T7 RNA polymerase mutants and their applications

Abstract

The present invention obtains a series of T7 RNA polymerase mutants by mutating amino acids at one or more different sites on the basis of wild-type T7 RNA polymerase, which have lower dsRNA byproducts, higher product integrity, higher yield or transcription activity compared with the wild type. The T7 RNA polymerase mutant with reduced dsRNA byproducts of the present invention is more suitable for in vitro transcription system than wild-type T7 RNA polymerase, with extremely low byproducts and higher purity. It can meet the requirements of RNA drugs or mRNA vaccines after simple purification, which can greatly reduce the difficulty and cost of downstream purification operations, and the transcription product is of high quality and has lower immune side effects. The T7 RNA polymerase mutant of the present invention may also include one or more of the following properties: higher product integrity, higher transcription yield, higher transcription activity, etc.

Description

Technical Field

[0001] The patent of this invention relates to a T7 RNA polymerase mutant and its application, belonging to the field of biotechnology. Background Art

[0002] Ribonucleic acid (RNA) is an extremely important biological macromolecule that plays an important role in the transmission of genetic information. It is widely present in nature and has always been a hot research field. In recent years, with the deepening of RNA research, the role of RNA in disease detection and treatment has begun to emerge, and RNA therapy has emerged. RNA therapy refers to the use of RNA-based molecules to treat or prevent diseases. It has high target specificity and low genetic toxicity, and has high potential for a wide range of genetic molecules and some rare diseases. With the successful development of mRNA COVID-19 vaccines and the approval of many other new RNA drugs, RNA drugs have leapt to the forefront of drug research, and the supply of high-quality RNA molecules of specific length and specific sequence has also posed a huge challenge. There are two main methods for the in vitro synthesis of RNA: chemical synthesis and enzymatic synthesis. Chemical synthesis is mainly suitable for short-chain RNA. For long-chain RNA, the synthesis cost is too high, which limits its practicality. The mRNA encoding protein usually has several thousand nucleotides, so for long-chain mRNA, enzymatic synthesis is currently the common and best solution.

[0003] In vitro synthesis of long-chain mRNA is generally done by using a double-stranded DNA template and transcribing mRNA through RNA polymerase. In this process, T7 RNA polymerase is the most commonly used RNA polymerase. T7 RNA polymerase is a single-subunit RNA polymerase derived from Escherichia coli T7 bacteriophage. It was identified in the 1970s and has since been widely used in in vivo protein expression and in vitro RNA synthesis. T7 RNA polymerase has a total of 883 amino acids and is about 99 kDa. It uses double-stranded DNA as a template and recognizes a specific promoter sequence to transcribe and synthesize mRNA. T7 RNA polymerase has a mature and stable heterologous expression purification process, and does not require any other auxiliary factors to exercise normal transcription activity. It can also produce high-fidelity full-length RNA transcripts, making it the most mainstream tool enzyme for in vitro transcription. Although T7 RNA polymerase has significant advantages as an in vitro RNA synthetase, there are still some disadvantages that cannot be ignored in its application. For example, in the process of synthesizing RNA in vitro, in addition to the target product - complete single-stranded RNA, many by-products such as dsRNA, single-stranded short RNA, and 3' terminal extension products will also be produced. However, mRNA vaccines or RNA drugs have strict requirements on RNA purity and integrity in order to minimize the immune stress response in the body and achieve the best efficacy and safety. In application, although the quality of RNA can be improved by removing transcription by-products through purification and other means, it also increases the cost and difficulty of operation, which is not conducive to large-scale production. Therefore, it is a more direct, economical and effective solution to modify T7 RNA polymerase to reduce by-products in the transcription reaction and obtain high-quality RNA products.

[0004] In view of these problems, the present invention provides a T7 RNA polymerase mutant capable of obtaining higher quality RNA in in vitro synthesis and its application. Summary of the invention

[0005] The purpose of the present invention is to provide a T7 RNA polymerase mutant.

[0006] The technical solution adopted by the present invention is:

[0007] A T7 RNA polymerase mutant, which is a protein described in any one of the following a1-a4:

[0008] a1: a protein having amino acid substitutions at one or at least two of the following positions of the wild-type T7 RNA polymerase as shown in SEQ ID NO.1: I6, F11, I19, K60, N67, A70, I82, N86, D87, F162, K180, V186, V214, N233, A247, P277, M369, Y457, A465, H523, K610, A615, L651, S686, K740, N764;

[0009] a2: a protein having substantially the same enzyme activity and function obtained by replacing and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutation in the amino acid sequence shown in a1;

[0010] a3: a protein that has more than 90% sequence homology with the protein in a1 and whose enzyme activity and function are basically equivalent to those of the protein in a1;

[0011] a4: A fusion protein obtained by connecting the N-terminus and / or C-terminus of any of the amino acid sequences described in a1-a3 to a tag or an enzyme cleavage site, wherein the tag or enzyme cleavage site does not affect the function of the T7 RNA polymerase mutant.

[0012] Preferably, the T7 RNA polymerase mutant is a protein described in any one of the following a1-a4:

[0013] a1: a protein having one or at least two substitutions in the following positions of the wild-type T7 RNA polymerase as shown in SEQ ID NO.1: I6, I19, K60, N67, A70, N86, F162, K180, V186, N233, A247, P277, A465, L651, N764;

[0014] a2: a protein having substantially the same enzyme activity and function obtained by replacing and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutation in the amino acid sequence shown in a1;

[0015] a3: a protein that has more than 90% sequence homology with the protein in a1 and whose enzyme activity and function are basically equivalent to those of the protein in a1;

[0016] a4: A fusion protein obtained by connecting the N-terminus and / or C-terminus of any of the amino acid sequences described in a1-a3 to a tag or an enzyme cleavage site, wherein the tag or enzyme cleavage site does not affect the function of the T7 RNA polymerase mutant.

[0017] Preferably, the amino acid is replaced by any one of the following or a combination of at least two of them: I6G, F11L, I19T, K60I, N67S, A70Q or T, I82V, N86D, D87G, F162S, K180E or D, V186I, V214A, N233D, A247T or I, P277L, M369S or T, Y457H, A465T, H523R, K610R, A615T, L651M, S686G, K740R, N764D.

[0018] Preferably, the amino acid is replaced by any one of the following or a combination of at least two: I6G, F11L, I19T, K60I, N67S, A70Q, N86D, F162S, K180E or D, V186I, V214A, N233D, A247I, P277L, M369S, Y457H, A465T, H523R, L651M, N764D. Preferably, it is a protein described in any one of b1-b3 below:

[0019] b1: A protein obtained by replacing any of the following combinations of amino acids with the wild-type T7 RNA polymerase shown in SEQ ID NO.1:

[0020] (1)N86D / A615T;

[0021] (2) F162S;

[0022] (3) F162S / K180E;

[0023] (4) A247I;

[0024] (5) P277L;

[0025] (6)A70Q / F162S / K180E;

[0026] (7)N233D / A465T;

[0027] (8)D87G / N233D / V186I;

[0028] (9)A70T / P277L;

[0029] (10)N67S / I82V / Y457H / K610R / L651M / N764D;

[0030] (11) F162S / A247T;

[0031] (12) F162S / N233D / A247T;

[0032] (13) V214A;

[0033] (14)I19T / K180D / M369T;

[0034] (15) F162S / K180E / A247I;

[0035] (16)A70Q / V214A;

[0036] (17)A465T;

[0037] (18) F11L;

[0038] (19) K180E;

[0039] (20)M369S / S686G;

[0040] (21)K60I / A465T / K740R;

[0041] (22)H523R;

[0042] (23)A70T / K180D;

[0043] (24)I6G;

[0044] (25)A70Q / K180E;

[0045] (26)A70Q;

[0046] (27) V214A / A465T;

[0047] (28)A70Q / A247T;

[0048] b2: a protein having substantially the same enzyme activity and performance obtained by replacing and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutation in the amino acid sequence shown in b1;

[0049] b3: A fusion protein obtained by connecting the N-terminus and / or C-terminus of any of the amino acid sequences described in b1-b2 to a tag or an enzyme cleavage site, wherein the tag or enzyme cleavage site does not affect the function of the T7 RNA polymerase mutant.

[0050] The above-mentioned T7 RNA polymerase mutant has one or more of the following characteristics: lower dsRNA byproducts (usually ≤0.01 ng / μg; or ≤0.001 ng / μg; or ≤0.0001 ng / μg;), higher product integrity (which may be increased by ≥5%, or ≥9%), higher yield or transcription activity, and lower usage of cap analogs.

[0051] Preferably, a His tag and a thrombin cleavage site are connected to the N-terminus of the mutant.

[0052] The gene encoding the T7 RNA polymerase mutant described above.

[0053] The expression vector of the above-mentioned T7 RNA polymerase mutant.

[0054] The expression host bacteria of the above-mentioned T7 RNA polymerase mutant.

[0055] The use of the T7 RNA polymerase mutant in preparing in vivo transcription or in vitro transcription reagents.

[0056] The invention also discloses a transcription kit containing the T7 RNA polymerase mutant.

[0057] The use of the above-mentioned T7 RNA polymerase mutants in catalyzing the co-transcriptional capping reaction.

[0058] The invention also discloses a co-transcription capping kit, comprising the above-mentioned T7 RNA polymerase mutant, a capping analogue and a buffer system.

[0059] Preferably, the buffer system comprises: 30-50 mM Tris, 5-20 mM DTT, 1-5 mM spermidine, 20-100 mM MgCl 2 .

[0060] The present invention also discloses a method, comprising: contacting a nucleic acid template with the T7 RNA polymerase mutant mentioned above, and performing transcription to obtain a transcription product.

[0061] Preferably, the method further comprises contacting the transcription product with a pharmaceutically acceptable additive to produce a pharmaceutical dosage form.

[0062] Preferably, the method further comprises contacting the transcription product with a capping enzyme to form a capped transcription product.

[0063] Preferably, the method further comprises contacting the capped transcript with a pharmaceutically acceptable additive to produce a pharmaceutical dosage form.

[0064] The present invention also discloses a method, comprising: contacting a nucleic acid template, a cap analog, the above-mentioned T7 RNA polymerase mutant, and a buffer system, performing transcription and capping, and obtaining a capped transcription product.

[0065] Preferably, the method further comprises contacting the capped transcript with a pharmaceutically acceptable additive to produce a pharmaceutical dosage form.

[0066] The present invention obtains a series of T7 RNA polymerase mutants by mutating amino acids at one or more different sites on the basis of wild-type T7 RNA polymerase, which have lower dsRNA byproducts, higher product integrity, higher yield or transcription activity compared with the wild type. The T7 RNA polymerase mutant with reduced dsRNA byproducts of the present invention is more suitable for in vitro transcription system than wild-type T7 RNA polymerase, with extremely low byproducts and higher purity. It can meet the requirements of RNA drugs or mRNA vaccines after simple purification, which can greatly reduce the difficulty and cost of downstream purification operations, and the transcription product is of high quality and has lower immune side effects. The T7 RNA polymerase mutant of the present invention may also include one or more of the following properties: higher product integrity, higher transcription yield, higher transcription activity, lower usage of cap analogs, etc.

[0067] Compared with the prior art, the present invention has the following beneficial effects:

[0068] (1) The present invention can significantly reduce the content of dsRNA byproducts in in vitro transcription by T7 RNA polymerase by designing mutations at one or more different amino acid sites.

[0069] (2) The T7 RNA polymerase mutant of the present invention has a higher product integrity.

[0070] (3) The T7 RNA polymerase mutant of the present invention has higher transcription activity, and the best effect can reach 2 times that of the wild type.

[0071] (4) The T7 RNA polymerase mutant of the present invention is suitable for low-concentration cap analogs, which can significantly reduce the amount of cap analogs used and save the cost of in vitro transcription. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 : Electrophoresis of purified proteins of mutants Mut-022, Mut-025, Mut-029, Mut-049, Mut-050, and Mut-057. In the figure, M022 refers to Mut-022; 1#, 2#, and 3# represent different loading volumes, the same below.

[0073] Figure 2 : Electrophoresis of purified proteins of mutants Mut-077, Mut-082, Mut-083, Mut-103, and Mut-119.

[0074] Figure 3 : Electrophoresis of purified proteins of mutants Mut-134, Mut-139, Mut-144, Mut-161, Mut-162, and Mut-168.

[0075] Figure 4 : Electrophoresis of purified proteins of mutants Mut-176, Mut-180, Mut-184, Mut-195, Mut-197, Mut-207, Mut-218, and Mut-231.

[0076] Figure 5 : Electrophoresis of purified proteins of mutants Mut-246, Mut-258, and Mut-271.

[0077] Figure 6 : dsRNA dot hybridization results of mutants Mut-022, Mut-025, Mut-029, Mut-049, Mut-050, Mut-057, Mut-077, Mut-082. WT represents wild-type T7 RNA polymerase, and 4 parallel tests were performed for each mutant, the same below.

[0078] Figure 7 : dsRNA dot hybridization results of mutants Mut-083, Mut-103, Mut-119, and Mut-134. WT represents wild-type T7 RNA polymerase.

[0079] Figure 8 : dsRNA dot hybridization results of mutants Mut-139, Mut-144, Mut-161, Mut-162, and Mut-168. WT represents wild-type T7 RNA polymerase.

[0080] Fig. 9 : dsRNA dot hybridization results of mutants Mut-176, Mut-180, Mut-184, Mut-195, Mut-197, Mut-207, Mut-218. WT represents wild-type T7 RNA polymerase.

[0081] Fig.10 : dsRNA dot hybridization results of mutants Mut-231, Mut-246, Mut-258, and Mut-271. WT represents wild-type T7 RNA polymerase.

[0082] Fig.11 : Quantification results of 4k template dsRNA. WT represents wild-type T7 RNA polymerase.

[0083] Fig.12 : Quantification results of 8k template dsRNA. WT represents wild-type T7 RNA polymerase.

[0084] Fig.13 : Results of template purity test for mutant 4k. WT represents wild-type T7 RNA polymerase.

[0085] Fig.14 : The results of the purity test of mutant 8k template. WT represents wild-type T7 RNA polymerase.

[0086] Fig.15 : The yield of GMP-grade co-transcription products of mutants Mut-029, Mut-057, Mut-119, Mut-258 and WT with the addition of cap analog m7G(5')ppp(5')(2'OMeA)pU. WT represents wild-type T7 RNA polymerase. DETAILED DESCRIPTION

[0087] The specific implementation modes of the present invention are further described below in conjunction with the accompanying drawings, but the description of the embodiments does not impose any limitation on the protection scope of the present invention.

[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0089] Unless otherwise specified, the materials and instruments used in the following examples can be obtained from conventional commercial channels.

[0090] Embodiment 1:

[0091] 1. The following mutants were obtained by directed evolution. The DNA sequences of the T7 RNA polymerase mutant and the N-terminal 6*His tag and the thrombin cleavage site were constructed into the pAR1219 vector (pAR1219 was purchased from Sigma-Aldrich, Cat#T2076) by homologous recombination, restriction ligation, sequence synthesis, etc., and transformed into Escherichia coli BL21. The wild-type T7 RNA polymerase sequence is shown in Seq ID NO.1, and its corresponding nucleotide sequence is shown in Seq ID NO.2.

[0092] Mutant number Mutation site (based on Seq ID NO.1) Mut-022 N86D / A615T Mut-025 F162S Mut-029 F162S / K180E Mut-049 A247I Mut-050 P277L Mut-057 A70Q / F162S / K180E Mut-077 N233D / A465T Mut-082 D87G / N233D / V186I Mut-083 A70T / P277L Mut-103 N67S / I82V / Y457H / K610R / L651M / N764D Mut-119 F162S / A247T Mut-134 F162S / N233D / A247T Mut-139 V214A Mut-144 I19T / K180D / M369T Mut-161 F162S / K180E / A247I Mut-162 A70Q / V214A Mut-168 A465T Mut-176 F11L Mut-180 K180E Mut-184 M369S / S686G Mut-195 K60I / A465T / K740R Mut-197 H523R Mut-207 A70T / K180D Mut-218 I6G Mut-231 A70Q / K180E Mut-246 A70Q Mut-258 V214A / A465T Mut-271 A70Q / A247T

[0093] 2. Inoculate the mutant with correct sequence verification into 4 mL of fresh LB liquid medium and culture it in a shaker at 37°C and 200 rpm overnight;

[0094] 3. Inoculate each mutant bacterial suspension into three 800 mL fresh 2×YT liquid culture medium at a ratio of 1%, and culture at 37°C and 250 rpm until OD 600 When the value was about 0.6, 0.5 mM IPTG was added and cultured at 16 °C and 250 rpm for about 16 h to induce the expression of exogenous proteins;

[0095] 4. Collect the bacterial suspension of each mutant, centrifuge at 4°C, 5000 rpm for 10 min, and discard the supernatant;

[0096] 5. Weigh about 4 g of wet cells and dissolve them in about 40 mL of 20 mM Tris-HCl buffer at pH 8.0. Store the remaining cells in a -20°C refrigerator.

[0097] 6. Clean the ultrasonic disruptor and ultrasonically disrupt the mutants in batches at 60% power, 3s ON / 5s OFF for about 30 minutes until the solution is basically clear;

[0098] 7. Centrifuge the ultrasonicated solution at 4°C and 15,000 rpm for 30 min and collect the supernatant;

[0099] 8. Debug the AKTA Pure-150 equipment and use a nickel column with a column volume of 10 mL to purify the mutant. The purification procedure and purification buffer are shown in the following table:

[0100] step Buffer Flow rate (mL / min) Volume (mL) balance Buffer A 5 50 Loading Buffer A 5 40 Wash 1 Buffer A 5 50 Wash 2 20%Buffer B 5 50 Elution 60%Buffer B 5 40 Wash 3 100%Buffer B 5 100

[0101] Buffer A formula: 20 mM Tris-HCl, 100 mM NaCl, 10% glycerol (pH 8.0);

[0102] Buffer B formula: 20 mM Tris-HCl, 100 mM NaCl, 10% glycerol, 500 mM imidazole (pH 8.0);

[0103] 9. Collect the eluate and dialyze into 1L Buffer C. Debug the AKTA Pure-150 equipment and use a 5mL DEAE column to purify the mutant. The purification procedure and purification buffer are shown in the following table:

[0104] step Buffer Flow rate (mL / min) Volume (mL) balance Buffer C 5 50 Loading Buffer C 5 1000 Wash 1 Buffer C 5 100 Wash 2 10%Buffer D 5 50 Elution 30%Buffer D 5 20 Wash 3 100%Buffer D 5 50

[0105] Buffer C formula: 20mM Tris-HCl, 10% glycerol, 1mM EDTA, 2mM DTT (pH 7.5);

[0106] Buffer D formula: 20mM Tris-HCl, 10% glycerol, 1mM EDTA, 2mM DTT, 1M NaCl (pH 7.5);

[0107] 10. Collect the eluate, add 50% volume of glycerol, mix thoroughly and filter through a 0.22 μm filter membrane, and use electrophoresis to detect the purity of each mutant and estimate the protein concentration. Figure 1-5 As shown, the purity of each mutant exceeded 90%, which could meet the needs of subsequent characterization.

[0108] Embodiment 2:

[0109] 1. Dilute each mutant protein to about 0.5 mg / mL, and then grade dilute to about 0.4 mg / mL, 0.2 mg / mL, 0.1 mg / mL, and 0.05 mg / mL;

[0110] 2. Take the T7 RNA polymerase in the commercial kit 10623ES60 of Yisheng Biological, and dilute 250U / μL to 100U / μL, 50U / μL, 25U / μL, 10U / μL, 5U / μL, 2U / μL, and 1U / μL for later use;

[0111] 3. According to the kit components and reaction conditions, the above enzymes were respectively subjected to in vitro transcription reaction, and the transcription template was the 4K template shown in SEQ ID No. 3;

[0112] 4. Prepare the working solution according to the requirements of AAT Bioquest pyrophosphate detection kit AAT-21611, mix each gradient mutant and commercial T7 RNA polymerase gradient dilution sample with the pyrophosphate quantitative working solution, incubate at room temperature for 20 minutes, and measure the fluorescence value in an ELISA instrument under the conditions of Ex / Em=316nm / 456nm (Cutoff 420nm);

[0113] 5. Plot a curve between the enzyme activity of the commercial T7 RNA polymerase after dilution and the fluorescence signal, and convert the enzyme activity of the diluted mutant according to the signal value of the mutant. The volume activity and specific activity of each mutant are as follows:

[0114] Mutant number Volume activity (U / μL) Protein concentration (mg / mL) Specific activity (U / μg) WT 654.3 1.73 378.21 Mut-022 2641.0 5.34 494.20 Mut-025 700.0 1.33 524.74 Mut-029 495.0 0.87 568.97 Mut-049 444.6 1.25 356.82 Mut-050 518.3 1.23 422.76 Mut-057 490.6 1.31 375.36 Mut-077 754.9 1.77 427.10 Mut-082 1063.5 2.65 401.70 Mut-083 777.5 1.43 542.28 Mut-103 990.0 2.01 492.29 Mut-119 418.0 1.11 376.29 Mut-134 318.0 1.03 310.24 Mut-139 732.0 1.62 451.85 Mut-144 396.1 1.24 318.37 Mut-161 413.6 1.44 286.89 Mut-162 713.5 1.66 430.25 Mut-168 645.1 1.98 325.81 Mut-176 1401.0 3.26 429.75 Mut-180 1401.0 3.26 429.75 Mut-184 317.9 0.56 567.61 Mut-195 457.8 0.92 497.64 Mut-197 995.4 1.92 518.46 Mut-207 1555.0 1.98 785.34 Mut-218 448.0 1.12 400.00 Mut-231 492.0 1.14 431.58 Mut-246 940.0 1.66 566.27 Mut-258 628.8 1.27 495.13 Mut-271 561.5 1.41 398.25

[0115] The specific activity of the mutants obtained was equivalent to or higher than that of the wild-type T7 RNA polymerase, among which the mutant Mut-207 had the highest specific activity, which was about twice that of the wild-type T7 RNA polymerase.

[0116] Embodiment 3:

[0117] 1. Prepare the in vitro transcription system of T7 RNA polymerase according to the following table, dilute the wild-type T7 RNA polymerase or mutant to 250U / μL, or add the corresponding volume according to the enzyme activity, and react at 37℃ for 3h;

[0118] Components volume Final concentration <![CDATA[10×Transcription Buffer(Mg 2+ Plus)]]> 2.0μL 1× CTP / GTP / ATP / UTP (100mM each) 0.4μL each 2mM each T7 RNA Polymerase 250U - RNase inhibitor (40U / μL) 40U - <![CDATA[RNase free H 2 The]]> Make up to 18 μL - Template DNA 500ng -

[0119] The in vitro transcription template was the test 4k template (SEQ ID No. 3), the RNase inhibitor was Yisheng Biological Product Cat#10603, and the CTP / GTP / ATP / UTP was Yisheng Biological Product Cat#10133;

[0120] 2. Add 115 μL RNase free H to 20 μL of reaction mixture. 2 O and 15 μL 3M sodium acetate (pH 5.2), mixed well;

[0121] 3. Extract once with an equal volume of phenol-chloroform (1:1), then extract twice with an equal volume of chloroform, collect the supernatant, and transfer to a new RNase free EP tube;

[0122] 4. Add 2 volumes of anhydrous ethanol to precipitate RNA. Mix well and place at -20℃ for more than 30 minutes, centrifuge at 4℃ and 16500rpm for 15 minutes, and collect the precipitate;

[0123] 5. Add 500 μL of ice-cold 70% ethanol to wash the RNA precipitate twice;

[0124] 6. Use 20 μL RNase free H 2 O dissolves RNA pellet;

[0125] 7. Dilute the wild-type and mutant transcripts by 50 and 100 times, respectively, to ensure that the RNA concentration does not exceed the Nanodrop calibration range. Test the RNA concentration of each mutant under two dilution conditions. When the numerical deviation is not large after multiplying the 50-fold and 100-fold sample concentrations by the dilution multiple, take the mean as the average value of the mutant transcripts. If the difference between the two dilution multiples is large, the RNA concentration needs to be retested. The results of the wild-type and mutant RNA yields are as follows:

[0126]

[0127]

[0128] Embodiment 4:

[0129] 1. Take an appropriate amount of RNA product into a new RNase free EP tube and use RNase free H 2 O accurately diluted to 1200ng / μL and mixed thoroughly. To prevent RNA degradation, the dilution process must be performed on ice;

[0130] 2. After instant centrifugation, heat the diluted RNA sample at 95°C for 3 minutes and immediately place it on ice to ensure that the RNA secondary structure is opened;

[0131] 3. Cut the NC membrane into appropriate size, and sequentially drop 2 μL of each mutant product onto the NC membrane with a pipette, and spot each sample 2-4 times. During the spotting process, be careful not to let the tip of the pipette touch the NC membrane, and wait for the sample to diffuse freely, and then drop the next sample after it is half dry;

[0132] 4. Transfer the spotted NC membrane to a 37°C oven and treat for 30 minutes to crosslink RNA to the membrane;

[0133] 5. Take out the NC membrane and transfer it to a clean plastic box. Wash it with 10 mL TBST for 5 minutes to wash away the unbound RNA.

[0134] 6. Discard the TBST supernatant and add 10 mL TBSTw blocking solution (QuickBlock TM Blocking solution, Bio-Tech P0231), incubate at room temperature and 30 rpm for 1 h;

[0135] 7. Discard the blocking solution, add the blocking solution containing 1% primary antibody Mouse anti double-stranded RNA (J2) (10010500-Nordic-Mubio), and incubate at 4°C overnight;

[0136] 8. Remove the primary antibody solution, wash the NC membrane with 10 mL of TBST solution, incubate at room temperature at 30 rpm for 10 min, and repeat 3 times;

[0137] 9. Add 10 mL of TBST containing 1% goat anti-mouse antibody (Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L), Yisheng Biological Products Cat#33201) and incubate at room temperature at 30 rpm for 1 hour;

[0138] 10. Repeat step 8 to clean the NC membrane;

[0139] 11. Perform color development according to the instructions of the BCIP / NBT color development kit (Solarbio, PR1100) and conduct qualitative analysis on the dsRNA content in the products of different mutants.

[0140] like Figure 6-10As shown, the gray intensities of the spots of mutants Mut-025, Mut-029, Mut-049, Mut-057, Mut-077, Mut-103, Mut-119, Mut-134, Mut-162, Mut-231, Mut-246, Mut-258, and Mut-271 on the NC membrane were significantly lower than those of the wild type, while the gray intensities of the spots of mutants Mut-022, Mut-050, Mut-081, Mut-083, Mut-139, Mut-144, Mut-161, Mut-168, Mut-176, Mut-180, Mut-184, Mut-195, Mut-197, Mut-207, and Mut-218 were comparable to those of the wild type, and quantitative analysis was required. According to the kit principle, the higher the gray value of the mutant product after spot hybridization, the higher the dsRNA content.

[0141] Embodiment 5:

[0142] 1. Referring to Example 3, an 8k self-replicating template (SEQ ID No. 4) was used for in vitro transcription and the product RNA was purified. At the same time, the 4k template transcription product in Example 3 was taken and the IVT product was quantitatively analyzed using a dsRNA quantitative kit;

[0143] 2. According to the Yisheng Bio dsRNA Quantification Kit (ELISA method, Cat#36717), dilute the STD1 standard sample step by step;

[0144] 3. Add each mutant and diluted standard to the ELISA plate at 100 μL / well, with two replicates for each sample, ensuring that the sample addition is completed within 15 minutes. After sealing the plate with a sealing film, incubate at room temperature at 500 rpm for 1 hour;

[0145] 4. Discard the liquid in the wells, add 1× Wash Buffer (250 μL / well) and wash the plate five times. After each addition of the wash buffer, let it stand for 30 seconds, and then pat the ELISA plate dry;

[0146] 5. Add the biotin-coupled detection antibody prepared to the working concentration into the ELISA plate, 100 μL / well, and incubate at room temperature at 500 rpm for 1 hour;

[0147] 6. Repeat step 4;

[0148] 7. Add the pre-prepared Streptavidin-HRP to the ELISA plate, 100 μL / well, and incubate at room temperature at 500 rpm for 30 min;

[0149] 8. Repeat step 4;

[0150] 9. 10 minutes before use, restore the substrate solution to room temperature, add the substrate solution TMB to the ELISA plate, 100 μL / well, and incubate at room temperature in the dark for 30 minutes;

[0151] 10. Add 50 μL / well stop solution to the ELISA plate and gently shake the plate until the color is evenly developed;

[0152] 11. Immediately read the absorbance value at 450nm / 650nm wavelength (450nm as the detection wavelength, 650nm as the reference wavelength).

[0153] like Fig.11 As shown, the dsRNA content in the total RNA of the mutants was significantly lower than that of the wild type (0.9070ng / μg) when transcribed with a 4k template, and the dsRNA in the products of the mutants Mut-029, Mut-057, Mut-103, Mut-134, and Mut-162 was less than 0.01ng / μg of total RNA, and the dsRNA in the products of the Mut-258 mutant was less than 0.001ng / μg of total RNA. Fig.12 As shown, when transcribed with 8k template, the dsRNA of mutants Mut-029, Mut-057, Mut-077, Mut-103, Mut-134, Mut-162, and Mut-258 was less than 0.01ng / μg total RNA. Among them, the dsRNA of Mut-258 was as low as 0.0003ng / μg total RNA, that is, the dsRNA yield was less than one millionth.

[0154] Embodiment 6:

[0155] 1. Prepare diluent, Lower Marker and other reagents according to the relevant procedures of Bioptic R1 RNA cartridge, and dilute the above RNA sample to 10 ng / μL;

[0156] 2. Treat the RNA sample at 70°C for 2 minutes and then place it on ice for more than 5 minutes;

[0157] 3. Set Qsep-100 equipment program parameters as needed;

[0158] 4. Quickly add the processed RNA sample to the microplate and run the program;

[0159] 5. Determine the purity range of each sample based on the equipment's recommended range.

[0160] like Fig.13As shown in the figure, when transcribed with a 4k template, the purity (i.e., integrity) of the products of the mutants Mut-168, Mut-082, Mut-258, Mut-119, and Mut-077 was increased by more than 5% compared to the wild type, among which the integrity of the Mut-077 product could reach 89.4%. Fig.14 As shown in the figure, the mutants Mut-258, Mut-119, Mut-246, and Mut-134 retained the advantage of improved product integrity when transcribing 8k templates, and the highest transcription product with about 85% integrity was obtained, which was more than 9% higher than the wild type. Product integrity refers to the ratio of the number of full-length mRNA to the total number of mRNA in the transcription product.

[0161] Embodiment 7:

[0162] 1. Prepare the following reaction buffer: 40mM Tris (pH=7.9±0.2), 10mM DTT, 2mM spermidine, 46mM MgCl 2 , used for T7 RNA polymerase in vitro co-transcription experiments;

[0163] 2. Remove the cap analog (Cat. No.: Yisheng Biological: 10679ES80) stock solution with RNase free H 2 O is diluted to a suitable concentration for later use;

[0164] 3. According to the method of Example 3, an in vitro transcription system was prepared. Each mutant and wild-type T7 RNA polymerase were respectively configured with four cap analog concentration gradients, so that the cap analog concentrations in the final reaction system were 10 mM, 5 mM, 2.5 mM and 0 mM. The concentrations of other components in the in vitro transcription system remained unchanged and RNase free H was used. 2 O replenishes the system to 20 μL;

[0165] 4. Perform in vitro transcription according to the T7 High Yield RNA Synthesis Kit (Yisheng Bio: 10623ES50) operation manual. Purify the product RNA and use 20 μL RNase free H 2 O and test the RNA yield of each reaction tube using Nanodrop one.

[0166] like Fig.15 As shown, in the above reaction buffer, the RNA yields of the mutants Mut-029, Mut-057, Mut-119, and Mut-258 in a reaction system without adding cap analogs were comparable to those of the wild type, indicating that the above mutants retained the selectivity of the wild-type T7 RNA polymerase for the cap analogs.

[0167] The yield of the mutants at the recommended cap analog concentration (10mM) was reduced compared to the wild type, with Mut-029 yielding only 2.6mg / mL; however, when the cap analog concentration was gradually reduced, the transcription yield of the mutants gradually increased, with the yields of the mutants Mut-029, Mut-057, and Mut-119 reaching the wild type's 10mM yield level at 2.5mM cap analog concentration. The above results show that in this reaction buffer, the mutants Mut-029, Mut-057, and Mut-119 are more suitable for low-concentration cap analogs, which can significantly reduce the amount of cap analogs used and save the cost of in vitro transcription.

[0168] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A T7 RNA polymerase mutant, characterized in that The following proteins: A protein obtained by replacing any of the following combinations of amino acids based on the wild-type T7 RNA polymerase with the amino acid sequence shown in SEQ ID NO.1: (2) F162S; (3) F162S / K180E; (6) A70Q / F162S / K180E; (8) D87G / N233D / V186I; (11) F162S / A247T; (12) F162S / N233D / A247T; (19) K180E; (23) A70T / K180D; (25) A70Q / K180E.

2. The T7 RNA polymerase mutant according to claim 1, characterized in that A His tag and a thrombin cleavage site were attached to the N-terminus of the mutant.

3. A gene encoding the T7 RNA polymerase mutant according to claim 1 or 2.

4. The expression vector of the T7 RNA polymerase mutant according to claim 1 or 2.

5. An expression host bacterium for the T7 RNA polymerase mutant according to claim 1 or 2.

6. Use of the T7 RNA polymerase mutant according to claim 1 or 2 in the preparation of an in vivo transcription or in vitro transcription reagent.

7. A transcription kit, characterized in that Contains the T7 RNA polymerase mutant according to claim 1 or 2.

8. Use of the T7 RNA polymerase mutant according to claim 1 or 2 in in vitro catalytic co-transcriptional capping.

9. A co-transcriptional capping kit comprising the T7 RNA polymerase mutant according to claim 1 or 2, a cap analog and a buffer system.

10. The kit according to claim 9, characterized in that The buffer system includes: 30-50 mM Tris, 5-20 mM DTT, 1-5 mM spermidine, and 20-100 mM MgCl2.

11. A method, characterized in that include: The nucleic acid template is contacted with the T7 RNA polymerase mutant according to claim 1 or 2, and in vitro transcription is performed to obtain a transcription product.

12. The method of claim 11, further comprising contacting the transcription product with a pharmaceutically acceptable additive to produce a pharmaceutical dosage form.

13. The method of claim 11, further comprising contacting the transcription product with a capping enzyme to form a capped transcription product.

14. The method of claim 13, further comprising contacting the capped transcript with a pharmaceutically acceptable additive to produce a pharmaceutical dosage form.

15. A method, characterized in that include: The nucleic acid template, the cap analog, the T7 RNA polymerase mutant according to claim 1 or 2, and the buffer system are contacted, and in vitro transcription and capping are performed to obtain a capped transcription product.

16. The method of claim 15, further comprising contacting the capped transcript with a pharmaceutically acceptable additive to produce a pharmaceutical dosage form.

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