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] The present application relates to the field of biotechnology, in particular to RNA polymerase variants, methods for preparing the same and uses thereof. BACKGROUND

[0002] mRNA therapy refers to the use of mRNA-based drugs developed to treat or prevent diseases, and by introducing mRNA as a vaccine or therapeutic agent, it is possible to make in vitro transcribed (IVT) mRNA as an information carrier to guide the production of functional proteins or peptides in the human body. The mRNA vaccine development cycle is relatively short, which can quickly develop new vaccine candidates to respond to viral mutations, and through the dual mechanisms of humoral immunity and T cell immunity, its immunogenicity is strong, the effect is significant, and the production process is simple, easy to develop and mass-produce.

[0003] According to the latest news, in addition to mRNA, the research of circular RNA (circRNA) related drugs has also made a breakthrough. Orna company (Orna Therapeutics) uses circRNA to develop in vivo cell therapy products, and in the research report published at the 2022 American Society of Gene and Cell Therapy (ASGCT) annual meeting, it has been proved that it also has great application potential in other fields such as tumor treatment.

[0004] RNA has made a big difference in the field of drug development such as vaccines, but in the actual production process, the catalyst-T7 RNA polymerase (T7 RNAP) is an important part of cost control in production. The modification of high specific activity enzyme mutants can significantly reduce the amount of enzyme added, thereby reducing production costs, so it is urgent to develop effective high specific activity T7 RNAP. SUMMARY

[0005] In a first aspect, the present application provides a class of RNA polymerase variants, which has an amino acid sequence comprising at least one mutation selected from the following amino acid positions: N370, M306 or A382, compared with SEQ ID NO: 1.

[0006] In a second aspect, the present application provides a class of biological materials selected from one or more of the following:

[0007] 1) a polynucleotide molecule encoding the above-mentioned variant;

[0008] 2) an expression vector comprising the polynucleotide molecule as described in 1);

[0009] 3) a host cell comprising the polynucleotide molecule as described in 1), or a host cell comprising the expression vector as described in 2).

[0010] In a third aspect, the present application provides a method for preparing the above-mentioned RNA polymerase variant.

[0011] In a fourth aspect, the present application provides a composition comprising at least one RNA polymerase variant as described herein.

[0012] In a fifth aspect, the present application provides a kit comprising at least one RNA polymerase variant as described herein.

[0013] In a sixth aspect, the present application provides use of the above-mentioned RNA polymerase variant in preparing RNA in vitro.

[0014] In a seventh aspect, the present application also provides a method for preparing RNA. DETAILED DESCRIPTION

[0016] RNA polymerase variant

[0017] The RNA polymerase variant provided by the present application comprises at least one mutation at an amino acid position selected from N370, M306 or A382, compared to the amino acid sequence of SEQ ID NO: 1, and the type of mutation is selected from substitution or deletion.

[0018] In some embodiments, the substitution at position N370 of the variant can be selected from K, Q, R, Y, T, L or P. In some embodiments, the substitution at position A382 of the variant is K. In some embodiments, the substitution at position M306 of the variant can be selected from K.

[0019] In some embodiments, the amino acid sequence of the variant comprises any one mutation selected from N370K, N370Q, N370R, N370Y, N370T, N370L, N370P, M306K, M306K+N370P, A382K, N370L+A382K or N370P+A382K, relative to SEQ ID NO: 1.

[0020] In some embodiments, the amino acid sequence of the variant is as set forth in any one of SEQ ID NOs: 2-13.

[0021] In some embodiments, the T7 RNA polymerase provided by the present disclosure has higher specific activity than 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 to wild-type polymerase.

[0022] biomaterials

[0023] The present application provides polynucleotides encoding RNA polymerase variants. The polynucleotide sequences are any polynucleotide sequences encoding variants without changing the amino acid sequence due to the degeneracy of codons or the preference of codons of host cells expressing polypeptides. In some embodiments, the polynucleotide sequences encoding the RNA polymerase variants of the present application can be selected from SEQ ID NO: 15-26.

[0024] The expression vector provided by the present application comprises a polynucleotide molecule encoding the RNA polymerase variants of the present application. In some embodiments, the expression vector further comprises one or more regulatory sequences, such regulatory sequences include but are not limited to enhancers, promoters, leader peptide sequences, signal peptide sequences, terminator sequences; wherein the regulatory sequences are operably linked to the polynucleotide molecule encoding the variants.

[0025] In some embodiments, the expression vector can be a linear or circular DNA molecule, usually comprising elements such as a multiple cloning site, a resistance gene, a replication initiation site, etc. In some embodiments, the expression vector described in the present application is preferably pQE-80L.

[0026] The host cell provided by the present application refers to any cell that is conducive to the expression of the variants of the present application, i.e. any cell that is susceptible to transformation, transfection or transduction with the expression vector described in the present application, covering any progeny cells that are not identical to the parent cell due to mutations that occur during replication.

[0027] In some embodiments, the host cell is a prokaryotic cell, which can be selected from a gram-positive bacterium or a gram-negative bacterium. In some embodiments, the host cell is a gram-positive bacterium, including but not limited to: Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, 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, Limnobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma. In some embodiments, the host cell is Escherichia coli BL21 (DE3).

[0028] Methods of making RNA polymerase variants

[0029] The present application provides methods of making RNA polymerase variants, comprising 1) culturing the host cell described herein under conditions suitable for expression of the variant; and 2) recovering the variant.

[0030] In some embodiments, the method of recovering the variant can be any method known in the art, such as centrifugation, filtration, treatment with a crystallizing protein precipitant (salting out), extraction, sonication, ultrafiltration, dialysis, various chromatographic methods such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, affinity chromatography, HPLC, and combinations thereof.

[0031] In some embodiments, the method of making further comprises a step of purifying the variant, which can be any method known in the art, such as chromatography (e.g., ion-exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing, and size-exclusion chromatography), isoelectric focusing electrophoresis, ammonium sulfate precipitation, SDS-PAGE, and the like.

[0032] Compositions

[0033] The present application provides compositions comprising at least one RNA polymerase variant described herein.

[0034] The compositions described herein can be a composition storing the RNA polymerase variant. In some embodiments, the compositions described herein can optionally comprise, in addition to the RNA polymerase variant described above, 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 the like. In some embodiments, the compositions storing the RNA polymerase variant described herein comprise the RNA polymerase variant, Tris-HCl, NaCl, EDTA, DTT, Triton X-100, and glycerol.

[0035] In some embodiments, the compositions further comprise a template DNA. In some embodiments, the compositions further comprise 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 pyrophosphatases, magnesium ions, and the like.

[0036] Kits

[0037] The kits provided herein comprise at least one RNA polymerase variant as described herein.

[0038] In some embodiments, the kits further comprise 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 pyrophosphatases, magnesium ions, and the like. In one embodiment, the in vitro transcription components can be selected from commercially available RNA in vitro transcription reagents.

[0039] Uses

[0040] The present application provides uses of at least one variant as described herein in the preparation of RNA in vitro. In some embodiments, the preparation of RNA in vitro comprises contacting a DNA template, modified or unmodified nucleoside triphosphates with at least one RNA polymerase variant as described herein, incubating in an in vitro transcription reaction system, and obtaining the target product. In some embodiments, the preparation of RNA in vitro further comprises magnesium ions.

[0041] The present application also provides uses of at least one RNA polymerase variant as described herein in the synthesis of RNA drugs.

[0042] Methods of preparation

[0043] The present application provides a method of preparing RNA, comprising contacting a DNA template, modified or unmodified nucleoside triphosphates with at least one RNA polymerase variant as described herein, incubating in an in vitro transcription reaction system, and obtaining the target product.

[0044] 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 produced using the RNA polymerase variant described herein 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% improved integrity relative to the product produced using wild-type RNA polymerase (SEQ ID NO: 1). In some embodiments, the target product produced using the RNA polymerase variant described herein has improved yield relative to the product produced using wild-type RNA polymerase (SEQ ID NO: 1).

[0045] In some embodiments, the RNA product produced using the method described herein has higher yield, and / or has higher integrity, and / or has less dsRNA impurity content, and / or more capped mRNA product, etc. relative to the product produced using wild-type RNA polymerase (SEQ ID NO: 1).

[0046] 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 RNAse inhibitor, inorganic pyrophosphatase, magnesium ion. In some embodiments, the in vitro transcription buffer system further comprises water (e.g., 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 comprises a cap analog, which can be selected from unmethylated cap analog, dimethylated cap analog, trimethylated cap analog, dimethylated symmetric cap analog, or anti reverse cap analog.

[0047] Other embodiments

[0048] 1. An RNA polymerase variant, the variant comprising a mutation at any one of the positions selected from N370, M306, or A382 relative to SEQ ID NO: 1.

[0049] 2. The variant of item 1, wherein:

[0050] (1) the substitution at the N370 position is selected from K, Q, R, Y, T, L, or P;

[0051] (2) the substitution at position A382 is K;

[0052] (3) the substitution at position M306 is K;

[0053] 3. The variant of item 1, having an amino acid sequence comprising any one mutation selected from the group consisting of N370K, N370Q, N370R, N370Y, N370T, N370L, N370P, M306K, M306K+N370P, A382K, N370L+A382K, or N370P+A382K, relative to SEQ ID NO: 1.

[0054] 4. The variant of item 1, having an amino acid sequence as set forth in any one of SEQ ID NOs: 2-13.

[0055] 5. A biological material selected from one or more of the following:

[0056] 1) a polynucleotide molecule encoding the RNA polymerase variant of any one of items 1-4;

[0057] 2) an expression vector comprising the polynucleotide molecule of item 1);

[0058] 3) a host cell comprising the polynucleotide molecule of item 1), or a host cell comprising the expression vector of item 2).

[0059] 6. A method of producing the variant of any one of items 1-4, comprising: (1) culturing the host cell of item 5; and (2) recovering the variant.

[0060] 7. A composition comprising the variant of any one of items 1-4.

[0061] 8. A kit comprising the variant of any one of items 1-4.

[0062] 9. Use of the variant of any one of items 1-4, the composition of item 7, or the kit of item 8, in in vitro transcription.

[0063] 10. Use of an RNA polymerase variant having an amino acid sequence as set forth in any one of SEQ ID NOs: 2-13, in the preparation of RNA by in vitro transcription.

[0064] 11. Use of the RNA polymerase variant of any one of items 1-4, in the synthesis of an RNA drug.

[0065] 12. A method for preparing RNA, wherein the method comprises contacting a DNA template, modified or unmodified nucleoside triphosphates with the RNA polymerase variant of any one of the first to fourth items, incubating in an in vitro transcription reaction system, and obtaining a target RNA product.

[0066] Advantages

[0067] The present application provides an RNA polymerase variant, compared with wild-type T7 RNA polymerase, the polymerase variant has high catalytic efficiency, high specific activity, can significantly reduce the amount of enzyme added, and further 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 DRAWINGS

[0068] Figure 1 Figure 1 is a schematic diagram for construction of a recombinant plasmid;

[0069] Figure 2 Figure 3 is the specific activity of different RNA polymerase variants;

[0070] Figure 3 Figure 4 is the RNA yield generated by different RNA polymerase variants in an in vitro transcription reaction. DETAILED DESCRIPTION

[0071] The technical solutions of the present application will be further described below in combination with specific examples. However, the following examples 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 examples, if not specifically stated, 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.

[0072] The enzyme activity is defined as: under the condition of 37℃, pH 8.0, the amount of enzyme required for incorporating 1 nmol of [H]ATP into acid-insoluble precipitate within 1 hour is defined as 1 unit of activity. 3 The enzyme activity is defined as: under the condition of 37℃, pH 8.0, the amount of enzyme required for incorporating 1 nmol of [H]ATP into acid-insoluble precipitate within 1 hour is defined as 1 unit of activity.

[0073] Example 1 Preparation of RNA polymerase variant

[0074] DNA fragments according to SEQ ID NO: 14-26 (amino acid sequence corresponding to SEQ ID NO: 1-13) were synthesized, and after PCR amplification, the BseRI and HindIII enzyme digestion sites of the expression vector pQE-80L were introduced to obtain a recombinant expression vector. The constructed vector was introduced into E. coli BL21 (DE3) by transformation technology, and after coating and screening on an antibiotic (ampicillin) resistant plate, a clonal strain was obtained. The obtained strain was cultured in a 37°C incubator overnight, and the single colonies that grew out were subjected to plasmid extraction and sequencing, and finally a recombinant engineering bacterium containing the target gene was obtained. The E. coli recombinant strain with successful sequencing was inoculated into LB medium for overnight activation culture, and then 1-5% V / V was inoculated into fermentation broth (LB medium). The culture was incubated to an OD 600 value of 0.6-0.8, and then 0.5 mol / L IPTG was added at a final concentration. After 4-6 h of incubation at 37°C, the strain was collected by centrifugation at 12,000 rpm at 5°C, and the collected strain was washed with 0.2M PBS buffer with a pH value of 7.0 to obtain the bacterial body. After ultrasonic crushing, affinity chromatography (His trap HP, 29-0510-21, Cytiva) purification was performed to obtain the RNA polymerase stock solution. The correspondence between the wild type and variants of the RNA polymerase and the amino acid sequence is shown in Tables 1-1 to 1-3:

[0075] Table 1-1

[0076]

[0077] Table 1-2

[0078]

[0079]

[0080] Table 1-3

[0081]

[0082]

[0083] Example 2: T7 RNAP activity assay

[0084] Take two rows of eight rows and place them in the ice box, assuming that the above purified enzyme activity is 500 U / μL, and gradient dilution is carried out 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, and add blank control 0 U / μL; the T7 RNA polymerase standard (Vazyme, item number DD4101R-01 is diluted 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, and add blank control 0 U / μL, the enzyme activity determination system is shown in Table 2.

[0085] Table 2: Enzyme activity detection system

[0086]

[0087] After mixing the above solution, centrifuge for 15 s, react at 37℃-50℃ for 30 min (ABIPCR instrument), configure fluorescent dye (Vazyme, item number EQ212 fluorescent dye component) mixture, add 180 μL fluorescent dye mixture to the eight-row array after reaction, mix well, and take 180 μL and add to a black enzyme label plate, use an enzyme label instrument for detection, set parameters excitation wavelength 630 nm, emission wavelength 680 nm, and use plotting software to calculate the regression equation (slope k) of T7 RNA polymerase standard and enzyme variant sample enzyme activity.

[0088] Y (standard) = k2X + b

[0089] Y (sample) = k1X + b

[0090] Actual enzyme activity: sample actual enzyme activity = k1 / k2 x 500 U

[0091] Actual enzyme specific activity: sample actual enzyme specific activity = sample actual enzyme activity / protein concentration (μg)

[0092] The results are shown in Figure 2 The above mutant T7 RNAP has significantly improved enzyme specific activity at 37℃, especially mutant A382K, which significantly improves the enzyme specific activity to 1822.13 U / μg, with a >5-fold improvement effect.

[0093] Example 3: In vitro transcription reaction

[0094] 1) The above enzyme stock solution was diluted with storage buffer (Vazyme, item number: GMP4101PB) to an enzyme activity of 300 U / μL. The reaction components (20 μL) in Table 3-1 were loaded into an eight-row array and mixed, and centrifuged; the eight-row array was placed in a PCR instrument for 1 h of reaction at 37°C, then 36 μL of magnetic beads (Vazyme, item number: N412) were added and mixed, followed by incubation at room temperature for 2-5 min; the mixture was placed on a magnetic stand to purify the mRNA, and after purification, the purified mRNA was transferred to an RNase-free centrifuge tube, and the concentration was detected using One drop (RNA yield (μg) = concentration (ng / μL) * volume (μL) / 1000);

[0095] 2) 200 ng of RNA was taken and subjected to capillary electrophoresis detection using Qsep400 automatic nucleic acid analyzer, R1 clamp (injection and separation 4KV, 20 nt Marker), and the integrity of the mRNA was detected (intact RNA product peak area / total RNA product peak area).

[0096] Table 3-1: Reaction system ratio

[0097]

[0098] Table 3-2

[0099]

[0100] As Figure 3 As shown in Table 3-2, the high specific activity mutant was reacted according to the same enzyme activity, the protein dosage was reduced, and at the same time, the yield did not decrease significantly (all >180 μg), and the integrity of the RNA product was improved.

Claims

1. A variant of RNA polymerase characterized in that, The amino acid sequence of the variant is set forth in SEQ ID NO:

11.

2. Biomaterial, characterized in that, The biological material is selected from one or more of the following: 1) a polynucleotide molecule encoding the RNA polymerase variant of claim 1; 2) an expression vector comprising the polynucleotide molecule as set forth in 1); 3) a host cell comprising the polynucleotide molecule as set forth in 1), or a host cell comprising the expression vector as set forth in 2).

3. A method of producing a variant according to claim 1, c h a r a c t e r i s e d in that, Comprising: (1) culturing the host cell as set forth in claim 2; and (2) recovering the variant.

4. Composition, characterized in that, Comprising the variant as set forth in claim 1.

5. A kit, characterized in that, Comprising the variant as set forth in claim 1.

6. Use of the variant of claim 1, the composition of claim 4, or the kit of claim 5 in in vitro transcription.

7. Use of an RNA polymerase variant for the preparation of RNA by in vitro transcription, characterized in that, The amino acid sequence of the polymerase variant is set forth in SEQ ID NO:

11.

8. A method of preparing RNA, characterized by, The method comprises contacting a DNA template, modified or unmodified nucleoside triphosphates with the RNA polymerase variant of claim 1, incubating in an in vitro transcription reaction system, and obtaining a target RNA product.

9. Use of the RNA polymerase variant of claim 1 in synthesis of RNA drugs.

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