A functionally improved T7 RNA polymerase variant

Abstract

The present invention relates to the field of nucleic acid synthesis, and specifically provides a functionally improved T7 RNA polymerase variant, which, based on the wild-type T7 RNA polymerase, comprises one or more amino acid mutations at the 75th, 216th, 237th, 744th and 786th amino acid residues relative to the amino acid positions shown in SEQ ID NO: 1. The results of in vitro transcription assays show that, compared with the wild-type T7 RNA polymerase, the improved T7 RNA polymerase variant of the present invention exhibits better performance in synthesizing mRNA.

Description

[0001] Cross-reference information

[0002] This application claims priority to Chinese patent application No. 202310898803.2 filed on July 20, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of nucleic acid synthesis, and in particular to a T7 RNA polymerase variant with improved function. Background Art

[0004] T7 RNA polymerase (EC 2.7.7.6) is a monomeric bacteriophage-produced RNA polymerase that catalyzes the formation of RNA in the 5'→3' direction. During transcription initiation, T7 recognizes a specific promoter sequence, the T7 promoter. T7 RNA polymerase consists of 883 amino acids and has a molecular weight of 99 kDa. T7 RNA polymerase has many characteristics relative to other RNA polymerases: a) it is a single-subunit enzyme compared to multi-subunit prokaryotic and eukaryotic RNA polymerases; b) it is highly specific for the T7 promoter and has no affinity for unrelated DNA, including the highly homologous T3 promoter (Klement et al., 1990); c) it does not require any additional protein factors to perform a complete transcription cycle (Cheetham and Steitz, 2000); d) it elongates about 5 times faster than E. coli RNA polymerase and can produce very long transcripts (Pavco and Steege, 1991); f) transcription termination can only be performed by class I and class II termination signals, independent of the transcription termination factor of E. coli RNA polymerase (Lyakhov et al., 1998). These characteristics increase the advantages of this enzyme, and it is therefore widely used to express heterologous genes under the control of the T7 promoter for in vivo and in vitro experiments.

[0005] Synthetic messenger RNA (mRNA)-based therapeutics are increasingly used in gene and cell therapy, genome engineering, enzyme replacement therapy, and now vaccine development during the global SARS-CoV-2 pandemic. mRNA for such purposes can be generated by enzymatic in vitro transcription (IVT) reactions and formulated for in vivo delivery.

[0006] Enzymatic synthesis of modified RNA is a simple and cost-effective technical route. Unfortunately, the wild-type T7 RNA polymerase, a commonly used enzyme in this RNA in vitro transcription synthesis reaction, has a low efficiency in incorporating modified nucleotides. Therefore, protein modification of T7 RNA polymerase through genetic engineering to find T7 RNA polymerase mutants that can efficiently incorporate modified nucleotides and at the same time increase the yield of modified / unmodified RNA is the key to solving the current problem of being unable to generate large amounts of modified RNA by enzymatic synthesis in vitro (Jackson et al., 2020). In the "Technical Guidelines for Pharmaceutical Research of mRNA Vaccines for the Prevention of New Coronavirus" issued by NMPA in 2020, it is recommended to control the quality of DNA templates, mRNA stock solutions and finished LNP-mRNA, including the unique characterization of mRNA: capping rate, polyA tail length, dsRNA residue, etc. How to improve these key indicators plays a key role in the generation of mRNA vaccines. Summary of the invention

[0007] In a first aspect, the present invention provides a T7 RNA polymerase variant, which comprises one or more amino acid mutations at amino acid residues 75, 216, 237, 744 and 786 relative to the amino acid position of SEQ ID NO: 1 based on the wild-type T7 RNA polymerase.

[0008] In some embodiments, the amino acid sequence of the wild-type T7 RNA polymerase is shown in SEQ ID NO:1.

[0009] In some embodiments, the T7 RNA polymerase variant comprises one or more mutations of T75L, C216L, V237K, Q744R, and Q786M.

[0010] In other embodiments, the T7 RNA polymerase variant comprises a mutation selected from the group consisting of:

[0011] (1) T75L, C216L, V237K and Q786M; (2) T75L, C216L, Q744R and Q786M; and (3) T75L, C216L and Q786M.

[0012] In some embodiments, the T7 RNA polymerase variant comprises the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0013] In a second aspect, the present invention also provides an isolated nucleic acid encoding the above-mentioned T7 RNA polymerase variant.

[0014] In some embodiments, the nucleic acid encoding the T7 RNA polymerase variant comprises the nucleotide sequence set forth in SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0015] In a third aspect, the present invention also provides a recombinant vector comprising the above-mentioned isolated nucleic acid.

[0016] In a fourth aspect, the present invention further provides a host cell comprising the isolated nucleic acid or the recombinant vector, wherein the host cell is capable of expressing the T7 RNA polymerase variant under suitable conditions.

[0017] In a fifth aspect, the present invention further provides a kit comprising the aforementioned T7 RNA polymerase variant, the aforementioned isolated nucleic acid, the aforementioned recombinant vector or the aforementioned host cell.

[0018] In a sixth aspect, the present invention further provides use of the aforementioned T7 RNA polymerase variant, the aforementioned isolated nucleic acid, the aforementioned recombinant vector, the aforementioned host cell or the aforementioned kit in in vitro transcription synthesis of mRNA.

[0019] In a seventh aspect, the present invention also provides a method for producing RNA, comprising using the above-mentioned T7 RNA polymerase variant to transcribe and synthesize RNA.

[0020] The results of in vitro transcription assays show that, compared with the wild-type T7 RNA polymerase, the improved T7 RNA polymerase variant of the present invention exhibits better performance in synthesizing mRNA. DETAILED DESCRIPTION

[0021] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0022] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0023] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0024] In the present invention, “preferred”, “better”, “more preferred” and “suitable” are only used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of the present invention.

[0025] The present invention first provides a T7 RNA polymerase variant, which, based on a wild-type T7 RNA polymerase, comprises one or more amino acid mutations selected from the following 1) to 5): 1) an amino acid corresponding to the 75th amino acid of the amino acid sequence shown in SEQ ID NO: 1; 2) an amino acid corresponding to the 216th amino acid of the amino acid sequence shown in SEQ ID NO: 1; 3) an amino acid corresponding to the 237th amino acid of the amino acid sequence shown in SEQ ID NO: 1; 4) an amino acid corresponding to the 744th amino acid of the amino acid sequence shown in SEQ ID NO: 1; 5) an amino acid corresponding to the 786th amino acid of the amino acid sequence shown in SEQ ID NO: 1.

[0026] In the present invention, "mutation" refers to the substitution, deletion or insertion of amino acid residues at one or more sites in the amino acid sequence of a protein (such as T7 RNA polymerase). In some embodiments of the present invention, the mutation on the amino acid residue can be an amino acid residue substitution.

[0027] In the present invention, "correspondence" means that the compared proteins (enzymes) make equal contributions to the performance of their functions. For example, for a reference amino acid sequence (i.e., the amino acid sequence of SEQ ID NO: 1), when the amino acid sequence of the comparison object is arranged in a manner that allows for optimal comparison while taking into account the partial homology of the primary structure (amino acid sequence) (at this time, gaps can be introduced as needed to optimize the alignment), the amino acid at the position corresponding to the specific amino acid in the reference amino acid sequence can be determined as the "corresponding amino acid". Instead of comparing the primary structures, the comparison between the three-dimensional structures can be used, or not only the comparison between the primary structures but also the comparison between the three-dimensional structures can be used to determine the "corresponding amino acid". By using the three-dimensional structure information, a highly reliable comparison result can be obtained. In this case, a method can be used to compare while comparing the atomic coordinates of the three-dimensional structures of multiple enzymes. The three-dimensional structure information of the mutant target enzyme can be obtained from a common protein database in the art.

[0028] The present invention found that the above mutation sites are highly correlated with the ability of T7 RNA polymerase to synthesize mRNA. When mutations occur at the above sites, the T7 RNA polymerase variants show better performance in synthesizing mRNA.

[0029] In some embodiments, those skilled in the art can select one of the above-mentioned sites for mutation, or can select multiple (such as 2, 3, 4; or 5) amino acids for combined mutation, both of which can enable T7 RNA polymerase to obtain better performance.

[0030] In a preferred embodiment of the present invention, the amino acid sequence of the wild-type T7 RNA polymerase is shown as SEQ ID NO:1.

[0031] The following nomenclature is used for amino acid residue substitution in the present invention: original amino acid, position, substituted amino acid. As an example, threonine (T) at position T75 is substituted with lysine (L) and represented as T75L. Multiple amino acid residue substitutions are separated by a comma (""), such as T75L, C216L, V237K.

[0032] In a preferred embodiment of the present invention, the T7 RNA polymerase variant may contain one or more mutations selected from T75L, C216L, V237K, Q744R and Q786M. When the T7 RNA polymerase variant undergoes the above amino acid residue substitutions, its ability to synthesize mRNA is better.

[0033] As a preferred embodiment of the present invention, the T7 RNA polymerase variant may include mutations selected from the following groups: (1) T75L, C216L, V237K and Q786M; (2) T75L, C216L, Q744R and Q786M; and (3) T75L, C216L and Q786M. When the mutations are combined according to the above scheme, the comprehensive performance of the T7 RNA polymerase variant is significantly better. Among them, it is further preferred that the T7 RNA polymerase variant includes mutations of T75L, C216L, Q744R and Q786M.

[0034] In a more preferred embodiment, the T7 RNA polymerase variant comprises the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0035] As a preferred embodiment, the amino acid sequence of the T7 RNA polymerase variant is shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0036] In addition, in some embodiments, the T7 RNA polymerase variant may have one or more conservative substitutions of amino acids compared to the solutions provided by the present invention (such as SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4). Preferably, the above-mentioned "conservative substitutions" occur at positions other than the mutation sites mentioned in the present invention.

[0037] The "conservative substitution" mentioned herein is also referred to as substitution by "homologous" amino acid residues, and refers to substitutions in which the amino acid residues are replaced by amino acid residues having similar side chains, for example, amino acids with basic side chains (such as lysine, arginine and histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), non-charged polar side chain amino acids (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chain amino acids (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chain amino acids (such as threonine, valine, isoleucine) and aromatic side chain amino acids (such as tyrosine, phenylalanine, tryptophan, histidine). Conservative amino acid substitutions usually have little effect on the activity of the resulting protein.

[0038] In general, when a part of the amino acid sequence of a certain protein is mutated, sometimes the mutated protein has the same function as the protein before the mutation. That is, sometimes the mutation of the amino acid sequence does not have a substantial effect on the function of the protein, and the function of the protein is maintained before and after the mutation. If this technical common sense is taken into account, when compared with the T7 RNA polymerase variant consisting of an amino acid sequence obtained by mutation of one or more amino acids selected from the above 1) to 5), although the slight difference in the amino acid sequence is not confirmed (wherein, the difference in the amino acid sequence occurs at a position other than the mutation site mentioned in the present invention), it can be considered to be an enzyme substantially the same as the above T7 RNA polymerase variant. Here, "slightly different amino acid sequences" typically refer to the deletion, substitution, or addition, insertion, or combination of one or more (upper limit is, for example, 3, 5, 7 or 10) amino acids constituting the amino acid sequence. Mutation is preferably a conservative substitution of amino acids. The amino acid sequence of the "substantially identical enzyme" preferably has an identity (%) of 90% or more, more preferably 95% or more, further preferably 98% or more, and most preferably 99% or more to the amino acid sequence of the T7 RNA polymerase variant used as a benchmark. The amino acid sequence may differ at multiple positions.

[0039] Those skilled in the art can prepare the above-mentioned T7 RNA polymerase variants in combination with common sense, and the preparation method thereof is not particularly limited in the present application. In some embodiments, the preparation method of the T7 RNA polymerase variant comprises: expressing a gene encoding the T7 RNA polymerase variant in a host. In a preferred embodiment, the gene expressing the T7 RNA polymerase variant can be first inserted into a pQE-80L vector to obtain a recombinant plasmid; then the recombinant plasmid is introduced into Escherichia coli BL21 (DE3) to obtain a recombinant bacterium; and then the recombinant bacterium is cultured to obtain the T7 RNA polymerase variant.

[0040] The present invention also provides an isolated nucleic acid encoding the above-mentioned T7 RNA polymerase variant.

[0041] In a preferred embodiment of the present invention, the isolated nucleic acid comprises the nucleotide sequence shown in SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

[0042] In one embodiment, the isolated nucleic acid has a nucleotide sequence as shown in SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

[0043] In some embodiments, when the isolated nucleic acid is compared with SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, although the function of the protein encoded by it is the same, a part of the base sequence is different, and it is composed of a base sequence containing one or more base substitutions, deletions, insertions, additions, or inversions. Preferably, the isolated nucleic acid has at least about 60%, 70%, 80%, 90%, 95%, 99%, 99.9% similarity with SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 or its complementary sequence, or can hybridize with SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 under stringent conditions.

[0044] The nucleic acid of the present invention can be prepared in an isolated state by using standard genetic engineering methods, molecular biology methods, biochemical methods, etc., with reference to the sequence information disclosed in this specification or the attached sequence listing.

[0045] The isolated nucleic acid can be used to prepare T7 RNA polymerase variants, or as an experimental tool for the purpose of explaining the mechanism of action of T7 RNA polymerase variants, or as a tool for designing or preparing further mutants of T7 RNA polymerase.

[0046] The present invention also provides a recombinant vector comprising the isolated nucleic acid.

[0047] The "vector" of the present invention refers to a nucleic acid molecule capable of transporting a nucleic acid inserted therein to a target. Those skilled in the art can select an appropriate vector according to the purpose of use (cloning, expression of protein) and the type of host cell. The vector of the present invention is preferably an expression vector. "Expression vector" refers to a vector capable of introducing a nucleic acid inserted therein into a target cell (host cell) and capable of expressing it in the cell. An expression vector generally comprises a promoter sequence necessary for the expression of the inserted nucleic acid, an enhancer sequence that promotes expression, and the like. In addition, an expression vector comprising a selection marker can also be used. When using the expression vector, the presence or absence (and degree) of the introduction of the expression vector can be confirmed by utilizing the selection marker.

[0048] The present invention also provides a host cell, which comprises the above-mentioned isolated nucleic acid or the above-mentioned recombinant vector. The host cell can express the above-mentioned T7 RNA polymerase variant under suitable conditions.

[0049] As host cells, microorganisms such as Escherichia coli and budding yeast are preferably used from the perspective of ease of operation, but any host cell can be used in the present invention as long as the recombinant vector can replicate and the gene of the T7 RNA polymerase variant can be expressed.

[0050] The present invention also provides a kit, which comprises the above-mentioned T7 RNA polymerase variant, the above-mentioned isolated nucleic acid, the above-mentioned recombinant vector or the above-mentioned host cell.

[0051] In one embodiment, the kit further contains one or more selected from the group consisting of a buffer reagent, ribonucleoside triphosphates, a nuclease inhibitory protein, a DNA enzyme, an RNA purification reagent, and water.

[0052] The present invention also provides the use of the above-mentioned T7 RNA polymerase variant, the above-mentioned isolated nucleic acid, the above-mentioned recombinant vector, the above-mentioned host cell or the above-mentioned kit in in vitro transcription synthesis of mRNA.

[0053] The present invention also provides a method for producing RNA, comprising using the above-mentioned T7 RNA polymerase variant to transcribe and synthesize RNA.

[0054] In some embodiments, the method for producing RNA comprises: using ribonucleoside triphosphates as raw materials, using a template DNA encoding the target RNA and the T7 RNA polymerase variant to transcribe and synthesize the target RNA.

[0055] As a specific embodiment, the method for producing RNA comprises the following steps: S1, incubating the template DNA with the T7 RNA polymerase variant at 35-40° C. for 1-3 hours, then removing the template DNA and purifying to obtain the target RNA.

[0056] In a preferred embodiment, the RNA is mRNA.

[0057] Combining the findings of the present invention and the experimental data of the following specific examples, it can be concluded that the performance of the above-mentioned T7 RNA polymerase variants in synthesizing RNA is significantly improved. Specifically, compared with the wild-type T7 RNA polymerase, the quality of RNA synthesized by the above-mentioned T7 RNA polymerase variants is significantly improved, and better performance is shown in one or more indicators such as capping rate, integrity rate, PolyA integrity and dsRNA residual rate.

[0058] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0059] Example

[0060] The sequence information of T7 RNA polymerase variants used in the examples is shown in Table 1 below:

[0061] Table 1 T7 RNA polymerase variant sequence information

[0062] name Mutation information Corresponding amino acid sequence Corresponding DNA sequence wild type none SEQ ID NO:1 SEQ ID NO:5 Mut_1 T75L, C216L, V237K, Q786M SEQ ID NO:2 SEQ ID NO:6 Mut_2 T75L, C216L, Q744R, Q786M SEQ ID NO:3 SEQ ID NO:7 Mut_3 T75L, C216L, Q786M SEQ ID NO:4 SEQ ID NO:8

[0063] Example 1: Expression and purification of T7 RNA polymerase

[0064] 1. Gene synthesis and recombinant vector construction

[0065] The pQE-80L vector (GenScript) was selected as the expression vector, and the complete wild-type and mutant DNA sequences of T7 RNA polymerase (SEQ ID NO: 5-8, the mutation sites of the mutants are shown in Table 1) were inserted into the pQE-80L vector through the restriction endonuclease NdeI / HindIII restriction site. After Sanger sequencing verification, the wild-type, Mut_1, Mut_2, and Mut_3 recombinant plasmids containing the wild-type and mutant DNA sequences were obtained. The vector construction scheme is shown in Table 2.

[0066] Table 2 Vector construction scheme

[0067] Protein name Carrier Cloning scheme wild type pQE-80L ATG--His tag--BamHI--wild--Stop codon--HindIII Mut_1 pQE-80L ATG--His tag--BamHI--Mut_1--Stop codon--HindIII Mut_2 pQE-80L ATG--His tag--BamHI--Mut_2--Stop codon--HindIII Mut_3 pQE-80L ATG--His tag--BamHI--Mut_3--Stop codon--HindIII

[0068] 2. Protein expression and purification

[0069] Transformation steps: Take out the BL21 (DE3) competent cells from the ultra-low temperature refrigerator, place them on ice to melt, add the above-constructed plasmid (100ng), gently pipette to mix thoroughly, place on ice for 30 minutes, heat shock in a water bath at 42 degrees for 90 seconds, and place on ice for 3 minutes. Add 100μl of room temperature LB liquid culture medium, shake and culture at 37°C, 200 rpm in a shaker for 60 minutes. Mix the bacterial solution and apply it to the ampicillin resistance plate, invert the plate, and culture it at 37°C overnight.

[0070] Small test culture: Pick 3 monoclones respectively, inoculate them into 4 ml LB test tubes containing 100 μg / ml ampicillin, and culture them in a shaker at 37°C and 200 rpm. When OD600 reaches 0.6-0.8, add 0.5 mM IPTG to 2 test tubes respectively, culture at 15°C for 16 hours and 37°C for 4 hours. The last test tube is a negative reference. SDS-PAGE and Western blot are used to detect protein expression and solubility. According to the test results, monoclones with high protein expression are preserved in glycerol tubes.

[0071] Expanded culture: Inoculate the stored BL21 (DE3) glycerol bacteria into TB medium containing ampicillin and culture at 37°C. When OD600 reaches about 1.2, add 0.5mM IPTG to the medium and culture at 37°C for 4 hours. Collect the cells by centrifugation.

[0072] 3. Purification

[0073] The harvested E. coli cells were resuspended in lysis buffer (50 mM Tris-HCl, 1 M NaCl, 10% glycerol, pH 8.0, 1 mM DTT, 1 mM PMSF, 0.5% Triton X-100, nuclease) and then sonicated. The supernatant after centrifugation was purified by six steps using nickel column, PEI precipitation, ammonium sulfate precipitation, CHT (ceramic hydroxyapatite) chromatography, and Superdex 200 (specific purification conditions are as follows) to obtain the target protein. After sterilization by filtration with a 0.22 μm filter membrane, it was stored in aliquots.

[0074] 3.1 Nickel column purification

[0075] The supernatant of the cell lysate was loaded onto a pre-equilibrated nickel column. After washing with buffer A (50 mM Tris-HCl, 1 M NaCl, 10% glycerol, pH 8.0, 1 mM DTT, 0.5% TritonX-114 and 0.5% TritonX-100, pH 8.0) and buffer B (50 mM Tris-HCl, 500 mM NaCl, pH 8.0, 1 mM DTT), His-tagged proteins were eluted with buffers C to F (all containing 50 mM Tris-HCl, 500 mM NaCl, pH 8.0, 1 mM DTT, wherein buffers C to F contained imidazole at final concentrations of 20 mM, 50 mM, 250 mM, and 500 mM, respectively).

[0076] 3.2 PEI precipitation

[0077] The proteins eluted with 50 mM and 250 mM imidazole were combined, PEI was added at a final concentration of 0.1%, the mixture was mixed and centrifuged at 13000 rpm for 20 min, and the supernatant was collected.

[0078] 3.3 Ammonium sulfate precipitation

[0079] Add ammonium sulfate with a final saturation of 60% to the supernatant of 3.2, stir on ice water for 15 min, centrifuge at 12000 rpm for 20 min, collect the precipitate, and redissolve it with buffer B (50 mM Tris-HCl, 500 mM NaCl, 1 mM DTT, pH 8.0).

[0080] 3.4 Nuclease removal

[0081] The protein re-dissolved in 3.3 was digested with nuclease at a final concentration of 1 μg / ml for 2 hours, and then passed through a nickel column to remove the nuclease. The protein solution after nuclease digestion was loaded onto a pre-equilibrated nickel column. After washing with buffer G (50mM Tris-HCl, 500mMNaCl, 1mM DTT, 0.5% Triton X-100, 0.5% Triton X-114, pH 8.0) and buffer H (50mM Tris-HCl, 150mM NaCl, pH 8.0, 1mM DTT), His-tagged proteins were eluted with buffer I (50mM Tris-HCl, 150mM NaCl, pH8.0, 1mM DTT, 250mM imidazole).

[0082] 3.5 CHT purification

[0083] The protein solution eluted with 250 mM imidazole was loaded onto a pre-equilibrated (equilibrium buffer containing 10 mM Na3PO4, 150 mM NaCl, pH 7.5, 1 mM DTT) CHT column, and then linearly eluted with elution buffer (500 mM Na3PO4, 150 mM NaCl, pH 7.5, 1 mM DTT) for 20 CV to collect the target protein.

[0084] 3.6SEC purification

[0085] The target protein collected in 3.5 was purified by SEC (Superdex 200 column, 100mM Tris-HCl, 200mM NaCl, 2mM DTT, pH 7.9). The target protein was then further dialyzed into the final buffer (50mM Tris-HCl, 100mM NaCl, 1mM DTT, 50% glycerol, pH 7.9) and filtered with a 0.22μm filter before aliquoting and storage.

[0086] Example 2 Preparation of mRNA samples

[0087] 1. DNA template preparation

[0088] UPP1 gene (1.96 kb, SEQ ID NO: 9) and IGF2R gene (8.5 kb, SEQ ID NO: 10) were selected as short DNA template and long DNA template for testing respectively. Two gene fragments were synthesized, and linear plasmid DNA containing T7 promoter sequence and 5'UTR, UPP1 gene or IGF2R gene, 3'UTR and 100A tail was used as template DNA for in vitro transcription. The first 50 nucleotide sequences of the 5' end of the two DNA templates were the same, and the same cleavage probe could be used.

[0089] 2. In vitro transcription

[0090] Purified T7 RNA polymerase, template DNA prepared in Example 1 and other components were added according to Table 3, the reaction solution was mixed evenly, centrifuged briefly, and incubated at 37°C for 2h (reaction buffer: 40mM Tris-HCl, 6mMMgCl2, 1mM DTT, 2mM spermidine, pH 7.9). N7-methyl G-triphosphate-AG capping analog was used to prepare mRNA with cap1 structure.

[0091] Table 3

[0092] Components volume <![CDATA[H2O]]> Make up to 20 μL 1x reaction buffer 2μL NTP (A, U, G, C) 2μL (0.5mM each) N7-methyl G-triphosphate-AG capped analog 10mM Template DNA 1μL (1μg / μL) RNase inhibitors 0.5μL (1U / μl) T7 RNA polymerase 2μL Total volume 20μL

[0093] 3. Remove DNA template

[0094] After the transcription reaction was completed, 1 μL DNase I (RNase-free) was added to each tube, mixed, centrifuged briefly, and incubated at 37°C for 15-30 min to remove the template DNA.

[0095] 4. RNA Purification

[0096] (1) Add 115 μL RNase-free H2O and 15 μL 3 M sodium acetate (pH 5.2) to 20 μL of reaction mixture and mix well.

[0097] (2) Extract once with an equal volume of phenol / chloroform (1:1), and then extract twice with an equal volume of chloroform. Collect the supernatant and transfer it to a new RNase-free EP tube.

[0098] (3) Add 2 volumes of anhydrous ethanol to precipitate RNA. Mix well and place at -20°C for at least 30 min. Centrifuge at maximum speed at 4°C for 15 min and collect the precipitate.

[0099] (4) Add 500 μL of ice-cold 70% ethanol to wash the RNA precipitate.

[0100] (5) Dissolve the RNA precipitate in 20 μL RNase-free H2O. The purified RNA is then tested for quality.

[0101] Example 3: mRNA quality detection

[0102] 1. Capping rate detection

[0103] The cap structure of mRNA can maintain stability in vivo and promote protein translation. The proportion of mRNA with a cap structure can affect the immunogenicity and translation efficiency of mRNA.

[0104] 1.1 Annealing

[0105] The annealing reaction system includes 500 pmol RNase H probe (biotin labeled) (NEB, M0297S), 10×RNase H buffer (NEB, M0297S) and 100 pmol mRNA (mRNA prepared and purified in Example 2), and 5 times the amount of probe is used to ensure that all mRNA is bound to the probe. The reaction conditions are: the probe and mRNA are mixed, annealed at 95°C for 5 minutes, then reduced to 65°C, 55°C, 40°C at 2 minute intervals, and finally reduced to 22°C.

[0106] 1.2 Combination

[0107] Add the annealed mRNA and probe to the pre-treated streptavidin magnetic beads and incubate at room temperature for 30 minutes. Add 50% RNase H, mix well by pipetting, incubate at 37°C for 3 hours, then place on a magnetic stand and discard the supernatant.

[0108] 1.3 Cleaning

[0109] The cells were washed three times with a wash buffer (5 mM Tris-HCl, 0.5 mM EDTA, pH 7.5) containing 1 M NaCl to remove the enzyme, mRNA, and reaction buffer; and then washed three times with distilled water to remove excess salt.

[0110] 1.4 Elution

[0111] Add 75% methanol (preheated at 80°C) and incubate at 80°C for 1 min to elute the mRNA cleavage fragments.

[0112] 1.5 Fluid exchange

[0113] The eluted product was dried by rotary evaporation and resuspended in 0.1 mM EDTA solution containing 1% methanol for LC-MS (Thermo Fisher) analysis.

[0114] 1.6 Detection

[0115] Liquid chromatography-mass spectrometry (LC-MS) was used to quantify the mass spectrometry peak area of ​​components with or without cap structures, thereby obtaining the 5' capping rate. The results are shown in Table 4.

[0116] Table 4 Cap1 mRNA capping rate

[0117]

[0118] As shown in Table 4 above, the three designed T7 RNA polymerase variants showed higher capping rates than the wild type in the Cap1 co-transcriptional capping rate test.

[0119] 2. Integrity detection

[0120] The Agilent System capillary electrophoresis system was used to detect the integrity of mRNA. The experimental process was as shown in the literature (Vogel et al., 2021), and the results are shown in Table 5.

[0121] Table 5 Cap1 mRNA integrity

[0122]

[0123] The mRNA transcript integrity test showed that the integrity of short mRNA transcribed by the designed T7 RNA polymerase variants was significantly higher, especially Mut_2; while the integrity of long mRNA transcribed was comparable to that of the wild type.

[0124] 3. Residual dsRNA detection

[0125] The process of preparing mRNA by in vitro transcription using T7 RNA polymerase will produce byproducts such as double-stranded RNA (dsRNA). dsRNA can be recognized by RIG-I like receptors (RLRs), which then triggers the body's natural immune response. The detection method is based on the enzymatic immunoblotting method in the draft "mRNA Vaccine Quality Analysis Method" (https: / / www.uspnf.com / notices / analytical-procedures-mrna-vaccines-20220210) formulated by the United States Pharmacopeia (USP) in 2022 for dsRNA detection. The test results are shown in Table 6.

[0126] Table 6 dsRNA residual rate of Cap1 mRNA

[0127]

[0128] The results of dsRNA detection residues showed that the dsRNA residues in samples of short mRNA and long mRNA produced using T7 RNA polymerase variants were significantly lower than those in samples produced using wild-type T7 RNA polymerase.

[0129] 4. PolyA tail detection

[0130] PolyA can protect the cap structure from degradation, and cooperate with polyA binding protein, 5'Cap (cap structure) and translation initiation factor protein to initiate protein translation.

[0131] 4.1 Preparation of mRNA samples

[0132] Long mRNA products were selected to test the integrity of the Poly A tail.

[0133] 4.2 Enzyme Digestion

[0134] Endonuclease RNAse T1 (Biyuntian, R7096S) can specifically cut after guanine ribonucleotide (G) of single-stranded RNA. 100 pmol of mRNA was heated to 95°C and then quickly cooled to 25°C. Then, 10×RNase H buffer and 2μl RNase T1 enzyme (100μl system) were added and incubated at 37°C for 3 hours.

[0135] 4.3 Purification

[0136] The polyA tail was specifically bound to oligo dT magnetic beads (Thermo Fisher, 61002) for purification. The beads were washed with 0.1 M ammonium acetate solution, and then 75% methanol was added and incubated at 80°C for 1 min to elute the polyA fragment, and the eluate was collected.

[0137] 4.4 Fluid exchange

[0138] The eluate was then rotary evaporated to remove methanol and resuspended in 0.1 mM EDTA solution containing 1% methanol for LC-MS analysis.

[0139] 4.5 Detection

[0140] The distribution of the polyA tail was quantitatively analyzed based on the peak areas of different absorption peaks. The results are shown in Table 7.

[0141] Table 7 PolyA integrity of Cap1 long mRNA

[0142] Protein name PolyA integrity of Cap1 long mRNA (%) wild type 92.8 Mut_1 92.5 Mut_2 94.5 Mut_3 91.6

[0143] The results showed that the mRNA polyA integrity of the product produced by the T7 RNA polymerase variant Mut_2 was higher than that of the wild type, while the mRNA polyA integrity of the product produced by other variants was basically the same as that of the wild type.

[0144] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0145] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0146] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

[0147] References:

[0148] Boulain, J.-C. et al. 2013. “Mutants with Higher Stability and Specific Activity from a Single Thermosensitive Variant of T7 RNA Polymerase”. Protein Engineering Design and Selection 26(11):725-34.

[0149] Cheetham, Graham MT, and Thomas A Steitz. 2000. “Insights into Transcription: Structure and Function of Single-Subunit DNA-Dependent RNA Polymerases”. Current Opinion in Structural Biology 10(1):117-23.

[0150] Chelliserrykattil, Jijumon, and Andrew D Ellington. 2004. “Evolution of a T7 RNA Polymerase Variant That Transcribes 2’-O-Methyl RNA”. Nature Biotechnology 22(9):1155-60.

[0151] Jackson, Nicholas A.C. et al. 2020. “The Promise of MRNA Vaccines: A Biotech and Industrial Perspective”. npj Vaccines 5(1):11.

[0152] Klement, John F. et al. 1990. “Discrimination between Bacteriophage T3 and T7 Promoters by the T3 and T7 RNA Polymerases Depends Primarily upon a Three-Base-Pair Region Located 10 to 12 Base-Pairs Upstream from the Start Site”. Journal of Molecular Biology 215(1):21–29.

[0153] Lyakhov, Dmitry L et al. 1998. “Pausing and Termination by Bacteriophage T7 RNA Polymerase”. Journal of Molecular Biology 280(2):201-13.

[0154] Pavco, Pamela A., and Deborah A. Steege. 1991. ”Characterization of Elongating T7 and SP6 RNA Polymerases and Their Response to a Roadblock Generated by a Site-Specific DNA Binding Protein”. Nucleic Acids Research 19(17):4639-46.

[0155] Vogel, Annette B. et al. 2021. “BNT162b Vaccines Protect Rhesus Macaques from SARS-CoV-2”. Nature 592(7853):283-89。

Claims

1. A T7 RNA polymerase variant, wherein: The amino acid sequence of the T7 RNA polymerase variant is shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:

4.

2. An isolated nucleic acid encoding the T7 RNA polymerase variant of claim 1.

3. The isolated nucleic acid according to claim 2, wherein the nucleotide sequence is shown in SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO:

8. A recombinant vector comprising the isolated nucleic acid according to claim 2 or 3.

5. A host cell comprising the isolated nucleic acid according to claim 2 or 3, or comprising the recombinant vector according to claim 4.

6. A kit comprising the T7 RNA polymerase variant of claim 1, the isolated nucleic acid of claim 2 or 3, the recombinant vector of claim 4 or the host cell of claim 5.

7. Use of the T7 RNA polymerase variant of claim 1, the isolated nucleic acid of claim 2 or 3, the recombinant vector of claim 4, the host cell of claim 5 or the kit of claim 6 in in vitro transcription synthesis of mRNA.

8. A method for producing RNA, the method comprising using the T7 RNA polymerase variant of claim 1 to transcribe and synthesize RNA.

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