RNA polymerase variants and uses thereof

Abstract

This application provides an RNA polymerase variant and its applications, relating to the field of biotechnology. This variant exhibits a significantly increased specific activity compared to the wild type, reducing the amount of polymerase required and thus saving costs. Simultaneously, the mutant effectively reduces the generation of incomplete fragments and improves RNA integrity. Furthermore, this application also provides a method for in vitro transcription to generate RNA.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to RNA polymerase variants, their preparation methods, and their applications. Background of the Invention

[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, it becomes possible to use in vitro transcribed (IVT) mRNA as a carrier to guide the production of functional proteins or peptides in the human body. mRNA vaccines have a relatively short development cycle, enabling the rapid development of novel candidate vaccines to address viral mutations. Through a dual mechanism of humoral and T-cell immunity, they exhibit strong immunogenicity and significant efficacy. Furthermore, their production process is simple, facilitating efficient research and large-scale production.

[0003] 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.

[0004] RNA has played a significant role in the development of vaccines and other drugs. However, in the actual production process, the catalyst T7 RNApolymerase (T7 RNAP) is an important part of cost control. Modifying enzyme mutants with high specific activity can significantly reduce the amount of enzyme added, thereby reducing production costs. Therefore, there is an urgent need to develop effective high specific activity T7 RNAP. 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 to SEQ ID NO: 1: N370, M306 or A382.

[0006] Secondly, this application provides a class of biological materials selected from one or more of the following:

[0007] 1) The polynucleotide molecule encoding the above variants;

[0008] 2) Expression vectors containing polynucleotide molecules as described in 1);

[0009] 3) Host cells containing the polynucleotide molecules described in 1), or host cells containing the expression vectors described in 2).

[0010] Thirdly, this application provides a method for preparing the aforementioned RNA polymerase variant.

[0011] Fourthly, this application provides a composition comprising at least one RNA polymerase variant as described in this application.

[0012] Fifthly, this application provides a kit comprising at least one RNA polymerase variant as described in this application.

[0013] Sixthly, this application provides the application of the above-mentioned RNA polymerase variant in the in vitro transcription preparation of RNA.

[0014] Seventhly, this application also provides a method for preparing RNA. Invention Details

[0016] RNA polymerase variants

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

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

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

[0020] In some embodiments, the amino acid sequence of the variant is as shown in any of SEQ ID NO: 2-13.

[0021] In some embodiments, the T7 RNA polymerase provided in this disclosure has a higher specific activity compared to the wild-type T7 RNA polymerase. In some embodiments, the specific activity of the polymerase variant is increased by 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, and 4 times compared to the wild-type polymerase.

[0022] biomaterials

[0023] This application provides a polynucleotide encoding an RNA polymerase variant. Due to codon degeneracy or codon bias in host cells expressing the polypeptide, the polynucleotide sequence can be any polynucleotide sequence encoding the variant without altering the amino acid sequence. In some embodiments, the polynucleotide sequence encoding the RNA polymerase variant of this application may be selected from SEQ ID NO: 15-26.

[0024] The expression vector provided in this application comprises a multinucleotide molecule encoding a variant of the RNA polymerase of this application. 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.

[0025] 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 described in this application is preferably pQE-80L.

[0026] The host cell provided in this application refers to any cell that is favorable for the expression of the variants of this application, that is, any cell that is susceptible after being transformed, transfected or transduced with the expression vector described in this application, and includes any daughter cells that are different from the parent cells due to mutations that occur during replication.

[0027] 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).

[0028] Preparation methods of RNA polymerase variants

[0029] The method for preparing RNA polymerase variants provided in this application includes 1) culturing the host cells described in this application under suitable variant expression conditions; and 2) recovering the variant.

[0030] 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.

[0031] 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.

[0032] Composition

[0033] The composition provided in this application comprises at least one RNA polymerase variant described in this application.

[0034] The composition described in this application can be a composition for storing RNA polymerase variants. In some embodiments, the composition described in this application may optionally contain, in addition to the aforementioned RNA polymerase variants, components such as buffering agents (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), and stabilizers (e.g., glycerol). In some embodiments, the composition described in this application for storing RNA polymerase variants comprises: RNA polymerase variant, 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 may be selected from one or more buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatases, magnesium ions, etc.

[0036] Reagent test kit

[0037] The kit provided in this application contains at least one RNA polymerase variant as described in this application.

[0038] In some embodiments, the kit further comprises at least one in vitro transcription component, which may be selected from one or more buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatases, magnesium ions, etc. In one embodiment, the in vitro transcription system component may be selected from commercially available RNA in vitro transcription reagents.

[0039] application

[0040] This application provides the use of at least one variant as described herein in the in vitro transcription preparation of RNA. In some embodiments, the in vitro transcription preparation of RNA comprises contacting a DNA template, a modified or unmodified nucleoside triphosphate, with at least one RNA polymerase variant as described herein, incubating in an in vitro transcription reaction system to obtain the target product. In some embodiments, the in vitro transcription preparation of RNA further includes magnesium ions.

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

[0042] Preparation method

[0043] This application provides a method for preparing RNA, comprising contacting a DNA template, a modified or unmodified nucleoside triphosphate with at least one RNA polymerase variant described in this application, incubating in an in vitro transcription reaction system to obtain a 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 prepared using the RNA polymerase variant described in this application exhibits improved product integrity by 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% compared to the product prepared using wild-type RNA polymerase (SEQ ID NO: 1). In some embodiments, the target product prepared using the RNA polymerase variant described in this application exhibits increased yield compared to the product prepared using wild-type RNA polymerase (SEQ ID NO: 1).

[0045] In some embodiments, the RNA product prepared using the method described in this application has higher yield, and / or higher integrity, and / or less dsRNA impurity content, and / or more capped mRNA product compared to that 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 may 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 analogue, which may be selected from unmethylated cap analogues, dimethylated cap analogues, trimethylated cap analogues, dimethylated symmetrical cap analogues, or anti-reverse cap analogues.

[0047] Other implementation plans

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

[0049] 2. A variant as described in 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 paragraph 1, wherein the amino acid sequence of the variant contains a 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 variants described in item 1, having an amino acid sequence as shown in any of SEQ ID NO: 2-13.

[0055] 5. Biological materials, selected from one or more of the following:

[0056] 1) Encoding a polynucleotide molecule of any of the RNA polymerase variants described in items 1-4;

[0057] 2) Expression vectors containing polynucleotide molecules as described in 1);

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

[0059] 6. A method for preparing any of the variants described in items 1-4, characterized in that it comprises: (1) culturing host cells as described in item 5; and (2) recovering the variant.

[0060] 7. A composition comprising any of the variants described in items 1-4.

[0061] 8. A kit containing any of the variants described in items 1-4.

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

[0063] 10. Application of RNA polymerase variants in the in vitro transcription preparation of RNA, wherein the amino acid sequence of the variant is shown in any of SEQ ID NO: 2-13.

[0064] 11. The use of any of the RNA polymerase variants described in items 1-4 in the synthesis of RNA drugs.

[0065] 12. A method for preparing RNA, characterized in that the method comprises contacting a DNA template, a modified or unmodified nucleoside triphosphate, and 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.

[0066] Beneficial effects

[0067] This application provides an RNA polymerase variant that, compared to wild-type T7 RNA polymerase, exhibits higher catalytic efficiency and higher specific activity, significantly reducing the amount of enzyme required and thus lowering production costs. Furthermore, the RNA variant provided in this application can effectively improve the integrity of RNA products. Attached Figure Description

[0068] Figure 1 This is a schematic diagram illustrating the construction of recombinant plasmids;

[0069] Figure 2 Specific activities of different RNA polymerase variants;

[0070] Figure 3 RNA yield generated by different RNA polymerase variants in in vitro transcription reactions. Detailed Implementation

[0071] 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.

[0072] Enzyme activity is defined as: the ability of 1 nmol of enzyme to produce an enzyme activity at 37°C and pH 8.0 within 1 hour. 3 The amount of enzyme required to incorporate H]ATP into an acid-insoluble precipitate is defined as one active unit.

[0073] Example 1: Preparation of RNA polymerase variants

[0074] DNA fragments were synthesized based on the DNA sequences shown in SEQ ID NO: 14–26 (encoding amino acid sequences corresponding to SEQ ID NO: 1–13). 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 then transformed into E. coli BL21(DE3). After screening by anti-antibiotic (ampicillin) plate plating, cloned strains were obtained. The obtained strains were incubated overnight at 37°C. The single colonies that grew were subjected to plasmid extraction and sequencing to finally obtain recombinant engineered bacteria containing the target gene. After successful sequencing of the recombinant E. coli strain, the culture was activated overnight in LB medium. The culture was then inoculated into the fermentation broth (LB medium) at 1-5% v / v and cultured until the OD600 value reached 0.6-0.8. IPTG was added to a final concentration of 0.5 mol / L, and the culture was continued at 37°C for 4-6 h. The strain was collected by centrifugation at 12,000 rpm and 5°C. The collected strain was washed with 0.2 M PBS buffer (pH 7.0) to obtain the bacterial cells. After sonication, the cells were purified by affinity chromatography (His trap HP, 29-0510-21, Cytiva) to obtain the RNA polymerase stock solution. The correspondence between the wild-type and variant 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] Two rows of eight-unit packs were placed in an ice box. Assuming the activity of the purified enzyme was 500 U / μL, serial dilutions were performed 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, with a blank control of 0 U / μL added. The T7 RNA polymerase standard (Vazyme, catalog number 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 accurate enzyme activity, with a blank control of 0 U / μL added. The enzyme activity assay system is shown in Table 2.

[0085] Table 2: Enzyme Activity Detection System

[0086]

[0087] After mixing the above solution, centrifuge for 15 seconds and react at 37℃-50℃ for 30 minutes (ABI PCR instrument). Prepare a fluorescent dye (Vazyme, fluorescent dye component in product number EQ212) mixture. Add 180 μL of the fluorescent dye mixture to the eight-pack after reaction and mix well. Pipette 180 μL into a black ELISA plate and use an ELISA reader for detection. Set the parameters to excitation wavelength 630 nm and emission wavelength 680 nm. Use graphing software to calculate the regression equation (slope k) for the enzyme activity of T7 RNA polymerase standard and enzyme variant samples.

[0088] Y(standard product) = k2X + b

[0089] Y(sample) = k1X + b

[0090] Actual enzyme activity: Sample actual enzyme activity = k1 / k2 × 500U

[0091] Actual enzyme specific activity: Sample actual enzyme specific activity = Sample actual enzyme activity / Protein concentration (μg)

[0092] The results are as follows Figure 2 As shown, the specific activity of the above mutant T7 RNAP at 37℃ was significantly increased, especially the mutant A382K, which significantly increased the specific activity to 1822.13 U / μg, an increase of >5 times.

[0093] Example 3: In vitro transcription reaction

[0094] 1) Dilute the above enzyme stock solution with storage buffer (Vazyme, catalog number: GMP4101PB) to an enzyme activity of 300 U / μL. Add the reaction components (20 μL) from Table 3-1 to an octet, mix well, and centrifuge. Place the octet on a PCR instrument at 37℃ 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 rack to purify the mRNA. After purification, transfer to an RNase-free centrifuge tube to obtain the purified mRNA. Detect the concentration using a one-drop assay (RNA yield (μg) = concentration (ng / μL) * volume (μL) / 1000).

[0095] 2) Take 200 ng of RNA and perform capillary electrophoresis using the Qsep400 fully automated nucleic acid analyzer. Use the R1 clip (injection and separation 4KV, 20nt Marker) to detect the integrity of the mRNA (peak area of ​​intact RNA product / peak area of ​​total RNA product).

[0096] Table 3-1: Reaction System Proportions

[0097]

[0098] Table 3-2

[0099]

[0100] like Figure 3 As shown in Table 3-2, the high specific activity mutants, when reacted with the same enzyme activity, reduced the amount of protein fed without a significant decrease in yield (all >180 μg), and the integrity of the RNA products was improved to some extent.

Claims

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

11.

2. Biomaterial, characterized in that, The biomaterial is selected from one or more of the following: 1) Encoding a polynucleotide molecule of the RNA polymerase variant 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. The method for preparing the variant according to claim 1, characterized in that, include: (1) Culturing the host cells as described in claim 2; and (2) recycled variants.

4. A composition, characterized in that, Includes the variant as described in claim 1.

5. A reagent kit, characterized in that, Includes the variant as described in claim 1.

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

7. The application of RNA polymerase variants in the in vitro transcription preparation of RNA, characterized in that, The amino acid sequence of the polymerase variant is shown in SEQ ID NO:

11.

8. A method for preparing RNA, characterized in that, The method comprises 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, and obtaining the target RNA product.

9. The use of the RNA polymerase variant of claim 1 in RNA drug synthesis.

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