Modified RNA polymerase

Abstract

To provide a modified RNA polymerase having enhanced heat resistance or specific activity.SOLUTION: The foregoing problem is solved by a modified RNA polymerase having a modified amino acid at a predetermined position.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a modified RNA polymerase. More specifically, the present invention relates to a modified RNA polymerase having improved thermal stability or specific activity as compared with the wild type, a method for producing the RNA polymerase, and uses thereof.

Background Art

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

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

[0004] In recent years, RNA synthesized by RNA polymerase has also been applied to mRNA pharmaceuticals including mRNA vaccines, and RNA polymerase is widely used in the field of pharmaceutical production. Patent Document 1 describes a modified T7 RNA polymerase with improved thermal stability, but there is a desire for an RNA polymerase with further improved performance.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] As described above, as the uses of RNA polymerase expand, further improvement in the performance of RNA polymerase is desired. The present inventors have regarded the low thermal stability and specific activity of conventional RNA polymerases as problems. Therefore, an object of the present invention is to provide a modified RNA polymerase having higher thermal stability or specific activity than conventional ones.

Means for Solving the Problems

[0007] As a result of intensive studies to solve the above problems, the present inventors have found that an RNA polymerase capable of achieving improved thermal stability or specific activity can be obtained by modifying the amino acid at a predetermined position in the amino acid sequence of wild-type RNA polymerase, and have completed the present invention.

[0008] That is, the representative invention of the present application is as follows. [Item 1] An RNA polymerase having an amino acid sequence with 90% or more identity to the amino acid sequence of SEQ ID NO: 1 and having the amino acid at the position corresponding to position 197 in SEQ ID NO: 1 modified. [Item 2] An RNA polymerase having an amino acid sequence with 90% or more identity to the amino acid sequence of SEQ ID NO: 1 and having the amino acid at the position corresponding to position 197 in SEQ ID NO: 1 modified to asparagine. [Item 3] A polynucleotide encoding the RNA polymerase according to Item 1 or 2. [Item 4] A vector containing the polynucleotide encoding the RNA polymerase according to Item 1 or 2. [Item 5] A recombinant host cell transformed with a vector containing a polynucleotide encoding the RNA polymerase according to item 1 or 2. [Item 6] A method for producing RNA polymerase using the polynucleotide encoding the RNA polymerase according to item 1 or 2, the vector containing the polynucleotide, or the recombinant host cell transformed with the vector. [Item 7] A reagent comprising the RNA polymerase according to item 1 or 2, the polynucleotide encoding the RNA polymerase, the vector containing the polynucleotide, or the recombinant host cell transformed with the vector. [Item 8] A method for performing an in vitro transcription reaction using the RNA polymerase according to item 1 or 2. [Item 9] The method according to item 8, wherein the amount of RNA after the in vitro transcription reaction is 110% or more as compared with the case where the in vitro transcription reaction is performed using the corresponding wild-type RNA polymerase. [Item 10] A method for synthesizing mRNA using the RNA polymerase according to item 1 or 2. [Item 11] The method according to item 10, wherein the amount of mRNA is 110% or more as compared with the case where mRNA synthesis is performed using the corresponding wild-type RNA polymerase. [Item 12] A method for synthesizing non-coding RNA using the RNA polymerase according to item 1 or 2. [Item 13] The method according to item 12, wherein the non-coding RNA is at least one selected from microRNA, siRNA, piRNA, rRNA, tRNA, snRNA, snoRNA, SLRNA, SRPRNA, mRNA-like non-coding RNA, and oligonucleotide. [Item 14] A method for synthesizing a guide RNA for gene editing using the RNA polymerase according to item 1 or 2. [Item 15] A method for synthesizing an RNA pharmaceutical using the RNA polymerase according to item 1 or 2. [Item 16] A method for performing an isothermal amplification reaction using the RNA polymerase according to item 1 or 2. [Item 17] A method for performing a gene test using the RNA polymerase according to item 1 or 2. [Item 18] A method for performing protein expression in vivo or cell-free protein synthesis in vitro using the RNA polymerase according to item 1 or 2. [Advantages of the Invention]

[0009] The modified RNA polymerase provided by the present invention has improved thermal stability or specific activity compared to the wild type, and can achieve a higher yield than the conventional wild type RNA polymerase during RNA synthesis. [Modes for Carrying Out the Invention]

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

[0011] The present invention provides a modified RNA polymerase. The modified RNA polymerase (hereinafter also referred to as "modified RNA polymerase") refers to a wild type RNA polymerase in which at least one or more amino acid residues are mutated, that is, substituted, deleted or added with another amino acid.

[0012] As used herein, the term "wild-type RNA polymerase" (hereinafter also referred to as "wild-type" or "WT") refers to an RNA polymerase into which no artificial mutation has been introduced. Examples of wild-type RNA polymerases include T7 RNA polymerase, T3 RNA polymerase, K11 RNA polymerase, SP6 RNA polymerase, Syn5 RNA polymerase, KP34 RNA polymerase, VSW-3 RNA polymerase, and the like. A preferred RNA polymerase is, for example, the wild-type T7 RNA polymerase consisting of the amino acid sequence shown in SEQ ID NO: 1. T7 RNA polymerase means an RNA polymerase derived from T7 phage, as is well known to those skilled in the art.

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

[0014] The RNA polymerase of the present invention has an amino acid sequence different from that of a conventionally known wild-type RNA polymerase. Therefore, in this specification, the RNA polymerase of the present invention may sometimes be referred to as a mutant RNA polymerase or a modified RNA polymerase. In this specification, the terms "mutant" or "modified" in the case of "mutant RNA polymerase" or "modified RNA polymerase" are used interchangeably and mean having an amino acid sequence different from that of a conventionally known RNA polymerase, without distinguishing whether it is due to artificial mutation or natural mutation. Therefore, the mutant RNA polymerase of the present invention is an RNA polymerase in which one or more amino acids are modified in the amino acid sequence shown in SEQ ID NO: 1 representing the wild-type T7 RNA polymerase sequence and has an amino acid sequence different from SEQ ID NO: 1, regardless of whether the mutant RNA polymerase is an RNA polymerase obtained by artificial mutation or an RNA polymerase obtained by natural mutation in nature.

[0015] In this specification, for base sequences, amino acid sequences, and their individual constituent factors, simplified symbols using alphabetical notation may sometimes be used, all in accordance with the common practice in the fields of molecular biology and genetic engineering. Also, in this specification, to simply indicate mutations in amino acid sequences, notations such as "G197N" are used. "G197N" indicates the substitution of glycine (G) at the 197th position with asparagine (N). That is, it indicates the type of amino acid residue before substitution, its location, and the type of amino acid residue after substitution. Also, unless otherwise specified, the sequence numbers correspond to the sequence numbers described in the sequence listing.

[0016] In one embodiment, the mutant RNA polymerase may have modifications at a certain ratio in the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the mutant RNA polymerase preferably has an identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 1. The mutant RNA polymerase is not particularly limited as long as its RNA polymerase activity and / or thermal stability are not lost. For example, those composed of amino acid sequences having an identity of 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more with the amino acid sequence set forth in SEQ ID NO: 1 are preferred. Here, the identity of the amino acid sequences can be evaluated by any means known in the art. For example, it can be calculated using commercially available or analytical tools available through telecommunications lines (Internet). As an example, it is possible to calculate the identity of the amino acid sequences by using the default (initial setting) parameters in the homology algorithm BLAST (Basic local alignment search tool) of the National Center for Biotechnology Information (NCBI) of the United States at http: / / www.ncbi.nlm.nih.gov / BLAST / . Furthermore, the amino acid sequence of the mutant RNA polymerase may be an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 1. Here, "one or several" is not particularly limited as long as the RNA polymerase activity and / or thermal stability are not lost. For example, it is 1 to 30, more preferably 1 to 20, still more preferably 1 to 10, even more preferably 1 to 5, and still more preferably 1 to 3. The amino acid sequences as described above may be artificially prepared, for example, by genetic engineering techniques, or may be amino acid sequences of naturally occurring proteins.

[0017] In one embodiment, when the mutant RNA polymerase has a substitution in the amino acid sequence set forth in SEQ ID NO: 1, the substitution is preferably a substitution between amino acids that are structurally and / or chemically similar (so-called conservative substitution). Examples of conservative substitutions include, for example, substitutions between hydrophilic amino acids (N, Q, S, T, Y, C, H, K, R, D, E), preferably substitutions between neutral polar amino acids (N, Q, S, T, Y, C), substitutions between basic amino acids (H, K, R) and substitutions between acidic amino acids (D, E), substitutions between neutral nonpolar amino acids (A, G, V, L, I, P, F, M, W), substitutions between aromatic amino acids (W, F, H, Y), substitutions between nitrogen-containing amino acids (K, R, N, Q, P), substitutions between sulfur-containing amino acids (C, M), substitutions between oxygen-containing amino acids (S, T), substitutions between β-branched chain amino acids (V, L, I), and substitutions between amino acids having a straight-chain alkyl or hydrogen side chain (A, G), but are not limited thereto.

[0018] The RNA polymerase of the present invention can be composed of an amino acid sequence containing the amino acid residues as described above. A person skilled in the art can use any genetic engineering technique known in the art, for example, by appropriately designing a base sequence encoding the target amino acid sequence, transforming a host cell with a construct in which the base sequence is incorporated into an arbitrary expression vector or the like, and expressing it, and manufacturing the RNA polymerase protein of the target amino acid sequence.

[0019] In a preferred embodiment, the RNA polymerase of the present invention has an identity of 90% or more, preferably 95% or more, more preferably 96% or more, still more preferably 97% or more, even more preferably 98% or more, and most preferably 99% or more with the amino acid sequence of SEQ ID NO: 1, and is an RNA polymerase in which the amino acid at the position corresponding to the 197th position in SEQ ID NO: 1 is modified. In SEQ ID NO: 1, the amino acid at the position corresponding to the 197th position is glycine, and the amino acids that can substitute for glycine at the position corresponding to the 197th position include, but are not particularly limited to, asparagine, alanine, proline, isoleucine, leucine, valine, phenylalanine, tryptophan, tyrosine, glutamine, cysteine, methionine, serine, threonine, aspartic acid, glutamic acid, arginine, histidine, and lysine. In a more preferred embodiment, the amino acid at the position corresponding to the 197th position is substituted with a hydrophilic amino acid in the RNA polymerase. Examples of hydrophilic amino acids include asparagine, arginine, lysine, serine, threonine, aspartic acid, glutamine, glutamic acid, cysteine, histidine, tyrosine, etc., preferably asparagine and glutamine, and particularly preferably asparagine.

[0020] As used herein, the "position corresponding to the 197th position in SEQ ID NO:1" means the 197th position in the amino acid sequence of wild-type T7 RNA polymerase (SEQ ID NO:1), or the position in the amino acid sequence of mutant RNA polymerase corresponding to the 197th position in the amino acid sequence of wild-type T7 RNA polymerase (SEQ ID NO:1). The "position corresponding to the 197th position in SEQ ID NO:1" in the amino acid sequence of mutant RNA polymerase can be easily identified, for example, by comparing and aligning the amino acid sequence of wild-type T7 RNA polymerase (SEQ ID NO:1) with the amino acid sequence of the mutant RNA polymerase by any known means. For example, the comparison and alignment of amino acid sequences can be performed using commercially available or analysis tools available through telecommunications lines (Internet). As an example, amino acid sequence alignment can be performed by using the default (initial setting) parameters in the multiple alignment program Clustal Omega (https: / / www.ebi.ac.uk / jdispatcher / msa / clustalo) provided by the European Bioinformatics Institute (EMBL).

[0021] The RNA polymerase of the present invention can have a high specific activity. The RNA polymerase of the present invention can have a specific activity of about 1.1 times or more, preferably about 1.2 times or more, about 1.3 times or more, about 1.4 times or more, about 1.5 times or more, about 1.6 times or more, about 1.7 times or more, about 1.8 times or more compared to the corresponding wild-type RNA polymerase. Specifically, the specific activity can be confirmed by the following measurement method.

[0022] [Activity measurement method] The activity of RNA polymerase can be measured by the following procedure. The measurement conditions can be appropriately set by those skilled in the art. For example, if the enzyme activity is too high, the sample containing the measurement target can be appropriately diluted and then measured. First, 45 μL of reaction solution (the final concentrations in 50 μL of the reaction solution after enzyme addition are shown below) (40 mM Tris-HCl (pH 8.0), 50 mM NaCl, 8 mM MgCl2, 5 mM DTT, 10 ng / μL template DNA, 0.4 mM ATP, 0.4 mM CTP, 0.4 mM GTP, 0.4 mM UTP) was added to a 0.2 mL PCR tube, and then 5 μL of wild-type RNA polymerase with known activity was further added, and the reaction was carried out at 37 °C for 10 minutes in a heat block. After 10 minutes, the reaction was stopped by adding 10 μL of 1 M EDTA. This reaction was performed for a dilution series of RNA polymerase. Then, the amount of RNA in each reaction solution was quantified using the Qubit® RNA BR Assay Kit (manufactured by Thermo Fisher Scientific), and a calibration curve was created from the activity values of the RNA polymerase added in each dilution series and the quantified amount of RNA. The same reaction was also performed for the RNA polymerase to be measured, and the activity was calculated using the calibration curve created above based on the obtained amount of RNA.

[0023] [Measurement of specific activity] Measure the absorbance at 280 nm (A280), and calculate the enzyme concentration assuming that the enzyme concentration is 1 mg / mL when A280 is 1. The specific activity is calculated by dividing the activity value calculated as described above by the enzyme concentration.

[0024] The RNA polymerase of the present invention may exhibit high thermal stability compared to the corresponding wild-type RNA polymerase. In a preferred embodiment, the RNA polymerase of the present invention may be an RNA polymerase that exhibits a residual activity greater than 50% when heat-treated at 50 °C for 5 minutes, and may further be an RNA polymerase that exhibits a residual activity rate of 60% or more, an RNA polymerase that exhibits a residual activity rate of 70% or more, or an RNA polymerase that exhibits a residual activity rate of 80% or more. In a specific embodiment, the RNA polymerase of the present invention may be an RNA polymerase that exhibits a residual activity rate of 90% or more when heat-treated at 50 °C for 5 minutes.

[0025] From yet another perspective, the RNA polymerase of the present invention can be, for example, a mutant RNA polymerase that exhibits a high residual activity rate when heat-treated under certain conditions as compared to the residual activity rate of the corresponding wild-type RNA polymerase. Specifically, for example, when heat-treated at 50°C for 5 minutes, it can be an RNA polymerase that exhibits a residual activity rate about 1.1-fold or more, preferably about 1.2-fold or more, preferably about 1.2-fold or more, preferably about 1.3-fold or more, preferably about 1.4-fold or more, preferably about 1.5-fold or more, preferably about 1.6-fold or more, preferably about 1.7-fold or more, preferably about 1.8-fold or more, preferably about 1.9-fold or more higher than the residual activity rate of the corresponding wild-type RNA polymerase.

[0026] [Measurement of Thermal Stability (Residual Activity Rate after Heat Treatment)] Specifically, the thermal stability can be confirmed by the following measurement method. First, each mutant RNA polymerase to be measured is diluted to 50 U / μL with a storage buffer (20 mM KPO4 (pH 7.7), 100 mM NaCl, 50% glycerol, 0.1 mM EDTA, 5 mM DTT, 0.01% Triton® X-100), and then the activity value before storage is measured according to the procedure described in the above activity measurement method. Next, each mutant RNA polymerase to be measured diluted in the above storage buffer is stored under predetermined storage conditions (for example, under the condition of storing in an incubator at 40°C to 60°C for 1 to 20 minutes, and in a preferred embodiment, under the condition of storing in an incubator at 50°C for 5 minutes). After a predetermined time has elapsed since the start of storage (for example, after 1 to 20 minutes, and in a preferred embodiment, after 5 minutes), the activity value after storage is measured according to the procedure described in the above activity measurement method in the same manner as before storage. Then, the residual activity rate can be calculated by dividing the activity value after storage by the activity value before storage as described in the following formula I. Residual activity rate (%) = (Activity value after storage / Activity value before storage) × 100... (Formula I)

[0027] In a further embodiment, the present invention provides a polynucleotide encoding the RNA polymerase of the present invention as described above. Here, the polynucleotide encoding the RNA polymerase refers to, for example, a polynucleotide from which the protein of the RNA polymerase of the present invention can be obtained when it is expressed by a conventional method. That is, it refers to a polynucleotide composed of a base sequence corresponding to the amino acid sequence of the protein of the RNA polymerase of the present invention. A person skilled in the art can easily determine the base sequence corresponding to a given amino acid sequence according to the codon table well-known in the art. In addition, the polynucleotide encoding the RNA polymerase of the present invention also includes polynucleotides that differ due to codon degeneracy. The polynucleotide can be any nucleic acid polymer such as DNA, RNA, etc.

[0028] In a further embodiment, the present invention provides a vector containing the polynucleotide. Specifically, the polynucleotide encoding the above RNA polymerase is transferred to a vector (for example, an expression vector, a cloning vector, etc.) as needed. The vector can be any vector as long as it enables cloning and / or expression of the RNA polymerase of the present invention, and examples include plasmids. Examples of plasmids include, but are not limited to, pUC118, pUC18, pBR322, pBluescript, pLED-M1, p73, pGW7, pkk223-3, etc.

[0029] In a further embodiment, the present invention provides a cell transformed with the vector. Such a cell can be suitably used for expressing the protein encoding the RNA polymerase of the present invention. In certain preferred embodiments, the recombinant host cell of the present invention is obtained by transforming a host cell with the above expression vector. Examples of the host cell include Escherichia coli and yeast, and Escherichia coli is particularly preferred. Examples of Escherichia coli include Escherichia coli DH5α, JM109, HB101, XL1Blue, PR1, BL21, etc. That is, in the present invention, it is preferable to insert the gene encoding the above RNA polymerase into the above vector to obtain an expression vector, and further transform the host cell with the expression vector.

[0030] In one embodiment, the expression vector of the present invention may contain elements for facilitating the purification of RNA polymerase, such as extracellular secretion signals, protein purification tags (His tag, Flag tag, Strep tag, GST tag, MBP tag), etc.

[0031] In a further embodiment, a method for producing the RNA polymerase using the polynucleotide, the vector, the recombinant host cell, and / or one or more of these is also provided. For example, after transforming a host cell with the expression vector, it is spread on an agar medium containing a drug such as ampicillin to form colonies. The colonies are inoculated into a nutrient medium, such as LB medium, 2×YT medium, TB medium, etc., cultured at 37°C for 12 to 20 hours, and then the cells are disrupted to extract a crude enzyme solution. As a method for disrupting the cells, any known method may be used. For example, physical disruption methods such as ultrasonic treatment, French press, or glass bead disruption, or lytic enzymes such as lysozyme can be used. As a method for obtaining the purified RNA polymerase from the obtained crude enzyme solution, any method may be used. For example, by subjecting it to centrifugation, ultracentrifugation, ultrafiltration, nucleic acid removal treatment, salting out, dialysis, ion exchange column chromatography, hydrophobic column chromatography, affinity chromatography, gel filtration column chromatography, etc., the RNA polymerase of the present invention can be isolated.

[0032] In a further embodiment, a reagent containing the RNA polymerase, the polynucleotide, the vector, the recombinant host cell, and / or one or more of these is also provided.

[0033] The present invention further provides a method for performing an in vitro transcription reaction using the RNA polymerase. Examples of the in vitro transcription reaction include, but are not particularly limited to, a method of synthesizing RNA using the RNA polymerase with linear or circular DNA as a template. Examples of the in vitro transcription reaction include, but are not particularly limited to, a method of synthesizing RNA using the RNA polymerase with linear or circular DNA as a template in the presence of a buffer and ribonucleotides. The concentration of the RNA polymerase added during the reaction can be, for example, in the range of 0.1 U / μL to 20 U / μL, but is not particularly limited. The concentration of linear or circular DNA can be, for example, in the range of 1 ng / μL to 200 ng / μL, but is not particularly limited. Examples of the ribonucleotides used in the in vitro transcription reaction include, in addition to ATP, CTP, GTP, and UTP, modified bases such as those modified by deamination, methylation, pseudouridylation, etc., but are not particularly limited. The concentration of ribonucleotides can be, for example, in the range of 0.1 mM to 20 mM, but is not particularly limited. During the in vitro transcription reaction, cap analogs such as ARCA, mCAP, CleanCap® Reagent AG, CleanCap® Reagent M6, CleanCap® Reagent AU (manufactured by TriLink) can be added, but are not particularly limited. The concentration of the cap analog can be, for example, in the range of 0.1 mM to 20 mM, but is not particularly limited. Examples of the buffer used in the in vitro transcription reaction include Tris buffer, phosphate buffer, Good's buffer, etc., but are not particularly limited. The concentration of the buffer can be, for example, in the range of 5 mM to 500 mM, but is not particularly limited. During the in vitro transcription reaction, RNase inhibitor, pyrophosphatase, etc. can be added, but are not particularly limited. During the in vitro transcription reaction, salts such as NaCl and metal ions such as magnesium ions can be added, but are not particularly limited. The concentration of NaCl can be, for example, in the range of 1 mM to 200 mM, but is not particularly limited.The concentration of magnesium ions can be added, for example, in the range of 0.1 mM to 20 mM, but is not particularly limited. During the in vitro transcription reaction, EDTA, dithiothreitol, glycerol, surfactant, spermidine, etc. can be added, but are not particularly limited. The concentration of EDTA can be added, for example, in the range of 0.0001 mM to 2 mM, but is not particularly limited. The concentration of dithiothreitol can be added, for example, in the range of 0.1 mM to 20 mM, but is not particularly limited. The concentration of glycerol can be added, for example, in the range of 0.1% to 20%, but is not particularly limited. The concentration of surfactant can be added, for example, in the range of 0.0001% to 1%, but is not particularly limited. The concentration of spermidine can be added, for example, in the range of 0.1 mM to 20 mM, but is not particularly limited.

[0034] In addition, as a specific embodiment, the present invention further provides a method for performing an in vitro transcription reaction in which the yield of RNA after the in vitro transcription reaction in the transcription reaction is at least 110% or more compared to the case where the in vitro transcription reaction is performed using the corresponding wild-type RNA polymerase. The ratio can preferably be 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more. The ratio can be calculated, for example, by performing an in vitro transcription reaction with the corresponding wild-type RNA polymerase and each mutant RNA polymerase to be measured by the method described in [Activity Measurement Method], and comparing the amounts of RNA obtained.

[0035] In a specific embodiment, the present invention further provides a method for synthesizing mRNA using the RNA polymerase. Examples of the method for synthesizing mRNA include, but are not particularly limited to, a method of synthesizing mRNA using linear or circular DNA as a template in the presence of a buffer and ribonucleotides, using the RNA polymerase. The concentration of the RNA polymerase added during the reaction can be, for example, in the range of 0.1 U / μL to 20 U / μL, but is not particularly limited. The concentration of the linear or circular DNA can be, for example, in the range of 1 ng / μL to 200 ng / μL, but is not particularly limited. Examples of the ribonucleotides used for mRNA synthesis include, in addition to ATP, CTP, GTP, and UTP, modified bases such as those modified by deamination, methylation, pseudouridylation, etc., but are not particularly limited. The concentration of the ribonucleotides can be, for example, in the range of 0.1 mM to 20 mM, but is not particularly limited. Examples of the buffer used for mRNA synthesis include Tris buffer, phosphate buffer, Good's buffer, etc., but are not particularly limited. The concentration of the buffer can be, for example, in the range of 5 mM to 500 mM, but is not particularly limited. During mRNA synthesis, an RNase inhibitor, pyrophosphatase, etc. can be further added, but are not particularly limited. The concentration of the RNase inhibitor can be, for example, in the range of 0.1 U / μL to 20 U / μL, but is not particularly limited. The concentration of the pyrophosphatase can be, for example, in the range of 0.0001 U / μL to 0.02 U / μL, but is not particularly limited. During mRNA synthesis, salts such as NaCl and metal ions such as magnesium ions can be further added, but are not particularly limited. The concentration of NaCl can be, for example, in the range of 1 mM to 200 mM, but is not particularly limited. The concentration of the magnesium ions can be, for example, in the range of 0.1 mM to 20 mM, but is not particularly limited. During mRNA synthesis, EDTA, dithiothreitol, glycerol, surfactant, spermidine, etc. can be further added, but are not particularly limited. The concentration of EDTA can be, for example, in the range of 0.0001 mM to 2 mM, but is not particularly limited.The concentration of dithiothreitol can be added, for example, in the range of 0.1 mM to 20 mM, but is not particularly limited. The concentration of glycerol can be added, for example, in the range of 0.1% to 20%, but is not particularly limited. The concentration of the surfactant can be added, for example, in the range of 0.0001% to 1%, but is not particularly limited. The concentration of spermidine can be added, for example, in the range of 0.1 mM to 20 mM, but is not particularly limited. The 5'-end of the mRNA contains a cap structure. As a method for adding the cap structure at that time, post-transcriptional capping and co-transcriptional capping can be mentioned, but are not particularly limited. Examples of post-transcriptional capping include methods using a capping enzyme, GTP, and SAM, but are not particularly limited. Examples of co-transcriptional capping include methods using cap analogs such as ARCA, mCAP, CleanCap (registered trademark) Reagent AG, CleanCap (registered trademark) Reagent M6, and CleanCap (registered trademark) Reagent AU (manufactured by TriLink), but are not particularly limited. The concentration of the cap analog can be added, for example, in the range of 0.1 mM to 20 mM, but is not particularly limited. PolyA exists at the 3'-end of the mRNA. When the template DNA does not contain polyA, examples of methods for adding polyA to the 3'-end of the synthesized RNA include methods using polyA polymerase, but are not particularly limited.

[0036] Also, as a specific embodiment, the present invention further provides an mRNA synthesis method in which the yield of mRNA in the transcription reaction is at least 110% or more compared to the case where mRNA synthesis is performed using the corresponding wild-type RNA polymerase. The ratio can preferably be 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more. The ratio can be calculated, for example, by performing mRNA synthesis using the corresponding wild-type RNA polymerase and each mutant RNA polymerase to be measured by the method described in [Activity measurement method], and comparing the obtained RNA amounts.

[0037] Also, as a specific embodiment, the present invention further provides a method for synthesizing non-coding RNA using the RNA polymerase. Examples of non-coding RNA include, but are not particularly limited to, microRNA, siRNA, piRNA, rRNA, tRNA, snRNA, snoRNA, SLRNA, SRPRNA, mRNA-like non-coding RNA, oligonucleotides, etc.

[0038] Also, as a specific embodiment, the present invention further provides a method for synthesizing guide RNA for gene editing using the RNA polymerase. Examples of gene editing methods include, but are not particularly limited to, the CRISPER / Cas9 system, etc.

[0039] Also, as a specific embodiment, the present invention further provides a method for using the RNA polymerase in the synthesis of RNA pharmaceuticals. Examples of RNA pharmaceuticals include, but are not particularly limited to, mRNA, siRNA, microRNA, antisense RNA, those using aptamers, etc.

[0040] Also, as a specific embodiment, the present invention further provides a method for using the RNA polymerase in isothermal amplification reactions. Examples of isothermal amplification reactions include, but are not particularly limited to, the NASBA method, the TMA method, etc.

[0041] Also, as a specific embodiment, the present invention further provides a method for performing gene testing using the RNA polymerase. Examples of methods for performing gene testing include those in which the gene of a target pathogen is amplified and detected by the above-mentioned isothermal amplification reaction, etc., to confirm the presence or absence of the pathogen, but are not particularly limited to this.

[0042] Also, as a specific embodiment, the present invention further provides a method for performing in vivo protein expression or cell-free protein synthesis in vitro using the RNA polymerase.

Examples

[0043] Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following examples.

[0044] Preparation of Plasmid for T7 RNA Polymerase Expression in Example 1 A gene encoding wild-type T7 RNA polymerase with a 6×His tag added to the N-terminus (SEQ ID NO: 2) was artificially synthesized and cloned into the EcoRI-PstI cloning site of pkk223-3 to create a plasmid incorporating the gene encoding wild-type T7 RNA polymerase with a 6×His tag added to the N-terminus. Hereinafter, this expression plasmid will be referred to as pkk223-3-T7RNAP. Using pkk223-3-T7RNAP as a template, site-directed mutagenesis was performed using the primers shown in Table 1 and the KOD-Plus-Mutagenesis Kit (manufactured by Toyobo) to create an expression plasmid for modified T7 RNA polymerase.

[0045]

Table 1

[0046] The obtained plasmid has a nucleotide sequence encoding a modified T7 RNA polymerase with a 6×His tag added to the N-terminus.

[0047] Preparation of T7 RNA Polymerase in Example 2 The wild-type T7 RNA polymerase and the modified T7 RNA polymerase with a 6×His tag added to the N-terminus were all prepared by the same method. Hereinafter, as an example, a preparation example of the wild-type T7 RNA polymerase is shown. Escherichia coli JM109 was transformed with pkk223-3-T7RNAP and statically cultured at 37 °C for 20 hours on an LB agar medium containing 100 μg / mL ampicillin. A single colony on the agar medium was inoculated into 3 mL of LB liquid medium containing 100 μg / mL ampicillin and shake-cultured at 37 °C for 20 hours in a 15 mL test tube. 1 mL of this culture solution was inoculated into 80 mL of TB liquid medium containing 100 μg / mL ampicillin, shake-cultured at 37 °C for 20 hours in a 500 mL Sakaguchi flask, and then the cells were recovered from the culture solution by centrifugation. 1 g of the obtained cells was suspended in 10 mL of disruption buffer (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10% glycerol, 1 mM DTT, 20 mM imidazole), and the cells were disrupted on ice using an ultrasonic disruptor. This cell disruption solution was centrifuged at 20,000×g for 20 minutes at 4 °C, and after the supernatant was recovered, the supernatant was used for purification with His GraviTrap (manufactured by GE Healthcare). The equilibration and washing buffer was (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10% glycerol, 1 mM DTT, 20 mM imidazole), and the elution buffer was (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10% glycerol, 1 mM DTT, 300 mM imidazole). The eluted fraction was recovered and replaced with a storage buffer (20 mM KPO4 (pH 7.7), 100 mM NaCl, 50% glycerol, 0.1 mM EDTA, 5 mM DTT, 0.01% Triton® X-100) to obtain a wild-type T7 RNA polymerase with a 6×His tag added to the N-terminus. In the above preparation example, a modified T7 RNA polymerase with a 6×His tag added to the N-terminus was obtained by using the plasmid for expressing the modified T7 RNA polymerase prepared in Example 1 during transformation.

[0048] Measurement of Specific Activity of T7 RNA Polymerase in Example 3 The activities were measured using wild-type T7 RNA polymerase with an added His-tag at the N-terminus and modified T7 RNA polymerase (G197N) with an added His-tag at the N-terminus in the above-described activity measurement method. As the template DNA at that time, approximately 4 kbp of dsDNA (SEQ ID NO: 5) containing the T7 promoter sequence was used. The length downstream of the T7 promoter of this dsDNA is approximately 1 kbp, and the length of the transcribed RNA is approximately 1 kb. The protein concentration of each modified T7 RNA polymerase was calculated by measuring the absorbance at A280, and the specific activity was calculated by dividing by the activity and the protein concentration. The results are shown in Table 2.

[0049]

Table 2

[0050] From the results in Table 2, it was confirmed that the specific activity of the modified T7 RNA polymerase (G197N) was improved compared to the wild type.

[0051] Measurement of Thermostability of T7 RNA Polymerase in Example 4 The wild-type T7 RNA polymerase with an added His-tag at the N-terminus and the modified T7 RNA polymerase (G197N) with an added His-tag at the N-terminus were diluted to 50 U / μL with a storage buffer (20 mM KPO4 (pH 7.7), 100 mM NaCl, 50% glycerol, 0.1 mM EDTA, 5 mM DTT, 0.01% Triton® X-100), and then the activity values before storage were measured according to the procedure described in the above activity measurement method. Each T7 RNA polymerase diluted in the above storage buffer was stored in an incubator at 50 °C for 5 minutes, and then the activity was measured, and the residual activity rate was calculated from the activity values before and after storage. The results are shown in Table 3.

[0052]

Table 3

[0053] From the results in Table 3, it was confirmed that the modified T7 RNA polymerase (G197N) had a higher residual activity rate after heat treatment compared to the wild type and its heat resistance was improved.

Industrial Applicability

[0054] The present invention can be suitably used for RNA synthesis for various purposes.

Claims

1. An RNA polymerase having at least 90% identity with the amino acid sequence of SEQ ID NO: 1 and having the amino acid at the position corresponding to the 197th position in SEQ ID NO: 1 modified.

2. An RNA polymerase having at least 90% identity with the amino acid sequence of SEQ ID NO: 1 and having the amino acid at the position corresponding to the 197th position in SEQ ID NO: 1 modified to asparagine.

3. A polynucleotide encoding the RNA polymerase according to Claim 1 or 2.

4. A vector containing the polynucleotide encoding the RNA polymerase according to Claim 1 or 2.

5. A recombinant host cell transformed with the vector containing the polynucleotide encoding the RNA polymerase according to Claim 1 or 2.

6. A method for producing an RNA polymerase using the polynucleotide encoding the RNA polymerase according to Claim 1 or 2, the vector containing the polynucleotide, or the recombinant host cell transformed with the vector.

7. A reagent comprising the RNA polymerase according to Claim 1 or 2, the polynucleotide encoding the RNA polymerase, the vector containing the polynucleotide, or the recombinant host cell transformed with the vector.

8. A method for performing an in vitro transcription reaction using the RNA polymerase according to Claim 1 or 2.

9. The method according to Claim 8, wherein the amount of RNA after the in vitro transcription reaction is 110% or more as compared with the case where the in vitro transcription reaction is performed using the corresponding wild-type RNA polymerase.

10. A method for synthesizing mRNA using the RNA polymerase according to Claim 1 or 2.

11. The method according to Claim 10, wherein the amount of mRNA is 110% or more as compared with the case where mRNA synthesis is performed using the corresponding wild-type RNA polymerase.

12. A method for synthesizing non-coding RNA using the RNA polymerase according to Claim 1 or 2.

13. The method according to Claim 12, wherein the non-coding RNA is at least one selected from microRNA, siRNA, piRNA, rRNA, tRNA, snRNA, snoRNA, SLRNA, SRPRNA, mRNA-like non-coding RNA, and oligonucleotide.

14. A method for synthesizing guide RNA for gene editing using the RNA polymerase according to claim 1 or 2.

15. A method for synthesizing RNA pharmaceuticals using the RNA polymerase according to claim 1 or 2.

16. A method for performing an isothermal amplification reaction using the RNA polymerase according to claim 1 or 2.

17. A method for performing gene testing using the RNA polymerase according to claim 1 or 2.

18. A method for performing protein expression in vivo or cell-free protein synthesis in vitro using the RNA polymerase according to claim 1 or 2.

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