RNA polymerase variant and RNA synthesis

Abstract

Provided is a variant of an RNA polymerase, the variant being characterized in that an amino acid corresponding to arginine at position 386 in the amino acid sequence of a wild-type T7 RNA polymerase represented by SEQ ID NO: 1 is substituted with an aromatic amino acid.

Description

RNA polymerase mutants and RNA synthesis

[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 medicines are attracting attention as a new medical modality. mRNA medicines are artificially produced drugs containing mRNA, which encodes a protein, as their active ingredient. By producing the protein in the body after administration, the drug exerts medical effects based on the function of that protein. During the global spread of SARS-CoV-2 infection that occurred in 2020, mRNA vaccines, a type of mRNA medicine, were put into practical use and administered to many people, making a significant contribution to preventing infection. In addition to being able to be designed in a short period of time based on the genetic information of the virus, the possibility of mass-producing different types of vaccines using the same manufacturing process, i.e., common equipment, is cited as an advantage over other medicines.

[0003] In vivo, RNA is synthesized by a transcription reaction catalyzed by RNA polymerase, using double-stranded DNA as a template. RNA transcription can be reproduced in vitro by preparing the necessary components for the reaction. However, many RNA polymerases present in prokaryotic and eukaryotic cells are composed of multiple subunits, making the production of each subunit an obstacle to practical application. On the other hand, RNA polymerases derived from bacteriophages (e.g., T7, T3, and K11), chloroplasts, and mitochondria are composed of a single polypeptide (single-subunit DNA-dependent RNA polymerases: Non-Patent Document 1).

[0004] RNA polymerases are useful tools in genetic engineering research, and RNA polymerases derived primarily from T7 and SP6 phages have been commercially available as research reagents. Typical applications include the preparation of labeled RNA for use as hybridization probes and nucleic acid amplification methods combined with reverse transcription and double-stranded cDNA synthesis. Because the basic enzymatic properties of bacteriophage-derived RNA polymerases have already been elucidated, they are now also used in the industrial production of RNA.

[0005] In the industrial production of RNA, it is advantageous to use a highly stable RNA polymerase. For this reason, mutants of T7 RNA polymerase with enhanced thermostability have been created (Patent Document 1). Attempts have also been made to create mutants 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 must have a cap structure at the 5' end for efficient translation. It is known that double-stranded RNA (dsRNA) is generated as a by-product in transcription reactions catalyzed by RNA polymerase. For example, double-stranded RNA administered to a human body can induce therapeutically undesirable immune responses via Toll-like receptors and other pathways. Therefore, a transcription reaction system capable of synthesizing RNA with a cap structure while suppressing the generation of double-stranded RNA is advantageous for the production of mRNA pharmaceuticals composed of single-stranded RNA.

[0007] International Publication Pamphlet WO2001 / 066705 International Publication Pamphlet WO2019 / 036682

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

[0009] An object of the present invention is to provide an RNA polymerase mutant that is more suitable than conventional RNA polymerases for producing single-stranded RNA having a cap structure and has a reduced ability to produce double-stranded RNA, in order to stably supply high-purity single-stranded RNA that can be used as a medicine, etc.

[0010] As a result of intensive efforts to solve the above-mentioned problems, the present inventors discovered a mutant RNA polymerase that can efficiently transcribe RNA in the presence of a cap analog by introducing a new mutation into a specific position of the RNA polymerase, and further constructed a high-purity single-stranded RNA synthesis system using the mutant, thereby completing the present invention.

[0011] For example, the following aspects are provided: (1) A mutant RNA polymerase, characterized in that the amino acid corresponding to arginine at position 386 in the amino acid sequence of wild-type T7 RNA polymerase shown in SEQ ID NO: 1 is substituted with an aromatic amino acid. (2) The RNA polymerase mutant according to (1), wherein the substitution mutation has been introduced into an RNA polymerase comprising an amino acid sequence 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 of (1) to (3), further comprising an amino acid corresponding to an amino acid selected from the group consisting of glutamic acid at position 48, arginine at position 50, phenylalanine at position 51, arginine at position 52, and proline at position 72 in the amino acid sequence shown in SEQ ID NO: 1 substituted with an amino acid different from the original amino acid. (5) The RNA polymerase mutant according to (4), in which an amino acid at a position selected from the group consisting of serine at position 48, 50, 51, 52, and 72 in the amino acid sequence shown in SEQ ID NO: 1 has been substituted with alanine. (6) The RNA polymerase mutant according to any of (1) to (5), in which an amino acid at a position 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 has been substituted with an amino acid different from the original amino acid. (7) The RNA polymerase mutant according to (6), in which the amino acid at position 430 in the amino acid sequence shown in SEQ ID NO: 1 has been substituted with proline, the amino acid at position 633 in the amino acid sequence shown in SEQ ID NO: 1 has been substituted with proline, the amino acid at position 849 in the amino acid sequence shown in SEQ ID NO: 1 has been substituted with isoleucine, or the amino acid at position 880 in the amino acid sequence shown in SEQ ID NO: 1 has been substituted with tyrosine. (8) A nucleic acid encoding the RNA polymerase mutant according to any one of (1) to (7).(9) A vector carrying the nucleic acid according to (8). (10) A method for producing an RNA polymerase mutant, comprising the step of culturing a cell into which the nucleic acid according to (8) has been introduced. (11) A method for producing single-stranded RNA, characterized by using a reaction solution containing the RNA polymerase mutant according to any one of (1) to (7). (12) The production method according to (11), characterized in that the reaction solution further contains a single-stranded DNA-binding protein, a single-stranded RNA-binding protein, or a cold shock protein. (13) The production method according to (12), wherein the single-stranded DNA-binding protein is T4 phage gene 32 protein. (14) The production method according to (12), characterized in that the reaction solution contains a cold shock protein. (15) The production method according to (14), wherein the cold shock protein is CspA.

[0012] The present invention provides an RNA polymerase mutant having RNA transcription activity suitable for in vitro production of highly pure RNA, as well as a method for producing the same. According to the present invention, an RNA polymerase with a mutation at a specific amino acid can synthesize single-stranded RNA more efficiently in a reaction solution containing a cap analog, and further suppresses the production of double-stranded RNA. Therefore, the RNA polymerase mutant of the present invention is highly effective in providing RNA of higher purity than conventional methods.

[0013] Figure 1 shows the amount of total RNA synthesized using the RNA polymerase mutant of the present invention in the presence or absence of a cap analog. Figure 2 shows the amount of protein expressed from RNA synthesized using the RNA polymerase mutant of the present invention. Figure 3 shows the dsRNA content in RNA of different lengths synthesized using the RNA polymerase mutant of the present invention in the presence of a cap analog. Figure 4 shows the cap retention rate of RNA synthesized using the RNA polymerase mutant of the present invention.

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

[0015] As used herein, the identity of amino acid sequences refers to the percentage of identical amino acid residues in two amino acid sequences when the two sequences are optimally aligned with each other. If a position in a first sequence is occupied by the same amino acid residue as the corresponding position in a second sequence, the sequences are said to be identical at that residue position. Appropriate alignment of two sequences can be performed using various algorithms, such as the BLAST algorithm.

[0016] As used herein, amino acid mutations include 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 mutants of the present invention The present invention provides mutants of single-subunit DNA-dependent RNA polymerases suitable for producing single-stranded RNAs used in mRNA pharmaceuticals and the like.

[0019] RNA polymerase refers to an enzyme that has the activity of synthesizing an RNA strand complementary to the sequence of DNA as a template, and among these, those consisting of a single polypeptide chain are called single-subunit DNA-dependent RNA polymerases. The RNA polymerase mutants of the present invention are those in which a mutation has been introduced into the amino acid sequence of this type of RNA polymerase. Although not particularly limited, the RNA polymerase mutants provided by the present invention have improved transcription efficiency in a reaction solution containing a cap analog.

[0020] The RNA polymerase mutant of the present invention (hereinafter sometimes referred to as the mutant of the present invention) is preferably a mutant of an RNA polymerase derived from a bacteriophage, such as a mutant of T7 phage RNA polymerase, or an RNA polymerase with high amino acid sequence identity to said enzyme (e.g., an RNA polymerase derived from T3 phage, K11 phage, or SP6 phage). While not limiting the present invention, an example of the RNA polymerase mutant of the present invention is an RNA polymerase mutant comprising an amino acid sequence with 70% or more sequence identity to the amino acid sequence of wild-type T7 RNA polymerase shown in SEQ ID NO: 1. The amino acid sequences of the RNA polymerases of T3 phage (NCBI ACCESSION_CAC86264) and K11 phage (NCBI ACCESSION_P18147) share 70% or more identity with the amino acid sequence of T7 RNA polymerase. Therefore, it is possible to easily identify amino acids in T3 polymerase and K11 polymerase that correspond to specific amino acids present in the amino acid sequence of wild-type T7 RNA polymerase shown in SEQ ID NO: 1 (see Non-Patent Document 1). For example, R386 in T7 RNA polymerase corresponds to position 387 in T3 polymerase and position 409 in K11 polymerase. In one embodiment 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 SEQ ID NO: 1. The mutant polypeptide has RNA polymerase activity, i.e., the activity of transcribing RNA using DNA as a template.

[0021] The present invention will be explained below using mutants derived from T7 RNA polymerase as examples. Mutants in which R386 in the amino acid sequence of wild-type T7 RNA polymerase shown in SEQ ID NO: 1 is substituted with an aromatic amino acid synthesize single-stranded RNA having a cap structure more efficiently than the wild-type in a reaction solution containing a cap analog. Preferably, in the mutants of the present invention, R386 is substituted with phenylalanine, tyrosine, or tryptophan.

[0022] The RNA polymerase mutant of the present invention may further comprise one or more mutations, e.g., amino acid substitutions, in addition to the amino acid substitution corresponding to position R386 in the amino acid sequence of wild-type T7 RNA polymerase. There are no particular limitations on the additional mutations, as long as they do not significantly reduce the RNA transcription activity of the mutant of the present invention. Examples of additional substitutions include substitutions of amino acids corresponding to amino acids selected from E48, R50, F51, R52, or P72 in T7 RNA polymerase. Substitution of one or more of these five amino acids with other amino acids can reduce the ability of the mutant of the present invention to produce double-stranded RNA. While there are no particular limitations on the amino acids to be substituted, nonpolar aliphatic amino acids are preferred, and alanine is more preferred.

[0023] Furthermore, the RNA polymerase mutant of the present invention may be one into which a known mutation has been introduced. Patent Document 1 discloses an RNA polymerase mutant with improved thermostability, in which four amino acid substitutions (serine at position 430, serine at position 633, phenylalanine at position 849, and phenylalanine at position 880 are replaced with proline, proline, isoleucine, and tyrosine, respectively) have been introduced into T7 RNA polymerase, a single-subunit DNA-dependent RNA polymerase (the amino acid sequence of wild-type T7 RNA polymerase is shown in SEQ ID NO: 1). The RNA polymerase mutant of the present invention may be substituted with one or more amino acids corresponding to the amino acids S430, S633, F849, and 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.

[0024] A mutant of T7 RNA polymerase in which R386 is substituted with tryptophan and S430, S633, F849, and F880 are substituted with proline, proline, isoleucine, and tyrosine, respectively, has been designated T7-a2-a27. The amino acid sequence of T7-a2-a27 is shown in SEQ ID NO: 6. That is, a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6 is an example of a mutant of the present invention. Compared to wild-type T7 RNA polymerase, T7-a2-a27 has improved RNA transcription efficiency, particularly in the presence of a cap analog. Furthermore, mutants in which R50 or F51 of T7-a2-a27 is substituted with alanine have been designated T7-b7 and T7-b8, respectively. These two mutants are examples of mutants of the present invention in which the ability to produce double-stranded RNA is significantly reduced compared to wild-type T7 RNA polymerase.

[0025] In one embodiment of the present invention, the mutant of the present invention may have another peptide added thereto. Examples of such another peptide include, but are not limited to, a signal peptide required for the secretory expression of the mutant of the present invention and an affinity tag useful for purifying the mutant. The signal peptide may be selected from known secretory proteins or modified versions thereof depending on the host used to produce the mutant of the present invention, and is typically added to the N-terminus of the mutant. Furthermore, the affinity tag may be selected from known tags as long as the ligand that can be used for its capture is known. Examples of affinity tags include histidine (His) tag, HN tag, HAT tag, glutathione S-transferase (GST) tag, maltose binding protein (MBP) tag, C-Myc tag, and Strep(II) tag consisting of eight amino acid residues (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys). These tags may be added to either the N-terminus or C-terminus of the mutant. Furthermore, the affinity tag can be removed from the mutant of the present invention after purification. For this purpose, affinity tags are used that allow a protease recognition sequence, such as a recognition sequence for Factor Xa, PreScission Protease, thrombin, enterokinase, or TEV protease (Tobacco Etch Virus Protease), to be inserted between the RNA polymerase mutant of the present invention and the affinity tag.

[0026] The RNA polymerase mutants of the present invention can be produced using known recombinant protein production methods. Based on the nucleotide sequence information of the gene for a wild-type single-subunit DNA-dependent RNA polymerase, the codon for the amino acid corresponding to R386 in the amino acid sequence of T7 RNA polymerase is identified and then changed to a codon for an aromatic amino acid. A nucleic acid having such a designed nucleotide sequence, i.e., a nucleic acid encoding a mutant of the present invention, is prepared, and a mutant can be obtained using an appropriate expression vector and host, as described below. If it is desired to introduce a mutation at a position other than the R386 position, a nucleotide sequence encoding the desired mutant can be designed using similar procedures. Nucleic acids encoding the mutants of the present invention can be chemically synthesized or can be produced by site-specific mutagenesis of a nucleic acid encoding a wild-type RNA polymerase.

[0027] The RNA polymerase mutant of the present invention can be used not only for producing RNA, but also for nucleic acid amplification methods that include RNA polymerase as a component (e.g., the NASBA method (Nucleic Acid Sequence-based Amplification) described in U.S. Pat. No. 5,130,238, the 3SR method (Self-sustained sequence replication reaction) described in WO 90 / 06995, the SMART method (Signal-mediated amplification of RNA technology) described in WO 99 / 37806, and the TMA method (Transcription-mediated amplification) described in WO 91 / 01384). These are methods that can efficiently amplify target nucleic acids under isothermal conditions.

[0028] 2. Nucleic Acids Encoding the RNA Polymerase Mutants of the Present Invention The present invention provides nucleic acids encoding the RNA polymerase mutants described in 1. (hereinafter, sometimes referred to as nucleic acids of the present invention).

[0029] The nucleic acid of the present invention is not limited to a nucleic acid having a specific base sequence, as long as it encodes the RNA polymerase mutant of the present invention. As described above, the base sequence of the nucleic acid of the present invention is designed by identifying 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 for other amino acids. In order to increase the expression level of the RNA polymerase mutant of the present invention in a 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 mutant of the present invention may have a signal peptide or an affinity tag added thereto. Therefore, the nucleic acid of the present invention encompasses nucleic acids encoding the mutant of the present invention to which a signal peptide or an affinity tag has been added. The nucleotide sequence of the nucleic acid encoding the signal peptide or affinity tag may be designed according to the amino acid sequence thereof, and in this case, codons may be selected taking into consideration the host to be used.

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

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

[0033] 3. Vectors Comprising Nucleic Acids Encoding the RNA Polymerase Mutants of the Present Invention The present invention provides vectors carrying nucleic acids encoding the RNA polymerase mutants of the present invention. The vectors of the present invention are useful for introducing nucleic acids encoding the RNA polymerase mutants of the present invention into appropriate hosts for the production of the RNA polymerase mutants of the present invention or for other purposes.

[0034] There are no particular limitations on the vector into which a nucleic acid encoding an RNA polymerase mutant of the present invention is inserted. Vectors capable of autonomous replication in host cells or vectors that can be integrated into host chromosomes can be used. For example, plasmid vectors, phage vectors, viral vectors, artificial chromosomes, etc. can be used. A vector appropriate for the host to be used is selected. Vectors suitable for various hosts (Escherichia coli, Bacillus bacteria, yeast, filamentous fungi, insect cells, mammalian cells, etc.) are well known to those skilled in the art, and many are commercially available. These known vectors and their modifications can be used in the present invention. Numerous expression vectors have also been constructed for expressing recombinant proteins in hosts.

[0035] An expression vector has an appropriate promoter that can function in the host and other elements involved in transcription and translation (operators, terminators, enhancers, ribosome binding sites, etc.), and is suitable for producing the product of the gene carried in the host. For example, expression vectors for E. coli use promoters such as the trp promoter, lac promoter, PL promoter, and PR promoter, as well as their variants, but are not limited to these. If the promoter is inducible, expression of the gene product can be induced by appropriate manipulation.

[0036] The expression vector used in the present invention may further contain a nucleic acid encoding a signal peptide or an affinity tag. The nucleic acids encoding these peptides are positioned so as to be expressed as a fusion protein of the RNA polymerase mutant of the present invention and the affinity tag. Since vectors into which a nucleic acid encoding an affinity tag has been inserted are also known, the vector of the present invention can also be produced by inserting the nucleic acid of the present invention into such a vector. The expression vector thus constructed, carrying the nucleic acid of the present invention, is useful in producing the RNA polymerase mutant of the present invention.

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

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

[0039] Representative hosts used in industrial recombinant protein production, such as Bacillus bacteria such as Escherichia coli and Bacillus subtilis, are well known to those skilled in the art, and many strains are commercially available. An appropriate host strain can be selected from these strains, taking into consideration the production scale and other conditions. Furthermore, methods for producing recombinant proteins using yeast (genus Saccharomyces or Schizosaccharomyces) or insect cells as hosts are also known. The RNA polymerase mutant can be expressed and produced by introducing a nucleic acid encoding the RNA polymerase mutant of the present invention into these host cells, or by preparing host cells in which the nucleic acid has been integrated into the chromosome, and culturing the host.

[0040] The nucleic acid encoding the RNA polymerase mutant of the present invention can be incorporated into an appropriate vector and introduced into a host. For example, a vector that can be used as an expression vector can be selected. When a plasmid vector is used, the introduction method can be appropriately selected depending on the host, and examples of methods that can be used include calcium ion-based methods, lipofection, electroporation, spheroplast methods, and lithium acetate methods. Phage vectors and viral vectors can be used to infect host cells using methods appropriate for the vector, thereby obtaining cells into which nucleic acid encoding the RNA polymerase mutant has been introduced. Furthermore, the PET system described above can be used to produce the mutant of the present invention.

[0041] The cells thus obtained, harboring a nucleic acid encoding the RNA polymerase mutant, can be cultured, and the mutant of the present invention can be isolated from the culture. Culture conditions are not particularly limited as long as they are suitable for the host, expression vector, etc. Furthermore, known protein purification methods can be used to isolate and purify the RNA polymerase mutant. The following describes the case where Escherichia coli is used as the host. E. coli into which a 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, expression of the RNA polymerase mutant can be induced during culture by performing an appropriate manipulation of the promoter. After collecting and washing the E. coli cells from the culture, an E. coli lysate containing the mutant of the present invention can be obtained by ultrasonic disruption, lysozyme treatment, or other lysis treatment. If the mutant of the present invention is secreted and expressed, the culture supernatant is collected. Using this lysate or culture supernatant as a starting material, the RNA polymerase mutant of the present invention is purified by an appropriate combination of purification methods used in the field, such as ammonium sulfate precipitation, anion exchange columns, cation exchange columns, gel filtration columns, affinity chromatography columns, filtration, dialysis, etc. Mutants with affinity tags can be easily purified using an affinity support appropriate to the properties of the affinity tag. For example, mutants of the present invention having affinity tags containing multiple histidines (such as histidine tags, HN tags, and HAT tags) can be purified using a support bound to a metal such as nickel.

[0042] 5. Methods 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 an RNA polymerase mutant of the present invention. In the presence of double-stranded DNA as a template and four types of ribonucleotide triphosphates, the mutant of the present invention synthesizes single-stranded RNA with a sequence complementary to the template DNA. The RNA produced by the method of the present invention is not limited, and examples include mRNA, RNA probes, ribozymes, and guide RNAs (used in genome editing using Cas9, etc.).

[0043] The double-stranded DNA that can be used as a template in the method for producing single-stranded RNA of the present invention has a promoter sequence recognized by the mutant of the present invention. Promoter sequences recognized by bacteriophage-derived RNA polymerases are known (see, for example, FEBS Letters, 4, pp. 264-267, 1998). Furthermore, the promoter 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 nucleic acid amplification. For example, double-stranded DNA that can be used as a template can be prepared by inserting DNA of a base sequence encoding the desired RNA downstream of a plasmid carrying a promoter recognized by RNA polymerase. It is preferable to use circular DNA such as a plasmid after linearization (e.g., digestion with a restriction enzyme).

[0044] Although four types of ribonucleotide triphosphates (ATP, CTP, GTP, and UTP), which are substrates for natural RNA, are typically used, one or more of these four types can be replaced with a ribonucleotide triphosphate analog. There are no particular limitations on the ribonucleotide triphosphate analog. 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 to be administered to a living body, pseudouridine triphosphate, 1-methylpseudouridine triphosphate, or other UTP analogs may be used instead of UTP. The concentration of ribonucleotide triphosphate or its analog can be appropriately set depending on the purpose, etc., and is typically in the range of 0.2 to 15 mM.

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

[0046] The 5' end of eukaryotic mRNA contains a modification called a cap structure. This modification consists of a 7-methylguanosine attached to the 5' end via a 5'-5' triphosphate structure and methylation of the 2'-O position of the adjacent nucleotide. The cap structure not only contributes to mRNA stabilization, but is also said to increase translation efficiency by being recognized by translation initiation factors in eukaryotic cells.

[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 vaccinia virus-derived capping enzyme, 7-methylguanosine is added to the 5'-end of the single-stranded RNA (Cap0). Then, by the action of mRNA Cap 2'-O-methyltransferase, the 2'-O position of the nucleotide adjacent to the 7-methylguanosine is methylated (Cap1).

[0048] In the method of the present invention, a cap structure can be added in parallel with transcription of single-stranded RNA. In this case, transcription of single-stranded RNA using a mutant of the present invention is carried out in a reaction solution containing a compound known as a cap analog. The cap analog is not particularly limited, and cap analogs composed of dinucleotides, trinucleotides, or higher 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 the CleanCap (registered trademark) series of 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 by-product, and pyrophosphate inhibits the transcription reaction. Since inorganic pyrophosphatase has the activity of catalyzing the hydrolysis of pyrophosphate to produce orthophosphate, the RNA synthesis reaction is promoted by the decomposition of pyrophosphate by the enzyme.

[0050] Furthermore, the present invention provides a method for reducing the production of double-stranded RNA in an RNA transcription reaction, characterized by using a reaction solution containing a protein with affinity for nucleic acids. Examples of proteins with affinity for nucleic acids include single-stranded DNA-binding protein (SSB), single-stranded RNA-binding protein, and cold shock protein. When a reaction solution for RNA transcription containing a protein with affinity for nucleic acids is used, the production of double-stranded RNA is suppressed compared to when the reaction solution does not contain the protein. Examples of single-stranded DNA-binding proteins include T4 phage gene 32 protein (T4 gp32), T7 phage single-stranded DNA-binding protein, and Escherichia coli single-stranded DNA-binding protein. Cold shock proteins are a general term for proteins that are transiently expressed at high levels in bacteria and the like when the growth temperature is lowered. A cold shock protein named CspA is known in Escherichia coli. Eight proteins, CspB to CspI, are known to have high amino acid sequence identity with CspA, and of these, CspB, CspG, and CspI are cold shock proteins [J. Bacteriol., 181, pp. 1603-1609, 1999]. Furthermore, homologs of these cold shock proteins are also present in microorganisms such as Bacillus subtilis (CspB), Bacillus caldolyticus (CspB), Thermotoga maritima (CspB, CspL), and Lactobacillus plantarum (CspL). While not limiting the present invention, CspA from Escherichia coli is suitable for RNA transcription reactions. In the method for reducing the production of double-stranded RNA in the RNA transcription reaction, any RNA polymerase may be used in the RNA transcription reaction, and wild-type or mutant RNA polymerases may be used. The mutant RNA polymerase is not limited to the RNA polymerase mutant of the present invention. Furthermore, in the method for producing single-stranded RNA of the present invention, a reaction solution containing a protein having affinity for nucleic acids can also be used.

[0051] 6. Kits of the Present Invention The present invention provides kits containing the RNA polymerase mutants of the present invention for use in synthesizing desired RNA.

[0052] In addition to the mutant of the present invention, the kit of the present invention may contain 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 as individual components so that they can be mixed at the time of use to prepare a reaction solution, or multiple components may be packaged as a mixture to facilitate preparation of a reaction solution. Examples of kits include kits containing a premix solution prepared so that a reaction solution can be completed by adding only template DNA and water (e.g., sterile water) at the time of use, and kits containing the premix in a dry state. Other components that may be contained in the kit include ribonuclease inhibitors, cap analogs, inorganic pyrophosphatase, bovine serum albumin, and other proteins (e.g., single-stranded DNA-binding proteins, single-stranded RNA-binding proteins, and cold shock proteins).

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

[0054] Experimental Method (1) Method for Preparing T7 RNA Polymerase Mutant The nucleotide sequence of the gene encoding wild-type RNA polymerase derived from Escherichia phage T7 (Bacteriophage T7) strain is disclosed in NCBI Reference Sequence No. [NC_001604 Gene ID: 1261050]. The amino acid sequence of wild-type T7 RNA polymerase encoded by this nucleotide sequence is shown in SEQ ID NO: 1 in the Sequence Listing. An artificial gene having a nucleotide sequence encoding an amino acid sequence into which a desired mutation was introduced was chemically synthesized. The obtained artificial gene was introduced into the plasmid pET6xHN-N (manufactured by Takara Bio USA) using In-Fusion (registered trademark) HD ​​Cloning Kit (manufactured by Takara Bio USA). The resulting plasmid has a nucleotide sequence encoding a T7 RNA polymerase mutant tagged with histidine at the N-terminus.

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

[0056] The resulting bacterial cells 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 suspension was shaken at 4°C for 1 hour. After shaking, the suspension was centrifuged at 15,000 x g for 30 minutes at 4°C, and the supernatant was collected. This supernatant was applied to a Ni-NTA Agarose (Qiagen) column to adsorb the RNA polymerase mutant. After washing the column, the adsorbed material was eluted with a buffer containing imidazole. The resulting eluate was then applied to a POROS HQ (Thermo Fisher Scientific) column to adsorb the RNA polymerase mutants. After washing the column, the adsorbed material was eluted with a buffer containing sodium chloride. The resulting 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-substituted 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 was measured using a reaction solution with the following composition. The RNA polymerase activity (units) contained in the solution was measured at 37° C. for 1 hour at 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 the Amount of dsRNA Produced The prepared T7 RNA polymerase mutants were tested for RNA transcription reaction by the following method. The 10X T7 RNA Polymerase Buffer included with T7 RNA Polymerase ver. 2.0 (product number 2541, Takara Bio Inc.) was used, with a final concentration of 10 mM NTP, 1 μg of Positive Control Template (FLuc) (linearized DNA in which a sequence encoding firefly luciferase is located downstream of the T7 promoter) included with Takara IVTpro™ mRNA Synthesis System (product number 6141, Takara Bio Inc.) as template DNA, 0.1 U of Pyrophosphatase (inorganic) (product number 2450, Takara Bio Inc.), and 20 U of RNase Inhibitor ver. A reaction mixture with a final volume of 20 μL was prepared containing 200 U of T7 RNA polymerase mutant 2.0 (product number 2315, manufactured by Takara Bio Inc.) prepared in Experimental Method (1). As a control, a reaction mixture 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 thermal cycler TP-990 ThermalCycler Dice® Real Time System III (manufactured by Takara Bio Inc.). Then, 10 U of DNase I (product number 2270, manufactured by Takara Bio Inc.) was added and treated at 37°C for 15 minutes to degrade the template DNA. After treatment, the reaction solution was stopped on ice.

[0061] The DNase I-treated reaction solution was purified by precipitation with a final concentration of 3M lithium chloride, and the precipitates were finally 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 RNA concentration thus obtained represents the sum of the amount of single-stranded RNA and the amount of double-stranded RNA (total RNA amount). The amount of double-stranded RNA was measured using the EasyAna dsRNA (Modified) Quantitative Detection Kit (ELISA) 2.0 (product number DD3509EN, manufactured by Vazyme) according to the instructions.

[0062] Example 1 Preparation of T7 RNA polymerase mutants Artificial genes encoding mutant proteins in which the amino acid substitutions shown in Table 1 were introduced into the amino acid sequence of wild-type T7 RNA polymerase were designed and chemically synthesized using known methods. Recombinant plasmids carrying the obtained artificial genes were prepared according to experimental method (1), and the mutants were expressed and purified. The names of the thus-obtained T7 RNA polymerase mutants into which each mutation was introduced and the SEQ ID NOs of their amino acid sequences (SEQ ID NOs: 2 to 17) are shown in Table 1.

[0063] In Table 1, "SY43-44AA" indicates a combination of two amino acid substitutions, S43A and Y44A.

[0064] Example 2: Effect of Substitution at R386 Using the mutants a27, a28, and a29 prepared in Example 1, which are wild-type T7 RNA polymerase mutants with a substitution at R386, luciferase mRNA encoded by the Positive Control Template was synthesized. The reaction mixture had the composition described in Experimental Methods (2), and three types were used: one containing a cap analog [CleanCap® Reagent AG (3'OMe) (Trilink)] at a final concentration of 2 mM or 8 mM, and one containing no cap analog. As a control, mRNA synthesis was also performed using wild-type T7 RNA polymerase. The reaction mixture was incubated at 37°C for 120 minutes, treated with DNase I, and the RNA was purified, followed by measurement of RNA concentration. The measured total RNA amounts are shown in Figure 1.

[0065] As shown in Figure 1, the total RNA amount of the three mutants was reduced compared to that of the wild-type RNA polymerase. Furthermore, the total RNA amount of each mutant correlated with the cap analog concentration in the reaction mixture, whereas the total RNA amount of the wild-type RNA polymerase was not affected by the cap analog.

[0066] Protein expression from the RNA obtained in each reaction mixture was examined. HEK293T cells (1 x 10^5 cells / well) grown on a 24-well plate were seeded and cultured for 18-24 hours. A mixture of the obtained RNA (500 ng / well) and a transfection reagent (TransIT-mRNA Transfection Reagent) was added to the cells to transfect them. After transfection, the cells were incubated at 37°C for 24 hours, and luciferase activity in the cells was measured using the ONE-Glo™ Luciferase Assay System (Promega) according to the instructions. The results (measured values ​​for each test) are shown in Figure 2.

[0067] As shown in Figure 2, the RNAs obtained from mutants a27, a28, and a29 in a reaction mixture containing a cap analog showed higher luciferase expression levels in cells than those obtained from wild-type T7 RNA polymerase. This suggests that substituting W, F, or Y for R386 of T7 RNA polymerase allows for more efficient synthesis of capped mRNA.

[0068] Example 3: Evaluation of RNA Transcribed by T7 RNA Polymerase Mutants The T7 RNA polymerase mutants and wild-type T7 RNA polymerase prepared in Example 1 were subjected to an evaluation test of the transcribed RNA. The test was performed using the reaction solution, reaction conditions, and measurement method described in Experimental Method (2), except that two types of reaction solution were used: one containing CleanCap (registered trademark) Reagent AG (3'OMe) at a final concentration of 8 mM, and one not. The total RNA amount and double-stranded RNA content obtained in each reaction solution were measured, and the mutants and wild-type were compared. The results are shown in Table 2. Note that "T7-WT" in the table refers to wild-type T7 RNA polymerase.

[0069]

[0070] As shown in Table 2, the mutant of SEQ ID NO: 6 (T7-a2_a27) produced a reduced amount of double-stranded RNA in the presence of a cap analog compared to the wild-type polymerase. The heavy mutants T7-b6, b7, b8, b9, and T7-a2_a7_a27, which were obtained by introducing additional mutations into this mutant, resulted in a further suppression of dsRNA production compared to T7-a2_a27.

[0071] Example 5: Inhibitory effect of cold shock protein (CspA) on dsRNA production According to the method described in Chatterjee et al. (Journal of Biochemistry. 114, p. 663-669, 1993), E. coli CspA protein was prepared and used in the following experiment. Various concentrations of CspA and T7 RNA polymerase contained in Takara IVTpro™ T7 mRNA Synthesis Kit (product number 6144, manufactured by Takara Bio Inc.) were added to the reaction solution described in Experimental Method (2), and the reaction was carried out at 37 ° C for 120 minutes. The total amount of RNA produced after the reaction and the content of double-stranded RNA were measured, and the results are shown in Table 3.

[0072]

[0073] As shown in Table 3, it was shown that the production of dsRNA was suppressed by adding CspA to the transcription reaction system at a final concentration of 0.8 to 4.8 μg / μl. The total RNA amount was not significantly affected by CspA.

[0074] Example 6: Evaluation of dsRNA production inhibition by T4 Gene 32 Protein The effect of T4 gp32 on the mutant T7-b7 was examined. Various concentrations of T4 Gene 32 Protein (NEB) and T7 RNA polymerase included in the Takara IVTpro™ T7 mRNA Synthesis Kit (6144, Takara Bio Inc.) were added to the reaction solution described in Experimental Method (2) to prepare a reaction mixture. The reaction was carried out at 37°C and 42°C for 120 minutes. The total amount of RNA produced after the reaction and the content of double-stranded RNA were measured, and the results are shown in Table 4.

[0075]

[0076] As shown in Table 4, it was shown that the amount of double-stranded RNA produced was suppressed by adding 0.2 to 0.8 μg / μl of T4 Gene 32 Protein to the reaction system.

[0077] Example 7 Evaluation of dsRNAs Produced by Transcription of RNAs of Different Chain Lengths Template DNAs encoding RNAs of different chain lengths were prepared for the T7 RNA polymerase mutant T7-b7 prepared in Example 1 and wild-type T7 RNA polymerase, and an evaluation test of the transcribed RNAs was performed.

[0078] As template DNA encoding RNA of different chain lengths, seven types of DNA sequences were prepared that would generate RNA of 0.7 kb (ZsGreen), 1.2 kb (B-actin), 1.7 kb (FLuc), 4.2 kb (Cas9), 7.5 kb (FASN), 10 kb (Lambda DNA), and 12 kb (Lambda DNA) by transcription. Next, plasmid DNA was constructed in which each DNA was inserted downstream of the T7 promoter, and each plasmid was linearized with a restriction enzyme to prepare seven types of template DNA. Next, an RNA transcription test was performed using the reaction solution, reaction conditions, and measurement method of experimental method (2). The linearized plasmid was used as the template DNA, and CleanCap (registered trademark) Reagent AG (3'OMe) was added to all reaction solutions at a final concentration of 4 mM. The dsRNA content (ng) per μg of total RNA was calculated from the total RNA and double-stranded RNA amounts obtained in each reaction mixture, and the results were compared between mutant T7-b7 and wild-type RNA polymerase. The results are shown in Figure 3. Note that "T7-WT" in the figure refers to wild-type T7 RNA polymerase.

[0079] As shown in Figure 3, in the presence of CleanCap (registered trademark) Reagent AG (3'OMe) at a final concentration of 4 mM, mutant T7-b7 was shown to produce RNA with a lower dsRNA content than wild-type T7 RNA polymerase, regardless of the length of the template DNA.

[0080] Example 8: Confirmation of Cap Analog Retention Rate by LC-MS. The cap retention rates of RNA prepared with the T7 RNA polymerase mutant T7-b7 prepared in Example 1 and wild-type T7 RNA polymerase were confirmed using liquid chromatography-mass spectrometry (LC-MS). The positive control template included in the Takara IVTpro™ mRNA Synthesis System was used as a template to synthesize the luciferase RNA encoded therein. The reaction solution had the composition described in Experimental Method (2), and five types were used: one containing a cap analog [CleanCap® Reagent AG (3'OMe) (Trilink)] at final concentrations of 2 mM, 4 mM, 6 mM, or 8 mM, and one containing no cap analog. The reaction mixture was incubated at 37°C for 120 minutes and then treated with DNase I to purify the RNA. The cap (Cap1) retention rate of the purified RNA was measured using LC-MS analysis by the method of Michael Beverly et al. (Anal Bioanal Chem. 2016 Jul; 408(18):5021-30). The results are shown in Figure 4. Note that "T7-WT" in the figure refers to wild-type T7 RNA polymerase.

[0081] As is clear from Figure 4, the cap retention rate of RNA obtained with mutant T7-b7 was equal to or greater than that of wild-type polymerase under all cap analog concentration conditions tested. Even when the cap analog concentration was reduced to 2 mM, T7-b7 retained a high cap of 95.3%, equivalent to 8 mM for the wild-type. There was no significant difference in the amount of total RNA produced by either enzyme at each cap analog concentration. On the other hand, when no cap analog was added, the total RNA amount produced by wild-type T7 RNA polymerase exceeded that of T7-b7. This is consistent with the results shown in Figure 1.

[0082] The present invention, which is highly effective in producing highly pure mRNA, is useful in a wide range of fields, including genetic engineering, biology, and medicine.

[0083] <h2 style=";text-align:left;direction:ltr">SEQ ID NO1: T7-WT SEQ ID NO2: a27 SEQ ID NO3: a28 SEQ ID NO4: a29 SEQ ID NO5: T7-a2 SEQ ID NO6: T7-a2_a27 SEQ ID NO7: T7-b2 SEQ ID NO8: T7-b3 SEQ ID NO9: T7-b4 SEQ ID NO10: T7-b5 SEQ ID NO11: T7-a2_a5_a27 SEQ ID NO12: T7-b1 SEQ ID NO13: T7-b6 SEQ ID NO14: T7-b7 SEQ ID NO15: T7-b8 SEQ ID NO16: T7-b9 SEQ ID NO17: T7-a2_a7_a27

Claims

1. A mutant RNA polymerase, characterized in that the amino acid corresponding to arginine at position 386 in the amino acid sequence of wild-type T7 RNA polymerase shown in SEQ ID NO:1 is substituted with an aromatic amino acid.

2. The RNA polymerase mutant described in claim 1, wherein the RNA polymerase mutant is a mutant in which the substitution mutation has been introduced into an RNA polymerase comprising an amino acid sequence having 70% or more sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

3. The RNA polymerase mutant of claim 1 or 2, which comprises 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 claims 1 to 3, further comprising an amino acid corresponding to an amino acid selected from the group consisting of glutamic acid at position 48, arginine at position 50, phenylalanine at position 51, arginine at position 52, and proline at position 72 in the amino acid sequence shown in SEQ ID NO:1, substituted with an amino acid different from the original amino acid.

5. The RNA polymerase mutant of claim 4, in which amino acids corresponding to positions selected from the group consisting of 48, 50, 51, 52 and 72 in the amino acid sequence shown in SEQ ID NO:1 are substituted with alanine.

6. The RNA polymerase mutant according to any one of claims 1 to 5, further comprising 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 substituted with an amino acid different from the original amino acid.

7. The RNA polymerase mutant of claim 6, in which the amino acid corresponding to serine at position 430 in the amino acid sequence shown in SEQ ID NO:1 is replaced with proline, the amino acid corresponding to serine at position 633 in the amino acid sequence shown in SEQ ID NO:1 is replaced with proline, the amino acid corresponding to phenylalanine at position 849 in the amino acid sequence shown in SEQ ID NO:1 is replaced with isoleucine, or the amino acid corresponding to phenylalanine at position 880 in the amino acid sequence shown in SEQ ID NO:1 is replaced with tyrosine.

8. A nucleic acid encoding the RNA polymerase mutant of any one of claims 1 to 7.

9. A vector carrying the nucleic acid according to claim 8.

10. A method for producing an RNA polymerase mutant, comprising the step of culturing a cell into which the nucleic acid according to claim 8 has been introduced.

11. A method for producing single-stranded RNA, comprising using a reaction solution containing the RNA polymerase mutant according to any one of claims 1 to 7.

12. The method according to claim 11, wherein the reaction solution further contains a single-stranded DNA binding protein, a single-stranded RNA binding protein, or a cold shock protein.

13. The method of claim 12, wherein the single-stranded DNA binding protein is T4 phage gene 32 protein.

14. The method according to claim 12, wherein the reaction solution contains a cold shock protein.

15. The method of claim 14, wherein the cold shock protein is CspA.

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