A t7-rna polymerase mutant capable of reducing dsrna production and uses thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nucleic acid tools and nucleic acid organisms, and particularly relates to a T7-RNA polymerase mutant capable of reducing the generation of dsRNA and application thereof. BACKGROUND

[0002] T7-RNA polymerase is a single subunit protein of 99 kDa (883 amino acids) found in bacteriophage T7. Like multi-subunit RNA polymerases (RNAPs), T7 RNAP often releases its transcript over 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 rate of transcription, it is the rate-limiting step for efficient transcription, and is partly responsible for the generation of dsRNA. In vivo, the transcription of target genes by T7 RNAP is effectively abolished due to the large number of abortive cycles.

[0003] Unlike multi-subunit RNA polymerases (RNAPs), the monomeric RNAP from bacteriophage T7, upon recognition of its cognate promoter, creates an "open complex" by locally unwinding the DNA and uses a single nucleotide to initiate template-directed RNA synthesis. During the synthesis of the first 10 nucleotides, the RNAP maintains contact with the promoter (the region of DNA is referred to as the "initial transcription sequence" (ITS) and the corresponding transcription complex is referred to as the "initial complex" (IC)). Upon removal of the promoter, the IC undergoes a major structural rearrangement, transitioning to the elongation complex (EC). In comparison to the EC, the IC is very unstable and often 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 exploit the abortive cycle of DNA-dependent RNA polymerases, which pre-dates the "DNA World" and the evolution of the original RNA polymerases can have triggered the replication of DNA. Thus, the abortive cycle can have played an important role in the evolution of the "DNA World". In vitro studies on primer-dependent transcription initiation have shown that RNA polymerases from both eukaryotes and prokaryotes can use oligonucleotides of varying lengths from 2 to 8 nt to initiate transcription. Abortive transcripts have a transcriptional repressive effect. In vitro transcription experiments have shown that the length of abortive transcripts is mainly 2-10 nt, with the longest being 19 nt, and is mainly concentrated at 2 nt, 4 nt, 6 nt and 7 nt.

[0004] T7-RNA polymerase has high transcription efficiency and strong elongation ability, but it also has some non-negligible shortcomings as a tool for in vitro RNA synthesis. It can produce many by-products during RNA synthesis, including oligonucleotides produced during transcription initiation, interrupted RNA products caused by termination signals, 3' end extension products caused by RdRp activity, etc. (Jain N et al., 2020). dsRNA of in vitro synthesized RNA products can cause the activation of innate immunity after RNA drugs are delivered into vertebrates, which is a key problem that needs to be solved for current RNA targeted therapy. Although multiple purification steps can meet the requirements of drug use, they increase the production cost and are not conducive to the stability of RNA drugs. Therefore, it is of great application value to develop new RNA synthesis tool enzymes that maintain high transcription efficiency while reducing the proportion of dsRNA.

[0005] In the prior art, the serine at position 43 from the N terminus of the wild-type T7-RNA polymerase is replaced by a class A or class B amino acid to obtain a S43Y mutant, which can effectively reduce the generation of dsRNA. Jean Guillerez et al. reported a P266L mutant (Guillerez et al., 2005), which can promote the structural transition of T7 enzyme from initiation to elongation, reduce the generation of abortion RNA, and thus reduce the generation of dsRNA. SUMMARY

[0006] The technical problem to be solved is that the prior art has the above-mentioned deficiencies. The purpose of the present application is to provide a T7-RNA polymerase mutant capable of reducing the generation of dsRNA and its application. The T7-RNA polymerase mutant can be used for in vitro RNA production, which is significantly different from the existing T7-RNA polymerase, and provides an effective candidate enzyme tool for RNA research and application.

[0007] Technical solution: A T7-RNA polymerase mutant capable of reducing the generation of dsRNA, which is obtained by changing the amino acid in the wild-type T7-RNA polymerase sequence shown in SEQ ID NO: 1. The nucleotide sequence encoding the above-mentioned sequence is shown in SEQ ID NO: 2, i.e. the tryptophan at position 328 from the N terminus of the wild-type T7-RNA polymerase amino acid sequence is replaced by methionine, phenylalanine, asparagine, alanine or lysine.

[0008] Further, the T7-RNA polymerase mutant is used in in vitro transcription.

[0009] Further, the T7-RNA polymerase mutant is used in non-therapeutic RNA synthesis.

[0010] Further, the application provides the T7-RNA polymerase mutant for use in non- therapeutic purposes in non-coding RNA synthesis.

[0011] Further, the application provides the T7-RNA polymerase mutant for use in non- therapeutic purposes in non-coding RNA synthesis, wherein the non-coding RNA is microRNA, siRNA, piRNA, rRNA, tRNA, snRNA, snoRNA, S RNA, SRP RNA, mRNA-like non-coding RNA, non-polyA-tailed non-coding RNA or oligonucleotide.

[0012] Further, the application provides the T7-RNA polymerase mutant for use in non- therapeutic purposes in gene editing.

[0013] Further, the application provides the T7-RNA polymerase mutant for use in non- therapeutic purposes in RNA drug synthesis.

[0014] Further, the application provides the T7-RNA polymerase mutant for use in non- therapeutic purposes in in vivo protein expression or in vitro translation system of cell-free protein expression.

[0015] Further, the application provides the T7-RNA polymerase mutant for use in non- therapeutic purposes in synthesis of biological transcriptional regulatory element.

[0016] The present application analyzes the mutation sites that can promote the transition from the initial structure complex (IC) to the extended structure complex (EC) according to the results of the kinetic simulation by computer-aided design. In addition to the sites that have been reported to affect the transcription activity and the sites that are significantly associated with the active sites in the kinetic simulation results, the candidate mutants that can reduce the generation of dsRNA are selected to screen the amino acid mutations. Specifically, the wild-type tryptophan at position 328 is mutated to asparagine (W328N mutant, SEQ ID NO: 3), or the wild-type tryptophan at position 328 is mutated to cysteine (W328C mutant, SEQ ID NO: 4), the wild-type tryptophan at position 328 is mutated to aspartic acid (W328D mutant, SEQ ID NO: 5), the wild-type tryptophan at position 328 is mutated to glycine (W328G mutant, SEQ ID NO: 6), the wild-type tryptophan at position 328 is mutated to alanine (W328A mutant, SEQ ID NO: 7), the wild-type tryptophan at position 328 is mutated to histidine (W328H mutant, SEQ ID NO: 8), the wild-type tryptophan at position 328 is mutated to lysine (W328K mutant, SEQ ID NO: 9), the wild-type tryptophan at position 328 is mutated to phenylalanine (W328F mutant, SEQ ID NO: 10), and the wild-type tryptophan at position 328 is mutated to methionine (W328M mutant, SEQ ID NO: 11). The mutant genes are inserted into a prokaryotic expression vector by sequencing the mutants and by the method of molecular cloning, and the protein expression is performed in E. coli.

[0017] The wild-type T7-RNA polymerase is used as a control for in vitro transcription, and the transcription product is precipitated with lithium chloride. The transcription activity is determined according to the concentration of the transcription product, and the dsRNA detection is performed using the Nuclisens dsRNA detection kit. It is found through screening that the Trp328>Met mutant (indicating that the Trp at position 328 is mutated to Met) has a significant effect of reducing the dsRNA byproduct, and the proportion of the generated dsRNA is much lower than that of the wild-type enzyme. The HPLC detection of the abortive RNA (the detection results are shown in Table 1) shows that the Trp328>Met mutant has a significant effect of reducing the dsRNA byproduct. 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 indeed promotes the structural conversion during the enzyme transcription process. The present application performs saturation mutation on the 328 site, and finds that replacing the 328 site with phenylalanine, asparagine, alanine or lysine can significantly reduce the generation of dsRNA, but individual mutants require higher enzyme dosage to ensure that the IVT yield does not decrease, such as W328A and W328K. Although individual mutants require the use of high-concentration enzymes, the proportion of dsRNA generation is still significantly lower than that of wild-type enzymes. The reason for the low yield of individual mutants may be that the transcription system of wild-type enzymes is not fully compatible with the properties of mutant enzymes, and a transcription system suitable for mutant enzymes needs to be developed in the later stage to enhance the activity of mutant enzymes. While most of the other mutants do not have a significant decrease in transcription activity. Under the same transcription conditions and transcription time, this mutant can generate purer target product, reducing the difficulty of later purification.

[0018] Beneficial effects:

[0019] 1. The present application reduces a large amount of screening work through computer simulation, improves the speed of enzyme evolution, and greatly reduces the cost and time of enzyme modification;

[0020] 2. The T7-RNA polymerase mutant of the present application can significantly reduce the dsRNA generated during IVT while maintaining high transcription efficiency;

[0021] 3. The T7-RNA polymerase mutant of the present application reduces the complexity of the later purification work, reduces the purification cost of RNA production, and can quickly prepare RNA drugs that meet the use requirements, providing a high-efficiency tool enzyme for large-scale production of RNA drugs. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Figure 1 is an electrophoresis result diagram of RNA products of T7 RNA polymerase mutant and wild type T7 RNA polymerase transcribing p19 gene, 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.

[0023] Figure 2 Figure 3 is a HPLC detection result diagram of IVT product aborted RNA of wild type T7-RNA polymerase, P266L mutant, W328F mutant, S43Y mutant and W328M mutant. DETAILED DESCRIPTION

[0024] The application will be further described in conjunction with the accompanying drawings and examples. The following examples are illustrative of specific embodiments of the application, but the application is not to be limited to the embodiments.

[0025] Example 1

[0026] Method for preparing T7-RNA polymerase mutants for reducing dsRNA production

[0027] Expression and purification of T7-RNA polymerase mutants

[0028] The expression vector of T7-RNA polymerase mutants was constructed by molecular cloning. The expression plasmid was pMAL-c4X, and a 6xHis tag was added at the front of the expression sequence, and the original MBP tag of the plasmid was removed. The prokaryotic expression vector containing the mutants was transformed into E. coli BL21 (DE3) expression strain, and the bacteria were picked and cultured. The bacteria were placed in LB medium containing 100 μg / ml ampicillin, and cultured at 37°C for 3-4 hours. When the OD value was close to 1.0, isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.3 mM to induce expression at 16°C for 15 hours. Then the bacteria were collected by centrifugation at 4°C and 8000 rpm for 15 min, washed once in a lysis buffer containing 20 mM Tris-Hcl and 100 mM Nacl (pH = 7.9), and the bacteria were collected by centrifugation, and the bacteria were resuspended in a lysis buffer containing 20 mM Tris-Hcl (pH = 7.9) and 100 mM Nacl at 5 times w / v. 600

[0029] The resuspended bacterial solution was crushed in a French press, with a pressure control of 900 psi and a control of the drop speed interval of more than 2 seconds, and the collection tube was in ice water bath. The crushed solution was centrifuged at high speed for 30 min at 12000 rpm / min, and the supernatant was filtered through a needle filter with a pore size of 1 μm, 0.45 μm and 0.22 μm, respectively. The filtered supernatant was purified by nickel column.

[0030] First, the nickel column was equilibrated with more than 10 volumes of equilibration buffer (20 mM Tris-Hcl (pH 7.9), 100 mM Nacl). The filtered supernatant was added to the equilibrated nickel column at a speed of 1 mL / min. After all the protein solution passed through the nickel column, the equilibration buffer was washed to the baseline. The buffer containing 50 mM imidazole was used for washing, and the flow rate was 1 mL / min. After washing, 300 mM imidazole was added for elution, and the flow rate was 1 mL / min. The eluate was collected and concentrated using a 30 kDa ultrafiltration tube from Sartorius.

[0031] ​The concentrated solution was subjected to SDS-PAGE electrophoresis, stained with a fast staining solution for 20 min, and then destained. The concentration and purity of the concentrated protein were observed by comparing with a protein marker. The concentrated protein was subjected to buffer exchange using a Sartorius 30 kDa ultrafiltration tube. The buffer exchange buffer was 40 mM Tris-HCl, 100 mM NaCl, 20 mM β-ME, 2 mM EDTA, 0.1% (w / v) X-100, pH 7.9. The protein solution after buffer exchange was mixed with an equal volume of glycerol, and then stored at -20°C for later use.

[0032] Obtaining of a transcription template

[0033] The transcription template was obtained by amplifying a vector available in the laboratory by PCR. The vector contained a T7 promoter and a P19 mRNA coding sequence (see SEQ ID NO: 12). The purified PCR product was used as a transcription template. The in vitro transcription reaction was performed in a 20 ul system containing 40 mM Tris-HCL (pH = 8.0), 200 nM RNA polymerase, 0.1 ul RNAase inhibitor, 0.2 uL pyrophosphatase, 20 ng / uL PCR template, 4 mM ATP, GTP, CTP, UTP, 12 mM MgCl2, 2 mM spermidine, 2 mM DTT. After incubation at 37°C for 2 h, 1 ul of DNAase I was added to digest the template at 37°C for 30 min.

[0034] 1 ul of T7-RNA polymerase mutant was added to 4 uL DEPC water, followed by the addition of 5 uL 2*RNA loading buffer. After mixing, the mixture was heated at 80°C for 2 min, and then placed on ice. A 1.5% agarose gel was prepared, and electrophoresis was performed at 190 V for 20 min. EB detection was performed, and the results are shown in Figure 1 The T7-RNA polymerase mutant had a single band of interest, i.e., a higher purity of the product of interest.

[0035] Example 2

[0036] Comparison of the amounts of aborted RNA generated by the W328M mutant, the W328F mutant and wild-type T7 RNA polymerase transcription products

[0037] The transcription template was obtained and the in vitro transcription reaction was performed as in Example 1. The IVT system was 100 uL. The IVT product was subjected to agarose electrophoresis to detect whether transcription was normal. The mRNA concentration was adjusted to be consistent, and the aborted RNA was detected by HPLC. The length of the aborted RNA generated by IVT was generally 2-10 nt. The HPLC detection method is as follows:

[0038] (1) Preparation of a mobile phase

[0039] Mobile phase A is 100 mM triethylamine acetate, pH 7.0; mobile phase B is mobile phase A + 25% acetonitrile.

[0040] (2) Chromatographic conditions

[0041] The chromatographic column was a Hypersil GOLD™ (4.6 x 250 mm; particle size 5 μm) reversed-phase column (C18 column); the flow rate was 1.0 mL / min; the column temperature was 60 °C; the detection wavelength was 260 nm; and the injection volume was 10 μL.

[0042] (3) Gradient separation procedure

[0043] Starting solution: 99% solution A + 1% solution B

[0044] 2 min: 99% solution A + 1% solution B

[0045] 25.5 min: 50% solution A + 50% solution B

[0046] 26.0 min: 0% solution A + 100% solution B

[0047] 31.0 min: 0% solution A + 100% solution B

[0048] 31.5 min: 99% solution A + 1% solution B

[0049] 45.0 min: 99% solution A + 1% solution B

[0050] (4) Analysis of abortion RNA detection results

[0051] The results of the miscarriage RNA test are as follows Figure 2 As shown in the results, the Yangshen P226L mutant can significantly reduce the production of abortion RNA, while the Yangshen S43Y mutant cannot reduce the production of abortion RNA. The W328M and W328F mutants can significantly reduce the production of abortion RNA during IVT.

[0052] Example 3

[0053] Comparing the production of dsRNA during p19 gene transcription by wild-type T7-RNA polymerase and mutants.

[0054] The transcription template was obtained and the in vitro transcription reaction method was the same as in Example 1. dsRNA detection was performed using the Nanjing dsRNA (modified) quantitative detection kit (enzyme-linked immunoassay) 2.0, item number DD3509-01. For specific experimental operations, refer to the kit instructions. Using wild-type T7-RNA polymerase as a reference, S43Y mutant and P266L mutant as double positive controls, three IVT parallels were performed for each T7 mutant, and each parallel was detected three times. The samples were diluted to the same detection concentration, and the experiment was repeated three times. The results of the three detections 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 was 50-60 pg / ug, which was lower than the 60-70 pg / ug of the positive control S43Y. The dsRNA generated by the W328F, W328N, W328K, and W328A mutants was 100-170 pg / ug, which was higher than the 60-70 pg / ug of the S43Y mutant and lower than the 153-168 pg / ug of the P266L mutant. The amount of dsRNA generated during the IVT process of the five T7-RNA polymerase mutants protected by the present patent was significantly lower than that of the wild-type T7-RNA polymerase (341-400 pg / ug).

[0055] The above description is only the preferred embodiment of the present application, and does not limit the present application in any form. Any person skilled in the art can make many possible changes and modifications to the technical solutions of the present application, or modify equivalent embodiments, without departing from the spirit and technical solutions of the present application, using the methods and technical contents disclosed above. Therefore, any simple modification, equivalent replacement, equivalent change and modification of the above embodiments made in accordance with the technical essence of the present application, without departing from the technical solutions of the present application, still falls within the scope of protection of the technical solutions of the present application.

Claims

1. A mutant of T7-RNA polymerase capable of reducing dsRNA production, characterized in that: A mutant in which the tryptophan at position 328 from the N-terminus of the amino acid sequence of wild-type T7-RNA polymerase shown in SEQ ID NO. 1 is substituted with 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. Use according to claim 4, characterized in that: The non-coding RNA is microRNA, siRNA, piRNA, rRNA, tRNA, snRNA, snoRNA, SIRNA, SRPRNA, mRNA-like non-coding RNA.

6. Use according to claim 4, characterized in that: The non-coding RNA is non-coding RNA without polyA tail.

7. Use according to claim 4, characterized in that: The non-coding RNA is oligonucleotide.

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

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

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

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

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