RNA polymerase variants and uses thereof

Abstract

The invention provides an RNA polymerase variant and application thereof, and relates to the technical field of biology, the enzyme specific activity of the variant is obviously improved compared with that of a wild type, and the addition amount of polymerase is reduced, so that the cost is saved. Meanwhile, the mutant also effectively reduces the generation of incomplete fragment impurities and improves the integrity. In addition, the invention further provides a method for generating RNA through in-vitro transcription.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and particularly to RNA polymerase variants, methods for preparing the same, and their applications. Background Art

[0002] 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. mRNA vaccines have a relatively short R & D cycle, can quickly develop new candidate vaccines to cope with virus mutations, and through the dual mechanisms of humoral immunity and T cell immunity, they have strong immunogenicity, remarkable effects, and a simple production process, making it easy to conduct efficient R & D and large-scale production.

[0003] According to the latest news, in addition to mRNA, breakthroughs have also been made in the research of circular RNA (circRNA) - related drugs. Orna Therapeutics has developed an in vivo cell therapy product using circRNA, and in the research report announced at the 2022 annual meeting of the American Society of Gene and Cell Therapy (ASGCT), its great application potential in other fields such as tumor treatment has been demonstrated.

[0004] RNA has shown great promise in the R & D field of drugs such as vaccines, but in the actual production process, the catalyst - T7 RNA polymerase (T7 RNAP) is an important part of cost control. The modification of highly specific activity enzyme mutants can significantly reduce the enzyme addition amount, thereby reducing production costs. Therefore, there is an urgent need to develop effective T7 RNAP with high specific activity. Summary of the Invention

[0005] In a first aspect, this application provides a class of RNA polymerase variants, whose amino acid sequences contain at least one mutation selected from the following amino acid sites compared with SEQ ID NO: 1: N370, M306, or A382.

[0006] In a second aspect, this application provides a class of biological materials, which are selected from one or more of the following: 1) Polynucleotide molecules encoding the above - mentioned variants; 2) Expression vectors containing the polynucleotide molecules described in 1); 3) Host cells containing the polynucleotide molecules described in 1), or host cells containing the expression vectors described in 2).

[0007] In a third aspect, this application provides a method for preparing the above - mentioned RNA polymerase variants.

[0008] Fourth aspect, the present application provides a composition comprising at least one RNA polymerase variant as described in the present application.

[0009] Fifth aspect, the present application provides a kit comprising at least one RNA polymerase variant as described in the present application.

[0010] Sixth aspect, the present application provides the use of the above-mentioned RNA polymerase variant in the preparation of RNA by in vitro transcription.

[0011] Seventh aspect, the present application further provides a method for preparing RNA. DETAILED DESCRIPTION OF THE INVENTION

[0013] RNA POLYMERASE VARIANT The RNA polymerase variant provided by the present application, compared with the amino acid sequence SEQ ID NO: 1, contains at least one mutation selected from the following amino acid sites: N370, M306 or A382, and the mutation type is selected from substitution or deletion.

[0014] In some embodiments, the substitution at the N370 position of the variant can be selected from K, Q, R, Y, T, L or P. In some embodiments, the substitution at the A382 position of the variant is K. In some embodiments, the substitution at the M306 position of the variant can be selected from K.

[0015] In some embodiments, the amino acid sequence of the variant relative to SEQ ID NO: 1 contains any one of the following mutations: N370K, N370Q, N370R, N370Y, N370T, N370L, N370P, M306K, M306K + N370P, A382K, N370L + A382K or N370P + A382K.

[0016] In some embodiments, the amino acid sequence of the variant is as shown in any one of SEQ ID NOs: 2-13.

[0017] In some embodiments, the T7 RNA polymerase provided by the present disclosure has a higher specific activity compared with the wild-type T7 RNA polymerase. In some embodiments, the specific activity of the polymerase variant is increased by 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 4-fold compared with the wild-type polymerase.

[0018] BIOMATERIAL The present application provides polynucleotides encoding RNA polymerase variants. Due to the degeneracy of codons or the codon preferences of the host cells expressing the polypeptide, without changing the amino acid sequence, the polynucleotide sequence is any polynucleotide sequence encoding the variant. In some embodiments, the polynucleotide sequences encoding the RNA polymerase variants of the present application may be selected from SEQ ID NO: 15 - 26.

[0019] The expression vectors provided by the present application contain polynucleotide molecules encoding the RNA polymerase variants of the present application. In some embodiments, the expression vectors further contain one or more regulatory sequences, such regulatory sequences including but not limited to enhancers, promoters, leader peptide sequences, signal peptide sequences, terminator sequences; wherein the regulatory sequences are operably linked to the polynucleotide molecules encoding the variants.

[0020] In some embodiments, the expression vectors may be linear or circular DNA molecules, usually containing elements such as multiple cloning sites, resistance genes, replication origins, etc. In some embodiments, the expression vectors described in the present application are preferably pQE - 80L.

[0021] The host cells provided by the present application refer to any cells that are favorable for the expression of the variants of the present application, that is, any cells that are susceptible after being transformed, transfected or transduced with the expression vectors described in the present application, covering any progeny cells that are different from the parental cells due to mutations occurring during replication.

[0022] In some embodiments, the host cells are prokaryotic cells, and may be selected from Gram - positive bacteria or Gram - negative bacteria. In some embodiments, the host cells are Gram - positive bacteria, including but not limited to: Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. In some embodiments, the host cells are Gram - negative bacteria, including but not limited to: Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Pelobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma. In some embodiments, the host cell is Escherichia coli BL21(DE3).

[0023] Method for preparing RNA polymerase variants The method for preparing RNA polymerase variants provided by the present application includes: 1) culturing the host cells described in the present application under conditions suitable for variant expression; and 2) recovering the variant.

[0024] In some embodiments, the method for recovering variants may be methods well-known in the art, such as centrifugation, filtration, treatment with a protein precipitating agent by crystallization (salting out), extraction, sonication, ultrafiltration, dialysis, various chromatographies such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, and combinations of the above methods.

[0025] In some embodiments, the preparation method further includes a step of purifying the variant, and the purification step may be methods well-known in the art, such as chromatography (such as ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing, and size exclusion chromatography), isoelectric focusing electrophoresis, ammonium sulfate precipitation, SDS-PAGE, etc.

[0026] Composition The composition provided by the present application comprises at least one RNA polymerase variant as described in the present application.

[0027] The composition described in the present application may be a composition for storing RNA polymerase variants. In some embodiments, in addition to the above RNA polymerase variants, the composition described in the present application 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 composition for storing RNA polymerase variants described in the present application contains: RNA polymerase variant, Tris-HCl, NaCl, EDTA, DTT, Triton X-100, and glycerol.

[0028] In some embodiments, the composition further contains template DNA. In some embodiments, the composition further contains at least one in vitro transcription component, and the in vitro transcription component may be selected from one or more buffer components, modified or unmodified ribonucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatase, magnesium ions, etc.

[0029] Kit The kit provided by the present application contains at least one RNA polymerase variant as described in the present application.

[0030] In some embodiments, the kit further contains at least one in vitro transcription component, and the in vitro transcription component may be selected from one or more buffer components, modified or unmodified ribonucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatase, magnesium ions, etc. In one embodiment, the in vitro transcription system components may be selected from commercially available in vitro RNA transcription reagents.

[0031] Application The present application provides the use of at least one variant as described in the present application in the in vitro transcription for preparing RNA. In some embodiments, the in vitro transcription for preparing RNA includes contacting a DNA template, modified or unmodified nucleoside triphosphates with at least one RNA polymerase variant as described in the present application, and incubating in an in vitro transcription reaction system to obtain a target product. In some embodiments, the in vitro transcription for preparing RNA further includes magnesium ions.

[0032] The present application also provides the use of at least one RNA polymerase variant as described in the present application in the synthesis of RNA drugs.

[0033] Preparation method The present application provides a method for preparing RNA, which includes contacting a DNA template, modified or unmodified nucleoside triphosphates with at least one RNA polymerase variant as described in the present application, and incubating in an in vitro transcription reaction system to obtain a target product.

[0034] In some embodiments, the target product includes but is not limited to mRNA, siRNA, gRNA, saRNA, dsRNA, ssRNA, miRNA, piRNA, shRNA, etc. In some embodiments, the target product prepared by using the RNA polymerase variant as described in the present application has 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% higher integrity compared to the product prepared by using the wild-type RNA polymerase (SEQ ID NO: 1). In some embodiments, the yield of the target product prepared by using the RNA polymerase variant as described in the present application is increased compared to the product prepared by using the wild-type RNA polymerase (SEQ ID NO: 1).

[0035] In some embodiments, the RNA product prepared by using the method as described in the present application has a higher yield, and / or higher integrity, and / or less dsRNA impurity content, and / or more capped mRNA products, etc. compared to the product prepared by using the wild-type RNA polymerase (SEQ ID NO: 1).

[0036] In some embodiments, the in vitro transcription reaction system contains one or more buffer components. In some embodiments, the buffer components can be selected from Tris-HCl, Hepes, citric acid, or commercially available buffer components. In some embodiments, the in vitro transcription buffer system further contains an RNase inhibitor, inorganic pyrophosphatase, and magnesium ions. In some embodiments, the in vitro transcription buffer system further contains water (such as DEPC-water, RNase-free water, DNase-free water, sterilized purified water, deionized water, distilled water, etc.). In some embodiments, the in vitro transcription buffer system further includes a cap analog, and the cap analog 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.

[0037] Other embodiments 1. An RNA polymerase variant, the amino acid sequence of which contains a mutation at any one of the following sites relative to SEQ ID NO: 1: N370, M306, or A382.

[0038] 2. The variant according to item 1, wherein: (1) The substitution at the N370 position is selected from K, Q, R, Y, T, L, or P; (2) The substitution at the A382 position is K; (3) The substitution at the M306 position is K; 3. The variant according to item 1, the amino acid sequence of which contains a mutation at any one of the following sites relative to SEQ ID NO: 1: N370K, N370Q, N370R, N370Y, N370T, N370L, N370P, M306K, M306K+N370P, A382K, N370L+A382K, or N370P+A382K.

[0039] 4. The variant according to item 1, the amino acid sequence of which is as shown in any one of SEQ ID NOs: 2-13.

[0040] 5. A biological material, which is selected from one or more of the following: 1) A polynucleotide molecule encoding the RNA polymerase variant according to any one of items 1-4; 2) An expression vector containing the polynucleotide molecule as described in 1); 3) A host cell containing the polynucleotide molecule as described in 1), or a host cell containing the expression vector as described in 2).

[0041] 6. A method for preparing any of the variants described in items 1-4, characterized by comprising: (1) culturing the host cell as described in item 5; and (2) recovering the variant.

[0042] 7. A composition comprising any of the variants described in items 1-4.

[0043] 8. A kit comprising any of the variants described in items 1-4.

[0044] 9. Use of any of the variants described in items 1-4, the composition described in item 7, or the kit described in item 8 in in vitro transcription.

[0045] 10. Use of an RNA polymerase variant in the preparation of RNA by in vitro transcription, wherein the amino acid sequence of the variant is as shown in any of SEQ ID NO: 2-13.

[0046] 11. Use of any of the RNA polymerase variants described in items 1-4 in the synthesis of RNA drugs.

[0047] 12. A method for preparing RNA, characterized in that the method comprises contacting a DNA template, modified or unmodified nucleoside triphosphates with any of the RNA polymerase variants described in items 1-4, and incubating in an in vitro transcription reaction system to obtain a target RNA product.

[0048] Advantageous Effects The present application provides an RNA polymerase variant. Compared with the wild-type T7 RNA polymerase, the polymerase variant has high catalytic efficiency, has a high specific activity, can significantly reduce the enzyme addition amount, and thus reduce the production cost. In addition, the RNA variant provided by the present application can effectively improve the integrity of the RNA product. Brief Description of the Drawings

[0049] Figure 1 It is a schematic diagram for the construction of a recombinant plasmid; Figure 2 It is the specific activity of different RNA polymerase variants; Figure 3 It is the RNA yield generated by different RNA polymerase variants in an in vitro transcription reaction. Detailed Embodiments

[0050] The technical solutions of the present application will be further described below in conjunction with specific embodiments. However, the following embodiments are only examples of the present application and do not represent or limit the protection scope of the present application. The protection scope of the present application 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.

[0051] The definition of enzyme activity is as follows: Under the conditions of 37 °C and pH 8.0, the amount of enzyme required to incorporate 1 nmol 3 of [H] ATP into acid-insoluble precipitate within 1 hour is defined as 1 activity unit.

[0052] Example 1 Preparation of RNA polymerase variants DNA fragments were synthesized according to the DNA sequences shown in SEQ ID NO: 14-26 (the corresponding amino acid sequences are SEQ ID NO: 1-13). After PCR amplification, they were introduced into the BseRI and HindIII restriction sites of the expression vector pQE-80L to obtain recombinant expression vectors. The constructed vectors were introduced into E. coli BL21(DE3) by transformation technology. After screening by spreading on an antibiotic (ampicillin) resistance plate, clone strains were obtained. The obtained strains were cultured overnight in a 37 °C incubator. The single colonies that grew out were subjected to plasmid extraction and sequencing. Finally, recombinant engineering bacteria containing the target gene were obtained. The successfully sequenced E. coli recombinant strains were inoculated into LB medium for overnight activation culture, and then inoculated at 1-5% V / V into the fermentation broth (LB medium). They were cultured until the OD600 value reached 0.6-0.8. IPTG with a final concentration of 0.5 mol / L was added and the culture was continued at 37 °C for 4-6 h. Then, the strains were collected by centrifugation at 12,000 rpm and 5 °C. The collected strains were washed with 0.2 M PBS buffer with a pH value of 7.0 to obtain cell pellets. After ultrasonic disruption, affinity chromatography (His trap HP, 29-0510-21, Cytiva) was used for purification to obtain the crude RNA polymerase solution. The corresponding relationships between the wild-type RNA polymerase and its variants and the amino acid sequences are shown in Tables 1-1 to 1-3: Table 1-1

[0053] Table 1-2

[0054] Table 1-3

[0055] Example 2: T7 RNAP activity assay Take two rows of eight-well strips and place them in an ice box. Assume that the purified enzyme activity is 500 U / μL as described above, and perform serial dilutions until the enzyme activities are 0.1 U / μL, 0.2 U / μL, 0.3 U / μL, 0.4 U / μL, 0.5 U / μL, 0.6 U / μL, and add a blank control of 0 U / μL; Dilute the T7 RNA polymerase standard (Vazyme, catalog number DD4101R-01) to 0.1 U / μL, 0.2 U / μL, 0.3 U / μL, 0.4 U / μL, 0.5 U / μL, 0.6 U / μL according to its accurate enzyme activity, add a blank control of 0 U / μL, and the enzyme activity assay system is shown in Table 2.

[0056] Table 2: Enzyme Activity Detection System

[0057] Mix the above solutions and centrifuge for 15 s, react at 37 °C - 50 °C for 30 min (ABI PCR instrument), prepare a mixed solution of fluorescent dye (fluorescent dye component in Vazyme, catalog number EQ212), add 180 μL of the fluorescent dye mixed solution to the reacted eight-well strip and mix well, pipette 180 μL and add it to a black microplate, and use a microplate reader for detection. Set the parameters: excitation wavelength 630 nm, emission wavelength 680 nm, and use graphing software to calculate the regression equation (slope is k) of the enzyme activities of the T7 RNA polymerase standard and the enzyme variant samples.

[0058] Y (standard) = k2X + b Y (sample) = k1X + b Actual enzyme activity: Sample actual enzyme activity = k1 / k2 × 500 U Actual specific enzyme activity: Sample actual specific enzyme activity = sample actual enzyme activity / protein concentration (μg) The results are as Figure 2 shown. The specific enzyme activities of the above mutant T7 RNAP at 37 °C were all significantly increased. In particular, for mutant A382K, the specific enzyme activity was significantly increased to 1822.13 U / μg, and the improvement effect was > 5-fold.

[0059] Example 3: In Vitro Transcription Reaction 1) Dilute the above enzyme stock solution to an enzyme activity of 300 U / μL with storage buffer (Vazyme, catalog number: GMP4101PB). Aliquot 20 μL of the reaction components shown in Table 3-1 into an eight-strip tube, mix well, and centrifuge. Incubate the eight-strip tube in a PCR instrument at 37 °C for 1 h, then add 36 μL of magnetic beads (Vazyme, catalog number: N412), mix well, and incubate at room temperature for 2 - 5 min. Place the mixture on a magnetic stand to purify the mRNA. After purification, transfer it to an RNase-free centrifuge tube to obtain purified mRNA, and use One drop to measure the concentration (RNA yield (μg) = concentration (ng / μL) × volume (μL) / 1000). 2) Take 200 ng of RNA and perform capillary electrophoresis using a Qsep400 fully automated nucleic acid analyzer with an R1 cassette (injection and separation at 4 KV, 20 nt Marker) to detect the integrity of the mRNA (integrated RNA product peak area / total RNA product peak area).

[0060] Table 3-1: Reaction system ratio

[0061] Table 3-2

[0062] As Figure 3 shown in Table 3-2, when the high specific activity mutants are fed in the reaction with the same enzyme activity, the protein feed amount is reduced while the yield does not decrease significantly (both > 180 μg), and at the same time, the integrity of the RNA products is improved to a certain extent.

Claims

1. An RNA polymerase variant, characterized in that, The amino acid sequence of said variant contains a mutation at any site selected from the following with respect to SEQ ID NO: 1: N370, M306 or A382.

2. The variant according to claim 1, characterized in that, Wherein: (1) The substitution at position N370 is selected from K, Q, R, Y, T, L or P; (2) The substitution at position A382 is K; (3) The substitution at position M306 is K.

3. The variant according to claim 1, characterized in that, The amino acid sequence of said variant contains a mutation at any site selected from the following with respect to SEQ ID NO: 1: N370K, N370Q, N370R, N370Y, N370T, N370L, N370P, M306K, M306K+N370P, A382K, N370L+A382K or N370P+A382K.

4. The variant according to claim 1, characterized in that, The amino acid sequence of said variant is as shown in any one of SEQ ID NOs: 2-13.

5. 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 1-4; 2) An expression vector containing the polynucleotide molecule as described in 1); 3) A host cell containing the polynucleotide molecule as described in 1), or a host cell containing the expression vector as described in 2).

6. A method for preparing a variant according to any one of claims 1-4, characterized in that, Including: (1) Culturing the host cell as described in claim 5; and (2) Recovering the variant.

7. A composition, characterized in that, Containing a variant as described in any one of claims 1-4.

8. Kit, characterized in that, Containing a variant as described in any one of claims 1-4.

9. Use of the variant as described in any one of claims 1-4, the composition as described in claim 7 or the kit as described in claim 8 in in vitro transcription.

10. Use of an RNA polymerase variant in the preparation of RNA by in vitro transcription, characterized in that, The amino acid sequence of said polymerase variant is as shown in any one of SEQ ID NOs: 2-13.

11. A method for preparing RNA, characterized in that, The method includes contacting a DNA template, modified or unmodified nucleoside triphosphates with an RNA polymerase variant as described in any one of claims 1-4, incubating in an in vitro transcription reaction system to obtain a target RNA product.

12. Use of the RNA polymerase variant as described in any one of claims 1-4 in the synthesis of RNA drugs.

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