RNA polymerase variants and their applications

Abstract

The present application provides an RNA polymerase variant and its application, relating to the field of biotechnology. The variant exhibits significantly improved enzyme specific activity compared to the wild-type, reducing the amount of polymerase added and thus saving costs. Furthermore, the variant effectively reduces the production of incomplete fragment impurities, thereby improving integrity. Furthermore, the present application provides a method for generating RNA through in vitro transcription.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to RNA polymerase variants, preparation methods and applications thereof. Background Art

[0002] mRNA therapy refers to the use of mRNA-based drugs to treat or prevent diseases. By introducing mRNA as a vaccine or therapeutic agent, in vitro transcribed (IVT) mRNA acts as an information carrier to guide the production of functional proteins or peptides in the human body. mRNA vaccines have a relatively short R&D cycle, enabling the rapid development of new vaccine candidates to respond to viral mutations. Through dual mechanisms of humoral and T-cell immunity, they are highly immunogenic and effective, and their simple production process facilitates efficient R&D and large-scale production.

[0003] According to the latest news, in addition to mRNA, research on circular RNA (circRNA)-related drugs has also made breakthroughs. Orna Therapeutics has developed an in vivo cell therapy product using circRNA. In a research report presented at the 2022 American Society of Gene and Cell Therapy (ASGCT) Annual Meeting, it has demonstrated its huge potential for application in other fields, such as cancer treatment.

[0004] RNA has made great progress in the research and development of vaccines and other drugs, but in the actual production process, the catalyst - T7 RNApolymerase (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. Therefore, there is an urgent need to develop an effective high-specific-activity T7 RNAP. Summary of the Invention

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

[0006] In a second aspect, the present application provides a type of biomaterial, which is selected from one or more of the following:

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

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

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

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

[0014] In a seventh aspect, the present application also provides a method for preparing RNA. Detailed Description of the Invention

[0016] RNA polymerase variants

[0017] The RNA polymerase variant provided herein comprises, compared with the amino acid sequence of SEQ ID NO: 1, at least one mutation selected from the following amino acid sites: N370, M306 or A382, wherein the mutation type is selected from substitution or deletion.

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

[0019] In some embodiments, the amino acid sequence of the variant comprises 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.

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

[0021] In some embodiments, the T7 RNA polymerase provided herein has a higher specific activity than wild-type T7 RNA polymerase. In some embodiments, the polymerase variant has a specific activity that is 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, or 4-fold higher than wild-type polymerase.

[0022] Biomaterials

[0023] The present application provides polynucleotides encoding RNA polymerase variants. Due to codon degeneracy or the codon preference of the host cell expressing the polypeptide, the polynucleotide sequence can be 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 can be selected from SEQ ID NOs: 15-26.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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).

[0028] Method for preparing RNA polymerase variants

[0029] 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.

[0030] 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.

[0031] 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), isoelectric focusing electrophoresis, ammonium sulfate precipitation, SDS-PAGE, etc.

[0032] Composition

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

[0034] The compositions described herein may be compositions for storing RNA polymerase variants. In some embodiments, in addition to the aforementioned RNA polymerase variants, the compositions described herein may optionally include: buffer components (e.g., Tris base, Tris-HCl, HEPES, MOPS), salts (e.g., NaCl), enzyme inhibitors (e.g., EDTA), reducing agents (e.g., DTT), surfactants (e.g., Triton X-100), stabilizers (e.g., glycerol), and other components. In some embodiments, the compositions described herein for storing RNA polymerase variants include: RNA polymerase variants, Tris-HCl, NaCl, EDTA, DTT, Triton X-100, and glycerol.

[0035] 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.

[0036] Reagent test kit

[0037] The kit provided herein comprises at least one RNA polymerase variant as described herein.

[0038] In some embodiments, the kit 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. In one embodiment, the in vitro transcription system components can be selected from commercially available RNA in vitro transcription reagents.

[0039] application

[0040] The present application provides the use of at least one variant as described herein in in vitro transcription to prepare RNA. 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, incubating in an in vitro transcription reaction system, and obtaining a target product. In some embodiments, the in vitro transcription to prepare RNA further comprises magnesium ions.

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

[0042] Preparation method

[0043] 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.

[0044] In some embodiments, the target products include, but are not limited to, mRNA, siRNA, gRNA, saRNA, dsRNA, ssRNA, miRNA, piRNA, shRNA, etc. In some embodiments, the target products produced by the RNA polymerase variants described herein have an improved integrity of 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% relative to the product produced by the wild-type RNA polymerase (SEQ ID NO: 1). In some embodiments, the target products produced by the RNA polymerase variants described herein have an improved yield relative to the product produced by the wild-type RNA polymerase (SEQ ID NO: 1).

[0045] 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).

[0046] In some embodiments, the in vitro transcription reaction system includes 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 also includes an RNase inhibitor, an inorganic pyrophosphatase, and magnesium ions. In some embodiments, the in vitro transcription buffer system also includes 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 also includes 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.

[0047] Other implementation plans

[0048] 1. An RNA polymerase variant, wherein the amino acid sequence of the variant comprises a mutation at any one of the following sites relative to SEQ ID NO: 1: N370, M306 or A382.

[0049] 2. A variant according to item 1, wherein:

[0050] (1) The substitution at position N370 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 as described in item 1, wherein the amino acid sequence of the variant comprises any mutation selected from 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.

[0054] 4. The variant as described in item 1, whose amino acid sequence is shown in any one of SEQ ID NOs: 2-13.

[0055] 5. Biomaterials selected from one or more of the following:

[0056] 1) A polynucleotide molecule encoding the RNA polymerase variant according to any one of items 1 to 4;

[0057] 2) an expression vector comprising the polynucleotide molecule described in 1);

[0058] 3) A host cell comprising the polynucleotide molecule described in 1), or a host cell comprising the expression vector described in 2).

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

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

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

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

[0063] 10. Use of an RNA polymerase variant in preparing RNA by in vitro transcription, wherein the amino acid sequence of the variant is shown in any one of SEQ ID NOs: 2-13.

[0064] 11. Use of any RNA polymerase variant described in items 1 to 4 in the synthesis of RNA drugs.

[0065] 12. A method for preparing RNA, characterized in that the method comprises contacting a DNA template, modified or unmodified nucleoside triphosphates with the RNA polymerase variant described in any one of items 1 to 4, and incubating them in an in vitro transcription reaction system to obtain a target RNA product.

[0066] Beneficial effects

[0067] The present application provides an RNA polymerase variant. Compared with the wild-type T7 RNA polymerase, the polymerase variant has high catalytic efficiency and high specific activity, which can significantly reduce the amount of enzyme added, thereby reducing production costs. In addition, the RNA variant provided by the present application can effectively improve the integrity of the RNA product. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 Schematic diagram of the construction of recombinant plasmid;

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

[0070] Figure 3 RNA yields generated in in vitro transcription reactions for different RNA polymerase variants. DETAILED DESCRIPTION

[0071] 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.

[0072] The enzyme activity is defined as the amount of enzyme that can be converted to 1 nmol at 37°C and pH 8.0 within 1 hour. 3 The amount of enzyme required to incorporate H] ATP into the acid-insoluble precipitate was defined as 1 activity unit.

[0073] Example 1 Preparation of RNA polymerase variants

[0074] DNA fragments were synthesized according to the DNA sequences shown in SEQ ID NOs: 14 to 26 (the encoded amino acid sequences correspond to SEQ ID NOs: 1 to 13). After PCR amplification, the fragments were introduced into the BseRI and HindIII restriction sites of the expression vector pQE-80L to obtain a recombinant expression vector. The constructed vector was introduced into E. coli BL21 (DE3) by transformation technology. After screening by antibiotic (ampicillin) resistance plate coating, cloned strains were obtained. The obtained strains were incubated in a 37°C incubator overnight, and the grown single colonies were subjected to plasmid extraction and sequencing to ultimately obtain recombinant engineered bacteria containing the target gene. Successfully sequenced recombinant E. coli strains were inoculated into LB medium for overnight activation. A 1-5% v / v inoculation was then performed into the fermentation broth (LB medium) and cultured to an OD600 value of 0.6-0.8. IPTG was added to a final concentration of 0.5 mol / L and cultured at 37°C for 4-6 hours. The strains were then harvested by centrifugation at 12,000 rpm at 5°C and washed with 0.2 M PBS buffer (pH 7.0) to obtain the collected cells. After ultrasonic disruption, the cells were purified by affinity chromatography (His trap HP, 29-0510-21, Cytiva) to obtain a stock RNA polymerase solution. The corresponding amino acid sequences of wild-type RNA polymerases and their variants are shown in Tables 1-1 to 1-3.

[0075] Table 1-1

[0076]

[0077] Table 1-2

[0078]

[0079] Table 1-3

[0080]

[0081] Example 2: T7 RNAP activity assay

[0082] Two rows of eight strips were placed in an ice box. Assuming the enzyme activity of the purified enzyme was 500 U / μL, a gradient dilution was performed to an enzyme activity of 0.1 U / μL, 0.2 U / μL, 0.3 U / μL, 0.4 U / μL, 0.5 U / μL, and 0.6 U / μL. A blank control of 0 U / μL was added. T7 RNA polymerase standard (Vazyme, Cat. No. DD4101R-01) was diluted to 0.1 U / μL, 0.2 U / μL, 0.3 U / μL, 0.4 U / μL, 0.5 U / μL, and 0.6 U / μL according to its exact enzyme activity. A blank control of 0 U / μL was added. The enzyme activity assay system is shown in Table 2.

[0083] Table 2: Enzyme activity detection system

[0084]

[0085] Mix the above solution and centrifuge for 15 seconds. Incubate at 37°C-50°C for 30 minutes (ABI PCR instrument). Prepare a fluorescent dye mixture (Vazyme, the fluorescent dye component in Catalog No. EQ212). Add 180 μL of the fluorescent dye mixture to the eight-well strip after the reaction and mix thoroughly. Pipette 180 μL into a black microplate and detect using a microplate reader with the excitation wavelength set to 630 nm and the emission wavelength set to 680 nm. Use graphing software to calculate the regression equation (slope k) for the enzyme activity of the T7 RNA polymerase standard and enzyme variant samples.

[0086] Y (standard product) = k2X + b

[0087] Y(sample)=k1X+b

[0088] Actual enzyme activity: Actual enzyme activity of sample = k1 / k2×500U

[0089] Actual enzyme specific activity: Actual enzyme specific activity of sample = Actual enzyme activity of sample / protein concentration (μg)

[0090] The results are as follows Figure 2 As shown, the enzyme specific activity of the above mutants T7 RNAP at 37°C was significantly improved, especially the mutant A382K, which significantly increased the enzyme specific activity to 1822.13 U / μg, with an improvement effect of >5 times.

[0091] Example 3: In vitro transcription reaction

[0092] 1) Dilute the enzyme stock solution with storage buffer (Vazyme, Catalog No. GMP4101PB) to an enzyme activity of 300 U / μL. Add the reaction components (20 μL) listed in Table 3-1 to eight strips, mix thoroughly, and centrifuge. Place the strips in a PCR instrument and incubate at 37°C for 1 hour. Then add 36 μL of magnetic beads (Vazyme, Catalog No. N412), mix thoroughly, and incubate at room temperature for 2-5 minutes. Place the mixture on a magnetic rack to purify mRNA. After purification, transfer the purified mRNA to an RNase-free centrifuge tube and determine its concentration using a one-drop assay (RNA yield (μg) = concentration (ng / μL) * volume (μL) / 1000).

[0093] 2) 200 ng of RNA was subjected to capillary electrophoresis using a Qsep400 fully automated nucleic acid analyzer with an R1 cartridge (injection and separation 4 kV, 20 nt marker) to determine mRNA integrity (peak area of ​​intact RNA product / peak area of ​​total RNA product).

[0094] Table 3-1: Reaction system ratio

[0095]

[0096] Table 3-2

[0097]

[0098] like Figure 3 As shown in Table 3-2, when the high specific activity mutants were fed with the same enzyme activity, the protein feed amount was reduced, while the yield did not decrease significantly (all >180μg), and the integrity of the RNA product was improved to a certain extent.

Claims

1. RNA polymerase variant, characterized in that The amino acid sequence of the variant comprises a substitution at position N370 relative to SEQ ID NO: 1, and the amino acid sequence of the variant is shown in any one of SEQ 2-8, 10, 12-13.

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 according to claim 1; 2) an expression vector comprising the polynucleotide molecule described in 1); 3) A host cell comprising the polynucleotide molecule described in 1), or a host cell comprising the expression vector described in 2).

3. The method for preparing the variant according to claim 1, characterized in that: include: (1) Cultivating the host cell as claimed in claim 2; and (2) recycling variants.

4. A composition, characterized in that Contains the variant as claimed in claim 1.

5. A kit, characterized in that Contains the variant as claimed in claim 1.

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

7. Use of an RNA polymerase variant in preparing RNA by in vitro transcription, characterized in that: The amino acid sequence of the polymerase variant is shown in any one of SEQ 2-8, 10, 12-13.

8. A method for preparing RNA, characterized in that: The method comprises contacting a DNA template, modified or unmodified nucleoside triphosphates with the RNA polymerase variant according to claim 1, and incubating them in an in vitro transcription reaction system to obtain a target RNA product.

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

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