEQ ID NO: 1 being modified, and one or more amino acids at positions corresponding to the 61st, 156th, 189th, 165th, and 661st positions in SEQ ID NO: 1 being modified.

[0031] In a preferred embodiment, the RNA polymerase of the present invention has 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more identity with the amino acid sequence of SEQ ID NO: 1, satisfies one or more of the following (i) to (iv), and one or more of the amino acids at the positions corresponding to the 61st, 156th, 189th, 165th, and 661st positions in SEQ ID NO: 1 are modified: (i) The amino acid at position 430 in Sequence ID No. 1 is proline, alanine, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (ii) The amino acid at position 633 in Sequence ID No. 1 is proline, alanine, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (iii) The amino acid at position 849 in Sequence ID No. 1 is isoleucine, proline, alanine, glycine, leucine, methionine, valine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (iv) The amino acid at position 880 in Sequence ID No. 1 is tyrosine, isoleucine, proline, alanine, glycine, leucine, methionine, valine, tryptophan, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid.

[0032] In a preferred embodiment, the RNA polymerase of the present invention may have an identity of 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more of the amino acid sequence of SEQ ID NO: (i) The amino acid at position 430 in Sequence ID No. 1 is proline, alanine, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (ii) The amino acid at position 633 in Sequence ID No. 1 is proline, alanine, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (iii) The amino acid at position 849 in Sequence ID No. 1 is isoleucine, proline, alanine, glycine, leucine, methionine, valine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (iv) The amino acid at position 880 in Sequence ID No. 1 is tyrosine, isoleucine, proline, alanine, glycine, leucine, methionine, valine, tryptophan, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (v) The amino acid at position 61 in Sequence ID No. 1 is proline, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (vi) The amino acid at position 156 in Sequence ID No. 1 is asparagine, glutamine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, serine, threonine, arginine, histidine, lysine, or glutamic acid; (vii) The amino acid at position 189 in Sequence ID No. 1 is asparagine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, glutamine, serine, threonine, arginine, histidine, lysine, or glutamic acid; (viii) The amino acid at position 165 in Sequence ID No. 1 is serine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (ix) The amino acid at position 661 in Sequence ID No. 1 is asparagine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid.

[0033] In a preferred embodiment, the RNA polymerase of the present invention may have an identity of 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more of the amino acid sequence of SEQ ID NO: (i) The amino acid at position 430 in Sequence ID No. 1 is proline; (ii) The amino acid at position 633 in Sequence ID No. 1 is proline; (iii) The amino acid at position 849 in Sequence ID No. 1 is isoleucine; (iv) The amino acid at position 880 in Sequence ID No. 1 is tyrosine; (v) The amino acid at position 61 in Sequence ID No. 1 is proline, glycine, serine, aspartic acid, or glutamic acid; (vi) The amino acid at position 156 in Sequence ID No. 1 is asparagine; (vii) The amino acid at position 189 in Sequence ID No. 1 is asparagine; (viii) The amino acid at position 165 in Sequence ID No. 1 is serine; (ix) The amino acid at position 661 in sequence number 1 is asparagine.

[0034] In a more preferred embodiment, the RNA polymerase of the present invention is an RNA polymerase that has 90% or more identity, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more identity with the amino acid sequence of SEQ ID NO: (i) The amino acid at position 430 in Sequence ID No. 1 is proline; (ii) The amino acid at position 633 in Sequence ID No. 1 is proline; (iii) The amino acid at position 849 in Sequence ID No. 1 is isoleucine; (iv) The amino acid at position 880 in SEQ ID NO: 1 is tyrosine; and (v) The amino acid at position 61 in Sequence ID No. 1 is one of glutamic acid, aspartic acid, serine, glycine, or proline, with proline being particularly preferred.

[0035] In a preferred embodiment, the RNA polymerase of the present invention is an RNA polymerase capable of synthesizing RNA at 48°C or higher, wherein the proportion of dsRNA in the RNA synthesized at 48°C or higher is about 70% or less, preferably about 60% or less, preferably about 50% or less, preferably about 40% or less, more preferably about 30% or less, even more preferably about 20% or less, even more preferably about 10% or less, even more preferably about 5% or less, even more preferably about 3% or less, and even more preferably about 1% or less, compared to the proportion of dsRNA in the RNA synthesized at 37°C using the corresponding wild-type RNA polymerase. In the present invention, "RNA polymerase capable of synthesizing RNA at 48°C or higher" is an RNA polymerase capable of synthesizing 25 μg or more of RNA when RNA is synthesized by the following method.

[0036] [Methods for RNA synthesis at temperatures above 48°C] 2.5 μL of each mutant RNA polymerase to be measured (200 U / μL) was added to 47.5 μL of reaction mixture prepared on ice (the final concentration in 50 μL of reaction mixture after enzyme addition is shown below) (30 mM Tris-HCl (pH 8.0), 27 mM magnesium acetate, 3 mM DTT, 10 ng / μL template DNA, 5 mM ATP, 5 mM CTP, 5 mM GTP, 5 mM UTP, 1 U / μL RNase inhibitor, 0.002 U / μL pyrophosphatase). The reaction was then carried out for 1 hour at any temperature of 48°C or higher using a heat block to synthesize RNA.

[0037] The lower limit of the reaction temperature in RNA synthesis may be 48°C or higher, preferably 49°C or higher, preferably 50°C or higher, preferably 51°C or higher, preferably 52°C or higher, preferably 53°C or higher, preferably 54°C or higher, preferably 55°C or higher, preferably 56°C or higher, preferably 57°C or higher, preferably 58°C or higher, preferably 59°C or higher, and preferably 60°C or higher. The upper limit of the reaction temperature in RNA synthesis may be 95°C or lower, preferably 90°C or lower, preferably 85°C or lower, preferably 80°C or lower, preferably 75°C or lower, and preferably 70°C or lower. The "proportion of dsRNA in RNA" can be calculated, for example, by performing an in vitro transcription reaction at 37°C with the corresponding wild-type RNA polymerase, performing an in vitro transcription reaction at 48°C or higher with each mutant RNA polymerase to be measured, calculating the proportion of dsRNA in each RNA, and then comparing the values.

[0038] The RNA polymerase of the present invention can reduce RNA impurities that are longer than the RNA size predicted from the template DNA by high-temperature reactions (e.g., 48°C or higher). The length of RNA impurities that are longer than the RNA size predicted from the template DNA may be 10 nt or more, 20 nt or more, 30 nt or more, 40 nt or more, 50 nt or more, 60 nt or more, 70 nt or more, 80 nt or more, 90 nt or more, 100 nt or more, 200 nt or more, 300 nt or more, 400 nt or more, 500 nt or more, or 1000 nt or more than the RNA size predicted from the template DNA. Examples of such RNA impurities include impurities produced by further elongation of the 3' end of RNA generated after the transcription reaction (3' elongated RNA). One possible mechanism for generating 3' elongated RNA is that the 3' end folds back to form a loop structure, after which RNA is synthesized using RNA as a template by RNA-dependent RNA polymerase activity. However, at high temperatures, even if the 3' end folds back, annealing becomes less likely, inhibiting the formation of the loop structure and preventing 3' end elongation.

[0039] In a preferred embodiment, the RNA polymerase of the present invention may be an RNA polymerase capable of synthesizing RNA at 48°C or higher, wherein the proportion of dsRNA in the RNA synthesized at 48°C or higher is 70% or less compared to the proportion of dsRNA in the RNA synthesized at 37°C using the corresponding wild-type RNA polymerase. In a more preferred embodiment, the RNA polymerase may further have mutations in predetermined amino acid residues. Examples of such amino acid residue mutations include, as mentioned above, (i) the amino acid at position 430 in SEQ ID NO: 1, (ii) the amino acid at position 633 in SEQ ID NO: 1, (iii) the amino acid at position 849 in SEQ ID NO: 1, (iv) the amino acid at position 880 in SEQ ID NO: 1, (v) the amino acid at position 61 in SEQ ID NO: 1, (vi) the amino acid at position 156 in SEQ ID NO: 1, (vii) the amino acid at position 189 in SEQ ID NO: 1, (viii) the amino acid at position 165 in SEQ ID NO: 1, and (ix) the amino acid at position 661 in SEQ ID NO: 1. The types of amino acids that replace these residues are also as mentioned above.

[0040] In a preferred embodiment, the RNA polymerase of the present invention is an RNA polymerase capable of synthesizing RNA at 48°C or higher, wherein the proportion of dsRNA in the RNA synthesized at 48°C or higher is 70% or less, preferably 67% or less, preferably 65% ​​or less, preferably 60% or less, preferably 55% or less, and preferably 50% or less, compared to the proportion of dsRNA in the RNA synthesized at 37°C using the corresponding wild-type RNA polymerase, and may satisfy one or more of the following (i) to (iv), and optionally one or more of the following (v) to (ix): (i) The amino acid at position 430 in SEQ ID NO: 1 is proline, alanine, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid, preferably proline; (ii) The amino acid at position 633 in SEQ ID NO: 1 is proline, alanine, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid, preferably proline; (iii) The amino acid at position 849 in SEQ ID NO: 1 is isoleucine, proline, alanine, glycine, leucine, methionine, valine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid, preferably isoleucine; (iv) The amino acid at position 880 in SEQ ID NO: 1 is tyrosine, isoleucine, proline, alanine, glycine, leucine, methionine, valine, tryptophan, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid, preferably tyrosine; (v) The amino acid at position 61 in SEQ ID NO: 1 is proline, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid, preferably proline, glycine, serine, aspartic acid, or glutamic acid, and more preferably proline; (vi) The amino acid at position 156 in Sequence ID No. 1 is asparagine, glutamine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, serine, threonine, arginine, histidine, lysine, or glutamic acid, preferably asparagine; (vii) The amino acid at position 189 in Sequence ID No. 1 is asparagine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, glutamine, serine, threonine, arginine, histidine, lysine, or glutamic acid, preferably asparagine; (viii) The amino acid at position 165 in Sequence ID No. 1 is serine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid, preferably serine; (ix) The amino acid at position 661 in Sequence ID No. 1 is asparagine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid, preferably asparagine.

[0041] In a preferred embodiment, the RNA polymerase of the present invention is an RNA polymerase capable of synthesizing RNA at 48°C or higher, wherein the proportion of dsRNA in the RNA synthesized at 48°C or higher is 70% or less, preferably 67% or less, preferably 65% ​​or less, preferably 60% or less, preferably 55% or less, and preferably 50% or less, compared to the proportion of dsRNA in the RNA synthesized at 37°C using the corresponding wild-type RNA polymerase, and satisfies all of the following (i) to (iv), or satisfies all of the following (i) to (v): (i) The amino acid at position 430 in Sequence ID No. 1 is proline; (ii) The amino acid at position 633 in Sequence ID No. 1 is proline; (iii) The amino acid at position 849 in Sequence ID No. 1 is isoleucine; (iv) The amino acid at position 880 in Sequence ID No. 1 is tyrosine; (v) The amino acid at position 61 in sequence number 1 is proline.

[0042] The RNA polymerase of the present invention is characterized by a reduction in the amount of dsRNA after the transcription reaction. The RNA polymerase of the present invention may be an RNA polymerase in which the proportion of dsRNA in the RNA after the transcription reaction is about 90% or less, preferably about 80% or less, more preferably about 70% or less, even more preferably about 60% or less, even more preferably about 50% or less, even more preferably about 40% or less, even more preferably about 30% or less, even more preferably about 20% or less, and particularly preferably about 10% or less, compared to the corresponding wild-type RNA polymerase.

[0043] [Method for measuring the proportion of dsRNA in RNA after transcription] The proportion of dsRNA in the RNA after the transcription reaction can be specifically determined by the following measurement method: Add 1 μL of RNA polymerase (50 U / μL) to 49 μL of reaction solution (the final concentrations in 50 μL of reaction solution after enzyme addition are shown below) (40 mM Tris-HCl (pH 8.0), 50 mM NaCl, 8 mM MgCl2, 5 mM DTT, 10 ng / μL template DNA, 0.4 mM ATP, 0.4 mM CTP, 0.4 mM GTP, 0.4 mM UTP), 0.4 U / μL RNase inhibitor), and then allow the reaction to proceed for 1 hour at 37°C using a heat block. The template DNA used is a dsDNA containing a promoter corresponding to the RNA polymerase to be measured (one of the following: T7 promoter, T3 promoter, SP6 promoter, etc.), a 5' UTR, a 3' UTR, and poly(A) (100nt) in that order from the 5' end, with the firefly luciferase gene (Fluc) as the coding sequence (CDS) between the 5' UTR and 3' UTR. After the reaction, 2.5 μL of Turbo® DNase (2 U / μL) (Thermo Fisher Scientific) is added, and the reaction is carried out at 37°C for 15 minutes. This reaction mixture is purified using the Monarch® RNA Cleanup Kit (NEB) to obtain purified RNA. The absorbance at 260 nm (A260) of the obtained purified RNA is measured, and the RNA concentration is calculated assuming that an A260 of 1 corresponds to an RNA concentration of 40 ng / μL. A calibration curve is created using the Double-stranded RNA (dsRNA) ELISA kit (K1 based) (Exalpha Biologicals) and the ELISA results obtained from the attached dsRNA control. Purified RNA is diluted to 100 ng / μL, then further diluted to multiple concentrations, and ELISA is performed using the Double-stranded RNA (dsRNA) ELISA kit (K1 based). The dsRNA concentration is calculated from the calibration curve using the values ​​obtained that fall within the range of the calibration curve. Furthermore, by dividing the dsRNA concentration by the RNA concentration, the percentage (%) (w / w) of dsRNA in the RNA can be calculated.

[0044] The RNA polymerase of the present invention may have high specific activity. Compared to the corresponding wild-type RNA polymerase, the RNA polymerase of the present invention may have a specific activity of about 1.1 times or more, preferably about 1.2 times or more, about 1.3 times or more, about 1.4 times or more, about 1.5 times or more, about 1.6 times or more, about 1.7 times or more, or about 1.8 times or more. The specific activity can be specifically confirmed by the following measurement method.

[0045] [Activity measurement method] RNA polymerase activity can be measured by the following procedure. Measurement conditions can be appropriately set by those skilled in the art; for example, if the enzyme activity is too high, the sample containing the target of measurement can be appropriately diluted before measurement. First, 45 μL of reaction solution (the final concentration in 50 μL of reaction solution after enzyme addition is shown below) (40 mM Tris-HCl (pH 8.0), 50 mM NaCl, 8 mM MgCl2, 5 mM DTT, 10 ng / μL template DNA, 0.4 mM ATP, 0.4 mM CTP, 0.4 mM GTP, 0.4 mM UTP) was added to a 0.2 mL PCR tube. Then, 5 μL of wild-type RNA polymerase with known activity was added, and the reaction was carried out at 37°C for 10 minutes using a heat block. After 10 minutes, the reaction was stopped by adding 10 μL of 1 M EDTA. This reaction was repeated to create a dilution series of RNA polymerase. Subsequently, the amount of RNA in each reaction solution was quantified using the Qubit® RNA BR Assay Kit (Thermo Fisher Scientific), and a calibration curve was created from the activity values ​​of the RNA polymerase added in each dilution series and the amount of RNA quantified. The same reaction is performed on the RNA polymerase to be measured, and the activity is calculated using the calibration curve created above based on the amount of RNA obtained.

[0046] [Measurement of specific activity] The absorbance at 280 nm (A280) is measured, and the enzyme concentration is calculated assuming that an A280 value of 1 corresponds to an enzyme concentration of 1 mg / mL. The specific activity is calculated by dividing the activity value calculated in the above manner by the enzyme concentration.

[0047] The RNA polymerase of the present invention may exhibit higher thermal stability compared to the corresponding wild-type RNA polymerase. In preferred embodiments, the RNA polymerase of the present invention may exhibit a residual activity of more than 50% when heat-treated at 50°C for 5 minutes, for example, and may exhibit a residual activity of 60% or more, 70% or more, or 80% or more. In specific embodiments, the RNA polymerase of the present invention may exhibit a residual activity of 90% or more when heat-treated at 50°C for 5 minutes.

[0048] From yet another perspective, the RNA polymerase of the present invention may be a mutant RNA polymerase that, for example, exhibits a higher residual activity rate compared to the residual activity rate of the corresponding wild-type RNA polymerase when heat-treated under predetermined conditions. Specifically, for example, when heat-treated at 50°C for 5 minutes, it may be an RNA polymerase that exhibits a residual activity rate that is about 1.1 times or more, preferably about 1.2 times or more, preferably about 1.3 times or more, preferably about 1.4 times or more, preferably about 1.5 times or more, preferably about 1.6 times or more, preferably about 1.7 times or more, preferably about 1.8 times or more, and preferably about 1.9 times or more, compared to the residual activity rate of the corresponding wild-type RNA polymerase.

[0049] [Measurement of thermal stability (residual activity rate after heat treatment)] Thermal stability can be specifically confirmed by the following measurement methods. First, each mutant RNA polymerase to be measured is diluted to 50 U / μL using a storage buffer (20 mM KPO4 (pH 7.7), 100 mM NaCl, 50% glycerol, 0.1 mM EDTA, 5 mM DTT, 0.01% Triton® X-100), and then its activity value before storage is measured according to the procedure described in the activity measurement method. Next, each mutant RNA polymerase to be measured, diluted in the above storage buffer, is stored in a 50°C incubator for 5 minutes. After storage, the activity value after storage is measured according to the procedure described in the activity measurement method, in the same manner as before storage. Then, the residual activity rate can be calculated by dividing the activity value after storage by the activity value before storage, as shown in Equation I below. Remaining activity rate (%) = (Activity value after storage / Activity value before storage) × 100 ... (Equation I)

[0050] In a further embodiment, the present invention provides a polynucleotide encoding the RNA polymerase of the present invention as described above. Such a polynucleotide can be suitably used for the expression of the RNA polymerase of the present invention and / or the creation of vectors for expression and / or the creation of transformed cells. Here, the polynucleotide encoding the RNA polymerase refers to a polynucleotide that, when expressed by a conventional method, yields the RNA polymerase protein of the present invention. That is, it refers to a polynucleotide composed of a base sequence corresponding to the amino acid sequence of the RNA polymerase protein of the present invention. Those skilled in the art can easily determine the base sequence corresponding to a predetermined amino acid sequence according to a codon table well known in the art. Furthermore, the polynucleotide encoding the RNA polymerase of the present invention also includes polynucleotides that differ due to codon degeneracy. The polynucleotide can be any nucleic acid such as DNA or RNA, but DNA is preferred. The polynucleotide can be synthesized, for example, by artificial gene synthesis. One example of a method for synthesizing DNA in artificial gene synthesis is to chemically synthesize two or more 10-100 nt oligoDNAs with partially overlapping sequences, then ligate them using PCA (polymerase chain assembly) or other enzyme-based methods, design primers for both ends of the final target sequence, and perform PCR to obtain double-stranded DNA, but this method is not particularly limited. When synthesizing RNA in artificial gene synthesis, one example is to synthesize double-stranded DNA such that the 5' end contains a sequence of an arbitrary promoter (T7 promoter, T3 promoter, SP6 promoter, etc.) during the synthesis of the double-stranded DNA described above, and then perform an in vitro transcription reaction using that DNA as a template with an RNA polymerase (T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, etc.) that specifically recognizes the arbitrary promoter to synthesize RNA.

[0051] In a further embodiment, the present invention provides a vector comprising the polynucleotide. Such a vector can be suitably used for the expression of the RNA polymerase of the present invention and / or the creation of transformed cells. The vector can be created by introducing the polynucleotide encoding the RNA polymerase into a vector (e.g., an expression vector, a cloning vector, etc.). The introduction of the polynucleotide into the vector can be carried out by any method, such as restriction enzyme treatment and ligation, or seamless cloning such as Gibson assembly. In a particular embodiment, the vector of the present invention may further contain elements such as a promoter, terminator, ribosome binding site, and drug resistance genes that function in a host cell. Examples of drug resistance genes include resistance genes to drugs such as ampicillin, kanamycin, and tetracycline. In a particular embodiment, the vector of the present invention may contain elements that enable homologous recombination with the genome of a host cell. The vector can be anything that enables the cloning and / or expression of the RNA polymerase of the present invention, such as a plasmid. Examples of plasmids include pUC118, pUC18, pBR322, pBluescript, pLED-M1, p73, pGW7, and pkk223-3, but are not limited to these.

[0052] In a further embodiment, the present invention provides cells transformed with the vector. Such cells can be suitably used to express the RNA polymerase-encoding protein of the present invention. In a particular preferred embodiment, recombinant host cells of the present invention are obtained by transforming host cells with the vector. Examples of host cells include Escherichia coli, Bacillus subtilis, filamentous fungi, and yeast, but Escherichia coli is particularly preferred. Examples of Escherichia coli include Escherichia coli DH5α, JM109, HB101, BL21, and BL21(DE3). That is, in the present invention, it is preferable to insert the gene encoding the RNA polymerase into the vector to make an expression vector, and then further transform host cells with the expression vector.

[0053] In a further embodiment, a method for producing RNA polymerase using the polynucleotide, the vector, the recombinant host cells, and / or one or more thereof is also provided. In a particular embodiment, after transforming the host cells with the vector, the resulting recombinant host cells are cultured using any culture apparatus. Examples of culture apparatus include, but are not limited to, test tubes, flasks, jar fermenters, etc. Examples of culture media include, but are not limited to, LB medium, 2×YT medium, TB medium, etc. Culture conditions can be set as appropriate, the culture temperature may be 10 to 40°C, and the culture time may be 1 to 100 hours. Aeration and / or stirring may be performed during culture, and culture may be performed by batch culture or fed-batch culture. After culture, the cells are collected by centrifugation or the like, and the crude enzyme solution is extracted by disrupting or lysing the collected cells. Any known method may be used to disrupt or lysing the cells. Examples of disruption methods include sonication, physical disruption methods such as French press or glass bead disruption, and examples of lysing methods include, but are not limited to, methods using enzymes such as lysozyme. Any method may be used to obtain purified RNA polymerase from the obtained crude enzyme solution. For example, the RNA polymerase of the present invention can be isolated by centrifugation, ultracentrifugation, ultrafiltration, nucleic acid removal, salting out, dialysis, or various column chromatography methods (ion exchange column chromatography, hydrophobic chromatography, affinity chromatography, gel filtration column chromatography, etc.).

[0054] In further embodiments, reagents comprising the RNA polymerase, the polynucleotide, the vector, the recombinant host cell, and / or one or more thereof are also provided. In certain embodiments, the reagent may be a liquid or a solid from which water has been removed by methods such as freeze-drying. In certain embodiments, the reagent may be contained in any container. In certain embodiments, the reagent may be divided into any number of one or more bottles. In certain embodiments, the reagent may contain a substance that enables the expression of the RNA polymerase from the polynucleotide, the vector, and the recombinant host cell. Examples of such substances include, but are not limited to, cell-free protein synthesis reagents. Examples of cell-free protein synthesis reagents include, but are not limited to, those using cell extracts and reconstituted cell-free protein synthesis systems in which factors involved in the translation reaction are purified and mixed. The substances included in the cell-free protein synthesis reagent include, but are not limited to, cell extracts or purified factors involved in translation reactions, 20 types of amino acids, ATP, GTP, creatine phosphate, creatine kinase, buffers (Tris buffer, phosphate buffer, Good's buffer, etc.), salts (potassium acetate, magnesium acetate), spermidine, dithiothreitol, etc. Examples of reagents using cell extracts include Taiyo Nippon Sanso's Cell-Free-Kun (registered trademark), which are derived from extracts of rabbit reticulocytes, wheat germ, insect cells, Escherichia coli, and human cells, but are not particularly limited. Examples of reconstituted cell-free protein synthesis systems include, but are not particularly limited to, PUREflex (registered trademark) 1.0 from Scene Frontier. In certain embodiments, the reagent may contain substances necessary for in vitro transcription reactions.Substances necessary for the in vitro transcription reaction include, but are not limited to, buffers (such as Tris buffer, phosphate buffer, and Good's buffer), ribonucleotides (such as ATP, CTP, GTP, UTP, and modified bases (bases modified by deamination, methylation, methoxylation, pseudouridineation, etc.)), capping analogs (such as ARCA, mCAP, CleanCap® Reagent AG, CleanCap® Reagent M6, and CleanCap® Reagent AU (manufactured by TriLink)), RNase inhibitors, pyrophosphatases, salts such as NaCl, metal ions such as magnesium ions, EDTA, dithiothreitol, glycerol, surfactants, spermidine, and linear or circular DNA. In certain embodiments, the reagents may further contain substances necessary for post-transcription capping. Substances necessary for post-transcription capping include, but are not limited to, capping enzymes and GTP, mRNA Cap 2'-O-methyltransferase, SAM (S-adenosylmethionine), buffers (such as Tris buffer, phosphate buffer, and Good's buffer), salts such as NaCl and KCl, metal ions such as magnesium ions, EDTA, dithiothreitol, glycerol, and surfactants. In certain embodiments, the reagent may further contain substances necessary for adding poly(A) to the 3' end of RNA. Substances necessary for poly(A) addition include, but are not limited to, ATP, poly(A) polymerase, buffers (such as Tris buffer, phosphate buffer, and Good's buffer), salts such as NaCl, metal ions such as magnesium ions and manganese ions, EDTA, dithiothreitol, glycerol, and surfactants. In certain embodiments, the reagent may further contain substances necessary for preparing template DNA for the in vitro transcription reaction.Substances necessary for preparing template DNA for in vitro transcription reactions include, but are not limited to, restriction enzymes, buffers (such as Tris buffer, phosphate buffer, and Good's buffer), salts such as NaCl, metal ions such as magnesium ions, EDTA, dithiothreitol, glycerol, surfactants, PCR enzymes, and oligoDNA. In certain embodiments, the reagent may further contain substances necessary for purifying the template DNA for in vitro transcription reactions. Substances necessary for purifying the template DNA include, but are not limited to, DNA purification beads and DNA purification columns. In certain embodiments, the reagent may further contain substances necessary for purifying RNA. Substances necessary for purifying RNA include, but are not limited to, RNA purification beads and RNA purification columns.

[0055] The present invention further provides a method for performing an in vitro transcription reaction using the RNA polymerase. The in vitro transcription reaction includes, but is not limited to, a method of synthesizing RNA using the RNA polymerase with linear or circular DNA as a template. The in vitro transcription reaction also includes, but is not limited to, a method of synthesizing RNA using the RNA polymerase with linear or circular DNA as a template in the presence of a buffer and ribonucleotides. The concentration of RNA polymerase added during the reaction can be, for example, in the range of 0.1 U / μL to 20 U / μL, but is not particularly limited. The concentration of linear or circular DNA can be, for example, in the range of 1 ng / μL to 200 ng / μL, but is not particularly limited. The ribonucleotides used in the in vitro transcription reaction include, but is not particularly limited to, ATP, CTP, GTP, UTP, and modified bases that have undergone modifications such as deamination, methylation, methoxylation, and pseudouridineization. The concentration of ribonucleotides can be, for example, in the range of 0.1 mM to 20 mM, but is not particularly limited. During the in vitro transcription reaction, cap analogs such as ARCA, mCAP, CleanCap® Reagent AG, CleanCap® Reagent M6, and CleanCap® Reagent AU (manufactured by TriLink) can be added, but are not particularly limited. The concentration of the cap analog can be added in the range of, for example, 0.1 mM to 20 mM, but are not particularly limited. Examples of buffers used in the in vitro transcription reaction include Tris buffer, phosphate buffer, and Good's buffer, but are not particularly limited. The concentration of the buffer can be added in the range of, for example, 5 mM to 500 mM, but are not particularly limited. During the in vitro transcription reaction, RNase inhibitors and pyrophosphatases can be added, but are not particularly limited. The concentration of the RNase inhibitor can be added in the range of, for example, 0.1 U / μL to 20 U / μL, but are not particularly limited. The concentration of the pyrophosphatase can be added in the range of, for example, 0.0001 U / μL to 0.02 U / μL, but are not particularly limited.During the in vitro transfer reaction, salts such as NaCl and metal ions such as magnesium ions can be added, but are not particularly limited. The concentration of NaCl can be added in the range of 1 mM to 200 mM, for example, but are not particularly limited. The concentration of magnesium ions can be added in the range of 0.1 mM to 20 mM, for example, but are not particularly limited. During the in vitro transfer reaction, EDTA, dithiothreitol, glycerol, surfactants, spermidine, etc. can be added, but are not particularly limited. The concentration of EDTA can be added in the range of 0.0001 mM to 2 mM, for example, but are not particularly limited. The concentration of dithiothreitol can be added in the range of 0.1 mM to 20 mM, for example, but are not particularly limited. The concentration of glycerol can be added in the range of 0.1% to 20%, for example, but are not particularly limited. The concentration of surfactants can be added in the range of 0.0001% to 1%, for example, but are not particularly limited. The spermidine concentration can be added in a range of, for example, 0.1 mM to 20 mM, but is not particularly limited.

[0056] Furthermore, as a specific embodiment, the present invention further provides a method for performing an in vitro transcription reaction in which the proportion of dsRNA in the RNA after the in vitro transcription reaction is 90% or less compared to when the in vitro transcription reaction is performed using the corresponding wild-type RNA polymerase. The proportion may preferably be 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less. The proportion can be calculated, for example, by the method described in [Method for measuring the proportion of dsRNA in RNA after transcription reaction], by performing an in vitro transcription reaction with the corresponding wild-type RNA polymerase and each mutant RNA polymerase to be measured, calculating the proportion of dsRNA in the RNA, and then comparing the values.

[0057] Furthermore, in a specific embodiment, the present invention also provides a method for synthesizing mRNA using the RNA polymerase. The method for synthesizing mRNA includes, but is not particularly limited to, a method in which mRNA is synthesized using the RNA polymerase with linear or circular DNA as a template in the presence of a buffer and ribonucleotides. The concentration of RNA polymerase added during the reaction can be, for example, in the range of 0.1 U / μL to 20 U / μL, but is not particularly limited. The concentration of linear or circular DNA can be, for example, in the range of 1 ng / μL to 200 ng / μL, but is not particularly limited. The ribonucleotides used for mRNA synthesis include, but are not particularly limited to, ATP, CTP, GTP, UTP, and modified bases that have undergone modifications such as deamination, methylation, methoxylation, and pseudouridineization. The concentration of ribonucleotides can be, for example, in the range of 0.1 mM to 20 mM, but is not particularly limited. The buffer used for mRNA synthesis includes, but is not particularly limited to, Tris buffer, phosphate buffer, and Good's buffer. The buffer solution can be added in a range of, for example, 5 mM to 500 mM, but is not particularly limited. During mRNA synthesis, RNase inhibitors, pyrophosphatases, etc., can be added, but are not particularly limited. The concentration of the RNase inhibitor can be added in a range of, for example, 0.1 U / μL to 20 U / μL, but is not particularly limited. The concentration of the pyrophosphatase can be added in a range of, for example, 0.0001 U / μL to 0.02 U / μL, but is not particularly limited. During mRNA synthesis, salts such as NaCl and metal ions such as magnesium ions can be added, but are not particularly limited. The concentration of NaCl can be added in a range of, for example, 1 mM to 200 mM, but is not particularly limited. The concentration of magnesium ions can be added in a range of, for example, 0.1 mM to 20 mM, but is not particularly limited. During mRNA synthesis, EDTA, dithiothreitol, glycerol, surfactants, spermidine, etc., can be added, but are not particularly limited.The concentration of EDTA can be added in a range of, for example, 0.0001 mM to 2 mM, but is not particularly limited. The concentration of dithiothreitol can be added in a range of, for example, 0.1 mM to 20 mM, but is not particularly limited. The concentration of glycerol can be added in a range of, for example, 0.1% to 20%, but is not particularly limited. The concentration of surfactant can be added in a range of, for example, 0.0001% to 1%, but is not particularly limited. The concentration of spermidine can be added in a range of, for example, 0.1 mM to 20 mM, but is not particularly limited. The 5' end of mRNA contains a cap structure, and methods for adding the cap structure include, but are not particularly limited, post-transcriptional capping and co-transcriptional capping. Post-transcriptional capping methods include, but are not particularly limited, methods for adding Cap-0 to the 5' end of mRNA using a capping enzyme, GTP, and SAM, and further, if necessary, converting Cap-0 to Cap-1 using mRNA Cap 2'-O-methyltransferase and SAM. Co-transcription capping methods include, but are not limited to, methods using cap analogs such as ARCA, mCAP, CleanCap® Reagent AG, CleanCap® Reagent M6, and CleanCap® Reagent AU (manufactured by TriLink). The concentration of the cap analog can be added in the range of, for example, 0.1 mM to 20 mM, but is not limited to that range. If poly(A) is present at the 3' end of mRNA but not in the template DNA, methods for adding poly(A) to the 3' end of the synthesized RNA include, but are not limited to, methods using poly(A) polymerase.

[0058] Furthermore, in a specific embodiment, the present invention further provides an mRNA synthesis method in which the proportion of dsRNA in mRNA during the transcription reaction is 90% or less compared to the case where mRNA synthesis is performed using the corresponding wild-type RNA polymerase. The proportion may preferably be 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less. The proportion can be calculated, for example, by the method described in [Method for measuring the proportion of dsRNA in RNA after transcription reaction], by performing mRNA synthesis with the corresponding wild-type RNA polymerase and each mutant RNA polymerase to be measured, calculating the proportion of dsRNA in the RNA, and then comparing the values.

[0059] Furthermore, in a specific embodiment, the present invention also provides a method for synthesizing non-coding RNA using the RNA polymerase described above. Examples of non-coding RNAs include, but are not limited to, microRNA, siRNA, piRNA, rRNA, tRNA, snRNA, snoRNA, SLRNA, SRPRNA, mRNA-like non-coding RNA, oligonucleotides, etc. As an example of a method for synthesizing non-coding RNA, one method is to perform an in vitro transcription reaction using, for example, the method described above, with a template DNA containing DNA corresponding to the target non-coding RNA, but is not particularly limited. The template DNA may be any DNA as long as it contains DNA corresponding to the target non-coding RNA, but it is preferable that it contains a promoter sequence (T7 promoter, T3 promoter, SP6 promoter, etc.) recognized by the RNA polymerase of the present invention upstream of the corresponding DNA. The template DNA may be prepared by any method, but is not particularly limited, for example, by restriction enzyme treatment of a plasmid or by using DNA synthesized by artificial gene synthesis. The plasmid may be any plasmid as long as it contains DNA corresponding to the target non-coding RNA, but may also contain the promoter sequence, restriction enzyme site, drug resistance gene, etc. described above. As an example of a method for obtaining the plasmid described above, one method involves artificially synthesizing DNA corresponding to the target non-coding RNA, cloning it into any plasmid containing a promoter sequence recognized by the RNA polymerase of the present invention (such as the T7 promoter, T3 promoter, or SP6 promoter) upstream of the site where the DNA is inserted, a restriction enzyme site downstream, and optionally a drug resistance gene at any position, and then transforming the plasmid into any host, preferably E. coli, by any method, culturing the resulting recombinant host cells by any method, and then extracting the plasmid. However, the method is not particularly limited. The obtained plasmid can be made linear by restriction enzyme treatment of the restriction enzyme sites contained in the plasmid with any usable restriction enzyme, and then purified by any method to obtain a template DNA.Methods using DNA synthesized by artificial gene synthesis include, for example, artificially synthesizing double-stranded DNA such that it contains DNA corresponding to the target non-coding RNA and a promoter sequence recognized by the RNA polymerase of the present invention (T7 promoter, T3 promoter, SP6 promoter, etc.) upstream of it, amplifying it by PCR or other means as needed, and purifying it as needed to use as template DNA, but are not particularly limited.

[0060] Furthermore, as a specific embodiment, the present invention also provides a method for using the RNA polymerase in the synthesis of RNA drugs. Examples of RNA drugs include, but are not limited to, mRNA, siRNA, microRNA, antisense RNA, and aptamers. An example of a method for using the RNA polymerase in the synthesis of RNA drugs is a method in which an in vitro transcription reaction is performed using the method described above, for example, with the RNA polymerase and template DNA containing DNA corresponding to any RNA (mRNA, siRNA, microRNA, antisense RNA, aptamer, etc.) to be used as an RNA drug. However, the method is not particularly limited. The template DNA containing DNA corresponding to any RNA (mRNA, siRNA, microRNA, antisense RNA, aptamer, etc.) can be any DNA that contains DNA corresponding to any RNA, but it may also contain a promoter sequence recognized by the RNA polymerase of the present invention (T7 promoter, T3 promoter, SP6 promoter, etc.) upstream of the DNA, and optionally a 5' UTR upstream, a 3' UTR and poly(A), restriction enzyme sites, etc. downstream. The template DNA may be prepared by any method, and methods well known to those skilled in the art can be used. For example, the above-described method can be appropriately modified and used for the synthesis of non-coding RNA.

[0061] In a specific embodiment, the present invention further provides a method for performing in vivo protein expression or in vitro cell-free protein synthesis using the RNA polymerase. An example of a method for performing in vivo protein expression using the RNA polymerase is, but is not limited to, a method of transfecting any cells with mRNA synthesized using the RNA polymerase in any way. In a specific embodiment, the mRNA may be any mRNA that enables the expression of the target protein, but preferably contains RNA encoding the target protein, and may contain a 5'UTR upstream and a 3' UTR and poly(A) downstream. In a specific embodiment, the method for synthesizing mRNA may be, but is not limited to, performing an in vitro transcription reaction using, for example, the method described above, with template DNA containing DNA encoding the target protein as a template. The template DNA may be prepared by any method, and methods well known to those skilled in the art can be used, and for example, the above method can be appropriately modified for the synthesis of non-coding RNA. In a specific embodiment, when performing protein expression, culture or the like may be performed after transfection as needed, but is not particularly limited. One example of a method for performing in vitro cell-free protein synthesis using the RNA polymerase is, but is not limited to, adding the RNA polymerase, the polynucleotide, the vector, the recombinant host cell, and / or one or more of these to any cell-free protein synthesis reagent. [Examples]

[0062] The present invention will be specifically described below with reference to examples. However, the present invention is not limited to the following examples.

[0063] Example 1: Preparation of a plasmid for T7 RNA polymerase expression A gene encoding wild-type T7 RNA polymerase with a 6×His tag at the N-terminus (SEQ ID NO: 2) was artificially synthesized and cloned into the EcoRI-PstI cloning site of pkk223-3 to create a plasmid incorporating the gene encoding wild-type T7 RNA polymerase with a 6×His tag at the N-terminus. Hereafter, this expression plasmid will be referred to as pkk223-3-T7RNAP. Using pkk223-3-T7RNAP as a template, site-directed mutagenesis was performed using the primers listed in Table 1 and the KOD-Plus-Mutagenesis Kit (Toyobo Co., Ltd.) to create expression plasmids for each modified T7 RNA polymerase. Expression plasmids for each modified T7 RNA polymerase, which have multiple amino acid modifications, were created by repeating site-directed mutagenesis using the modified T7 RNA polymerase expression plasmids obtained by site-directed mutagenesis as a template.

[0064] [Table 1]

[0065] Each obtained plasmid contains a nucleotide sequence encoding a modified T7 RNA polymerase with a 6×His tag at its N-terminus.

[0066] Example 2: Preparation of T7 RNA polymerase Wild-type T7 RNA polymerase with a 6×His tag at the N-terminus and each modified T7 RNA polymerase were all prepared using the same method. An example of wild-type T7 RNA polymerase preparation is shown below. Escherichia coli JM109 was transformed with pkk223-3-T7RNAP and cultured statically on LB agar medium containing 100 μg / mL ampicillin at 37°C for 20 hours. Single colonies from the agar medium were inoculated into 3 mL of LB liquid medium containing 100 μg / mL ampicillin and cultured with shaking in a 15 mL test tube at 37°C for 20 hours. 1 mL of this culture was inoculated into 80 mL of TB liquid medium containing 100 μg / mL ampicillin and cultured with shaking in a 500 mL Sakaguchi flask at 37°C for 20 hours. The bacterial cells were then collected from the culture by centrifugation. One g of the obtained bacterial cells was suspended in 10 mL of lysation buffer (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10% glycerol, 1 mM DTT, 20 mM imidazole), and the cells were lysed on ice using an ultrasonic lysator. The lysate was centrifuged at 20,000 × g for 20 minutes at 4°C, and the supernatant was collected. This supernatant was then used for purification using a His GraviTrap (GE Healthcare). The equilibration and washing buffer used was (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10% glycerol, 1 mM DTT, 20 mM imidazole), and the elution buffer used was (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10% glycerol, 1 mM DTT, 300 mM imidazole). The eluted fraction was collected and replaced with storage buffer (20 mM KPO4 (pH 7.7), 100 mM NaCl, 50% glycerol, 0.1 mM EDTA, 5 mM DTT, 0.01% Triton® X-100) to obtain wild-type T7 RNA polymerase with a 6×His tag at the N-terminus. In the above preparation example, each modified T7 RNA polymerase with a 6×His tag at the N-terminus was obtained by using the expression plasmid for each modified T7 RNA polymerase prepared in Example 1 during transformation. The activity of each modified T7 RNA polymerase was measured using the activity measurement method described above, and the obtained activity values ​​were diluted to the desired concentration in storage buffer in the following examples.For activity measurement, a 4kbp dsDNA (SEQ ID NO: 13) containing the T7 promoter sequence was used as the template DNA. The length downstream of the T7 promoter in this dsDNA is approximately 1kbp, and the length of the transcribed RNA is approximately 1kb.

[0067] Example 3: RNA synthesis under high-temperature conditions Using wild-type T7 RNA polymerase with a His tag added to the N-terminus and modified T7 RNA polymerases (A61P / S430P / S633P / F849I / F880Y) similarly with a His tag added to the N-terminus, 2.5 μL of each T7 RNA polymerase (200 U / μL) was prepared on ice to make 47.5 μL of reaction solution (the final concentration in 50 μL of reaction solution after enzyme addition is shown below) (30 mM Tris-HCl (pH 8.0), 27 mM magnesium acetate, 3 mM DTT, 10 ng / μL template DNA, 5 mM ATP, 5 mM CTP, 5 mM GTP, 5 mM UTP, 1 U / μL RNase inhibitor, 0.002 U / μL) After adding the compound (pyrophosphatase), the reaction was carried out for 1 hour at each temperature of 40°C, 42.5°C, 45°C, 47.5°C, 50°C, and 52.5°C using a heat block. The template DNA used was dsDNA (SEQ ID NO: 14) containing the T7 promoter, 5' UTR, 3' UTR, and poly(A) (105 nt), with the firefly luciferase gene (Fluc) as the coding sequence (CDS). The generated RNA is an approximately 1.9 kb mRNA without a cap structure, composed of 5' UTR, CDS (Fluc), 3' UTR, and poly(A) from the 5' end. After the reaction, analysis was performed using a 4200 TapeStation (Agilent Technologies), RNA Screen Tape (Agilent Technologies), RNA Screen Tape (Agilent Technologies), and RNA Sample Buffer (Agilent Technologies) to confirm the amount of target RNA (approximately 1900 nt) produced, the amount of impurity RNA (2000-2500 nt) longer than the RNA size predicted from the template DNA, and their respective percentages. The results are shown in Table 2.

[0068] [Table 2]

[0069] The results in Table 2 show that when using wild-type T7 RNA polymerase, a sufficient amount (more than 25 μg) of target RNA could not be obtained under high-temperature conditions of 50°C and 52.5°C. On the other hand, modified T7 RNA polymerases (A61P / S430P / S633P / F849I / F880Y) were able to synthesize RNA under high-temperature conditions of 50°C and 52.5°C. Furthermore, when RNA synthesis was performed using modified T7 RNA polymerase under high-temperature conditions of 50°C and 52.5°C, it was confirmed that the proportion of impurity RNA (2000-2500 nt) longer than the target RNA predicted from the template DNA size (approximately 1900 nt) was reduced. This suggests that performing the transcription reaction under high-temperature conditions using modified T7 RNA polymerase suppresses the generation of impurity RNA (3' elongated RNA) due to further elongation of the 3' end of the synthesized RNA.

[0070] Example 4: Measurement of dsRNA quantity after transcription reaction Using wild-type T7 RNA polymerase with a His tag at the N-terminus, and various modified T7 RNA polymerases (S430P / S633P / F849I / F880Y, A61P / S430P / S633P / F849I / F880Y) with similarly added His tags at the N-terminus, and Hi-T7 RNA Polymerase (NEB), 1 μL of each T7 RNA polymerase (50 U / μL) was added to 49 μL of reaction solution (the final concentration in 50 μL of reaction solution after enzyme addition is shown below) (40 mM Tris-HCl (pH 8.0), 50 mM NaCl, 8 mM MgCl2, 5 mM DTT, 10 ng / μL template DNA, 0.4 mM ATP, 0.4 mM CTP, 0.4 mM GTP, 0.4 mM UTP), 0.4 U / μL After adding the RNA to an RNase inhibitor, the reaction was carried out for 1 hour at 37°C or 48°C using a heat block. The template DNA used was dsDNA (SEQ ID NO: 15) containing the T7 promoter, 5' UTR, 3' UTR, and poly(A) (100nt), with the firefly luciferase gene (Fluc) as the coding sequence (CDS). The generated RNA is approximately 1.9kb mRNA without a cap structure, composed of 5'UTR, CDS (Fluc), 3'UTR, and poly(A) from the 5' end. After the reaction, 2.5 μL of Turbo® DNase (2U / μL) (Thermo Fisher Scientific) was added, and the reaction was carried out for another 15 minutes at 37°C. This reaction mixture was purified using the Monarch® RNA Cleanup Kit (NEB) to obtain purified RNA. The absorbance (A260) at 260 nm of the obtained purified RNA was measured, and the RNA concentration was calculated assuming that an A260 value of 1 corresponds to an RNA concentration of 40 ng / μL. A calibration curve was created from the measured values ​​obtained by performing ELISA on the attached dsRNA control using the Double-stranded RNA (dsRNA) ELISA kit (K1 based) (Exalpha Biologicals).Each purified RNA was diluted to 100 ng / μL, and then further diluted to multiple concentrations. ELISA was performed using a Double-stranded RNA (dsRNA) ELISA kit (K1 based). The dsRNA concentration was calculated from the calibration curve created above, using the values ​​that fell within the range of the calibration curve. Furthermore, the percentage (%) (w / w) of dsRNA in the RNA was calculated by dividing the dsRNA concentration by the RNA concentration. The results are shown in Table 3.

[0071] [Table 3]

[0072] First, under high-temperature conditions of 48°C, a sufficient amount of the target RNA could not be synthesized using wild-type T7 RNA polymerase (data not shown). As shown in Table 3, when RNA synthesis was performed using the modified T7 RNA polymerase (S430P / S633P / F849I / F880Y quadruple mutant), the ratio of dsRNA to RNA after transcription at 48°C was reduced compared to the ratio after transcription at 37°C. Furthermore, when RNA synthesis was performed using the modified T7 RNA polymerase (A61P / S430P / S633P / F849I / F880Y quintuple mutant), the ratio of dsRNA to RNA after transcription was reduced both after the 48°C and 37°C reactions, and it was confirmed that the ratio of dsRNA to RNA after transcription was even further reduced compared to the quadruple mutant. On the other hand, when RNA synthesis was performed using NEB Hi-T7, no reduction in the ratio of dsRNA to RNA after transcription was observed. These results indicate that performing RNA synthesis at a high temperature of 48°C using a quadruple mutant of the modified T7 RNA polymerase reduces the ratio of dsRNA to RNA after transcription. Furthermore, it was shown that performing RNA synthesis using a quintuple mutant of the modified T7 RNA polymerase reduces the ratio of dsRNA to RNA after transcription in both cases, both after transcription at 37°C and 48°C. [Industrial applicability]

[0073] The present invention can be suitably used for RNA synthesis for various purposes.

Claims

1. It has more than 90% identity with the amino acid sequence of Sequence ID No. 1, One or more amino acids at positions 430, 633, 849, and 880 in Sequence ID No. 1 have been modified, and RNA polymerase in which one or more amino acids at positions corresponding to the 61st, 156th, 189th, 165th, and 661st positions in Sequence ID No. 1 have been modified.

2. The RNA polymerase according to claim 1, having 90% or more identity with the amino acid sequence of SEQ ID NO: 1, and satisfying one or more of (i) to (iv) and one or more of (v) to (ix) below: (i) The amino acid at position 430 in Sequence ID No. 1 is proline, alanine, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (ii) The amino acid at position 633 in Sequence ID No. 1 is proline, alanine, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (iii) The amino acid at position 849 in Sequence ID No. 1 is isoleucine, proline, alanine, glycine, leucine, methionine, valine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (iv) Whether the amino acid at position 880 in Sequence ID No. 1 is tyrosine, isoleucine, proline, alanine, glycine, leucine, methionine, valine, tryptophan, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (v) The amino acid at position 61 in SEQ ID NO: 1 is proline, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (vi) The amino acid at position 156 in Sequence ID No. 1 is asparagine, glutamine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, serine, threonine, arginine, histidine, lysine, or glutamic acid; (vii) The amino acid at position 189 in Sequence ID No. 1 is asparagine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, glutamine, serine, threonine, arginine, histidine, lysine, or glutamic acid; (viiii) Whether the amino acid at position 165 in Sequence ID No. 1 is serine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (ix) The amino acid at position 661 in Sequence ID No. 1 is asparagine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid.

3. The RNA polymerase according to claim 1 or 2, satisfying all of the following (i) to (v): (i) The amino acid at position 430 in Sequence ID No. 1 is proline; The amino acid at position 633 in (ii) Sequence ID 1 is proline; The amino acid at position 849 in (iii) Sequence ID 1 is isoleucine; The amino acid at position 880 in (iv) Sequence ID No. 1 is tyrosine; The amino acid at the position corresponding to the 61st position in (v) Sequence ID No. 1 is proline.

4. An RNA polymerase capable of synthesizing RNA at 48°C or higher, wherein the proportion of dsRNA in the RNA synthesized at 48°C or higher is 70% or less compared to the proportion of dsRNA in the RNA synthesized at 37°C using the corresponding wild-type RNA polymerase.

5. The RNA polymerase according to claim 4, which satisfies one or more of the following (i) to (iv), and optionally one or more of the following (v) to (ix): (i) The amino acid at position 430 in Sequence ID No. 1 is proline, alanine, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (ii) The amino acid at position 633 in Sequence ID No. 1 is proline, alanine, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (iii) The amino acid at position 849 in Sequence ID No. 1 is isoleucine, proline, alanine, glycine, leucine, methionine, valine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (iv) The amino acid at position 880 in SEQ ID NO: 1 is tyrosine, isoleucine, proline, alanine, glycine, leucine, methionine, valine, tryptophan, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (v) The amino acid at position 61 in SEQ ID NO: 1 is proline, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (vi) The amino acid at position 156 in Sequence ID No. 1 is asparagine, glutamine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, serine, threonine, arginine, histidine, lysine, or glutamic acid; (vii) The amino acid at position 189 in Sequence ID No. 1 is asparagine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, glutamine, serine, threonine, arginine, histidine, lysine, or glutamic acid; (viiii) The amino acid at position 165 in Sequence ID No. 1 is serine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid; (ix) The amino acid at position 661 in Sequence ID No. 1 is asparagine, glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, or glutamic acid.

6. An RNA polymerase according to claim 4 or 5 that satisfies all of (i) to (iv) below, or satisfies all of (i) to (v) below: (i) The amino acid at position 430 in Sequence ID No. 1 is proline; (ii) The amino acid at position 633 in SEQ ID NO: 1 is proline; (iii) The amino acid at position 849 in Sequence ID No. 1 is isoleucine; (iv) The amino acid at position 880 in Sequence ID No. 1 is tyrosine; (v) The amino acid at position 61 in Sequence ID No. 1 is proline.

7. A method for producing RNA polymerase using a polynucleotide encoding RNA polymerase according to claim 1 or 4, a vector containing the polynucleotide, or recombinant host cells transformed using the vector.

8. A reagent comprising an RNA polymerase according to claim 1 or 4, a polynucleotide encoding the RNA polymerase, a vector containing the polynucleotide, or a recombinant host cell transformed using the vector.

9. A method for performing an in vitro transcription reaction using the RNA polymerase described in claim 1 or 4.

10. The method according to claim 9, wherein the proportion of dsRNA in the RNA after the in vitro transcription reaction is 90% or less compared to when the in vitro transcription reaction is performed using the corresponding wild-type RNA polymerase.

11. A method for synthesizing mRNA using the RNA polymerase described in claim 1 or 4.

12. The method according to claim 11, wherein the proportion of dsRNA in mRNA is 90% or less compared to when mRNA synthesis is performed using the corresponding wild-type RNA polymerase.

13. A method for synthesizing non-coding RNA using the RNA polymerase described in claim 1 or 4.

14. A method for synthesizing an RNA drug using the RNA polymerase described in claim 1 or 4.

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