T7 RNA polymerase mutant capable of not generating immunogenic by-product and having high transcriptional activity, and use thereof

Abstract

Provided are a T7 RNA polymerase mutant capable of not generating an immunogenic by-product and having high transcriptional activity, and the use thereof, belonging to the technical field of biology. The amino acid sequence of the T7 RNA polymerase mutant is as shown in SEQ ID NO: 2. The T7 RNA polymerase mutant which is constructed by transformation of an original T7 RNA polymerase generates much less immunogenic RNAs than wild-type T7 RNA polymerases during a transcription phase, thus simplifying nucleic acid production processes, reducing the content of immunogenic by-products generated during the nucleic acid production processes, and shortening the time for synthesizing nucleic acids. The T7 RNA polymerase mutant has wide application prospects in the fields of RNA vaccines and drugs, in-vitro transcription, gene editing, virus RNA probe detection, etc.

Description

A T7 RNA polymerase mutant that does not produce immunogenic byproducts and possesses high transcriptional activity and its applications. Technical Field

[0001] This invention relates to a T7 RNA polymerase mutant that does not produce immunogenic byproducts and has high transcriptional activity and its applications, belonging to the field of biotechnology. Background Technology

[0002] In recent years, there has been a growing demand for research on viral genetic information RNA and the development of vaccines such as mRNA. Meanwhile, small-molecule nucleic acids offer more advantages than large-molecule biological agents and small-molecule drugs as vaccines or drugs. They can be used not only for the prevention of infectious diseases, the treatment of tumors, and protein replacement therapy, but also for standardized manufacturing processes. Currently, the main sources of mRNA are chemical synthesis and enzymatic synthesis. However, chemical synthesis requires a large number of RNA molecules of specific length and sequence as raw materials, and the cost of chemical synthesis increases dramatically with the increase of nucleotide length. When the number of nucleotides exceeds one hundred, chemical synthesis becomes unsuitable. Protein-encoding mRNA typically contains several thousand nucleotides; therefore, enzymatic synthesis is currently the only method for preparing long-chain RNA. The T7 RNA polymerase in vitro transcription system provides a rapid method for synthesizing pure, single-stranded RNA molecules, catalyzing the formation of RNA in the 5′–3′ direction. This enzyme has high promoter specificity and only transcribes DNA or DNA replicas downstream of the T7 promoter in bacteriophages. In vivo, T7 RNA polymerase can almost completely transcribe all DNA sequences downstream of the T7 promoter. However, various byproducts are generated during transcription, including immunogenic double-stranded RNAs (dsRNAs) transcribed from the product template; these byproducts can severely affect drug efficacy and safety. The generation of dsRNA and other byproducts is influenced by the conformation of T7 RNA polymerase in the catalytic cycle. To ensure high mRNA purity for repeated or long-term therapeutic applications and reduce nucleic acid degradation, quality loss, or yield reduction during purification steps, a more efficient synthesis process is needed to reduce the impurity burden in downstream purification steps. Therefore, the development of novel RNA synthesis enzymes that can maintain efficient transcriptional activity while reducing the heterogeneity of byproducts such as dsRNA has significant application value.

[0003] Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a T7 RNA polymerase mutant that does not produce immunogenic byproducts and has high transcriptional activity, along with its applications. This T7 RNA polymerase mutant significantly reduces dsRNA transcription byproducts while maintaining RNA yield and purity, reducing the burden on downstream purification processes and controlling transcription-related dsRNA impurities.

[0005] To achieve the above objectives, the technical solution of the present invention includes:

[0006] In a first aspect, the present invention provides a T7 RNA polymerase mutant, wherein the mutated amino acid of the T7 RNA polymerase mutant is one or a combination of two or more of I14K, A18E, K53E, R57Y, A83K, D87E, E90K, R96T, M190T, E249S, K332Q, N410Q, A491G, V554T, A558V, E565D, A584H, I587T, K614R, L719E, K765E or A822V.

[0007] The T7 RNA polymerase mutant of the present invention is obtained by mutating the wild-type T7 RNA polymerase shown in SEQ ID NO: 1. The T7 RNA polymerase mutant is obtained by mutating one or more combinations of the above-mentioned amino acids.

[0008] Furthermore, the amino acid sequence of the T7 RNA polymerase mutant is shown in SEQ ID NO: 2.

[0009] The T7 RNA polymerase mutant of the present invention can be used for in vitro RNA synthesis. Compared with wild-type T7 RNA polymerase, its transcriptional synthesis activity of modified mRNA is significantly improved, and the content of byproduct (dsRNA) is significantly reduced. It can effectively solve the technical problems of high cost, cumbersome steps and low yield in the preparation of nucleic acids. In addition, the T7 RNA polymerase mutant provided by the present invention is fused with a His tag, which greatly facilitates the subsequent protein purification steps. Therefore, the T7 RNA polymerase mutant provided by the present invention can be applied to the large-scale industrial production of modified mRNA.

[0010] In a second aspect, the present invention provides a nucleotide sequence encoding the T7 RNA polymerase mutant, the nucleotide sequence being shown in SEQ ID NO: 3.

[0011] Thirdly, the present invention provides a recombinant plasmid containing the T7 RNA polymerase mutant.

[0012] Furthermore, the recombinant plasmid is expressed using a pET series vector.

[0013] Furthermore, the pET series vectors include pET-3a(+), pET-21a(+), pET-28a(+), or pET-30a(+).

[0014] Fourthly, the present invention provides recombinant microbial cells expressing the T7 RNA polymerase variant or containing the nucleotide sequence described above.

[0015] Furthermore, the recombinant microbial cells are derived from Escherichia coli, Bacillus subtilis, or Pichia pastoris.

[0016] Fifthly, the present invention provides the application of T7 RNA polymerase mutants in in vitro transcription and synthesis of RNA. Beneficial effects:

[0017] Compared with the prior art, the technical solution of this application has the following advantages: (1) The T7 RNA polymerase mutant provided by this invention is fused with a His tag, which can obtain high-purity protein through one-step nickel column purification, reduce the purification process, and increase the total amount of protein. (2) In the application of in vitro transcription synthesis of mRNA, the T7 RNA polymerase mutant provided by this invention has the advantages of high transcription efficiency, less by-product production, high synthesis yield, high purity, strong specificity, and good storage stability compared with wild-type T7 RNA polymerase. It can achieve the purpose of accurate, simple, low-cost and efficient and stable purification of mRNA, and has important application value in mRNA-related vaccine and drug research. Attached Figure Description

[0018] Figure 1 shows the purification results of the T7 RNA polymerase mutant. In the figure, M represents the protein molecular weight standard, 1 represents the flow-through buffer, 2 represents the washing buffer, 3 represents the elution buffer, and 4 represents the elution with 1M imidazole.

[0019] Figure 2 shows the condensation results of the T7 RNA polymerase mutant. In the figure, M represents the protein molecular weight standard, and 1 represents the T7 RNA polymerase mutant.

[0020] Figure 3 shows the amplification results of the cDNA template. In the figure, M represents the protein molecular weight standard and 1 represents the cDNA template.

[0021] Figure 4 shows the transcription results of the T7 RNA polymerase mutant. In the figure, M represents the protein molecular weight standard, and 1 represents the transcription of RNA.

[0022] Figure 5 shows the standard curves for detecting the activity of wild-type and T7 RNA polymerase mutants.

[0023] Figure 6 shows the standard curve of the dsRNA byproduct detection kit.

[0024] Figure 7 shows a comparison of byproduct results between wild-type and T7 RNA polymerase mutant. Detailed Implementation

[0025] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0026] Example 1

[0027] (1) Codon optimization and plasmid construction of T7 RNA polymerase mutant

[0028] The modified mutant did not produce immunogenic byproducts during transcription and possessed extremely high transcriptional activity. Codon optimization and full-gene synthesis of the T7 RNA polymerase mutant were performed at Sangon Biotech (Shanghai) Co., Ltd. A 6×His restriction enzyme was designed at the N-terminus of the T7 RNA polymerase mutant, and the mutant was ligated into the *Escherichia coli* expression vector pET-28a(+) via double digestion at BamHI and NdeI restriction sites to obtain a plasmid containing the target gene fragment. The plasmid construct was transformed into *E. coli* strain BL21 for preservation.

[0029] (2) Expression and purification of T7 RNA polymerase mutant

[0030] The plasmid containing the target gene fragment was transformed into Escherichia coli strain BL21 and cultured in LB medium containing kanamycin at 180 rpm and 37°C until OD was reached. 600After reaching a pH of 0.6 to 1.2, 0.5 mM isopropyl thiogalactoside was added, and protein expression was induced for 12 h at 180 rpm and 20 °C. The bacterial pellet was collected by centrifugation at 4 °C and 6000 rpm for 20 min. The culture medium was discarded, and the pellet was resuspended in protein binding buffer (pH 8.0, 50 mM Tris-HCl, 0.1 M NaCl) to obtain a supernatant containing the target protein. The bacterial pellet was thoroughly lysed by low-temperature (ice bath) sonication throughout the process. The T7 RNA polymerase mutant protein was purified based on the affinity between the His tag and the Ni-NTA agarose gel purification medium. A 5 ml nickel column was connected to a protein purification instrument to purify the His-labeled T7 RNA polymerase. First, equilibrate the nickel column with 10 column volumes of wash buffer (corresponding to the wash buffer in Figure 1; pH 8.0, 50 mM Tris-HCl, 0.1 M NaCl). After centrifuging the lysed turbid solution at 12000 rpm for 30 min, load the supernatant containing the target protein through the nickel column packing material. Then, equilibrate the nickel column with wash buffer (corresponding to the elution buffer in the figure; pH 8.0, 50 mM Tris-HCl, 0.1 M NaCl). Next, elute the T7 RNA polymerase from the column with imidazole solutions of different concentration gradients (0 M-1 M), and collect the liquid according to the elution order. Identify the collected proteins by SDS-PAGE electrophoresis. Based on the electrophoresis results, select proteins with high purity and concentration for dialyzing into imidazole-free buffer and store at -80°C.

[0031] The protein expression and purification results are shown in Figure 1. First, the protein was purified by nickel ion affinity chromatography. The SDS-PAGE results showed that the bands of the elution buffer rich in the target protein were relatively simple, indicating that the protein purity was high.

[0032] The protein concentration results are shown in Figure 2. The protein samples purified by Ni column were collected, dialyzed into a stabilization buffer, and the protein samples were detected by SDS-PAGE. The electrophoresis results showed that the purity of the T7 RNA polymerase mutant reached more than 95%.

[0033] The purified protein of this invention exhibits a single, uncontaminated protein band, resulting in efficient expression and high yield.

[0034] The amino acid sequence of the obtained T7 RNA polymerase mutant is shown in SEQ ID NO: 2, and the nucleotide sequence encoding the T7 RNA polymerase mutant is shown in SEQ ID NO: 3.

[0035] (3) Transcription reaction

[0036] 1) Template acquisition - Design universal primers, primer sequences are:

[0037] F:5'-AGATCTCGATCCCGCGAAATTA-3'

[0038] R:5”-GCTCAGCGGTGGCAGCAGC-3”

[0039] 2) Template sequence

[0040] PCR amplification technology is used to amplify the target DNA fragment. The target fragment, along with the 5' and 3' primers, undergoes a three-step reaction of "high-temperature denaturation-low-temperature annealing-primer extension" for 25-33 cycles. The number of DNA fragments increases exponentially, thus obtaining the desired target gene in a short time. The PCR instrument settings are shown in Table 1.

[0041] After the PCR reaction of the target gene, electrophoresis was performed using a 1% agarose gel. The electrophoresis apparatus was set to 110V for 21 minutes. Electrophoresis was considered complete when the marker bands had completely separated, and the bands were observed under UV light. If the target band matched the molecular weight of the target gene, the band was cut off with a clean, sterilized blade and placed in a sterile EP tube.

[0042] Table 1 PCR instrument setup procedure

[0043] The target gene was recovered using a gel extraction kit, following the instructions in the kit's manual. The target gene was then eluted with 25 μL of preheated (65°C) elution buffer. The eluted sample was labeled and stored at -20°C for later use.

[0044] The results are shown in Figure 3. After the PCR reaction was completed, the PCR products were examined by 1% agarose gel electrophoresis. A clear band was detected at a position of about 328 bp. The size of the band was consistent with the molecular weight of the template, which initially indicated that the cDNA template was successfully amplified. The target band was cut off under UV light and the gel was recovered.

[0045] 3) The transcription reaction system is as follows:

[0046] Table 2 Transcription reaction system

[0047] Reaction conditions: 37℃ for 2 hours.

[0048] The transcription results are shown in Figure 4. After the transcription reaction was completed, the transcription products were examined by 1% agarose gel electrophoresis. The transcribed RNA bands were clearly visible between 100bp and 300bp, indicating that the transcription was successful and that the T7 RNA polymerase mutant had transcriptional activity.

[0049] 4) Stability test

[0050] Open the website: http: / / tm.life.nthu.edu.tw / , enter the amino acid sequence, and click confirm to obtain the result.

[0051] Table 3 Stability Comparison

[0052] The results are shown in Table 3. The stability of the mutant T7 RNA polymerase was increased by 0.18% compared with the wild type, which greatly improved the stability of the wild-type T7 RNA polymerase (amino acid sequence shown in SEQ ID NO.1).

[0053] 5) Activity detection

[0054] Activity detection mainly uses PhosphoWorks TM The Fluorimetric Pyrophosphate Assay Kit*Blue Fluorescence* (AAT BIO quest, 21611) is used for detection. The detection principle is as follows: a double-stranded DNA template containing the T7 promoter sequence is transcribed by T7 RNA polymerase in the presence of A / U / C / GTP, synthesizing an RNA chain and producing pyrophosphate (PPi). This kit provides the most powerful spectrophotometric method for measuring pyrophosphate. By adding a PPi sensor, the fluorescence intensity is directly proportional to the PPi yield. Since the PPi yield is positively correlated with the activity of T7 RNA polymerase, its activity can be determined using the fluorescence intensity of the PPi sensor (refer to the kit instructions for specific detection procedures).

[0055] The results are shown in Table 4 and Figure 5, based on PhosphoWorks. TM The Fluorimetric Pyrophosphate Assay Kit*Blue Fluorescence* (AAT BIO quest, 21611) was used to create a standard curve for PPi and accurately measure the amount of PPi produced by T7 RNA polymerase. The yield of PPi was positively correlated with the activity of T7 RNA polymerase. The fluorescence results showed that the activity of the T7 RNA polymerase mutant was higher than that of the wild-type T7 RNA polymerase, indicating that the T7 RNA polymerase mutant has higher activity.

[0056] 6) Detection of dsRNA byproducts generated during transcription

[0057] The detection of dsRNA byproducts mainly relies on the dsRNA Quantitative Detection Kit (ELISA method) (manufacturer: Hanhai New Enzyme). This kit utilizes a double-antibody sandwich method coupled with a biotin-streptavidin system to quantitatively detect the content of double-stranded RNA (dsRNA) in samples. Anti-dsRNA antibodies are coated in the microwells of the ELISA plate. After adding the sample and incubating and washing, biotinylated detection antibodies are added for further incubation, forming an antibody-antigen-antibody complex. After washing again, horseradish peroxidase (HRP)-labeled streptavidin (SA) is added. After thorough washing, the substrate TMB (3,3',5,5'-tetramethylbenzidine) is added for color development. TMB is converted to blue under the catalysis of peroxidase, and then to yellow after acid termination. The color intensity is positively correlated with the dsRNA content in the sample. The absorbance (OD value) is measured at 450 nm using an ELISA reader, and the dsRNA concentration in the sample is calculated based on the standard curve (refer to the kit instructions for specific detection steps).

[0058] Table 4 Comparison of T7 RNA polymerase activity assays

[0059] The results are shown in Table 5, Figure 6, and Figure 7. A standard curve for dsRNA was constructed using the dsRNA quantification kit (ELISA method), and the amount of dsRNA in the transcription product was measured. As shown in Figure 6, the higher the dsRNA yield, the yellower the color. Observation of the color after the reaction indicates that the T7 RNA polymerase mutant basically does not produce dsRNA. Combined with the comparison of the measured fluorescence value, the transcription product was diluted by different factors. The measured value of the diluted sample was close to the 0 value. This result proves that the T7 RNA polymerase mutant basically does not produce the transcription byproduct dsRNA after the transcription reaction.

[0060] Table 5 Comparison of dsRNA content, a byproduct of transcription reaction.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A T7 RNA polymerase mutant, characterized in that, The mutated amino acids of the T7 RNA polymerase mutant are one or more of the following: I14K, A18E, K53E, R57Y, A83K, D87E, E90K, R96T, M190T, E249S, K332Q, N410Q, A491G, V554T, A558V, E565D, A584H, I587T, K614R, L719E, K765E, or A822V.

2. The T7 RNA polymerase mutant according to claim 1, characterized in that, The amino acid sequence of the T7 RNA polymerase mutant is shown in SEQ ID NO:

2.

3. A nucleotide sequence encoding the T7 RNA polymerase mutant of claim 2, characterized in that, The nucleotide sequence is shown in SEQ ID NO:

3.

4. A recombinant plasmid containing the T7 RNA polymerase mutant of claim 2.

5. The recombinant plasmid according to claim 4, characterized in that, The recombinant plasmid was expressed using pET series vectors.

6. The recombinant plasmid according to claim 5, characterized in that, The pET series vectors include pET-3a(+), pET-21a(+), pET-28a(+), or pET-30a(+).

7. A recombinant microbial cell expressing the T7 RNA polymerase variant of claim 2 or containing the nucleotide sequence of claim 3.

8. The recombinant microbial cell according to claim 7, characterized in that, The recombinant microbial cells used Escherichia coli, Bacillus subtilis, or Pichia pastoris as the starting strain.

9. The use of the T7 RNA polymerase mutant according to claim 1 or 2 in in vitro transcription and synthesis of RNA.

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