RNA polymerase mutant

Abstract

Provided is an RNA polymerase mutant in which an amino acid residue selected from the group consisting of glutamic acid at position 48 and arginine at position 50 in the amino acid sequence of a wild-type T7 RNA polymerase set forth in SEQ ID NO: 1 is substituted with an aliphatic amino acid residue.

Description

RNA polymerase variant

[0001] The present invention relates to an RNA polymerase useful for producing single-stranded RNA, a nucleic acid encoding the RNA polymerase, and a method for producing single-stranded RNA using the RNA polymerase.

[0002] mRNA pharmaceuticals have attracted attention as a new pharmaceutical modality. An mRNA pharmaceutical is a pharmaceutical containing, as an active ingredient, artificially produced mRNA encoding a protein, and exhibits a medical effect based on the function of the protein by producing the protein in the administered living body. In the worldwide spread of the SARS-CoV-2 infection that occurred in 2020, an mRNA vaccine, which is one of the mRNA pharmaceuticals, was put into practical use and contributed greatly to preventing infection by being administered to many people. In addition to being able to be designed in a short period based on the genetic information of the virus, the possibility of mass-producing different types of vaccines in the same manufacturing process, that is, using common facilities, is cited as an advantage compared with other pharmaceuticals.

[0003] RNA is synthesized in vivo by a transcription reaction using double-stranded DNA catalyzed by RNA polymerase as a template. Although it is possible to reproduce the transcription of RNA in vitro by preparing the elements necessary for the reaction, many RNA polymerases present in prokaryotic cells and eukaryotic cells are composed of multiple subunits, and the production of each of them has become an obstacle to practical use. On the other hand, RNA polymerases derived from bacteriophages (T7 phage, T3 phage, K11 phage, etc.), chloroplasts, and mitochondria are composed of a single polypeptide (single-subunit DNA-dependent RNA polymerase: Non-Patent Document 1).

[0004] RNA polymerase is a useful tool in genetic engineering research, and RNA polymerases derived mainly from T7 phage and SP6 phage have been commercially available as research reagents. Typical applications include the preparation of labeled RNA used as hybridization probes and nucleic acid amplification methods combined with reverse transcription and double-stranded cDNA synthesis. Since its basic enzymatic properties had already been elucidated, bacteriophage-derived RNA polymerase is now also used in the industrial production of RNA.

[0005] In the industrial production of RNA, it is advantageous to use highly stable RNA polymerases. For this reason, variants of T7 RNA polymerase with enhanced thermal stability have been created (Patent Document 1). Attempts have also been made to create variants focusing on E42, S43, Y44, E45, M46, G47, A255, R257, A258, G259, A260, L261, and A262 of T7 RNA polymerase (Patent Document 2, Non-Patent Document 2).

[0006] On the other hand, when used in pharmaceuticals administered to humans, RNA needs to have a cap structure at its 5' end for efficient translation. Furthermore, it is known that double-stranded RNA (dsRNA) is produced as a byproduct in the transcription reaction by RNA polymerase, but double-stranded RNA administered into the human body, for example, can induce therapeutically undesirable immune responses via Toll-like receptors and other pathways. Therefore, for the production of mRNA drugs composed of single-stranded RNA, a transcription reaction system that can suppress the production of double-stranded RNA and synthesize RNA with a cap structure is advantageous.

[0007] International public brochure WO2001 / 066705 International public brochure WO2019 / 036682

[0008] Biotechnology Journal International, 20(3), p1-35, 2017NATURE BIOTECHNOLOGY VOLUME 41, p560-568, 2023

[0009] The object of the present invention is to provide a variant of RNA polymerase with reduced double-stranded RNA production ability, which is useful for supplying high-purity single-stranded RNA that can be used as a pharmaceutical or the like.

[0010] As a result of diligent efforts to solve the above problems, the inventors of the present invention have discovered a variant of RNA polymerase that can suppress the production of double-stranded RNA and efficiently transcribe single-stranded RNA by introducing a novel mutation at a specific position in RNA polymerase. Furthermore, they have constructed a high-purity single-stranded RNA synthesis system using this variant, thereby completing the present invention.

[0011] For example, the following embodiments are provided: (1) An RNA polymerase mutant in which an amino acid selected from the group consisting of glutamic acid at position 48 and arginine at position 50 in the amino acid sequence of wild-type T7 RNA polymerase shown in SEQ ID NO: 1 is substituted with another amino acid, provided that the amino acid corresponding to arginine at position 386 is not substituted. (2) The RNA polymerase mutant according to (1), wherein a mutation is introduced into an RNA polymerase having 70% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1. (3) The RNA polymerase mutant according to (1) or (2), comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1. (4) The RNA polymerase mutant according to any one of (1) to (3), wherein an amino acid selected from the group consisting of the group consisting of positions 48 and 50 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with alanine. (5) An RNA polymerase variant according to any one of (1) to (4), wherein an amino acid corresponding to an amino acid selected from the group consisting of serine at position 430, serine at position 633, phenylalanine at position 849, and phenylalanine at position 880 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with an amino acid different from the original amino acid. (6) An RNA polymerase variant according to (5), having an amino acid substitution selected from the group consisting of a substitution of the amino acid corresponding to serine at position 430 in the amino acid sequence shown in SEQ ID NO: 1 with proline, a substitution of the amino acid corresponding to serine at position 633 in the amino acid sequence shown in SEQ ID NO: 1 with proline, a substitution of the amino acid corresponding to phenylalanine at position 849 in the amino acid sequence shown in SEQ ID NO: 1 with isoleucine, and a substitution of the amino acid corresponding to phenylalanine at position 880 in the amino acid sequence shown in SEQ ID NO: 1 with tyrosine. (7) A nucleic acid encoding an RNA polymerase variant according to any one of (1) to (6). (8) A vector holding the nucleic acid according to (7). (9) A method for producing RNA polymerase mutants, comprising the step of culturing cells into which the nucleic acids described in (7) have been introduced.(10) A method for producing single-stranded RNA, characterized by using a reaction solution containing an RNA polymerase variant described in any of (1) to (6). (11) The method according to (10), characterized in that the reaction solution contains a protein selected from a single-stranded RNA-binding protein or a cold shock protein. (12) The method according to (11), wherein the single-stranded RNA-binding protein is the T4 phage gene 32 protein. (13) The method according to (11), wherein the cold shock protein is CspA.

[0012] This invention provides an RNA polymerase variant having RNA transcription activity suitable for the in vitro production of high-purity single-stranded RNA, as well as a method for producing the same. According to this invention, RNA polymerase in which a mutation has been introduced into a specific amino acid can synthesize single-stranded RNA more efficiently while suppressing the production of double-stranded RNA. Therefore, it exhibits a superior effect in supplying RNA of higher purity than conventional methods.

[0013] Figure 1 shows the total amount of RNA synthesized using the RNA polymerase variant of the present invention, either in the absence or in the presence of the capping analog. Figure 2 shows the content of double-stranded RNA in the RNA synthesized using the RNA polymerase variant of the present invention, either in the absence or in the presence of the capping analog.

[0014] (Definitions of Terms) In this specification, nonpolar aliphatic amino acids refer to glycine, alanine, leucine, isoleucine, valine, and proline, and may be written as G, A, L, I, V, and P, respectively. Polar amino acids refer to serine, threonine, glutamine, and asparagine, and may be written as S, T, Q, and N, respectively. Acidic amino acids refer to glutamic acid and aspartic acid, and may be written as E and D, respectively. Basic amino acids refer to lysine, arginine, and histidine, and may be written as K, R, and H, respectively. Aromatic amino acids refer to phenylalanine, tyrosine, and tryptophan, and may be written as F, Y, and W, respectively. Sulfur-containing amino acids refer to cysteine ​​and methionine, and may be written as C and M, respectively.

[0015] In this specification, amino acid sequence identity refers to the percentage of the number of identical amino acid residues in two amino acid sequences when they are optimally aligned with each other. Sequences are said to exhibit identity at a residue position if a position in a first sequence is occupied by the same amino acid residue as the corresponding position in a second sequence. Proper alignment of two sequences can be performed using various algorithms, such as the BLAST algorithm.

[0016] In this specification, an amino acid mutation includes the deletion, addition, insertion, or substitution of one or more amino acids.

[0017] The present invention will be described in detail below.

[0018] 1. RNA Polymerase Variants of the Present Invention The present invention provides variants of single-subunit DNA-dependent RNA polymerase suitable for the production of single-stranded RNA used in mRNA pharmaceuticals and the like.

[0019] RNA polymerase is an enzyme that uses DNA as a template to synthesize RNA strands complementary to its sequence. Those consisting of a single polypeptide chain are called single-subunit DNA-dependent RNA polymerases. The RNA polymerase variant of the present invention is obtained by introducing a mutation into the amino acid sequence of this type of RNA polymerase. While not particularly limited, the RNA polymerase variant provided by the present invention produces less double-stranded RNA compared to wild-type RNA polymerase.

[0020] The RNA polymerase variants of the present invention (hereinafter sometimes referred to as "the variants of the present invention") are preferably variants of bacteriophage-derived RNA polymerases, such as T7 phage RNA polymerase, and RNA polymerase variants with high amino acid sequence identity to said enzyme (for example, RNA polymerase derived from T3 phage, K11 phage, or SP6 phage). While not particularly limiting the present invention, examples of RNA polymerase variants of the present invention include variants in which mutations are introduced into RNA polymerase having 70% or more sequence identity with the wild-type T7 RNA polymerase shown in SEQ ID NO: 1. The amino acid sequences of T3 phage and K11 phage RNA polymerase have 70% or more identity with the amino acid sequence of T7 RNA polymerase. Therefore, the amino acids of T3 RNA polymerase and K11 RNA polymerase corresponding to specific amino acids present in the amino acid sequence of wild-type T7 RNA polymerase shown in SEQ ID NO: 1 can be easily identified (see Non-Patent Literature 1). For example, the R50 position in T7 RNA polymerase corresponds to position 51 in T3 RNA polymerase and position 50 in K11 RNA polymerase. In one aspect of the present invention, a mutant polypeptide is provided that has 90% or more, preferably 95% or more, and more preferably 98% or more sequence identity with the amino acid sequence of T7 RNA polymerase shown in Sequence ID No. 1. This mutant polypeptide has RNA polymerase activity, that is, activity to transcribe RNA using DNA as a template.

[0021] The present invention will be described below using mutants derived from T7 RNA polymerase as an example. In mutants of wild-type T7 RNA polymerase shown in Sequence ID No. 1, E48 and / or R50 are replaced with aliphatic amino acids, and the production of double-stranded RNA is suppressed compared to the wild type. Preferably, in the mutants of the present invention, E48 and R50 are replaced with alanine, which is a nonpolar aliphatic amino acid.

[0022] The RNA polymerase variant of the present invention may have one or more additional mutations, such as amino acid substitutions, in addition to the amino acid substitutions corresponding to positions E48 and / or R50 in the amino acid sequence of wild-type T7 RNA polymerase. These additional mutations are not particularly limited as long as they do not result in a significant decrease in the RNA transcription activity of the variant of the present invention.

[0023] Furthermore, the RNA polymerase variant of the present invention may also be one into which known mutations have been introduced. Patent Document 1 discloses an RNA polymerase variant with improved heat resistance, in which four amino acid substitutions (serine at position 430, serine at position 633, phenylalanine at position 849, and phenylalanine at position 880 are substituted with proline, proline, isoleucine, and tyrosine, respectively) have been introduced into T7 RNA polymerase, a single-subunit DNA-dependent RNA polymerase (SEQ ID NO: 1 shows the amino acid sequence of wild-type T7 RNA polymerase). For example, the RNA polymerase variant of the present invention may have substitutions introduced into one or more amino acids corresponding to amino acids selected from the above amino acids S430, S633, F849, or F880. Preferably, one or more mutations selected from the group consisting of substitution of the amino acid corresponding to S430 with proline, substitution of the amino acid corresponding to S633 with proline, substitution of the amino acid corresponding to F849 with isoleucine, and substitution of the amino acid corresponding to F880 with tyrosine can be introduced. However, in the mutant of the present invention, the amino acid corresponding to arginine at position 386 of SEQ ID NO: 1 is not mutated.

[0024] Mutants of wild-type T7 RNA polymerase in which E48 or R50 is replaced with alanine are named T7-d3 and T7-d4, respectively. These two mutants are examples of mutants of the present invention, exhibiting significantly reduced double-stranded RNA production ability compared to wild-type T7 RNA polymerase. The amino acid sequences of T7-d3 and T7-d4 are shown in SEQ ID NOs: 3 and 4, respectively. Furthermore, a mutant in which R50 of T7 RNA polymerase is replaced with alanine, and in addition, S430, S633, F849, and F880 are replaced with proline, proline, isoleucine, and tyrosine, respectively, is named T7-c3. The amino acid sequence of T7-c3 is shown in SEQ ID NO: 8. That is, the polypeptide containing the amino acid sequence shown in SEQ ID NO: 8 is an example of a mutant of the present invention. T7-c3 exhibits particularly reduced double-stranded RNA production ability compared to wild-type T7 RNA polymerase.

[0025] In one aspect of the present invention, the mutant of the present invention may have other peptides attached. Examples of such peptides include, but are not limited to, signal peptides necessary for the secretory expression of the mutant of the present invention and affinity tags useful for the purification of the mutant. The signal peptide can be selected from a suitable one derived from a known secretory protein or a variant thereof, depending on the host used to produce the mutant of the present invention, and is usually attached to the N-terminus of the mutant. The affinity tag can be any one whose ligand is known to be used for capture, and can be selected from known ones. Examples of affinity tags include histidine (His) tags, HN tags, HAT tags, glutathione S-transferase (GST) tags, maltose-binding protein (MBP) tags, C-Myc tags, and the Strep tag (Strep-tag II) consisting of eight amino acid residues (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys). These tags may be attached to either the N-terminus or the C-terminus of the mutant. Furthermore, affinity tags can be removed from the mutant of the present invention after purification. For this purpose, an affinity tag is used that allows for the insertion of a protease recognition sequence, such as Factor Xa, PreScission Protein, thrombin, enterokinase, or TEV protease (Tobacco Etch Virus Protein), between the RNA polymerase variant of the present invention and the affinity tag.

[0026] The RNA polymerase variant of the present invention can be prepared using known recombinant protein preparation methods. Based on the nucleotide sequence information of the wild-type single-subunit DNA-dependent RNA polymerase gene, the amino acid codons corresponding to positions E48 and R50 in the amino acid sequence of T7 RNA polymerase can be identified and replaced with codons of other amino acids, such as nonpolar aliphatic amino acids. A nucleic acid having the nucleotide sequence thus designed, i.e., a nucleic acid encoding the variant of the present invention, can be prepared and, using an appropriate expression vector and host, the variant can be obtained as described below. If it is desired to introduce a mutation at a position other than those corresponding to E48 and R50, the nucleotide sequence encoding the desired variant can be designed using a similar procedure. The nucleic acid encoding the variant of the present invention may be chemically synthesized, or it may be prepared by introducing site-directed mutagenesis into the nucleic acid encoding wild-type RNA polymerase.

[0027] The RNA polymerase variant of the present invention can be used not only for the production of RNA, but also for nucleic acid amplification methods that include RNA polymerase as a component (for example, the NASBA method described in U.S. Patent No. 5,130,238: Nucleic Acid Sequence-based Amplification, the 3SR method described in WO90 / 06995: Self-sustained sequence replication reaction, the SMART method described in WO99 / 37806: Signal mediated amplification of RNA technology, the TMA method described in WO91 / 01384: Translation-mediated amplification, etc.). These are methods that can efficiently amplify target nucleic acids under isothermal conditions.

[0028] 2. Nucleic acids encoding RNA polymerase variants of the present invention The present invention provides nucleic acids encoding the RNA polymerase variants described in 1. (hereinafter sometimes referred to as "nucleic acids of the present invention").

[0029] The nucleic acid of the present invention may be any nucleic acid that encodes the RNA polymerase variant of the present invention, and is not limited to nucleic acids with a specific base sequence. As described above, the base sequence of the nucleic acid of the present invention is designed by identifying the codons corresponding to the amino acids to be substituted based on the base sequence information of the wild-type RNA polymerase gene, and changing them to codons of other amino acids. In order to increase the expression level of the RNA polymerase variant of the present invention in the host, the codons of the designed base sequence may be changed to those suitable for the host without changing the amino acid sequence encoded by the sequence (codon optimization).

[0030] As described above, the RNA polymerase variant of the present invention may have a signal peptide or affinity tag attached. Therefore, the nucleic acid of the present invention includes nucleic acids encoding the variant of the present invention to which a signal peptide or affinity tag has been attached. The base sequence of the nucleic acid encoding the signal peptide or affinity tag may be designed according to their amino acid sequences, and in doing so, codon selection may be made considering the host to be used.

[0031] The nucleic acids encoding the RNA polymerase variant of the present invention may be chemically synthesized, or they may be prepared by introducing site-directed mutagenesis into nucleic acids encoding wild-type RNA polymerase. Methods for introducing site-directed mutagenesis into nucleic acids are well known to those skilled in the art, and kits for such methods are commercially available.

[0032] The PET system, a recombinant protein expression system using E. coli as a host, utilizes bacteriophage-derived RNA polymerase. By introducing a nucleic acid encoding the desired protein downstream of a promoter recognized by RNA polymerase into E. coli, and then expressing RNA polymerase within the same E. coli, the expression of the desired protein is strongly induced. In this system, RNA polymerase is supplied from an RNA polymerase gene integrated into the E. coli chromosome or from an expression vector introduced into E. coli. The nucleic acid of the present invention can be used as a source of RNA polymerase in the PET system or similar expression systems.

[0033] 3. Vectors Containing Nucleic Acids Encoding the RNA Polymerase Variant of the Present Invention The present invention provides vectors that contain nucleic acids encoding the RNA polymerase variant of the present invention. The vectors of the present invention are useful for introducing nucleic acids encoding the RNA polymerase variant of the present invention into a suitable host for the production of the RNA polymerase variant of the present invention or for other purposes.

[0034] The vectors that hold the nucleic acids encoding the RNA polymerase variant of the present invention are not particularly limited. Vectors that can automate in host cells or vectors that can be incorporated into host chromosomes can be used. For example, plasmid vectors, phage vectors, viral vectors, artificial chromosomes, etc., can be used. The vector should be selected to suit the host to which it will be used. Vectors suitable for various hosts (E. coli, Bacillus bacteria, yeast, filamentous fungi, insect cells, mammalian cells, etc.) are well known to those skilled in the art, and many are also commercially available. These known vectors and their modifications can be used in the present invention. Many expression vectors for expressing recombinant proteins in a host have also been constructed.

[0035] Expression vectors contain appropriate promoters and other transcription and translation-related factors (operators, terminators, enhancers, ribosome binding sites, etc.) that can function within the host, making them suitable for producing the gene product in the host. For example, expression vectors for E. coli use promoters such as the trp promoter, lac promoter, PL promoter, PR promoter, and their modifications, but are not limited to those mentioned above. If the promoter is inducible, gene product expression can be induced by appropriate manipulation.

[0036] The expression vector used in the present invention may further contain nucleic acids encoding a signal peptide or an affinity tag. These nucleic acids encoding the peptides are positioned so that a fusion protein of the RNA polymerase variant of the present invention and the peptide is expressed. Since vectors with inserted nucleic acids encoding affinity tags are also known, the vector of the present invention can be constructed by inserting the nucleic acids of the present invention into such a vector. The expression vector thus constructed, carrying the nucleic acids of the present invention, is useful in the production of the RNA polymerase variant of the present invention.

[0037] 4. Method for Producing the RNA Polymerase Mutant of the Present Invention The present invention provides a method for producing the RNA polymerase mutant of the present invention. The method comprises the step of culturing cells into which nucleic acids encoding the mutant of the present invention have been introduced, and then collecting the RNA polymerase mutant from the resulting culture.

[0038] The cells (hosts) used to produce the mutants of the present invention are not particularly limited, as long as they are hosts used in the production of recombinant proteins. For example, bacteria (such as Escherichia coli and Bacillus subtilis), yeast, filamentous fungi, insect cells, eukaryotic cells, and animal cells (such as mammalian cells including human cells) can be used.

[0039] Typical hosts used in the industrial production of recombinant proteins, such as Escherichia coli and Bacillus subtilis bacteria, are well known to those skilled in the art, and many strains are commercially available. From these, an appropriate host strain can be selected and used, taking into account the production scale and other conditions. Furthermore, methods for producing recombinant proteins using yeast (Saccharomyces and Schizosaccharomyces genera) and insect cells are also known. By introducing the nucleic acid encoding the RNA polymerase variant of the present invention into these host cells, or by creating host cells in which the nucleic acid is incorporated on the chromosome, and culturing the host, the RNA polymerase variant can be expressed and produced.

[0040] The nucleic acid encoding the RNA polymerase variant of the present invention can be introduced into a host by being loaded onto a suitable vector. For example, any vector suitable for expression can be selected. When using a plasmid vector, the introduction method can be appropriately selected depending on the host; for example, methods using calcium ions, lipofection, electroporation, spheroplasty, lithium acetate, etc., can be used. Phage vectors and viral vectors can be used to infect host cells using methods appropriate to the vector, thereby obtaining cells into which the nucleic acid encoding the RNA polymerase variant has been introduced. Furthermore, the PET system described above may be used in the production of the variant of the present invention.

[0041] Cells containing the nucleic acid encoding the RNA polymerase mutant obtained in this way can be cultured, and the mutant of the present invention can be obtained from the culture. The culture conditions are not particularly limited as long as they are suitable for the host, expression vector, etc., used. In addition, known protein purification methods can be used for the isolation and purification of the RNA polymerase mutant. The following describes the case in which Escherichia coli is used as the host. Escherichia coli into which the nucleic acid encoding the mutant of the present invention has been introduced is cultured at a temperature suitable for its growth, for example, 37°C. If the nucleic acid encoding the RNA polymerase mutant is located downstream of an inducible promoter, the expression of the RNA polymerase mutant can be induced by performing an operation suitable for the promoter during culture. After recovering the Escherichia coli cells from the culture and washing them, an Escherichia coli lysate containing the mutant of the present invention can be obtained by sonication, lysozyme treatment, or other lysis treatments. If the mutant of the present invention is secretorially expressed, the supernatant of the culture is recovered. Using this lysate or culture supernatant as a starting material, the RNA polymerase variant of the present invention is purified by appropriately combining purification methods used in this field, such as ammonium sulfate precipitation, anion exchange column, cation exchange column, gel filtration column, affinity chromatography column, filtration, dialysis, etc. Mutants with affinity tags can be easily purified using affinity carriers appropriate to the properties of the affinity tags. For example, mutants of the present invention having affinity tags containing multiple histidines (histidine tags, HN tags, HAT tags, etc.) can be purified using carriers to which a metal such as nickel is bound.

[0042] 5. Method for Producing Single-Stranded RNA of the Present Invention The present invention provides a method for producing single-stranded RNA (in vitro transcription method) using the RNA polymerase variant of the present invention. In the presence of a template double-stranded DNA and four types of ribonucleotide triphosphates, the variant of the present invention synthesizes single-stranded RNA with a sequence complementary to the template DNA. The single-stranded RNA produced by the method of the present invention is not limited to mRNA, and examples include mRNA, RNA probes, ribozymes, guide RNA (used for genome editing with Cas9, etc.).

[0043] In the method for producing single-stranded RNA of the present invention, the double-stranded DNA that can be used as a template has a promoter sequence recognized by the variant of the present invention. The promoter sequences recognized by bacteriophage-derived RNA polymerases are known (see, for example, FEBS Letters, 4, p264-267, 1998). Further, the template double-stranded DNA may contain a sequence encoding a poly(A) tail at the 3'-end of the region encoding the RNA to be transcribed. The origin of the template double-stranded DNA is not particularly limited, and it may be plasmid DNA or a DNA fragment obtained by a nucleic acid amplification method. For example, a double-stranded DNA that can be used as a template can be prepared by inserting a DNA having a base sequence encoding a desired RNA downstream of the promoter into a plasmid carrying a promoter recognized by RNA polymerase. Note that circular DNA such as a plasmid is preferably used after linearization (for example, digestion with a restriction enzyme).

[0044] For ribonucleotide 3-phosphates, usually the four types (ATP, CTP, GTP, UTP) that are substrates of natural-type RNA are used, but one or more of the four types can be replaced with analogs of ribonucleotide 3-phosphates. There is no particular limitation on the analogs of ribonucleotide 3-phosphates. Known analogs known as substrates for RNA polymerase can be used in the method of the present invention. For example, in the production of RNA administered to a living body, pseudouridine 3-phosphate, 1-methylpseudouridine 3-phosphate, or other UTP analogs may be used in place of UTP. The concentration of ribonucleotide 3-phosphate or its analog can be appropriately set according to the purpose and the like, and is usually in the range of 0.2 to 15 mM.

[0045] The reaction solution used for in vitro transcription contains, in addition to a buffer component for maintaining the pH at an appropriate value, magnesium ions and a reducing agent (such as dithiothreitol). It can also contain various other components, such as salts (e.g., NaCl, KCl), polyamines (e.g., spermidine), ribonuclease inhibitors, proteins (e.g., bovine serum albumin), surfactants, etc. The concentrations of these components can be set by referring to the composition of known reaction solutions. The pH of the reaction solution is usually adjusted to the range of 7.0 to 9.0.

[0046] At the 5'-end of eukaryotic mRNA, there is a modification called a cap structure. This modification consists of 7-methylguanosine linked via a 5'-5' triphosphate structure to the 5'-end and methylation at the 2'-O position of the adjacent nucleotide. The cap structure is said to contribute to the stabilization of mRNA and to increase the efficiency of translation by being recognized by eukaryotic translation initiation factors.

[0047] A cap structure can be added to the single-stranded RNA produced by the method of the present invention. By treating the single-stranded RNA with a capping enzyme, such as a capping enzyme derived from vaccinia virus, 7-methylguanosine is added to the 5'-end of the single-stranded RNA (Cap0). Then, when mRNA Cap 2'-O-methyltransferase acts, the 2'-O position of the nucleotide adjacent to 7-methylguanosine is methylated (Cap1).

[0048] In the method of the present invention, a cap structure can be added in parallel with the transcription of single-stranded RNA. In this case, transcription of single-stranded RNA using the variant of the present invention is carried out in a reaction solution containing a compound called a cap analog. The cap analog is not particularly limited, and cap analogs composed of dinucleotides, trinucleotides, or more nucleotides can be used. For example, commercially available cap analogs such as m7G(5')ppp(5')G (standard cap), ARCA (Anti-Reverse Cap Analog), and CleanCap (registered trademark) series cap analogs (manufactured by TriLink) can also be used in the present invention.

[0049] The reaction solution of the present invention may contain inorganic pyrophosphatase. When RNA is synthesized by RNA polymerase, pyrophosphate accumulates as a byproduct, and pyrophosphate inhibits the transcription reaction. Inorganic pyrophosphatase has the activity to catalyze the hydrolysis of pyrophosphate to produce orthophosphate, and therefore, the RNA synthesis reaction is promoted by the degradation of pyrophosphate by this enzyme.

[0050] Furthermore, in the method for producing single-stranded RNA of the present invention, the generation of double-stranded RNA in the RNA transcription reaction can be reduced by using a reaction solution containing a protein that has affinity for nucleic acids. Examples of proteins that have affinity for nucleic acids include single-stranded RNA-binding proteins and cold shock proteins. When a reaction solution for RNA transcription containing a protein that has affinity for nucleic acids is used, the generation of double-stranded RNA is suppressed compared to when these proteins are not included. Examples of single-stranded RNA-binding proteins include the T4 phage gene 32 protein (T4 gp32), the T7 phage gene 2.5 protein (T7 gp2.5), and the single-stranded DNA-binding protein of E. coli. Cold shock proteins are a general term for proteins that are transiently expressed at high levels in bacteria and other organisms when the growth temperature is reduced. In E. coli, a cold shock protein named CspA is known. Furthermore, eight proteins, CspB to CspI, are known to have high amino acid sequence identity with CspA, of which CspB, CspG, and CspI are cold shock proteins [J. Bacteriol., 181, pp. 1603-1609, 1999]. In addition, homologs of these cold shock proteins are also present in microorganisms such as Bacillus subtilis (CspB), Bacillus cardritiscus (CspB), Thermotogamaritima (CspB, CspL), and Lactobacillus plantarum (CspL). While not particularly limited to the present invention, CspA from Escherichia coli is preferred for the RNA transcription reaction.

[0051] 6. The present invention provides a kit containing the RNA polymerase variant of the present invention for use in the synthesis of a desired single-stranded RNA.

[0052] The kit of the present invention may contain, in addition to the variant of the present invention, components necessary for preparing a reaction solution for RNA transcription, such as buffer components, magnesium salts, four types of NTPs or NTP analogs, polyamines, reducing agents, surfactants, and other components. These components may be packaged individually so that they can be mixed at the time of use to prepare the reaction solution, or multiple components may be packaged as a mixture to facilitate the preparation of the reaction solution. Examples include a kit containing a premix solution prepared so that the reaction solution can be completed by adding only template DNA and water (sterilized water, etc.) at the time of use, and a kit containing the premix in a dry state. Other components that may be included in the kit include ribonuclease inhibitors, cap analogs, inorganic pyrophosphatases, bovine serum albumin, and other proteins (single-stranded DNA binding proteins, single-stranded RNA binding proteins, cold shock proteins, etc.).

[0053] The present invention will be described in detail below with reference to examples, but the scope of the present invention is not limited to these examples.

[0054] Experimental Method (1) Preparation of T7 RNA Polymerase Mutant The nucleotide sequence of the gene encoding wild-type RNA polymerase from the Escherichia phage T7 (Bacteriophage T7) strain is disclosed in NCBI Reference Sequence No. NC_001604 [GeneID: 1261050]. The amino acid sequence of the wild-type T7 RNA polymerase encoded by this nucleotide sequence is shown as Sequence ID No. 1 in the sequence listing. An artificial gene having a nucleotide sequence encoding an amino acid sequence in which a desired mutation has been introduced into the said amino acid sequence was chemically synthesized. The obtained artificial gene was introduced into plasmid pET6xHN-N (Takara Bio USA) using the In-Fusion® HD Cloning Kit (Takara Bio USA). The resulting plasmid contains a nucleotide sequence encoding a T7 RNA polymerase variant with a histidine tag attached to its N-terminus.

[0055] Next, Escherichia coli BL21 DE3 strain (manufactured by Takara Bio Inc.) was transformed with the plasmid and incubated overnight at 37°C on a 1.5% agarose LB plate containing 100 μg / mL ampicillin. Single colonies were collected from this plate and inoculated into LB medium containing 100 μg / mL ampicillin (hereinafter referred to as LB-AP medium), and incubated overnight with shaking at 37°C. 300 μL of this culture was inoculated into 25 mL of LB-AP medium and incubated overnight with shaking at 37°C. When the OD600 value reached 0.6, 1 mM IPTG was added to the culture medium and induction culture was performed for 4 hours at 30°C, and bacterial cells were collected.

[0056] The bacterial cells obtained above were suspended in 2 mL of a solution containing 50 mM Tris·HCl pH 8.0, 100 mM NaCl, 1 mM EDTA (pH 8.0), and 5% glycerol. Lysozyme (Sinopharm Chemical Reagent Co., Ltd.) was added to a final concentration of 0.1 mg / mL, and the mixture was shaken at 4°C for 1 hour. After shaking, the mixture was centrifuged at 15,000 × g at 4°C for 30 minutes, and the supernatant was collected. This supernatant was applied to a Ni-NTA Agarose (Qiagen) column, and RNA polymerase mutants were adsorbed onto it. After washing the column, the adsorbed material was eluted with a buffer containing imidazole. Next, the obtained eluate was applied to a POROS HQ (Thermo Fisher Scientific) column to adsorb the RNA polymerase mutant. After washing the column, the adsorbed material was eluted with a buffer containing sodium chloride. The obtained eluate was dialyzed, and the buffer was replaced with 20 mM potassium phosphate pH 7.9, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, and 50% glycerol. The resulting buffer-replaced solution was used as the RNA polymerase mutant solution in each test.

[0057] The activity of the polymerase contained in the RNA polymerase mutant solution described above was measured using a reaction solution with the following composition. The RNA polymerase activity (number of units) contained in the solution was measured at 37°C with 1 nmol [ ３ The amount of enzyme that incorporates [H]GMP into the acid-insoluble precipitate was calculated as 1 U.

[0058] [Composition of reaction solution for activity measurement (50 μl)] 40 mM Tris-HCl (pH 8.0) 8 mM MgCl ２ 2 mM spermidine 5 mM DTT 0.4 mM ATP / UTP / CTP 0.4 mM [ ３ H]GTP 1 μg / 50 μl pT7-2 DNA (manufactured by USB)

[0059] (2) Method for evaluating the amount of transcribed RNA and double-stranded RNA produced The RNA transcription reaction of the prepared T7 RNA polymerase mutant was tested using the following method. Using the 10X T7 RNA Polymerase Buffer included with T7 RNA Polymerase ver2.0 (product number 2541, manufactured by Takara Bio Inc.), a final concentration of 10 mM NTP was prepared. The template DNA consisted of 1 μg of Positive Control Template (FLuc) (linear DNA with a sequence encoding firefly luciferase located downstream of the T7 promoter) included with Takara IVTpro™ mRNA Synthesis System (product number 6141, manufactured by Takara Bio Inc.), 0.1 U of Pyrophosphate (inorganic) (product number 2450, manufactured by Takara Bio Inc.), and 20 U of RNAse Inhibitor ver. A reaction solution with a final volume of 20 μL was prepared containing 200 U of the T7 RNA polymerase mutant prepared according to experimental method (1) using 2.0 (product number 2315, manufactured by Takara Bio Inc.). For comparison, a reaction solution containing wild-type T7 RNA polymerase was also prepared.

[0060] The RNA transcription reaction was carried out at 37°C for 120 minutes using a ThermalCycler TP-990 ThermalCycle Dice® Real Time System III (manufactured by Takara Bio Inc.). Subsequently, 10 U equivalent of DNase I (product number 2270, manufactured by Takara Bio Inc.) was added, and the mixture was treated at 37°C for 15 minutes to degrade the template DNA. The reaction mixture was then stopped on ice.

[0061] The DNase I-treated reaction solutions were precipitated and purified with 3 M lithium chloride, and finally, these precipitates were dissolved in RNase-free water. The RNA concentration in the resulting RNA solution was measured using a spectrophotometer (1 OD at 260 nM = 40 μg / ml RNA). The amount of RNA in the reaction solution calculated from the obtained RNA concentration represents the sum of single-stranded RNA and double-stranded RNA (total RNA). The amount of double-stranded RNA was measured using the EasyAna dsRNA (Modified) Quantitative Detection Kit (ELISA) 2.0 (product number DD3509EN, Vazyme), following the instructions in its manual.

[0062] Example 1 Preparation of T7 RNA polymerase mutants An artificial gene encoding a mutant protein in which the amino acid substitutions shown in Table 1 were introduced into the amino acid sequence of wild-type T7 RNA polymerase was designed using a known method and chemically synthesized. Recombinant plasmids containing the obtained artificial gene were prepared according to experimental method (1), and the mutants were expressed and purified. The names and amino acid sequence numbers (SEQ ID NOs. 2-8) of the T7 RNA polymerase mutants into which each mutation was introduced are shown in Table 1.

[0063]

[0064] Example 2 RNA Synthesis with Each Mutant Using the T7 RNA polymerase mutants of SEQ ID NOs. 2-8 prepared in Example 1, mRNA of the luciferase encoded in the Positive Control Template was synthesized. The reaction mixture had the composition described in experimental method (2), and two types were used: one containing a final concentration of 8 mM cap analog [CleanCap® Reagent AG (3'OMe) (Trilink)] and one without the cap analog. mRNA synthesis using wild-type T7 RNA polymerase was also performed as a control. After incubation at 37°C for 120 minutes, the reaction mixture was treated with DNase I to purify the RNA, and then the RNA concentration was measured. The measured total RNA amount is shown in Figure 1.

[0065] As is clear from Figure 1, all mutants except T7-d5 (R51A) and T7-d6 (R52A) were able to synthesize total RNA at a level equivalent to or greater than that of wild-type RNA polymerase.

[0066] Example 3 Evaluation of Transcript RNA using T7 RNA Polymerase Mutants The T7 RNA polymerase mutant and wild-type T7 RNA polymerase prepared in Example 1 were evaluated for their transcription RNA. The tests were conducted using the reaction mixture, reaction conditions, and measurement methods described in Experimental Method (2). Two types of reaction mixtures were used: one containing CleanCap® Reagent AG (3'OMe) at a final concentration of 8 mM, and one without. The total RNA amount and double-stranded RNA content obtained from each reaction mixture were measured, and the mutant and wild-type were compared. The double-stranded RNA content is shown in Figure 2.

[0067] As is clear from Figure 2, in the presence or absence of Cap analogs, the T7-d3 (E48A) and T7-d4 (R50A) mutants showed significantly suppressed double-stranded RNA production compared to the wild type. Surprisingly, the T7-c3 (R50A, S430P, S633P, F849I, F880Y) mutant, which combines the heat-stable mutants T7-a2 and T7-d4, showed the greatest suppression of double-stranded RNA production.

[0068] This invention, which exhibits excellent efficacy in producing high-purity single-stranded RNA, is useful in a wide range of fields, including genetic engineering, biology, and medicine.

[0069] SEQ ID NO1: T7-WT SEQ ID NO2: T7-a2 SEQ ID NO3: T7-d3 SEQ ID NO4: T7-d4 SEQ ID NO5: T7-d5 SEQ ID NO6: T7-d6 SEQ ID NO7: T7-a7 SEQ ID NO8: T7-c3

Claims

1. A variant of RNA polymerase in which an amino acid selected from the group consisting of glutamic acid at position 48 and arginine at position 50 in the amino acid sequence of wild-type T7 RNA polymerase shown in Sequence ID No. 1 is substituted with another amino acid, provided that the amino acid corresponding to arginine at position 386 is not substituted.

2. The RNA polymerase mutant according to claim 1, wherein a mutation has been introduced into an RNA polymerase having 70% or more sequence identity with the amino acid sequence shown in Sequence ID No.

1.

3. The RNA polymerase variant according to claim 1 or 2, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No.

1.

4. The RNA polymerase variant according to any one of claims 1 to 3, wherein an amino acid selected from the group consisting of the 48th and 50th positions in the amino acid sequence shown in Sequence ID No. 1 is substituted with alanine.

5. The RNA polymerase mutant according to any one of claims 1 to 4, wherein an amino acid selected from the group consisting of serine at position 430, serine at position 633, phenylalanine at position 849, and phenylalanine at position 880 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with an amino acid different from the original amino acid.

6. The RNA polymerase variant according to claim 5, having an amino acid substitution selected from the group consisting of a substitution of the amino acid corresponding to serine at position 430 in the amino acid sequence shown in SEQ ID NO: 1 with proline, a substitution of the amino acid corresponding to serine at position 633 in the amino acid sequence shown in SEQ ID NO: 1 with proline, a substitution of the amino acid corresponding to phenylalanine at position 849 in the amino acid sequence shown in SEQ ID NO: 1 with isoleucine, and a substitution of the amino acid corresponding to phenylalanine at position 880 in the amino acid sequence shown in SEQ ID NO: 1 with tyrosine.

7. A nucleic acid encoding an RNA polymerase variant according to any one of claims 1 to 6.

8. A vector that holds the nucleic acid according to claim 7.

9. A method for producing an RNA polymerase mutant, comprising the step of culturing cells into which the nucleic acid described in claim 7 has been introduced.

10. A method for producing single-stranded RNA, characterized by using a reaction solution containing an RNA polymerase variant described in any one of claims 1 to 6.

11. The method for producing a protein according to claim 10, characterized in that the reaction solution contains a protein selected from single-stranded RNA-binding proteins or cold shock proteins.

12. The method for producing the product according to claim 11, wherein the single-stranded RNA-binding protein is the T4 phage gene 32 protein.

13. The manufacturing method according to claim 11, wherein the cold shock protein is CspA.

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