Modified RNA polymerase

Abstract

The present invention addresses the problem of providing a novel modified RNA polymerase. The problem is solved by a modified RNA polymerase in which at least one amino acid residue at a specific position is modified.

Description

Modified RNA polymerase

[0001] The present invention relates to modified RNA polymerases.

[0002] DNA-dependent RNA polymerases are enzymes that recognize promoter sequences in DNA and synthesize RNA using DNA as a template. They have been the subject of extensive research because they are responsible for the transcription reaction that synthesizes mRNA from genomic DNA in vivo. Among these, phage-derived RNA polymerases have a relatively simple structure and function as a single subunit, making them the subject of biochemical and structural biological research for a long time. These RNA polymerases are also widely used for in vitro RNA synthesis, and the RNAs synthesized by them are used as RNA probes for molecular biology techniques, various functional RNAs, or template RNAs for intracellular protein expression or cell-free protein synthesis systems.

[0003] Furthermore, isothermal amplification reactions such as the NASBA method and the TMA method have been developed by utilizing 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 also been applied to mRNA medicines, including mRNA vaccines, and RNA polymerase is also widely used in the field of pharmaceutical manufacturing. One of the problematic impurities in the manufacturing process of mRNA medicines is dsRNA. Because dsRNA induces inflammatory reactions in vivo, it is desirable to remove it as much as possible from mRNA medicines. Non-Patent Document 1 describes a modified T7 RNA polymerase that can reduce the production of dsRNA during the transcription reaction.

[0005] Dousis, A., Ravichandran, K., Hobert, EM et al., Nat. Biotechnol., 41:560-568, 2023

[0006] As described above, as the applications of RNA polymerases expand, further improvements in the performance of RNA polymerases are desired. Therefore, an objective of the present invention is to provide a novel modified RNA polymerase.

[0007] As a result of intensive research to solve the above problems, the present inventors have found that the production of dsRNA in a transcription reaction can be reduced by modifying at least one amino acid at a specific position in the amino acid sequence of a wild-type RNA polymerase. Based on this finding, the present inventors have conducted further intensive research, and as a result have completed the present invention.

[0008] That is, the present invention encompasses the following aspects: [Item 1] An RNA polymerase having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 1, in which at least one amino acid selected from the group consisting of amino acids at positions 61, 156, 189, 165, and 661 of the amino acid sequence of SEQ ID NO: 1 has been modified. [Item 2] An RNA polymerase having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 1 and that satisfies at least one of the following conditions (i) to (v): (i) the amino acid at position 61 of the amino acid sequence of SEQ ID NO: 1 is glutamic acid, aspartic acid, serine, glycine, or proline; (ii) the amino acid at position 156 of the amino acid sequence of SEQ ID NO: 1 is asparagine; (iii) the amino acid at position 189 of the amino acid sequence of SEQ ID NO: 1 is asparagine; (iv) the amino acid at position 165 of the amino acid sequence of SEQ ID NO: 1 is serine, glutamic acid, arginine, or proline; and (v) the amino acid at position 661 of the amino acid sequence of SEQ ID NO: 1 is asparagine. [Item 3] A nucleic acid having 90% or more identity with the amino acid sequence of SEQ ID NO: 1, wherein: (1) the amino acid at the position corresponding to position 61 of the amino acid sequence of SEQ ID NO: 1 is glutamic acid, aspartic acid, serine, glycine, or proline; (2) the amino acid at the position corresponding to position 156 of the amino acid sequence of SEQ ID NO: 1 is asparagine; (3) the amino acid at the position corresponding to position 189 of the amino acid sequence of SEQ ID NO: 1 is asparagine; (4) the amino acid at the position corresponding to position 165 of the amino acid sequence of SEQ ID NO: 1 is serine, glutamic acid, arginine, or proline; (5) the amino acid at the position corresponding to position 661 of the amino acid sequence of SEQ ID NO: 1 is asparagine; (6) the amino acid at the position corresponding to position 61 of the amino acid sequence of SEQ ID NO: 1 is glutamic acid, and the amino acid at the position corresponding to position 156 of the amino acid sequence of SEQ ID NO: 1 is asparagine;(7) the amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is glutamic acid, and the amino acid at the position corresponding to the 165th position in the amino acid sequence of SEQ ID NO: 1 is serine; (8) the amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is glutamic acid, and the amino acid at the position corresponding to the 189th position in the amino acid sequence of SEQ ID NO: 1 is asparagine; (9) the amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is glutamic acid, and the amino acid at the position corresponding to the 661st position in the amino acid sequence of SEQ ID NO: 1 is asparagine; (10) the amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is proline, and the amino acid at the position corresponding to the 165th position in the amino acid sequence of SEQ ID NO: 1 is serine; (11) the amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is proline, and the amino acid at the position corresponding to the 189th position in the amino acid sequence of SEQ ID NO: 1 is asparagine; (12) The amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is proline, and the amino acid at the position corresponding to the 661st position in the amino acid sequence of SEQ ID NO: 1 is asparagine; (13) The amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is glutamic acid, the amino acid at the position corresponding to the 156th position in the amino acid sequence of SEQ ID NO: 1 is asparagine, and the amino acid at the position corresponding to the 165th position in the amino acid sequence of SEQ ID NO: 1 is serine; (14) The amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is glutamic acid, the amino acid at the position corresponding to the 165th position in the amino acid sequence of SEQ ID NO: 1 is serine, and the amino acid at the position corresponding to the 189th position in the amino acid sequence of SEQ ID NO: 1 is asparagine; (15) The amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is glutamic acid, the amino acid at the position corresponding to the 165th position in the amino acid sequence of SEQ ID NO: 1 is serine, and the amino acid at the position corresponding to the 661st position in the amino acid sequence of SEQ ID NO: 1 is asparagine;(16) The amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is proline, the amino acid at the position corresponding to the 156th position in the amino acid sequence of SEQ ID NO: 1 is asparagine, and the amino acid at the position corresponding to the 165th position in the amino acid sequence of SEQ ID NO: 1 is serine; (17) The amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is proline, the amino acid at the position corresponding to the 165th position in the amino acid sequence of SEQ ID NO: 1 is serine, and the amino acid at the position corresponding to the 189th position in the amino acid sequence of SEQ ID NO: 1 is asparagine; (18) The amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is proline, the amino acid at the position corresponding to the 165th position in the amino acid sequence of SEQ ID NO: 1 is serine, and the amino acid at the position corresponding to the 661st position in the amino acid sequence of SEQ ID NO: 1 is asparagine; (19) The amino acid at the position corresponding to position 61 of the amino acid sequence of SEQ ID NO: 1 is glutamic acid, the amino acid at the position corresponding to position 156 of the amino acid sequence of SEQ ID NO: 1 is asparagine, the amino acid at the position corresponding to position 165 of the amino acid sequence of SEQ ID NO: 1 is serine, and the amino acid at the position corresponding to position 189 of the amino acid sequence of SEQ ID NO: 1 is asparagine; (20) The amino acid at the position corresponding to position 61 of the amino acid sequence of SEQ ID NO: 1 is glutamic acid, the amino acid at the position corresponding to position 165 of the amino acid sequence of SEQ ID NO: 1 is serine, the amino acid at the position corresponding to position 189 of the amino acid sequence of SEQ ID NO: 1 is asparagine, and the amino acid at the position corresponding to position 661 of the amino acid sequence of SEQ ID NO: 1 is asparagine; (21) The amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is proline, the amino acid at the position corresponding to the 156th position in the amino acid sequence of SEQ ID NO: 1 is asparagine, the amino acid at the position corresponding to the 165th position in the amino acid sequence of SEQ ID NO: 1 is serine, and the amino acid at the position corresponding to the 189th position in the amino acid sequence of SEQ ID NO: 1 is asparagine;(22) The amino acid at the position corresponding to position 61 of the amino acid sequence of SEQ ID NO: 1 is proline, the amino acid at the position corresponding to position 165 of the amino acid sequence of SEQ ID NO: 1 is serine, the amino acid at the position corresponding to position 189 of the amino acid sequence of SEQ ID NO: 1 is asparagine, and the amino acid at the position corresponding to position 661 of the amino acid sequence of SEQ ID NO: 1 is asparagine; (23) The amino acid at the position corresponding to position 61 of the amino acid sequence of SEQ ID NO: 1 is glutamic acid, the amino acid at the position corresponding to position 156 of the amino acid sequence of SEQ ID NO: 1 is asparagine, the amino acid at the position corresponding to position 165 of the amino acid sequence of SEQ ID NO: 1 is serine, the amino acid at the position corresponding to position 189 of the amino acid sequence of SEQ ID NO: 1 is asparagine, and the amino acid at the position corresponding to position 661 of the amino acid sequence of SEQ ID NO: 1 is asparagine; or (24) The RNA polymerase according to Item 1 or 2, having an amino acid sequence in which the amino acid at position 61 in the amino acid sequence of SEQ ID NO: 1 is proline, the amino acid at position 156 in the amino acid sequence of SEQ ID NO: 1 is asparagine, the amino acid at position 165 in the amino acid sequence of SEQ ID NO: 1 is serine, the amino acid at position 189 in the amino acid sequence of SEQ ID NO: 1 is asparagine, and the amino acid at position 661 in the amino acid sequence of SEQ ID NO: 1 is asparagine. [Item 4] A polynucleotide encoding the RNA polymerase according to any one of Items 1 to 3. [Item 5] A vector comprising the polynucleotide according to Item 4. [Item 6] A recombinant host cell transformed with the vector according to Item 5. [Item 7] A method for producing an RNA polymerase using the polynucleotide according to Item 4, the vector according to Item 5, and / or the recombinant host cell according to Item 6. [Item 8] A reagent comprising the RNA polymerase according to any one of Items 1 to 3, the polynucleotide according to Item 4, the vector according to Item 5, and / or the recombinant host cell according to Item 6. [Item 9] A method for synthesizing RNA, comprising allowing the RNA polymerase according to any one of Items 1 to 3 to act on template DNA. [Item 10]The method of Aspect 9, wherein the proportion of dsRNA in the synthesized RNA is less than 90% of the proportion of dsRNA in RNA synthesized using a corresponding wild-type RNA polymerase under the same conditions. [Aspect 11] The method of Aspect 9 or Aspect 10, wherein the amount of synthesized RNA is 110% or more of the amount of RNA synthesized using a corresponding wild-type RNA polymerase under the same conditions. [Aspect 12] The method of any of Aspects 9 to 11, wherein the RNA synthesis is mRNA synthesis. [Aspect 13] The method of Aspect 12, wherein the proportion of dsRNA in the synthesized mRNA is less than 90% of the proportion of dsRNA in mRNA synthesized using a corresponding wild-type RNA polymerase under the same conditions. [Aspect 14] The method of Aspect 12 or Aspect 13, wherein the amount of synthesized mRNA is 110% or more of the amount of mRNA synthesized using a corresponding wild-type RNA polymerase under the same conditions. [Aspect 15] The method of any of Aspects 9 to 11, wherein the RNA synthesis is non-coding RNA synthesis. [Item 16] The method according to Item 15, wherein the non-coding RNA is at least one selected from the group consisting of microRNA, siRNA, piRNA, rRNA, tRNA, snRNA, snoRNA, SLRNA, SRPRNA, mRNA-like non-coding RNA, and oligonucleotide. [Item 17] A method for synthesizing guide RNA for gene editing using the RNA polymerase according to any one of Items 1 to 3. [Item 18] A method for synthesizing an RNA drug using the RNA polymerase according to any one of Items 1 to 3. [Item 19] The method according to Item 9, wherein the RNA synthesis is carried out by an isothermal amplification reaction. [Item 20] A method for genetic testing using the RNA polymerase according to any one of Items 1 to 3. [Item 21] A method for in vivo protein expression or in vitro cell-free protein synthesis using the RNA polymerase according to any one of Items 1 to 3.

[0009] According to the present invention, a novel modified RNA polymerase can be provided.

[0010] The present invention will be described in further detail below while illustrating 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, the term "to" in this specification means "at least or equal to, at most or equal to", for example, "X to Y" in this specification means "at least X and at most Y". In this specification, "and / or" means any one or any possible combination of two or more of the listed elements. In this specification, "comprising" encompasses the concepts of "consisting essentially of" and "consisting only of".

[0011] In one aspect, the present invention provides a modified RNA polymerase. As used herein, "modification" refers to a mutation of an amino acid residue, i.e., a substitution, deletion, insertion, or addition. A modified RNA polymerase (hereinafter also referred to as a "modified RNA polymerase") refers to an RNA polymerase in which at least one amino acid residue in the amino acid sequence of a wild-type RNA polymerase has been mutated, i.e., substituted, deleted, inserted, or added.

[0012] As used herein, "wild-type RNA polymerase" (hereinafter also referred to as "wild-type" or "WT") refers to an RNA polymerase into which no mutation has been artificially introduced. 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 wild-type RNA polymerase is, for example, a wild-type T7 RNA polymerase consisting of the amino acid sequence of SEQ ID NO: 1. As is well known to those skilled in the art, T7 RNA polymerase refers to an RNA polymerase derived from T7 phage.

[0013] The RNA polymerase of the present invention may have an improved specific activity compared to the corresponding wild-type RNA polymerase. Here, "specific activity" refers to the enzymatic activity per unit protein weight when the enzymatic activity is measured at a temperature of, for example, 20°C to 100°C. Furthermore, "corresponding wild-type RNA polymerase" refers to the wild-type of an RNA polymerase of the same type as a certain modified RNA polymerase. For example, the "corresponding wild-type RNA polymerase" for a modified T7 RNA polymerase is the wild-type T7 RNA polymerase. Furthermore, for example, the "corresponding wild-type RNA polymerase" for a modified T3 RNA polymerase is the wild-type T3 RNA polymerase.

[0014] The RNA polymerase of the present invention has an amino acid sequence different from that of conventionally known wild-type RNA polymerases. Therefore, herein, the RNA polymerase of the present invention may be referred to as a mutant RNA polymerase or a modified RNA polymerase. As used herein, "mutant" and "modified" when referring to a "mutant RNA polymerase" or a "modified RNA polymerase" are used interchangeably and refer to an amino acid sequence different from that of conventionally known wild-type RNA polymerases, without distinguishing between an artificial mutation and a natural mutation. Thus, the mutant RNA polymerase of the present invention is an RNA polymerase in which at least one amino acid in the amino acid sequence of SEQ ID NO: 1, which represents the wild-type T7 RNA polymerase sequence, has been modified, thereby having an amino acid sequence different from that of SEQ ID NO: 1, regardless of whether the mutant RNA polymerase is an RNA polymerase obtained by artificial mutation or a natural mutation.

[0015] In this specification, simplified symbols using alphabetical notation may be used for nucleotide sequences, amino acid sequences, and their individual components, but all follow the conventions in the fields of molecular biology and genetic engineering. Furthermore, in this specification, to simply indicate mutations in amino acid sequences, notations such as "A61E" are used. "A61E" indicates the substitution of alanine (A) at position 61 of the amino acid sequence with glutamic acid (E). Specifically, it indicates the type of amino acid residue before substitution, the position of the substituted amino acid in the amino acid sequence, and the type of amino acid residue after substitution. Furthermore, unless otherwise specified, SEQ ID NOs correspond to those listed in the sequence listing. Furthermore, in the case of multiple mutants, the above notations can be connected with " / " (for example, it can be expressed as A61E / D156N / N165S).

[0016] In one embodiment, the amino acid sequence of the mutant RNA polymerase may have a certain percentage of modifications in the amino acid sequence of SEQ ID NO: 1. In one embodiment, the amino acid sequence of the mutant RNA polymerase preferably has 90% or more identity to the amino acid sequence of SEQ ID NO: 1. The mutant RNA polymerase is not particularly limited as long as it does not lose its RNA polymerase activity. For example, it is composed of an amino acid sequence that has, for example, 90% or more identity to the amino acid sequence of SEQ ID NO: 1, preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. 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 an analysis tool that is commercially available or available via telecommunications lines (Internet), and one example is the homology algorithm BLAST (Basic local alignment search tool) of the National Center for Biotechnology Information (NCBI) available at http: / / www.ncbi.nlm.nih. It is possible to calculate the identity of amino acid sequences by using default (initial setting) parameters in .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 have been deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 1. Here, "one or more" is not particularly limited as long as the RNA polymerase activity is not lost, and may be, for example, 1 to 30, preferably 1 to 20, 1 to 10, 1 to 5, or 1 to 3. Such an amino acid sequence may be, for example, artificially produced by genetic engineering techniques, or may be the amino acid sequence of a naturally occurring protein.

[0017] In one embodiment, when the mutant RNA polymerase has a substitution in the amino acid sequence of SEQ ID NO: 1, the substitution is preferably between structurally and / or chemically similar amino acids (so-called conservative substitution). Examples of conservative 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 amino acids (V, L, I), and substitutions between amino acids with linear alkyl or hydrogen side chains (A, G).

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

[0019] The RNA polymerase of the present invention can be composed of an amino acid sequence containing the amino acid residues described above. Those skilled in the art can produce an RNA polymerase protein having a desired amino acid sequence by any genetic engineering technique known in the art, for example, by appropriately designing a base sequence encoding the desired amino acid sequence, incorporating the sequence into an expression vector, etc., and transforming the vector into a host cell to express the vector.

[0020] In a preferred embodiment, the RNA polymerase of the present invention has, for example, 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of SEQ ID NO: 1, and is, for example, an RNA polymerase in which the amino acid at the position corresponding to the 61st amino acid in the amino acid sequence of SEQ ID NO: 1 has been modified, preferably an RNA polymerase in which the amino acid at the position corresponding to the 61st amino acid has been substituted with another amino acid. The amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is alanine, and the amino acid that replaces the alanine residue at the position corresponding to the 61st position can be, for example, glycine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, aspartic acid or glutamic acid, preferably glutamic acid, aspartic acid, serine, threonine, glycine or proline, more preferably glutamic acid, aspartic acid, serine, glycine or proline.Without intending to be bound by any particular theory, it is believed that the amino acid mutation at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 contributes to reducing the proportion of dsRNA in the RNA produced by the transcription reaction using modified RNA polymerase.

[0021] As used herein, the phrase "a position corresponding to position 61 in the amino acid sequence of SEQ ID NO: 1" refers to the 61st position in the amino acid sequence of wild-type T7 RNA polymerase (SEQ ID NO: 1), or the position in the amino acid sequence of a mutant RNA polymerase corresponding to position 61 in the amino acid sequence of wild-type T7 RNA polymerase (SEQ ID NO: 1). The "position corresponding to position 61 in the amino acid sequence of SEQ ID NO: 1" in the amino acid sequence of a mutant RNA polymerase can be easily identified, for example, by comparing and aligning the amino acid sequence of the wild-type T7 RNA polymerase (SEQ ID NO: 1) with the amino acid sequence of the mutant RNA polymerase using any known means. For example, comparison and alignment of amino acid sequences can be performed using commercially available analytical tools or those available via telecommunications lines (Internet). For example, amino acid sequence alignment can be performed using the multiple alignment program Clustal Omega (https: / / www.ebi.ac.uk / jdispatcher / msa / clustalo) provided by the European Bioinformatics Institute (EMBL) with default (initial setting) parameters. In this specification, the same applies when a position corresponding to an amino acid at any position other than the 61st position in the amino acid sequence of SEQ ID NO: 1 is described.

[0022] In a preferred embodiment, the RNA polymerase of the present invention has, for example, 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of SEQ ID NO: 1, and is, for example, an RNA polymerase in which the amino acid at position 156 of the amino acid sequence of SEQ ID NO: 1 has been modified, preferably substituted with another amino acid. The amino acid at position 156 of the amino acid sequence of SEQ ID NO: 1 is aspartic acid, and examples of the amino acid substituting for 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 of the amino acid sequence of SEQ ID NO: 1 is modified to a basic amino acid or a neutral amino acid.Basic amino acids or neutral amino acids include, for example, asparagine, glycine, alanine, proline, isoleucine, leucine, valine, phenylalanine, tryptophan, tyrosine, glutamine, cysteine, methionine, serine, threonine, arginine, histidine, or lysine, and asparagine or glutamine is preferred, and asparagine is more preferred.Without intending to be bound by any particular theory, it is believed that the amino acid mutation at position 156 of the amino acid sequence of SEQ ID NO: 1 contributes to reducing the proportion of dsRNA in the RNA produced by transcription reaction using modified RNA polymerase.

[0023] In a preferred embodiment, the RNA polymerase of the present invention has, for example, 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of SEQ ID NO: 1, and is, for example, an RNA polymerase in which the amino acid at the position corresponding to position 165 of the amino acid sequence of SEQ ID NO: 1 has been modified, preferably substituted with another amino acid. The amino acid at position 165 of the amino acid sequence of SEQ ID NO: 1 is asparagine, and examples of the amino acid substituting 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, glutamic acid, arginine, and proline being preferred, and serine being more preferred. Without intending to be bound by any particular theory, it is believed that the amino acid mutation at the position corresponding to amino acid 165 of the amino acid sequence of SEQ ID NO: 1 contributes to reducing the proportion of dsRNA in the RNA produced by a transcription reaction using the modified RNA polymerase.

[0024] In a preferred embodiment, the RNA polymerase of the present invention has, for example, 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of SEQ ID NO: 1, and is, for example, an RNA polymerase in which the amino acid at the position corresponding to position 189 of the amino acid sequence of SEQ ID NO: 1 has been modified, preferably substituted with another amino acid. The amino acid at position 189 of the amino acid sequence of SEQ ID NO: 1 is aspartic acid, and examples of amino acids substituting 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 is preferably substituted with a basic amino acid or a neutral amino acid at the amino acid position corresponding to the 189th amino acid in the amino acid sequence of SEQ ID NO: 1, and examples of basic amino acid or a neutral amino acid include glycine, alanine, proline, isoleucine, leucine, valine, phenylalanine, tryptophan, tyrosine, asparagine, glutamine, cysteine, methionine, serine, threonine, arginine, histidine, or lysine, and is preferably substituted with asparagine or glutamine, and more preferably substituted with asparagine.Without intending to be bound by any particular theory, it is believed that the amino acid mutation at the position corresponding to the 189th amino acid in the amino acid sequence of SEQ ID NO: 1 contributes to reducing the proportion of dsRNA in the RNA produced by the transcription reaction using modified RNA polymerase.

[0025] In a preferred embodiment, the RNA polymerase of the present invention has, for example, 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of SEQ ID NO: 1, and is, for example, an RNA polymerase in which the amino acid at the position corresponding to position 661 of the amino acid sequence of SEQ ID NO: 1 has been modified, preferably substituted with another amino acid. The amino acid at position 661 of the amino acid sequence of SEQ ID NO: 1 is serine, and examples of the amino acid substituting for serine at position 661 of the amino acid sequence of SEQ ID NO: 1 include glycine, alanine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, threonine, arginine, histidine, lysine, aspartic acid, and glutamic acid. Preferably, the substitution is with asparagine or glutamine, and more preferably with asparagine. Without intending to be bound by any particular theory, it is believed that the amino acid mutation at the position corresponding to amino acid 661 of the amino acid sequence of SEQ ID NO: 1 contributes to reducing the proportion of dsRNA in the RNA produced by a transcription reaction using the modified RNA polymerase.

[0026] In a preferred embodiment, the RNA polymerase of the present invention has, for example, 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of SEQ ID NO: 1, and is an RNA polymerase in which at least one amino acid selected from the group consisting of amino acids at positions corresponding to 61, 156, 189, 165, and 661 of the amino acid sequence of SEQ ID NO: 1 has been modified.

[0027] In a preferred embodiment, the RNA polymerase of the present invention may be an RNA polymerase that has, for example, 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of SEQ ID NO: 1, and has an amino acid sequence that satisfies at least one of the following (i) to (v): (i) the amino acid at position 61 of the amino acid sequence of SEQ ID NO: 1 is glutamic acid, aspartic acid, serine, glycine, or proline; (ii) the amino acid at position 156 of the amino acid sequence of SEQ ID NO: 1 is asparagine; (iii) the amino acid at position 189 of the amino acid sequence of SEQ ID NO: 1 is asparagine; (iv) the amino acid at position 165 of the amino acid sequence of SEQ ID NO: 1 is serine, glutamic acid, arginine, or proline; and (v) the amino acid at position 661 of the amino acid sequence of SEQ ID NO: 1 is asparagine.

[0028] In a preferred embodiment, the RNA polymerase of the present invention has, for example, 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of SEQ ID NO: 1, and is, for example, (1) the amino acid at position 61 of the amino acid sequence of SEQ ID NO: 1 is glutamic acid, aspartic acid, serine, glycine, or proline; (2) the amino acid at position 156 of the amino acid sequence of SEQ ID NO: 1 is asparagine; (3) the amino acid at position 189 of the amino acid sequence of SEQ ID NO: 1 is asparagine; (4) the amino acid at position 165 of the amino acid sequence of SEQ ID NO: 1 is serine, glutamic acid, arginine, or proline; (5) the amino acid at position 661 of the amino acid sequence of SEQ ID NO: 1 is asparagine; (6) the amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is glutamic acid, and the amino acid at the position corresponding to the 156th position in the amino acid sequence of SEQ ID NO: 1 is asparagine; (7) the amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is glutamic acid, and the amino acid at the position corresponding to the 165th position in the amino acid sequence of SEQ ID NO: 1 is serine; (8) the amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is glutamic acid, and the amino acid at the position corresponding to the 189th position in the amino acid sequence of SEQ ID NO: 1 is asparagine; (9) the amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is glutamic acid, and the amino acid at the position corresponding to the 661st position in the amino acid sequence of SEQ ID NO: 1 is asparagine; (10) the amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is proline, and the amino acid at the position corresponding to the 165th position in the amino acid sequence of SEQ ID NO: 1 is serine; (11) The amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is proline, and the amino acid at the position corresponding to the 189th position in the amino acid sequence of SEQ ID NO: 1 is asparagine; (12) The amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is proline, and the amino acid at the position corresponding to the 661st position in the amino acid sequence of SEQ ID NO: 1 is asparagine;(13) The amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is glutamic acid, the amino acid at the position corresponding to the 156th position in the amino acid sequence of SEQ ID NO: 1 is asparagine, and the amino acid at the position corresponding to the 165th position in the amino acid sequence of SEQ ID NO: 1 is serine; (14) The amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is glutamic acid, the amino acid at the position corresponding to the 165th position in the amino acid sequence of SEQ ID NO: 1 is serine, and the amino acid at the position corresponding to the 189th position in the amino acid sequence of SEQ ID NO: 1 is asparagine; (15) The amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is glutamic acid, the amino acid at the position corresponding to the 165th position in the amino acid sequence of SEQ ID NO: 1 is serine, and the amino acid at the position corresponding to the 661st position in the amino acid sequence of SEQ ID NO: 1 is asparagine; (16) The amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is proline, the amino acid at the position corresponding to the 156th position in the amino acid sequence of SEQ ID NO: 1 is asparagine, and the amino acid at the position corresponding to the 165th position in the amino acid sequence of SEQ ID NO: 1 is serine; (17) The amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is proline, the amino acid at the position corresponding to the 165th position in the amino acid sequence of SEQ ID NO: 1 is serine, and the amino acid at the position corresponding to the 189th position in the amino acid sequence of SEQ ID NO: 1 is asparagine; (18) The amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is proline, the amino acid at the position corresponding to the 165th position in the amino acid sequence of SEQ ID NO: 1 is serine, and the amino acid at the position corresponding to the 661st position in the amino acid sequence of SEQ ID NO: 1 is asparagine; (19) The amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is glutamic acid, the amino acid at the position corresponding to the 156th position in the amino acid sequence of SEQ ID NO: 1 is asparagine, the amino acid at the position corresponding to the 165th position in the amino acid sequence of SEQ ID NO: 1 is serine, and the amino acid at the position corresponding to the 189th position in the amino acid sequence of SEQ ID NO: 1 is asparagine;(20) The amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is glutamic acid, the amino acid at the position corresponding to the 165th position in the amino acid sequence of SEQ ID NO: 1 is serine, the amino acid at the position corresponding to the 189th position in the amino acid sequence of SEQ ID NO: 1 is asparagine, and the amino acid at the position corresponding to the 661st position in the amino acid sequence of SEQ ID NO: 1 is asparagine; (21) The amino acid at the position corresponding to the 61st position in the amino acid sequence of SEQ ID NO: 1 is proline, the amino acid at the position corresponding to the 156th position in the amino acid sequence of SEQ ID NO: 1 is asparagine, the amino acid at the position corresponding to the 165th position in the amino acid sequence of SEQ ID NO: 1 is serine, and the amino acid at the position corresponding to the 189th position in the amino acid sequence of SEQ ID NO: 1 is asparagine; (22) The amino acid at the position corresponding to position 61 of the amino acid sequence of SEQ ID NO: 1 is proline, the amino acid at the position corresponding to position 165 of the amino acid sequence of SEQ ID NO: 1 is serine, the amino acid at the position corresponding to position 189 of the amino acid sequence of SEQ ID NO: 1 is asparagine, and the amino acid at the position corresponding to position 661 of the amino acid sequence of SEQ ID NO: 1 is asparagine; (23) The amino acid at the position corresponding to position 61 of the amino acid sequence of SEQ ID NO: 1 is glutamic acid, the amino acid at the position corresponding to position 156 of the amino acid sequence of SEQ ID NO: 1 is asparagine, the amino acid at the position corresponding to position 165 of the amino acid sequence of SEQ ID NO: 1 is serine, the amino acid at the position corresponding to position 189 of the amino acid sequence of SEQ ID NO: 1 is asparagine, and the amino acid at the position corresponding to position 661 of the amino acid sequence of SEQ ID NO: 1 is asparagine;or (24) an RNA polymerase having an amino acid sequence in which the amino acid at position 61 of the amino acid sequence of SEQ ID NO: 1 is proline, the amino acid at position 156 of the amino acid sequence of SEQ ID NO: 1 is asparagine, the amino acid at position 165 of the amino acid sequence of SEQ ID NO: 1 is serine, the amino acid at position 189 of the amino acid sequence of SEQ ID NO: 1 is asparagine, and the amino acid at position 661 of the amino acid sequence of SEQ ID NO: 1 is asparagine, preferably (10) an RNA polymerase having an amino acid sequence in which the amino acid at position 61 of the amino acid sequence of SEQ ID NO: 1 is proline, and the amino acid at position 165 of the amino acid sequence of SEQ ID NO: 1 is serine;

[0029] In one embodiment, the RNA polymerase of the present invention is characterized by being able to reduce the generation of dsRNA in transcription reaction.Compared with corresponding wild-type RNA polymerase, the RNA polymerase of the present invention can be the RNA polymerase in which the proportion of dsRNA in the RNA after transcription reaction is about 90% or less, preferably about 80% or less, about 70% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, or about 10% or less.

[0030] [Method for measuring the proportion of dsRNA in RNA after transcription reaction] The proportion of dsRNA in RNA after transcription reaction can be specifically confirmed by the following measurement method: 1 μL of 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 MgCl ２, 5 mM DTT, 10 ng / μL template DNA, 0.4 mM ATP, 0.4 mM CTP, 0.4 mM GTP, 0.4 mM UTP), and 0.4 U / μL RNase inhibitor) and then incubated in a heat block at 37°C for 1 hour. The template DNA used is dsDNA containing, in this order from the 5' end, a promoter corresponding to the RNA polymerase to be measured (one of the T7 promoter, T3 promoter, SP6 promoter, etc.), a 5'UTR, a 3'UTR, and poly(A) (100 nt), and the firefly luciferase gene (Fluc) as a coding sequence (CDS) between the 5'UTR and 3'UTR. After the reaction, 2.5 μL of Turbo (registered trademark) DNase (2 U / μL) (manufactured by Thermo Fisher Scientific) is added, and the reaction is continued at 37 ° C for 15 minutes. This reaction solution is purified using Monarch (registered trademark) RNA Cleanup Kit (manufactured by 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 the RNA concentration is 40 ng / μL when A260 is 1. A calibration curve is created from the measured values ​​obtained by performing ELISA on the attached dsRNA control using a double-stranded RNA (dsRNA) ELISA kit (K1 based) (Exalpha Biologicals). The purified RNA was diluted to 100 ng / μL and further diluted to multiple concentrations, followed by ELISA using a double-stranded RNA (dsRNA) ELISA kit (K1 based). The dsRNA concentration was calculated from the calibration curve obtained using the values ​​within the calibration curve. Furthermore, the dsRNA concentration was divided by the RNA concentration to calculate the percentage (w / w) of dsRNA in the RNA.

[0031] In one embodiment, the RNA polymerase of the present invention may have a high specific activity. Compared to the corresponding wild-type RNA polymerase, the RNA polymerase of the present invention may have a specific activity that is 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. Specifically, the specific activity can be confirmed by the following measurement method.

[0032] [Method for measuring activity] The activity of RNA polymerase can be measured by the following procedure. Measurement conditions can be set appropriately by those skilled in the art. For example, if the enzyme activity is too high, the sample containing the target to be measured can be appropriately diluted before measurement. First, 45 μL of reaction solution (the final concentration in 50 μL of reaction solution after adding the enzyme is shown below) (40 mM Tris-HCl (pH 8.0), 50 mM NaCl, 8 mM MgCl ２ , 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, followed by the addition of 5 μL of wild-type RNA polymerase with known activity, and the reaction was carried out in a heat block at 37°C for 10 minutes. After 10 minutes, the reaction was stopped by adding 10 μL of 1 M EDTA. This reaction was carried out using a dilution series of the RNA polymerase. The amount of RNA in each reaction solution was then quantified using a Qubit® RNA BR Assay Kit (manufactured by Thermo Fisher Scientific), and a calibration curve was created using the activity value of the RNA polymerase added in each dilution series and the quantified RNA amount. The same reaction was carried out for the RNA polymerase to be measured, and the activity was calculated using the calibration curve created above based on the obtained RNA amount.

[0033] [Measurement of specific activity] The absorbance at 280 nm (A280) is measured, and the enzyme concentration is calculated assuming that the enzyme concentration is 1 mg / mL when A280 is 1. The specific activity is calculated by dividing the activity value calculated as above by the enzyme concentration.

[0034] In one embodiment, the RNA polymerase of the present invention may exhibit higher thermostability than the corresponding wild-type RNA polymerase. In a preferred embodiment, the RNA polymerase of the present invention may be an RNA polymerase that exhibits a residual activity of more than 50% when heat-treated at 50°C for 5 minutes, for example, an RNA polymerase that exhibits a residual activity rate of 60% or more, an RNA polymerase that exhibits a residual activity rate of 70% or more, or an RNA polymerase that exhibits a residual activity rate of 80% or more. In a specific embodiment, the RNA polymerase of the present invention may be an RNA polymerase that exhibits a residual activity rate of 90% or more when heat-treated at 50°C for 5 minutes.

[0035] From another perspective, the RNA polymerase of the present invention may be a mutant RNA polymerase that, for example, when heat-treated under predetermined conditions, exhibits a higher residual activity rate than that of the corresponding wild-type RNA polymerase. Specifically, for example, when heat-treated at 50° C. for 5 minutes, the mutant RNA polymerase may exhibit a residual activity rate that is at least about 1.1 times, preferably at least about 1.2 times, at least about 1.3 times, at least about 1.4 times, at least about 1.5 times, at least about 1.6 times, at least about 1.7 times, at least about 1.8 times, or at least about 1.9 times higher than that of the corresponding wild-type RNA polymerase.

[0036] [Measurement of Thermal Stability (Residual Activity Rate After Heat Treatment)] Thermal stability can be specifically confirmed by the following measurement method. First, each mutant RNA polymerase to be measured is diluted to 50 U / μL with a storage buffer (20 mM KPO4 (pH 7.7), 100 mM NaCl, 50% glycerol, 0.1 mM EDTA, 5 mM DTT, 0.01% Triton (registered trademark) X-100), and the 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 with the storage buffer is stored in an incubator at 50°C for 5 minutes. After storage, the activity value after storage is measured according to the procedure described in the activity measurement method, similar to that before storage. Next, the residual activity rate can be calculated by dividing the activity value after storage by the activity value before storage, as shown in the following formula I. Residual activity rate (%) = (activity value after storage / activity value before storage) × 100 (Equation I)

[0037] In a further embodiment, the present invention provides a polynucleotide encoding the RNA polymerase of the present invention. Such polynucleotides can be suitably used for expressing the RNA polymerase of the present invention and / or for constructing expression vectors and / or for constructing transformed cells. Here, a polynucleotide encoding an RNA polymerase refers to a polynucleotide that, when expressed by conventional methods, yields the RNA polymerase protein of the present invention. That is, it refers to a polynucleotide composed of a nucleotide 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 nucleotide sequence corresponding to a given amino acid sequence using codon tables well known in the art. Furthermore, polynucleotides encoding the RNA polymerase of the present invention also include 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. Examples of methods for synthesizing DNA by artificial gene synthesis include, but are not limited to, chemical synthesis of two or more 10-100 nt oligo-DNAs having partially overlapping sequences, ligating them by PCA (polymerase chain assembly) or other enzymatic methods, designing primers at both ends of the final target sequence, and performing PCR to obtain double-stranded DNA. When synthesizing RNA by artificial gene synthesis, for example, during the synthesis of the double-stranded DNA, double-stranded DNA is synthesized in such a way that it contains the sequence of an arbitrary promoter (T7 promoter, T3 promoter, SP6 promoter, etc.) at the 5' end, and RNA can be synthesized by performing an in vitro transcription reaction using the DNA as a template with an RNA polymerase that specifically recognizes the arbitrary promoter (T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, etc.).

[0038] In a further embodiment, the present invention provides a vector containing the polynucleotide. Such a vector can be suitably used for expressing the RNA polymerase of the present invention and / or creating transformed cells. The vector can be created by introducing a polynucleotide encoding the RNA polymerase into a vector (e.g., an expression vector, a cloning vector, etc.). Introduction of the polynucleotide into the vector can be performed by any method, including restriction enzyme digestion and ligation, or seamless cloning such as Gibson assembly. In certain embodiments, the vector of the present invention may further contain elements that function in host cells, such as a promoter, terminator, ribosome binding site, and drug resistance gene. Examples of drug resistance genes include resistance genes for drugs such as ampicillin, kanamycin, and tetracycline. In certain embodiments, the vector of the present invention may contain elements that enable homologous recombination with the genome of the host cell. The vector may be any type that enables cloning and / or expression of the RNA polymerase of the present invention, such as a plasmid. Examples of plasmids include, but are not limited to, pUC118, pUC18, pBR322, pBluescript, pLED-M1, p73, pGW7, and pkk223-3.

[0039] In a further embodiment, the present invention provides a cell transformed with the vector. Such a cell can be suitably used to express a protein encoding the RNA polymerase of the present invention. In a particularly preferred embodiment, the recombinant host cell of the present invention is obtained by transforming a host cell with the expression vector. Examples of the host cell include Escherichia coli and yeast, with Escherichia coli being particularly preferred. Examples of Escherichia coli include Escherichia coli DH5α, JM109, HB101, XL1Blue, PR1, and BL21. That is, in the present invention, it is preferable to insert a gene encoding the RNA polymerase into the vector to form an expression vector, and then transform a host cell with the expression vector.

[0040] In a further embodiment, a method for producing the RNA polymerase using the polynucleotide, the vector, the recombinant host cell, and / or one or more of these is also provided. In a specific embodiment, after transforming a host cell with the vector, the resulting recombinant host cell is cultured using any culture device. Examples of culture devices include, but are not limited to, test tubes, flasks, and jar fermenters. Examples of media used for culture include, but are not limited to, LB medium, 2xYT medium, and TB medium. Culture conditions can be set appropriately, with the culture temperature ranging from 10 to 40°C and the culture time ranging from 1 to 100 hours. Aeration and / or stirring may be performed during culture, and either batch culture or fed-batch culture may be used. After culture, the cells are recovered by centrifugation or the like, and a crude enzyme solution is extracted by disrupting or lysing the recovered cells. Any known method may be used to disrupt or lyse the cells. Examples of disruption methods include physical disruption methods such as sonication, French press, or glass bead disruption, and examples of lysis methods include, but are not limited to, methods using enzymes such as lysozyme. Any method may be used to obtain the purified RNA polymerase from the obtained crude enzyme solution, but the RNA polymerase of the present invention can be isolated by, for example, centrifugation, ultracentrifugation, ultrafiltration, nucleic acid removal treatment, salting out, dialysis, or various column chromatography methods (ion exchange column chromatography, hydrophobic chromatography, affinity chromatography, gel filtration column chromatography, etc.).

[0041] In further embodiments, a reagent containing the RNA polymerase, the polynucleotide, the vector, the recombinant host cell, and / or one or more of these is also provided. In certain embodiments, the reagent may be a liquid or a solid from which water has been removed by techniques such as lyophilization. In certain embodiments, the reagent may be contained in any container. In certain embodiments, the reagent may be divided into any number of bottles, one or more. In certain embodiments, the reagent may contain a substance that enables expression of the RNA polymerase from the polynucleotide, the vector, or 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 that use cell extracts and reconstituted cell-free protein synthesis systems in which factors involved in translation reactions are purified and mixed. Substances contained in cell-free protein synthesis reagents 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 that use cell extracts include, but are not limited to, Fusetsu-kun (registered trademark) from Taiyo Nippon Sanso Co., Ltd., and reagents derived from extracts of rabbit reticulocytes, wheat germ, insect cells, Escherichia coli, and human cells. Examples of reconstituted cell-free protein synthesis systems include, but are not limited to, PUREflex (registered trademark) 1.0 from Scene Frontier Co., Ltd. In certain embodiments, the reagents may contain substances necessary for in vitro transcription reactions.Substances necessary for in vitro transcription reactions include, but are not limited to, buffer solutions (Tris buffer, phosphate buffer, Good's buffer, etc.), ribonucleotides (ATP, CTP, GTP, UTP, modified bases (bases modified by deamination, methylation, methoxylation, pseudouridylation, etc.), cap analogs (ARCA, mCAP, CleanCap® Reagent AG, CleanCap® Reagent M6, CleanCap® Reagent AU (TriLink), etc.), RNase inhibitors, pyrophosphatase, salts such as NaCl, metal ions such as magnesium ions, EDTA, dithiothreitol, glycerol, surfactants, spermidine, linear or circular DNA, etc. In certain embodiments, the reagents may further contain substances necessary for post-transcriptional capping. Substances necessary for post-transcriptional capping include, but are not limited to, a capping enzyme and GTP, mRNA Cap 2'-O-methyltransferase, SAM (S-adenosylmethionine), a buffer solution (e.g., Tris buffer, phosphate buffer, Good's buffer), salts such as NaCl and KCl, metal ions such as magnesium ions, EDTA, dithiothreitol, glycerol, surfactants, etc. In certain embodiments, the reagent may further contain a substance 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, a buffer solution (e.g., Tris buffer, phosphate buffer, Good's buffer), salts such as NaCl, metal ions such as magnesium ions, manganese ions, EDTA, dithiothreitol, glycerol, surfactants, etc. In certain embodiments, the reagent may further contain a substance necessary for preparing template DNA for an in vitro transcription reaction. Substances necessary for preparing template DNA for in vitro transcription reaction include, but are not limited to, restriction enzymes, buffer solutions (Tris buffer, phosphate buffer, Good's buffer, etc.), salts such as NaCl, metal ions such as magnesium ions, EDTA, dithiothreitol, glycerol, surfactants, PCR enzymes, oligo-DNAs, etc.In certain embodiments, the reagent may further contain a substance necessary for purifying template DNA for in vitro transcription reactions. Examples of substances necessary for purifying template DNA include, but are not limited to, beads for DNA purification, columns for DNA purification, etc. In certain embodiments, the reagent may further contain a substance necessary for purifying RNA. Examples of substances necessary for purifying RNA include, but are not limited to, beads for RNA purification, columns for RNA purification, etc.

[0042] The present invention further provides a method for synthesizing RNA, comprising allowing the RNA polymerase to act on template DNA. RNA synthesis is carried out by an in vitro transcription reaction. Examples of in vitro transcription reactions include, but are not limited to, methods in which RNA is synthesized using the RNA polymerase and linear or circular DNA as a template. Examples of in vitro transcription reactions include, but are not limited to, methods in which RNA is synthesized using the RNA polymerase and linear or circular DNA as a template in the presence of a buffer solution and ribonucleotides. The concentration of RNA polymerase added during the reaction can be, for example, 0.1 U / μL to 20 U / μL, but is not particularly limited. The concentration of linear or circular DNA can be, for example, 1 ng / μL to 200 ng / μL, but is not particularly limited. Ribonucleotides used in the in vitro transcription reaction include, but are not limited to, ATP, CTP, GTP, UTP, and modified bases modified by deamination, methylation, methoxylation, pseudouridylation, etc. 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, for example, in the range of 0.1 mM to 20 mM, but is not particularly limited. Examples of buffers used in the in vitro transcription reaction include, but are not particularly limited to, Tris buffer, phosphate buffer, Good's buffer, etc. The concentration of the buffer can be, for example, in the range of 5 mM to 500 mM, but is not particularly limited. During the in vitro transcription reaction, RNase inhibitors, pyrophosphatase, etc. can be added, but are not particularly limited. The concentration of the RNase inhibitor may be, for example, in the range of 0.1 U / μL to 20 U / μL, but is not particularly limited.The concentration of pyrophosphatase may be, for example, within the range of 0.0001 U / μL to 0.02 U / μL, but is not particularly limited thereto. During the in vitro transcription reaction, salts such as NaCl and metal ions such as magnesium ions may be further added, but is not particularly limited thereto. The concentration of NaCl may be, for example, within the range of 1 mM to 200 mM, but is not particularly limited thereto. The concentration of magnesium ions may be, for example, within the range of 0.1 mM to 20 mM, but is not particularly limited thereto. During the in vitro transcription reaction, EDTA, dithiothreitol, glycerol, surfactants, spermidine, etc. may be further added, but is not particularly limited thereto. The concentration of EDTA may be, for example, within the range of 0.0001 mM to 2 mM, but is not particularly limited thereto. The concentration of dithiothreitol may be, for example, within the range of 0.1 mM to 20 mM, but is not particularly limited thereto. The concentration of glycerol may be, for example, within the range of 0.1% to 20%, but is not particularly limited thereto. The surfactant may be added at a concentration in the range of, for example, 0.0001% to 1%, but is not particularly limited thereto, and the spermidine may be added at a concentration in the range of, for example, 0.1 mM to 20 mM, but is not particularly limited thereto.

[0043] In addition, in a specific embodiment, the present invention further provides an RNA synthesis method, wherein the proportion of dsRNA in the RNA synthesized by the RNA synthesis method is less than 90% of the proportion of dsRNA in the RNA synthesized by the corresponding wild-type RNA polymerase under the same conditions.The proportion can be preferably 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 25% or less, 20% or less, 15% or less, 13% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% 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 RNA synthesis under the same conditions using the corresponding wild-type RNA polymerase and each mutant RNA polymerase to be measured, calculating the proportion of dsRNA in RNA, and then comparing the values.

[0044] In a specific embodiment, the present invention further provides an RNA synthesis method in which the amount of RNA synthesized by the RNA synthesis method (RNA yield) is at least 110% or more of the amount of mRNA synthesized using a corresponding wild-type RNA polymerase under the same conditions. This ratio can preferably be 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, or 190% or more. The ratio can be calculated, for example, by the method described in "Method for measuring the proportion of dsRNA in RNA after transcription reaction," by performing the steps up to measuring the concentration of purified RNA using the corresponding wild-type RNA polymerase and each mutant RNA polymerase to be measured under the same conditions, calculating the RNA yield by multiplying the liquid volume of purified RNA by the RNA concentration, and then comparing the RNA yields.

[0045] In a specific embodiment, the present invention also provides a method for synthesizing mRNA using the RNA polymerase. That is, in the RNA synthesis method, the RNA synthesis may be mRNA synthesis. Examples of mRNA synthesis methods include, but are not limited to, methods in which linear or circular DNA is used as a template using the RNA polymerase in the presence of a buffer solution and ribonucleotides. The concentration of the RNA polymerase added during the reaction can be, for example, 0.1 U / μL to 20 U / μL, but is not particularly limited. The concentration of the linear or circular DNA can be, for example, 1 ng / μL to 200 ng / μL, but is not particularly limited. Ribonucleotides used in mRNA synthesis include, but are not limited to, ATP, CTP, GTP, UTP, and modified bases such as those modified by deamination, methylation, methoxylation, and pseudouridination. The concentration of the ribonucleotides added can be, for example, 0.1 mM to 20 mM, but is not particularly limited. Buffers used for mRNA synthesis include, but are not limited to, Tris buffer, phosphate buffer, Good's buffer, etc. The buffer concentration can be, for example, within the range of 5 mM to 500 mM, but is not particularly limited. During mRNA synthesis, RNase inhibitors, pyrophosphatase, etc. can also be added, but are not particularly limited. The RNase inhibitor concentration can be, for example, within the range of 0.1 U / μL to 20 U / μL, but is not particularly limited. The pyrophosphatase concentration can be, for example, within the range of 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 also be added, but are not particularly limited. The NaCl concentration can be, for example, within the range of 1 mM to 200 mM, but is not particularly limited. The magnesium ion concentration can be, for example, within the range of 0.1 mM to 20 mM, but is not particularly limited. During mRNA synthesis, EDTA, dithiothreitol, glycerol, a surfactant, spermidine, etc. may be further added, but are not particularly limited thereto.The concentration of EDTA can be, for example, in the range of 0.0001 mM to 2 mM, but is not particularly limited. The concentration of dithiothreitol can be, for example, in the range of 0.1 mM to 20 mM, but is not particularly limited. The concentration of glycerol can be, for example, in the range of 0.1% to 20%, but is not particularly limited. The concentration of surfactant can be, for example, in the range of 0.0001% to 1%, but is not particularly limited. The concentration of spermidine can be, for example, in the range of 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 limited to, post-transcriptional capping and co-transcriptional capping. Examples of post-transcriptional capping include, but are not limited to, methods in which Cap-0 is added to the 5' end of mRNA using a capping enzyme, GTP, and SAM, and then, if necessary, converting Cap-0 to Cap-1 using mRNA Cap 2'-O-methyltransferase and SAM. Examples of co-transcriptional capping include, but are not limited to, methods using cap analogs such as ARCA, mCAP, CleanCap (registered trademark) Reagent AG, CleanCap (registered trademark) Reagent M6, and CleanCap (registered trademark) 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 particularly limited. When poly A is present at the 3' end of mRNA but the template DNA does not contain poly A, methods for adding poly A to the 3' end of synthesized RNA include, but are not particularly limited to, methods using poly A polymerase.

[0046] In a specific embodiment, the present invention further provides an mRNA synthesis method in which the proportion of dsRNA in the mRNA synthesized by the mRNA synthesis method is less than 90% of the proportion of dsRNA in the mRNA synthesized using a corresponding wild-type RNA polymerase under the same conditions. The proportion can 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 synthesizing mRNA using the corresponding wild-type RNA polymerase and each mutant RNA polymerase to be measured under the same conditions, calculating the proportion of dsRNA in the mRNA, and then comparing the values.

[0047] In a specific embodiment, the present invention further provides an mRNA synthesis method in which the amount of mRNA synthesized by the mRNA synthesis method (mRNA yield) is at least 110% or more of the amount of mRNA synthesized using a corresponding wild-type RNA polymerase under the same conditions. This ratio can preferably be 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, or 190% or more. The ratio can be calculated, for example, by the method described in "Method for measuring the proportion of dsRNA in RNA after transcription reaction," by performing the steps up to measuring the concentration of purified mRNA using the corresponding wild-type RNA polymerase and each mutant RNA polymerase to be measured under the same conditions, calculating the mRNA yield by multiplying the volume of purified mRNA by the mRNA concentration, and then comparing the mRNA yields.

[0048] In a specific embodiment, the present invention also provides a method for synthesizing non-coding RNA using the RNA polymerase. That is, in the RNA synthesis method, the RNA synthesis may be non-coding RNA synthesis. Examples of non-coding RNA include, but are not limited to, microRNA, siRNA, piRNA, rRNA, tRNA, snRNA, snoRNA, SLRNA, SRPRNA, mRNA-like non-coding RNA, and oligonucleotides. Examples of methods for synthesizing non-coding RNA include, but are not limited to, RNA synthesis using a template DNA containing DNA corresponding to the non-coding RNA of interest, for example, by the method described above. The template DNA may be any DNA as long as it contains DNA corresponding to the non-coding RNA of interest, but preferably contains a promoter sequence (e.g., T7 promoter, T3 promoter, SP6 promoter) recognized by the RNA polymerase of the present invention upstream of the corresponding DNA. The template DNA may be prepared by any method, including, but not limited to, methods such as restriction enzyme treatment of a plasmid and methods using DNA synthesized by artificial gene synthesis. The plasmid may be any one containing DNA corresponding to the non-coding RNA of interest, but may also contain the promoter sequence, restriction enzyme site, drug resistance gene, etc. described above. One example of a method for obtaining the plasmid is to artificially synthesize DNA corresponding to the non-coding RNA of interest, and then clone it into any plasmid containing a promoter sequence (T7 promoter, T3 promoter, SP6 promoter, etc.) recognized by the RNA polymerase of the present invention upstream of the site where the DNA is to be inserted, a restriction enzyme site downstream, and, if necessary, a drug resistance gene or the like at any position. The plasmid is then transformed into, for example, any host, preferably Escherichia coli, by any method, and the resulting recombinant host cells are cultured by any method, followed by extraction of the plasmid. The resulting plasmid can be linearized by restriction enzyme treatment with any restriction enzyme that can utilize the restriction enzyme site contained in the plasmid, and then purified by any method to obtain template DNA.Examples of methods using DNA synthesized by artificial gene synthesis include, but are not limited to, artificial gene synthesis of double-stranded DNA in a form that includes DNA corresponding to the non-coding RNA of interest and a promoter sequence (T7 promoter, T3 promoter, SP6 promoter, etc.) upstream thereof that is recognized by the RNA polymerase of the present invention, and further amplifying the DNA by PCR or the like as needed, and further purifying the resulting DNA as needed, and using the resulting DNA as template DNA.

[0049] In a specific embodiment, the present invention further provides a method for synthesizing guide RNA for gene editing using the RNA polymerase. Examples of gene editing methods include, but are not limited to, the CRISPER / Cas9 system.

[0050] In a specific embodiment, the present invention also provides a method for using the RNA polymerase in the synthesis of an RNA drug. Examples of RNA drugs include, but are not limited to, those using mRNA, siRNA, microRNA, antisense RNA, and aptamers. One example of a method for using the RNA polymerase in the synthesis of an RNA drug includes, but is not limited to, an in vitro transcription reaction using the RNA polymerase and a template DNA containing DNA corresponding to any RNA used in the RNA drug (e.g., mRNA, siRNA, microRNA, antisense RNA, aptamer), for example, by the method described above. The template DNA containing DNA corresponding to any RNA (e.g., mRNA, siRNA, microRNA, antisense RNA, aptamer) may be any DNA containing DNA corresponding to any RNA. The template DNA may include, upstream of the DNA, a promoter sequence recognized by the RNA polymerase of the present invention (e.g., T7 promoter, T3 promoter, SP6 promoter), and, if necessary, a 5'UTR upstream and a 3'UTR, polyA, restriction enzyme site, etc. downstream. The template DNA may be prepared by any method known to those skilled in the art, and for example, the method described above for the synthesis of non-coding RNA can be used with appropriate modifications.

[0051] In a specific embodiment, the present invention further provides a method for using the RNA polymerase in an isothermal amplification reaction, including, but not limited to, the NASBA method and the TMA method.

[0052] In a specific embodiment, the present invention also provides a method for genetic testing using the RNA polymerase, including, but not limited to, a method for confirming the presence or absence of a pathogen by amplifying and detecting the gene of the pathogen of interest using the isothermal amplification reaction or the like.

[0053] In certain embodiments, the present invention also provides a method for in vivo protein expression or in vitro cell-free protein synthesis using the RNA polymerase. An example of a method for in vivo protein expression using the RNA polymerase includes, but is not limited to, transfecting any cell with mRNA synthesized using the RNA polymerase by any method. In certain embodiments, the mRNA may be any mRNA that enables expression of the target protein. Preferably, the mRNA contains RNA encoding the target protein, and may contain a 5'UTR upstream and a 3'UTR and polyA downstream. In certain embodiments, the mRNA can be synthesized by performing an in vitro transcription reaction using a template DNA containing DNA encoding the target protein as a template, for example, by the method described above, but is not limited thereto. The template DNA can be prepared by any method known to those skilled in the art, including, for example, the method described above for synthesizing non-coding RNA, with appropriate modifications. In certain embodiments, when expressing a protein, culturing or the like can be performed as needed after transfection, but is not particularly limited thereto. An example of a method for performing in vitro cell-free protein synthesis using the RNA polymerase includes, but is not particularly limited to, a method of 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.

[0054] The present invention will be specifically described below with reference to examples, although the present invention is not limited to the following examples.

[0055] Example 1: Preparation of Plasmids for Expression of T7 RNA Polymerase A gene (SEQ ID NO: 2) encoding wild-type T7 RNA polymerase with a 6xHis tag attached to the N-terminus was artificially synthesized and cloned into the EcoRI-PstI cloning site of pkk223-3 to prepare a plasmid incorporating a gene encoding wild-type T7 RNA polymerase with a 6xHis tag attached to the N-terminus. Hereinafter, this expression plasmid will be referred to as pkk223-3-T7RNAP. Expression plasmids for each modified T7 RNA polymerase were prepared by site-directed mutagenesis using pkk223-3-T7RNAP as a template, the primers listed in Table 1, and a KOD-Plus-Mutagenesis Kit (Toyobo). The expression plasmids for each modified T7 RNA polymerase in which multiple amino acids were modified were prepared by repeating site-directed mutagenesis in the same manner using the expression plasmid for the modified T7 RNA polymerase obtained by site-directed mutagenesis as a template.

[0056]

[0057] Each of the obtained plasmids has a base sequence encoding a modified T7 RNA polymerase with a 6xHis tag attached to the N-terminus.

[0058] Example 2: Preparation of T7 RNA Polymerase Wild-type T7 RNA polymerase with a 6xHis tag added to the N-terminus and each modified T7 RNA polymerase were all prepared using the same method. An example of the preparation of wild-type T7 RNA polymerase is shown below. Escherichia coli JM109 was transformed with pkk223-3-T7RNAP and cultured statically at 37°C for 20 hours on LB agar medium containing 100 μg / mL ampicillin. A single colony on the agar medium was inoculated into 3 mL of LB liquid medium containing 100 μg / mL ampicillin and cultured with shaking at 37°C in a 15 mL test tube 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 at 37°C in a 500 mL Sakaguchi flask for 20 hours, after which the cells were collected from the culture by centrifugation. 1 g of the obtained bacterial cells was suspended in 10 mL of disruption buffer (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10% glycerol, 1 mM DTT, 20 mM imidazole), and the cells were disrupted on ice using an ultrasonic disrupter. The disrupted bacterial cell solution was centrifuged at 20,000 × g for 20 minutes at 4°C, and the supernatant was collected and then used for purification using His GraviTrap (manufactured by GE Healthcare). The equilibration and washing buffer was 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10% glycerol, 1 mM DTT, and 20 mM imidazole. The elution buffer was 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10% glycerol, 1 mM DTT, and 300 mM imidazole. The eluted fractions were collected and replaced with storage buffer (20 mM KPO (pH 7.7), 100 mM NaCl, 50% glycerol, 0.1 mM EDTA, 5 mM DTT, and 0.01% Triton® X-100) to obtain wild-type T7 RNA polymerase with an N-terminal 6xHis tag. In the above preparation examples, the expression plasmids for each modified T7 RNA polymerase prepared in Example 1 were used during transformation to obtain each modified T7 RNA polymerase with a 6xHis tag attached to the N-terminus.

[0059] Example 3: Measurement of specific activity of T7 RNA polymerase. Wild-type T7 RNA polymerase with an N-terminal His tag and each of the modified T7 RNA polymerases (A61E, A61D, A61S, A61P, D156N, N165S, D189N, S661N, A61E / D156N, A61E / N165S, A61E / D189N, A61E / S661N, A61P / N165S, A61P / D189N, A61P / S661N) with a similar N-terminal His tag were used to measure activity using the above-described activity measurement method. A 4 kbp dsDNA (SEQ ID NO: 20) containing the T7 promoter sequence was used as the template DNA. The length of the dsDNA downstream of the T7 promoter was approximately 1 kbp, resulting in a transcribed RNA of approximately 1 kb. The protein concentration of each modified T7 RNA polymerase was calculated by measuring the absorbance at A280, and the specific activity was calculated by dividing the activity by the protein concentration. The results are shown in Table 2.

[0060]

[0061] The results in Table 2 confirmed that all of the modified T7 RNA polymerases had improved specific activities compared to the wild type.

[0062] Example 4 Measurement of Thermostability of T7 RNA Polymerase Wild-type T7 RNA polymerase with a His tag added to its N-terminus and each of the modified T7 RNA polymerases (A61E, D156N, N165S, D189N, S661N) with a similar His tag added to their N-terminus were each diluted to 50 U / μL with storage buffer (20 mM KPO (pH 7.7), 100 mM NaCl, 50% glycerol, 0.1 mM EDTA, 5 mM DTT, 0.01% Triton® X-100), and the activity before storage was measured according to the procedure described in the activity measurement method above. Each T7 RNA polymerase diluted in the storage buffer was heat-treated for 5 minutes in an incubator at 50°C, and then its activity was measured. The residual activity was calculated from the activity before and after storage. The results are shown in Table 3.

[0063]

[0064] The results in Table 3 confirm that all of the modified T7 RNA polymerases had higher residual activity after heat treatment than the wild type.

[0065] Example 5 Measurement of the amount of dsRNA after transcription reaction Wild-type T7 RNA polymerase with a His tag added to the N-terminus and each modified T7 with a His tag added to the N-terminus RNA polymerase (A61E, A61D, A61S, A61G, A61P, D156N, N165S, N165E, N165R, N165P, D189N, S661N, A61E / D156N, A61E / N165S, A61E / D189N, A61E / S661N, A61P / N165S, A61P / D189N, A61P / S661N, A61E / D156N / N165S, A61E / N165S / D189N, A61E / N165S / S661N, A61P / D156N / N165S , A61P / N165S / D189N, A61P / N165S / S661N, A61E / D156N / N165S / D189N, A61E / N165S / D189N / S661N, A61P / D156N / N165S / D189N, A61P / N165S / D189N / S661N, A61E / D156N / N165S / D189N / S661N, A61 / D156N / N165S / D189N / S661N) were measured for activity by the same method as in Example 3, and the activity of each of the storage buffers (20 mM After diluting the T7 RNA polymerase to 50 U / μL with 40 mM Tris-HCl (pH 7.7), 100 mM NaCl, 50% glycerol, 0.1 mM EDTA, 5 mM DTT, and 0.01% Triton® X-100, 1 μL of each T7 RNA polymerase (50 U / μL) was added 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 MgCl ２, 5 mM DTT, 10 ng / μL template DNA, 0.4 mM ATP, 0.4 mM CTP, 0.4 mM GTP, 0.4 mM UTP), and 0.4 U / μL RNase inhibitor), and then the reaction was carried out in a heat block at 37°C for 1 hour. The template DNA used was a dsDNA (SEQ ID NO: 21) containing a T7 promoter, 5'UTR, 3'UTR, and poly(A) (100 nt), and the firefly luciferase gene (Fluc) as the coding sequence (CDS). The generated RNA is an approximately 1.9 kb mRNA without a cap structure, consisting of, from the 5' end, the 5'UTR, CDS (Fluc), 3'UTR, and poly(A). After the reaction, 2.5 μL of Turbo (registered trademark) DNase (2 U / μL) (manufactured by Thermo Fisher Scientific) was added, and the reaction was further carried out at 37 ° C for 15 minutes. This reaction solution was purified using Monarch (registered trademark) RNA Cleanup Kit (manufactured by NEB) to obtain purified RNA. The absorbance at 260 nm (A260) of the obtained purified RNA was measured, and the RNA concentration was calculated assuming that the RNA concentration was 40 ng / μL when A260 was 1. A calibration curve was created from the measured values ​​obtained by performing ELISA on the attached dsRNA control using a double-stranded RNA (dsRNA) ELISA kit (K1 based) (Exalpha Biologicals). Each purified RNA was diluted to 100 ng / μL and further diluted to multiple concentrations, followed by ELISA using a double-stranded RNA (dsRNA) ELISA kit (K1 based). The measured values ​​obtained were used within the range of the calibration curve, and the dsRNA concentration was calculated from the calibration curve created above. Furthermore, the dsRNA concentration was divided by the RNA concentration to calculate the percentage (w / w) of dsRNA in the RNA. The results are shown in Table 4.

[0066]

[0067] The results in Table 4 confirmed that for all of the modified T7 RNA polymerases, the ratio of the amount of dsRNA to the amount of RNA after the transcription reaction was lower than that for the wild type.

[0068] The present invention can be suitably used for RNA synthesis for various purposes.

Claims

1. An RNA polymerase having an amino acid sequence with 90% or more identity to the amino acid sequence of SEQ ID NO: 1, and at least one amino acid corresponding to positions 61, 156, 189, 165, and 661 of the amino acid sequence of SEQ ID NO: 1 being modified.

2. An RNA polymerase having an amino acid sequence with 90% or more identity to the amino acid sequence of SEQ ID NO: 1, and satisfying at least one of the following (i) to (v): (i) the amino acid at the position corresponding to position 61 of the amino acid sequence of SEQ ID NO: 1 is glutamic acid, aspartic acid, serine, glycine, or proline; (ii) the amino acid at the position corresponding to position 156 of the amino acid sequence of SEQ ID NO: 1 is asparagine; (iii) the amino acid at the position corresponding to position 189 of the amino acid sequence of SEQ ID NO: 1 is asparagine; (iv) the amino acid at the position corresponding to position 165 of the amino acid sequence of SEQ ID NO: 1 is serine, glutamic acid, arginine, or proline; and (v) the amino acid at the position corresponding to position 661 of the amino acid sequence of SEQ ID NO: 1 is asparagine.

3. Having an amino acid sequence with 90% or more identity to the amino acid sequence of SEQ ID NO: 1, and (1) the amino acid at the position corresponding to the 61st position of the amino acid sequence of SEQ ID NO: 1 is glutamic acid, aspartic acid, serine, glycine, or proline; (2) the amino acid at the position corresponding to the 156th position of the amino acid sequence of SEQ ID NO: 1 is asparagine; (3) the amino acid at the position corresponding to the 189th position of the amino acid sequence of SEQ ID NO: 1 is asparagine; (4) the amino acid at the position corresponding to the 165th position of the amino acid sequence of SEQ ID NO: 1 is serine, glutamic acid, arginine, or proline; (5) the amino acid at the position corresponding to the 661st position of the amino acid sequence of SEQ ID NO: 1 is asparagine; (6) the amino acid at the position corresponding to the 61st position of the amino acid sequence of SEQ ID NO: 1 is glutamic acid and the amino acid at the position corresponding to the 156th position of the amino acid sequence of SEQ ID NO: 1 is asparagine; (7) the amino acid at the position corresponding to the 61st position of the amino acid sequence of SEQ ID NO: 1 is glutamic acid and the amino acid at the position corresponding to the 165th position of the amino acid sequence of SEQ ID NO: 1 is serine; (8) the amino acid at the position corresponding to the 61st position of the amino acid sequence of SEQ ID NO: 1 is glutamic acid and the amino acid at the position corresponding to the 189th position of the amino acid sequence of SEQ ID NO: 1 is asparagine; (9) the amino acid at the position corresponding to the 61st position of the amino acid sequence of SEQ ID NO: 1 is glutamic acid and the amino acid at the position corresponding to the 661st position of the amino acid sequence of SEQ ID NO: 1 is asparagine; (10) the amino acid at the position corresponding to the 61st position of the amino acid sequence of SEQ ID NO: 1 is proline and the amino acid at the position corresponding to the 165th position of the amino acid sequence of SEQ ID NO: 1 is serine; (11) the amino acid at the position corresponding to the 61st position of the amino acid sequence of SEQ ID NO: 1 is proline and the amino acid at the position corresponding to the 189th position of the amino acid sequence of SEQ ID NO: 1 is asparagine; (12) the amino acid at the position corresponding to the 61st position of the amino acid sequence of SEQ ID NO: 1 is proline and the amino acid at the position corresponding to the 661st position of the amino acid sequence of SEQ ID NO: 1 is asparagine;(13) The amino acid at the position corresponding to the 61st position of the amino acid sequence of SEQ ID NO: 1 is glutamic acid, the amino acid at the position corresponding to the 156th position of the amino acid sequence of SEQ ID NO: 1 is asparagine, and the amino acid at the position corresponding to the 165th position of the amino acid sequence of SEQ ID NO: 1 is serine; (14) The amino acid at the position corresponding to the 61st position of the amino acid sequence of SEQ ID NO: 1 is glutamic acid, the amino acid at the position corresponding to the 165th position of the amino acid sequence of SEQ ID NO: 1 is serine, and the amino acid at the position corresponding to the 189th position of the amino acid sequence of SEQ ID NO: 1 is asparagine; (15) The amino acid at the position corresponding to the 61st position of the amino acid sequence of SEQ ID NO: 1 is glutamic acid, the amino acid at the position corresponding to the 165th position of the amino acid sequence of SEQ ID NO: 1 is serine, and the amino acid at the position corresponding to the 661st position of the amino acid sequence of SEQ ID NO: 1 is asparagine; (16) The amino acid at the position corresponding to the 61st position of the amino acid sequence of SEQ ID NO: 1 is proline, the amino acid at the position corresponding to the 156th position of the amino acid sequence of SEQ ID NO: 1 is asparagine, and the amino acid at the position corresponding to the 165th position of the amino acid sequence of SEQ ID NO: 1 is serine; (17) The amino acid at the position corresponding to the 61st position of the amino acid sequence of SEQ ID NO: 1 is proline, the amino acid at the position corresponding to the 165th position of the amino acid sequence of SEQ ID NO: 1 is serine, and the amino acid at the position corresponding to the 189th position of the amino acid sequence of SEQ ID NO: 1 is asparagine; (18) The amino acid at the position corresponding to the 61st position of the amino acid sequence of SEQ ID NO: 1 is proline, the amino acid at the position corresponding to the 165th position of the amino acid sequence of SEQ ID NO: 1 is serine, and the amino acid at the position corresponding to the 661st position of the amino acid sequence of SEQ ID NO: 1 is asparagine; (19) The amino acid at the position corresponding to the 61st position of the amino acid sequence of SEQ ID NO: 1 is glutamic acid, the amino acid at the position corresponding to the 156th position of the amino acid sequence of SEQ ID NO: 1 is asparagine, the amino acid at the position corresponding to the 165th position of the amino acid sequence of SEQ ID NO: 1 is serine, and the amino acid at the position corresponding to the 189th position of the amino acid sequence of SEQ ID NO: 1 is asparagine;(20) The amino acid at the position corresponding to the 61st position of the amino acid sequence of SEQ ID NO: 1 is glutamic acid, the amino acid at the position corresponding to the 165th position of the amino acid sequence of SEQ ID NO: 1 is serine, the amino acid at the position corresponding to the 189th position of the amino acid sequence of SEQ ID NO: 1 is asparagine, and the amino acid at the position corresponding to the 661st position of the amino acid sequence of SEQ ID NO: 1 is asparagine; (21) The amino acid at the position corresponding to the 61st position of the amino acid sequence of SEQ ID NO: 1 is proline, the amino acid at the position corresponding to the 156th position of the amino acid sequence of SEQ ID NO: 1 is asparagine, the amino acid at the position corresponding to the 165th position of the amino acid sequence of SEQ ID NO: 1 is serine, and the amino acid at the position corresponding to the 189th position of the amino acid sequence of SEQ ID NO: 1 is asparagine; (22) The amino acid at the position corresponding to the 61st position of the amino acid sequence of SEQ ID NO: 1 is proline, the amino acid at the position corresponding to the 165th position of the amino acid sequence of SEQ ID NO: 1 is serine, the amino acid at the position corresponding to the 189th position of the amino acid sequence of SEQ ID NO: 1 is asparagine, and the amino acid at the position corresponding to the 661st position of the amino acid sequence of SEQ ID NO: 1 is asparagine; (23) The amino acid at the position corresponding to the 61st position of the amino acid sequence of SEQ ID NO: 1 is glutamic acid, the amino acid at the position corresponding to the 156th position of the amino acid sequence of SEQ ID NO: 1 is asparagine, the amino acid at the position corresponding to the 165th position of the amino acid sequence of SEQ ID NO: 1 is serine, the amino acid at the position corresponding to the 189th position of the amino acid sequence of SEQ ID NO: 1 is asparagine, and the amino acid at the position corresponding to the 661st position of the amino acid sequence of SEQ ID NO: 1 is asparagine; or (24) The amino acid at the position corresponding to the 61st position of the amino acid sequence of SEQ ID NO: 1 is proline, the amino acid at the position corresponding to the 156th position of the amino acid sequence of SEQ ID NO: 1 is asparagine, the amino acid at the position corresponding to the 165th position of the amino acid sequence of SEQ ID NO: 1 is serine, the amino acid at the position corresponding to the 189th position of the amino acid sequence of SEQ ID NO: 1 is asparagine, and the amino acid at the position corresponding to the 661st position of the amino acid sequence of SEQ ID NO: 1 is asparagine, having the amino acid sequence, the RNA polymerase according to claim 1 or 2.; 4. A polynucleotide encoding the RNA polymerase according to claim 1.

5. A vector comprising the polynucleotide according to claim 4.

6. A recombinant host cell transformed with the vector according to claim 5.

7. A method for producing RNA polymerase using the polynucleotide according to claim 4, the vector according to claim 5, and / or the recombinant host cell according to claim 6.

8. A reagent comprising the RNA polymerase according to claim 1, the polynucleotide according to claim 4, the vector according to claim 5, and / or the recombinant host cell according to claim 6.

9. An RNA synthesis method comprising causing the RNA polymerase according to claim 1 to act on template DNA.

10. The method according to claim 9, wherein the proportion of dsRNA in the synthesized RNA is less than 90% of the proportion of dsRNA in the RNA synthesized using the corresponding wild-type RNA polymerase under the same conditions.

11. The method according to claim 9 or 10, wherein the RNA synthesis is mRNA synthesis.

12. The method according to claim 11, wherein the proportion of dsRNA in the synthesized mRNA is less than 90% of the proportion of dsRNA in the mRNA synthesized using the corresponding wild-type RNA polymerase under the same conditions.

13. A method for synthesizing an RNA pharmaceutical using the RNA polymerase according to claim 1.

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