A t7 rna polymerase mutant, preparation method and application thereof

Abstract

The application provides a T7 RNA polymerase mutant, a preparation method and application thereof, and relates to the technical field of biology. The T7 RNA polymerase mutant is based on a wild-type T7 RNA polymerase amino acid sequence shown as SEQ ID NO. 1, and amino acid substitution is carried out at at least one site selected from M54, P266, F268, F755 and R50. Compared with the wild-type T7 RNA polymerase, the mutant can realize safer and more efficient in-vitro synthesis of high-quality RNA by greatly reducing the double-stranded RNA byproduct with immunogenicity, and simultaneously improving the transcription yield and product integrity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a T7 RNA polymerase mutant, its preparation method, and its application. Background Technology

[0002] The development of genetic engineering and molecular biology techniques has spurred the emergence of novel drugs and diagnostic tools based on nucleic acid molecules. Among these, messenger RNA (mRNA), as a key molecule connecting genetic information and protein function, plays a central role in the life sciences. By synthesizing specific sequences of mRNA in vitro and delivering them to target cells, it is possible to guide the cells in synthesizing the necessary proteins, thereby achieving the goal of treating or preventing diseases. This technological approach has not only achieved breakthroughs in the field of infectious disease vaccines, such as the successful application in the COVID-19 vaccine, but has also provided entirely new solutions for cutting-edge fields such as cancer vaccines, rare disease treatments, and gene editing, greatly expanding the intervention capabilities and treatment scope of modern medicine.

[0003] Currently, the mainstream technology for large-scale industrial production of high-quality RNA molecules is in vitro transcription (ITT) based on bacteriophage RNA polymerase. Among these, T7 RNA polymerase is widely used due to its high catalytic efficiency, strict promoter specificity, and flexible adaptability to template length. This technology can efficiently synthesize target single-stranded RNAs, including mRNA, self-replicating RNA (saRNA), guide RNA (gRNA) for CRISPR-Cas gene editing systems, and highly sensitive diagnostic probes, using linearized DNA as a template in a reaction system. Because of its relatively mature production process and controllable cost, T7 RNA polymerase-based in vitro transcription has become a key platform technology supporting the development of the entire RNA drug and diagnostic industry.

[0004] However, a long-standing technical challenge in in vitro transcription using wild-type T7 RNA polymerase is the generation of double-stranded RNA (dsRNA) byproducts. The formation mechanism of dsRNA is complex, and as a process-related impurity in the final RNA product, it poses serious adverse effects. From a biosafety perspective, dsRNA is a potent pathogen-associated molecular pattern (PAMP) in the body, capable of being recognized by intracellular pattern recognition receptors (such as PKR and OAS), thereby activating downstream innate immune signaling pathways. This unintended immune activation not only inhibits the translation efficiency of target mRNA and reduces drug efficacy but may also trigger excessive inflammatory responses, posing unpredictable safety risks to the clinical application of the drug.

[0005] Therefore, to ensure the safety and efficacy of RNA drugs, the residual amount of dsRNA in the final product must be strictly controlled. Existing technical solutions mainly focus on downstream purification processes, such as using reversed-phase high-performance liquid chromatography (RP-HPLC) or cellulose affinity chromatography to remove dsRNA. However, these purification methods generally have many drawbacks, including cumbersome procedures and high equipment and consumable costs. More importantly, the purification process often leads to significant loss of the target RNA product, with yields typically only between 40% and 60%, directly increasing production costs. Furthermore, these methods struggle to completely remove all lengths and types of dsRNA subtypes, and the harsh conditions during purification can damage the integrity of mRNA molecules. In summary, existing technologies have failed to fundamentally solve the dsRNA contamination problem, posing significant challenges in ensuring product safety, improving production efficiency, and controlling costs.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a T7 RNA polymerase mutant, its preparation method, and its application. Compared with wild-type T7 RNA polymerase, these mutants achieve safer, more efficient, and higher-quality RNA synthesis in vitro by significantly reducing immunogenic double-stranded RNA byproducts and simultaneously increasing transcription yield and product integrity.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0009] In a first aspect, the present invention provides a T7 RNA polymerase mutant, wherein the T7 RNA polymerase mutant is based on the amino acid sequence of the wild-type T7 RNA polymerase shown in SEQ ID NO.1, with amino acid substitutions at at least one site selected from M54, P266, F268, F755 and R50.

[0010] In some embodiments, the amino acid substitution is selected from at least one of M54A, P266G, and F268L;

[0011] In some embodiments, the amino acid sequence of the amino acid mutant M54A is shown in SEQ ID NO.2;

[0012] In some embodiments, the amino acid sequence of the amino acid mutant P266G is shown in SEQ ID NO.3;

[0013] In some embodiments, the amino acid sequence of the amino acid mutant F268L is shown in SEQ ID NO.4.

[0014] Secondly, the present invention provides a polynucleotide molecule encoding a T7 RNA polymerase mutant as described in the foregoing embodiments.

[0015] Thirdly, the present invention provides a recombinant expression vector comprising the polynucleotide molecule as described in the foregoing embodiments.

[0016] Fourthly, the present invention provides a host cell containing the recombinant expression vector described in the foregoing embodiments; or, the chromosome is integrated with the polynucleotide molecule described in the foregoing embodiments.

[0017] Fifthly, the present invention provides a kit comprising the T7 RNA polymerase mutant as described in the foregoing embodiments.

[0018] Sixthly, the present invention provides a method for preparing a T7 RNA polymerase mutant as described in the foregoing embodiments, comprising:

[0019] An expression vector containing a gene encoding the T7 RNA polymerase mutant was constructed, and the expression vector was transformed into a host cell;

[0020] The host cells were cultured under induction conditions to obtain a culture expressing the T7 RNA polymerase mutant;

[0021] The T7 RNA polymerase mutant was isolated and purified from the culture.

[0022] In a seventh aspect, the present invention provides a method for preparing a T7 RNA polymerase mutant as described in the foregoing embodiments, wherein the host cell is *Escherichia coli*; and / or,

[0023] The host cell is *Escherichia coli* strain BL21; and / or,

[0024] The expression vector is a pCold vector; and / or,

[0025] The induction conditions are induction using isopropyl-β-D-thiogalactopyranoside; and / or,

[0026] The purification step includes purification by nickel column affinity chromatography.

[0027] Eighthly, the present invention provides a method for in vitro synthesis of RNA, comprising:

[0028] In vitro transcription was performed using the T7 RNA polymerase mutant as described in the foregoing embodiments to obtain RNA products.

[0029] Ninthly, the present invention provides an application of the T7 RNA polymerase mutant as described in the foregoing embodiments in the synthesis of RNA in an in vitro transcription system.

[0030] This invention provides a T7 RNA polymerase mutant, its preparation method, and its application. Compared with the wild-type T7 RNA polymerase shown in SEQ ID NO.1, the T7 RNA polymerase mutant contains amino acid substitutions at at least one of the sites M54, P266, F268, F755, and R50, and exhibits several significant positive effects during in vitro transcription.

[0031] The most significant improvement lies in the fact that these mutants can substantially reduce the production of immunogenic double-stranded RNA (dsRNA) byproducts. Experimental data show that, under the same transcriptional conditions, compared to the wild-type enzyme, the TRM1 (M54A) mutant and the TRM2 (P266G) mutant produced dsRNA levels reduced to 6.84% and 10.51% of the wild-type, respectively, while the TRM3 (F268L) mutant produced dsRNA levels reduced even further to 2.74% of the wild-type. Since dsRNA is a known potent immune activator that can trigger unwanted immune responses and interfere with mRNA translation, significantly reducing its content at the source can directly improve the biosafety of the final RNA product and reduce its potential immunogenicity risk.

[0032] Secondly, these mutants exhibited higher transcriptional activity and product yield. Data showed that all three mutants synthesized RNA products with higher concentrations and total yields than the wild-type enzyme, with the TRM2 (P266G) mutant achieving more than three times the yield of the wild-type. This indicates that these mutations not only solved the byproduct problem but also improved the synthesis efficiency of the target RNA, contributing to reduced costs and increased throughput in industrial production.

[0033] Furthermore, these mutants can improve the integrity of the transcription products. Electrophoresis results showed that the main band of the RNA products synthesized by these mutants was clear, with significantly reduced diffusion and tailing. This indicates that the fidelity and specificity of the transcription process are improved, resulting in fewer truncated or non-specific transcripts, thus yielding higher quality and more uniform RNA products. Attached Figure Description

[0034] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0035] Figure 1 The structure of the T7 RNA polymerase protein in Example 1 of this application;

[0036] Figure 2 The results of transcriptional integrity testing for mutants TRM1, TRM2, and TRM3 in Example 3 of this application;

[0037] Figure 3 This is the fitting result of the standard curve for the detection of double-stranded RNA products in Example 4 of this application;

[0038] Figure 4 The results are for the detection of double-stranded RNA products of mutants TRM1, TRM2 and TRM3 in Example 4 of this application. Detailed Implementation

[0039] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0040] In this embodiment of the application, a T7 RNA polymerase mutant is provided, wherein the T7 RNA polymerase mutant is based on the amino acid sequence of the wild-type T7 RNA polymerase shown in SEQ ID NO.1, and amino acid substitutions are made at at least one site selected from M54, P266, F268, F755 and R50.

[0041] This embodiment provides a modified protein, specifically an enzyme named "T7 RNA polymerase mutant". It is essentially a biological macromolecule. Unlike the naturally occurring wild-type T7 RNA polymerase with the sequence shown in SEQ ID NO.1, this is an artificially designed protein with predetermined alterations to the amino acid sequence at specific positions. It should be noted that the coding gene sequence for the wild-type T7 RNA polymerase is also shown in SEQ ID NO.1.

[0042] The basic backbone of this substance is the amino acid sequence of T7 RNA polymerase, whose reference sequence is SEQ ID NO.1. Its core components are specific amino acid substitutions introduced on this backbone.

[0043] In this embodiment, through a strategy combining directed evolution and rational design, and after extensive screening, specific sites in the T7 RNA polymerase protein sequence were found to play a crucial role in controlling the generation of dsRNA byproducts. Specifically, this embodiment identified M54, P266, F268, F755, and R50 sites in wild-type T7 RNA polymerase (whose amino acid sequence is shown in SEQ ID NO.1) as key amino acid residue sites affecting dsRNA generation.

[0044] It should be noted that, through directed evolution and rational design, and after extensive screening, this embodiment has for the first time identified the M54, P266, F268, F755, and R50 sites in wild-type T7 RNA polymerase (whose amino acid sequence is shown in SEQ ID NO.1) as key amino acid residue sites affecting dsRNA generation. The beneficial effects of this embodiment are not limited to the specific substitution methods verified in the embodiments (such as M54A, P266G, or F268L), but are based on the discovery of the crucial role of these specific sites in controlling dsRNA generation. Therefore, the scope covered in this embodiment includes not only substitution of a single site, but also the combination of substitutions of any number of these sites. Specifically, each of these five key sites can be substituted by any one of the other 19 natural amino acids (i.e., saturation mutation), which can also achieve the beneficial effect of reducing dsRNA byproducts.

[0045] The core technical solution in this embodiment lies in effectively reducing the amount of dsRNA byproducts generated during transcription by substituting amino acids at at least one of the five key sites mentioned above. This technical solution covers not only substitution of a single site but also the combination of substitutions of any number of sites. The beneficial effects of this embodiment are not limited to the specific substitution methods verified in the embodiment (such as M54A, P266G, or F268L), but are based on the first discovery of the contribution of these specific sites in controlling dsRNA generation. Therefore, replacing the amino acid residues at these sites with other types of amino acid residues can also achieve the beneficial effect of reducing dsRNA byproducts. By substituting amino acids at at least one key site, the resulting T7 RNA polymerase mutant can significantly improve transcription specificity and product integrity while maintaining or improving transcription efficiency, providing a more efficient and safer RNA synthesis tool for mRNA drugs, gene therapy, and diagnostics.

[0046] In some implementations, through extensive screening and verification, three amino acid substitution methods with particularly outstanding performance were further identified: M54A (replacing methionine at position 54 with alanine), P266G (replacing proline at position 266 with glycine), and F268L (replacing phenylalanine at position 268 with leucine). These three specific substitution methods are named mutants TRM1, TRM2, and TRM3, respectively, in this embodiment.

[0047] The experimental results strongly demonstrate the beneficial effects of these specific substitutions. All three substitutions exhibited extremely significant effects in reducing dsRNA byproducts. Compared to the wild-type T7 RNA polymerase's dsRNA yield (set at 100%), the mutants containing the M54A substitution (TRM1) and the P266G substitution (TRM2) showed significantly reduced dsRNA byproduct content to 6.84% and 10.51%, respectively. The most outstanding effect was observed in the mutant containing the F268L substitution (TRM3), which reduced dsRNA byproduct content to 2.74% of the wild-type, greatly improving the purity and safety of the transcript.

[0048] In addition to their significant advantages in reducing impurities, these preferred substitutions also maintained or even improved the enzyme's transcriptional efficiency. Compared to the wild type, mutants containing M54A, P266G, and F268L substitutions showed significantly increased concentrations and yields of transcripts, with the P266G (TRM2) mutant producing more than three times the RNA yield of the wild type. Furthermore, the integrity of the transcripts was improved, with electrophoresis results showing a higher proportion of the main band and a significant reduction in diffusion and tailing.

[0049] The complete amino acid sequences of the T7 RNA polymerase variants containing the M54A, P266G, and F268L mutations are shown in SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively. In summary, these three specific amino acid substitutions are the best examples of the efficient, low-dsRNA, high-yield T7 RNA polymerase variants of this invention. Therefore, this mutant is highly similar to the wild-type enzyme in its overall structure; the decisive difference lies in the alteration of amino acids at one or more specific positions.

[0050] This mutant was designed based on the principles of protein engineering. The specific three-dimensional structure of a protein determines its biological function. The function of T7 RNA polymerase is to synthesize RNA using DNA as a template during in vitro transcription. By altering a single amino acid in its amino acid sequence, a small change can be caused at that location, or even throughout the protein's spatial structure. This structural change further affects the way the enzyme binds to its substrate (DNA template, nucleotide) or the catalytic process, thereby altering its catalytic properties. For example, changing an amino acid at a certain position may affect the enzyme's catalytic activity, product fidelity, or the tendency to produce byproducts.

[0051] This specific structural alteration endows the T7 RNA polymerase mutant with the following beneficial effects compared to the wild-type enzyme (SEQ ID NO. 1): Significantly reduced generation of double-stranded RNA (dsRNA) byproducts: During in vitro transcription, the mutant produces a substantial decrease in dsRNA impurities. The mutant exhibits higher catalytic efficiency, enabling the synthesis of more desired target RNA molecules under the same reaction conditions. The RNA product synthesized by this mutant has a clearer main band with reduced diffusion and tailing, indicating better product uniformity and integrity.

[0052] In this embodiment, a polynucleotide molecule is provided that encodes the T7 RNA polymerase mutant as described in the foregoing embodiments.

[0053] In this embodiment, a recombinant expression vector is provided, which comprises the polynucleotide molecule as described in the foregoing embodiments.

[0054] In this application embodiment, a host cell is provided, the host cell containing the recombinant expression vector described in the foregoing embodiments; or, the chromosome is integrated with the polynucleotide molecule described in the foregoing embodiments.

[0055] In this application embodiment, a kit is provided, the kit comprising the T7 RNA polymerase mutant as described in the foregoing embodiments.

[0056] The core component of the above kit is a T7 RNA polymerase mutant. This mutant is a protein based on the sequence of SEQ ID NO.1 with amino acid substitutions at specific positions, and also includes proteins with sequences such as at least one of SEQ ID NO.2-4.

[0057] As a functional "kit," in addition to the core enzyme, it typically includes, but is not limited to, other supporting reagents required for in vitro transcription reactions, so that users can use them directly. Depending on the technical background and implementation scheme, these supporting components may include:

[0058] (1) Buffer: Provides the most suitable chemical environment (such as pH value and ion concentration) for enzyme activity.

[0059] (2) Nucleotide triphosphates (NTPs): namely ATP, CTP, GTP and UTP, which are the raw materials for synthesizing RNA chains.

[0060] (3) DNA template: A DNA sequence used to guide RNA synthesis.

[0061] (4) Other auxiliary enzymes or reagents: such as RNase inhibitors (used to prevent product degradation), pyrophosphatase, etc.

[0062] It should be clarified that the essential characteristic of this description is that it must include the aforementioned T7 RNA polymerase mutant, and the other components are configured to make it a complete, ready-to-use product.

[0063] In this embodiment of the application, a method for preparing the T7 RNA polymerase mutant as described in the foregoing embodiments is provided, comprising:

[0064] Step S1: Construct an expression vector containing a gene encoding the T7 RNA polymerase mutant, and transform the expression vector into a host cell.

[0065] In this step, we construct an expression vector containing the gene encoding the T7 RNA polymerase mutant: First, we need to obtain the DNA sequence (i.e., the gene) encoding the target mutant protein. Then, we insert this gene into a circular DNA molecule called an "expression vector." This vector acts like a transport vehicle, safely delivering the gene into the cell and instructing the cell to read it.

[0066] The constructed expression vector carrying the target gene is then introduced into a rapidly multiplying "host cell" (such as E. coli). This host cell then becomes a miniature factory capable of producing the target protein.

[0067] Step S2: The host cells are cultured under induction conditions to obtain a culture expressing the T7 RNA polymerase mutant.

[0068] In this step, host cells containing the expression vector are placed in a suitable culture medium for mass culture and propagation. Once the cells reach a certain density, an "inducer" (such as IPTG) is added. This inducer acts like a switch, activating mechanisms within the host cells to enable them to read the gene from the vector in large quantities and synthesize the T7 RNA polymerase mutant protein.

[0069] The T7 RNA polymerase mutant is expressed, and the result of this process is the production of large quantities of the target protein within each host cell. The mixture containing all the cells and the culture medium is called a "culture".

[0070] Step S3: Isolate and purify the T7 RNA polymerase mutant from the culture.

[0071] After expression, the target protein mixes with thousands of proteins, nucleic acids, lipids, and other components of the host cell. This step first requires cell disruption to release all contents. Then, using protein separation and purification techniques (such as nickel column affinity chromatography mentioned in the examples), the target protein is precisely separated from the complex mixture based on its unique properties (such as tags, size, charge, etc.), removing all impurities and ultimately obtaining a high-purity T7 RNA polymerase mutant protein.

[0072] This method is based on the principles of modern genetic engineering and recombinant protein expression technology. Its core is to use the central dogma (DNA→RNA→protein) to introduce the gene (DNA) encoding the target protein into an easily cultured organism (host cell), and "borrow" the organelles and metabolic system of the organism to use it as a bioreactor to produce the exogenous target protein on a large scale.

[0073] The main advantage of this method is that it provides a feasible, standardized, and reproducible route to obtain functional T7 RNA polymerase mutants. Without this preparation method, the proteins defined in the aforementioned embodiments would merely be theoretical amino acid sequences. This method transforms the gene sequence design blueprint into a practically usable protein product with specific superior properties, which is a necessary prerequisite and foundation for realizing all downstream applications of this enzyme (such as for RNA synthesis, preparation kits, etc.).

[0074] In this embodiment, a method for preparing the T7 RNA polymerase mutant as described in the foregoing embodiments is provided, wherein the host cell is Escherichia coli.

[0075] In some embodiments, the host cell is Escherichia coli BL21 strain.

[0076] In some embodiments, the expression vector is a pCold vector.

[0077] In some embodiments, the induction condition is induction using isopropyl-β-D-thiogalactopyranoside.

[0078] In some embodiments, the purification step includes purification by nickel column affinity chromatography.

[0079] In this embodiment of the application, a method for in vitro synthesis of RNA is provided, comprising:

[0080] In vitro transcription was performed using the T7 RNA polymerase mutant as described in the foregoing embodiments to obtain RNA products.

[0081] In this application embodiment, an application of the T7 RNA polymerase mutant as described in the foregoing embodiments in the synthesis of RNA in an in vitro transcription system is provided.

[0082] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0083] Example 1: Crystal structure of T7 RNA polymerase

[0084] In this embodiment, the crystal structure of T7 RNA polymerase (see...) Figure 1 ) to conduct analysis.

[0085] Based on the crystal analysis results, the T7 RNA polymerase (amino acid sequence shown in SEQ ID NO.1) was modified to alter its conformation during the transition from promoter binding to DNA template binding. The modified T7 RNA polymerase (amino acid sequences shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4) can be found in Table 1.

[0086] Table 1. Information and sequence of T7 RNA polymerase

[0087]

[0088] Example 2

[0089] In this embodiment, crude purified T7 RNA polymerase mutant was prepared.

[0090] Experimental methods:

[0091] (1) Construction and expression of T7 RNA polymerase mutant:

[0092] T7 RNA polymerase mutants TRM1, TRM2, and TRM3 were constructed using molecular cloning methods. The prokaryotic expression vector pCold containing these mutants was then transformed into E. coli BL21 expression strain.

[0093] Select bacteria for expansion culture. Place the bacteria in LB medium containing 100 μg / mL kanamycin and incubate at 37°C in a shaker until OD reaches 100%. 600When the value was close to 1, isopropyl-β-D-thiopyranogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM and expression was induced in a shaker at 20 °C for 15 h.

[0094] (2) Purification of crude pure enzyme from T7 RNA polymerase mutant:

[0095] Cells induced by T7 RNA polymerase mutants TRM1, TRM2, and TRM3 were centrifuged at 5000 rpm for 20 min at 4 °C to collect the cell pellet. The cells were thoroughly resuspended in lysis buffer containing 500 mM NaCl, 40 mM Tris-HCl (pH 7.5), and 0.1% Triton, and stored at -80 °C for 30 min. After thawing on ice, lysozyme was added to a final concentration of 0.5 mg / mL, and the cells were digested at 37 °C for 30 min–1 h to induce cell lysis. The protein lysis buffer was centrifuged at 12000 rpm for 30 min at 4 °C in a high-speed refrigerated centrifuge to separate the supernatant.

[0096] The entire nickel column purification process must be performed on ice or at 4°C. Before passing the protein through the nickel column, the column should be equilibrated with 10 volumes of Binding Buffer (500 mM NaCl, 20 mM Tris-HCl, 25 mM Imidazole). Then, the supernatant of the protein lysis buffer is loaded into the nickel column. After loading, the column is washed with 5-10 column volumes of Wash Buffer (500 mM NaCl, 20 mM Tris-HCl, 75 mM Imidazole) with a low imidazole concentration. The target protein is then eluted with Elute Buffer (500 mM NaCl, 20 mM Tris-HCl, 500 mM Imidazole). The protein samples from each step of the nickel column purification process are detected by SDS-PAGE electrophoresis. The sample with the best concentration and purity of the target protein band is selected for dialysis to remove imidazole. The dialyzed sample is then transferred to enzyme preservation solution.

[0097] Example 3

[0098] In this embodiment, the integrity of the T7 RNA polymerase mutant was tested.

[0099] (1) In vitro transcription of T7 RNA polymerase mutant:

[0100] The in vitro transcription reaction consisted of 40 mM Tris-HCl (pH 8.0), 16 mM MgCl2, 2 mM spermidine, 5 mM DTT, 4 mM ATP, 4 mM GTP, 4 mM CTP, 4 mM UTP, 0.3 μL RNase inhibitor, 0.2 μL pyrophosphatase, 200 ng RNA polymerase, and 14 nM PCR template (3000 bp). DEPC water was added to a final volume of 20 μL. After adding all components according to the transcription system, the reaction was carried out at 37°C for 2 h using a heated apparatus. Transcription verification was performed according to Table 2 below.

[0101] Table 2. Transcription System

[0102]

[0103] (2) Transcription product integrity detection:

[0104] After the transcription reaction was completed, 1 μL of DNase I and 2 μL of 10×DNase I Reaction Buffer were added to the reaction system. The reaction was continued at 37℃ for 15 min to digest the DNA template. Finally, the reaction was terminated by heating at 75℃ for 20 min. After the transcription product was recovered, the RNA concentration was measured. 200 ng of RNA was added to 2×RNA loading buffer, mixed well, heated at 65℃ for 5 min, and then placed on ice for verification by 2% agarose gel electrophoresis (120 V for 30 min). The integrity of the product was judged by the uniformity of the electrophoretic bands of the recovered transcription product. The results are as follows: Figure 2 The concentrations and yields of the recovered transcripts are shown in Table 3.

[0105] Table 3. Concentration and yield of transcripts after recovery

[0106]

[0107] Integrity testing results showed that, compared to wild-type T7 RNA polymerase, the concentrations and yields of transcripts from mutants TRM1, TRM2, and TRM3 were all higher than those from the wild type, with the TRM2 mutant showing more than three times the concentration and yield of the wild type. Integrity testing of the transcripts from mutants TRM1, TRM2, and TRM3 showed that the wild-type transcripts had a clear main band but were diffuse and exhibited tailing, while the mutant transcripts had a higher proportion of the main band and a significantly reduced amount of diffuse bands.

[0108] Example 4

[0109] In this embodiment, the content of the transcription product dsRNA was detected.

[0110] Experimental methods:

[0111] The dsRNA content of the T7 mutant transcript was detected using a dsRNA residual quantification kit. The steps are as follows:

[0112] (1) Plate coating: Add 10 μL of capture antibody to 3.4 mL of coating solution, invert and mix at least 30 times, then mix thoroughly with a mixer. Add 100 μL of the above diluted 1× capture antibody to each well and incubate overnight at 4°C.

[0113] (2) Residual detection: Discard the liquid in the wells, add at least 300 μL of 1× washing buffer to each well, let it stand for 30 s, wash the plate twice, and remove as much residual liquid as possible after washing. Add 180 μL of blocking buffer to each well of the microplate and incubate at 37℃ for 2 h.

[0114] (3) Preparation of standards: The self-made dsRNA standard (UTP) was diluted with SD buffer to 3, 1.5, 0.75, 0.375, 0.1875, 0.09375, 0.046875 and 0 ng / mL.

[0115] (4) Sample addition: Discard all liquid from the sealed microplate, ensuring no liquid residue remains in the wells. Then add 100 μL of the prepared standards and samples to each well of the microplate.

[0116] (5) Incubation and washing: After sealing the plate with the sealing film, incubate it in a constant temperature incubator at 37℃ for 1 h. After incubation, carefully remove the sealing film, discard the liquid in the wells, add at least 300 μL of 1× washing solution to each well, let it stand and soak for 30 s, and wash the plate 4 times in a row. The last time, try to remove as much residual liquid as possible.

[0117] (6) Add detection antibody: Add 1× detection antibody solution at a sample volume of 100 μL per well.

[0118] (7) Incubation and washing: After sealing the plate with the sealing film, incubate it in a constant temperature incubator at 37℃ for 1 h. After incubation, carefully remove the sealing film, discard the liquid in the wells, add at least 300 μL of 1× washing solution to each well, let it stand and soak for 30 s, and wash the plate 4 times in a row. The last time, try to remove as much residual liquid as possible.

[0119] (8) Add enzyme-labeled secondary antibody: Add 1× enzyme-labeled secondary antibody solution at a sample volume of 100 μL per well.

[0120] (9) Incubation and washing: After sealing the plate with the sealing film, incubate it in a constant temperature incubator at 37℃ for 1 h. After incubation, carefully remove the sealing film, discard the liquid in the wells, add at least 300 μL of 1× washing solution to each well, let it stand and soak for 30 s, and wash the plate 4 times in a row. The last time, try to remove as much residual liquid as possible.

[0121] (10) Color development: Add 100 μL of color development solution to each well of the microplate, seal the plate with sealing film, and incubate in a 37°C constant temperature incubator for 14 min in the dark.

[0122] (11) Termination / Reading: Add 50 μL of stop solution to each well, mix gently, and read the value within 10 minutes. Detect the absorbance of each well at a single wavelength of 450 nm using a microplate reader. Plot the standard curve data. The double-stranded RNA product detection standard curve system and corresponding OD values ​​are shown in Table 4. The standard curve fitting curve is shown in... Figure 3 (Refer to Table 5 for the fitting equation and fitting parameters).

[0123] Table 4. Standard curve system for double-stranded RNA product detection

[0124]

[0125] Table 5. Logistic curve fitting with parameters ( )

[0126]

[0127] The results showed a good relationship between the standard curve and R. 2 The value was 0.99999. The content of double-stranded RNA product in the corresponding sample and the proportion of double-stranded RNA product in the mutant relative to the wild type were calculated. The results are shown in Table 6. The data in Table 6 were plotted as follows. Figure 4 .

[0128] Table 6. Statistical Table of Double-Stranded RNA Product Content Detection

[0129]

[0130] Based on the calculated dsRNA content, the dsRNA content from highest to lowest is: WT > TRM2 > TRM1 > TRM3. Compared to the wild type, the dsRNA content in the mutant transcripts is significantly reduced. The content of double-stranded RNA (dsRNA) transcriptional byproducts in mutant TRM1 (M54A) and mutant TRM2 (P266G) is reduced to 6.84% and 10.51%, respectively. The content of double-stranded RNA (dsRNA) transcriptional byproducts in mutant TRM3 (F268L) is reduced to 2.74%, greatly improving transcription specificity and product integrity.

[0131] In summary, the T7 RNA polymerase variants TRM1, TRM2, and TRM3, which provide a series of low dsRNA transcriptional byproducts in the embodiments of this application, have amino acid sequences as shown in any one of SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4. Compared with the wild type, the content of double-stranded RNA (dsRNA) transcriptional byproducts in mutant TRM1 (M54A) and mutant TRM2 (P266G) is reduced to 6.84% and 10.51%, respectively. The content of double-stranded RNA (dsRNA) transcriptional byproducts in mutant TRM3 (F268L) is reduced to 2.74%, greatly improving transcription specificity and product integrity. The T7 RNA polymerase and its variant encoding genes, expression vectors, and expression host bacteria, as well as their applications in mRNA transcription, are also disclosed. Compared with the wild type, the T7 RNA polymerase variants designed in this invention can significantly reduce dsRNA production while maintaining or improving transcription efficiency, specificity, and product integrity, providing an efficient and safe RNA synthesis tool for mRNA technology, gene therapy, and diagnostics.

[0132] 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 therein. Such 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 amino acid sequence of the T7 RNA polymerase mutant is shown in SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.

4.

2. A polynucleotide molecule, characterized in that, Encodes the T7 RNA polymerase mutant as described in claim 1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the polynucleotide molecule as described in claim 2.

4. A host cell, characterized in that, The host cell contains the recombinant expression vector of claim 3; or, the chromosome is integrated with the polynucleotide molecule of claim 2.

5. A reagent kit, characterized in that, The kit includes the T7 RNA polymerase mutant as described in claim 1.

6. A method for preparing the T7 RNA polymerase mutant as described in claim 1, characterized in that, include: An expression vector containing the gene encoding the T7 RNA polymerase mutant was constructed, and the expression vector was transformed into a host cell; The host cells were cultured under induction conditions to obtain a culture expressing the T7 RNA polymerase mutant; The T7 RNA polymerase mutant was isolated and purified from the culture.

7. The method for preparing the T7 RNA polymerase mutant as described in claim 6, characterized in that, The host cell is Escherichia coli.

8. The method for preparing the T7 RNA polymerase mutant as described in claim 7, characterized in that, The host cell was Escherichia coli strain BL21.

9. The method for preparing the T7 RNA polymerase mutant as described in claim 6, characterized in that, The expression vector is the pCold vector.

10. The method for preparing the T7 RNA polymerase mutant as described in claim 6, characterized in that, The induction conditions were performed using isopropyl-β-D-thiogalactopyranoside.

11. The method for preparing the T7 RNA polymerase mutant as described in claim 6, characterized in that, The purification step includes purification by nickel column affinity chromatography.

12. A method for synthesizing RNA in vitro, characterized in that, include: In vitro transcription was performed using the T7 RNA polymerase mutant as described in claim 1 to obtain the RNA product.

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