WO2024235228A1 - Rna polymerase mutants with improved performance - Google Patents

Abstract

Provided are a series of RNA polymerase mutants with improved performance. The mutants, compared with an amino acid sequence of a parental RNA polymerase, have one or more amino acid mutations at positions 121, 223, 632, 217, 397, 369, and 579 or have one or more amino acid mutations that occur at amino acid residues at equivalent positions in the parental RNA polymerase. The mutants are at least improved in terms of any of the following: enzymatic activity, thermal stability, 3' terminal consistency, in-vitro transcription yield, dsRNA byproducts, immunogenicity, and the like, and can meet various requirements of practical application and research.

Description

A RNA polymerase mutant with improved performance Technical Field

The invention relates to an RNA polymerase mutant with improved performance, belonging to the technical field of genetic engineering and enzyme engineering.

Background Art

T7 RNA polymerase (T7 RNAP) is derived from Escherichia coli bacteriophage T7 and is a DNA-dependent single-subunit RNA polymerase that highly specifically recognizes the T7 promoter sequence. The enzyme has a wide range of applications in in vitro RNA synthesis, including the synthesis of gRNA, siRNA, and isotope-labeled or non-isotope-labeled RNA probes, and the synthesis of capped mRNA with the participation of Cap analog for intracellular or in vivo expression and mRNA vaccine and drug research. In addition, it can also be used for isothermal amplification, such as sequence-specific nucleic acid in vitro amplification (NASBA) and transcription-mediated amplification (TMA).

However, wild-type T7 RNAP has some shortcomings in practical applications, such as the generation of byproducts during RNA synthesis, including short fragments generated during transcription initiation, 3' end extension products, and dsRNA byproducts, which can affect the effects of mRNA vaccine and drug research. With the development of mRNA drugs and molecular diagnostic technology, the wild-type T7 RNA polymerase currently on the market cannot fully meet the growing research needs and needs to be optimized and improved to achieve better application value.

At present, some technologies have been used to improve T7 RNAP, such as mutants with improved enzyme activity, mutants with reduced dsRNA contaminants, and mutants with improved thermal stability. For example, US20150024435 A1 provides a mutant of T7 RNA polymerase with improved enzyme activity, CN 111212905 A provides a T7 RNAP mutant that reduces dsRNA contaminants and concatenated transcripts produced during in vitro transcription reactions; and CN 112831484 B provides a method suitable for the synthesis of RNA containing internal termination signals and RNA with hairpin structures at the ends, which can synthesize Cas9 sgRNA with higher purity than T7 RNAP-WT; CN 108779446 A and US 9193959 B2 provide T7 RNAP mutants with improved thermal stability. However, these mutants in the prior art often need to be improved in performance, such as insufficient performance improvement, balance between thermal stability and activity, or failure to effectively reduce byproducts.

Therefore, the present invention aims to develop a T7 RNAP mutant with improved performance, which has at least any of the following performance improvements: enzyme activity, thermal stability, 3' end consistency, in vitro transcription yield, dsRNA byproducts and immunogenicity, etc., so as to meet the various needs of practical applications and research.

Summary of the invention

To solve the above problems, the present invention provides a series of RNA polymerase mutants with improved performance, which have at least any of the following performance improvements: enzyme activity, thermal stability, 3' end consistency, in vitro transcription yield, dsRNA byproducts and immunogenicity, etc., and can meet various needs of practical applications and research.

The first object of the present invention is to provide an RNA polymerase mutant with improved performance, wherein the RNA polymerase mutant has one or more amino acid mutations at positions 121, 223, 632, 217, 397, 369, and 579, or one or more amino acid mutations occur at amino acid residues at equivalent positions in the parent RNA polymerase, based on the amino acid sequence corresponding to the parent RNA polymerase; the amino acid sequence of the parent RNA polymerase has at least 80% identity with the amino acids shown in SEQ ID NO.1 and has RNA polymerase activity;

In one embodiment, the property is selected from one or more of enzyme activity, thermal stability, 3' end consistency, in vitro transcription yield, dsRNA byproducts and immunogenicity.

In one embodiment, when the mutation site contains position 121, the amino acid sequence of the parent RNA polymerase has at least 90% identity with the amino acid shown in SEQ ID NO.1 and has RNA polymerase activity; when the mutation site contains position 369, the amino acid sequence of the parent RNA polymerase has at least 98.41% identity with the amino acid shown in SEQ ID NO.1 and has RNA polymerase activity.

In one embodiment, the parent RNA polymerase is a wild-type T7 RNA polymerase whose amino acid sequence is shown in SEQ ID NO.1.

In one embodiment, the parent RNA polymerase having at least 80% identity with the amino acid shown in SEQ ID NO.1 is from different Escherichia coli phages or other phages, including but not limited to: Escherichia phage T7, Enterobacteria phage vB EcoP IMME390, Enterococcus phage EFA-2, Escherichia phage 13a, Escherichia phage 64795ec1, Escherichia phage ClCC 80001, Escherichia phage Ebrios, Escherichia phage EG1, Escherichia phage HZ2R8, Escherichia phage HZP2, Escherichia phage JacobBu rckhardt, Escherichia phage JeanTinguely, Escherichia phage LL2, Escherichia phage N30, Escherichia phage NC-A, Klebsiella p hage Patroon、Salmonella phage 3A_8767、Salmonella phage Vi06、Serratia phage 2050H2、Serratia phage Pila、Serratia phage S lM9-3Y, Escherichia phage JB01, Yersinia phage phiA1122, Yersinia phage vB_YpYeO9, Yersinia phage YpP-R, Aeromonas phage PZL The amino acid sequence of any one of the RNA polymerases selected from the group consisting of Citrobacter phage SH2, Escherichia phage E-2, Yersinia phage phiYe-F10, Citrobacter phage phiCFP-1, Citrobacter phage SH1, Salmonella phage phiSG-JL2, Yersinia phage phiYeO3-12, Klebsiella phage 31, and Enterobacteria phage T3. The 223rd, 632nd, 217th, 397th, and 579th positions of the amino acid sequences of the above parent RNA polymerases are conserved sequences.

Optionally, the accession numbers of the amino acid sequence of the parent RNA polymerase are QZB83517.1, YP 009814072.1, QJT70354.1, YP 002003938.1, YP 009291482.1, YP 009152465.1, YP 009799383.1, YP 009795804.1, YP 009798010.1, YP 009820203.1, QXV80637.1, QXV80862.1, YP 009812318.1, YP 009813771.1, YP 009820155.1, QBQ72872.1, YP 009804754.1, YP 004306655.1, YP 009791749.1, QFG06779.1, YP 009786924.1, QOV06378.1, NP 848264.1, WMX18675.1, AFK13398.1, QMP81608.1, YP_009289300.1, YP_009226209.1, YP_009785350.1, YP_009205657.1, YP_009286631.1, YP_001949750.1, NP_052071.1, QGH73719.1, NP_523301.1, corresponding to SEQ ID NO.1, SEQ ID NO.29-62, respectively; wherein SEQ ID NO.29-62 and SEQ ID Compared with the amino acid of NO.1, it has 97.96%, 98.07%, 98.64%, 98.07%, 98.19%, 90.26%, 97.51%, 97.40%, 97.40%, 98.19%, 98.53%, 82.35%, 98.75%, 97.73%, 82.47%, 98.41%, 92 .98%, 82.47%, 98.41%, 82.47%, 98.30%, 98.41%, 98.19%, 98.53%, 89.92%, 82.69%, 82.58%, 82.47%, 82.47%, 82.35%, 82.47%, 82.24%, 82.13%, 82.01% sequence identity.

Optionally, the amino acid sequence of the parent RNA polymerase has at least 90% identity with the amino acid shown in SEQ ID NO.1 and has RNA polymerase activity; optionally, the amino acid sequence of the parent RNA polymerase corresponds to SEQ ID NO.1, SEQ ID NO.30-39, SEQ ID NO.41-42, SEQ ID NO.44-45, SEQ ID NO.47, SEQ ID NO.49-53, respectively, and the 121st, 223rd, 632nd, 217th, 397th, and 579th positions of its amino acid sequence are conserved sequences.

Optionally, the amino acid sequence of the parent RNA polymerase has at least 98.41% identity with the amino acid shown in SEQ ID NO.1 and has RNA polymerase activity; optionally, the amino acid sequence of the parent RNA polymerase is as shown in SEQ ID NO.1 or SEQ ID NO.44, and the amino acids at positions 121, 223, 632, 217, 397, 369, and 579 in these two sequences are consistent.

In one embodiment, the amino acid at position 217 is mutated to L, A or V, preferably, mutated to L;

The amino acid at position 397 is mutated to W, A or P, preferably, to W;

The amino acid at position 121 is mutated to M, L or S, preferably, mutated to M;

The amino acid at position 223 is mutated to M, P, L, V or F, preferably, mutated to M;

The amino acid at position 632 is mutated to G or E, preferably, mutated to G;

The amino acid at position 369 is mutated to T or E;

The amino acid at position 579 mutates to A, W or D; preferably, mutates to W.

In one embodiment, the RNA polymerase mutant also includes mutations at one or more of the following positions corresponding to the amino acid sequence of the parent RNA polymerase: position 430, position 633, position 786, position 744, position 724, position 43, and position 723.

In one embodiment, the amino acid at position 430 is mutated to a non-polar hydrophobic amino acid; alternatively, it is mutated to P, L, V, M, F; preferably, it is mutated to P;

The amino acid at position 633 is mutated to a non-polar hydrophobic amino acid; optionally, it is mutated to P, L, V, M or F, A, I, W; preferably, it is mutated to P;

The amino acid at position 786 is mutated to a non-polar hydrophobic amino acid or Y; alternatively, it is mutated to P, I, A, V, Y, L, M, W or F; preferably, it is mutated to L;

The amino acid at position 744 is mutated to R, P or L; preferably, mutated to R;

The amino acid at position 43 mutates to Y or A; preferably, mutates to Y;

The amino acid at position 723 is mutated to E or S; preferably, mutated to E;

The amino acid at position 724 is mutated to N.

In one embodiment, the RNA polymerase mutant comprises at least any one of the following mutations that is equivalent to or corresponds to the parent polymerase mutant: I217L, I217A, I217V, S397W, S397A, S397P, T121M, T121L, T121S, S223M, S223P, S223L, S223V, S223F, R632G, R632E, M369E, M369T, N579A, N579W, and N579D mutations.

In one embodiment, the RNA polymerase mutant simultaneously has at least the following mutations equivalent to or corresponding to the parent polymerase mutant: I217L, S397W, S430P, S633P and Q786L, named I217L/S397W/S430P/S633P/Q786L (hereinafter referred to as M7).

In one embodiment, the RNA polymerase mutant simultaneously has at least the following mutations equivalent to or corresponding to the parent polymerase mutant: I217L, S397W, S430P, S633P, Q786L, N579W, named I217L/S397W/S430P/S633P/Q786L/N579W.

In one embodiment, the RNA polymerase mutant simultaneously has at least the following mutations equivalent to or corresponding to the parent polymerase mutant: I217L, S397W, S430P, S633P, Q786L, Q744R, named I217L/S397W/S430P/S633P/Q786L/Q744R.

In one embodiment, the RNA polymerase mutant simultaneously has at least the following mutations equivalent to or corresponding to the parent polymerase mutant: I217L, S397W, S430P, S633P, Q786L, C723E, A724N, named I217L/S397W/S430P, S633P/Q786L/C723E/A724N.

In one embodiment, the RNA polymerase mutant simultaneously has at least the following mutations equivalent to or corresponding to the parent polymerase mutant: I217L, S397W, S430P, S633P, Q786L, Q744R, S43Y, and is named I217L/S397W/S430P/S633P/Q786L/Q744R/S43Y (hereinafter referred to as M8).

In one embodiment, the amino acid sequence of the RNA polymerase mutant is such as SEQ ID NO.2 (corresponding to I217L), SEQ ID NO.3 (corresponding to S397W), SEQ ID NO.4 (corresponding to T121M), SEQ ID NO.5 (corresponding to S223M), SEQ ID NO.6 (corresponding to R632G), SEQ ID NO.7 (corresponding to I217L/S397W/S430P/S633P/Q786L) or SEQ ID NO. SEQ ID NO.8 (corresponding to I217L/S397W/S430P/S633P/Q786L/Q744R/S43Y), SEQ ID NO.79 (corresponding to M369T), SEQ ID NO.80 (corresponding to N579W), SEQ ID NO.81 (corresponding to I217L/S397W/S430P/S633P/Q786L/N579W), SEQ ID NO.82 (corresponding to I217L/S397W/S430P/S633P/Q786L/Q744R), and SEQ ID NO.83 (corresponding to I217L/S397W/S430P/S633P/Q786L/C723E/A724N).

The second object of the present invention is to provide a polynucleotide encoding the aforementioned RNA polymerase mutant.

In one embodiment, the sequence of the polynucleotide is as shown in SEQ ID NO.16 (corresponding to I217L), SEQ ID NO.17 (corresponding to S397W), SEQ ID NO.18 (corresponding to T121M), SEQ ID NO.19 (corresponding to S223M), SEQ ID NO.20 (corresponding to R632G), SEQ ID NO.21 (corresponding to I217L/S397W/S430P/S633P/Q786L), and SEQ ID NO.22 (corresponding to I217L/S397W/S430P/S633P/Q786L/Q744R/S43Y).

The third object of the present invention is to provide a vector carrying the aforementioned polynucleotide.

Optionally, the vector is pQE-80L, or may be pET-28a(+) or pPIC9K, etc.

The fourth object of the present invention is to provide a cell, which carries the aforementioned polynucleotide or the aforementioned vector, or expresses the aforementioned RNA polymerase mutant. The cell may refer to a single cell, a cell line, or a cell culture. Cells herein include their offspring, which may not necessarily be identical to the primary cell due to natural, accidental or deliberate mutations, and may differ from the primary cell in morphology and/or in genomic DNA. The cell may be a natural cell or a transformant.

A fifth object of the present invention is to provide a method for synthesizing a polynucleotide, wherein the method uses the RNA polymerase mutant of the present invention.

In one embodiment, the synthesis method comprises using an enzyme system comprising the RNA polymerase mutant to catalyze the synthesis of polynucleotides or using the whole cells of the above-mentioned cells to catalyze the synthesis of polynucleotides.

Optionally, the synthesis method comprises in vitro transcription synthesis, isothermal amplification or transcription-mediated amplification.

The sixth object of the present invention is to provide an application of the above-mentioned RNA polymerase mutant or the above-mentioned polynucleotide synthesis method in at least one of mRNA vaccine preparation, nucleic acid drug preparation, gene editing and protein expression system construction, and the protein expression system construction includes but is not limited to in vivo protein expression system construction or cell-free protein expression in vitro translation system construction.

A seventh object of the present invention is to provide a kit for polynucleotide synthesis, which comprises the aforementioned RNA polymerase mutant.

The eighth object of the present invention is to provide a method for improving at least one performance of an RNA polymerase; the performance is selected from the group consisting of improved enzyme activity, improved thermal stability, improved 3' end consistency, higher in vitro transcription yield, fewer dsRNA byproducts and lower immunogenicity, the method comprising: based on the amino acid sequence corresponding to the parent RNA polymerase, one or more amino acid mutations are present at positions 121, 223, 632, 217, 397, 369 and 579, or one or more amino acid mutations occur at amino acid residues at equivalent positions in the parent RNA polymerase; the amino acid sequence of the parent RNA polymerase has at least 80% identity with the amino acids shown in SEQ ID NO.1 and has RNA polymerase activity.

In one embodiment, when the mutation site contains position 121, the amino acid sequence of the parent RNA polymerase has at least 90% identity with the amino acid shown in SEQ ID NO.1 and has RNA polymerase activity; when the mutation site contains position 369, the amino acid sequence of the parent RNA polymerase has at least 98.41% identity with the amino acid shown in SEQ ID NO.1 and has RNA polymerase activity.

Beneficial effects of the present invention:

The present invention provides a series of RNA polymerase mutants with improved performance, which have at least any of the following performance improvements: enzyme activity, thermal stability, 3' end consistency, dsRNA byproducts and immunogenicity, etc., and can meet various needs of practical applications and research.

in:

(1) Based on the RNA polymerase with the amino acid sequence shown in SEQ ID NO.1, the present invention mutates the 121st amino acid to construct single-point mutants T121M, T121A, and T121V, which have an enzyme activity increased by more than 47% compared with the wild-type WT; among them, T121M has the highest enzyme activity, which is increased by 123% compared with the wild-type WT;

The amino acid at position 217 was mutated to construct single-point mutants I217L, I217A, and I217V, which increased the enzyme activity by more than 47% compared with the wild-type WT. Among them, I217L had the highest enzyme activity, which increased by 34% compared with the wild-type WT.

The amino acid at position 223 was mutated to construct single-point mutants S223M, S223L, and S223V, which increased the enzyme activity by more than 18% compared with the wild-type WT. Among them, S223M had the highest enzyme activity, which increased by 25% compared with the wild-type WT.

The single-point mutants S397W and S397P were constructed by mutating the amino acid at position 397. The enzyme activity of S397W was increased by more than 36% compared with the wild-type WT. Among them, the enzyme activity of S397W was the highest, which was increased by 62% compared with the wild-type WT.

The amino acid at position 632 was mutated to construct single-point mutants R632G and R632E, which showed an enzyme activity increase of more than 20% compared with the wild-type WT. Among them, R632G had the highest enzyme activity, which increased by 22% compared with the wild-type WT.

The amino acid at position 579 was mutated to construct a single-point mutant N579W, which showed an enzyme activity increase of 46% compared with the wild-type WT;

Among them, the relative enzyme activities of the single point mutant I217L, single point mutant S397W, single point mutant T121M, single point mutant S223M and single point mutant R632G at 37°C are significantly better than those of the single point mutant S430P, single point mutant G47A+884G, single point mutant S633P and single point mutant Q786L reported in the prior art.

(2) The present invention combines mutations based on single-point mutations to construct combined mutants I217L/S397W/S430P/S633P/Q786L (i.e., M7), I217L/S397W/S430P/S633P/Q786L/Q744R/S43Y (i.e., M8), I217L/S397W/S430P/S633P/Q786L/Q744R, and I217L/S397W/S430P/S633P/Q786L/C723E/A724N. Compared with the wild-type WT, I217L/S397W/ The enzyme activity of S430P/S633P/Q786L/Q744R increased by 95%, the enzyme activity of I217L/S397W/S430P/S633P/Q786L/C723E/A724N increased by 83%, and the enzyme activity of M8 increased by 336%; the Tm values of M7, M8, I217L/S397W/S430P/S633P/Q786L/Q744R and I217L/S397W/S430P/S633P/Q786L/C723E/A724N were significantly higher than those of the wild type and other mutants reported in the prior art.

(3) The combined mutant M8 of the present invention has significantly better in vitro transcription yield and 3' end consistency product at 50°C than the wild-type WT and other mutants reported in the prior art, indicating that the combined mutant M8 has higher IVT yield and 3' end consistency; the IVT yields of I217L/S397W/S430P/S633P/Q786L/Q744R and I217L/S397W/S430P/S633P/Q786L/C723E/A724N at 37°C are 43% and 10.7% higher than those of the wild-type WT, respectively, and the purities reach 79.4% and 77.4%, respectively.

(4) The combined mutants M8, I217L/S397W/S430P/S633P/Q786L/Q744R and I217L/S397W/S430P/S633P/Q786L/C723E/A724N of the present invention have significantly lower dsRNA transcription byproduct contents than the wild-type WT, mutants reported in the prior art, and the M7 combined mutant. The M8 combined mutant has a lower dsRNA content per unit mass of IVT product under reaction conditions of 50°C, which can reach the same level as the G47A+884G mutant reported in CN 111212905A that can reduce dsRNA contaminants and concatenated transcripts in in vitro transcription products.

(5) The in vitro transcription product of the combined mutant M8 of the present invention has lower immunogenicity than the wild type.

(6) The present invention successfully established a high-throughput T7 RNAP mutant library screening platform by using the FADS platform to encapsulate single cells and reaction systems. Using this platform, the present invention screened the following T7 RNA polymerase mutants, which correspond to the amino acid sequence shown in SEQ ID NO.1, and have at least one of the following amino acid residues: L at position 217, A or W at position 397, and A or W at position 579; and have at least 80% identity with SEQ ID NO.1. The T7 RNA polymerase mutant provided by the present invention has good thermal stability and a good temperature adaptability range. It can react not only under conventional 37°C conditions, but also under high temperature 50°C conditions. The T7 RNAP mutant provided by the present invention also has good application performance under 37°C conditions. The T7 RNA polymerase combination mutant containing multiple mutation sites has a purity of in vitro transcription product that is better than that of the wild-type T7 RNA polymerase or the reported T7 RNA polymerase mutant. The T7 RNA polymerase mutant provided by the present invention still has very good application performance under 50°C conditions, the yield of synthetic RNA is significantly better than that of the wild type and reported mutants, the product purity is not worse than the existing technology, and the synthesized mRNA has fewer dsRNA by-products, which will produce lower immunogenicity, and the 3' consistency of the product is significantly improved compared with the existing technology. These indicators are very beneficial for the research and production of mRNA vaccines and drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

Figure 1 is a schematic diagram of the T7 RNAP recombinant expression vector;

FIG2 is a flow chart of microdroplet sorting based on fluorescence activation of aptamers;

FIG3 is a graph showing the change in protein concentration of wild-type T7 RNAP with fluorescence intensity when the relative activities of different mutants were determined by real-time fluorescence method;

FIG4A shows the dsRNA content detection results of IVT products of T7 RNAP wild type and single mutants S430P, S633P, Q786L, I217L, S397W, combined mutants I217L/S397W/S430P/S633P/Q786L, I217L/S397W/S430P/S633P/Q786L/N579W, I217L/S397W/S430P/S633P/Q786L/Q744R, and I217L/S397W/S430P/S633P/Q786L/C723E/A724N;

Figure 4B shows the dsRNA content detection results of IVT products of T7 RNAP wild type and single mutants I217L, S397W, S430P, S633P, Q786L, combined mutants I217L/S397W/S430P/S633P/Q786L, combined mutants I217L/S397W/S430P/S633P/Q786L/N579W, combined mutants I217L/S397W/S430P/S633P/Q786L/Q744R, and combined mutants I217L/S397W/S430P/S633P/Q786L/C723E/A724N;

Figure 5A shows the 3' end consistency test results of the IVT product of T7 RNAP wild type at 37°C;

Figure 5B shows the 3' end consistency test results of the IVT product of T7 RNAP mutant Q744R at 37°C;

Figure 5C shows the 3' end consistency test results of the IVT product of T7 RNAP mutant S43Y at 37°C;

Figure 5D shows the 3' end consistency test results of the IVT product of T7 RNAP mutant G47A+884G at 37°C;

Figure 5E shows the 3' end consistency test results of the IVT product of T7 RNAP mutant M7: I217L/S397W/S430P/S633P/Q786L at 37°C;

Figure 5F shows the 3' end consistency test results of the IVT product of T7 RNAP mutant M7: I217L/S397W/S430P/S633P/Q786L at 50°C;

Figure 5G shows the 3' end consistency test results of the IVT product of T7 RNAP mutant M8: I217L/S397W/S430P/S633P/Q786L/Q744R/S43Y at 37°C;

Figure 5H shows the 3’ end consistency test results of the IVT product of T7 RNAP mutant M8: I217L/S397W/S430P/S633P/Q786L/Q744R/S43Y at 50°C;

Figure 5I shows the 3’ end consistency test results of the IVT product of T7 RNAP mutant I217L at 50°C;

Figure 5J shows the 3’ end consistency test results of the IVT product of T7 RNAP mutant S397W at 50°C;

Figure 6A shows the detection results of IFN-α, a cellular immune response factor, in the IVT products of T7 RNAP wild type and mutant M8;

Figure 6B shows the detection results of IL-6, a cellular immune response factor, in the IVT products of wild-type T7 RNAP and mutant M8;

FIG7A is a sequence conservation analysis of T7 RNAP mutation sites 1 to 129 in RNA polymerase amino acid sequences from different sources;

FIG7B is a sequence conservation analysis of T7 RNAP mutation sites 130 to 259 in RNA polymerase amino acid sequences from different sources;

FIG7C is a sequence conservation analysis of T7 RNAP mutation sites 260 to 389 in RNA polymerase amino acid sequences from different sources;

FIG7D is a sequence conservation analysis of T7 RNAP mutation sites 390 to 519 in RNA polymerase amino acid sequences from different sources;

Figure 7E is a sequence conservation analysis of T7 RNAP mutation sites 520 to 650 in the amino acid sequences of RNA polymerases from different sources.

DETAILED DESCRIPTION

The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than 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.

Some definitions or terms related to the present invention:

Improved performance: The term "improved performance" refers to characteristics associated with a variant that is improved relative to the parent. Such improved performance includes, but is not limited to, one or more of the following: improved enzymatic activity of the RNA polymerase mutant, improved thermal stability, higher in vitro transcription yield of the RNA polymerase mutant in an in vitro transcription (IVT) reaction, improved 3' end consistency, fewer dsRNA byproducts, or RNA transcripts produced by the RNA polymerase mutant have lower immunogenicity, etc.

Corresponding to: As used herein, the term "corresponding to" refers to a manner of determining a specific amino acid in a sequence where a specific amino acid sequence is referenced. For example, for the purposes of the present invention, when a specific amino acid position is referenced, a skilled person can compare another amino acid sequence with the amino acid sequence that has been referenced to determine which specific amino acid may be of interest in the other amino acid sequence. Alternative alignment methods may be used and are well known to those skilled in the art.

Mutant: As used herein, the term "mutant" or "polypeptide variant" or "polypeptide" or "RNA polymerase mutant" when used in relation to variants of the present invention means a polypeptide having RNA polymerase enzymatic activity comprising an alteration (i.e., a substitution, insertion and/or deletion) at one or more (e.g., several) positions relative to a "parent" RNA polymerase. Substitution means replacing the amino acid occupying a position with a different amino acid; deletion means removing the amino acid occupying a position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position. In describing the variants of the present invention, the nomenclature described below has been adapted for ease of reference. Accepted IUPAC single-letter or three-letter abbreviations for amino acids are used. Substitution: For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, a substitution of glutamic acid at position 49 by arginine is represented as "E49R". Multiple mutations are separated by a symbol ("/"), for example "E49R/M358F" represents that glutamic acid (E) and methionine (M) at position 49 and position 358 are replaced by arginine (R) and phenylalanine (F), respectively.

Parent or parent RNA polymerase: The term "parent" RNA polymerase as used herein means an RNA polymerase that is altered to produce an RNA polymerase mutant of the present invention. The term also refers to a polypeptide to which a mutant of the present invention is compared. A parent can be a naturally occurring (wild-type) polypeptide, or it can even be a variant thereof prepared by any suitable means. For example, a parent protein can be a variant of a naturally occurring polypeptide that has been modified or altered in terms of amino acid sequence. Thus, a parent RNA polymerase can have one or more (or one or several) amino acid substitutions, deletions and/or insertions. Thus, a parent RNA polymerase can be a variant of a parent RNA polymerase. A parent can also be an allelic variant, a polypeptide encoded by any of two or more alternative forms of a gene occupying the same chromosomal locus.

Wild-type enzyme: The term "wild-type" when referring to an amino acid sequence or a nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a natural or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance (e.g., a protein, amino acid, or nucleic acid sequence) found in nature. In contrast, the term "non-naturally occurring" refers to any substance not found in nature (e.g., a recombinant nucleic acid and protein sequence produced in a laboratory, or a modification of a wild-type sequence). When the parent enzyme is not a variant enzyme, the terms "wild-type enzyme" and "parent enzyme" are used interchangeably.

Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

For the purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. [Journal of Molecular Biology] 48: 443-453), as implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite [European Molecular Biology Open Software Suite], Rice et al., 2000, Trends Genet. [Genetics Trend] 16: 276-277) (preferably 5.0.0 version or later). The parameters used can be a gap opening penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of the "longest identity" marked with Needle (obtained using the -nobrief option) is used as the identity percentage and is calculated as follows:

(number of identical residues x 100)/(length of alignment - total number of gaps in the alignment)

Alternatively, the parameters used can be a gap opening penalty of 10, a gap extension penalty of 0.5 and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of the "longest identity" of the Needle tag (obtained using the -nobrief option) is used as the identity percentage and is calculated as follows:

(number of identical deoxyribonucleotides × 100)/(length of alignment – total number of gaps in alignment)

Expression: As used herein, the term "expression" refers to any step involved in the production of a variant including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: As used herein, the term "expression vector" refers to a linear or circular DNA molecule that comprises a polynucleotide encoding a variant and is operably linked to control sequences that provide for its expression.

Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication, as well as recombinant host cells, isolated host cells (e.g., isolated recombinant host cells), heterologous host cells.

Recombinant: When used in reference to a cell, nucleic acid, protein, or vector, the term "recombinant" means that it has been modified from its native state. Thus, for example, a recombinant cell expresses genes not found in the native (non-recombinant) form of the cell, or expresses native genes at different levels or under different conditions than found in nature. A recombinant nucleic acid differs from a native sequence in one or more nucleotides and/or is operably linked to a heterologous sequence (e.g., a heterologous promoter in an expression vector). A recombinant protein may differ from a native sequence in one or more amino acids and/or be fused to a heterologous sequence. A vector comprising a nucleic acid encoding a polypeptide is a recombinant vector. The term "recombinant" is synonymous with "genetically modified" and "transgenic."

The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than 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.

It should be noted that the abbreviations of amino acid residues herein are the same as the general definitions in the art, and the full names are still indicated in brackets after the abbreviations in some records herein. "Amino acid residue" and "amino acid" can be used interchangeably herein. The amino acid sequence herein starts from the nitrogen terminus.

In some optional embodiments, a method for screening T7 RNA polymerase mutants is provided, comprising amplifying a polynucleotide encoding T7 RNA polymerase in a reaction system containing manganese ions, then transforming a vector carrying the amplified product into cells, and inducing expression to sort target cells using a droplet microfluidic cell sorting method; the manganese ion concentration in the reaction system is 0.1 to 0.6 mM, preferably 0.2 mM to 0.3 mM. Droplet microfluidic cell sorting is based on a microfluidic ultra-high throughput fluorescence-activated droplet sorting platform (Fluorescence-Activated Droplet Sorting, FADS), and the FADS screening process includes three steps: Droplet encapsulation and incubation of single cells, incubation and reaction, and ultra-high throughput screening of single cell droplets after reaction. FADS can encapsulate and culture single cells, use specific fluorescent substrates to perform fluorescence quantification of the production of secondary metabolites or specific enzymes of single cells, and use the screening platform to sort and collect droplets whose fluorescence intensity meets the screening threshold, and finally obtain a large number of candidate droplets. This screening method has the advantages of high throughput and strong specificity.

In an optional embodiment, the heat treatment condition is 40-50° C. for 5-10 min, preferably 45° C. for 7 min.

In an alternative embodiment, the target cells are the 0.4% to 0.45% of droplet-encapsulated cells that exhibit the highest viability.

In an optional embodiment, the amplified polynucleotide encoding T7 RNA polymerase and the nucleotide sequence are as shown in SEQ ID NO.15, and the expressed amino acid sequence is the wild-type T7 RNA polymerase as shown in SEQ ID NO.1.

In an optional embodiment, the method further includes at least one step of: isolating a target fragment from a target cell sorted by a droplet microfluidic cell sorting method, transforming the fragment into another cell, and screening the cell again after culturing the fragment.

In an optional embodiment, T7 RNA polymerase mutants expressed by positive cells are collected, the system containing the T7 RNA polymerase mutants is heat-treated, and then the enzyme activity is determined, and the positive clones whose enzyme activity is at least 1.5 times that of the wild type are sequenced.

In one embodiment, T7 RNA polymerase mutants were screened using the following protocol:

First, PCR amplification of the T7 RNAP gene (SEQ ID NO.15) was performed using the recombinant vector pQE-80L-T7 RNAP containing the wild-type T7 RNAP gene as a template, and amplification products with different mutation degrees were obtained by changing the Mn ²⁺ concentration in the reaction system; then the product was connected with the vector pQE-80L to construct a recombinant expression vector, which was electroporated into competent E.coli 10G, and the library capacity was calculated to be more than 106 based on the grown clones, and the Mn ²⁺ concentration for introducing a mutation frequency of 2 to 3 bases per gene was preferably 0.2mM-0.3mM.

Then, the obtained mixed plasmid mutant library was electroporated into fresh E. coli BL21 (DE3) competent cells to obtain a mutant library containing about 107 clones. According to the steps of protein expression, E. coli was induced and expressed, and the single cell and reaction system were encapsulated using the FADS platform. The single cell encapsulation rate was controlled at about 10% to improve the accuracy of screening. During in vitro incubation, the droplets were treated at a temperature of about 45°C for an appropriate time, and the activity was reduced to about one-third of the original to achieve the effect of high temperature treatment, and then incubated at 37°C for 3h.

Before using FADS to sort the mutant library, the most suitable laser spot position, sorting speed and other conditions are determined by sorting FITC and ink. Then the incubated microdroplets are injected into the detection and sorting chip. The droplets of about 0.42% showing the highest activity are collected in a 1.5mL centrifuge tube, and the positive gene is recovered by PCR. The target fragment is treated with BamHI and HindIII double enzymes and reconstructed into the pQE-80L linearized vector. The ligated vector is transformed into the BL21 (DE3) competent state again, and the single clone grown on the plate is picked for rescreening.

During the sorting process, several rounds of sorting were performed on different batches of transformed mutant libraries, and the obtained positive genes were mixed and recovered to ensure that more beneficial mutation points could be sorted. From the clones obtained in the previous round, fresh single clones were picked in a 96-well plate, and the wild-type BL21 (DE3)-T7 RNAP single clone was used as a control. After induction and expression, the crude enzyme solution was prepared by lysing the cell with cell lysis solution, and the crude enzyme solution was heat treated at 45°C for 5 minutes, and then quickly placed on ice. The crude enzyme solution was transferred to a 384-well plate and the enzyme activity was measured using the STAR system (iSpinach aptamer-based monitoring of transcription activity in real-time, STAR, see application number: CN202211274914.8, the invention name is "A detection method for real-time monitoring of in vitro transcribed RNA synthesis", the introduction and use of the system are detailed in CN115896213A text). Then the mutants whose residual activity after heat treatment is at least 1.5 times that of the wild type are sequenced, and the distribution of mutation points is analyzed by sequencing results.

According to the distribution of the above mutation points, a recombinant expression vector carrying a nucleotide sequence encoding a T7 RNAP mutant was constructed and transformed into BL21 (DE3) Escherichia coli competent cells to obtain recombinant strains of T7 RNP wild type and mutant, induced expression and prepared T7 RNAP mutants by protein purification. Then, a single-stranded RNA aptamer was synthesized by in vitro transcription reaction. The single-stranded RNA can bind to a specific chemical small molecule DFHBI to produce a significant fluorescence signal enhancement. The relative activity of different mutants and T7 RNAP-WT was determined by monitoring the change of real-time fluorescence during the reaction. Further, the Tm values of different mutants were determined by differential scanning fluorimetry by combining the T7 RNAP mutant protein solution with Sypro Orange dye. The application performance of T7 RNAP mutants is achieved by analyzing the products synthesized by in vitro transcription (IVT), including IVT yield, product purity, dsRNA content in the product and 3' end consistency index of the product.

In the above embodiment, multiple mutation sites that can improve the thermal stability of the T7 RNA polymerase mutant are screened out. Therefore, the present invention also provides a T7 RNA polymerase mutant screened out by the above screening process. The T7 RNA polymerase mutant described herein is a polypeptide that has at least one amino acid residue difference compared to the wild-type T7 RNA polymerase. In an optional embodiment, the amino acid sequence of the wild-type T7 RNA polymerase is shown in SEQ ID NO.1.

The T7 RNA polymerase mutant provided by the present invention has at least one of the following amino acid residues: corresponding to the amino acid sequence shown in SEQ ID NO.1, position 217 is L (leucine), position 397 is A (alanine) or W (tryptophan), and position 579 is A (alanine) or W (tryptophan). The T7 RNA polymerase mutant containing the aforementioned amino acid residues has good thermal stability.

The amino acid sequence corresponding to SEQ ID NO.1 as described herein refers to the amino acid residue determined by the position number of the position in the amino acid sequence shown in SEQ ID NO.1 when the T7 RNA polymerase mutant is aligned with the amino acid sequence shown in SEQ ID NO.1. For example, when one or more amino acid residues are missing at the nitrogen end of the T7 RNA polymerase mutant, the position number of a certain amino acid residue in the amino acid sequence shown in SEQ ID NO.1 of the T7 RNA polymerase mutant corresponding to the amino acid sequence shown in SEQ ID NO.1 is different from the position number of the position in the T7 RNA polymerase mutant. For example, when the nitrogen end of the T7 RNA polymerase mutant lacks N amino acid residues, an amino acid residue is located at the i-th position in the amino acid sequence shown in SEQ ID NO.1 and is located at the i-N position in the amino acid sequence of the T7 RNA polymerase mutant. In this article, the amino acid residue is defined by its position in the amino acid sequence corresponding to SEQ ID NO.1, rather than its position in the mutant.

Furthermore, the T7 RNA polymerase mutant provided by the present invention has at least 80% identity with SEQ ID NO.1, for example, it may be but is not limited to at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1. "Identity" or similar terms herein refer to the percentage of identical nucleotides or amino acids between the sequences to be compared under optimal alignment. The percentage is purely statistical, and the differences between the two sequences may be (but not necessarily) randomly distributed over the entire length of the sequences to be compared. The comparison of two sequences is typically performed by identifying local regions of corresponding sequences relative to fragments or "comparison windows" after optimal alignment.

It is understood that a T7 RNA polymerase mutant having at least 80% identity with SEQ ID NO.1 contains at least one of the amino acid residues at position 217 being L, at position 397 being A or W, and at position 579 being A or W.

In an optional embodiment, the T7 RNA polymerase mutant contains an amino acid residue corresponding to L at position 217 of the amino acid sequence shown in SEQ ID NO.1, and has at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1.

In an optional embodiment, the T7 RNA polymerase mutant contains an amino acid residue that is A or W at position 397 of the amino acid sequence shown in SEQ ID NO.1, and has at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1, wherein preferably position 397 is W.

In an optional embodiment, the T7 RNA polymerase mutant contains an amino acid residue that is A or W at position 579 of the amino acid sequence shown in SEQ ID NO.1, and has at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1, wherein preferably position 579 is W.

In an optional embodiment, the T7 RNA polymerase mutant removes at least one of the aforementioned amino acid residues and also includes at least one of the following: corresponding to the amino acid sequence shown in SEQ ID NO.1, at least one of the positions 369, 430, 633, 786, 744, 723 and 724 is different from SEQ ID NO.1.

In an optional embodiment, the 633rd position corresponding to the amino acid sequence shown in SEQ ID NO.1 is a non-polar hydrophobic amino acid, further preferably P (proline), L (leucine), V (valine), M (methionine) or F (phenylalanine); further preferably P, and has at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1. The mutation at position 633 is beneficial to improve the stability of T7 RNA polymerase.

In an optional embodiment, the 744th position corresponding to the amino acid sequence shown in SEQ ID NO.1 is R (arginine), P (proline) or L (leucine); more preferably R; and has at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1. The mutation at position 744 is beneficial to the improvement of T7 RNA polymerase activity.

In an optional embodiment, the 786th position corresponding to the amino acid sequence shown in SEQ ID NO.1 is Y (tyrosine) or a non-polar hydrophobic amino acid; more preferably Y, L (leucine), M (methionine), W (tryptophan) or F (phenylalanine); more preferably L; and has at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1. The mutation at position 786 is beneficial to improve the stability of T7 RNA polymerase.

In an optional embodiment, the 723rd position corresponding to the amino acid sequence shown in SEQ ID NO.1 is E (glutamic acid) or S (serine); more preferably E; and has at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1. The mutation at position 723 is beneficial to improve protein aggregation.

In an optional embodiment, the 369th position corresponding to the amino acid sequence shown in SEQ ID NO.1 is T (threonine) or E (glutamate); and it has at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1.

In an optional embodiment, the 579th position corresponding to the amino acid sequence shown in SEQ ID NO.1 is W (tryptophan) or A (alanine); further preferably W; and it has at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1.

In an optional embodiment, the 430th position corresponding to the amino acid sequence shown in SEQ ID NO.1 is P (proline); and it has at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1.

In an optional embodiment, the 724th position corresponding to the amino acid sequence shown in SEQ ID NO.1 is N (asparagine); and it has at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1.

In an optional embodiment, the T7 RNA polymerase mutant is selected from any one of the following:

(a) There is at least an I217L mutation in the amino acid sequence shown in SEQ ID NO.1; that is, position 217 of SEQ ID NO.1 mutates from isoleucine to leucine.

(b) There is at least S397W mutation in the amino acid sequence shown in SEQ ID NO.1; that is, position 397 of SEQ ID NO.1 mutates from serine to tryptophan.

(c) There is at least N579W mutation in the amino acid sequence shown in SEQ ID NO.1; that is, position 579 of SEQ ID NO.1 mutates from asparagine to tryptophan.

(d) at least I217L, S397W, S430P, S633P and Q786L mutations exist in the amino acid sequence shown in SEQ ID NO.1; that is, position 217 of SEQ ID NO.1 mutates from isoleucine to leucine, position 397 mutates from serine to tryptophan, position 430 mutates from serine to proline, position 633 mutates from serine to proline, and position 786 of glutamine mutates from leucine.

(e) in the amino acid sequence shown in SEQ ID NO.1, there are at least I217L, S397W, S430P, S633P, Q786L and N579W mutations; that is, position 217 of SEQ ID NO.1 mutates from isoleucine to leucine, position 397 mutates from serine to tryptophan, position 430 mutates from serine to proline, position 633 mutates from serine to proline, position 786 mutates from glutamine to leucine, and position 579 mutates from asparagine to tryptophan.

(f) in the amino acid sequence shown in SEQ ID NO.1, there are at least I217L, S397W, S430P, S633P, Q786L and Q744R mutations; that is, position 217 of SEQ ID NO.1 mutates from isoleucine to leucine, position 397 mutates from serine to tryptophan, position 430 mutates from serine to proline, position 633 mutates from serine to proline, position 786 mutates from glutamine to leucine, and position 744 mutates from glutamine to arginine.

(g) at least I217L, S397W, S430P, S633P, Q786L, C723E and A724N mutations exist in the amino acid sequence shown in SEQ ID NO.1. That is, the 217th position of SEQ ID NO.1 mutates from isoleucine to leucine, the 397th position mutates from serine to tryptophan, the 430th position mutates from serine to proline, the 633rd position mutates from serine to proline, the 786th position of glutamine mutates from leucine, the 723rd position mutates from cysteine to glutamic acid, and the 724th position of alanine mutates from asparagine.

In an optional embodiment, the amino acid sequence of the T7 RNA polymerase mutant is SEQ ID NO.2 (corresponding to I217L), SEQ ID NO.3 (corresponding to S397W), SEQ ID NO.4 (corresponding to T121M), SEQ ID NO.5 (corresponding to S223M), SEQ ID NO.6 (corresponding to R632G), SEQ ID NO.7 (corresponding to I217L/S397W/S430P/S633P/Q786L) or SEQ ID NO.8 (corresponding to I217L/S397W/S430P/S633P/Q786L /Q744R/S43Y), SEQ ID NO.79 (corresponding to M369T), SEQ ID NO.80 (corresponding to N579W), SEQ ID NO.81 (corresponding to I217L/S397W/S430P/S633P/Q786L/N579W), SEQ ID NO.82 (corresponding to I217L/S397W/S430P/S633P/Q786L/Q744R), and SEQ ID NO.83 (corresponding to I217L/S397W/S430P/S633P/Q786L/C723E/A724N).

SEQ ID NO.2 is a T7 RNA polymerase mutant with a single point mutation I217L, whose Tm value is 50.5±0.21℃. The residual activity after treatment at 50℃ for 5 minutes is 52%. Taking the enzyme activity of the wild type as 100% as a reference, the activity of this mutant is 134%.

SEQ ID NO.3 is a T7 RNA polymerase mutant with a single point mutation S397W, whose Tm value is 49.2±0.13℃. The residual activity after treatment at 50℃ for 5 minutes is 47%. Taking the enzyme activity of the wild type as 100% as a reference, the activity of this mutant is 162%.

SEQ ID NO.80 is a T7 RNA polymerase mutant with a single point mutation N579W, whose Tm value is 48.0±0.23℃ and the residual activity after treatment at 50℃ for 5 minutes is 38%.

SEQ ID NO.7 is a T7 RNA polymerase mutant with combined mutations I217L, S397W, S430P, S633P and Q786L, with a Tm value of 54.52±0.20℃ and a residual activity of 58% after heat treatment at 58℃ for 15min.

SEQ ID NO.81 is a T7 RNA polymerase mutant with combined mutations I217L, S397W, S430P, S633P, Q786L and N579W, with a Tm value of 55.56±0.16°C and a residual activity of 65% after heat treatment at 58°C for 15 minutes.

SEQ ID NO.82 is a T7 RNA polymerase mutant with combined mutations I217L, S397W, S430P, S633P, Q786L and Q744R. The Tm value is 54.50±0.18℃. The residual activity after heat treatment at 58℃ for 15min is 87%. Taking the enzyme activity of the wild type as 100% as a reference, the activity of this mutant is 195%.

SEQ ID NO.83 is a polymerase mutant with combined mutations I217L, S397W, S430P, S633P, Q786L, C723E and A724N. The Tm value is 54.54±0.23℃. The residual activity after heat treatment at 58℃ for 15min is 81%. Taking the enzyme activity of the wild type as 100% as a reference, the activity of this mutant is 183%.

According to another aspect of the present invention, the present invention also provides a polynucleotide encoding the above-mentioned T7 RNA polymerase mutant.

As used herein, "polynucleotide" refers to a polymeric form of nucleotides of any length, including ribonucleotides and/or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. The polynucleotide encodes the T7 RNA polymerase mutant described above, optionally encoding the sense strand or the antisense strand. The polynucleotide may be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

According to another aspect of the present invention, the present invention also provides a vector, which carries a polynucleotide encoding the above-mentioned T7 RNA polymerase mutant. The vector is well known to those skilled in the art, including but not limited to: plasmid; phagemid; cosmid; artificial chromosome, such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); bacteriophage such as λ phage or M13 phage and animal virus, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses. In some embodiments, the vector of the present invention contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

According to another aspect of the present invention, the present invention further provides a cell, which carries the aforementioned polynucleotide or the aforementioned vector, or expresses the aforementioned T7 RNA polymerase mutant. The cell may refer to a single cell, a cell line, or a cell culture. The cell herein includes its progeny, which may not necessarily be completely identical to the primary cell due to natural, accidental or intentional mutations, and may differ from the primary cell in morphology and/or in genomic DNA. The cell may be a natural cell or a transformant.

According to another aspect of the present invention, the present invention also provides a method for synthesizing a polynucleotide, wherein the method uses the RNA polymerase mutant of the present invention. The method for synthesizing the polynucleotide comprises using an enzyme system comprising the aforementioned T7 RNA polymerase mutant to catalyze a synthesis reaction. The synthesis method comprises in vitro transcription synthesis (In Vitro Transcription, IVT), isothermal amplification, such as sequence-specific nucleic acid in vitro amplification (Nucleic Acid Specific-Based Amplification, NASBA) or transcription-mediated amplification (Transcription mediated amplification, TMA)

According to another aspect of the present invention, the present invention also provides the use of the aforementioned T7 RNA polymerase mutant or the aforementioned polynucleotide synthesis method in any one of the following: at least one of mRNA vaccine preparation, nucleic acid drug preparation, gene editing and protein expression system construction, and the protein expression system construction includes but is not limited to in vivo protein expression system construction or cell-free protein expression in vitro translation system construction.

According to another aspect of the present invention, the present invention also provides a kit for polynucleotide synthesis, which comprises the aforementioned T7 RNA polymerase mutant. It is understandable that the kit for polynucleotide synthesis provided by the present invention may also comprise other detection reagents or consumables acceptable in the art, and those skilled in the art may select corresponding reagents and/or consumables based on the contents well known in the art and recorded in various general and more specific teaching materials, references, process manuals, product descriptions, standard documents and equipment manuals. Specific optional reagents and consumables in the kit include, but are not limited to, one or more of buffers, salts, metal ions, dNTPs, enzymes, primers, probes, fluorescent dyes, luminescent substrates, reference substances, quality control substances and calibrators.

The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

The following describes the preferred embodiments of the present invention.

The examples are for better explanation of the present invention and are not used to limit the present invention.

Example 1: Construction and screening of mutation library

1. Construction of mutation library

The wild-type T7 RNA polymerase gene nucleotide sequence (corresponding amino acid sequence such as SEQ ID NO.1) was optimized according to the Escherichia coli codon to obtain the nucleotide sequence SEQ ID NO.15, and then the optimized nucleotide sequence was inserted between the two restriction endonuclease sites of BamHI and HindIII of the vector pQE-80L (Qiagen, item number HG-VYQ0254), that is, the recombinant vector pQE-80L-T7 RNAP containing the wild-type T7 RNAP gene was obtained. The plasmid structure is shown in Figure 1. The recombinant vector pQE-80L-T7 RNAP was used as a template to amplify the T7 RNAP gene, and the degree of mutation was mainly achieved by changing the Mn ²⁺ concentration in the reaction system. 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM Mn ²⁺ concentration gradients were set for gene amplification, and the mutation rate was optimal with 1 to 3 mutation points per gene.

The amplification system was as follows: pQE-80L-T7 RNAP plasmid (0.2-1 ng/μL), dATP (0.25 mM), dGTP (0.25 mM), dCTP (1.05 mM), dTTP (1.05 mM), DreamTaq Buffer (Takara), DreamTaq (Takara, 0.1 U/μL), MnCl ₂ (0.1 mM-0.6 mM), T7 RNAP-F (0.2 μM), T7 RNAP-R (0.2 μM), and the total PCR reaction system was 50 μL.

Among them, the DNA sequences of amplification primers T7 RNAP-F and T7 RNAP-R are shown in Table 1, and the amplification PCR program is shown in Table 2:

Table 1 Primer sequences for construction of mutation library

Table 2 PCR amplification program

The target fragment size was analyzed by agarose gel electrophoresis, recovered by the kit, and then double-digested with BamHI and HindIII. The digested product was then purified and recovered, and ligated with the linearized pQE-80L vector (double-digested with BamHI and HindIII) overnight under the action of T4 DNA ligase.

The recovered ligation product was electrotransformed into electrocompetent E. coli 10G, and the library capacity was calculated based on the clones grown, and the library capacity was more than 10 ^6. The frequency of introduction under 0.2 mM Mn ²⁺ was 1.7 base mutations/gene, and the frequency of introduction under 0.3 mM Mn ²⁺ was 3.5 base mutations/gene. Therefore, in this example, the mutant plasmids obtained under 0.2 mM Mn ²⁺ and 0.3 mM Mn ²⁺ were mixed to obtain a mutant library in which an average of 2.6 mutant bases were introduced into each gene.

2. Screening of mutant library

The obtained mixed plasmid mutant library was electroporated into fresh E. coli BL21 (DE3) competent cells to obtain a clone mutant library containing about 10 ^7. The whole screening process is shown in Figure 2. According to the steps of protein expression, E. coli was induced and expressed, and the single cell and reaction system were encapsulated using the FADS platform (droplet microfluidic cell sorting based on microfluidic ultra-high throughput fluorescence-activated droplet sorting platform, Fluorescence-Activated Droplet Sorting, FADS). The reaction system was published in patent CN 115896213 A, including single clones, 40-100nM DNA template ispinach, 100μM DFHBI, 0.5-6mM NTPs, 30mM MgCl ₂ , 5Mm NaCl, 20mM DTT, 0.002U/μL inorganic pyrophosphatase, and 0.2U/μL mouse ribonuclease inhibitor. The single-cell encapsulation rate is controlled at about 10% to improve the accuracy of screening. During in vitro incubation, the droplets need to be heat-treated at 45°C for 7 minutes, and the activity will drop to about one-third of the original to achieve the effect of high-temperature treatment, and then incubated at 37°C for 3 hours. Before using FADS to sort the mutant library, the optimal laser spot position, sorting speed and other conditions are determined by sorting FITC and ink. The incubated microdroplets are then re-injected into the detection and sorting chip.

According to the signal distribution of the mutant library, the PMT was set to 0.43V, the small and large thresholds of sorting were set to 0.2V and 0.6V respectively, and the sorting voltage was 1.6V. The droplets of about 0.42% showing the highest activity were collected in a 1.5mL centrifuge tube, and the positive genes were recovered by PCR. The target fragments were double-digested with SacI and HindIII, reconstructed into the pQE-80L linearized vector, and the ligated vector was transformed into BL21 (DE3) competent cells again, and the single clones grown on the plate were picked for rescreening. In the actual sorting process, we carried out several rounds of sorting on different batches of transformed mutant libraries, and mixed and recovered the obtained positive genes to ensure that more beneficial mutations could be sorted.

From the clones obtained in the previous round, about 393 fresh monoclones were picked in a 96-well plate, and three wild-type BL21 (DE3)-T7 RNAP monoclones were used as controls. After induction and expression, the crude enzyme solution was prepared by lysing the cell lysate, and the crude enzyme solution was heat-treated at 45°C for 5 minutes, quickly placed on ice, and then transferred to a 384-well plate. The enzyme activity was measured using the STAR system (iSpinach aptamer-based monitoring of transcription activity in real-time, STAR, see application number: CN202211274914.8, the invention name is "A detection method for real-time monitoring of in vitro transcribed synthetic RNA", the introduction and use of the system are detailed in CN115896213A text). Mutants whose residual activity after heat treatment is at least 1.5 times that of the wild type were sequenced. Analysis of the sequencing results revealed that the frequencies of mutations at positions 217, 397, 121, 223, 632, 369 and 579 corresponding to the parental RNA polymerase (i.e., wild-type T7 RNAP, as shown in the amino acid sequence SEQ ID NO.1) were relatively high.

Example 2: Preparation of T7 RNAP

1. Construction of recombinant vector expressing T7 RNA polymerase mutant

According to the results obtained in Example 1, the wild-type T7 RNAP shown in the amino acid sequence SEQ ID NO.1 was determined as the parent, and the 217th amino acid, the 397th amino acid, the 121st amino acid, the 223rd amino acid and the 632nd amino acid of the parent amino acid sequence were used as key sites to design mutants and combination mutants with amino acid sequences as shown in SEQ ID NO.2 to 8; mutants S430P, S633P, Q786L, Q744R, S43Y and mutant combinations reported in the prior art were designed as controls, and the amino acid sequences were as shown in SEQ ID NO.9 to 14, namely the following mutants:

Single point mutant I217L (SEQ ID NO.2);

Single point mutant S397W (SEQ ID NO.3);

Single point mutant T121M (SEQ ID NO.4);

Single point mutant S223M (SEQ ID NO.5);

Single point mutant R632G (SEQ ID NO.6);

Combined mutant I217L/S397W/S430P/S633P/Q786L (SEQ ID NO. 7);

Combined mutant I217L/S397W/S430P/S633P/Q786L/Q744R/S43Y (SEQ ID NO. 8);

Single point mutant S430P (SEQ ID NO.9);

Single point mutant S633P (SEQ ID NO.10);

Single point mutant Q786L (SEQ ID NO.11);

Single point mutant Q744R (SEQ ID NO.12);

Single point mutant S43Y (SEQ ID NO.13);

Single point mutant G47A+884G (SEQ ID NO.14);

Single point mutant M369T (SEQ ID NO.79);

Single point mutant N579W (SEQ ID NO.80);

Combined mutant I217L/S397W/S430P/S633P/Q786L/N579W (SEQ ID NO.81);

Combined mutant I217L/S397W/S430P/S633P/Q786L/Q744R (SEQ ID NO.82);

Combined mutant I217L/S397W/S430P/S633P/Q786L/C723E/A724N (SEQ ID NO.83).

Among them, S430P (US7507567B2), S633P (US7507567B2) and Q786L (CN102177236B) are mutation points reported in the prior art to improve the thermal stability of T7 RNAP, Q744R (US20150024435A1) is a mutation point reported in the prior art to improve the activity of T7 RNAP, S43Y (CN112831484B) is a mutation point reported in the prior art to reduce the RdRp activity of T7 RNAP, G47A+884G refers to the addition of an additional amino acid G at the C-terminus of the mutant amino acid sequence on the basis of G47A (total length 883), so that the total length of the mutant is 884. This mutation method (CN111212905A) can reduce dsRNA contaminants and concatenated transcripts generated during in vitro transcription reactions.

The specific steps for constructing the mutant vector are as follows: reverse transcribe the above amino acid sequences (SEQ ID NO.1-14, SEQ ID NO.79-83) and optimize them according to the Escherichia coli codon to obtain nucleotide sequences (SEQ ID NO.15-28, SEQ ID NO.84-88); then insert the nucleotide sequences between the two restriction endonuclease sites of BamHI and HindIII of the vector pQE-80L to form the recombinant expression vectors of wild-type T7 RNAP and mutant T7 RNAP.

2. Construction of recombinant microbial cells expressing T7 RNA polymerase mutants

The recombinant expression vector constructed in the previous step was transformed into BL21 (DE3) Escherichia coli competent cells to obtain recombinant strains of T7 RNAP wild type and mutants, which were named as: T7 RNAP-WT (for expressing wild type T7 RNAP), T7 RNAP-I217L (for expressing single point mutant I217L), T7 RNAP-S397W (for expressing single point mutant S397W), 7 RNAP-T121M (for expressing mutant T121M), T7 RNAP-S223M (for expressing mutant S223M), T7 RNAP-R632G (for expressing mutant R632G), T7 RNAP-I217L/S397W/S430P/S633P/Q786L (for expressing combined mutant M7: I217L/S397W/S430P/S633P/Q786L), T7 RNAP-I217L/S397W/S430P/S633P/Q786L/Q744R/S43Y (used to express the combined mutant M8: I217L/S397W/S430P/S633P/Q786L/Q744R/S43Y), and the control recombinant bacteria T7 RNAP-S430P (used to express the single point mutant S430P), T7 RNAP-S633P (used to express the single point mutant S633P), T7 RNAP-Q786L (used to express the single point mutant Q786L), T7 RNAP-G47A+884G (used to express the mutant G47A+884G), T7 RNAP-M369T (used to express the single point mutant M369T), T7 RNAP-N579W (used to express the single point mutant N579W), T7 RNAP-I217L/S397W/S430P/S633P/Q786L/N579W (used to express the combined mutant I217L/S397W/S430P/S633P/Q786L/N579W), T7 RNAP-I217L/S397W/S430P/S633P/Q786L/Q744R was used to express the combined mutant (I217L/S397W/S430P/S633P/Q786L/Q744R), I217L/S397W/S430P/S633P/Q786L/C723E/A724N (used to express the combined mutant I217L/S397W/S430P/S633P/Q786L/C723E/A724N).

3. Preparation of T7 RNAP

The wild-type and mutant recombinant strains prepared in the previous step were placed in LB medium containing ampicillin resistance and cultured in a shaking incubator at 37°C until the OD600 value was close to 1.2. Then, isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 1 mM was added to induce expression in a shaking incubator at 25°C overnight (16 hours). The bacterial precipitate was then collected by centrifugation at 4°C and 5000 rpm for 20 min. The bacteria were fully resuspended in a lysis buffer containing 300 mM NaCl, 20 mM Tris HCl (pH 7.5), 0.5 mg/mL lysozyme, and 0.5 mM DTT, and the protein lysate was centrifuged in a high-speed refrigerated centrifuge at 4°C and 18000 rpm for 1 h. The supernatant was then separated and filtered to remove impurities. The filtered supernatant can be temporarily stored on ice for subsequent nickel column purification. Before the protein passes through the nickel column, it is necessary to equilibrate the nickel column with 10 times the volume of elution buffer (20mM Tris HCl (PH7.5), 300mM NaCl, 0.5mM DTT), and then add the previously filtered protein solution to the nickel column. After all the protein solution has passed through the nickel column filler, first use a certain concentration of Triton X-ray to wash the impurities, and then use different gradients of imidazole solution (50mM 100mM 150mM-200mM-500mM) to elute, and use several test tubes to number them in order of outflow to collect the outflow. All the above operations must be performed on ice or at 4°C, and then all the eluted protein outflows are detected by SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining. Finally, the protein collection liquid with higher concentration and better purity was selected for ultrafiltration and centrifugation, and the protein was replaced into enzyme storage buffer for storage. The enzyme storage buffer consisted of 50mM Tris-HCl, 100mM NaCl, 20mM 2-Mercaptoethanol, 1mM EDTA, 50% Glycerol, 0.1% Triton X-100, pH 7.9.

Example 3: Real-time fluorescence assay for enzyme activity of different mutants

The wild-type T7 RNA and the mutant T7 RNA prepared in Example 2 were taken and their enzyme activities were tested.

1. Relative enzyme activity detection

The reaction system for real-time fluorescence detection of the relative enzyme activity of T7 RNAP mutants is shown in Table 3. For specific experimental methods, please refer to the Chinese patent application with application number: CN202211274914.8, and the invention name is "A detection method for real-time monitoring of RNA synthesized by in vitro transcription", in which the added protein concentration and volume of the T7 RNAP mutant are consistent with the wild type with known enzyme activity and protein concentration. The reaction is carried out in a 96-well plate, and the fluorescence value generated by the binding of the aptamer produced by the reaction and DFHBI is measured in real time by an enzyme reader. The reaction system is determined according to the experimental needs. The reaction conditions are 37°C. When the enzyme reader is used for real-time measurement, it takes 1 hour to measure a value every 1 minute. After the reaction is completed, the Origin software is used to draw a curve graph, and the standard curve of the protein concentration and fluorescence intensity of the wild type is calculated at the time with the best linear relationship, as shown in Figure 3, so as to calculate the relative enzyme activity of the mutant. All reagents in the reaction system need to be prepared with Nuclease-free H ₂ O, and the consumables used are all RNase-free grade to prevent the generated RNA from being degraded by RNase.

Table 3 Reaction system for determining relative enzyme activity of different mutants by real-time fluorescence method

The relative enzyme activity detection of mutants is shown in Table 4-1, Table 4-2, and Table 4-3. The enzyme activities of single point mutant I217L, single point mutant N579W, single point mutant S397W, single point mutant T121M, single point mutant S223M, and single point mutant R632G at 37°C are significantly improved compared with T7 RNAP-WT, and are significantly better than the single point mutants S430P, G47A+884G, single point mutant S633P, and single point mutant Q786L reported in the prior art. The enzyme activity of the combined mutant M8 is significantly higher than that of WT and the single point mutants S430P, S633P, Q744R, Q786L, S43Y, and G47A+884G mutants reported in the prior art. The relative activities of the combined mutants M7 and I217L/S397W/S430P/S633P/Q786L/N579W at 37°C were not different from those of T7 RNAP-WT. The relative activities of the combined mutants I217L/S397W/S430P/S633P/Q786L/Q744R and I217L/S397W/S430P/S633P/Q786L/C723E/A724N at 37°C were significantly better than those of T7 RNAP-WT, and the residual activity after heat treatment at 58°C for 15 min was significantly higher than that of T7 RNAP-WT. The residual activity of the combined mutants I217L/S397W/S430P/S633P/Q786L/Q744R and I217L/S397W/S430P/S633P/Q786L/C723E/A724N was significantly higher than that of T7 RNAP-WT, and was significantly higher than the residual activity of T7-RNAP-WT, single point mutant S430P, single point mutant S633P and single point mutant Q786L after treatment at 50°C for 5 min. The residual relative activity after heat treatment indicated that the combined mutants I217L/S397W/S430P/S633P/Q786L/Q744R and the combined mutants I217L/S397W/S430P/S633P/Q786L/C723E/A724N had higher thermal stability than the existing technology.

Table 4-1 Relative enzyme activity detection

Table 4-2 Relative enzyme activity detection

Table 4-3 Relative enzyme activity detection

2. Determination of Tm value of T7 RNAP by differential scanning fluorimetry

Take 18 μL T7 RNAP protein solution (0.2 mg/mL) and 2 μL Sypro Orange dye (100×) respectively, and dilute the protein solution and Sypro Orange dye to the required concentration with PBS. First turn on the qPCR instrument to preheat for 20 minutes, set the lid temperature to 100°C, set the sample temperature starting point to 25°C, the heating rate to 0.5°C/s, 5s waiting time, and then cycle 120 times to 85°C. Set the filter to the Sypro Orange channel and take the average of 10 samples. The Tm of a protein is an indicator of thermal stability. Generally speaking, the higher the Tm, the more stable the protein.

The results are shown in Table 5. The Tm values of single point mutants M369T and N579W were higher than those of WT; single point mutant I217L, single point mutant S397W, combined mutants M7 and M8, combined mutants I217L/S397W/S430P/S633P/Q786L/N579W, and combined mutants I217L/S397W/S430P/S633P/Q786L The Tm values of I217L/S397W/S430P/S633P/Q786L/C723E/A724N were significantly higher than those of the wild type and other mutants (WT, S430P, S633P, Q786L) reported in the prior art, indicating that the above-mentioned combined mutants have higher thermal stability than the single point mutants and T7 RNAP-WT reported in the prior art.

Table 5 Tm values of wild type and mutants of T7 RNAP (℃)

3. Comparison of IVT yield and product purity at different temperatures

The in vitro transcription (IVT) reaction system is shown in Table 6. The reaction system can be scaled up in equal proportions. After the system is configured, it is placed at 37°C and 50°C for 2 hours, respectively. Then, 10U DNaseI is added to each reaction according to 1μg template and reacted at 37°C for 30 minutes to digest the DNA template. The IVT product is then recovered by LiCl precipitation, the RNA concentration is measured, and the mRNA yield (μg) corresponding to 1μg template DNA is calculated.

Table 6 In vitro transcription reaction system

The mRNA yield (μg) corresponding to 1μg template DNA is shown in Table 7-1. Under conventional 37°C reaction conditions, the IVT yield of the combined mutant M8 of the present invention is significantly improved compared with the wild-type WT and the single-point mutants S430P, S430P, Q786L, and G47A+884G reported in the prior art, and is comparable to the IVT yields of the mutants Q744R and S43Y reported in the prior art, and is also somewhat improved compared to the combined mutant M7 IVT yield; at the same time, under 50°C reaction conditions, the WT and Q744R reported in the prior art are significantly improved compared to the combined mutant M7. The mutants 744R, S43Y, and G74A+884G produced almost no IVT products, indicating that WT, Q744R, S43Y, and G47A+884G could not tolerate the reaction at 50°C; the IVT yield of the combined mutant I217L/S397W/S430P/S633P/Q786L/N579W at 50°C increased by 60% compared with the IVT yield of WT at 37°C; the combined mutant I217L/S397W/S430P/S633P/Q786L/N579W at 50°C increased by 60% compared with the IVT yield of WT at 37°C. The IVT yield of L/Q744R at 37℃ or 50℃ was significantly increased compared with WT and single-point mutants I217L, S397W, S430P, S633P, and Q786L. The IVT yield of the combined mutant I217L/S397W/S430P/S633P/Q786L/C723E/A724N at 37℃ was increased by about 20% compared with WT and single-point mutants I217L, S430P, S633P, and Q786L. Right, the IVT yield at 50°C was increased by about 120% compared with the IVT yield of WT and single point mutants I217L, S430P, S633P, and Q786L at 37°C; the IVT yield of the combined mutant M8 at 50°C was significantly higher than that of WT and mutants reported in the prior art, indicating that the combined mutants M8 and M7 have better IVT application performance, can produce higher yields of in vitro transcription products, can tolerate 50°C reactions, and have good thermal stability.

Table 7-1 IVT production

The product purities of the single mutants S430P, S633P, Q786L, I217L, S397W, the combined mutants M7, I217L/S397W/S430P/S633P/Q786L/N579W, I217L/S397W/S430P/S633P/Q786L/Q744R, and I217L/S397W/S430P/S633P/Q786L/C723E/A724N are shown in Table 7-2. The purity of the IVT product of N579W at 37°C was significantly improved compared with WT and single point mutants I217L, S397W, S430P, S633P, and Q786L, and the product purity improvement rate was all above 10%; the purity of the product of the combination mutants I217L/S397W/S430P/S633P/Q786L/Q744R and the combination mutants I217L/S397W/S430P/S633P/Q786L/C723E/A724N at 37°C was significantly improved compared with WT and single point mutants I217L, S397W, S430P, S633P, and Q786L, and the product purity improvement rate was all above 5%. The combined mutants I217L/S397W/S430P/S633P/Q786L, the combined mutants I217L/S397W/S430P/S633P/Q786L/N579W, the combined mutants I217L/S397W/S430P/S633P/Q786L/Q744R, and the combined mutants I217L/S397W/S430P/S633 The purity of the IVT product of P/Q786L/C723E/A724N at 50°C is within 5% relative to the purity of the IVT product of WT and single point mutants I217L, S397W, S430P, S633P, Q786L at 37°C and single point mutants I217L, S397W, S430P, S633P, Q786L at 50°C, and it is considered that there is no significant difference. This shows that the purity of the IVT product of the T7 RNAP mutant provided by the present invention is improved to a certain extent compared with the prior art.

Table 7-2 IVT product purity

4. Comparison of dsRNA content in IVT products at different temperatures

The wild type WT, single mutants Q744R, S43Y, G47+884G, T121M, S223M, R632G, S430P, S633P, Q786L, I217L, S397W, combined mutants M7 and M8 were tested using our company's self-developed dsRNA content ELISA quantitative detection kit. The purified IVT product RNA samples obtained in 3 were tested for dsRNA content. The specific operation method is as follows: (1) The dsRNA standard was diluted with STE Buffer to 8 concentrations of 2pg/μL, 1pg/μL, 0.5pg/μL, 0.25pg/μL, 0.125pg/μL, 0.0625pg/μL, 0.0312pg/μL, and 0pg/μL. The purified RNA samples to be tested were diluted to 3 different concentrations respectively. Gradient 10ng/μL, 5ng/μL, 1ng/μL; (2) Take 100μL of the diluted standard and the sample to be tested and add them to the microwells of the ELISA plate coated with dsRNA antibodies, and shake the plate at 500rpm for 60min at room temperature; (3) Add 250μL of washing solution and repeat the washing for 4 times, add 100μL of biotinylated detection antibody at working concentration to each well, and shake the plate at 500rpm for 60min at room temperature; (4) After washing, add 100μL of HRP-streptavidin at working concentration to each well, and react at 500rpm for 30min; (4) Finally, add 100μL of substrate colorimetric solution and react at room temperature in the dark for 30min, then add 50μL of stop solution to each well, and immediately perform on-machine detection, set the dual wavelength of the ELISA reader to 450nm/650nm, and calculate the dsRNA content of the sample to be tested using the curve fitted by the standard.

The experimental results are shown in FIG4A , wherein the dsRNA content of the combined mutant M8 of the present invention in the unit mass IVT product of the conventional 37° C. reaction of the wild-type WT, Q744R, S43Y and G47A+884G is significantly lower than that of the wild-type WT and the single point mutants Q744R, S43Y, S430P, S633P, Q786L and M7 combined mutants reported in the prior art; in the unit mass IVT product of the in vitro transcription reaction at 50° C., the combined mutant M The dsRNA content of 8 is significantly lower than that produced by the reaction at 37°C. The dsRNA content of the IVT product per unit mass under the reaction conditions of 50°C is significantly reduced compared to the single point mutants S430P, S633P, and Q786L reported in the prior art, and the dsRNA content of its 50°C in vitro transcription product can reach the same level as the G47A+884G mutant reported in CN111212905A that can reduce dsRNA contaminants and concatenated transcripts in the in vitro transcription product. Under the conditions of 50°C, the dsRNA content level of the combined mutant M8 of the present invention per unit mass of the IVT product is lower than that of the combined mutant M7. Since the wild type WT, mutants Q744R, S43Y, G47A+884G, T121M, S223M, and R632G produce almost no IVT products at 50°C, their dsRNA content at 50°C is not shown in the results.

Wild type WT, single mutants S430P, S633P, Q786L, I217L, S397W, combined mutants I217L/S397W/S430P/S633P/Q786L, I217L/S397W/S430P/S633P/Q786L/N579W, I217L/S397W/S430P/S633P/Q786L/Q744R, I217L/S397W/S430P/S633P/Q786L The dsRNA content in the IVT product of /C723E/A724N was detected by dot blot. The IVT RNA sample recovered by LiCl precipitation obtained in 3 was added dropwise to the positively charged Hybond N membrane (GE Amersham), and then the membrane was blocked in 5% (w/v) skim milk powder TBS-T buffer (20mM Tris, pH 7.4, 150mM NaCl, 0.1% v/v Tween-20). Then J2 anti-dsRNA antibody was diluted 1:5000; incubated overnight at 4°C. The blot was detected with HRP-conjugated secondary antibody (Cell Signaling Technologies). A dsRNA standard with a known concentration was used as a positive control.

The experimental results are shown in Figure 4B. When the mRNA loading amount was consistent, dot blot analysis showed that the single point mutants I217L, S397W, S430P, S633P, Q786L, the combined mutants I217L/S397W/S430P/S633P/Q786L, and the combined mutants I217L/S397W/S430P/S633P/Q786L/N579 The dsRNA byproduct content in the IVT products of W, combined mutants I217L/S397W/S430P/S633P/Q786L/Q744R, and combined mutants I217L/S397W/S430P/S633P/Q786L/C723E/A724N at 50°C was significantly lower than the dsRNA byproduct content in the IVT product of WT at 37°C. This indicates that the T7 RNAP mutants provided in the above examples have significantly improved dsRNA content in the IVT products compared to the wild type in the prior art through high temperature reaction.

5. 3' end consistency detection of IVT products at different temperatures

The 3' end consistency detection of IVT products is achieved through RNase T1 digestion experiment. RNase T1 can specifically degrade single-stranded RNA at the G residue, which forms the corresponding intermediate 2', 3'-cyclic phosphate (-mP) salt, cleaving the phosphodiester bond between the 3'-guanosine residue and the 5'-OH residue of the adjacent nucleotide; the reaction products are 3'-GMP and oligonucleotides containing terminal 3'-GMP. Then, the molecular weight of the 3' end product is analyzed by LC-MS and compared with the theoretical molecular weight. If the 3' end of the normal mRNA product is a G base ending with -OH, then the 3' end G base of the heterogeneous product ends with -mP, and the molecular weight of the heterogeneous product is 80 more than that of the normal product. The 3' end consistency (%) is calculated by analyzing the proportion of the theoretical molecular weight product.

The experimental results are shown in Figures 5A to 5J. The molecular weight of the pure product with 3' end consistency is 9663.1, and the molecular weight of the 3' end heterogeneous conjugation product is 9743.1. Figures 5A to 5J are the results of LC-MS analysis of 3' end products of IVT products of wild type WT (37°C), Q774R (37°C), S43Y (37°C), G47A+884G (37°C), M7 (37°C), M7 (50°C), M8 (37°C), M8 (50°C), I217L (50°C), and S397W (50°C). The detailed data analysis is shown in Table 8. Since the wild type WT, mutants Q744R, S43Y, and G47A+884G almost no IVT products are produced at 50°C, their 3' end consistency at 50°C is not shown in the results. The 3' end consistency product of the combined mutant M8 of the present invention at 37°C is significantly better than that of WT, single point mutant Q744R and combined mutant M7, and the 3' end consistency product of the combined mutant M8 at 50°C is significantly better than that of the single point mutant S43Y and mutant G47A+884G at 37°C. This indicates that the T7 RNAP combined mutant M8 provided by the present invention can not only withstand 50°C reaction, but also the 3' end consistency of the IVT product is significantly improved compared with the prior art.

Table 8 IVT product 3' end consistency

“/” indicates that the wild-type WT or mutant produced almost no IVT product at 50°C, so its 3' end identity at 50°C was not shown in the results.

6. Cellular response detection of IVT products at different temperatures

The immune response of cells to dsRNA byproducts may lead to adverse consequences. The purified IVT product RNA sample was used for cell response detection. The specific operation method is as follows: (1) Thaw the cryotube containing the Raw cell suspension and mix it evenly, and add the cell suspension to a culture flask or culture dish containing culture medium for overnight culture. When the RAW cell density reaches 80%-90%, subculture is performed. (2) Inoculate cells: Prepare IVT product-lipid complexes and transfect cells with cationic liposome transfection reagent Lipofectamine 3000. Among them, Polycytidylic acid (Poly (I: C)) and Resiquimod (R848) are used as positive controls, transfection reagent alone is used as negative control, and no transfection treatment is used as blank control. (3) After 48 hours of culture, serum was collected and IFN-α and IL-6 cytokines in cell serum were quantitatively detected using IFN alpha Mouse ELISA Kit (Thermo Scientific) and mouse IL-6 double antibody sandwich ELISA detection kit (Proteintech Group). The measured concentrations of the samples to be tested were calculated using the curves fitted by the standard samples, and finally the IL-6 and IFN-α expression of IVT products of T7 RNAP mutants at different temperatures was obtained. IFN-α and IL-6 are cytokines produced by the body's immune cells. They are immune response factors produced by immune cells through immune response when the body is infected. The levels of IFN-α and IL-6 detected in this study reflect the immunogenicity of the T7 RNA polymerase IVT products.

The experimental results are shown in Figures 6A and 6B. By comparing the intracellular IFN-α and IL-6 factor levels caused by the IVT products of the wild-type WT and mutant M8: I217L/S397W/S430P/S633P/Q786L/Q744R/S43Y, it was found that among the four different transcription templates, the IFN-α and IL-6 factor levels caused by the IVT products of M8 at 37°C and 50°C were lower than those of the wild-type WT, indicating that the mRNA obtained by the in vitro transcription reaction of the T7 RNA polymerase mutant M8 provided by the present invention has lower immunogenicity than that of the wild-type WT.

Example 4: T7 RNAP mutants with improved performance

According to the methods of Example 2 and Example 3, partial site-directed mutations were performed on the 217th amino acid, the 397th amino acid, the 121st amino acid, the 223rd amino acid and the 632nd amino acid based on the amino acid sequence of the wild-type T7 RNAP (shown in SEQ ID NO.1), and T7 RNAP mutants were prepared, and their activity changes were detected (the activity changes were detected using the relative activity detection method of Example 3, and the Tm values of each mutant were determined using the differential scanning fluorimetry of Example 3. The Tm value reflects the stability of each mutant protein. The higher the Tm value, the more stable the protein).

Table 9-1 Site-directed mutagenesis at different sites

Table 9-2 Site-directed mutations at different sites

The results are shown in Table 9-1 and Table 9-2. The results indicate that after the 786th position mutated to M or F, the 217th position mutated to A or V, the 397th position mutated to P, and the 579th position mutated to A or D, the enzyme activity and Tm value at 37°C were higher than those of the mutants S430P, S633P, Q786L, G47A+884G and T7 RNAP-WT reported in the prior art; after the 121st position mutated to L or S, the 223rd position mutated to L or V, and the 632nd position mutated to E, the enzyme activity at 37°C was significantly improved compared with the mutants reported in the prior art and T7 RNAP-WT, and the Tm value after the 369th position mutated to E was also improved to a certain extent compared with the wild-type T7 RNAP and the mutants reported in the prior art. The above results show that the T7 RNAP mutants with mutation sites Q786M, Q786F, I217A, I217V, S397P, N579A, and M369E have higher thermal stability than T7 RNAP-WT. Although the I121L, I121S, S223L, S223V, and R632E mutants have no obvious advantage in thermal stability over wild-type T7 RNAP, they have higher enzyme activity. These mutants will have broad application prospects in in vitro transcription, mRNA vaccine and drug research and production, gene editing, isothermal amplification, in vivo protein expression, or cell-free protein expression in vitro translation systems.

Example 5: T7-RNA mutation sites with high sequence similarity

RNA polymerases from different sources with an amino acid identity of more than 80% were compared, and the results are shown in Figures 7A to 7E. It was found that some sequences in the sequence are highly conserved, including positions 217, 223, 397, 579, and 632. When the amino acid substitutions at positions 217, 223, 397, 579, and 632 described in the present application are transferred to the corresponding positions in other closely related phage RNA polymerases (SEQ ID NO. 29 to SEQ ID NO. 62), they are expected to have the same effect. The closely related bacteriophage RNA polymerase amino acid sequences (SEQ ID NO. 29 to SEQ ID NO. 62) have 97.96%, 98.07%, 98.64%, 98.07%, 98.19%, 90.26%, 97.51%, 97.40%, 97.40%, 98.19%, 98.53% similarity with the wild-type amino acid sequence of T7 bacteriophage RNA polymerase shown in SEQ ID NO. 1 in Examples 1-4, respectively. %, 82.35%, 98.75%, 97.73%, 82.47%, 98.41%, 92.98%, 82.47%, 98.41%, 82.47%, 98.30%, 98.41%, 98.19%, 98.53%, 89.92%, 82.69%, 82.58%, 82.47%, 82.47%, 82.35%, 82.47%, 82.24%, 82.13%, 82.01% sequence identity.

The amino acid substitutions at positions 121 and 369 are expected to have the same effect when transferred to the corresponding positions in other closely related phage RNA polymerases. The amino acid sequences of the closely related phage RNA polymerases at position 121 (SEQ ID NO.29 to SEQ ID NO.39, SEQ ID NO.41 to SEQ ID NO.42, SEQ ID NO.44 to SEQ ID NO.45, SEQ ID NO.47, SEQ ID NO.49 to SEQ ID NO.53) have 97.96%, 98.07%, 98.64%, 98.07%, 98.66%, 98.67%, 98.68%, 98.69%, 98.70%, 98.71%, 98.72%, 98.73%, 98.74%, 98.76%, 98.77%, 98.78%, 98.80%, 98.81%, 98.82%, 98.83%, 98.84%, 98.86%, 98.87%, 98.83%, 98.84%, 98.85 ...3%, 98.83%, 98.83%, 98.83%, 98.8 98.19%, 90.26%, 97.51%, 97.40%, 97.40%, 98.19%, 98.535%, 98.75%, 97.73%, 98.41%, 98.30%, 98.41%, 98.19%, 98.53%, 89.92% sequence identity. The closely related bacteriophage RNA polymerase amino acid sequence SEQ ID NO.44 at position 369 has a sequence identity of 98.41% with the wild-type amino acid sequence of T7 bacteriophage RNA polymerase shown in SEQ ID NO.1 in Examples 1-4.

The amino acid sequence of the wild-type parent T7 RNA polymerase of the present invention (SEQ ID NO.1):

Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims (11)

A RNA polymerase mutant with improved performance, characterized in that the RNA polymerase mutant has one or more amino acid mutations at positions 121, 223, 632, 217, 397, 369, and 579 based on the amino acid sequence corresponding to the parent RNA polymerase, or one or more amino acid mutations occur at the amino acid residues at equivalent positions in the parent RNA polymerase; the amino acid sequence of the parent RNA polymerase has at least 80% identity with the amino acids shown in SEQ ID NO.1 and has RNA polymerase activity. The RNA polymerase mutant according to claim 1, characterized in that the RNA polymerase mutant also has one or more amino acid mutations at position 430, 633, 786, 744, 724, 43 or 723. The RNA polymerase mutant according to any one of claims 1 to 2, characterized in that, corresponding to the amino acid sequence shown in SEQ ID NO.1, The amino acid at position 217 is mutated to L, A or V; preferably, mutated to L; The amino acid at position 397 is mutated to W, A or P; preferably, mutated to W; The amino acid at position 121 is mutated to M, L or S; preferably, it is mutated to M; The amino acid at position 223 is mutated to M, P, L, V or F; preferably, mutated to M; The amino acid at position 632 is mutated to G or E; preferably, mutated to G; The amino acid at position 369 is mutated to T or E; The amino acid at position 579 mutates to A, W or D; preferably, mutates to W. The RNA polymerase mutant according to claim 2, characterized in that, corresponding to the amino acid sequence shown in SEQ ID NO.1, The amino acid at position 430 is mutated to a non-polar hydrophobic amino acid; optionally, it is mutated to P, L, V, M, or F; preferably, it is mutated to P; The amino acid at position 633 is mutated to a non-polar hydrophobic amino acid; optionally, it is mutated to P, L, V, M or F, A, I, W; preferably, it is mutated to P; The amino acid at position 786 is mutated to a non-polar hydrophobic amino acid or Y; alternatively, it is mutated to P, I, A, V, Y, L, M, W or F; preferably, it is mutated to L; The amino acid at position 744 is mutated to R, P or L; preferably, mutated to R; The amino acid at position 43 is mutated to Y or A; preferably, mutated to Y; The amino acid at position 723 is mutated to E or S; preferably, mutated to E; The amino acid at position 724 is mutated to N; Preferably, the RNA polymerase mutant simultaneously has at least the following mutations equivalent to or corresponding to the parent polymerase mutant: I217L, S397W, S430P, S633P and Q786L, named I217L/S397W/S430P/S633P/Q786L; Preferably, the RNA polymerase mutant simultaneously has at least the following mutations equivalent to or corresponding to the parent polymerase mutant: I217L, S397W, S430P, S633P, Q786L, N579W, named I217L/S397W/S430P/S633P/Q786L/N579W; Preferably, the RNA polymerase mutant simultaneously has at least the following mutations equivalent to or corresponding to the parent polymerase mutant: I217L, S397W, S430P, S633P, Q786L, Q744R, named I217L/S397W/S430P/S633P/Q786L/Q744R; Preferably, the RNA polymerase mutant simultaneously has at least the following mutations equivalent to or corresponding to the parent polymerase mutant: I217L, S397W, S430P, S633P, Q786L, C723E, A724N, named I217L/S397W/S430P, S633P/Q786L/C723E/A724N; Preferably, the RNA polymerase mutant simultaneously has at least the following mutations equivalent to or corresponding to the parent polymerase mutant: I217L, S397W, S430P, S633P, Q786L, Q744R, S43Y, named I217L/S397W/S430P/S633P/Q786L/Q744R/S43Y. A polynucleotide encoding the RNA polymerase mutant according to any one of claims 1 to 4. A vector carrying the polynucleotide of claim 5 or a cell expressing the RNA polymerase mutant of any one of claims 1 to 4. A method for synthesizing a polynucleotide, characterized by using the RNA polymerase mutant described in any one of claims 1 to 4; wherein the synthesis method includes in vitro transcription synthesis, isothermal amplification or transcription-mediated amplification. Application of the RNA polymerase mutant according to any one of claims 1 to 4 or the method for synthesizing the polynucleotide according to claim 6 in the preparation of mRNA vaccines, the preparation of nucleic acid drugs, gene editing or the construction of protein expression systems. A polynucleotide synthesis kit, characterized in that it comprises the RNA polymerase mutant according to any one of claims 1 to 4. A method for improving at least one performance of an RNA polymerase, wherein the performance is selected from enzyme activity, thermal stability, 3' end consistency, in vitro transcription yield, dsRNA byproducts and immunogenicity, characterized in that, based on the amino acid sequence corresponding to a parent RNA polymerase, one or more amino acid mutations occur at positions 121, 223, 632, 217, 397, 369, and 579, or one or more amino acid mutations occur at amino acid residues at equivalent positions in the parent RNA polymerase; the amino acid sequence of the parent RNA polymerase has at least 80% identity with the amino acids shown in SEQ ID NO.1 and has RNA polymerase activity. The method for screening T7 RNA polymerase mutants is characterized by comprising amplifying a polynucleotide encoding T7 RNA polymerase in a reaction system containing 0.1 to 0.6 mM manganese ions, then transforming a vector carrying the amplified product into cells, inducing expression in the cells and sorting target cells using a droplet microfluidic cell sorting method; in the in vitro incubation step of the droplet microfluidic cell sorting, heat treating the droplets; Preferably, the heat treatment is carried out at 40-50°C for 5-10 minutes; Preferably, the heat treatment condition is 45°C for 7 minutes; Preferably, the manganese ion concentration in the reaction system is 0.2 mM to 0.3 mM; Preferably, the target cells are 0.4% to 0.45% of the droplet-encapsulated cells that exhibit the highest viability; Preferably, the amplified polynucleotide encoding T7 RNA polymerase and the nucleotide sequence are shown as SEQ ID NO.15; Preferably, the method further comprises at least one step: separating the target fragments from the target cells sorted by the droplet microfluidic cell sorting method, transforming the fragments into another cell, and screening the cells again after culturing; Preferably, the T7 RNA polymerase mutants expressed by the positive cells are collected, the system containing the T7 RNA polymerase mutants is heat-treated, and then the enzyme activity is determined, and the positive clones whose enzyme activity is at least 1.5 times that of the wild type are sequenced.