T7-RNA polymerase mutant and application thereof

Abstract

The invention discloses a T7-RNA polymerase mutant and application thereof, and relates to the field of nucleic acid tool enzymes and nucleic acid biology. The T7-RNA polymerase mutant is obtained by mutating glycine at the 47th site from the N end of a full-length amino acid sequence of wild type T7-RNA polymerase into phenylalanine, tryptophan or tyrosine; or, the amino acid sequence of the T7-RNA polymerase mutant and the amino acid sequence of the wild type T7-RNA polymerase have at least 80% homology, and glycine in a peptide fragment as shown in SEQ ID NO.10 in the amino acid sequence is mutated into phenylalanine, tryptophan or tyrosine; the full-length amino acid sequence of the wild type T7-RNA polymerase is as shown in SEQ ID NO. 1. The T7-RNA polymerase mutant is suitable for enzymatic synthesis of RNA, and has strong application potential in the aspects of in-vitro transcription, RNA synthesis, RNA drug synthesis, RNA vaccine manufacturing, gene editing, in-vivo protein expression or in-vitro translation systems and the like.

Description

Technical Field

[0001] The present invention relates to the fields of nucleic acid tool enzymes and nucleic acid biology, and particularly to T7-RNA polymerase mutants and their applications. Background Art

[0002] RNA (ribonucleic acid) is an extremely important type of biological macromolecule in the process of genetic information transmission. With the gradual in-depth research on RNA, the value of RNA in disease treatment has gradually emerged. Driven by the COVID-19 pandemic, mRNA vaccines have moved from concept to reality and made outstanding contributions to safeguarding the health of people worldwide (Fernando P. Polack et al., 2020; L. R. Baden et al., 2020). The success of mRNA vaccines demonstrates the great potential of mRNA as a technical platform for vaccine or drug delivery. In vitro synthesized mRNA has the advantages of transiently expressing proteins in vivo, convenient production, safety, and high efficiency, and it is expected to become an excellent alternative to protein drugs. Some large pharmaceutical companies such as Moderna, Pfizer, etc. have already started to develop RNA drugs.

[0003] RNA in vitro synthesis mainly relies on two methods: chemical synthesis and enzymatic synthesis. Among them, chemical synthesis is only applicable to the synthesis of short-chain RNAs less than 100 nucleotides, and its synthesis cost will increase sharply with the increase in RNA length. However, the mRNA encoding proteins is often more than 1000 nucleotides. Therefore, enzymatic synthesis is currently the best solution for preparing long-chain mRNA. The single-subunit RNA polymerases encoded by short-tailed phages have significant advantages such as simple structure and high in vitro transcription efficiency, and they have now been widely used in in vitro transcription to synthesize RNA. Among them, the most widely used is the single-subunit RNA polymerase from Escherichia coli phage T7. However, although T7-RNA polymerase has significant advantages such as high yield and convenient operation, it also has some unavoidable disadvantages. For example, it will produce a lot of by-products during the process of synthesizing RNA, including oligonucleotides produced during the transcription initiation process, interrupted RNA products produced when encountering termination signals, 3'-terminal extension products caused by RdRp activity (RNA-dependent RNA polymerase activity) (Katalin et al., 2011), full-length complementary double-stranded RNA (dsRNA) produced by transcription initiation independent of promoters (Xin Mu et al., 2018), etc. Among them, the dsRNA produced by RdRp activity and transcription initiation independent of promoters is the most harmful, which will cause a strong immune response in the human body.

[0004] Given the harmfulness of dsRNA, it is extremely necessary to remove dsRNA from transcription products, especially for mRNA therapeutic drugs with a much larger injection dose than mRNA vaccines. Previous studies have shown that adding modified nucleotides (such as N1-methylpseudouridine, me1ψ) to the transcription system can greatly reduce the production of dsRNA (Kariko, K et al., 2005, 2010), but at the same time, it will greatly increase the production cost, and the safety of mRNA overmodified is also in doubt. Other methods, such as adding competitive DNA (Gholamalipour, Y et al., 2022), denaturants (Piao, X et al., 2022) or reducing the magnesium ion concentration in the transcription system, can also reduce dsRNA to a certain extent, but these methods will introduce new contaminants or greatly reduce the yield of the target RNA. In addition, there are also some purification methods, such as high performance liquid chromatography (HPLC) (Kariko, K et al., 2011) and cellulose membrane chromatography (Baiersdorfer, M et al., 2022), etc., which can remove dsRNA by-products, but using these purification methods in large-scale production will greatly increase the production cost, and increasing the purification process will also reduce the stability of RNA drugs. Therefore, developing new RNA synthetic tool enzymes to reduce dsRNA by-products while maintaining efficient transcription has very important application value.

[0005] In the prior art, the international authorized patent WO2020239144A1 discloses a novel RNA polymerase VSW-3 from a Pseudomonas phage. It has been found through research that the proportion of by-product dsRNA in the RNA produced by this RNA polymerase during transcription at 25°C for 14 hours is extremely low, and the 3'-end homogeneity of the RNA synthesized by it is relatively high. That is to say, the RNA synthesized by the VSW-3 RNA polymerase has a significant advantage in purity compared with the T7 RNA polymerase.

[0006] The Chinese authorized patent CN112831484B discloses a T7-RNA polymerase mutant, which is obtained by substituting the serine at the 43rd position from the N-terminus of the amino acid sequence of the wild-type T7-RNA polymerase with an amino acid of class A or class B. The amino acids of class A are tyrosine, phenylalanine, leucine, lysine or aspartic acid, and the amino acids of class B are tryptophan, isoleucine, arginine, asparagine, glutamine, glutamic acid or proline. The above-mentioned T7-RNA polymerase mutant is suitable for the synthesis of RNA containing a termination signal inside and RNA with a hairpin structure formed at the end, and this mutant can weaken the RNA-dependent RNA polymerase activity of the T7-RNA polymerase, thereby achieving the effect of reducing the production of dsRNA.

[0007] However, these techniques still cannot significantly reduce the dsRNA by-products generated by transcription initiation independent of the promoter while maintaining the efficient transcription of RNA polymerase. SUMMARY OF THE INVENTION

[0008] In view of the above deficiencies of the prior art, the present invention provides a T7-RNA polymerase mutant and its application. The T7-RNA polymerase mutant can significantly reduce the dsRNA by-products generated by transcription initiation independent of the promoter while maintaining the efficient transcription of RNA polymerase, which is specifically achieved through the following techniques:

[0009] The T7-RNA polymerase mutant provided by the present invention is obtained by mutating the glycine at the 47th position from the N-terminus of the full-length amino acid sequence of wild-type T7-RNA polymerase to phenylalanine, tryptophan or tyrosine; alternatively, the amino acid sequence of the T7-RNA polymerase mutant has at least 80% homology with the amino acid sequence of the wild-type T7-RNA polymerase, and the glycine in the peptide segment shown in SEQ ID NO.10 in its amino acid sequence is mutated to phenylalanine, tryptophan or tyrosine; the full-length amino acid sequence of the wild-type T7-RNA polymerase is as shown in SEQ ID NO.1.

[0010] Further, the amino acid sequence of the above T7-RNA polymerase mutant has at least 85% homology with the amino acid sequence of the wild-type T7-RNA polymerase, and the glycine in the peptide segment shown in SEQ ID NO.10 in its amino acid sequence is mutated to phenylalanine, tryptophan or tyrosine.

[0011] Further, the amino acid sequence of the above T7-RNA polymerase mutant has at least 90% homology with the amino acid sequence of the wild-type T7-RNA polymerase, and the glycine in the peptide segment shown in SEQ ID NO.10 in its amino acid sequence is mutated to phenylalanine, tryptophan or tyrosine.

[0012] Further, the amino acid sequence of the above T7-RNA polymerase mutant has at least 95% homology with the amino acid sequence of the wild-type T7-RNA polymerase, and the glycine in the peptide segment shown in SEQ ID NO.10 in its amino acid sequence is mutated to phenylalanine, tryptophan or tyrosine.

[0013] Further, the amino acid sequence of the above T7-RNA polymerase mutant has at least 99% homology with the amino acid sequence of the wild-type T7-RNA polymerase, and the glycine in the peptide segment shown in SEQ ID NO.10 in its amino acid sequence is mutated to phenylalanine, tryptophan or tyrosine.

[0014] Furthermore, the above-mentioned T7-RNA polymerase mutant is obtained by mutating the glycine at the 47th position from the N-terminus of the full-length amino acid sequence of the wild-type T7-RNA polymerase to phenylalanine; alternatively, the above-mentioned T7-RNA polymerase mutant has at least 80% homology with the amino acid sequence of the wild-type T7-RNA polymerase, and the glycine in the peptide segment shown in SEQ ID NO.10 in its amino acid sequence is mutated to phenylalanine.

[0015] Still further, the above-mentioned T7-RNA polymerase mutant has at least 85% homology with the amino acid sequence of the wild-type T7-RNA polymerase, and the glycine in the peptide segment shown in SEQ ID NO.10 in its amino acid sequence is mutated to phenylalanine.

[0016] Still further, the above-mentioned T7-RNA polymerase mutant has at least 90% homology with the amino acid sequence of the wild-type T7-RNA polymerase, and the glycine in the peptide segment shown in SEQ ID NO.10 in its amino acid sequence is mutated to phenylalanine.

[0017] Still further, the above-mentioned T7-RNA polymerase mutant has at least 95% homology with the amino acid sequence of the wild-type T7-RNA polymerase, and the glycine in the peptide segment shown in SEQ ID NO.10 in its amino acid sequence is mutated to phenylalanine.

[0018] Still further, the above-mentioned T7-RNA polymerase mutant has at least 99% homology with the amino acid sequence of the wild-type T7-RNA polymerase, and the glycine in the peptide segment shown in SEQ ID NO.10 in its amino acid sequence is mutated to phenylalanine.

[0019] Among the amino acid sequences with at least 80%, 85%, 90%, 95% or 99% homology to the amino acid sequence of the above-mentioned wild-type T7-RNA polymerase, they all contain the peptide segment sequence shown in SEQ ID NO.10. It should be noted that the position of the peptide segment shown in SEQ ID NO.10 in the amino acid sequence may change, but the amino acid sequence with the same function as the T7-RNA polymerase mutant still belongs to the protection scope of the present invention.

[0020] The applicant mutated the amino acid residues at positions 42-48 in the C-helix region of wild-type T7-RNA polymerase into tyrosine respectively, so as to screen mutants that can reduce the activity of T7-RNA polymerase initiating transcription without a promoter, and detected the effect of the T7-RNA polymerase mutants in reducing the activity of T7-RNA polymerase initiating transcription without a promoter. It was found that the mutant G47Y (formed by mutating glycine at the 47th position in the amino acid sequence of wild-type T7-RNA polymerase into tyrosine) had the most significant effect. Therefore, the G47 site is the key site affecting the activity of T7-RNA polymerase initiating transcription without a promoter.

[0021] The applicant further constructed mutants G47F (formed by mutating glycine at the 47th position in the amino acid sequence of wild-type T7-RNA polymerase into phenylalanine), G47W (formed by mutating glycine at the 47th position in the amino acid sequence of wild-type T7-RNA polymerase into tryptophan), G47H (formed by mutating glycine at the 47th position in the amino acid sequence of wild-type T7-RNA polymerase into histidine), G47Y and G47A (formed by mutating glycine at the 47th position in the amino acid sequence of wild-type T7-RNA polymerase into alanine). The effects of the above mutants in reducing the activity of T7-RNA polymerase initiating transcription without a promoter were detected by in vitro transcription, and the content of by-product dsRNA in the RNA transcribed by each mutant was detected. The results showed that compared with the mutant G47A, the mutants G47F, G47Y, and G47W had more prominent effects in reducing dsRNA generated by the activity of T7 RNA polymerase initiating transcription without a promoter, and among them, the effect of G47W was the most significant.

[0022] In order to explore the mechanism by which G47W reduces the activity of T7-RNA polymerase initiating transcription without a promoter, the applicant designed a 40bp DNA without a T7 promoter and incubated it with wild-type T7-RNA polymerase or mutant G47W at different concentrations. The results showed that compared with wild-type T7-RNA polymerase, the non-specific binding of mutant G47W to DNA without a promoter was significantly reduced. That is to say, after the 47th amino acid residue of T7-RNA polymerase was mutated into an aromatic amino acid, its larger side chain group had a large steric hindrance, which blocked the non-specific binding of RNA polymerase to DNA, thereby reducing its activity of initiating transcription without a promoter.

[0023] The applicant also compared the translation efficiency and immunogenicity of mRNAs synthesized by wild-type T7-RNA polymerase and the mutant G47W. The results showed that the GFP mRNA synthesized by the mutant G47W had higher translation efficiency and lower immunogenicity compared to that synthesized by wild-type T7-RNA polymerase. That is to say, the mutant G47W is more suitable for the production of mRNA vaccines and mRNA drugs.

[0024] The present invention also provides the application of the above-mentioned T7-RNA polymerase mutant in in vitro transcription.

[0025] The application of the above-mentioned T7-RNA polymerase mutant in the synthesis of non-coding RNA or mRNA for non-disease diagnosis and treatment purposes.

[0026] Furthermore, the above non-coding RNA is sgRNA, tRNA, siRNA, snoRNA or oligonucleotide.

[0027] The application of the above-mentioned T7-RNA polymerase mutant in gene editing for non-disease diagnosis and treatment purposes.

[0028] The application of the above-mentioned T7-RNA polymerase mutant in RNA vaccine manufacturing and RNA drug synthesis.

[0029] The application of the above-mentioned T7-RNA polymerase mutant in in vivo protein expression or cell-free protein expression in vitro translation systems for non-disease diagnosis and treatment purposes.

[0030] The application of the above-mentioned T7-RNA polymerase mutant in the synthesis of biological transcriptional regulatory elements for non-disease diagnosis and treatment purposes.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows: By research, the key amino acid sites affecting the promoter-independent transcription initiation of T7-RNA polymerase were discovered and identified, thereby providing a T7-RNA polymerase mutant that can ensure efficient transcription of RNA polymerase while significantly reducing the dsRNA by-products generated by promoter-independent transcription initiation. As a tool enzyme, this T7-RNA polymerase mutant is more suitable for in vitro RNA synthesis in terms of yield and immunogenicity. Description of the Drawings

[0032] Figure 1 Electrophoresis diagram of the transcription products of wild-type T7 RNA polymerase and its mutants at amino acid sites 42-48 described in Example 1;

[0033] Figure 2 Comparison diagram of the effects of reducing dsRNA by wild-type T7 RNA polymerase and its mutants at site G47 described in Example 2. Among them, Figure 2 A Gel imaging result diagram,Figure 2 Figure B is the imaging analysis result diagram of ChemiSciope6000; Figure 2 Figure C is the statistical analysis result diagram;

[0034] Figure 3 It is a comparison diagram of the non-specific binding of the wild-type T7 RNA polymerase and its mutant G47W to DNA described in Example 3. Among them, Figure 3 Figure A is the gel imaging result diagram, Figure 3 Figure B is the statistical analysis result diagram;

[0035] Figure 4 It is a comparison diagram of the translation efficiency and immunogenicity of the GFP mRNA transcribed by the wild-type T7 RNA polymerase and its mutant G47W described in Example 4. Among them, Figure 4 Figure A is the comparison diagram of translation efficiency, Figure 4 Figure B is the comparison diagram of immunogenicity;

[0036] Figure 5 It is a comparison diagram of the in vitro transcription yields of the wild-type T7 RNA polymerase and its mutants G47W and G47A+884G described in Example 5. Among them, Figure 5 Figure A is the result of the three of them transcribing GFP RNA, Figure 5 Figure B is the result of the three of them transcribing Cas9 RNA, Figure 5 Figure C is the result of the three of them transcribing S-gene RNA. Detailed implementation manners

[0037] Next, the technical solutions of the present invention will be described clearly and completely. Obviously, the described embodiments are only a 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 those of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

[0038] In the present invention, the applicant obtained a T7-RNA polymerase mutant by substituting glycine in the peptide segment shown in SEQ ID NO.10 in the amino acid sequence having at least 80% homology with SEQ ID NO.1 with phenylalanine, tryptophan or tyrosine. This T7-RNA polymerase mutant can also significantly reduce the dsRNA by-products generated by transcription starting without relying on a promoter.

[0039] Furthermore, a T7-RNA polymerase mutant was obtained by substituting glycine in the peptide segment shown in SEQ ID NO.10 in the amino acid sequence having at least 85% homology with SEQ ID NO.1 with phenylalanine, tryptophan or tyrosine. This T7-RNA polymerase mutant can also significantly reduce the dsRNA by-products generated by transcription starting without relying on a promoter.

[0040] Furthermore, a T7-RNA polymerase mutant obtained by replacing glycine in the peptide segment shown in SEQ ID NO.10 in an amino acid sequence having at least 90% homology with SEQ ID NO.1 with phenylalanine, tryptophan or tyrosine can also significantly reduce the dsRNA by-products generated by transcription initiation independent of the promoter.

[0041] Furthermore, a T7-RNA polymerase mutant obtained by replacing glycine in the peptide segment shown in SEQ ID NO.10 in an amino acid sequence having at least 95% homology with SEQ ID NO.1 with phenylalanine, tryptophan or tyrosine can also significantly reduce the dsRNA by-products generated by transcription initiation independent of the promoter.

[0042] Furthermore, a T7-RNA polymerase mutant obtained by replacing glycine in the peptide segment shown in SEQ ID NO.10 in an amino acid sequence having at least 99% homology with SEQ ID NO.1 with phenylalanine, tryptophan or tyrosine can still bring the above beneficial effects during transcription.

[0043] Example 1: Detection of the effect of reducing dsRNA on wild-type T7-RNA polymerase and mutants at amino acid positions 42-48

[0044] 1. Expression and purification of T7-RNA polymerase mutants

[0045] (1) Respectively construct T7-RNA polymerase mutants E42Y, S43Y, E45Y, M46Y, G47Y, E48Y by molecular cloning. The amino acid sequence of wild-type T7-RNA polymerase is shown in SEQ ID NO.1.

[0046] (2) Transform the prokaryotic expression vector pQE82L containing T7-RNA polymerase mutants E42Y, S43Y, E45Y, M46Y, G47Y, E48Y into the E.coli BL21(DE3) expression strain. Pick colonies and culture them in LB medium containing 100 μg / ml ampicillin at 37 °C and 220 rpm in a shaker until the OD 600 value is close to 1.2.

[0047] (3) Add isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.5 mM and induce expression at 16 °C and 180 rpm in a shaker for 16 h. After completion, centrifuge at 4 °C and 5000 rpm for 10 min to collect the bacterial cells.

[0048] (4) Then, resuspend the cells thoroughly in a buffer containing 300 mM NaCl and 20 mM Tris-HCl (pH = 7.5). After disrupting the cells with an ultrasonic crusher, centrifuge at 14,000 rpm for 2 h. Filter the separated supernatant through a filter membrane with a pore size of 0.22 μm. Add the filtered supernatant to a nickel column equilibrated with 10 volumes of elution buffer (20 mM Tris-HCl (pH = 7.5), 300 mM NaCl). After all the protein solution has passed through the nickel column, elute with imidazole solutions of different gradients (20 mM - 40 mM - 60 mM - 80 mM - 120 mM), and collect the eluate with EP tubes. All operations in this step need to be carried out on ice or at 4°C.

[0049] (5) Finally, detect all the collected protein eluates by SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining. Comprehensively select the protein with a higher concentration and better purity, add it to a dialysis bag, and dialyze it in 1 L of dialysis solution (100 mM NaCl, 50 mM Tris-HCl (pH = 7.5), 1 mM DTT, 0.1 mM EDTA, 50% glycerol, and 0.1% Triton X-100). After 4 h, replace the fresh and clean dialysis solution. After two dialysis steps, collect the protein and store it at -20°C.

[0050] 2. Obtaining the transcription reaction template and detecting the effect of reducing dsRNA

[0051] Design primers for amplifying the DNA template. The primer sequences are as follows:

[0052] Template-F: 5’-agatgcgtaaggagaaaataccgcatcaggcgcca-3’ (as shown in SEQ ID NO.2)

[0053] GFP-R: 5’-tcagttgtacagttcatccatgccatgtgtaatccc-3’ (as shown in SEQ ID NO.3)

[0054] Use the above general primers to amplify a DNA template containing the T7 promoter, 5’UTR, and GFP coding gene, and purify the PCR product using the DNA Clean&Concentrator TM -5 (ZYMO RESEARCH).

[0055] The in vitro transcription reaction system contains 40 mM Tris-HCl (pH = 8.0), 15 mM MgCl 2, 2 mM spermidine, 10 mM DTT, 4 mM ATP, GTP, CTP, UTP, 0.3 μL RNase inhibitor, 0.2 μL pyrophosphatase, 200 nM RNA polymerase, and 14 nM DNA template, and make up to 10 μL with DEPC water. Incubate the reaction system at 37 °C for 1 h, remove the template with DNase I, purify the RNA product with an RNA purification kit (New England Biolabs), measure the concentration of the RNA product, then take 400 ng of RNA from each experimental group, add 2 μL of 3x RNA loading buffer and make up to 6 μL with water, mix and heat at 75 °C for 4 min, then electrophorese on a 1.5% agarose gel at 100 V for 30 min, stain with EB and analyze with a gel imager.

[0056] The imaging results are as Figure 1 shown, where WT represents wild-type T7-RNA polymerase. From Figure 1 it can be seen that the mutant G47Y has a very significant effect on dsRNA, reducing the proportion of dsRNA to almost 0. The mutant S43Y can also significantly reduce the proportion of dsRNA, while other mutants even increase the proportion of dsRNA.

[0057] Example 2: Comparison of the reduction of dsRNA proportion by wild-type T7-RNA polymerase and its mutants at the G47 site

[0058] 1. Detection of the proportion of dsRNA in in vitro transcription products

[0059] The mutant proteins of T7-RNA polymerase and the DNA templates for in vitro transcription were obtained by the same method as in Example 1. The in vitro transcription reaction system was 40 mM Tris-HCl (pH = 8.0), 15 mM MgCl 2 , 2 mM spermidine, 5 mM DTT, 4 mM ATP, GTP, CTP, UTP, 0.3 μL RNase inhibitor, 0.2 μL pyrophosphatase, 200 nM RNA polymerase, and 14 nM PCR template, and make up to 10 μL with DEPC water. Incubate the reaction system at 37 °C for 1 h, remove the template with DNase I, purify the RNA product with an RNA purification kit (New England Biolabs), measure the concentration of the RNA product, then take 400 ng of RNA from each experimental group, add 2 μL of 3x RNA loading buffer and make up to 6 μL with water, mix and heat at 75 °C for 4 min, then electrophorese on a 1.5% agarose gel at 100 V for 30 min, stain with EB and analyze with a gel imager. The results are as Figure 2 shown in A.

[0060] 2. Dot-blot detection of the transcription products using an antibody that specifically binds to dsRNA

[0061] (1) Drop 250 ng, 500 ng, and 1000 ng of the RNA transcribed from wild-type T7 RNA polymerase and its G47-site mutant onto a TM-Ny+ membrane respectively. After it dries, use forceps to place the membrane in a container, flatten the membrane, and place it in a 5% non-fat milk TBS-T solution (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween-20) on a rocking platform at 40 rpm for 1 hour.

[0062] (2) Pour out the solution and wash it 3 times with TBS solution (50 mM Tris-HCl, pH 7.4, 150 mM NaCl), 10 minutes for each wash.

[0063] (3) Dilute the J2 antibody (SCICONS) with TBS-T containing 2% BSA (bovine serum albumin) at a ratio of 1:2000, add the diluted J2 antibody to cover the TM-Ny+ membrane, and then incubate at 25 °C and 40 rpm for 30 minutes.

[0064] (4) After 30 minutes of incubation, remove the primary antibody solution and wash it 3 times with TBS-T buffer, 10 minutes for each wash. Then add the secondary antibody (Jackson Immunology) solution diluted with TBS-T buffer at a ratio of 1:2000 and incubate at 25 °C and 40 rpm for 1 hour.

[0065] (5) After 1 hour of incubation, pour out the solution, wash it 2 times with TBS-T buffer, and wash it 1 time with TBS buffer.

[0066] (6) Add the enhanced chemiluminescence detection reagent and analyze it using the Qinxiang ChemiSciope6000 imaging system. The results are as shown in Figure 2 Figure B. Finally, use the software ImageJ to scan the grayscale value of Figure 2 Figure B and calculate the relative proportion of dsRNA in the RNA transcribed from wild-type T7 RNA polymerase and its G47-site mutant. Use the software GraphPad Prism to statistically analyze the results. The results are as shown in Figure 2 Figure C.

[0067] The comprehensive results are as shown in Figure 2 Figure, where WT represents wild-type T7-RNA polymerase. As shown in Figure 2It can be seen that, compared with the wild-type T7 RNA polymerase, the mutants at the G47 site of T7-RNA polymerase have a very significant effect on the by-product dsRNA, and can reduce the proportion of dsRNA in the transcription products. When it is mutated into aromatic amino acids (phenylalanine, tryptophan or tyrosine), namely G47F, G47W, G47Y, the effect is more significant. Among them, the mutant G47W has the best effect on reducing the proportion of dsRNA in the transcription products. Compared with the wild-type T7-RNA polymerase, it can reduce the proportion of dsRNA in the transcription products by more than 90%.

[0068] Example 3: Mechanism study on the significant reduction of dsRNA generation by the T7-RNA polymerase mutant G47W

[0069] Study on the non-specific binding of wild-type T7-RNA polymerase and its mutant G47W to promoterless DNA: The applicant annealed two completely complementary 40-nt DNA oligonucleotides (nucleotide sequences are shown in SEQ ID NO.6), and then incubated 1 μM of DNA with 0, 0.5, 1, 2 μM of wild-type T7-RNA polymerase and its mutant G47W in a buffer containing 40 mM Tris-HCl (pH = 8.0), 15 mM MgCl2, 2 mM spermidine, and 10 mM DTT at 25 °C for 10 minutes. After incubation, 5 μl of 3× loading buffer was added and electrophoresed on a 10% non-denaturing PAGE gel at 100 V for 60 min, and analyzed with a gel imager after EB staining. The imaging results are as Figure 3 shown in A. Then we Figure 3 scanned the bands in A for gray value with the software ImageJ, and statistically analyzed the results with the GraphPad Prism software. The results are as Figure 3 shown in B.

[0070] The comprehensive results are as Figure 3 shown, where WT represents wild-type T7-RNA polymerase. As Figure 3 can be seen, compared with the wild-type T7 RNA polymerase, the non-specific binding of the mutant G47W to promoterless DNA is significantly weakened. This may be because tryptophan contains a relatively large side-chain group, which hinders the binding of the polymerase to the DNA template, thus weakening its activity of initiating transcription independent of the promoter, and thereby significantly reducing the proportion of dsRNA in its transcription products.

[0071] Example 4: Comparison of the translation efficiency and immunogenicity of GFP mRNA transcribed by wild-type T7 RNA polymerase and its mutant G47W

[0072] 1. Synthesis of GFP mRNA

[0073] The in vitro transcription reaction system contains 40 mM Tris-HCl (pH = 8.0), 15 mM MgCl2, 2 mM spermidine, 10 mM DTT, 4 mM ATP, GTP, CTP, UTP, 3.2 mM CleanCap AG(3′OMe)(Trilink), 0.3 μL RNase inhibitor, 0.2 μL pyrophosphatase, 200 nM wild-type T7-RNA polymerase or mutant G47W, and 14 nM GFP DNA template (nucleotide sequence as shown in SEQ ID NO.7), and DEPC water is added to make up to 10 μL. The reaction system is incubated at 37 °C for 1 h, the template is removed with DNase Ⅰ, and the RNA product is purified with an RNA purification kit (New England Biolabs), and the concentration of the RNA product is measured.

[0074] Then, 10 μg of the purified RNA is tailed with E.coil poly(A)polymerase from New England Biolabs in a 20 μL reaction system, and the reaction system also contains 1×Poly(A)Polymerase reaction buffer, 5 units E.coil poly(A)polymerase, 1 mM ATP. After incubation at 37 °C for 30 minutes, the RNA product is immediately purified with an RNA purification kit (New England Biolabs), and the concentration of the RNA product is measured.

[0075] 2. Comparison of the translation efficiency of GFP mRNA transcribed by wild-type T7 RNA polymerase and its mutant G47W

[0076] The applicant incubated 0.5 μg of the capped and tailed GFP mRNA synthesized by wild-type T7-RNA polymerase or mutant G47W respectively with 1 μL of lipofectamine2000 (Thermo Fisher Scientific) in OptiMEM buffer at room temperature for 15 minutes, and then dropped it into 293T cells with a cell density of about 80% cultured in a 24-well plate (NEST). After culturing in a CO 2 incubator for 4 hours, 8 hours, and 20 hours, the expression of GFP was observed and recorded with a fluorescence microscope respectively. The results are as Figure 4 shown in A. The results show that the GFP mRNA synthesized by mutant G47W has a higher translation efficiency than that synthesized by wild-type T7-RNA polymerase.

[0077] 3. Comparison of the translation immunogenicity of GFP mRNA transcribed by wild-type T7 RNA polymerase and its mutant G47W

[0078] The applicant incubated 2 μg of capped and polyadenylated GFP mRNA synthesized by wild-type T7-RNA polymerase or mutant G47W respectively with 4 μL of lipofectamine 2000 (Thermo Fisher Scientific) in OptiMEM buffer at room temperature for 15 minutes, and then dropped it into 293T cells with a cell density of about 80% cultured in a 6-well plate (NEST). After culturing in a CO 2 incubator for 24 hours, the IFN-β content in the cells of the blank group, WT group, and G47W group was detected using a Human IFN-β (Interferon Beta) ELISA Kit (Elabscience). The test results were statistically analyzed using GraphPad Prism software, and the results are as Figure 4 shown in B.

[0079] The results showed that the GFP mRNA synthesized by mutant G47W had lower immunogenicity compared to that synthesized by wild-type T7-RNA polymerase, enabling the full expression of GFP and being more suitable for the production of mRNA vaccines and mRNA drugs.

[0080] Example 5: Yield comparison of wild-type T7-RNA polymerase and mutants G47W and G47A+884G

[0081] To compare the yields of wild-type T7-RNA polymerase and its mutants G47W and G47A+884G in multiple dimensions, we obtained Cas9 (the nucleotide sequence of the Cas9 DNA template is shown in SEQ ID NO.8) and S-gene DNA template (the nucleotide sequence of the S-gene DNA template is shown in SEQ ID NO.9) by the same method as obtaining the GFP DNA template above. Among them, GFP represents a shorter mRNA, 877 nt, and Cas9 and S-gene represent longer mRNAs, 4314 nt and 3975 nt respectively. The sequences of the primers GFP-R, Cas9-R, and S-gene-R used are shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5 respectively.

[0082] The in vitro transcription reaction system contained 40 mM Tris-HCl (pH = 8.0), 15 mM MgCl 2, 2 mM spermidine, 10 mM DTT, 4 mM ATP, GTP, CTP, UTP, 0.3 μL RNase inhibitor, 0.2 μL pyrophosphatase, 200 nM RNA polymerase (wild type, mutant G47W, mutant G47A+884G) and 14 nM DNA template, and make up to 10 μL with DEPC water. After incubating the reaction system at 37°C for 10, 20, 40, and 80 min respectively, take 0.5 μl of the sample, add 2 μl of 3x RNA loading buffer and make up to 6 μl of water. After mixing, heat at 75°C for 4 min. After 80 min, perform electrophoresis on a 1.5% agarose gel at 100 V for 30 min. After EB staining, analyze with a gel imager, and statistically analyze the results using GraphPad Prism software.

[0083] The results are as Figure 5 shown. The left side of the figure is the gel imaging diagram, and the right side is the corresponding statistical result diagram, where WT represents wild-type T7-RNA polymerase. Figure 5 A shows the results of transcribing GFP RNA by the three, Figure 5 B shows the results of transcribing Cas9 RNA by the three, Figure 5 C shows the results of transcribing S-gene RNA by the three. It can be Figure 5 seen that when the mutants G47W and G47A+884G transcribe short-chain mRNA (GFP), the yields are slightly increased compared to the wild type; when transcribing long-chain mRNA (Cas9, S-gene), the yield of mutant G47W is almost the same as that of the wild type, while the yield of mutant G47A+884G is significantly lower than that of the wild type and mutant G47W, indicating that the transcription efficiency of mutant G47W is not affected.

[0084] Application Example

[0085] The T7-RNA polymerase mutants obtained in the above examples have great application potential in aspects such as in vitro transcription, non-coding RNA or mRNA synthesis, RNA drug synthesis, RNA vaccine manufacturing, gene editing, in vivo protein expression, or in vitro translation systems. These applications are explained in detail in the literature. For example, see the following literature:

[0086] The application of T7-RNA polymerase mutants in in vitro transcription can be found in the literature: Davanloo P, Rosenberg AH, Dunn JJ, Studier FW. Cloning and expression of the gene for bacteriophage T7RNApolymerase. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2035-9. doi:10.1073 / pnas.81.7.2035.

[0087] The application of T7-RNA polymerase mutants in the synthesis of non-coding RNA or mRNA for non-disease diagnosis and treatment purposes can be found in the literature: Milligan JF, Groebe DR, Witherell GW, Uhlenbeck OC. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 1987 Nov 11;15(21):8783-98. doi:10.1093 / nar / 15.21.8783.

[0088] The application of T7-RNA polymerase mutants in gene editing for non-disease diagnosis and treatment purposes can be found in the literature: Vaidyanathan S, Azizian KT, Haque AKMA, Henderson JM, Hendel A, Shore S, Antony JS, Hogrefe RI, Kormann MSD, Porteus MH, McCaffrey AP. Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce Immunogenicity without HPLC Purification. Mol Ther Nucleic Acids. 2018 Sep 7;12:530-542. doi:10.1016 / j.omtn.2018.06.010.

[0089] The application of T7-RNA polymerase mutants in RNA drug synthesis can be found in the literature: Granit V, Benatar M, Kurtoglu M, MD, Chahin N, Sahagian G, Feinberg MH, Slansky A, Vu T, Jewell CM, Singer MS, Kalayoglu MV, Howard JF Jr, Mozaffar T; MG-001 Study Team. Safety and clinical activity of autologous RNA chimeric antigen receptor T-cell therapy in myasthenia gravis (MG-001): a prospective, multicentre, open-label, non-randomised phase 1b / 2a study. Lancet Neurol. 2023 Jul;22(7):578-590. doi: 10.1016 / S1474-4422(23)00194-1.

[0090] The application of T7-RNA polymerase mutants in RNA vaccine manufacturing can be found in the literature: Baden LR, El Sahly HM, Essink B, Kotloff K, Frey S, Novak R, Diemert D, Spector SA, Rouphael N, Creech CB, McGettigan J, Khetan S, Segall N, Solis J, Brosz A, Fierro C, Schwartz H, Neuzil K, Corey L, Gilbert P, Janes H, Follmann D, Marovich M, Mascola J, Polakowski L, Ledgerwood J, Graham BS, Bennett H, Pajon R, Knightly C, Leav B, Deng W, Zhou H, Han S, Ivarsson M, Miller J, Zaks T; COVE Study Group. Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. N Engl J Med. 2021 Feb 4;384(5):403-416. doi: 10.1056 / NEJMoa2035389.

[0091] The application of the T7-RNA polymerase mutant in the synthesis of biological transcriptional regulatory elements for non-diagnostic and non-therapeutic purposes can be found in the literature: Tabor S, Richardson CC. A bacteriophage T7 RNA polymerase / promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb; 82(4): 1074-8. doi: 10.1073 / pnas.82.4.1074.

[0092] The above specific embodiments have described the implementation of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solutions of the present invention, and these simple variations all fall within the protection scope of the present invention.

Claims

1. T7-RNA polymerase mutant, characterized in that, the T7-RNA polymerase mutant is obtained by mutating the glycine at the 47th position from the N-terminus of the full-length amino acid sequence of wild-type T7-RNA polymerase into phenylalanine, tryptophan or tyrosine; alternatively, the T7-RNA polymerase mutant has at least 80% homology with the amino acid sequence of the wild-type T7-RNA polymerase, and the glycine in the peptide segment shown in SEQ ID NO. 10 in its amino acid sequence is mutated into phenylalanine, tryptophan or tyrosine; the full-length amino acid sequence of the wild-type T7-RNA polymerase is as shown in SEQ ID NO.

1.

2. The T7-RNA polymerase mutant according to claim 1, characterized in that, the T7-RNA polymerase mutant is obtained by mutating the glycine at the 47th position from the N-terminus of the full-length amino acid sequence of the wild-type T7-RNA polymerase into phenylalanine; alternatively, the T7-RNA polymerase mutant has at least 80% homology with the amino acid sequence of the wild-type T7-RNA polymerase, and the glycine in the peptide segment shown in SEQ ID NO. 10 in its amino acid sequence is mutated into phenylalanine.

3. Use of the T7-RNA polymerase mutant according to claim 1 or 2 in in vitro transcription.

4. Use of the T7-RNA polymerase mutant according to claim 1 or 2 in the synthesis of non-coding RNA or mRNA for non-disease diagnosis and treatment purposes.

5. Use of the T7-RNA polymerase mutant according to claim 1 or 2 in gene editing for non-disease diagnosis and treatment purposes.

6. Use of the T7-RNA polymerase mutant according to claim 1 or 2 in the synthesis of RNA drugs.

7. Use of the T7-RNA polymerase mutant according to claim 1 or 2 in the manufacture of RNA vaccines.

8. Use of the T7-RNA polymerase mutant according to claim 1 or 2 in in vivo protein expression or cell-free protein expression in vitro translation system for non-disease diagnosis and treatment purposes.

9. Use of the T7-RNA polymerase mutant according to claim 1 or 2 in the synthesis of biological transcriptional regulatory elements for non-disease diagnosis and treatment purposes.

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