RNA polymerase variants and uses thereof

Abstract

Provided is an RNA polymerase variant and a method for preparing the same, and the RNA polymerase variant is used in in vitro transcription to obtain an RNA product with low dsRNA contamination. Furthermore, a method for preparing RNA by in vitro transcription is also provided.

Description

RNA polymerase variants and their applications Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to RNA polymerase variants, preparation methods thereof, and applications thereof in RNA synthesis.

[0002] Background of the Invention

[0003] At the end of 2019, the novel coronavirus disease (COVID-19) broke out globally, infecting billions of people worldwide. Before the release of Paxlovid (a combination of namatevir and ritonavir tablets) (which received emergency approval from the FDA on November 22, 2021), vaccines became the most effective line of defense against infection. In this massive global battle against the pandemic, billions of people received COVID-19 vaccines, including BNT162b2 (mRNA vaccine) developed by BioNTech and Pfizer, mRNA-1273 (mRNA vaccine) developed by Moderna, and AZD1222 (adenovirus vector vaccine) developed by AstraZeneca. RNA vaccines have shown the highest protective efficacy. By preventing infection, reducing the incidence of severe illness, and curbing the spread of the epidemic, mRNA vaccines have made a significant contribution to the fight against the pandemic.

[0004] The research and development cycle of mRNA vaccines is relatively short, and new vaccine candidates can be quickly developed to respond to viral mutations. Through the dual mechanisms of humoral immunity and T cell immunity, it has strong immunogenicity and significant effects. The production process is simple, making it easy to develop and mass-produce efficiently, allowing for rapid global supply in the fight against epidemics similar to the new coronavirus.

[0005] In addition to mRNA, research on circular RNA (circRNA)-related drugs has also seen breakthroughs. Orna Therapeutics has developed an in vivo cell therapy product using circRNA. A research report presented at the 2022 American Society of Gene and Cell Therapy (ASGCT) Annual Meeting demonstrated its enormous potential for application in other fields, such as cancer treatment.

[0006] RNA has made great progress in the development of vaccines and other drugs. However, the removal of some impurities in the actual production process requires further study. Double-stranded RNA (dsRNA) impurities can cause strong immunogenicity (Goubau et al., 2014; Kato et al., 2006; Mu et al., 2018). Therefore, it is urgent to develop methods to effectively reduce double-stranded RNA impurities. Summary of the Invention

[0007] In the first aspect, the present application provides a class of RNA polymerase variants, whose amino acid sequence, compared with SEQ ID NO: 1, comprises at least one mutation selected from the following amino acid sites: R34, R52, F55, L59, I154, L170, K172, R173, M183, E187 or G753, wherein the mutation type is selected from substitution or deletion.

[0008] In a second aspect, the present application provides a type of biomaterial, which is selected from one or more of the following:

[0009] 1) a polynucleotide molecule encoding the above variant;

[0010] 2) an expression vector comprising the polynucleotide molecule described in 1);

[0011] 3) A host cell comprising the polynucleotide molecule described in 1), or a host cell comprising the expression vector described in 2).

[0012] In a third aspect, the present application provides a method for preparing the above-mentioned RNA polymerase variant.

[0013] In a fourth aspect, the present application provides a composition comprising at least one RNA polymerase variant as described herein.

[0014] In a fifth aspect, the present application provides a kit comprising at least one RNA polymerase variant as described in the present application.

[0015] In a sixth aspect, the present application provides the use of the above-mentioned RNA polymerase variants, compositions or kits in preparing RNA by in vitro transcription.

[0016] In a seventh aspect, the present application also provides a method for preparing RNA.

[0017] Detailed Description of the Invention

[0018] RNA polymerase variants

[0019] In the first aspect, the present application provides a class of RNA polymerase variants, whose amino acid sequence, compared with SEQ ID NO: 1, comprises at least one mutation selected from the following amino acid sites: R34, R52, F55, L59, I154, L170, K172, R173, M183, E187 or G753, wherein the mutation type is selected from substitution or deletion.

[0020] In some embodiments, the amino acid sequence of the variant has an amino acid mutation relative to SEQ ID NO: 1, and the mutated amino acid position is selected from R52, F55, L59, I154, L170, K172, M183, E187 or G753.

[0021] In some embodiments, the mutation at the R52 site is R52A;

[0022] In some embodiments, the mutation at the F55 site is selected from F55E, F55G, F55K, F55L or F55N;

[0023] In some embodiments, the mutation at the L59 site is L59A;

[0024] In some embodiments, the mutation at the I154 site is I154D;

[0025] In some embodiments, the mutation at the L170 site is selected from L170A or L170D;

[0026] In some embodiments, the mutation at the K172 site is a deletion;

[0027] In some embodiments, the mutation at the M183 site is M183E;

[0028] In some embodiments, the mutation at the E187 site is E187D;

[0029] In some embodiments, the mutation at the G753 site is G753D.

[0030] In some embodiments, the amino acid sequence of the variant, relative to SEQ ID NO: 1, comprises:

[0031] (1) a mutation at the R34 site, and further comprising a mutation of any one or two amino acids selected from the L170, K172, R173 or F182 site, wherein the mutation type is selected from substitution or deletion, wherein the mutation at the R34 site is selected from R34A; or

[0032] (2) a mutation at the R52 site, and further comprising a mutation of any one amino acid selected from the L170 or K179 site, wherein the mutation type is selected from substitution or deletion, wherein the mutation at the R52 site is R52A; or

[0033] (3) a mutation at the F55 site, and further comprising a mutation of any one amino acid selected from the group consisting of L170, Y178, F182, or M183, wherein the mutation type is selected from substitution or deletion, wherein the mutation at the F55 site is selected from F55G or F55L; or

[0034] (4) a mutation at the L59 site, and further comprising a mutation of any one or two amino acids selected from the K172, R173, or M183 site, wherein the mutation type is selected from substitution or deletion, wherein the mutation at the L59 site is L59A; or

[0035] (5) a mutation at the I154 site, and further comprising a mutation of any one or two amino acids selected from the group consisting of Q232, L446, C510, or C530, wherein the mutation type is selected from substitution or deletion, wherein the mutation at the I154 site is I154D; or

[0036] (6) a mutation at the L170 site, and further comprising a mutation of any amino acid selected from the group consisting of R34, R52, F55, or N871, wherein the mutation type is selected from substitution or deletion, wherein the mutation at the L170 site is selected from L170D or L170E; or

[0037] (7) a mutation at K172 and / or R173, and further comprising a mutation of any amino acid selected from R34, L59, E167, E168, Y178 or K387, wherein the mutation type is selected from substitution or deletion, wherein the mutation at K172 and / or R173 is deletion; or a mutation at K172 and R173, and further comprising a mutation of any amino acid selected from K387 or K389, wherein the mutation type is selected from substitution or deletion, wherein the mutation at K172 is K172A and the mutation at R173 is R172G; or

[0038] (8) A mutation at the M183 site, and further comprising a mutation of any one or two amino acids at sites selected from F55, L59, N171, I210, R386 or D388, wherein the mutation type is selected from substitution or deletion, wherein the mutation at the M183 site is M183E.

[0039] In some embodiments, the amino acid sequence of the variant comprises a mutation at any one of the following positions relative to SEQ ID NO: 1:

[0040] (1) R34A+Del172-173, R34A+F182E, R34A+L170E; or

[0041] (2) R52A+K179D, R52A+L170E; or

[0042] (3)F55G+Y178H, F55L+F182E, F55L+L170E, F55L+M183E,

[0043] (4) L59A+Del172-173, L59A+M183E; or

[0044] (5) I154D+C530S, I154D+L446F, I154D+Q232R+C510Q; or

[0045] (6) F55L+L170E, L170D+N871D, R34A+L170E, R52A+L170E; or

[0046] (7) Del172+E167D, Del172-173+E168D, Del172-173+K387S, Del172-173+Y178G, Del172-173+Y178P, K172A+R173G+K387A, K172A+R173G+K387Q, K172A+R173G+K389A, L59A+Del172-173, R34A+Del172-173; or

[0047] (8)F55L+M183E, L59A+M183E, M183E+I210V, M183E+N171K+D388N, R386H+M183E+I210V, R386N+M183E+I210V.

[0048] In some embodiments, the amino acid sequence of the variant comprises any mutation selected from the group consisting of R34A+F182E, R52A+K179D, F55G+Y178H, F55L+F182E, I154D+C530S, I154D+L446F, I154D+Q232R+C510Q, F55L+L170E, L170D+N871D, R34A+L170E, R52A+L170E, Del172+E167D, Del172-173+E168D, Del172-173+K387S, Del17 2-173+Y178G, Del172-173+Y178P, K172A+R173G+K387A, K172A+R173G+K387Q, K172A+R173G+K389A, L59A+Del172-173, R3 4A+Del172-173, F55L+M183E, L59A+M183E, M183E+I210V, M183E+N171K+D388N, R386H+M183E+I210V, R386N+M183E+I210V.

[0049] In some embodiments, the amino acid sequence of the variant is as shown in any one of SEQ ID NOs: 2-58.

[0050] In some embodiments, the amino acid sequence of the variant is set forth in any one of SEQ ID NOs: 3-9, SEQ ID NO: 11, SEQ ID NOs: 15-16, SEQ ID NOs: 19-20, SEQ ID NO: 22, or SEQ ID NOs: 32-58.

[0051] Biomaterials

[0052] The present application provides polynucleotides encoding RNA polymerase variants. Due to the degeneracy of codons or the codon preference of the host cell expressing the polypeptide, the polynucleotide sequence is any polynucleotide sequence encoding the variant without changing the amino acid sequence. In some embodiments, the polynucleotide sequence encoding the RNA polymerase variant of the present application is as shown in any one of SEQ ID NO: 60-116. In some embodiments, the polynucleotide sequence encoding the RNA polymerase variant of the present application can be selected from any one of SEQ ID NO: 61-67, SEQ ID NO: 69, SEQ ID NO: 73-74, SEQ ID NO: 77-78, SEQ ID NO: 80, and SEQ ID NO: 90-116.

[0053] The expression vectors provided herein comprise a polynucleotide encoding an RNA polymerase variant of the present invention. In some embodiments, the expression vectors further comprise one or more regulatory sequences, including but not limited to enhancers, promoters, leader peptide sequences, signal peptide sequences, and terminator sequences; wherein the regulatory sequences are operably linked to the polynucleotide encoding the variant.

[0054] In some embodiments, the expression vector can be a linear or circular DNA molecule, which generally contains elements such as a multiple cloning site, a resistance gene, a replication origin, etc. In some embodiments, the expression vector described herein is preferably pQE-80L.

[0055] The host cell provided in this application refers to any cell that is beneficial for the expression of the variant of this application, that is, any cell that can be transformed, transfected or transduced with the expression vector described in this application, and includes any progeny cells that are different from the parent cell due to mutations that occur during replication.

[0056] In some embodiments, the host cell is a prokaryotic cell, which can be selected from gram-positive bacteria or gram-negative bacteria. In some embodiments, the host cell is a gram-positive bacteria, including but not limited to: Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Ocean Bacillus, Staphylococcus, Streptococcus and Streptomyces. In some embodiments, the host cell is a gram-negative bacteria, including but not limited to: Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Silene Bacillus, Neisseria, Pseudomonas, Salmonella and Ureaplasma. In some embodiments, the host cell is Escherichia coli BL21 (DE3).

[0057] Method for preparing RNA polymerase variants

[0058] The method for preparing an RNA polymerase variant provided herein comprises 1) culturing the host cell described herein under conditions suitable for expression of the variant; and 2) recovering the variant.

[0059] In some embodiments, the method for recovering the variant can be a method well known in the art, such as centrifugation, filtration, treatment with a crystallization protein precipitant (salting out), extraction, ultrasonication, ultrafiltration, dialysis, various chromatography methods such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc., HPLC, and combinations of the above methods.

[0060] In some embodiments, the preparation method further includes a step of purifying the variant, and the purification step can be a method well known in the art, such as chromatography (such as ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing, and size exclusion chromatography), ammonium sulfate precipitation, etc.

[0061] Composition

[0062] The present application provides a composition comprising at least one RNA polymerase variant described herein.

[0063] The composition described herein may be a composition for storing RNA polymerase variants. In some embodiments, the composition described herein, in addition to the above-mentioned RNA polymerase variants, may optionally include: a buffer component (such as Tris base, Tris-HCl, HEPES, MOPS), a salt (such as NaCl), an enzyme inhibitor (such as EDTA), a reducing agent (such as DTT), a surfactant (such as Triton X-100), a stabilizer (such as glycerol), and the like. In some embodiments, the composition for storing RNA polymerase variants described herein comprises: RNA polymerase variants, Tris-HCl, NaCl, EDTA, DTT, Triton X-100, and glycerol.

[0064] In some embodiments, the composition further comprises template DNA. In some embodiments, the composition further comprises at least one in vitro transcription component, which can be selected from one or more buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatase, magnesium ions, etc.

[0065] Reagent test kit

[0066] The kit provided herein comprises at least one RNA polymerase variant as described herein.

[0067] In some embodiments, the kit may further comprise one or more in vitro transcription reaction reagents, such as: a buffer component, modified or unmodified nucleoside triphosphates, an RNase inhibitor, an inorganic pyrophosphatase, magnesium ions, water, and the like. In some embodiments, the kit may further comprise a cap analog, which may be selected from an unmethylated cap analog, a dimethylated cap analog, a trimethylated cap analog, a dimethylated symmetric cap analog, or an anti-reverse cap analog. In some embodiments, the kit may further comprise at least one in vitro transcription component, which may be selected from one or more buffer components, modified or unmodified nucleoside triphosphates, an RNase inhibitor, an inorganic pyrophosphatase, magnesium ions, and the like. In one embodiment, the in vitro transcription system components may be selected from commercially available RNA in vitro transcription reagents.

[0068] application

[0069] The present application provides the use of the above-mentioned RNA polymerase variants, compositions or kits in in vitro transcription to prepare RNA. The present application also provides the use of at least one variant as described herein to reduce the generation of dsRNA impurities in in vitro transcription to prepare RNA.

[0070] In some embodiments, the in vitro transcription to prepare RNA comprises contacting a DNA template, modified or unmodified nucleoside triphosphates with at least one RNA polymerase variant described herein, and incubating the mixture in an in vitro transcription reaction system to obtain a target product.

[0071] Preparation method

[0072] The present application provides a method for preparing RNA, comprising contacting a DNA template, modified or unmodified nucleoside triphosphates with at least one RNA polymerase variant described in the present application, incubating in an in vitro transcription reaction system, and obtaining a target product.

[0073] In vitro transcription reaction systems and incubation conditions suitable for generating RNA products are well known in the art. A person of ordinary skill in the art can determine the appropriate pH value, reaction temperature, reaction time, salt concentration, or whether to add exogenous auxiliary factors, etc. of the reaction system, taking into account the optimal activity of RNA polymerase. In some embodiments, the in vitro transcription reaction system described herein comprises in vitro transcription reaction reagents: one or more buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatase, magnesium ions, water, etc. In some embodiments, in the incubation step described herein, the incubation temperature is 30 to 50°C, preferably 37°C. In some embodiments, in the incubation step described herein, the incubation time is 20 to 240 minutes, preferably 60 minutes.

[0074] In some embodiments, the RNA products prepared using the methods described herein have higher yields, and / or higher integrity, and / or lower dsRNA impurity content, and / or more capped mRNA products, compared to those prepared using wild-type RNA polymerase (SEQ ID NO: 1).

[0075] In some embodiments, the relative residual amount of dsRNA impurities (ratio of residual dsRNA of the variant group / residual dsRNA of the wild-type (WT)) compared to an RNA product prepared using the protocols described herein using a wild-type RNA polymerase is less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1% or less.

[0076] In some embodiments, the in vitro transcription reaction system comprises one or more buffer components. In some embodiments, the buffer component can be selected from Tris-HCl, Hepes, citric acid or commercially available buffer components. In some embodiments, the in vitro transcription buffer system further comprises an RNase inhibitor, an inorganic pyrophosphatase, and magnesium ions. In some embodiments, the in vitro transcription buffer system further comprises water (e.g., DEPC-water, RNase-free water, DNase-free water, sterile purified water, deionized water, distilled water, etc.). In some embodiments, the in vitro transcription buffer system further comprises a cap analog, which can be selected from an unmethylated cap analog, a dimethylated cap analog, a trimethylated cap analog, a dimethylated symmetric cap analog, or an anti-reverse cap analog.

[0077] Other implementation plans

[0078] 1. An RNA polymerase variant, wherein the amino acid sequence of the variant comprises at least one mutation selected from the following amino acid positions relative to SEQ ID NO: 1: R34, R52, F55, L59, I154, E167, E168, L170, N171, K172, R173, Y178, K179, F182, M183, E187, I210, Q232, R386, K387, D388, K389, L446, C510, C530, G753, N871, wherein the mutation is selected from substitution or deletion.

[0079] 2. A variant as described in item 1, wherein:

[0080] (1) The substitution at the R34 position is A;

[0081] (2) The substitution at position R52 is A;

[0082] (3) the substitution at position F55 is selected from E, L, K, G, and N;

[0083] (4) the substitution at position L59 is A;

[0084] (5) the substitution at position I154 is D;

[0085] (6) the substitution at position E167 is D;

[0086] (7) the substitution at position E168 is D;

[0087] (8) the substitution at position L170 is selected from A, D, and E;

[0088] (9) The substitution at position N171 is K;

[0089] (10) The substitution at K172 is A, G, or P, or the mutation at K172 is a deletion;

[0090] (11) The substitution at the R173 site is G, or the mutation at the R173 site is a deletion;

[0091] (12) the substitution at position Y178 is selected from G, H, and P;

[0092] (13) The substitution at K179 is D;

[0093] (14) The substitution at position F182 is E;

[0094] (15) The substitution at position M183 is E;

[0095] (16) The substitution at position E187 is D;

[0096] (17) The substitution at position I210 is V; (18) The substitution at position Q232 is R;

[0097] (19) the substitution at position R386 is selected from H or N;

[0098] (20) the substitution at K387 is selected from A, Q, S, and Y;

[0099] (21) The substitution at position D388 is N;

[0100] (22) The substitution at K389 is A;

[0101] (23) The substitution at position L446 is F;

[0102] (24) The substitution at position C510 is Q;

[0103] (25) The substitution at position C530 is S;

[0104] (26) The substitution at position G753 is D;

[0105] (27) The substitution at position N871 is D.

[0106] 3. The variant according to item 1, wherein the amino acid sequence of the variant comprises any mutation selected from the group consisting of R52A, F55E, F55G, F55K, F55L, F55N, L59A, I154D, L170D, M183E, E187D, G753D, R34A+F182E, R52A+K179D, F55G+Y178H, F55L+F182E, I154D+C530S, I154D+L446F, I154D+Q232R+C510Q, F55L+L170E, L170D+N871D, R34A+L170E, R52A+L170E, Del172+E167D, Del172- 173+E168D, Del172-173+K387S, Del172-173+Y178G, Del172-173+Y178P, K172A+R173G+K387A, K172A+R173G+K387Q, K172A+R173G+K389A, L5 9A+Del172-173, R34A+Del172-173, F55L+M183E, L59A+M183E, M183E+I 210V, M183E+N171K+D388N, R386H+M183E+I210V, R386N+M183E+I210V.

[0107] 4. The variant according to item 1, wherein the amino acid sequence of the variant is shown in any one of SEQ ID NOs: 3-9, SEQ ID NO: 11, SEQ ID NOs: 15-16, SEQ ID NOs: 19-20, SEQ ID NO: 22, or SEQ ID NOs: 32-58.

[0108] 5. Biomaterials selected from one or more of the following:

[0109] 1) A polynucleotide molecule encoding an RNA polymerase variant as described in any one of items 1 to 4;

[0110] 2) an expression vector comprising the polynucleotide molecule described in 1);

[0111] 3) A host cell comprising the polynucleotide molecule described in 1), or a host cell comprising the expression vector described in 2).

[0112] 6. A method for preparing the variant according to any one of claims 1 to 4, comprising:

[0113] (1) culturing the host cell according to claim 5; and

[0114] (2) Recover variants.

[0115] 7. A composition comprising the variant according to any one of items 1 to 4.

[0116] 8. A kit comprising the variant according to any one of items 1 to 4.

[0117] 9. Use of the variant according to any one of items 1 to 4, the composition according to item 7, or the kit according to item 8 in in vitro transcription.

[0118] 10. Use of the variant according to any one of items 1 to 4, the composition according to item 7, or the kit according to item 8 for reducing the formation of dsRNA impurities during RNA preparation by in vitro transcription.

[0119] 11. The use as described in item 10, wherein the process for preparing RNA comprises contacting a DNA template with a variant as described in any one of item 4, and incubating the mixture in an in vitro transcription system.

[0120] 12. A method for preparing RNA, comprising contacting a DNA template, modified or unmodified nucleoside triphosphates, and an RNA polymerase variant, and incubating the mixture in an in vitro transcription reaction system to obtain a target RNA product; wherein the amino acid sequence of the variant is as shown in any one of SEQ ID NOs: 3-9, SEQ ID NO: 11, SEQ ID NOs: 15-16, SEQ ID NOs: 19-20, SEQ ID NO: 22, or SEQ ID NOs: 32-58.

[0121] Compared with the prior art, the present invention has the following advantages:

[0122] Compared with wild-type T7 RNA polymerase, the addition of the T7 RNA polymerase mutants of the present application to the in vitro transcription reaction system significantly reduced the production of double-stranded dsRNA contaminants, especially the mutant combinations F55L+F182E, L170D+N871D, L59A+M183E, M183E+I210V, R34A+F182E, R34A+L170E, R386H+M183E+I210V, R386N+M183E+I210V, R52A+K179D, and R52A+L170E were able to reduce dsRNA to less than 0.5% of the wild type. BRIEF DESCRIPTION OF THE DRAWINGS

[0123] Figure 1 is a schematic diagram of the construction of a recombinant plasmid; DETAILED DESCRIPTION

[0124] The technical solutions of the present application are further described below with reference to specific examples. However, the following examples are merely examples of the present application and do not represent or limit the scope of protection of the present application. The scope of protection of the present application shall be subject to the claims. In the following examples, unless otherwise specified, all reagents and consumables used were purchased from common suppliers in the field, and the experimental methods and technical means used were conventional methods and means in the field.

[0125] In the present application, the enzyme activity is defined as: at 37°C, pH 8.0, the enzyme activity is 1 nmol [ 3 The amount of enzyme required to incorporate [H]ATP into the acid-insoluble precipitate was defined as 1 activity unit.

[0126] Example 1: Preparation of RNA polymerase variants

[0127] The RNA polymerase and its variants shown in Table 1 were synthesized using DNA sequences (SEQ ID NOs: 60-118) and amplified by PCR. The DNA sequences were then introduced into the BseRI and HindIII restriction sites of the expression vector pQE-80L to obtain a recombinant expression vector. The constructed vector was then introduced into E. coli BL21 (DE3) by transformation technology, plated on an LB plate containing ampicillin resistance, and placed in a 37°C incubator overnight. The resulting single colonies were subjected to plasmid extraction and sequencing to obtain recombinant engineered bacteria containing the target gene. The successfully sequenced recombinant E. coli strain was inoculated into LB medium for overnight activation culture, and then 1-5% V / V was inoculated into fermentation broth (LB medium) and cultured to an OD600 value of 0.6-0.8. IPTG with a final concentration of 0.5 mol / L was added and cultured for 4-6 hours. The strain was then collected by centrifugation at 12000 rpm and 5°C. The collected strain was washed with PBS buffer (pH 7.0, 0.2 M) to obtain the bacterial cells; after ultrasonic disruption, the cells were purified by affinity chromatography to obtain an RNA polymerase stock solution.

[0128] WT is the wild-type T7 RNA polymerase, and its amino acid sequence is:

[0129] The corresponding relationship between RNA polymerase and its variants and amino acid sequences is shown in Table 1:

[0130] Table 1: RNA polymerase wild type and variant mutation sites and corresponding amino acid sequence numbers

[0131] Example 2: dsRNA impurity formation during in vitro transcription

[0132] (1) The enzyme stock solution obtained in Example 1 was diluted with storage buffer (Vazyme, Catalog No.: DD4101) to an enzyme activity of 300 U / μL. The reaction components (20 μL) listed in Table 2 were added to the eight-well strips, mixed, and centrifuged. The eight-well strips were placed in a PCR instrument and reacted at 37°C for 1 hour. 36 μL of magnetic beads (Vazyme, Catalog No.: N412) were added, mixed, and incubated at room temperature for 2-5 minutes. The mixture was placed on a magnetic rack to purify the RNA product. After purification, the mixture was transferred to an RNase-free centrifuge tube to obtain the purified RNA product.

[0133] (2) The dsRNA impurity content was tested using a dsRNA detection kit (Vazyme, catalog number: DD3509).

[0134] Table 2: Reaction system ratio

[0135] Table 3: dsRNA detection results

[0136] The dsRNA detection results are shown in Table 3. Compared with the WT group, the polymerase variants in Example 1 were all able to effectively reduce the generation of dsRNA impurities during in vitro transcription. Among them, the mutant combinations F55L+F182E, L170D+N871D, L59A+M183E, M183E+I210V, R34A+F182E, R34A+L170E, R386H+M183E+I210V, R386N+M183E+I210V, R52A+K179D, and R52A+L170E were able to reduce dsRNA production to less than 1% of the wild-type level.

Claims

1. An RNA polymerase variant, characterized in that, The amino acid sequence of the variant contains at least one mutation selected from the following amino acid sites relative to SEQ ID NO: 1: R34, R52, F55, L59, I154, L170, K172, R173, M183, E187 or G753, and the mutation type is selected from substitution or deletion.

2. The variant according to claim 1, characterized in that, The amino acid sequence of the variant has a mutation of one amino acid relative to SEQ ID NO: 1, and the site of the mutated amino acid is selected from R52, F55, L59, I154, L170, K172, M183, E187 or G753.

3. The variant according to claim 2, wherein: (1) The mutation at the R52 site is R52A: (2) The mutation at the F55 site is selected from F55E, F55G, F55K, F55L or F55N; (3) The mutation at the L59 site is L59A; (4) The mutation at the I154 site is I154D; (5) The mutation at the L170 site is selected from L170A or L170D; (6) The mutation at the K172 site is a deletion; (7) The mutation at the M183 site is M183E; (8) The mutation at the E187 site is E187D; (9) The mutation at the G753 site is G753D.

4. The variant according to claim 1, characterized in that, The amino acid sequence of the variant relative to SEQ ID NO: 1 contains: (1) A mutation at the R34 site, and also contains a mutation of one or two amino acids at any one of the sites selected from L170, K172, R173 or F182, and the mutation type is selected from substitution or deletion, wherein the mutation at the R34 site is selected from R34A; or (2) A mutation at the R52 site, and also contains a mutation of one amino acid at any one of the sites selected from L170 or K179, and the mutation type is selected from substitution or deletion, wherein the mutation at the R52 site is R52A; or (3) A mutation at the F55 site, and also contains a mutation of one amino acid at any one of the sites selected from L170, Y178, F182 or M183, and the mutation type is selected from substitution or deletion, wherein the mutation at the F55 site is selected from F55G or F55L; or (4) A mutation at the L59 site, and also contains a mutation of one or two amino acids at any one of the sites selected from K172, R173 or M183, and the mutation type is selected from substitution or deletion, wherein the mutation at the L59 site is L59A; or (5) A mutation at the I154 site, and also contains a mutation of one or two amino acids at any one of the sites selected from Q232, L446, C510 or C530, and the mutation type is selected from substitution or deletion, wherein the mutation at the I154 site is I154D; or (6) A mutation at the L170 site, and also contains a mutation of one amino acid at any one of the sites selected from R34, R52, F55 or N871, and the mutation type is selected from substitution or deletion, wherein the mutation at the L170 site is selected from L170D or L170E; or (7) A mutation at the K172 and / or R173 site, and further comprising a mutation of any one amino acid selected from the R34, L59, E167, E168, Y178 or K387 site, wherein the type of mutation is selected from substitution or deletion, and the mutation at the K172 and / or R173 site is a deletion; or a mutation at the K172 and R173 sites, and further comprising a mutation of any one amino acid selected from the K387 or K389 site, wherein the type of mutation is selected from substitution or deletion, and the mutation at the K172 site is K172A and the mutation at the R173 site is R172G; or (8) A mutation at the M183 site, and further comprising a mutation of any one or two amino acids selected from the F55, L59, N171, I210, R386 or D388 site, wherein the type of mutation is selected from substitution or deletion, and the mutation at the M183 site is M183E.

5. The variant according to claim 4, characterized in that, The amino acid sequence of the variant, relative to SEQ ID NO: 1, comprises a mutation at any one of the following sites: R34A+F182E, R52A+K179D, F55G+Y178H, F55L+F182E, I154D+C530S, I154D+L446F, I154D+Q232R+C510Q, F55L+L170E, L170D+N871D, R34A+L170E, R52A+L170E, Del172+E167D, Del172-173+E168D, Del172-173+K387S, Del172-173+Y178G, Del172-173+Y178P, K172A+R173G+K387A, K172A+R173G+K387Q, K172A+R173G+K389A, L59A+Del172-173, R34A+Del172-173, F55L+M183E, L59A+M183E, M183E+I210V, M183E+N171K+D388N, R386H+M183E+I210V, R386N+M183E+I210V.

6. The variant according to claim 1, characterized in that, The amino acid sequence of the variant is as shown in any one of SEQ ID NOs: 2-58.

7. A biological material, characterized in that, The biological material is selected from one or more of the following: 1) A polynucleotide molecule encoding an RNA polymerase variant as described in any one of claims 1-6; 2) An expression vector comprising the polynucleotide molecule as described in 1); 3) A host cell comprising the polynucleotide molecule as described in 1), or a host cell comprising the expression vector as described in 2).

8. A method for preparing a variant according to any one of claims 1-6, characterized in that, Comprising: (1) Culturing the host cell as claimed in claim 7; and (2) Recovering the variant.

9. A composition, characterized in that, Comprising a variant as described in any one of claims 1-6.

10. Kit, characterized in that, Comprising a variant as described in any one of claims 1-6.

11. Use of the variant as described in any one of claims 1-6, the composition as described in claim 9, or the kit as described in claim 10 in in vitro transcription.

12. Use of the variant as described in any one of claims 1-6, the composition as described in claim 9, or the kit as described in claim 10 in reducing the generation of dsRNA impurities during the preparation of RNA by in vitro transcription.

13. The use as described in claim 12, wherein the process of preparing RNA comprises contacting a DNA template with the variant as described in any one of claims 1-6 and incubating in an in vitro transcription system.

14. A method for preparing RNA, characterized in that, The method comprises contacting a DNA template, modified or unmodified nucleoside triphosphates with an RNA polymerase variant and incubating in an in vitro transcription reaction system to obtain a target RNA product; wherein the amino acid sequence of the polymerase variant is as shown in any one of SEQ ID NO: 2-58.

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