RNA polymerase variants

Abstract

The present application provides a class of RNA polymerase variants capable of significantly reducing the generation of dsRNA impurities in the process of in vitro transcription. The present application also relates to compositions and kits comprising the variants, and the use of the variants, compositions and kits described in the present application in in vitro transcription.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to RNA polymerase variants and their applications. Background Technology

[0002] In late 2019, the novel coronavirus disease 2019 (COVID-19) broke out globally, infecting billions of people worldwide. Before Paxlovid (a combination of nematriberi and ritonavir tablets) was available (it received FDA emergency approval on November 22, 2021), vaccines became the most effective line of defense against infection. In this massive global fight against the pandemic, billions of people were vaccinated with COVID-19 vaccines such as BNT162b2 (mRNA vaccine) developed by BioNTech and Pfizer, mRNA-1273 (mRNA vaccine) developed by Moderna, and AZD1222 (adenovirus vector vaccine) developed by AstraZeneca. Among these, RNA vaccines offered the highest protective efficacy. mRNA vaccines, by preventing infection and reducing the rate of severe illness, curbed the spread of the epidemic, making a significant contribution to this fight.

[0003] mRNA vaccines have a relatively short development cycle, enabling the rapid development of new candidate vaccines to address viral mutations. Through a dual mechanism of humoral and T-cell immunity, they exhibit strong immunogenicity and significant effects. Furthermore, their production process is simple, facilitating efficient research and development and large-scale production, thus enabling rapid and efficient global supply for combating pandemics like COVID-19.

[0004] According to the latest news, in addition to mRNA, research on circular RNA (circRNA)-related drugs has also made breakthrough progress. Orna Therapeutics has developed in vivo cell therapy products using circRNA, and a research report presented at the 2022 American Society for Gene and Cell Therapy (ASGCT) Annual Meeting has demonstrated its great application potential in other fields such as cancer treatment.

[0005] RNA has shown great promise in the development of vaccines and other drugs, but the removal of some impurities in the actual production process still needs further research. Among them, double-stranded RNA (dsRNA) impurities can cause strong immunogenicity (Goubau et al., 2014; Kato et al., 2006; Mu et al., 2018). Therefore, there is an urgent need to develop effective methods to reduce double-stranded RNA impurities. Summary of the Invention

[0006] In a first aspect, the present invention provides an RNA polymerase variant whose amino acid sequence, compared with SEQ ID NO: 1, contains at least one mutation selected from the following amino acid sites: R50, K53, F55, R57, Q58, L59, G62, V64, D66, N67, A68, K71, E90, E91, N131, I154, F162, K164, N171, M190, M267, N289, L296, K303, L305, F751, or Q754, wherein the mutation type may be self-substitution or deletion.

[0007] In a second aspect, the present invention provides a class of biological materials selected from one or more of the following:

[0008] 1) The polynucleotide molecule encoding the above variants;

[0009] 2) Expression vectors containing polynucleotide molecules as described in 1);

[0010] 3) Host cells containing the polynucleotide molecules described in 1), or host cells containing the expression vectors described in 2).

[0011] Thirdly, the present invention provides a method for preparing the above-mentioned RNA polymerase variant.

[0012] Fourthly, the present invention provides a composition comprising at least one RNA polymerase variant as described herein.

[0013] Fifthly, the present invention provides a kit comprising at least one RNA polymerase variant as described herein.

[0014] In a sixth aspect, the present invention also provides the use of the above-described RNA polymerase variants, compositions or kits in in vitro transcription.

[0015] In a seventh aspect, the present invention also provides a method for preparing RNA. Invention Details

[0017] RNA polymerase variants

[0018] The RNA polymerase variant provided by this invention contains, compared with the amino acid sequence SEQ ID NO: 1, at least one mutation selected from the following amino acid sites: R50, K53, F55, R57, Q58, L59, G62, V64, D66, N67, A68, K71, E90, E91, N131, I154, F162, K164, N171, M190, M267, N289, L296, K303, L305, F751, or Q754, wherein the mutation type may be a substitution or a deletion.

[0019] In some implementations, the variant is replaced with A at the R50 site.

[0020] In some implementations, the variant is replaced with A at the K53 site.

[0021] In some embodiments, the substitution at the F55 site of the variant is selected from A, H, S, Y, D, P, I, or M.

[0022] In some embodiments, the substitution at the R57 site of the variant is selected from A or G.

[0023] In some implementations, the substitution at the Q58 site of the variant is selected from A or G.

[0024] In some implementations, the variant is replaced with G at the L59 site.

[0025] In some embodiments, the substitution at the G62 site of the variant is selected from A, D, I, K, Q, or V.

[0026] In some implementations, the substitution at the V64 site of the variant is selected from A or G.

[0027] In some implementations, the variant is replaced with A at the D66 site.

[0028] In some implementations, the variant is replaced with A at the N67 site.

[0029] In some implementations, the substitution at the A68 site of the variant is selected from D or H.

[0030] In some implementations, the variant is replaced with A at the K71 site.

[0031] In some implementations, the variant is replaced with D at the E90 site.

[0032] In some implementations, the variant is replaced with D at the E91 site.

[0033] In some implementations, the variant is replaced with K at the N131 site.

[0034] In some implementations, the variant is replaced with A at the I154 site.

[0035] In some implementations, the variant is replaced with A at the F162 site.

[0036] In some implementations, the variant is replaced with D at the K164 site.

[0037] In some implementations, the mutation type at position 171 of the variant is deletion.

[0038] In some implementations, the variant is replaced by A at the M190 site.

[0039] In some implementations, the variant is replaced with A at the M267 site.

[0040] In some implementations, the variant is replaced with A at the N289 site.

[0041] In some implementations, the variant is replaced with A at the L296 site.

[0042] In some implementations, the variant is replaced with A at the K303 site.

[0043] In some implementations, the variant is replaced with A at the L305 site.

[0044] In some implementations, the variant is replaced with A at the F751 site.

[0045] In some implementations, the variant is replaced with D at the Q754 site.

[0046] In some embodiments, the amino acid sequence of the variant is relative to SEQ ID. NO:1 contains any mutation selected from the following: R50A, K53A, F55A, F55I, F55M, F55P, F55D, F55H, F55S, F55Y, R57G, R57A, Q58A, Q58G, L59G, G62D, G62I, G62K, G62Q, G62V, G62A, V64G, V64A, D66A, N67A, A68D, A68H, K71A, E90D, E91D, N131K, I154A, F162A, K164D, DEL171, M190A, M267A, N289A, L296A, K303A, L305A, F751A, or Q754D.

[0047] In some embodiments, the RNA polymerase variants provided by the present invention have an amino acid sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher compared to the sequences shown in any of SEQ ID NO: 2-44. In some embodiments, the amino acid sequence of the variant is as shown in any of SEQ ID NO: 2-44.

[0048] biomaterials

[0049] The present invention provides a polynucleotide molecule that encodes the RNA polymerase variant described herein; wherein the polynucleotide molecule may have various modifications in the coding region, as long as the amino acid sequence of the variant does not change with codon degeneracy or with the preferred codon in the organism expressing the variant. In some embodiments, the polynucleotide molecule is as shown in any of SEQ ID NO: 45-88.

[0050] The expression vector provided by this invention comprises a multinucleotide molecule encoding a variant of the RNA polymerase of this invention. In some embodiments, the expression vector further comprises 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 operatively linked to the multinucleotide molecule encoding the variant.

[0051] In some embodiments, the expression vector may be a linear or circular DNA molecule, typically containing elements such as a multiple cloning site, an antibiotic resistance gene, and a replication initiation site. In some embodiments, the expression vector of the present invention is preferably pQE-80L.

[0052] The host cell provided by this invention refers to any cell that is advantageous for the expression of the variants of this invention, that is, any cell that is susceptible after being transformed, transfected or transduced by the expression vector described in this invention, and includes any daughter cells that are different from the parent cells due to mutations that occur during replication.

[0053] In some embodiments, the host cell is a prokaryotic cell, selectable from Gram-positive or Gram-negative bacteria. In some embodiments, the host cell is a Gram-positive bacterium, including but not limited to: *Bacillus*, *Clostridium*, *Enterococcus*, *Bacillus aeruginosa*, *Lactobacillus*, *Lactococcus*, *Bacillus cereus*, *Staphylococcus*, *Streptococcus*, and *Streptomyces*. In some embodiments, the host cell is a Gram-negative bacterium, including but not limited to: *Campylobacter*, *Escherichia coli*, *Flavobacterium*, *Fusobacterium*, *Helicobacter*, *Selenobacter*, *Neisseria*, *Pseudomonas*, *Salmonella*, and *Ureaplasma*. In some embodiments, the host cell is *Escherichia coli* BL21(DE3).

[0054] Preparation methods of RNA polymerase variants

[0055] The method for preparing RNA polymerase variants provided by the present invention includes 1) culturing the host cells described in the present invention under suitable variant expression conditions; and 2) recovering the variant.

[0056] In some embodiments, the method for recovering variants can be a method known in the art, such as centrifugation, filtration, treatment with a crystalline protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatographic methods such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, and combinations thereof.

[0057] In some embodiments, the preparation method further includes a purification step of the variant, which can be a method known in the art, such as chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatographic focusing, and size exclusion chromatography), isoelectric point focusing electrophoresis, ammonium sulfate precipitation, SDS-PAGE, etc.

[0058] Composition

[0059] The composition provided by the present invention comprises at least one RNA polymerase variant described in the present invention.

[0060] The compositions described in this invention can be compositions for storing RNA polymerase variants. In some embodiments, in addition to the aforementioned RNA polymerase variants, the compositions of this invention may optionally contain: buffer components (such as Tris base, Tris-HCl, HEPES, MOPS), salts (such as NaCl), enzyme inhibitors (such as EDTA), reducing agents (such as DTT), surfactants (such as Triton X-100), stabilizers (such as glycerol), and other components. In some embodiments, the compositions of this invention for storing RNA polymerase variants contain: RNA polymerase variants, Tris-HCl, NaCl, EDTA, DTT, Triton X-100, and glycerol.

[0061] The compositions of the present invention can also be in vitro transcription reaction compositions. In some embodiments, in addition to the above-mentioned RNA polymerase variants, the compositions further comprise one or more in vitro transcription reaction reagents, such as: buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, etc. In some embodiments, the compositions further comprise a DNA template. In some embodiments, the compositions further comprise a cap analog. In some embodiments, the in vitro transcription reaction compositions of the present invention comprise: an RNA polymerase variant, a buffer component, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, and a cap analog. In some embodiments, the in vitro transcription reaction compositions of the present invention comprise: an RNA polymerase variant, a buffer component, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, a cap analog, and a DNA template.

[0062] Reagent test kit

[0063] The kit provided by this invention contains at least one RNA polymerase variant described herein. In some embodiments, the kit may further contain one or more in vitro transcription reaction reagents, such as buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, etc. In some embodiments, the kit also contains a cap analogue.

[0064] In some implementations, each component in the kit (if applicable) may be provided in liquid form (e.g., in solution) or in solid form (e.g., dry powder).

[0065] The kit described in this invention may include one or more containers containing one or more components described in this invention and optionally instructions for use.

[0066] Applications or uses

[0067] The present invention also provides the application of the above-mentioned RNA polymerase variants, compositions, or kits in various methods, including but not limited to RNA preparation, RNA probe preparation, RNA vaccine preparation, and protein preparation.

[0068] The present invention also provides the use of the above-mentioned RNA polymerase variants, compositions, or kits in in vitro transcription.

[0069] RNA preparation methods

[0070] This invention also 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 this invention, and incubating in an in vitro transcription reaction system to obtain a target RNA product. In some embodiments, the target RNA product may be selected from dsRNA, ssRNA, mRNA, siRNA, miRNA, piRNA, shRNA, or gRNA, etc. In some embodiments, the RNA product is mRNA.

[0071] Suitable in vitro transcription reaction systems and incubation conditions for generating RNA products are well known in the art. Those skilled in the art can determine appropriate reaction system pH, reaction temperature, reaction time, salt concentration, or whether to add exogenous cofactors, taking into account the optimal activity of RNA polymerase. In some embodiments, the in vitro transcription reaction system of the present invention includes in vitro transcription reaction reagents: one or more buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, etc. In some embodiments, the incubation step of the present invention uses an incubation temperature of 30–50°C, preferably 37°C. In some embodiments, the incubation step of the present invention uses an incubation time of 20–240 min, preferably 60 min.

[0072] In some embodiments, the RNA product prepared using the method described in this invention has higher yield, and / or higher integrity, and / or less dsRNA impurity content, and / or more capped mRNA product, compared to the RNA product prepared using wild-type RNA polymerase (SEQ ID NO: 1). In some embodiments, compared to the RNA product prepared using wild-type RNA polymerase, the relative residual amount of dsRNA impurities (the ratio of residual dsRNA in the variant group to the residual dsRNA in the WT group) of the RNA product prepared using the method described in this invention 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 lower.

[0073] In vitro transcription reaction reagents

[0074] The in vitro transcription reaction reagent described in this invention includes buffer components, nucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatase, magnesium ions, and water (such as DEPC-water, RNase-free water, DNase-free water, sterile purified water, deionized water, distilled water, etc.).

[0075] In some embodiments, the buffering component may be selected from one or more of the following: phosphate buffer, Tris buffer, MOPS buffer, HEPES buffer, citrate buffer, acetate buffer, malate buffer, MES buffer, histidine buffer, PIPES buffer, bis-tris buffer, and ethanolamine buffer.

[0076] In some embodiments, the nucleoside triphosphate may be selected from modified or unmodified nucleoside triphosphates (including analogues thereof). In some embodiments, the nucleoside triphosphate may be selected from unmodified ATP, GTP, CTP, or UTP. In some embodiments, the nucleoside triphosphate may be selected from modified nucleoside triphosphates, and the modification types on the nucleoside include, but are not limited to, m1A (N1-methyladenosine), m6A (N6-methyladenosine), m5C (5-methylcytidine), 5moU (5-methoxyuridine), ψ (pseudouridine), m1ψ (N1-methyl-pseudouridine), and labeled nucleoside triphosphates (the label may be biotin, fluorescent substances, digoxigenin, radioactive elements, etc.).

[0077] In some embodiments, the in vitro transcription reaction reagents described in this invention may be selected from any commercially available RNA in vitro transcription reagents.

[0078] Other implementation plans:

[0079] 1. An RNA polymerase variant, wherein the amino acid sequence of the variant relative to SEQ ID NO: 1 contains any mutation selected from the following: R50, K53, F55, R57, Q58, L59, G62, V64, D66, N67, A68, K71, E90, E91, N131, I154, F162, K164, N171, M190, M267, N289, L296, K303, L305, F751, or Q754, wherein the mutation type may be either substitution or deletion.

[0080] 2. A variant as described in item 1, wherein,

[0081] (1) The substitution at the R50 site is A;

[0082] (2) The substitution at the K53 site is A.

[0083] (3) The substitution at the F55 site is selected from A, H, S, Y, D, P, I or M.

[0084] (4) The substitution at the R57 site is selected from A or G.

[0085] (5) The substitution at the Q58 site is selected from A or G.

[0086] (6) The substitution at L59 site is G.

[0087] (7) The substitution at the G62 site is selected from A, D, I, K, Q, or V.

[0088] (8) The substitution at the V64 site is selected from A or G.

[0089] (9) The substitution at the D66 site is A.

[0090] (10) The substitution at the N67 site is A.

[0091] (11) The substitution at the A68 site is selected from D or H.

[0092] (12) The substitution at the K71 site is A.

[0093] (13) The substitution at the E90 site is D.

[0094] (14) The substitution at the E91 site is D.

[0095] (15) The substitution at the N131 site is K.

[0096] (16) The substitution at the I154 site is A.

[0097] (17) The substitution at the F162 site is A.

[0098] (18) The substitution at the K164 site is D.

[0099] (19) The mutation type at position 171 is deletion.

[0100] (20) The substitution at the M190 site is A.

[0101] (21) The substitution at the M267 site is A.

[0102] (22) The substitution at the N289 site is A.

[0103] (21) The substitution at L296 site is A.

[0104] (22) The substitution at the K303 site is A.

[0105] (23) The substitution at the L305 site is A.

[0106] (24) The substitution at the F751 site is A.

[0107] (25) The substitution at the Q754 site is D.

[0108] 3. The variant as described in item 2, whose amino acid sequence is relative to SEQ ID NO:1 contains any mutation selected from the following: R50A, K53A, F55A, F55I, F55M, F55P, F55D, F55H, F55S, F55Y, R57G, R57A, Q58A, Q58G, L59G, G62D, G62I, G62K, G62Q, G62V, G62A, V64G, V64A, D66A, N67A, A68D, A68H, K71A, E90D, E91D, N131K, I154A, F162A, K164D, DEL171, M190A, M267A, N289A, L296A, K303A, L305A, F751A, or Q754D.

[0109] 4. The variants described in item 3, having an amino acid sequence as shown in any of SEQ ID NO: 2-44.

[0110] 5. Biological materials, selected from one or more of the following:

[0111] 1) A polynucleotide molecule encoding any of the RNA polymerase variants described in items 1-4;

[0112] 2) Expression vectors containing polynucleotide molecules as described in 1);

[0113] 3) A host cell containing a polynucleotide molecule as described in 1), or a host cell containing an expression vector as described in 2).

[0114] 6. A composition comprising any of the variants described in items 1-4.

[0115] 7. A kit containing any of the variants described in items 1-4.

[0116] 8. The use of any variant described in items 1-4, the composition described in item 6, or the kit described in item 6 in in vitro transcription.

[0117] 9. A method for preparing RNA, comprising contacting a DNA template, a modified or unmodified nucleoside triphosphate, with any of the RNA polymerase variants described in items 1-4, incubating in an in vitro transcription reaction system to obtain a target RNA product. Beneficial effects

[0118] This invention mutates wild-type T7 RNA polymerase to obtain a class of RNA polymerase variants with high catalytic efficiency. These variants can significantly reduce the residual dsRNA impurities in in vitro transcribed RNA products, lower purification costs during RNA drug production, reduce the immunogenicity of RNA drugs, and decrease patient risk. Attached Figure Description

[0119] Figure 1 This is a schematic diagram of the construction of recombinant plasmids. Detailed Implementation

[0120] The technical solution of this application will be further described below with reference to specific embodiments. However, the following embodiments are merely examples of this application and do not represent or limit the scope of protection of this application. The scope of protection of this application is determined by 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 techniques used are conventional methods and techniques in the art.

[0121] Enzyme activity is defined as the ability of 1 nmol of enzyme to produce an enzyme activity at 37°C and pH 8.0 for 1 hour. 3 The amount of enzyme required to incorporate ATP into an acid-insoluble precipitate is defined as one active unit.

[0122] Example 1: Preparation of RNA polymerase variants

[0123] The RNA polymerases and their variants shown in Table 1 were synthesized using DNA sequences (SEQ ID NO: 45-88) and then amplified by PCR. The resulting DNA was 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 transformed into E. coli BL21(DE3) using transformation technology. The vector was plated on LB agar plates containing ampicillin and incubated overnight at 37°C. The resulting single colonies were subjected to plasmid extraction and sequencing to obtain recombinant engineered bacteria containing the target gene. The successfully sequenced E. coli recombinant strain was inoculated into LB medium for overnight activation culture, and then inoculated into fermentation broth (LB medium) at 1-5% v / v. The culture was continued until the OD 600 value reached 0.6-0.8. IPTG was added to a final concentration of 0.5 mol / L, and the culture was continued for 4-6 h. The cells were collected by centrifugation at 12000 rpm and 5°C. The cells were washed with 0.2 M PBS buffer (pH 7.0) to obtain the cell stock solution. After sonication, affinity chromatography was performed to purify the RNA polymerase stock solution.

[0124] WT is a wild-type T7 RNA polymerase variant, and its amino acid sequence is as follows:

[0125] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA (SEQ ID NO: 1)

[0126] The RNA polymerase variants and their mutation sites are shown in Table 1 as follows:

[0127] Table 1: Mutation sites of RNA polymerase variants and the corresponding amino acid sequence numbers

[0128]

[0129] Example 2: Validation of in vitro transcription reaction

[0130] 2.1 Unmodified NTP

[0131] (1) The enzyme stock solution obtained in Example 1 was diluted with storage buffer (50 mM Tris-HCl (25℃, pH 7.9), 100 mM NaCl, 0.1 mM EDTA, 2 mM DTT, 0.1% Triton X-100, 50% Glycerol) to an enzyme activity of 300 U / μL. The reaction components (20 μL) in Table 2 were loaded into an octet and mixed well, and centrifuged. The octet was placed on a PCR instrument and reacted at 37℃ for 1 h. Then, 36 μL of magnetic beads (Vazyme, catalog number: N412) were added, mixed well, and incubated at room temperature for 2-5 min. The mixture was placed on a magnetic rack to purify the mRNA. After purification, the mRNA was transferred to an RNase-free centrifuge tube to obtain the purified mRNA.

[0132] (2) The content of dsRNA impurities was tested using a dsRNA detection kit (Vazyme, catalog number: DD3509).

[0133] Table 2: Reaction System Proportions

[0134]

[0135] The dsRNA detection results are shown in Table 3. Compared with the WT group, all polymerase variants shown in Table 3 can effectively reduce the generation of dsRNA impurities during in vitro transcription. The dsRNA residue status (relative dsRNA residue) refers to the ratio of the dsRNA residue in the variant group to the dsRNA residue in the WT group.

[0136] Table 3: dsRNA Residual Status

[0137]

[0138] 2.2 Modified NTPs (m1ψTP)

[0139] (1) The RNA polymerase variant stock solution was diluted with storage buffer (50 mM Tris-HCl (25℃, pH 7.9), 100 mM NaCl, 0.1 mM EDTA, 2 mM DTT, 0.1% Triton X-100, 50% Glycerol) to an enzyme activity of 300 U / μL. The reaction components (20 μL) in Table 4 were loaded into an octet and mixed well, then centrifuged. The octet was placed on a PCR instrument and reacted at 37℃ for 1 h. Then 36 μL of magnetic beads (Vazyme, catalog number: N412) were added and mixed well. The mixture was incubated at room temperature for 2-5 min. The mixture was placed on a magnetic rack to purify the mRNA. After purification, the mRNA was transferred to an RNase-free centrifuge tube to obtain the purified mRNA.

[0140] (2) The content of dsRNA impurities was tested using a dsRNA detection kit (Vazyme, catalog number: DD3509).

[0141] Table 4: Reaction System Proportions

[0142]

[0143] The test results are shown in Table 5. Compared with the WT group, all polymerase variants shown in Table 5 can effectively reduce the generation of dsRNA impurities during in vitro transcription.

[0144] Table 5: dsRNA Residue Status

[0145]

Claims

1. An RNA polymerase variant, characterized in that, The amino acid sequence of the variant has undergone an F55 substitution mutation relative to SEQ ID NO: 1, and the substitution at the F55 site is selected from A, H, S, Y, D, P, I or M. The amino acid sequence of the variant is shown in any one of SEQ ID NO: 4-8, 42-44.

2. Biological materials, selected from one or more of the following: 1) Encoding a polynucleotide molecule that is a variant of the RNA polymerase as described in claim 1; 2) Expression vectors containing polynucleotide molecules as described in 1); 3) A host cell containing a polynucleotide molecule as described in 1), or a host cell containing an expression vector as described in 2).

3. A composition comprising the variant as claimed in claim 1.

4. A kit comprising the variant as described in claim 1.

5. The use of the variant of claim 1, the composition of claim 3, or the kit of claim 4 in in vitro transcription.

6. A method for preparing RNA, comprising contacting a DNA template, a modified or unmodified nucleoside triphosphate, with the RNA polymerase variant of claim 1, incubating in an in vitro transcription reaction system to obtain a target RNA product.

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