RNA polymerase variants and uses thereof

Abstract

The invention provides an RNA polymerase variant and a preparation method thereof. The integrity of an RNA product can be improved by utilizing the RNA polymerase variant disclosed by the invention, and the generation of truncated or over-extended impurities can be reduced. The present disclosure also provides a method of synthesizing RNA.

Description

Field of the Invention

[0001] The present disclosure relates to the field of biotechnology, and particularly to RNA polymerase variants and their applications. Background of the Invention

[0003] mRNA therapy refers to the use of drugs developed based on mRNA for treating or preventing diseases. By introducing mRNA as a vaccine or therapeutic agent, it becomes possible for in vitro transcribed (IVT) mRNA to serve as an information carrier to direct the production of functional proteins or peptides in the human body. After entering the 21st century, mRNA technology has become increasingly mature, and remarkable progress has been made in the research of cancer, rare diseases, genetic diseases, and infectious diseases.

[0004] Currently, mRNA vaccines are mainly divided into two types: one is non-replicating mRNA, which refers to delivering a complete gene sequence synthesized in vitro into the body through a vector and inducing a certain immune response. The mRNA vaccines Comirnaty (developed by Pfizer / BioNTech) and Spikevax (developed by Moderna), which were conditionally approved by the FDA for marketing in 2020, are non-replicating vaccines; the other is virus-derived self-amplifying RNA (saRNA). In addition to encoding the required target protein, saRNA also carries a sequence that can express an RNA polymerase (RNA-dependent RNA polymerase, RdRP). When this RNA polymerase is produced, it can use saRNA as a template to produce more saRNA. Compared with ordinary mRNA, saRNA can replicate autonomously in the body, and only a very small amount is needed to induce a strong immune response, prolonging the existence time of the antigen protein in the body. At the same time, since double-stranded RNA will be formed during the replication process of saRNA, it may stimulate the innate immune response of cells, further enhancing the effect of the vaccine and thus prolonging the therapeutic effect. Therefore, the use of saRNA vaccines can reduce the injection dose and frequency, and reduce the potential toxic and side effects of mRNA and the delivery carrier. Currently, several saRNA vaccines have entered clinical trials, but none of them have been approved for marketing. However, during the production process of saRNA, due to the relatively long self-replicating template (>8000bp), its integrity has always been a difficult problem to solve.

[0005] Compared with traditional vaccines, mRNA vaccines have the advantages of short R & D cycle, strong immunogenicity, simple production process, etc. However, the production of mRNA vaccines involves multiple biological processes and processing of raw materials, and quality assessments are required for various aspects such as the characteristics, purity, quantity, physical state (integrity), and safety of mRNA bulk drugs. Among them, the assessment of mRNA integrity is a crucial step. Since IVT mRNA will generate truncated or overextended impurities during synthesis, while reducing the integrity of mRNA, it will also lead to an increase in adverse immunogenicity. Therefore, it is crucial to ensure the integrity of mRNA during in vitro RNA synthesis. Summary of the Invention

[0006] In order to improve the integrity of RNA during the current RNA synthesis process, the present disclosure provides multiple RNA polymerase variants, which can significantly reduce the probability of mRNA truncation and overextension, and can be used to prepare high-integrity RNA products in in vitro transcription. The present disclosure also provides methods for preparing these variants and their applications in in vitro RNA synthesis.

[0007] In a first aspect, the present disclosure provides an RNA polymerase variant, the amino acid sequence of which has at least 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or higher sequence identity compared with SEQ NO: 1, wherein the polypeptide sequence of the RNA polymerase variant contains at least one mutation selected from the following amino acid sites: D137, V181, Y185, Y392, R393, K394, D395, K396, R398 or K399, and the mutation type is substitution or deletion, and the substituted amino acid can be selected from A, G, R, H, L, Y, S, E, D, W, F, I, M, V, P, K, T, C, N, Q.

[0008] In some embodiments, the RNA polymerase variant contains a substitution or deletion at the D137 site. In some embodiments, further, the variant also contains at least one substitution or deletion selected from the following amino acid sites: V181, Y185, Y392, R393, K394, D395, K396, R398 or K399.

[0009] In some embodiments, the RNA polymerase variant contains a substitution or deletion at the V181 site. In some embodiments, further, the variant also contains at least one substitution or deletion selected from the following amino acid sites: D137, Y185, Y392, R393, K394, D395, K396, R398 or K399.

[0010] In some embodiments, the RNA polymerase variant comprises a substitution or deletion at the Y185 position. In some embodiments, further, the variant further comprises a substitution or deletion at least one of the following amino acid positions: D137, V181, Y392, R393, K394, D395, K396, R398, or K399.

[0011] In some embodiments, the RNA polymerase variant comprises a substitution or deletion at the Y392 position. In some embodiments, further, the variant further comprises a substitution or deletion at least one of the following amino acid positions: D137, V181, Y185, R393, K394, D395, K396, R398, or K399.

[0012] In some embodiments, the RNA polymerase variant comprises a substitution or deletion at the R393 position. In some embodiments, further, the variant further comprises a substitution or deletion at least one of the following amino acid positions: D137, V181, Y185, Y392, K394, D395, K396, R398, or K399.

[0013] In some embodiments, the RNA polymerase variant comprises a substitution or deletion at the K394 position. In some embodiments, further, the variant further comprises a substitution or deletion at least one of the following amino acid positions: D137, V181, Y185, Y392, R393, D395, K396, R398, or K399.

[0014] In some embodiments, the RNA polymerase variant comprises a substitution or deletion at the D395 position. In some embodiments, further, the variant further comprises a substitution or deletion at least one of the following amino acid positions: D137, V181, Y185, Y392, R393, K394, K396, R398, or K399.

[0015] In some embodiments, the RNA polymerase variant comprises a substitution or deletion at the K396 position. In some embodiments, further, the variant further comprises a substitution or deletion at least one of the following amino acid positions: D137, V181, Y185, Y392, R393, K394, D395, R398, or K399.

[0016] In some embodiments, the RNA polymerase variant comprises a substitution or deletion at the R398 position. In some embodiments, further, the variant further comprises a substitution or deletion at least one of the following amino acid positions: D137, V181, Y185, Y392, R393, K394, D395, K396, or K399.

[0017] In some embodiments, the RNA polymerase variant comprises a substitution or deletion at the K399 position. In some embodiments, further, the variant further comprises a substitution or deletion at at least one amino acid position selected from the following: D137, V181, Y185, Y392, R393, K394, D395, K396 or R398.

[0018] In some embodiments, the mutation type at D137 is D137E. In some embodiments, the mutation type at V181 is V181A, V181D, V181E, V181G or DEL181 (i.e., deletion of V at position 181). In some embodiments, the mutation type at Y185 is Y185G, Y185D, Y185N, Y185E, Y185V, Y185H, Y185S, Y185F or Y185T. In some embodiments, the amino acid mutation type at Y392 is Y392A or Y392E. In some embodiments, the amino acid mutation type at R393 is R393A. In some embodiments, the amino acid mutation type at K394 is K394Q, K394Y or K394G. In some embodiments, the amino acid mutation type at D395 is D395Y, D395L or D395G. In some embodiments, the amino acid mutation type at K396 is K396A. In some embodiments, the amino acid mutation type at R398 is R398A. In some embodiments, the amino acid mutation type at K399 is K399A.

[0019] As used herein, "sequence identity" refers to the relationship between the sequences of two or more polypeptides, as determined by comparing (aligning) the sequences.

[0020] In some embodiments, the amino acid sequence of the RNA polymerase variant is selected from SEQ ID NO: 2-28.

[0021] In some embodiments, compared to any of the amino acid sequences shown in SEQ ID NO: 2-6, the variant comprises at least one mutation at an amino acid position selected from the following: D137E, Y185H / G / D / N / E / T / V / S / F, Y392A / E, R393A, D395G / Y / L, K396A, R398A, K399A, K394Y / Q / G.

[0022] In some embodiments, compared with any of the amino acid sequences shown in SEQ ID NOs: 7-15, the variant includes at least one mutation selected from the following amino acid sites: D137E, V181A / D / E / G / DEL, Y392A / E, R393A, D395G / Y / L, K396A, R398A, K399A, K394Y / Q / G.

[0023] In some embodiments, compared with the amino acid sequence shown in SEQ ID NO: 16, the variant includes at least one mutation selected from the following amino acid sites: V181A / D / E / G / DEL, Y185H / G / D / N / E / T / V / S / F, Y392A / E, R393A, D395G / Y / L, K396A, R398A, K399A, K394Y / Q / G.

[0024] In some embodiments, compared with the amino acid sequence shown in SEQ ID NO: 17 or 18, the variant includes at least one mutation selected from the following amino acid sites: D137E, V181A / D / E / G / DEL, Y185H / G / D / N / E / T / V / S / F, R393A, D395G / Y / L, K396A, R398A, K399A, K394Y / Q / G.

[0025] In some embodiments, compared with the amino acid sequence shown in SEQ ID NO: 19, the variant includes at least one mutation selected from the following amino acid sites: D137E, V181A / D / E / G / DEL, Y185H / G / D / N / E / T / V / S / F, Y392A / E, D395G / Y / L, K396A, R398A, K399A, K394Y / Q / G.

[0026] In some embodiments, compared with any of the amino acid sequences shown in SEQ ID NOs: 20-22, the variant includes at least one mutation selected from the following amino acid sites: D137E, V181A / D / E / G / DEL, Y185H / G / D / N / E / T / V / S / F, Y392A / E, R393A, D395G / Y / L, K396A, R398A, K399A.

[0027] In some embodiments, compared with any of the amino acid sequences shown in SEQ ID NOs: 23-25, the variant includes at least one mutation selected from the following amino acid sites: D137E, V181A / D / E / G / DEL, Y185H / G / D / N / E / T / V / S / F, Y392A / E, R393A, K396A, R398A, K399A, K394Y / Q / G.

[0028] In some embodiments, compared with the amino acid sequence shown in SEQ ID NO: 26, the variant includes at least one mutation selected from the following amino acid sites: D137E, V181A / D / E / G / DEL, Y185H / G / D / N / E / T / V / S / F, Y392A / E, R393A, D395G / Y / L, R398A, K399A, K394Y / Q / G.

[0029] In some embodiments, compared with the amino acid sequence shown in SEQ ID NO: 27, the variant includes at least one mutation selected from the following amino acid sites: D137E, V181A / D / E / G / DEL, Y185H / G / D / N / E / T / V / S / F, Y392A / E, R393A, D395G / Y / L, K396A, R399A, K394Y / Q / G.

[0030] In some embodiments, compared with the amino acid sequence shown in SEQ ID NO: 28, the variant includes at least one mutation selected from the following amino acid sites: D137E, V181A / D / E / G / DEL, Y185H / G / D / N / E / T / V / S / F, Y392A / E, R393A, D395G / Y / L, K396A, R398A, K394Y / Q / G.

[0031] The amino acid sequences of SEQ ID NOs: 1-28 described in the present disclosure all carry an MHHHHHH tag, which is a commonly used tag protein in the art for purposes such as facilitating purification and tracing. Those skilled in the art should understand that the RNA polymerase variants described in the present disclosure are protected by the present disclosure whether or not they carry a tag.

[0032] In a second aspect, the present disclosure provides a polynucleotide encoding an RNA polymerase variant. Due to codon degeneracy or the codon preference of the host cell expressing the polypeptide, without changing the amino acid sequence, the polynucleotide sequence is any polynucleotide sequence encoding the variant. In some embodiments, the polynucleotide encoding the RNA polymerase variant of the present disclosure can be selected from SEQ ID NOs: 30-56.

[0033] In a third aspect, the present disclosure provides a method for preparing an RNA polymerase variant, including generating at least one RNA polymerase variant described in the present disclosure in a host cell. In some embodiments, the host cell contains an expression vector carrying the polynucleotide of the RNA polymerase variant described in the present disclosure.

[0034] Fourth aspect, the present disclosure provides a method for generating RNA, which includes contacting a template DNA molecule with one or more RNA polymerase variants described in the present disclosure under conditions that result in the generation of an RNA transcript. In some embodiments, the RNA is mRNA. In some embodiments, the RNA is saRNA. In some embodiments, the RNA may also be siRNA, gRNA, dsRNA, ssRNA, miRNA, piRNA, shRNA, etc.

[0035] Fifth aspect, the present disclosure provides a method for in vitro transcription, which includes contacting a DNA template with one or more RNA polymerase variants described in the present disclosure under conditions that result in the generation of an RNA transcript.

[0036] In some embodiments, the method includes the following steps: 1) providing a DNA template containing a T7 promoter, wherein the T7 promoter is functionally linked to a target nucleotide sequence to be transcribed; 2) contacting the DNA template in step 1) with one or more RNA polymerase variants described in the present disclosure; 3) incubating the DNA template and the RNA polymerase variant in an in vitro transcription system.

[0037] In some embodiments, the length of the DNA template is 1000 - 10000 bp.

[0038] In some embodiments, the in vitro transcription system contains ribonucleoside triphosphates and buffer components. In some embodiments, the ribonucleoside triphosphates may be selected from modified or unmodified ribonucleoside triphosphates (including their analogs). In some embodiments, the ribonucleoside triphosphates may be selected from unmodified ATP, GTP, CTP, UTP. In some embodiments, the ribonucleoside triphosphates may be selected from modified ribonucleoside triphosphates, including but not limited to m1A (N1-methyladenosine), m6A (N6-methyladenosine), m5C (5-methylcytidine), 5moU (5-methoxyuridine), ψ (pseudouridine), m1ψ (N1-methyl-pseudouridine), ribonucleoside triphosphates with a label (the label may be biotin, a fluorescent substance, digoxin, a radioactive element, etc.).

[0039] In some embodiments, the in vitro transcription system in step 3) further contains an RNase inhibitor, inorganic pyrophosphatase, and magnesium ions. In some embodiments, the in vitro transcription system in step 3) further contains DEPC water.

[0040] In some embodiments, for the RNA produced by the in vitro transcription method described in the present disclosure, after purification, the integrity of the RNA product is improved; wherein, the integrity is the proportion of the complete RNA product obtained. In some embodiments, the RNA product may be coding RNA or non-coding RNA. In some embodiments, the RNA product includes but is not limited to mRNA, siRNA, gRNA, saRNA, dsRNA, ssRNA, miRNA, piRNA, shRNA, etc.

[0041] In some embodiments, for the mRNA produced by the in vitro transcription method described in the present disclosure, after purification, compared with using wild-type T7 RNA polymerase, the integrity of the mRNA product is improved by at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%.

[0042] In some embodiments, for the saRNA produced by the in vitro transcription method described in the present disclosure, after purification, compared with using wild-type T7 RNA polymerase, the integrity of the saRNA product is improved by at least about 3%, about 5%, about 10%, about 12%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25%.

[0043] In a sixth aspect, the present disclosure provides a method for co-transcriptional capping, which includes reacting a DNA template with one or more RNA polymerase variants, nucleoside triphosphates, and a cap analog described in the present disclosure under in vitro transcription reaction conditions. In some embodiments, the cap analog has the following structure:

[0044] Wherein,

[0045] B1-B n+1 are each independently a nucleobase; R1 is H or an alkyl group, and R2-R n+3 are each independently H, OH, an alkyl group, O-alkyl, S-alkyl, or NH-alkyl, and X1-X n+3 are each independently O or S, and Y1-Y n+4 are each independently O, S, NH, or CH2, and Z1-Zn+3 are each independently OH, SH, BH3, an alkyl group, or O-alkyl; M1-Mn are each independently O, S, NH, or CH2; l is 0, 1, or 2, n1 is 1, and n1-n n are each independently 0 or 1;

[0046] Seventh aspect, the present disclosure provides a composition or a kit, comprising one or more variants of the RNA polymerase described in the present disclosure. In one embodiment, the composition or the kit further comprises one or more components of an in vitro transcription system; wherein the in vitro transcription components may be selected from nucleoside triphosphates, RNase inhibitors, pyrophosphatases, buffer components, magnesium ions, etc. In one embodiment, the in vitro transcription system components may be selected from commercially available in vitro transcription reagents for RNA.

[0047] Eighth aspect, the RNA polymerase variant as described in the present disclosure is used in the synthesis of RNA drugs.

[0048] Ninth aspect, the present disclosure provides a composition, the composition comprising RNA and a pharmaceutically acceptable excipient, wherein the RNA is produced by the in vitro transcription method described in the present disclosure. In some embodiments, the RNA product is not chemically modified. In some embodiments, the RNA product is chemically modified.

[0049] Other embodiments:

[0050] 1. An RNA polymerase variant, the amino acid sequence of which has at least 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or higher sequence identity relative to SEQ NO: 1, and:

[0051] 1) includes a deletion or substitution of the amino acid at position D137, and the substituted amino acid may be selected from A, G, R, H, L, Y, S, E, F, I, M, V, P, K, T, C, N, Q; and / or

[0052] 2) includes a deletion or substitution of the amino acid at position V181, and the substituted amino acid may be selected from A, G, R, H, L, Y, S, E, D, W, F, I, M, P, K, T, C, N, Q; and / or

[0053] 3) includes a deletion or substitution of the amino acid at position Y185, and the substituted amino acid may be selected from A, G, R, H, L, S, E, D, W, F, I, M, V, P, K, T, C, N, Q; and / or

[0054] 4) includes a deletion or substitution of the amino acid at position Y392, and the substitution type may be selected from A, G, R, H, L, S, E, W, F, I, M, V, P, K, T, C, N, Q; and / or

[0055] 5) A deletion or substitution of the amino acid at position R393, where the substitution type is selected from A, G, H, S, E, D, W, F, I, M, V, P, K, T, C, N, Q; and / or

[0056] 6) A deletion or substitution of the amino acid at position D395, where the substitution type is selected from A, G, R, H, L, Y, S, W, F, I, M, V, P, K, T, C, N, Q; and / or

[0057] 7) A deletion or substitution of the amino acid at position K396, where the substituted amino acid is selected from A, G, R, H, L, Y, S, E, D, W, F, I, M, V, P, T, C, N, Q; and / or

[0058] 8) A deletion or substitution of the amino acid at position R398, where the substituted amino acid is selected from A, G, H, L, Y, S, E, D, W, F, I, M, V, P, K, T, C, N, Q; and / or

[0059] 9) A deletion or substitution of the amino acid at position K399, where the substituted amino acid is selected from A, G, R, H, L, Y, S, D, W, F, I, V, P, K, T, C, N, Q.

[0060] 2. The variant according to item 1, further comprising a deletion or substitution of the amino acid at position K394, where the substituted amino acid is selected from A, G, R, H, L, Y, S, E, D, W, F, I, M, V, P, T, C, N, Q.

[0061] 3. The variant according to item 1 or item 2, wherein:

[0062] 1) The amino acid at D137 is substituted with D137E; and / or

[0063] 2) The amino acid at V181 is substituted with V181A, V181D, V181E, V181G or DEL181; and / or

[0064] 3) The amino acid at Y185 is substituted with Y185G, Y185D, Y185N, Y185E, Y185V, Y185H, Y185S, Y185F or Y185T; and / or

[0065] 4) The amino acid at Y392 is substituted with Y392A or Y392E; and / or

[0066] 5) The amino acid at R393 is substituted with R393A; and / or

[0067] 6) The amino acid at K394 is substituted with K394Q, K394Y or K394G; and / or

[0068] 7) The amino acid substitution at D395 is D395Y, D395L or D395G; and / or

[0069] 8) The amino acid substitution at K396 is K396A; and / or

[0070] 9) The amino acid substitution at R398 is R398A; and / or

[0071] 10) The amino acid substitution at K399 is K399A.

[0072] 4. An RNA polymerase variant having an amino acid sequence with at least 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or higher sequence identity relative to SEQ NO: 1, and comprising a mutation at an amino acid site selected from at least one of the following: D137E, V181A / D / E / G / DEL, Y185H / G / D / N / E / T / V / S / F, Y392A / E, R393A, D395G / Y / L, K396A, R398A, K399A.

[0073] 5. The variant according to item 4, further comprising a mutation at K394Y / Q / G.

[0074] 6. An RNA polymerase variant having an amino acid sequence with at least 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or higher sequence identity with any one of the sequences shown in SEQ ID NO: 2-19, 23-28.

[0075] 7. An RNA polymerase variant having an amino acid sequence selected from SEQ ID NO: 2-19, 23-28.

[0076] 8. A polynucleotide comprising a nucleic acid sequence encoding an RNA polymerase variant as described in any one of items 1-7.

[0077] 9. A method for preparing an RNA polymerase variant, comprising introducing the polynucleotide as described in item 8 into a host cell.

[0078] 10. A method for generating RNA, comprising contacting a DNA template with at least one RNA polymerase variant as described in any one of items 1-7 under conditions that result in the production of an RNA transcript.

[0079] 11. A method for generating saRNA, comprising contacting a DNA template with at least one RNA polymerase variant as described in items 1-7 under conditions that result in the generation of an RNA transcript.

[0080] 12. A composition or kit, comprising at least one RNA polymerase variant as described in items 1-7 and at least one buffer component.

[0081] 13. Use of an RNA polymerase variant as described in any one of items 1-7 in the synthesis of RNA drugs.

[0082] 14. A composition, comprising RNA and a pharmaceutically acceptable excipient; wherein the RNA is produced by the method as described in any one of items 9-11. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 It is a schematic diagram for the construction of a recombinant plasmid.

[0085] ADVANTAGEOUS EFFECTS

[0086] 1. The RNA polymerase variant provided by the present disclosure has improved performance. During in vitro transcription, it can improve the integrity of RNA products, significantly reduce the probability of RNA truncation and overextension, reduce the generation of by-products, and has wide adaptability to templates and application scenarios.

[0087] 2. The RNA polymerase mutant provided by the present disclosure can reduce the purification cost during the production of RNA drugs, rapidly prepare RNA drugs meeting the use requirements, and provide a highly efficient tool enzyme for the large-scale production of RNA drugs. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0088] The technical solutions of the present disclosure will be further described below in conjunction with specific embodiments. However, the following embodiments are merely examples of the present disclosure and do not represent or limit the protection scope of the present disclosure. The protection scope of the present disclosure is subject to the claims. In the following embodiments, unless otherwise specified, the reagents and consumables used are purchased from ordinary suppliers in the art, and the experimental methods and technical means used are conventional methods and means in the art.

[0089] Example 1 Preparation of RNA Polymerase Variant

[0090] DNA fragments were synthesized according to the DNA sequences shown in SEQ ID NO: 29 - 56 (the corresponding amino acid sequences are SEQ ID NO: 1 - 28). After PCR amplification, they were introduced into the BseRI and HindIII restriction sites of the expression vector pQE-80L to obtain a recombinant expression vector. After screening by spreading on an antibiotic-resistant plate, a cloned strain was obtained. After successful recombination was verified, the obtained strain was overnight activated in LB medium at 37°C and then added to the fermentation broth (LB medium). It was cultured until the OD600 value reached 0.6 - 0.8, IPTG with a final concentration of 0.5 mol / L was added and culturing continued for 4 - 6 h. Then, the strain was collected by centrifugation at 12000 rmp at 5°C. The collected strain was washed with 0.2 M PBS buffer with a pH value of 7.0 to obtain the thallus. After ultrasonic disruption, affinity chromatography purification was carried out to obtain the RNA polymerase variant.

[0091] Example 2 In vitro transcription reaction verification I (DNA template I, NTP not modified)

[0092] The enzyme stock solution was diluted with the storage buffer to an enzyme activity of 300 U / μL. The MIX solution (except T7 RNA polymerase) was prepared in an EP tube according to the reaction system (20 μL) in Table 1, where the reaction buffer (Vazyme, product number: DD4101R); the MIX solution was aliquoted into an eight-well strip, and then 1 μL of the diluted T7 RNA polymerase and the sample were respectively pipetted into the eight-well strip containing the MIX solution, mixed well, and centrifuged; the eight-well strip was placed on a PCR instrument and reacted at 37°C for 1 h, then 36 μL of magnetic beads were added and mixed well, and incubated at room temperature for 2 - 5 min; the mixture was placed on a magnetic stand to purify the mRNA (Vazyme, product number: N412), and after purification, it was transferred to a RNase-free centrifuge tube to obtain the purified mRNA, and capillary electrophoresis was performed on a Qsep400 fully automatic nucleic acid analyzer to detect the integrity of the mRNA.

[0093] Table 1: Reaction system ratio

[0094]

[0095] The detection results are shown in Table 2. Using the wild-type T7 RNA polymerase (WT) as a control, the T7 RNA polymerase variant groups in Table 2 all showed enhanced mRNA product integrity, that is, the proportion of the complete product band. Among them, the Y185F, Y392A, D395G, K399A RNA polymerase variants greatly improved the integrity of the mRNA product (>90%).

[0096] Table 2: Comparison of the improvement effect of RNA polymerase variant integrity

[0097]

[0098]

[0099] Example 3 In Vitro Transcription Reaction Verification II (DNA Template I, m1ψ)

[0100] Dilute the stock enzyme solution with storage buffer to an enzyme activity of 300 U / μL. Prepare the MIX solution (except T7 RNA polymerase) according to the reaction system (20 μL) in Table 3 into an EP tube, where the reaction buffer (Vazyme, product number: DD4101R); dispense the MIX into an eight-strip, and then use a pipette to take 1 μL of the diluted T7 RNA polymerase and the sample into the eight-strip containing the MIX, mix well, and centrifuge; place the eight-strip on a PCR instrument and react at 37 °C for 1 h, then add 36 μL of magnetic beads and mix well, incubate at room temperature for 2 - 5 min; place the mixture on a magnetic stand to purify the mRNA (Vazyme, product number: N412), and transfer it to an RNase-free centrifuge tube after purification to obtain the purified mRNA, and detect its integrity by capillary electrophoresis.

[0101] Table 3: Reaction System Ratio

[0102]

[0103]

[0104] The detection results are shown in Table 4. Compared with the WT group, except for V181A, V181D, and Del181, the T7 RNA polymerase variant groups in Table 2 all showed enhanced mRNA product integrity, among which the RNA polymerase variants of D137E, K394Y, D395Y, and K396A greatly improved the integrity of the mRNA product (>90%).

[0105] Table 4: Comparison of the Improvement Effect of RNA Polymerase Variant Integrity

[0106]

[0107]

[0108] Example 4 In Vitro Transcription Reaction Verification III (saRNA Template, SEQ NO: 58)

[0109] Dilute the stock solutions of K394Y and K396A enzymes with storage buffer to an enzyme activity of 300 U / μL. Refer to the feeding ratio and experimental method of Example 2 to conduct an IVT experiment. After the experiment, detect its integrity by capillary electrophoresis. The detection results show that, with wild-type T7 RNA polymerase (WT) as a control, the integrity of the K394Y variant can be increased from 56.4% of WT to 73.1%, and that of K396 is increased to 75.5%, achieving a substantial improvement in the integrity of mRNA products.

Claims

1. An RNA polymerase variant having an amino acid sequence with at least 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or higher sequence identity relative to SEQ NO: 1, and: 1) includes a deletion or substitution of the amino acid at position D137, and the substituted amino acid is optionally selected from A, G, R, H, L, Y, S, E, F, I, M, V, P, K, T, C, N, Q; and / or 2) includes a deletion or substitution of the amino acid at position V181, and the substituted amino acid is optionally selected from A, G, R, H, L, Y, S, E, D, W, F, I, M, P, K, T, C, N, Q; and / or 3) includes a deletion or substitution of the amino acid at position Y185, and the substituted amino acid is optionally selected from A, G, R, H, L, S, E, D, W, F, I, M, V, P, K, T, C, N, Q; and / or 4) includes a deletion or substitution of the amino acid at position Y392, and the substitution type is optionally selected from A, G, R, H, L, S, E, W, F, I, M, V, P, K, T, C, N, Q; and / or 5) includes a deletion or substitution of the amino acid at position R393, and the substitution type is optionally selected from A, G, H, S, E, D, W, F, I, M, V, P, K, T, C, N, Q; and / or 6) includes a deletion or substitution of the amino acid at position D395, and the substitution type is optionally selected from A, G, R, H, L, Y, S, W, F, I, M, V, P, K, T, C, N, Q; and / or 7) includes a deletion or substitution of the amino acid at position K396, and the substituted amino acid is optionally selected from A, G, R, H, L, Y, S, E, D, W, F, I, M, V, P, T, C, N, Q; and / or 8) includes a deletion or substitution of the amino acid at position R398, and the substituted amino acid is optionally selected from A, G, H, L, Y, S, E, D, W, F, I, M, V, P, K, T, C, N, Q; and / or 9) includes a deletion or substitution of the amino acid at position K399, and the substituted amino acid is optionally selected from A, G, R, H, L, Y, S, D, W, F, I, V, P, K, T, C, N, Q.

2. The variant according to claim 1, further comprising a deletion or substitution of the amino acid at position K394, and the substituted amino acid is optionally selected from A, G, R, H, L, Y, S, E, D, W, F, I, M, V, P, T, C, N, Q.

3. The variant according to claim 1 or 2, wherein: 1) the amino acid substitution at D137 is D137E; and / or 2) the amino acid substitution at V181 is V181A, V181D, V181E, V181G or DEL181; and / or 3) The amino acid substitution at Y185 is Y185G, Y185D, Y185N, Y185E, Y185V, Y185H, Y185S, Y185F or Y185T; and / or 4) The amino acid substitution at Y392 is Y392A or Y392E; and / or 5) The amino acid substitution at R393 is R393A; and / or 6) The amino acid substitution at K394 is K394Q, K394Y or K394G; and / or 7) The amino acid substitution at D395 is D395Y, D395L or D395G; and / or 8) The amino acid substitution at K396 is K396A; and / or 9) The amino acid substitution at R398 is R398A; and / or 10) The amino acid substitution at K399 is K399A.

4. An RNA polymerase variant having an amino acid sequence with at least 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or higher sequence identity relative to SEQ NO: 1, and comprising a mutation at an amino acid site selected from at least one of the following: D137E, V181A / D / E / G / DEL, Y185H / G / D / N / E / T / V / S / F, Y392A / E, R393A, D395G / Y / L, K396A, R398A, K399A.

5. The variant according to claim 4, further comprising a mutation at K394Y / Q / G.

6. An RNA polymerase variant having an amino acid sequence with at least 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or higher sequence identity with any one of the sequences shown in SEQ ID NOs: 2-19, 23-28.

7. An RNA polymerase variant having an amino acid sequence selected from SEQ ID NOs: 2-19, 23-28.

8. A polynucleotide comprising a nucleic acid sequence encoding an RNA polymerase variant according to any one of claims 1-7.

9. A method for preparing an RNA polymerase variant, comprising introducing the polynucleotide according to claim 8 into a host cell.

10. A method for generating RNA, comprising contacting a DNA template with at least one RNA polymerase variant according to any one of claims 1-7 under conditions that result in the production of an RNA transcript.

11. A method for generating saRNA, comprising contacting a DNA template with at least one RNA polymerase variant according to any one of claims 1-7 under conditions that result in the production of an RNA transcript.

12. A composition or kit comprising at least one RNA polymerase variant according to any one of claims 1-7 and at least one buffer component.

13. Use of an RNA polymerase variant according to any one of claims 1-7 in the synthesis of RNA drugs.

14. A composition comprising RNA and a pharmaceutically acceptable excipient; wherein, The RNA is produced by the method as described in any one of claims 9-11.

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