T7-RNA polymerase mutant capable of reducing generation of dsRNA and application of T7-RNA polymerase mutant

Abstract

The invention provides a T7-RNA polymerase mutant capable of reducing generation of dsRNA and application of the T7-RNA polymerase mutant, tryptophan at the 328th site from the N terminal of an amino acid sequence of wild type T7-RNA polymerase is substituted by methionine, phenylalanine, asparagine, alanine or lysine, and a by-product dsRNA generated in an in-vitro transcription process can be reduced. Through computer simulation, a large amount of screening work is reduced, the enzyme evolution speed is increased, and the cost and time of enzyme modification are greatly reduced; the T7-RNA polymerase mutant maintains efficient transcription, reduces the complexity of later purification work, reduces the purification cost of RNA production, can quickly prepare RNA drugs meeting the use requirements, and provides an efficient tool enzyme for large-scale production of the RNA drugs.

Description

Technical Field

[0001] The invention belongs to the field of nucleic acid tool enzymes and nucleic acid biology, and specifically relates to a T7-RNA polymerase mutant capable of reducing dsRNA generation and an application thereof. Background Art

[0002] T7-RNA polymerase is a 99 kDa (883 amino acids) single subunit protein found in bacteriophage T7. Like multi-subunit RNA polymerases (RNAPs), T7 RNAP often releases its transcript within the first 8-12 transcribed nucleotides upon contact with a promoter. This abortive cycle, which results in the production of large amounts of abortive RNA, not only reduces the transcription rate, but is also the rate-limiting step for efficient transcription and is partly responsible for the generation of dsRNA. In vivo, transcription of target genes by T7 RNAP is virtually abolished due to the large number of abortive cycles. Unlike multi-subunit RNA polymerases (RNAPs), monomeric RNAP from bacteriophage T7, upon recognition of its cognate promoter, locally unwinds DNA to produce an "open complex" and uses a single nucleotide to initiate template-directed RNA synthesis. RNAP contacts the promoter are maintained during the synthesis of the first 10 nucleotides (the DNA region here is called the "initial transcribed sequence" (ITS), and the corresponding transcription complex is called the "initial complex" (IC)). After the removal of the promoter, the IC undergoes a major structural rearrangement and switches to the elongation complex (EC). Compared to the EC, the IC is very unstable and frequently dissociates before switching. As a result, small incomplete transcripts are repeatedly released until the polymerase finally starts productive transcription. Early DNA replication systems appear to have exploited abortive cycles of DNA-dependent RNA polymerases, which existed before the emergence of the "DNAWorld", and the evolution of primitive RNA polymerases could trigger DNA replication. Therefore, abortive cycles may have played an important role in the evolution of the "DNAWorld". In vitro studies on primer-dependent transcription initiation have shown that both eukaryotic and prokaryotic RNA polymerases can use oligonucleotides of varying lengths from 2 to 8 nt to initiate transcription. Abortive transcripts have a transcriptional inhibitory effect. In vitro transcription experiments have shown that the length of abortive transcripts is mainly 2 to 10 nt, with the longest being 19 nt, and is mainly concentrated in 2nt, 4nt, 6nt, and 7nt. T7-RNA polymerase has high transcription efficiency and strong extension ability, but it also has some disadvantages as an in vitro RNA synthesis tool that cannot be ignored. It may produce many by-products in the process of RNA synthesis, including oligonucleotides produced during transcription initiation, interrupted RNA products caused by termination signals, and 3'-terminal extension products caused by RdRp activity (Jain Netal., 2020). The dsRNA of RNA products synthesized in vitro will lead to the activation of innate immunity of RNA drugs after delivery into vertebrates, which is also a key problem to be solved in current RNA targeted therapies. Although the requirements for drug use can be met after multiple steps of purification, the production cost has been greatly increased in large-scale production and synthesis, and the increase in purification processes is not conducive to the stability of RNA drugs. Therefore, the current development of new RNA synthesis tool enzymes to maintain efficient transcription and reduce the proportion of dsRNA has very important application value. In the prior art, the Chinese patent CN112831484B discloses that the 43rd serine from the N-terminus is replaced by an A-type amino acid or a B-type amino acid, and the resulting S43Y mutant can effectively reduce the generation of dsRNA. The P266L mutant reported by Jean Guillerez et al. (Guillerez et al., 2005) can promote the structural conversion of the T7 enzyme from initiation to extension, reduce the generation of aborted RNA, and thus reduce the generation of dsRNA. Summary of the invention

[0003] Technical problem to be solved: In view of the above shortcomings of the prior art, the purpose of the present invention is to provide a T7-RNA polymerase mutant and its application that can reduce the generation of dsRNA. The T7-RNA polymerase mutant can be used for the production of RNA in vitro, and has obvious differences from the existing T7-RNA polymerase, providing an effective candidate enzyme tool for the research and application of RNA.

[0004] Technical solution: A T7-RNA polymerase mutant capable of reducing dsRNA generation, wherein an amino acid change is made in the wild-type T7-RNA polymerase sequence shown in SEQ ID NO: 1, and the nucleotide sequence encoding the above sequence is shown in SEQ ID NO: 2, that is, a mutant in which the tryptophan at position 328 starting from the N-terminus of the amino acid sequence of the wild-type T7-RNA polymerase is replaced by methionine, phenylalanine, asparagine, alanine or lysine. Furthermore, the T7-RNA polymerase mutant is used in in vitro transcription. Furthermore, the T7-RNA polymerase mutant is used in the synthesis of coding RNA for non-therapeutic purposes. Furthermore, the T7-RNA polymerase mutant is used in the synthesis of non-coding RNA for non-therapeutic purposes. Furthermore, the application in the synthesis of non-coding RNA is characterized in that the non-coding RNA is microRNA, siRNA, piRNA, rRNA, tRNA, snRNA, snoRNA, SLRNA, SRPRNA, mRNA-like non-coding RNA, non-coding RNA without a polyA tail, or oligonucleotide. Furthermore, the T7-RNA polymerase mutant is used in gene editing for non-therapeutic purposes. Furthermore, the T7-RNA polymerase mutant is used in the synthesis of RNA drugs. Furthermore, the T7-RNA polymerase mutant is used in an in vivo protein expression system for non-therapeutic purposes or an in vitro translation system for cell-free protein expression. Furthermore, the T7-RNA polymerase mutant is used in the synthesis of biological transcription regulatory elements for non-therapeutic purposes. The present invention uses computer-aided design and, based on kinetic simulation results, analyzes mutation sites that can promote the transition from an initial structural complex (IC) to an extended structural complex (EC). The sites that have been reported to affect transcriptional activity and the sites that are significantly associated with the active sites in the kinetic simulation results are removed, and candidate mutants that can reduce the generation of dsRNA are selected to screen amino acid mutations. Specifically, the tryptophan at position 328 of the wild type is mutated to asparagine (W328N mutant, SEQ ID NO: 3), or the tryptophan at position 328 of the wild type is mutated to cysteine ​​(W328C mutant, SEQ ID NO: 4), the tryptophan at position 328 of the wild type is mutated to aspartic acid (W328D mutant, SEQ ID NO: 5), the tryptophan at position 328 of the wild type is mutated to glycine (W328G mutant, SEQ ID NO: 6), the tryptophan at position 328 of the wild type is mutated to alanine (W328A mutant, SEQ ID NO: 7), the tryptophan at position 328 of the wild type is mutated to histidine (W328H mutant, SEQ ID NO: 8), and the tryptophan at position 328 of the wild type is mutated to cysteine ​​(W328C mutant, SEQ ID NO: 9). NO:8), mutating the tryptophan at position 328 of the wild type to lysine (W328K mutant, SEQ ID NO:9), mutating the tryptophan at position 328 of the wild type to phenylalanine (W328F mutant, SEQ ID NO:10), and mutating the tryptophan at position 328 of the wild type to methionine (W328M mutant, SEQ ID NO:11). By sequencing the mutants and using molecular cloning methods, the mutant genes were inserted into prokaryotic expression vectors and protein expression was performed in Escherichia coli. The wild-type T7-RNA polymerase was used as a control, and the purified enzyme was used for in vitro transcription. The transcription product was precipitated with lithium chloride, and the transcription activity was determined based on the concentration of the transcription product. The dsRNA detection was performed using the Novazon dsRNA detection kit. Screening revealed that the Trp328>Met mutant (indicates that the Trp at position 328 mutated to Met) had a significant effect in reducing dsRNA byproducts, and the proportion of dsRNA produced was much lower than that of the wild-type enzyme. The aborted RNA was detected by HPLC (see the test results). Figure 2 ), compared with the wild-type enzyme transcription product, the aborted RNA generated during the mutant transcription process is significantly reduced, proving that the mutation does promote the structural conversion during the enzyme transcription process. The present invention performs saturation mutation on site 328 and finds that replacing site 328 with phenylalanine, asparagine, alanine or lysine can significantly reduce the generation of dsRNA, but individual mutants require a higher enzyme dosage to ensure that the yield of IVT does not decrease, such as W328A and W328K. Although individual mutants require the use of high-concentration enzymes, the dsRNA production ratio is still significantly lower than that of the wild-type enzyme. The reason for the low yield of individual mutants may also be that the transcription system of the wild-type enzyme is not very consistent with the properties of the mutant enzyme. In the later stage, it is necessary to develop a transcription system suitable for the mutant enzyme to enhance the activity of the mutant enzyme. Most other mutants do not have a significant decrease in transcription activity. Under the same transcription conditions and transcription time, this mutant can generate a purer target product, reducing the difficulty of later purification. Beneficial effects: 1. The present invention reduces a lot of screening work through computer simulation, improves the speed of enzyme evolution, and greatly reduces the cost and time of enzyme modification; 2. The T7-RNA polymerase mutant of the present invention significantly reduces the dsRNA produced during the IVT process while maintaining efficient transcription; 3. The T7-RNA polymerase mutant of the present invention reduces the complexity of the later purification work, reduces the purification cost of RNA production, can quickly prepare RNA drugs that meet the use requirements, and provides an efficient tool enzyme for the large-scale production of RNA drugs. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The figure is the electrophoresis result of RNA products of p19 gene transcription by T7 RNA polymerase mutants and wild-type T7 RNA polymerase, wherein 1 is wild-type T7 RNA polymerase; 2 is P266L mutant; 3 is S43Y mutant; 4 is W328N mutant; 5 is W328C mutant; 6 is W328D mutant; 7 is W328G mutant; 8 is W328A mutant; 9 is W328H mutant; 10 is W328K mutant; 11 is W328F mutant; 12 is W328M mutant. Figure 2 This is a graph showing the results of RNA-HPLC detection of aborted IVT products of wild-type T7-RNA polymerase, P266L mutant, W328F mutant, S43Y mutant, and W328M mutant. DETAILED DESCRIPTION The present invention is further described below in conjunction with the accompanying drawings and embodiments. The following embodiments are explanations of the present invention and the present invention is not limited to the following embodiments: Example 1 Preparation method of T7-RNA polymerase mutant with reduced dsRNA production Expression and purification of T7-RNA polymerase mutants The T7-RNA polymerase mutant expression vector was constructed by molecular cloning. The expression plasmid was pMAL-c4X. A 6×His tag was added to the front of the expression sequence, and the original MBP tag of the plasmid was removed. The prokaryotic expression vector containing these mutants was transformed into the E. coli BL21 (DE3) expression strain, and the bacteria were selected for expansion and culture. The bacteria were placed in LB medium containing 100 μg / ml ampicillin and cultured at 37°C for 3-4 hours. The OD 600 The value was close to 1.0, and then isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.3 mM to induce expression at 16°C for 15 h, followed by centrifugation at 4°C and 8000 rpm for 15 min to collect the cells, wash once in a lysis buffer containing 20 mM Tris-HCl, 100 mM Nacl (pH = 7.9), collect the cells by centrifugation, and resuspend the cells in 5 times w / v of a lysis buffer containing 20 mM Tris-HCl (pH = 7.9) and 100 mM Nacl. The resuspended bacterial solution was crushed in a French press, the pressure was controlled at 900 psi, the dripping speed was controlled to be greater than 2 seconds, and the collection tube was placed in an ice water bath. The crushed liquid was centrifuged at 12000 rpm / min for 30 minutes, and the supernatant was filtered through a syringe filter with a pore size of 1μm, 0.45μm, and 0.22μm, respectively, and the filtered supernatant was purified by nickel column. First, the nickel column was balanced with more than 10 times the volume of the equilibration buffer (20mM Tris-Hcl (PH7.9), 100mM NaCl). The filtered supernatant was added to the balanced nickel column at a loading rate of 1mL / min. After all the protein solution passed through the nickel column, the equilibration buffer was rinsed to the baseline; the buffer containing 50mM imidazole was used for washing at a flow rate of 1mL / min; after washing, 300mM imidazole was added for elution at a flow rate of 1mL / min, and the eluate was collected and concentrated using a Sartorius 30kDa ultrafiltration tube. The concentrated solution was subjected to SDS-PAGE electrophoresis and dyed with a fast staining solution for 20 minutes. After decolorization, the concentration and purity of the concentrated protein were observed with a control protein. The concentrated protein was added to a Sartorius 30kDa ultrafiltration tube replacement buffer. The replacement buffer was 40mM Tris-HCl, 100mM NaCl, 20mMβ-ME, 2mM EDTA, 0.1% (w / v) X-100, pH 7.9. Add one volume of glycerol to the protein solution after replacing the buffer, mix thoroughly and store at -20°C for later use. Acquisition of transcription templates The transcription template was obtained by amplifying the vector already in the laboratory by PCR method. The vector contained T7 promoter and P19 mRNA coding sequence (see SEQ ID NO: 12). The purified PCR product was used as the transcription template. The in vitro transcription reaction was carried out in a 20ul system containing 40mMTris-HCL (PH=8.0), 200nM RNA polymerase, 0.1ul RNase inhibitor, 0.2uL pyrophosphatase, 20ng / uL PCR template, 4mM ATP, GTP, CTP, UTP, 12mM MgCl2, 2mM spermidine, and 2mM DTT. After incubation at 37°C for 2h, 1uL DNAase I was added to digest the template at 37°C for 30min. Comparison of transcription efficiency between T7-RNA polymerase mutants and wild-type T7-RNA polymerase Take 1uLT7-RNA polymerase mutant and add 4uLDEPC water, then add 5uL 2*RNA loading buffer, mix, heat at 80℃ for 2min, and place on ice. Prepare 1.5% agarose gel, run at 190V for 20min, and perform EB detection. The results are as follows: Figure 1 As shown in Figure 2, the T7-RNA polymerase mutant has a more single target band, which means a higher purity target product. Example 2 Comparison of the amount of abortive RNA produced by the transcription products of the W328M mutant, the W328F mutant and the wild-type T7 RNA polymerase The acquisition of transcription template and the in vitro transcription reaction method are the same as in Example 1. The IVT system is 100uL. The IVT product is tested by agarose electrophoresis to detect whether the transcription is normal. The mRNA concentration is adjusted to be consistent and the aborted RNA is detected by HPLC. The aborted RNA produced by IVT is generally 2 to 10 nt in length. The HPLC detection method is as follows: (1) Mobile phase preparation Mobile phase A was 100 mM triethylamine acetate, pH 7.0; mobile phase B was mobile phase A + 25% acetonitrile. (2) Chromatographic conditions The chromatographic column was a reverse phase chromatographic column (C18 column) of Hypersil GOLDTM (4.6X250mm; particle size 5um); the flow rate was 1.0mL / min; the chromatographic column temperature was 60°C; the detection wavelength was 260nm; and the injection volume was 10uL. (3) Separation gradient program Starting: 99% A solution + 1% B solution 2min: 99% A solution + 1% B solution 25.5min: 50% solution A + 50% solution B 26.0min: 0% solution A + 100% solution B 31.0min: 0% solution A + 100% solution B 31.5min: 99% A solution + 1% B solution 45.0min: 99% A solution + 1% B solution (4) Analysis of abortion RNA detection results Abortion RNA test results Figure 2 As shown, according to the test results, Yangshen P226L can significantly reduce the production of abortion RNA, Yangshen S43Y mutant cannot reduce the production of abortion RNA, W328M mutant and W328F mutant can significantly reduce the production of abortion RNA during IVT. Example 3 Comparison of dsRNA production during transcription of p19 gene by wild-type T7-RNA polymerase and mutants The acquisition of transcription template and the in vitro transcription reaction method are the same as in Example 1. The dsRNA detection adopts the Novazon dsRNA (modified) quantitative detection kit (enzyme-linked immunosorbent assay) 2.0, item number DD3509-01. For specific experimental operations, please refer to the kit instructions. With the wild-type T7-RNA polymerase as a reference, the S43Y mutant and the P266L mutant are double yang ginseng, three IVTs are parallel for each T7 mutant, and each parallel is tested three times. The sample is diluted to the same detection concentration, and the experiment is repeated three times. The results of the three tests were consistent, indicating that the W328M, W328F, W328N, W328A, and W328K mutant enzymes can reduce the amount of dsRNA generated to varying degrees. The dsRNA generated by the W328M mutant is 50-60pg / ug, which is lower than the 60-70pg / ug of Yangshen S43Y. The dsRNA generated by the W328F, W328N, W328K, and W328A mutants is 100-170pg / ug, which is higher than the 60-70pg / ug of Yangshen S43Y mutant and lower than the Yangshen P266L mutant (153-168pg / ug). The dsRNA generated by the five T7-RNA polymerase mutants protected by this patent during the IVT process is significantly lower than that of the wild-type T7-RNA polymerase (341-400pg / ug). The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A T7-RNA polymerase mutant capable of reducing dsRNA production, characterized in that: A mutant in which the tryptophan at position 328 starting from the N-terminus of the amino acid sequence of the wild-type T7-RNA polymerase shown in SEQ ID NO.1 is replaced by methionine, phenylalanine, asparagine, alanine or lysine.

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

3. Use of the T7-RNA polymerase mutant according to claim 1 in the synthesis of coding RNA for non-therapeutic purposes.

4. Use of the T7-RNA polymerase mutant according to claim 1 in the synthesis of non-coding RNA for non-therapeutic purposes.

5. The use in non-coding RNA synthesis according to claim 4, characterized in that: The non-coding RNA is microRNA, siRNA, piRNA, rRNA, tRNA, snRNA, snoRNA, SLRNA, SRPRNA, mRNA-like non-coding RNA, non-coding RNA without a polyA tail, or oligonucleotide.

6. Use of the T7-RNA polymerase mutant according to claim 1 in gene editing for non-therapeutic purposes.

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

8. Use of the T7-RNA polymerase mutant according to claim 1 in an in vivo protein expression system for non-therapeutic purposes or a cell-free protein expression in vitro translation system.

9. Use of the T7-RNA polymerase mutant according to claim 1 in the synthesis of biological transcription regulatory elements for non-therapeutic purposes.

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