A T7-RNA polymerase mutant capable of reducing IVT by-products

Abstract

The present invention discloses a T7-RNA polymerase mutant. By introducing mutations, an improved T7-RNA polymerase mutant is provided. The T7-RNA polymerase has a simple structure and high transcriptional efficiency, and is widely used in in vitro transcription for synthesizing RNA, which is an important tool enzyme for RNA-related research. The T7-RNA polymerase mutant of the present invention replaces the aspartic acid at position 240 of the wild-type T7-RNA polymerase with asparagine, and replaces the asparagine at position 762 with tryptophan. By mutating these two amino acid sites simultaneously, the by-products generated during in vitro transcription can be reduced. The T7-RNA polymerase mutant of the present invention not only maintains high-efficiency transcription, but also effectively reduces the generation of by-products such as oligonucleotides, truncated RNA products, and 3'-terminal extended products during in vitro transcription. At the same time, it can reduce the purification cost during the production of RNA drugs, providing an effective candidate enzyme tool for the research and application of RNA.

Description

Technical Field

[0001] The present invention belongs to the fields of nucleic acid tool enzymes and nucleic acid biology, and particularly relates to a mutant of phage T7-RNA polymerase and its applications. Background Art

[0002] T7-RNA polymerase is a single-subunit protein with a molecular weight of 99 kDa (883 amino acids) found in phage T7, mainly involved in transcriptional activity and not requiring any other auxiliary transcription factors. The specificity and stability of the transcriptional activity of T7-RNA polymerase come from the specificity of its promoter, which is a 23-nucleotide dsDNA sequence. This specificity for the promoter includes the promoter recognition loop and the N-terminal domain in the T7-RNA polymerase complex. T7-RNA polymerase can transcribe ssDNA oligonucleotide templates containing double-stranded T7 promoter sequences.

[0003] RNA (ribonucleic acid), as a type of biological macromolecule for genetic information transmission, widely exists in eukaryotes, prokaryotes, some viruses and viroids, and has many different types and functions. The gradual in-depth research on RNA reveals that RNA has very important application values in disease treatment. In vitro synthesized siRNA and mRNA can become important drugs for RNA-targeted therapy, and many large pharmaceutical companies are committed to developing RNA drugs for disease treatment. In addition, due to advantages such as transient protein expression in vivo, in vitro synthesized mRNA has currently been promoted and applied as a new type of vaccine - mRNA vaccine.

[0004] Based on its own advantages, mRNA can theoretically express any protein and can be explored to treat almost all protein-based diseases. Currently, the R & D of mRNA technology in COVID-19 vaccines fully demonstrates its unique advantages of strong protein expression ability and short R & D cycle. In addition, mRNA technology has now been gradually promoted to fields such as protein replacement therapy and cell therapy, and its future industrial application scenarios are very rich. With the help of the COVID-19 pandemic and the acceleration of technological changes, it is expected that the market scale of mRNA vaccines and drugs will also reach more than one trillion in the future. In such a huge market and a highly competitive environment, the commercialization process of mRNA will surely be further accelerated. The production of mRNA covers the processes of plasmid purification and mRNA preparation. Due to the complex production process, the large-scale commercial products are insufficient to meet the current market demand.

[0005] IVT (in vitro transcription) products are a mixture that contains not only the desired target mRNA product but also some impurities, including enzymes, residual NTPs, DNA templates, and abnormal mRNA products formed during the IVT process. Laboratory-level purification methods are based on DNAse digestion to remove DNA and then lithium chloride precipitation. However, these methods cannot remove some rare mRNA products, such as dsRNA and truncated RNA fragments, and the impurities associated with these products can reduce translation efficiency and are immunogenic. Therefore, the development of a complete and efficient industrial-level separation and purification process and the establishment of methods for characterizing product-related impurities are also one of the process challenges. Due to the high difficulty and cost of mRNA industrial purification, although it is important to develop a mature industrial purification process, it is a good strategy to reduce the generation of by-products during transcription if possible. If impurities can be reduced at the source, it will greatly reduce the difficulty of downstream purification and shorten the production cycle.

[0006] There are differences in the impurities of mRNA during the IVT process, so it is necessary to improve the selectivity of purification techniques so that process development scientists can formulate corresponding media according to the characteristics of the molecules. Currently, the mainstream mRNA purification processes all use oligo dT columns (which only selectively bind RNA with polyA tails). When using the oligo dT purification platform to purify mRNA, TFF (tangential flow filtration) or SEC (size exclusion chromatography) is used to remove a small amount of residual impurities, and some of these impurities are relatively complex. Finding a general purification platform is more difficult than we expected. The hybridization affinity between affinity chromatography Oligo (dT) and the Poly(A) tail has limitations. It cannot distinguish between ssRNA (single-stranded RNA) and dsRNA (double-stranded RNA) because both carry the Poly(A) tail, but removing trace amounts of dsRNA is very important, especially in the field of vaccine production. There will be trace amounts of dsRNA produced by IVT and co-transcriptional capping to produce mRNA, which can trigger the body's inherent immune response and reduce the efficacy of the vaccine. At high salt concentrations, the mutual repulsion between the negatively charged column ligands and RNA can be inhibited, promoting the formation of hydrogen bonds. After removing the salt ions, the repulsion between the negative charges of the column ligand-RNA is restored, destroying the hydrogen bonds and eluting the RNA. However, for some mRNA variants, it is found that there are very strong double-stranded RNA product impurities, which require a purification step to separate. Therefore, formulating chromatography according to the impurities will be an ideal way to obtain the final process.

[0007] In addition to the advantages of high transcriptional efficiency and strong elongation ability, T7-RNA polymerase also has some drawbacks that cannot be ignored as an in vitro RNA synthesis tool. During the process of synthesizing RNA, it may produce many by-products, including oligonucleotides generated during the transcriptional initiation process, interrupted RNA products caused by termination signals, and 3'-terminal elongation products caused by RdRp activity. Some of the by-products of in vitro synthesized RNA products can lead to the activation of innate immunity after the RNA drugs are delivered into vertebrates, which is also a key problem that needs to be solved in current RNA-targeted therapies. Although multi-step purification can meet the requirements for drug use, it causes a significant increase in production costs during large-scale production synthesis, and the increase in purification processes is also not conducive to the stability of RNA drugs. Therefore, developing new RNA synthesis tool enzymes to maintain high transcriptional efficiency while reducing non-specific transcriptional products has very important application value currently.

[0008] In the prior art, Chinese patent application CN107460177A provides an RNA polymerase mutant that can utilize chemically modified nucleotides. By replacing arginine at position 632 in the amino acid sequence of wild-type T7-RNA polymerase with cysteine, the transcriptional activity is improved, and 2'-triphosphonucleotides can be modified. Another example is Chinese authorized patent CN102177236B, which provides an RNA polymerase mutant with improved functions. By replacing the amino acid residues at at least one position among glutamine at position 786, lysine at position 179, and valine at position 685 in the amino acid sequence of wild-type T7-RNA polymerase with other amino acids, the thermal stability and / or specific activity of this T7-RNA polymerase mutant are improved. However, the prior art cannot reduce the by-products generated by IVT while maintaining high transcriptional efficiency. The by-products generated by IVT have caused the complexity of the subsequent purification work, increasing the time cost and labor cost of RNA production. Summary of the Invention

[0009] Technical problem to be solved: The object of the present invention is to provide a T7-RNA polymerase mutant that can reduce IVT by-products in view of the problem of generating many by-products during the in vitro synthesis of RNA. The present invention provides a mutant of T7-RNA polymerase, which can be used for the production of in vitro RNA. It is significantly different from the existing T7-RNA polymerase, providing an effective candidate enzyme tool for the research and application of RNA.

[0010] Technical solution: A method for producing a T7-RNA polymerase mutant, wherein the amino acids of the T7-RNA polymerase mutant are changed in the sequence shown in SEQ ID NO:2, and the nucleotide sequence encoding the above sequence is as shown in SEQ ID NO:3. The specific steps are as follows:

[0011] (a)Replacing the amino acids selected from Asp240 and Asn762 with different amino acids, which are numbered starting from the N-terminus of the wild-type reference, and wherein the different amino acid replacements are selected from Asp240Asn and Asn762Trp;

[0012] (b)Expressing a nucleic acid molecule in an expression system, which has a nucleotide sequence encoding the T7-RNA polymerase mutant of step (a), and isolating the expressed T7-RNA polymerase mutant from the expression system, thereby producing a T7-RNA polymerase mutant.

[0013] Insert the mutant gene into a prokaryotic expression vector, perform protein expression in E. coli, use the wild-type enzyme as a control, use the purified enzyme for in vitro transcription, and run a gel on the RNA product to verify the transcription effect. Through screening, it was found that the Asp240Asn and Asn762Trp double mutants have an obvious effect of reducing non-target transcription products. Surprisingly, the Asp240Asn and Asn762Trp double mutants can basically eliminate interrupted RNA transcription products. From the gel pattern of the transcription products, the bands are very bright and single, and basically no interrupted RNA is produced. Moreover, the concentration of the target product is higher than that of the wild-type enzyme, indicating that under the same transcription conditions and transcription time, the double mutants can produce RNA more efficiently.

[0014] Preferably, the application of the T7-RNA polymerase mutant in in vitro transcription.

[0015] Preferably, the application of the T7-RNA polymerase mutant in the synthesis of coding RNA.

[0016] Preferably, the application of the T7-RNA polymerase mutant in the synthesis of non-coding RNA.

[0017] Preferably, the application in the synthesis of non-coding RNA, characterized in that the non-coding RNA is microRNA, siRNA, piRNA, rRNA, tRNA, snRNA, snoRNA, SLRNA, SRPRNA, mRNA-like non-coding RNA, non-coding RNA without a polyA tail or oligonucleotide.

[0018] Preferably, the application of the T7-RNA polymerase mutant in gene editing.

[0019] Preferably, the application of the T7-RNA polymerase mutant in the synthesis of RNA drugs.

[0020] Preferably, the application of the T7-RNA polymerase mutant in in vivo protein expression or in vitro translation system of cell-free protein expression.

[0021] Preferably, the application of the T7-RNA polymerase mutant in the synthesis of biological transcriptional regulatory elements.

[0022] Beneficial effects:

[0023] 1. The T7-RNA polymerase mutant of the present invention can not only maintain efficient transcription, but also effectively reduce the generation of by-products such as oligonucleotides, interrupted RNA products, and 3'-terminal extended products during in vitro transcription, providing an effective candidate enzyme tool for the research and application of RNA.

[0024] 2. The T7-RNA polymerase mutant of the present invention can reduce the purification cost during the production of RNA drugs and rapidly prepare RNA drugs that meet the usage requirements, providing a highly efficient tool enzyme for the large-scale production of RNA drugs. Brief description of the drawings

[0025] Figure 1 It is an electrophoretogram of RNA products transcribed from the p19 gene by the T7-RNA polymerase mutant and the wild-type T7-RNA polymerase. Among them, 3 and 6 are the transcription products of the T7-RNA mutant enzyme, and 1, 2, 4, and 5 are the transcription products of the commercial T7-RNA polymerase. Detailed implementation manners

[0026] The embodiments described in the present invention are only partial embodiments of the present invention, not all embodiments. Other implementation contents based on the content of the present invention also fall within the protection scope of the present invention.

[0027] Example 1

[0028] Preparation method of the T7-RNA polymerase mutant for reducing IVT by-products

[0029] Expression and purification of the T7-RNA polymerase mutant

[0030] The T7-RNA polymerase mutants Asp240Asn and Asn762Trp were constructed by molecular cloning methods. The sequences are shown in SEQ ID NO:4, and the nucleotide sequences encoding the above T7-RNA polymerase mutants are shown in SEQ ID NO:5. The prokaryotic expression vector pTrcHis containing the mutant was transformed into the E. coli BL21 expression strain, and the bacteria were picked and cultured on a large scale. The bacteria were placed in an LB medium containing 100 μg / ml ampicillin and cultured in a shaker at 37°C for 3 to 4 hours, OD 600The value is close to 1.0, and then isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.3 mM is added to induce expression at 37 °C for 3 h. Subsequently, the cells are collected by centrifugation at 4 °C and 8000 rpm for 15 min, and the cell pellet is resuspended in a lysis buffer containing 100 mM NaCl, 40 mM Tris-HCl (pH = 7.5), 1 mg / ml lysozyme, and 0.5 mM DTT, and left standing on ice for 1 h.

[0031] The cells digested by lysozyme are sonicated. PMSF with a final concentration of 100 μg / ml is added before sonication. Sonication conditions: ice-water bath, sonication power 200 W, working for 3 s, interval 5 s, sonication time 20 min. After sonication, the supernatant is centrifuged at 16000 rpm / min for 30 min, and the supernatant is filtered through a 0.22 μm needle filter, and the filtered supernatant is purified by nickel column.

[0032] First, the nickel column is equilibrated with more than 10 volumes of equilibration buffer (40 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.5 mM DTT). The filtered supernatant is added to the equilibrated nickel column. After all the protein solution passes through the nickel column, 5 volumes of buffer containing 10 mM imidazole are slowly added for impurity washing. After impurity washing, 100 mM imidazole is added for elution, and the eluate is collected.

[0033] The eluted solution is subjected to SDS-PAGE electrophoresis, stained with Coomassie Brilliant Blue for 1 h, and after decolorization, the concentration and purity of the eluted protein are observed. The relatively pure protein that can meet the requirements of the next experiment is added to a 30 kDa ultrafiltration tube to replace the buffer. The replacement buffer is 40 mM Tris-HCl (pH = 7.9), 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, and 50% glycerol. The protein solution after replacing the buffer is stored at -20 °C for later use.

[0034] Example 2

[0035] Obtaining the transcription template

[0036] The transcription template was obtained by amplifying an existing vector in the laboratory using the PCR method. The vector contains the T7 promoter and the P19 mRNA coding sequence (see SEQ ID NO.1 in the sequence listing). The purified PCR product was used as the transcription template. The in vitro transcription reaction was carried out in a 20 μL system containing 40 mM Tris-Hcl (pH = 8.0), 200 nM RNA polymerase, 0.1 μL RNase inhibitor, 0.2 μL pyrophosphatase, 20 ng / μL PCR template, 4 mM ATP, GTP, CTP, UTP, 12 mM Mgcl2, 2 mM spermidine, and 2 mM DTT. After incubation at 37 °C for 2 h, 1 μL of DNAaseⅠ was added and the template was digested at 37 °C for 30 min.

[0037] Comparison of the transcription efficiency between the polymerase mutant and the wild-type T7-RNA polymerase

[0038] Take 1 μL of the T7-RNA polymerase mutant sample, add 4 μL of DEPC water, then add 5 μL of 2*RNA loading buffer, mix, heat at 80 °C for 2 min, and then place on ice. Prepare a 1.5% agarose gel, run the gel at 190 V for 20 min, and perform EB detection. The results are as Figure 1 shown. The double mutant of T7-RNA polymerase Asp240Asn and Asn762Trp has a higher yield of the target RNA, that is, it has a higher production efficiency.

[0039] Comparison of the transcription product specificity between the polymerase mutant and the wild-type T7-RNA polymerase

[0040] Four wild-type T7-RNA polymerases are commercially available single enzymes of T7-RNA polymerase. The method for obtaining the transcription template and the in vitro transcription reaction is the same as in Example 2. The transcription products were directly run on a 1.5% agarose gel at 190 V for 20 min and EB detection was performed. The results are as Figure 1 shown. The results show that the bands of the transcription products of the mutant enzyme are single, and non-target RNA products are hardly visible in the gel image, greatly reducing the workload of later purification and having higher product specificity.

[0041] Comparison of the situation of interrupted RNA generation during the transcription of the p19 gene by the polymerase mutant and the wild-type T7-RNA polymerase

[0042] Since the completely transcribed target RNA has Poly(A+), the proportion of the target RNA product can be judged by the high or low recovery rate of oligo(dT) purification. The method for obtaining the transcription template and the in vitro transcription reaction is the same as in Example 2, and the RNA product concentration was adjusted to be consistent before loading. The oligo(dT) purification method is as follows:

[0043] (1) Establishment of chromatographic conditions

[0044] Select a Monomix dT20 chromatographic column; the detection wavelength is 260 nm; the loading buffer is a mixed solution of 10 mM Tris, 1 mM EDTA, and 5 mM DTT at pH 7.4; the column volume V is 1.09 mL; the flow rate is 0.5 mL / min; the pressure is 1 - 3 PSI; the equilibration buffer is a mixed solution of 10 mM Tris, 1 mM EDTA, 5 mM DTT, and 1.6 mM Nacl at pH 7.4, 3 CV; the injection volume is 1 mL.

[0045] (2) RNA sample treatment: Add 0.5 ml of mRNA sample to 0.5 ml of 2× loading buffer, heat at 65 °C for 10 min, and place on ice for 10 min.

[0046] (3) Elution conditions

[0047] Equilibration buffer washing: 4 CV; Elution: The mobile phase is pure water, at room temperature, 8 CV; CIP: 0.1 M NaOH, at room temperature, 5 CV.

[0048] (4) Result analysis

[0049] The recovery rate of the transcription product of wild-type T7-RNA polymerase after purification by oligo(dT) is generally between 65% and 70%, while the recovery rate of the transcription product of the double mutant of T7-RNA polymerase D240N and N762W can be stably above 77%. The recovery rate of the transcription product of the double mutant after purification by oligo(dT) is significantly higher than that of the wild type, indicating that the proportion of the target RNA product that is completely transcribed is significantly higher than that of the wild type. When using wild-type T7-RNA polymerase for transcription, non-target product bands appear, including prematurely terminated RNA products. When using the double mutant polymerase of Asp240Asn and Asn762Trp for transcription, the conditions are single and clear, and non-target bands and prematurely terminated RNA products are not visible in the gel image and can be ignored. This result also reflects that the double mutant of Asp240Asn and Asn762Trp can produce a higher yield and higher purity of the target RNA compared to the wild-type enzyme.

[0050] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to list all implementation manners here. And the obvious changes or alterations derived therefrom are still within the protection scope of this invention.

Claims

1. A T7-RNA polymerase mutant, characterized in that: The sequence of the polymerase mutant is shown in SEQ ID NO.

4.

2. Use of the T7-RNA polymerase mutant according to claim 1 in in vitro transcription for non-diagnostic or non-therapeutic purposes.

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

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

5. Use according to claim 4 in the synthesis of non-coding RNA for non-diagnostic or non-therapeutic purposes, characterized in that, The non-coding RNA is microRNA, siRNA, piRNA, rRNA, tRNA, snRNA, snoRNA, SLRNA, SRPRNA, mRNA-like non-coding RNA, non-coding RNA without a polyA tail or oligonucleotide.

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

7. Use of the T7-RNA polymerase mutant according to claim 1 in the synthesis of RNA drugs for non-diagnostic or non-therapeutic purposes.

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

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

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