TW202536172A - Altered RNA polymerase, polynucleotide, vector, recombinant host cell, method for producing RNA polymerase, reagent, RNA synthesis method, and method for synthesizing RNA drug - Google Patents

Abstract

The problem of this invention is to provide a novel modified RNA polymerase. This invention solves the above problem by using a modified RNA polymerase in which at least one amino acid at a predetermined position is modified.

Description

Modified RNA polymerase, polynucleotides, vectors, recombinant host cells, methods for producing RNA polymerase, reagents, RNA synthesis methods, and methods for synthesizing RNA for pharmaceutical purposes.

This invention relates to a modified RNA (ribonucleic acid) polymerase.

DNA (deoxyribonucleic acid)-dependent RNA polymerases are enzymes that recognize promoter sequences in DNA and use DNA as a template to synthesize RNA. Because they are involved in the transcriptional reaction that synthesizes mRNA (messenger RNA) from genomic DNA in organisms, they have been the subject of much research. Among them, bacteriophage-derived RNA polymerases, due to their relatively simple structure and function as a single subunit, have been a subject of study in biochemistry and structural biology since ancient times. These RNA polymerases are also widely used for the synthesis of RNA in vitro. The RNA synthesized by RNA polymerases is used as RNA probes for molecular biology techniques or as various types of functional RNA, or as template RNA for intracellular protein expression or cellular protein synthesis systems.

In addition, taking advantage of the property of RNA polymerase to synthesize most RNA from template DNA, isothermal amplification reactions such as NASBA (nucleic acid sequence-based amplification) and TMA (transcription-mediated amplification) have been developed and applied in clinical diagnosis.

In recent years, RNA synthesized by RNA polymerase has been applied to mRNA drugs, primarily mRNA vaccines, and RNA polymerase is also widely used in pharmaceutical manufacturing. One of the problematic impurities in the manufacturing process of mRNA drugs is dsRNA (double-stranded RNA). Because dsRNA can cause inflammatory responses in organisms, its removal is sought in mRNA drugs. Non-Patent Literature 1 describes a modified T7 RNA polymerase that reduces the generation of dsRNA during transcription. [Previous Artwork][Non-Patent Literature]

[Non-Patent Reference 1] Dousis, A., Ravichandran, K., Hobert, E.M. et al., Nat. Biotechnol., 41:560-568, 2023.

[Problem to be Solved by the Invention] As mentioned above, with the expansion of the applications of RNA polymerase, further improvements in its performance are desired. Therefore, the problem of this invention is to provide a novel modified RNA polymerase. [Means for Solving the Problem]

The inventors of this invention, through repeated and meticulous research to solve the aforementioned problem, discovered that by altering at least one amino acid at a predetermined position in the amino acid sequence of wild-type RNA polymerase, the generation of dsRNA during transcription can be reduced. Based on this understanding, the inventors further repeated their meticulous research to complete this invention.

That is, the present invention includes the form described in the following items. [Item 1] An RNA polymerase having an amino acid sequence having more than 90% identity with the amino acid sequence of sequence number 1 and having at least one amino acid modified from a group consisting of amino acids at positions 61, 156, 189, 165 and 661 of the amino acid sequence of sequence number 1. [Item 2] An RNA polymerase having an amino acid sequence that is more than 90% identical to the amino acid sequence of Sequence No. 1 and satisfies at least one of the following groups (i) to (v): (i) the amino acid at position 61 of the amino acid sequence of Sequence No. 1 is glutamic acid, aspartic acid, serine, glycine, or proline; (ii) the amino acid at position 156 of the amino acid sequence of Sequence No. 1 is aspartic acid; (iii) the amino acid at position 189 of the amino acid sequence of Sequence No. 1 is aspartic acid; (iv) the amino acid at position 165 of the amino acid sequence of Sequence No. 1 is serine, glutamic acid, arginine, or proline; and (v) The amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is aspartic acid. [Item 3] The RNA polymerase described in Item 1 or Item 2, wherein the aforementioned RNA polymerase has an amino acid sequence that is more than 90% identical to the amino acid sequence of Sequence Number 1, and the aforementioned amino acid sequence is: (1) the amino acid at position 61 of the amino acid sequence of Sequence Number 1 is glutamic acid, aspartic acid, serine, glycine, or proline; (2) the amino acid at position 156 of the amino acid sequence of Sequence Number 1 is aspartic acid; (3) the amino acid at position 189 of the amino acid sequence of Sequence Number 1 is aspartic acid; (4) the amino acid at position 165 of the amino acid sequence of Sequence Number 1 is serine, glutamic acid, arginine, or proline; (5) (6) The amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; (7) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, and the amino acid at position 156 of the amino acid sequence corresponding to sequence number 1 is asparagine; (8) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, and the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine; (9) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, and the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine; The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, and the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; (10) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, and the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine; (11) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, and the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine; (12) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, and the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; (13) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, the amino acid at position 156 of the amino acid sequence corresponding to sequence number 1 is asparagine, and the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine; (14) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, and the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine; (15) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, and the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; (16) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, the amino acid at position 156 of the amino acid sequence corresponding to sequence number 1 is asparagine, and the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine; (17) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, and the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine; (18) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, and the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; (19) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, the amino acid at position 156 of the amino acid sequence corresponding to sequence number 1 is asparagine, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, and the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine; (20) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine, and the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; (21) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, the amino acid at position 156 of the amino acid sequence corresponding to sequence number 1 is asparagine, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, and the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine; (22) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine, and the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; (23) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, the amino acid at position 156 of the amino acid sequence corresponding to sequence number 1 is asparagine, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine, and the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; or (24) The amino acid at position 61 of the amino acid sequence corresponding to Sequence Number 1 is proline, the amino acid at position 156 of the amino acid sequence corresponding to Sequence Number 1 is asparagine, the amino acid at position 165 of the amino acid sequence corresponding to Sequence Number 1 is serine, the amino acid at position 189 of the amino acid sequence corresponding to Sequence Number 1 is asparagine, and the amino acid at position 661 of the amino acid sequence corresponding to Sequence Number 1 is asparagine. [Item 4] A polynucleotide that encodes an RNA polymerase as described in any one of Items 1 to 3. [Item 5] A vector containing the polynucleotide as described in Item 4. [Item 6] A recombinant host cell that has been transformed by a vector as described in Item 5. [Item 7] A method for producing RNA polymerase, comprising using a polynucleotide as described in Item 4, a vector as described in Item 5, and/or a recombinant host cell as described in Item 6 to produce RNA polymerase. [Item 8] A reagent comprising an RNA polymerase as described in any one of Items 1 to 3, a polynucleotide as described in Item 4, a vector as described in Item 5, and/or a recombinant host cell as described in Item 6. [Item 9] A method for RNA synthesis comprising acting an RNA polymerase as described in any one of Items 1 to 3 on template DNA. [Item 10] The method as described in Item 9, wherein the proportion of dsRNA in the synthesized RNA is less than 90% compared to the proportion of dsRNA in RNA synthesized under the same conditions using the corresponding wild-type RNA polymerase. [Item 11] The method as described in Item 9 or Item 10, wherein the amount of RNA synthesized is 110% or more compared to the amount of RNA synthesized under the same conditions using the corresponding wild-type RNA polymerase. [Item 12] The method as described in any one of Items 9 to 11, wherein the aforementioned RNA synthesis is mRNA synthesis. [Item 13] The method as described in Item 12, wherein the proportion of dsRNA in the synthesized mRNA is less than 90% compared to the proportion of dsRNA in mRNA synthesized under the same conditions using the corresponding wild-type RNA polymerase. [Item 14] The method as described in Item 12 or Item 13, wherein the amount of mRNA synthesized is more than 110% of the amount of mRNA synthesized under the same conditions using the corresponding wild-type RNA polymerase. [Item 15] The method as described in any one of Items 9 to 11, wherein the aforementioned RNA synthesis is non-coding RNA synthesis. [Item 16] The method as described in Item 15, wherein the aforementioned non-coding RNA is selected from at least one of the following groups: microRNA, siRNA, piRNA, rRNA, tRNA, snRNA, snoRNA, SLRNA, SRPRNA, mRNA-like noncoding RNA, and oligonucleotides. [Item 17] A method for synthesizing guide RNA for gene editing, wherein the guide RNA for gene editing is synthesized using an RNA polymerase as described in any one of items 1 to 3. [Item 18] A method for synthesizing an RNA medicine, wherein the RNA medicine is synthesized using an RNA polymerase as described in any one of items 1 to 3. [Item 19] The method as described in item 9, wherein the aforementioned RNA synthesis is performed by a constant-temperature amplification reaction. [Item 20] A method for performing gene testing, wherein gene testing is performed using an RNA polymerase as described in any one of items 1 to 3. [Item 21] A method for performing protein expression in vivo or cell-free protein synthesis in vitro, comprising using an RNA polymerase as described in any one of items 1 to 3 to perform protein expression in vivo or cell-free protein synthesis in vitro. [Invention Benefits]

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

The following describes various embodiments of the present invention and provides a further detailed explanation thereof, but the present invention is not limited to these embodiments. Furthermore, all non-patented and patented documents described in this specification are incorporated herein by reference in their entirety. In addition, the word "to" in this specification means "above and below," for example, "X to Y" in this specification means "above X and below Y." The word "and/or" in this specification means any one or more possible combinations of the listed elements. The word "containing" in this specification includes the concepts of "substantially constituted by" and "consisting solely of."

This invention provides a modified RNA polymerase as one embodiment of the invention. In this specification, "modification" refers to the mutation of an amino acid residue, i.e., substitution, deletion, insertion, or addition. The modified RNA polymerase (hereinafter also referred to as "modified RNA polymerase") refers to an RNA polymerase in which at least one amino acid residue in the amino acid sequence of the wild-type RNA polymerase has been mutated, i.e., substituted, deleted, inserted, or added.

In this specification, the term "wild-type RNA polymerase" (hereinafter also referred to as "wild-type" or "WT") refers to an RNA polymerase that has not been artificially mutated. Examples of wild-type RNA polymerases include: T7 RNA polymerase, T3 RNA polymerase, K11 RNA polymerase, SP6 RNA polymerase, Syn5 RNA polymerase, KP34 RNA polymerase, and VSW-3 RNA polymerase. A preferred wild-type RNA polymerase is, for example, wild-type T7 RNA polymerase, which consists of the amino acid sequence numbered 1. T7 RNA polymerase, as is well known to those skilled in the art, refers to an RNA polymerase derived from T7 bacteriophage.

The RNA polymerase of this invention can be an RNA polymerase with increased specific activity compared to a corresponding wild-type RNA polymerase. Here, "specific activity" refers to the enzyme activity per unit weight of protein when enzyme activity is measured, for example, at a temperature between 20°C and 100°C. Furthermore, "corresponding wild-type RNA polymerase" means the wild-type of an RNA polymerase of the same class as a particular modified RNA polymerase. For example, the "corresponding wild-type RNA polymerase" for modified T7 RNA polymerase is wild-type T7 RNA polymerase. Similarly, for example, the "corresponding wild-type RNA polymerase" for modified T3 RNA polymerase is wild-type T3 RNA polymerase.

The RNA polymerase of this invention has an amino acid sequence different from that of previously known wild-type RNA polymerases. Therefore, in this specification, the RNA polymerase of this invention is sometimes referred to as a mutant RNA polymerase or a modified RNA polymerase. In this specification, the terms "mutant" and "modified" are used interchangeably when referring to "mutant RNA polymerase" or "modified RNA polymerase," meaning that it has an amino acid sequence different from that of previously known wild-type RNA polymerases, rather than distinguishing between those obtained through artificial mutation and those obtained through natural mutation. Therefore, the mutant RNA polymerase of this invention is an RNA polymerase in which at least one amino acid in the amino acid sequence of Sequence Number 1, which displays the wild-type T7 RNA polymerase sequence, is modified to have an amino acid sequence different from that of Sequence Number 1, regardless of whether the mutant RNA polymerase is obtained through artificial mutation or through natural mutation.

In this specification, simplified notations using letters are sometimes used for base sequences, amino acid sequences, and their respective constituent factors, but these are all in accordance with conventions in the fields of molecular biology and genetic engineering. Furthermore, to concisely display amino acid sequence mutations, notations such as "A61E" are used. "A61E" indicates that the 61st amino acid in the sequence, alanine (A), is replaced by glutamic acid (E). That is, it shows the type of amino acid residue before substitution, the position of the substituted amino acid in the sequence, and the type of amino acid residue after substitution. In addition, unless otherwise specified, sequence numbers correspond to the sequence numbers recorded in the sequence listing. Furthermore, in the case of multiple mutants, the above markings can be connected by "/" (for example, they can be marked as A61E/D156N/N165S).

In one embodiment, the amino acid sequence of the mutant RNA polymerase may be a sequence of amino acids with a modified proportion in the amino acid sequence of Sequence Number 1. In one embodiment, the amino acid sequence of the mutant RNA polymerase preferably has a similarity of more than 90% to the amino acid sequence of Sequence Number 1. There are no particular limitations on the mutant RNA polymerase as long as it does not lose RNA polymerase activity; for example, it may consist of an amino acid sequence with a similarity of, for example, more than 90% to the amino acid sequence of Sequence Number 1, preferably more than 95%, 96%, 97%, 98%, or 99% to the amino acid sequence of Sequence Number 1. Here, the similarity of the amino acid sequence can be evaluated by any means known in the art. For example, commercially available analytical tools or those accessible via telecommunications lines (the Internet) can be used to calculate the amino acid sequence similarity. For instance, the similarity can be calculated using the BLAST (Basic Local Alignment Search Tool) algorithm from the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/BLAST/) with default (initial) parameters. Furthermore, the amino acid sequence of a mutant RNA polymerase can be an amino acid sequence obtained by deleting, substituting, inserting, and/or adding one or more amino acids in the sequence number 1. Here, "one or more" means that there is no particular limitation as long as RNA polymerase activity is not lost; for example, it can be 1 to 30, preferably 1 to 20, 1 to 10, 1 to 5, or 1 to 3. The amino acid sequence mentioned above can be an artificially created amino acid sequence through genetic engineering, or it can be an amino acid sequence derived from natural proteins.

In one embodiment, when the mutant RNA polymerase has a substitution in the amino acid sequence of sequence number 1, the substitution is preferably a substitution between structurally and/or chemically similar amino acids (so-called a retaining substitution). Examples of retained substitutions include, for example, 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 nitrogenous amino acids (K, R, N, Q, P), substitutions between sulfurous amino acids (C, M), substitutions between oxyamino acids (S, T), substitutions between β-branched amino acids (V, L, I), and substitutions between amino acids having a straight-chain alkyl or hydrogen side chain (A, G), but are not limited to these examples.

In certain embodiments, the RNA polymerase of this invention may also contain peptides other than RNA polymerase on either or both of the N-terminal and C-terminal sides. Examples of such peptides include: extracellular secretory information, protein purification tags (His tag, Flag tag, Strep tag, GST (glutathione S-transferase) tag, MBP (maltose-binding protein) tag, proteins with other functions, linker peptides, etc., but are not particularly limited.

The RNA polymerase of this invention can be composed of an amino acid sequence containing the amino acid residues described above. Those skilled in the art to which this invention pertains can use any genetic engineering method known in the art, such as appropriately designing and encoding the base sequence of the target amino acid sequence and inserting the base sequence into any expression vector, etc., to transform the resulting protein into a host cell and perform expression, to produce the RNA polymerase protein of the target amino acid sequence.

In a preferred embodiment, the RNA polymerase system of the present invention has, for example, more than 90% identity with the amino acid sequence of Sequence Number 1, more preferably more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% identity. For example, it is an RNA polymerase that is equivalent to an amino acid at position 61 of the amino acid sequence of Sequence Number 1 that has been modified, or more preferably an RNA polymerase that is equivalent to an amino acid at position 61 that has been substituted with other amino acids. If the amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is alanine, then the amino acid that replaces the alanine residue at position 61 can be, for example: glycine, proline, isoleucine, leucine, methionine, velamine, phenylalanine, tryptophan, tyrosine, cysteine, aspartic acid, glutamic acid, serine, threonine, arginine, histamine, lysine, aspartic acid, or glutamic acid. Preferably, it is glutamic acid, aspartic acid, serine, threonine, glycine, or proline, and even more preferably, it is glutamic acid, aspartic acid, serine, glycine, or proline. While not intended to be confined to a specific theory, it is believed that an amino acid mutation at position 61 of the amino acid sequence corresponding to sequence number 1 helps to reduce the proportion of dsRNA in RNA produced by transcription reactions using modified RNA polymerase.

In this specification, the term "position corresponding to the 61st amino acid sequence of sequence number 1" refers to the 61st amino acid sequence (sequence number 1) of the wild-type T7 RNA polymerase, or the position in the mutant RNA polymerase amino acid sequence corresponding to the 61st amino acid sequence (sequence number 1) of the wild-type T7 RNA polymerase. The position "corresponding to the 61st amino acid sequence of sequence number 1" in the mutant RNA polymerase amino acid sequence can be easily identified by, for example, by comparison and alignment of the amino acid sequence (sequence number 1) of the wild-type T7 RNA polymerase with the amino acid sequence of the mutant RNA polymerase using any known means. For example, the comparison and alignment of amino acid sequences can be performed using commercially available analytical tools or those accessible via telecommunications lines (the Internet). For instance, amino acid sequence alignment can be performed using the default (initial settings) parameters of Clustal Omega (https://www.ebi.ac.uk/jdispatcher/msa/clustalo), a multiplex sequence alignment program provided by the European Institute for Bioinformatics (EMBL). The same applies to the position of amino acids at any position other than the 61st amino acid in the sequence corresponding to sequence number 1, as described in this specification.

In a preferred embodiment, the RNA polymerase of the present invention has, for example, more than 90% identity with the amino acid sequence of Sequence Number 1, more preferably more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% identity. For example, it is an RNA polymerase in which the amino acid at position 156 of the amino acid sequence of Sequence Number 1 has been modified, and more preferably it is an RNA polymerase in which the amino acid has been substituted into another amino acid. The amino acid at position 156 of the amino acid sequence corresponding to Sequence Number 1 is aspartic acid. Examples of amino acids that can replace the aspartic acid residue at position 156 include: glycine, alanine, proline, isoleucine, leucine, methionine, volcanic acid, phenylalanine, tryptophan, tyrosine, cysteine, aspartic acid, glutamic acid, serine, threonine, arginine, histamine, lysine, or glutamic acid, but there is no particular limitation. In a preferred embodiment, the RNA polymerase of this invention is an RNA polymerase in which the amino acid at position 156 of the amino acid sequence corresponding to Sequence Number 1 is modified to be a basic or neutral amino acid. Examples of basic or neutral amino acids include aspartic acid, glycine, alanine, proline, isoleucine, leucine, volcanic acid, phenylalanine, tryptophan, tyrosine, glutamic acid, cysteine, methionine, serine, threonine, arginine, histamine, or lysine, with aspartic acid or glutamic acid being preferred, and aspartic acid being even more preferred. While not intended to be confined to a specific theory, it is believed that an amino acid mutation at position 156 of the amino acid sequence equivalent to sequence number 1 helps reduce the proportion of dsRNA in RNA produced by transcription reactions using modified RNA polymerases.

In a preferred embodiment, the RNA polymerase of the present invention has, for example, more than 90% identity with the amino acid sequence of Sequence Number 1, more preferably more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% identity. For example, it is an RNA polymerase in which the amino acid at position 165 of the amino acid sequence of Sequence Number 1 has been modified, and more preferably it is an RNA polymerase in which the amino acid has been substituted into another amino acid. The amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is aspartic acid. The amino acid that replaces the aspartic acid residue at position 165 can be listed as follows: glycine, alanine, proline, isoleucine, leucine, methionine, volcanic acid, phenylalanine, tryptophan, tyrosine, cysteine, glutamic acid, serine, threonine, arginine, histamine, lysine, aspartic acid, or glutamic acid, among which serine, glutamic acid, arginine, or proline are preferred, and serine is even more preferred. While not intended to be confined to a specific theory, it is believed that an amino acid mutation at position 165 of the amino acid sequence corresponding to sequence number 1 helps to reduce the proportion of dsRNA in RNA produced by transcription reactions using modified RNA polymerase.

In a preferred embodiment, the RNA polymerase of the present invention has, for example, more than 90% identity with the amino acid sequence of Sequence Number 1, more preferably more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% identity. For example, it is an RNA polymerase in which the amino acid at position 189 of the amino acid sequence of Sequence Number 1 has been modified, and more preferably it is an RNA polymerase in which the amino acid has been substituted into another amino acid. The amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is aspartic acid. Examples of amino acids that would replace the aspartic acid residue at position 189 include: glycine, alanine, proline, isoleucine, leucine, methionine, cellulose, phenylalanine, tryptophan, tyrosine, cysteine, aspartic acid, glutamic acid, serine, threonine, arginine, histamine, lysine, or glutamic acid, but there are no particular limitations. In a preferred embodiment, the RNA polymerase of the present invention preferably has an amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 replaced by a basic or neutral amino acid. Examples of basic or neutral amino acids include, for example, glycine, alanine, proline, isoleucine, leucine, volcanic acid, phenylalanine, tryptophan, tyrosine, aspartic acid, glutamic acid, cysteine, methionine, serine, threonine, arginine, histamine, or lysine. It is more preferably replaced by aspartic acid or glutamic acid, and even more preferably by aspartic acid. While not intended to be confined to a specific theory, it is believed that an amino acid mutation at position 189 of the amino acid sequence corresponding to sequence number 1 helps to reduce the proportion of dsRNA in RNA produced by transcription reactions using modified RNA polymerase.

In a preferred embodiment, the RNA polymerase of the present invention has, for example, more than 90% identity with the amino acid sequence of Sequence Number 1, more preferably more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% identity. For example, it is an RNA polymerase in which the amino acid at position 661 of the amino acid sequence of Sequence Number 1 has been modified, and more preferably it is an RNA polymerase in which the amino acid has been substituted into another amino acid. If the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is serine, then the amino acid that replaces the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 can be, for example: glycine, alanine, proline, isoleucine, leucine, methionine, volcanic acid, phenylalanine, tryptophan, tyrosine, cysteine, aspartic acid, glutamic acid, threonine, arginine, histamine, lysine, aspartic acid, or glutamic acid. It is preferred to replace it with aspartic acid or glutamic acid, and more preferably with aspartic acid. While not intended to be confined to a specific theory, it is believed that an amino acid mutation at position 661 of the amino acid sequence corresponding to sequence number 1 helps to reduce the proportion of dsRNA in RNA produced by transcription reactions using modified RNA polymerase.

In a preferred embodiment, the RNA polymerase of the present invention has, for example, more than 90% identity with the amino acid sequence of Sequence Number 1 (preferably more than 95%, more than 96%, more than 97%, more than 98%, or more than 99%) and is an RNA polymerase in which at least one amino acid has been modified from the group consisting of amino acids at positions 61, 156, 189, 165, and 661 of the amino acid sequence of Sequence Number 1.

In a preferred embodiment, the RNA polymerase of the present invention may be an RNA polymerase having an amino acid sequence having, for example, more than 90% identity with the amino acid sequence of Sequence Number 1 (preferably more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% identity) and satisfying an amino acid sequence selected from at least one of the following groups (i) to (v): (i) the amino acid at position 61 of the amino acid sequence of Sequence Number 1 is glutamic acid, aspartic acid, serine, glycine, or proline; (ii) the amino acid at position 156 of the amino acid sequence of Sequence Number 1 is aspartic acid; (iii) the amino acid at position 189 of the amino acid sequence of Sequence Number 1 is aspartic acid; (iv) The amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, glutamic acid, arginine, or proline; and (v) the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is aspartic acid.

In a preferred embodiment, the RNA polymerase of the present invention may have an amino acid sequence with an identity of, for example, more than 90%, preferably more than 95%, more than 96%, more than 97%, more than 98%, or more than 99%, such as the following amino acid sequences (1) to (24): (1) The amino acid at position 61 of the amino acid sequence of sequence number 1 is glutamic acid, aspartic acid, serine, glycine, or proline; (2) The amino acid at position 156 of the amino acid sequence of sequence number 1 is aspartic acid; (3) The amino acid at position 189 of the amino acid sequence of sequence number 1 is aspartic acid; (4) (5) The amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, glutamic acid, arginine, or proline; (6) The amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is aspartic acid; (7) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, and the amino acid at position 156 of the amino acid sequence corresponding to sequence number 1 is aspartic acid; (8) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, and the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine; The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, and the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine; (9) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, and the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; (10) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, and the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine; (11) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, and the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine; (12) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, and the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; (13) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, the amino acid at position 156 of the amino acid sequence corresponding to sequence number 1 is asparagine, and the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine; (14) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, and the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine; (15) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, and the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; (16) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, the amino acid at position 156 of the amino acid sequence corresponding to sequence number 1 is asparagine, and the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine; (17) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, and the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine; (18) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, and the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; (19) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, the amino acid at position 156 of the amino acid sequence corresponding to sequence number 1 is asparagine, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, and the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine; (20) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine, and the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; (21) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, the amino acid at position 156 of the amino acid sequence corresponding to sequence number 1 is asparagine, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, and the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine; (22) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine, and the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; (23) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, the amino acid at position 156 of the amino acid sequence corresponding to sequence number 1 is asparagine, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine, and the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; or (24) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, the amino acid at position 156 of the amino acid sequence corresponding to sequence number 1 is asparagine, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine, and the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; preferably, it can be an RNA polymerase having an amino acid sequence (10) where the amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline and the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine.

In one embodiment, the RNA polymerase of the present invention is characterized by its ability to reduce the generation of dsRNA during the transcription reaction. The RNA polymerase of the present invention may be an RNA polymerase that, compared with the corresponding wild-type RNA polymerase, produces RNA with a dsRNA content of approximately 90% or less, preferably approximately 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% or less.

[Methods for determining the proportion of dsRNA in RNA after transcription reaction] Specifically, the proportion of dsRNA in RNA after transcription reaction can be determined by the following methods. 1 μL of RNA polymerase (50 U/μL) was added to 49 μL of reaction solution (the following shows the final concentration in 50 μL of reaction solution after enzyme addition) (40 mM Tris-HCl (Tris(hydroxymethyl)aminomethane Hydrochloride) ( _pH 8.0), 50 mM NaCl, 8 mM MgCl2, 5 mM DTT (Dithiothreitol), 10 ng/μL template DNA, 0.4 mM ATP (adenosine triphosphate), 0.4 mM CTP (cytidine triphosphate), 0.4 mM GTP (guanosine triphosphate), 0.4 mM UTP (uridine triphosphate), 0.4 U/μL After being treated with an RNase (ribonuclease) inhibitor, the reaction was carried out at 37°C for 1 hour using a heating block. The template DNA was dsDNA containing, from the 5' end, a promoter corresponding to the RNA polymerase to be measured (any one of the T7 promoter, T3 promoter, SP6 promoter, etc.), a 5' UTR (untranslated region), a 3' UTR, a poly-A (polyadenylic acid) (100nt), and containing the firefly luciferase gene (Fluc) as the coding sequence (CDS) between the 5' UTR and 3' UTR. Following the reaction, 2.5 μL of Turbo 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 then purified using the Monarch RNA Cleanup Kit (manufactured by NEB) to obtain purified RNA. The absorbance (A260) of the purified RNA at 260 nm was measured, and the RNA concentration was calculated by assuming an A260 of 1 equals 40 ng/μL. A calibration curve was constructed using a double-stranded RNA (dsRNA) ELISA (enzyme-linked immunosorbent assay) kit (K1-based, Exalpha Biologicals) based on the ELISA results against the attached dsRNA control group. The purified RNA was diluted to 100 ng/μL, and then further diluted to multiple concentrations. An ELISA was then performed using a double-stranded RNA (dsRNA) ELISA kit (K1-based). The obtained measurement values were those within the detection range. The dsRNA concentration was calculated from the aforementioned detection range. By further dividing the dsRNA concentration by the RNA concentration, the percentage (%) (w/w) of dsRNA in the RNA could be calculated.

In one embodiment, the RNA polymerase of the present invention can exhibit high specific activity. The RNA polymerase of the present invention can have a specific activity of approximately 1.1 times or more compared to the corresponding wild-type RNA polymerase, preferably approximately 1.2 times or more, approximately 1.3 times or more, approximately 1.4 times or more, approximately 1.5 times or more, approximately 1.6 times or more, approximately 1.7 times or more, or approximately 1.8 times or more. Specific activity can be specifically confirmed by the following assay methods.

[Activity Assay Method] The activity of RNA polymerase can be measured by the following procedure. Those skilled in the art to which this invention pertains can appropriately set the assay conditions, for example, if the enzyme activity is too high, the sample containing the assay object can be appropriately diluted and then measured. First, 45 μL of reaction solution (hereinafter, showing the final concentration in 50 μL of reaction solution after enzyme addition) (40 mM Tris-HCl (pH 8.0), 50 mM NaCl, ₈ mM MgCl₂, 5 mM DTT, 10 ng/μL template DNA, 0.4 mM ATP, 0.4 mM CTP, 0.4 mM GTP, 0.4 mM UTP) was added to a 0.2 mL PCR (polymerase chain reaction) tube. Then, 5 μL of wild-type RNA polymerase with known activity was added, and the reaction was carried out at 37°C for 10 minutes. After 10 minutes, the reaction was stopped by adding 10 μL of 1 M EDTA (ethylenediaminetetraacetic acid). A series of diluted RNA polymerase solutions were prepared and this reaction was performed. Then, the amount of RNA in each reaction solution was quantified using the Qubit (registered trademark) RNA BR (Broad Range) analysis kit (manufactured by Thermo Fisher Scientific). Measurement lines were constructed from the activity values of the RNA polymerase added in each dilution series and the quantified amount of RNA. The same reaction was performed on the RNA polymerase for the assay target, and the activity was calculated using the constructed measurement lines based on the obtained RNA amount.

[Specific Activity Determination] The absorbance at 280 nm (A280) was measured. The enzyme concentration was calculated by assuming that an A280 of 1 indicates an enzyme concentration of 1 mg/mL. The specific activity was calculated by dividing the activity value calculated as described above by the enzyme concentration.

In one embodiment, the RNA polymerase of the present invention exhibits high thermal stability compared to the corresponding wild-type RNA polymerase. In a preferred embodiment, the RNA polymerase of the present invention may, for example, be an RNA polymerase exhibiting a residual activity rate of greater than 50% after heat treatment at 50°C for 5 minutes, an RNA polymerase exhibiting a residual activity rate of greater than 60%, an RNA polymerase exhibiting a residual activity rate of greater than 70%, or an RNA polymerase exhibiting a residual activity rate of greater than 80%. In a particular embodiment, the RNA polymerase of the present invention may be an RNA polymerase exhibiting a residual activity rate of greater than 90% after heat treatment at 50°C for 5 minutes.

From another perspective, the RNA polymerase of this invention can be, for example, a mutant RNA polymerase that, when heat-treated under predetermined conditions, exhibits a high residual activity rate compared to the residual activity rate of the corresponding wild-type RNA polymerase. Specifically, for example, it can be an RNA polymerase that, when heat-treated at 50°C for 5 minutes, exhibits a residual activity rate that is approximately 1.1 times higher than the residual activity rate of the corresponding wild-type RNA polymerase, preferably an RNA polymerase exhibiting a residual activity rate that is approximately 1.2 times higher, approximately 1.3 times higher, approximately 1.4 times higher, approximately 1.5 times higher, approximately 1.6 times higher, approximately 1.7 times higher, approximately 1.8 times higher, or approximately 1.9 times higher.

[Determination of Thermal Stability (Residual Activity After Heat Treatment)] Thermal stability can be specifically confirmed by the following method. First, each mutant RNA polymerase to be tested is diluted to 50 U/μL with a preservation buffer (20 mM _KPO4 (pH 7.7), 100 mM NaCl, 50% glycerol, 0.1 mM EDTA, 5 mM DTT, 0.01% Triton X-100). The activity value before preservation is then measured according to the procedure described in the aforementioned activity assay method. Second, each mutant RNA polymerase to be tested, diluted in the above preservation buffer, is stored in an incubator at 50°C for 5 minutes. After storage, the activity value is measured according to the same procedure described in the aforementioned activity assay method, just as before storage. Next, as described in Equation I below, the residual activity rate can be calculated by dividing the activity value after storage by the activity value before storage. Residual activity rate (%) = (Active value after storage / Activity value before storage) × 100…(Equation I)

In a further embodiment, the present invention provides a polynucleotide encoding the RNA polymerase of the present invention as described above. This polynucleotide is suitable for use in the expression of the RNA polymerase of the present invention and/or the fabrication of expression vectors and/or the fabrication of transformed cells. Here, the polynucleotide encoding the RNA polymerase refers to a polynucleotide that, for example, yields the protein of the RNA polymerase of the present invention when expressed by conventional methods. That is, it refers to a polynucleotide composed of the base sequence corresponding to the amino acid sequence of the protein of the RNA polymerase of the present invention. Those skilled in the art to which this invention pertains can easily determine the base sequence corresponding to a predetermined amino acid sequence using codon tables and other methods known in the art. Furthermore, the polynucleotides of the RNA polymerase of this invention also include polynucleotides that differ due to codon decomposition. Polynucleotides can be any nucleic acid such as DNA or RNA, but DNA is preferred. Polynucleotides can be synthesized, for example, by artificial gene synthesis. As an example of methods for synthesizing DNA by artificial gene synthesis, one can cite the following: synthesizing two or more oligoDNAs of 10 to 100 nt with partially overlapping sequences through chemical synthesis, then linking them using PCA (polymerase chain assembly) or other enzymatic methods, and finally designing primers at both ends of the final target sequence to obtain double-stranded DNA by PCR. However, there are no particular limitations. When RNA is synthesized by artificial gene synthesis, for example, double-stranded DNA can be synthesized in a form containing an arbitrary promoter sequence (T7 promoter, T3 promoter, SP6 promoter, etc.) at the 5' end during the synthesis of the aforementioned double-stranded DNA. Then, using this DNA as a template, RNA is synthesized by performing an in vivo transcription reaction using RNA polymerases (T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, etc.) that specifically recognize arbitrary promoters.

In a further embodiment, the present invention provides a vector containing the aforementioned polynucleotides. This vector is suitable for use in the expression of the RNA polymerase of the present invention and/or the production of transformed cells. The vector can be prepared by introducing the polynucleotide encoding the aforementioned RNA polymerase into a vector (e.g., an expression vector, a selection vector, etc.). The polynucleotide introduction vector can be performed by any method, such as restriction enzyme treatment and ligation, or seamless cloning such as Gibson assembly. In a particular embodiment, the vector of the present invention may further contain elements that function in the host cell, such as a promoter, a terminator, a ribosome-binding site, and a drug resistance gene. Regarding drug resistance genes, examples include those resistant to drugs such as ampicillin, kanamycin, and tetracycline. In certain embodiments, the vector of this invention may also contain elements that enable homologous recombination with the host cell's genome. If the vector enables selection and/or expression of the RNA polymerase of this invention, any vector may be used, such as plasmids. Examples of plasmids include pUC118, pUC18, pBR322, pBluescript, pLED-M1, p73, pGW7, and pkk223-3, but are not limited to these.

In a further embodiment, the present invention provides a cell that is transformed using the aforementioned vector. This cell is suitable for use in expressing genes encoding the RNA polymerase of the present invention. In a particular preferred embodiment, the recombinant host cell of the present invention can be obtained by transforming a host cell using the aforementioned expression vector. Examples of such host cells include Escherichia coli and yeast, with Escherichia coli being particularly preferred. Examples of Escherichia coli include, for example, Escherichia coli DH5α, JM109, HB101, XL1Blue, PR1, and BL21. That is, in this invention, it is preferable to insert the gene encoding the above-mentioned RNA polymerase into the above-mentioned vector as a expression vector, and further transform the host cell by means of the expression vector.

In a further embodiment, a method for manufacturing the aforementioned RNA polymerase is also provided, which involves using the aforementioned polynucleotide, the aforementioned vector, the aforementioned recombinant host cell, and/or one or more of the aforementioned polynucleotide, the aforementioned vector, and the aforementioned recombinant host cell to manufacture the aforementioned RNA polymerase. In a particular embodiment, after transforming the host cell using the aforementioned vector, the resulting recombinant host cell is cultured using any culture apparatus. Examples of culture apparatus include test tubes, flasks, and fermentation tanks, but there are no particular limitations. Regarding the culture medium used, examples include LB (Lysogeny broth) medium, 2×YT (Yeast extract tryptone) medium, and TB (Terrific broth) medium, but there are no particular limitations. Culture conditions can be appropriately set; the culture temperature can range from 10°C to 40°C, and the culture time can range from 1 hour to 100 hours. Aeration and/or stirring can be performed during culture, and it can be cultured using either batch culture or feed-batch culture. After culture, the cells are recovered by centrifugation or other methods. The crude enzyme solution is extracted by breaking or dissolving the recovered cells. Any known method can be used to break or dissolve the cells. For breaking methods, physical methods such as ultrasonic treatment, French press, or glass bead crushing can be listed. For dissolving methods, methods using enzymes such as lysozyme can be listed, but there are no particular limitations. The method for obtaining purified RNA polymerase from the crude enzyme solution can be any method, such as centrifugation, ultracentrifugation, ultrafiltration, nucleic acid removal treatment, salt precipitation, dialysis, and various column chromatography (ion exchange column chromatography, hydrophobic chromatography, affinity chromatography, gel filtration column chromatography, etc.) to isolate the RNA polymerase of this invention.

In a further embodiment, a reagent is provided that contains one or more of the aforementioned RNA polymerase, polynucleotide, vector, and recombinant host cell. In a specific embodiment, the reagent may be a liquid or a solid to which moisture has been removed by methods such as freeze-drying. In a specific embodiment, the reagent may be placed in any container. In a specific embodiment, the reagent may also be divided into one or more portions. In a specific embodiment, the reagent may contain a substance that enables the expression of the aforementioned RNA polymerase from the aforementioned polynucleotide, vector, and recombinant host cell. Examples of such substances include cell-free protein synthesis reagents, but there are no particular limitations. Regarding cell-free protein synthesis reagents, examples include cell-free protein synthesis reagents using cell extracts, and reconstituted cell-free protein synthesis systems that refine and mix the factors involved in the translation reaction, but there are no particular limitations. Regarding the substances contained in cell-free protein synthesis reagents, examples include: cell extracts, or refined factors involved in the translation reaction, as well as 20 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., but there are no particular limitations. Regarding cell-free protein synthesis reagents using cell extracts, examples include Musaibo Kun (registered trademark) from Taiyo Nippon Sanso Corporation, and cell-free protein synthesis reagents derived from extracts of rabbit reticulocytes, wheat germ, insect cells, Escherichia coli, and human cells, but there are no particular limitations. Regarding reconstitution-type cell-free protein synthesis systems, examples include PUREfrex (registered trademark) 1.0 manufactured by GeneFrontier, but there are no particular limitations. In certain embodiments, the above reagents may also contain substances required for in vitro transcription reactions. Regarding substances required for in vivo transcription reactions, examples include: buffers (such as Torres buffer, phosphate buffer, Goodyear buffer, etc.), ribonucleotides (ATP, CTP, GTP, UTP, modified bases (bases modified by deamination, methylation, methoxylation, pseudouridineation, etc.), cap analogs (ARCA (Anti-Reverse Cap Analog), mCAP, CleanCap Reagent AG, CleanCap Reagent M6, CleanCap Reagent). The reagents may contain AU (manufactured by 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., but are not particularly limited. In certain embodiments, the above reagents may also contain substances required for post-transcriptional capping. Regarding the substances required for post-transcriptional capping, the following can be listed: capping enzymes and GTP, mRNA Cap 2’-O-methyltransferase, SAM (S-adenosylmethionine), buffers (Torlis buffer, phosphate buffer, Goodyear buffer, etc.), salts such as NaCl and KCl, metal ions such as magnesium ions, EDTA, dithiothreitol, glycerol, surfactants, etc., but there are no particular limitations. In certain embodiments, the above reagents may also contain substances required for attaching poly-A to the 3’ end of RNA. Regarding the substances required for adding poly-A, the following can be listed: ATP, poly-A polymerase, buffers (such as Torres buffer, phosphate buffer, Goodyear buffer, etc.), salts such as NaCl, metal ions such as magnesium ions and manganese ions, EDTA, dithiothreitol, glycerol, surfactants, etc., but there are no particular limitations. In certain embodiments, the above reagents may also contain substances required for the preparation of template DNA for in vivo transcription reactions. The following substances are required for the preparation of template DNA for in vitro transcription reactions: restriction enzymes, buffers (such as Torres buffer, phosphate buffer, Goodyear buffer, etc.), salts such as NaCl, metal ions such as magnesium ions, EDTA, dithiothreitol, glycerol, surfactants, PCR enzymes, oligoDNA, etc., but there are no particular limitations. In certain embodiments, the above reagents may also contain additional substances required for the purification of template DNA for in vitro transcription reactions. The following substances are required for the purification of template DNA: DNA purification beads, DNA purification columns, etc., but there are no particular limitations. In certain embodiments, the above reagents may also contain additional substances required for RNA purification. Regarding the materials required for RNA purification, examples include RNA purification beads and RNA purification columns, but there are no particular limitations.

This invention further provides a method for RNA synthesis, comprising acting the aforementioned RNA polymerase on template DNA. RNA synthesis is carried out via an in vivo transcription reaction. Examples of in vivo transcription reactions include methods using straight-stranded or circular DNA as a template to synthesize RNA using the aforementioned RNA polymerase, but there are no particular limitations. Examples of in vivo transcription reactions include methods using straight-stranded or circular DNA as a template in the presence of a buffer and ribonucleotides to synthesize RNA using the aforementioned RNA polymerase, but there are no particular limitations. Regarding the concentration of RNA polymerase added during the reaction, it can be added in the range of 0.1 U/μL to 20 U/μL, for example, but there are no particular limitations. Regarding the concentration of linear or circular DNA, it can be added in the range of 1 ng/μL to 200 ng/μL, but there are no particular limitations. Regarding the ribonucleotides used in the in vivo transcription reaction, in addition to ATP, CTP, GTP, and UTP, modified bases such as those modified by deamination, methylation, methoxylation, and pseudouridineation can also be listed, but there are no particular limitations. Regarding the concentration of ribonucleotides, it can be added in the range of 0.1 mM to 20 mM, but there are no particular limitations. During in vitro transcription reactions, cap-like substances such as ARCA, mCAP, CleanCap (registered trademark) Reagent AG, CleanCap (registered trademark) Reagent M6, and CleanCap (registered trademark) Reagent AU (manufactured by TriLink) can be added, but there are no particular limitations. Regarding the concentration of these cap-like substances, for example, they can be added in the range of 0.1 mM to 20 mM, but there are no particular limitations. Regarding the buffers used in in vitro transcription reactions, examples include: Torres buffer, phosphate buffer, Goodyear buffer, etc., but there are no particular limitations. Regarding the concentration of the buffer, for example, it can be added in the range of 5 mM to 500 mM, but there are no particular limitations. During in vivo transcription, RNase inhibitors, pyrophosphatase, etc., may be added, but there are no particular limitations. Regarding the concentration of RNase inhibitors, for example, it can be added in the range of 0.1 U/μL to 20 U/μL, but there are no particular limitations. Regarding the concentration of pyrophosphatase, for example, it can be added in the range of 0.0001 U/μL to 0.02 U/μL, but there are no particular limitations. During in vivo transcription, salts such as NaCl, metal ions such as magnesium ions, etc., may be added, but there are no particular limitations. Regarding the concentration of NaCl, for example, it can be added in the range of 1 mM to 200 mM, but there are no particular limitations. Regarding the concentration of magnesium ions, for example, it can be added in the range of 0.1 mM to 20 mM, but there are no particular limitations. During in vitro transcription reactions, EDTA, dithiothreitol, glycerol, surfactants, spermidine, etc., may be added, but there are no particular limitations. Regarding the concentration of EDTA, for example, it can be added in the range of 0.0001 mM to 2 mM, but there are no particular limitations. Regarding the concentration of dithiothreitol, for example, it can be added in the range of 0.1 mM to 20 mM, but there are no particular limitations. Regarding the concentration of glycerol, for example, it can be added in the range of 0.1% to 20%, but there are no particular limitations. Regarding the concentration of surfactants, for example, it can be added in the range of 0.0001% to 1%, but there are no particular limitations. Regarding the concentration of spermidine, for example, it can be added in the range of 0.1 mM to 20 mM, but there are no particular limitations.

Furthermore, as a specific embodiment, the present invention provides an RNA synthesis method, wherein the proportion of dsRNA in the RNA synthesized by the aforementioned RNA synthesis method is less than 90% compared to the proportion of dsRNA in the RNA synthesized under the same conditions using the corresponding wild-type RNA polymerase. The aforementioned proportion is 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 aforementioned ratio can be calculated by comparing the values obtained by performing RNA synthesis under the same conditions using the corresponding wild-type RNA polymerase and the various mutant RNA polymerases that will be used as the test subjects, for example, according to the method described in "[Method for Determining the Ratio of dsRNA in RNA after Transcription Reaction]".

Furthermore, as a specific embodiment, the present invention provides an RNA synthesis method, wherein the amount of RNA synthesized by the aforementioned RNA synthesis method (RNA yield) is at least 110% of the amount of RNA synthesized under the same conditions using the corresponding wild-type RNA polymerase. Preferably, the aforementioned ratio is 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 aforementioned ratio can be calculated by, for example, using a method described in "[Method for Determining the Ratio of dsRNA in RNA After Transcription Reaction]", under the same conditions, performing steps with the corresponding wild-type RNA polymerase and each mutant RNA polymerase that will be used as the test target until the concentration of purified RNA is measured. The RNA yield is calculated by multiplying the volume of purified RNA by the RNA concentration, and then comparing these RNA yields.

Furthermore, as a specific embodiment, this invention also provides a method for synthesizing mRNA using the aforementioned RNA polymerase. That is, in the aforementioned RNA synthesis method, RNA synthesis can also be mRNA synthesis. Regarding the method for synthesizing mRNA, examples include: using linear or circular DNA as a template in the presence of a buffer and ribonucleotides to synthesize mRNA using the aforementioned RNA polymerase, but there are no particular limitations. Regarding the concentration of RNA polymerase added during the reaction, for example, it can be added in the range of 0.1 U/μL to 20 U/μL, but there are no particular limitations. Regarding the concentration of linear or circular DNA, for example, it can be added in the range of 1 ng/μL to 200 ng/μL, but there are no particular limitations. Regarding the ribonucleotides used in mRNA synthesis, in addition to ATP, CTP, GTP, and UTP, modified bases such as those modified by deamination, methylation, methoxylation, and pseudouridineation can also be included, but there are no particular limitations. Regarding the concentration of ribonucleotides, for example, a range of 0.1 mM to 20 mM can be added, but there are no particular limitations. Regarding the buffers used in mRNA synthesis, examples include Torres buffer, phosphate buffer, and Goodyear buffer, but there are no particular limitations. Regarding the concentration of the buffer, for example, a range of 5 mM to 500 mM can be added, but there are no particular limitations. RNase inhibitors, pyrophosphatases, etc., can also be added during mRNA synthesis, but there are no particular limitations. Regarding the concentration of RNase inhibitors, for example, a range of 0.1 U/μL to 20 U/μL can be added, but there are no particular limitations. Regarding the concentration of pyrophosphatase, for example, a range of 0.0001 U/μL to 0.02 U/μL can be added, but there are no particular limitations. Salts such as NaCl, metal ions such as magnesium ions, etc., can be added during mRNA synthesis, but there are no particular limitations. Regarding the concentration of NaCl, for example, a range of 1 mM to 200 mM can be added, but there are no particular limitations. Regarding the concentration of magnesium ions, for example, a range of 0.1 mM to 20 mM can be added, but there are no particular limitations. During mRNA synthesis, EDTA, dithiothreitol, glycerol, surfactants, spermidine, etc., can be added, but there are no particular limitations. Regarding the concentration of EDTA, for example, it can be added in the range of 0.0001 mM to 2 mM, but there is no particular limitation. Regarding the concentration of dithiothreitol, for example, it can be added in the range of 0.1 mM to 20 mM, but there is no particular limitation. Regarding the concentration of glycerol, for example, it can be added in the range of 0.1% to 20%, but there is no particular limitation. Regarding the concentration of surfactant, for example, it can be added in the range of 0.0001% to 1%, but there is no particular limitation. Regarding the concentration of spermidine, for example, it can be added in the range of 0.1 mM to 20 mM, but there is no particular limitation. If the 5' end of the mRNA contains a cap structure, the methods for attaching the cap structure include: post-transcriptional capping and co-transcriptional capping, but there is no particular limitation. Regarding post-transcriptional capping, examples include: using capping enzymes, GTP, or SAM to attach Cap-0 to the 5' end of mRNA; and further, as needed, using mRNA Cap 2'-O-methyltransferase or SAM to convert Cap-0 to Cap-1, etc., but there are no particular limitations. Regarding co-transcriptional capping, examples include: 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), etc., but there are no particular limitations. Regarding the concentration of cap analogs, for example, they can be added in the range of 0.1 mM to 20 mM, but there are no particular limitations. Regarding methods for adding poly-A to the 3' end of synthesized RNA when poly-A is present at the 3' end of mRNA but not in the template DNA, methods such as using poly-A polymerase can be listed, but there are no particular limitations.

Furthermore, as a specific embodiment, the present invention provides an mRNA synthesis method, wherein the proportion of dsRNA in the mRNA synthesized by the aforementioned mRNA synthesis method is less than 90% relative to the proportion of dsRNA in the mRNA synthesized under the same conditions using the corresponding wild-type RNA polymerase. Preferably, the aforementioned proportion is 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 aforementioned proportion can be calculated by, for example, using a method described in "[Method for Determining the Proportion of dsRNA in RNA After Transcription]", by synthesizing mRNA under the same conditions using the corresponding wild-type RNA polymerase and each mutant RNA polymerase that will be the test target, calculating the proportion of dsRNA in the mRNA, and then comparing the values.

Furthermore, as a specific embodiment, the present invention provides a method for mRNA synthesis, wherein the amount of mRNA synthesized by the aforementioned method (mRNA yield) is at least 110% of the amount of mRNA synthesized under the same conditions using the corresponding wild-type RNA polymerase. Preferably, the aforementioned ratio is 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 aforementioned ratio can be calculated by, for example, using a method described in "[Method for Determining the Ratio of dsRNA in RNA After Transcription Reaction]", under the same conditions, performing the steps with the corresponding wild-type RNA polymerase and each mutant RNA polymerase that will be used as the test object until the concentration of purified mRNA is measured. The mRNA yield is calculated by multiplying the volume of purified mRNA by the mRNA concentration, and these mRNA yields are then compared.

Furthermore, as a specific embodiment, this invention also provides a method for synthesizing non-coding RNA, which uses the aforementioned RNA polymerase to synthesize non-coding RNA. That is, in the aforementioned RNA synthesis method, RNA synthesis can also be non-coding RNA synthesis. Examples of non-coding RNA include: microRNA, siRNA, piRNA, rRNA, tRNA, snRNA, snoRNA, SLRNA, SRPRNA, mRNA-like non-coding RNA, oligonucleotides, etc., but there are no particular limitations. As for the method of non-coding RNA synthesis, as an example, a method can be listed where RNA is synthesized using template DNA containing DNA corresponding to the target non-coding RNA and by means such as the method described above, but there are no particular limitations. The template DNA can be any DNA containing DNA corresponding to the target non-coding RNA. Preferably, it contains a promoter sequence (T7 promoter, T3 promoter, SP6 promoter, etc.) recognized by the RNA polymerase of this invention upstream of the corresponding DNA. The template DNA can be prepared by any method, such as restriction enzyme treatment of plasmids or using DNA synthesized by artificial gene synthesis, but there are no particular limitations. As for the plasmid, any plasmid containing DNA corresponding to the target non-coding RNA can be used, and it can also be a plasmid containing the promoter sequence, restriction enzyme site, drug resistance gene, etc., as described above. Regarding the method for obtaining the aforementioned plasmids, as an example, the following can be listed: DNA corresponding to the target non-coding RNA is artificially synthesized and selectively colonized onto any plasmid (which contains a promoter sequence (T7 promoter, T3 promoter, SP6 promoter, etc.) recognized by the RNA polymerase of this invention upstream of the site where the DNA is to be inserted, a restriction enzyme site downstream, and, as needed, an antibiotic resistance gene at any position), and then the plasmid is transformed into any host (preferably Escherichia coli) by any method. The resulting recombinant host cells are cultured by any method, and the plasmids are extracted, but there are no particular limitations. The obtained plasmid can be made into a linear chain by restriction enzyme treatment with any restriction enzyme that can utilize the restriction enzyme sites contained in the plasmid, and then purified by any method to serve as template DNA. Regarding the method of using DNA synthesized by artificial gene synthesis, examples include: artificially synthesizing double-stranded DNA in the form of DNA containing DNA corresponding to the target non-coding RNA and containing a promoter sequence (T7 promoter, T3 promoter, SP6 promoter, etc.) recognized by the RNA polymerase of this invention upstream of that DNA; further amplifying it as needed using PCR, etc.; and using the further purified double-stranded DNA as template DNA, but there are no particular limitations.

Furthermore, as a specific embodiment, this invention also provides a method for synthesizing guide RNA for gene editing, which uses the aforementioned RNA polymerase to synthesize guide RNA for gene editing. Regarding gene editing methods, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR associated protein 9) systems can be listed, but there are no particular limitations.

Furthermore, as a specific embodiment, this invention also provides a method for using the aforementioned RNA polymerase in the synthesis of RNA pharmaceuticals. Regarding RNA pharmaceuticals, examples include those using mRNA, siRNA, microRNA, antisense RNA, and aptamers, but there are no particular limitations. As one example of the method for using the aforementioned RNA polymerase in the synthesis of RNA pharmaceuticals, examples include: using the aforementioned RNA polymerase and a template DNA containing DNA corresponding to any RNA (mRNA, siRNA, microRNA, antisense RNA, aptamers, etc.) used in RNA pharmaceuticals, and performing an in vivo transcription reaction by, for example, the method described above, but there are no particular limitations. The template DNA containing DNA corresponding to any RNA (mRNA, siRNA, microRNA, antisense RNA, adaptor, etc.) can be any template DNA. It can be a template DNA containing a promoter sequence (T7 promoter, T3 promoter, SP6 promoter, etc.) recognized by the RNA polymerase of this invention upstream, a 5' UTR upstream as needed, and a 3' UTR downstream, as well as poly-A, restriction enzyme sites, etc. The aforementioned template DNA can be prepared by any method, using methods known to those skilled in the art to which this invention pertains. For example, the method described above for the synthesis of non-coding RNA can be appropriately modified.

Furthermore, as a specific embodiment, this invention also provides a method for using the aforementioned RNA polymerase in a isothermal amplification reaction. Examples of isothermal amplification reactions include the NASBA method and the TMA method, but there are no particular limitations.

Furthermore, as a specific embodiment, this invention also provides a method for performing gene testing, which uses the aforementioned RNA polymerase to perform gene testing. Methods for performing gene testing include: methods such as amplifying and detecting the genes of a target pathogen using the aforementioned isothermal amplification reaction, thereby confirming the presence of the pathogen, etc., but there are no particular limitations.

Furthermore, as a specific embodiment, the present invention also provides a method for in vivo protein expression or in vitro cell-free protein synthesis, which uses the aforementioned RNA polymerase to perform in vivo protein expression or in vitro cell-free protein synthesis. As an example of the method for in vivo protein expression using the aforementioned RNA polymerase, one could include transfecting mRNA synthesized using the aforementioned RNA polymerase into any cell by any method, without particular limitation. In this specific embodiment, any mRNA can be used to enable the expression of the target protein; preferably, it is RNA containing the target protein, and it can also be mRNA containing a 5'UTR upstream and a 3'UTR downstream, as well as poly-A, etc. In a specific embodiment, regarding the method for synthesizing mRNA, template DNA containing DNA encoding the target protein can be used as a template, and in one example, in vitro transcription can be performed using the method described above, but there are no particular limitations. The aforementioned template DNA can be prepared by any method, and methods known to those skilled in the art to which this invention pertains can be used, such as appropriately modifying the method described above for the synthesis of non-coding RNA. In a specific embodiment, during protein expression, culture can be performed as needed after transfection, but there are no particular limitations. Regarding the method for in vitro cell-free protein synthesis using the aforementioned RNA polymerase, as an example, methods can be described by adding one or more of the aforementioned RNA polymerase, the aforementioned polynucleotide, the aforementioned vector, the aforementioned recombinant host cell, and/or the aforementioned RNA polymerase, the aforementioned polynucleotide, the aforementioned vector, and the aforementioned recombinant host cell to any cell-free protein synthesis reagent, but there are no particular limitations. [Example]

The present invention will now be described in detail with reference to embodiments. However, the present invention is not limited to the embodiments described below.

[Example 1: Preparation of Plastomeres for T7 RNA Polymerase Expression] The gene encoding the wild-type T7 RNA polymerase with a 6×His tag appended to the N-terminus (Sequence No. 2) was artificially synthesized and inserted into the EcoRI-PstI select site of pkk223-3 to create a plasomer containing the gene encoding the wild-type T7 RNA polymerase with a 6×His tag appended to the N-terminus. This expression plasomer will be referred to as pkk223-3-T7RNAP. Using pkk223-3-T7RNAP as a template, site-specific mutations were introduced using the primers described in Table 1 and the KOD-Plus-Mutagenesis Kit (manufactured by Toyoshoku) to prepare expression plasomers for various modified T7 RNA polymerases. The expression plastids of various modified T7 RNA polymerases with multiple amino acid alterations are produced by using the expression plastids of modified T7 RNA polymerases obtained by introducing site-specific mutations as templates and repeatedly introducing site-specific mutations in the same manner.

[Table 1] Table 1: List of primers used for mutation introduction mutation Forward induction sequence numbering Reverse inlet sequence numbering A61E 3 4 A61D 5 4 A61S 6 4 A61G 7 4 A61P 8 4 D156N 9 10 N165S 11 12 N165E 13 12 N165R 14 12 N165P 15 12 D189N 16 17 S661N 18 19

Each resulting plasmid has a base sequence encoding a modified T7 RNA polymerase with a 6×His tag appended to the N-terminus.

[Example 2: Preparation of T7 RNA Polymerase] Wild-type T7 RNA polymerase with a 6×His tag attached to its N-terminus and all modified T7 RNA polymerase systems were prepared using the same method. The following example illustrates the preparation of wild-type T7 RNA polymerase. pkk223-3-T7 RNAP was transformed into *Escherichia coli* JM109 and incubated statically at 37°C for 20 hours on LB agar medium containing 100 μg/mL ampicillin. A single colony from the agar medium was inoculated into 3 mL of LB liquid medium containing 100 μg/mL ampicillin and incubated in a 15 mL test tube at 37°C with shaking for 20 hours. 1 mL of this culture medium was inoculated into 80 mL of TB liquid medium containing 100 μg/mL ampicillin. After incubation in a 500 mL Sakaguchi flask at 37°C with shaking for 20 hours, the bacterial cells were recovered from the culture medium by centrifugation. 1 g of the obtained bacterial cells was suspended in 10 mL of a 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 using an ultrasonic disruptor on ice. The bacterial cell lysate was centrifuged at 20,000 × g at 4°C for 20 minutes. After recovering the supernatant, this supernatant was used for purification with His GraviTrap (manufactured by GE HealthCare). The equilibration and washing buffers were (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10% glycerol, 1 mM DTT, 20 mM imidazole), and the precipitation buffers were (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10% glycerol, 1 mM DTT, 300 mM imidazole). The elution fraction was recovered and replaced with a preservation buffer (20 mM _KPO4 (pH 7.7), 100 mM NaCl, 50% glycerol, 0.1 mM EDTA, 5 mM DTT, 0.01% Triton X-100) to obtain wild-type T7 RNA polymerase with a 6×His tag attached to the N-terminus. In the above preparation example, by using the expression plastids of each modified T7 RNA polymerase prepared in Example 1 during transformation, each modified T7 RNA polymerase with a 6×His tag attached to the N-terminus was obtained.

[Example 3: Determination of Specific Activity of T7 RNA Polymerase] Wild-type T7 RNA polymerase with a His tag attached to its N-terminus and various 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 the same His tag attached to their N-terminus were used, and their activity was determined by the aforementioned activity assay method. For the template DNA used, a 4kbp dsDNA (sequence number 20) containing the T7 promoter sequence was used. The length downstream of the T7 promoter of this dsDNA is approximately 1 kbp, and the length of the transcribed RNA is approximately 1 kb. The protein concentration of each variant of T7 RNA polymerase was calculated by measuring the absorbance of A280, and the specific activity was calculated by dividing the activity by the protein concentration. The results are shown in Table 2.

[Table 2] Table 2: Specific activity of various mutants Specific activity (KU/mg) Comparative activity relative to wild type wild type 149 1.00 A61E 279 1.87 A61D 316 2.12 A61S 254 1.70 A61P 292 1.96 D156N 208 1.40 N165S 234 1.57 D189N 201 1.35 S661N 231 1.55 A61E/D156N 356 2.39 A61E/N165S 402 2.70 A61E/D189N 450 3.02 A61E/S661N 399 2.68 A61P/N165S 377 2.53 A61P/D189N 296 1.99 A61P/S661N 320 2.15

The results in Table 2 confirmed that all modified T7 RNA polymerases showed increased activity compared to the wild type.

[Example 4: Determination of the thermostability of T7 RNA polymerase] Wild-type T7 RNA polymerase with a His tag attached to the N-terminus and various modified T7 RNA polymerases (A61E, D156N, N165S, D189N, S661N) with the same His tag attached to the N-terminus were diluted to 50 U/μL using a storage buffer (20 mM _KPO4 (pH 7.7), 100 mM NaCl, 50% glycerol, 0.1 mM EDTA, 5 mM DTT, 0.01% Triton (registered trademark) X-100). The activity value before storage was measured according to the procedure described in the aforementioned activity assay method. Each T7 RNA polymerase diluted in the aforementioned preservation buffer was heat-treated in an incubator at 50°C for 5 minutes, and its activity was measured. The residual activity rate was calculated from the activity values before and after preservation. The results are shown in Table 3.

[Table 3] Table 3: Residual activity rates of various mutants Residual activity rate (%) after heat treatment at 50°C for 5 minutes wild type 50 A61E 81 D156N 67 N165S 90 D189N 108 S661N 94

The results in Table 3 confirm that, for all modified T7 RNA polymerases, the residual activity after heat treatment is higher than that of the wild type.

[Example 5: Determination of dsRNA Levels After Transcription] The activity assay method used in Example 3 was employed to determine the levels of wild-type T7 RNA polymerase with a His tag attached to its N-terminus, as well as various modified T7 RNA polymerases with the same His tag attached to their N-terminus. RNA polymerases (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) The activities of 6N/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, A61P/D156N/N165S/D189N/S661N, were determined by using a 20mM buffer. The reaction mixture was diluted to 50 U/μL _{with KPO4} (pH 7.7), 100 mM NaCl, 50% glycerol, 0.1 mM EDTA, 5 mM DTT, and 0.01% Triton X-100. Then, 1 μL of each T7 RNA polymerase (50 U/μL) was added to 49 μL of reaction solution (the following shows the final concentration in 50 μL of reaction solution after enzyme addition): 40 mM Tris-HCl (pH 8.0), 50 mM NaCl, 8 mM MgCl2 _, 5 mM DTT, 10 ng/μL template DNA, 0.4 mM ATP, 0.4 mM CTP, 0.4 mM GTP, 0.4 mM UTP, 0.4 U/μL After adding the RNase inhibitor, the reaction was carried out at 37°C for 1 hour using a heating block. The template DNA was dsDNA (Sequence No. 21) containing the T7 promoter, 5'UTR, 3'UTR, poly-A (100nt), and the firefly luciferase gene (Fluc) as the coding sequence (CDS). The generated RNA was approximately 1.9kb mRNA without a cap, consisting of the 5'UTR, CDS (Fluc), 3'UTR, and poly-A from the 5' side. After the reaction, 2.5μL of Turbo DNase (2U/μL, manufactured by Thermo Fisher Scientific) was added, and the reaction was further carried out at 37°C for 15 minutes. The reaction solution was purified using the Monarch RNA Cleanup Kit (manufactured by NEB) to obtain purified RNA. The absorbance (A260) of the purified RNA at 260 nm was measured, and the RNA concentration was calculated by assuming that an A260 of 1 corresponds to an RNA concentration of 40 ng/μL. A calibration curve was constructed using a double-stranded RNA (dsRNA) ELISA kit (K1-based) (Exalpha Biologicals) from the ELISA results obtained against the attached dsRNA control group. Each purified RNA was diluted to 100 ng/μL, and then further diluted to multiple concentrations. ELISA was performed using a double-stranded RNA (dsRNA) ELISA kit (K1-based). The dsRNA concentration was calculated from the measured values that would form the detection line. The proportion (%) (w/w) of dsRNA in the RNA was then calculated by dividing the dsRNA concentration by the RNA concentration. The results are shown in Table 4.

[Table 4] Table 4: Proportion of dsRNA in RNA produced by various mutants RNA concentration (ng/μL) dsRNA concentration (ng/μL) The percentage (%) of dsRNA in RNA (w/w) The ratio of mutant dsRNA levels to wild-type dsRNA levels (%) wild type 100 0.157 0.157 100 A61E 100 0.018 0.018 11 A61D 100 0.020 0.020 13 A61S 100 0.039 0.039 25 A61G 100 0.020 0.020 13 A61P 100 0.002 0.002 1 D156N 100 0.051 0.051 32 N165S 100 0.043 0.043 27 N165E 100 0.040 0.040 25 N165R 100 0.081 0.081 52 N165P 100 0.008 0.008 5 D189N 100 0.044 0.044 28 S661N 100 0.093 0.093 59 A61E/D156N 100 0.015 0.015 10 A61E/N165S 100 0.006 0.006 4 A61E/D189N 100 0.010 0.010 6 A61E/S661N 100 0.014 0.014 9 A61P/N165S 100 0.001 0.001 1 A61P/D189N 100 0.002 0.002 1 A61P/S661N 100 0.003 0.003 2 A61E/D156N/N165S 100 0.013 0.013 8 A61E/N165S/D189N 100 0.024 0.024 15 A61E/N165S/S661N 100 0.018 0.018 11 A61P/D156N/N165S 100 0.001 0.001 1 A61P/N165S/D189N 100 0.002 0.002 1 A61P/N165S/S661N 100 0.001 0.001 1 A61E/D156N/N165S/D189N 100 0.017 0.017 11 A61E/N165S/D189N/S661N 100 0.032 0.032 20 A61P/D156N/N165S/D189N 100 0.001 0.001 1 A61P/N165S/D189N/S661N 100 0.001 0.001 1 A61E/D156N/N165S/D189N/S661N 100 0.016 0.016 10 A61P/D156N/N165S/D189N/S661N 100 0.001 0.001 1

The results in Table 4 confirm that for all modified T7 RNA polymerases, the ratio of dsRNA to RNA after transcription is reduced compared to the wild-type. [Industry Availability]

This invention is suitable for RNA synthesis for a wide variety of purposes.

TW202536172A_113150822_SEQL.xml

Claims (13)

A ribonucleic acid polymerase having an amino acid sequence that is more than 90% identical to the amino acid sequence of Sequence Number 1 and at least one amino acid in a group consisting of amino acids at positions 61, 156, 189, 165 and 661 of the amino acid sequence of Sequence Number 1 has been modified. A ribonucleic acid polymerase having an amino acid sequence that is more than 90% identical to the amino acid sequence of Sequence No. 1 and satisfies at least one of the following groups (i) to (v): (i) the amino acid at position 61 of the amino acid sequence of Sequence No. 1 is glutamic acid, aspartic acid, serine, glycine, or proline; (ii) the amino acid at position 156 of the amino acid sequence of Sequence No. 1 is aspartic acid; (iii) the amino acid at position 189 of the amino acid sequence of Sequence No. 1 is aspartic acid; (iv) the amino acid at position 165 of the amino acid sequence of Sequence No. 1 is serine, glutamic acid, arginine, or proline; and (v) The amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is aspartic acid. The ribonucleic acid polymerase described in claim 1 or 2, wherein the aforementioned ribonucleic acid polymerase has an amino acid sequence that is more than 90% identical to the amino acid sequence of sequence number 1, and the aforementioned amino acid sequence is: (1) the amino acid at position 61 of the amino acid sequence of sequence number 1 is glutamic acid, aspartic acid, serine, glycine, or proline; (2) the amino acid at position 156 of the amino acid sequence of sequence number 1 is aspartic acid; (3) the amino acid at position 189 of the amino acid sequence of sequence number 1 is aspartic acid; (4) the amino acid at position 165 of the amino acid sequence of sequence number 1 is serine, glutamic acid, arginine, or proline; (5) The amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; (6) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, and the amino acid at position 156 of the amino acid sequence corresponding to sequence number 1 is asparagine; (7) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, and the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine; (8) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, and the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine; (9) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, and the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; (10) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, and the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine; (11) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, and the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine; (12) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, and the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; (13) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, the amino acid at position 156 of the amino acid sequence corresponding to sequence number 1 is asparagine, and the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine; (14) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, and the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine; (15) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, and the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; (16) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, the amino acid at position 156 of the amino acid sequence corresponding to sequence number 1 is asparagine, and the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine; (17) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, and the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine; (18) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, and the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; (19) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, the amino acid at position 156 of the amino acid sequence corresponding to sequence number 1 is asparagine, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, and the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine; (20) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine, and the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; (21) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, the amino acid at position 156 of the amino acid sequence corresponding to sequence number 1 is asparagine, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, and the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine; (22) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine, and the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; (23) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is glutamic acid, the amino acid at position 156 of the amino acid sequence corresponding to sequence number 1 is asparagine, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine, and the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine; or (24) The amino acid at position 61 of the amino acid sequence corresponding to sequence number 1 is proline, the amino acid at position 156 of the amino acid sequence corresponding to sequence number 1 is asparagine, the amino acid at position 165 of the amino acid sequence corresponding to sequence number 1 is serine, the amino acid at position 189 of the amino acid sequence corresponding to sequence number 1 is asparagine, and the amino acid at position 661 of the amino acid sequence corresponding to sequence number 1 is asparagine. A polynucleotide that encodes a ribonucleic acid polymerase as described in Request 1. A carrier containing polynucleotides as described in claim 4. A recombinant host cell is transformed using a vector as described in claim 5. A method for producing ribonucleic acid polymerase, wherein the ribonucleic acid polymerase is produced using a polynucleotide as described in claim 4, a vector as described in claim 5, and/or a recombinant host cell as described in claim 6. A reagent comprising a ribonucleic acid polymerase as described in claim 1, a polynucleotide as described in claim 4, a vector as described in claim 5, and/or a recombinant host cell as described in claim 6. A method for synthesizing ribonucleic acid (RNA) comprises acting an RNA polymerase as described in claim 1 on a template deoxyribonucleic acid (DNA). As described in claim 9, the proportion of double-stranded ribonucleic acid (dsRNA) in the synthesized ribonucleic acid is less than 90% compared to the proportion of double-stranded ribonucleic acid in the ribonucleic acid synthesized under the same conditions using the corresponding wild-type ribonucleic acid polymerase. The method for synthesizing ribonucleic acid as described in claim 9 or 10, wherein the aforementioned ribonucleic acid synthesis is the synthesis of communication ribonucleic acid (mRNA). The method for synthesizing ribonucleic acid as described in claim 11, wherein the proportion of double-stranded ribonucleic acid in the synthesized communication ribonucleic acid is less than 90% relative to the proportion of double-stranded ribonucleic acid in the communication ribonucleic acid synthesized under the same conditions using the corresponding wild-type ribonucleic acid polymerase. A method for synthesizing a ribonucleic acid (RNA) drug, wherein the RNA polymerase described in claim 1 is used to synthesize the RNA drug.