RNA polymerase variants and uses thereof

Abstract

The application provides a T7 RNA polymerase variant and its application, the variant can significantly reduce the substrate high concentration inhibition in the in vitro transcription process, and improve the mRNA yield relative to the wild type T7 RNA polymerase. In addition, the application also provides a method for preparing RNA by using the variant, and the RNA molecules prepared by using the method can be obtained economically in high concentration.

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 in RNA synthesis. Background Technology

[0002] mRNA vaccines have a relatively short development cycle, enabling the rapid development of new candidate vaccines to address viral mutations. They utilize both humoral and T-cell immune mechanisms, exhibiting strong immunogenicity and significant efficacy. Furthermore, their production process is simple, facilitating efficient research and development and large-scale production.

[0003] Besides mRNA, engineered circular RNA (circRNA) has been shown to stably and efficiently express proteins in eukaryotic cells, opening up new applications for exogenous circRNA protein expression in eukaryotic cells and serving as an effective alternative to mRNA. Studies have shown that optimized circRNA and lipid nanoparticle (LNP) delivery systems can achieve higher protein expression, higher delivery efficiency, and lower immunogenicity. Currently, several companies, including Orna Therapeutics in the United States, have rapidly developed multiple circRNA drugs, which also have significant application potential in other fields such as cancer treatment.

[0004] Despite the promising prospects of RNA in vaccine and drug development, several common problems remain to be addressed in its actual production. Among these, double-stranded RNA (dsRNA) impurities, which can cause strong immunogenicity, have become a focal point of industry attention. Engineering the T7 RNA polymerase (T7 RNAP), a key catalytic enzyme in the in vitro transcription process, can significantly reduce dsRNA production at its source (CN 118922535A). Furthermore, the amount of NTPs used in the in vitro transcription process is also crucial. Due to the enzyme's limited tolerance to high substrate concentrations, excessive NTPs can significantly inhibit the in vitro transcription reaction and reduce yield. Similar to dsRNA impurity removal, if the T7 RNA polymerase can be significantly modified to improve its substrate tolerance and achieve efficient transcription under high NTP conditions, it will have significant implications for the economical production of mRNA and drug safety. Summary of the Invention

[0005] In a first aspect, this application provides a class of RNA polymerase variants whose amino acid sequence contains a mutation at position V634 relative to SEQ ID NO: 1, the mutation type being either substitution or deletion.

[0006] Secondly, this application provides one or more biological materials selected from the following: 1) A polynucleotide molecule encoding an RNA polymerase variant; 2) An expression vector containing the polynucleotide molecule 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).

[0007] Thirdly, this application provides a method for preparing the aforementioned RNA polymerase variant.

[0008] Fourthly, this application provides a composition comprising at least one RNA polymerase variant as described in this application.

[0009] Fifthly, this application provides a kit comprising at least one RNA polymerase variant as described in this application.

[0010] Sixthly, this application also provides the use of the above-mentioned RNA polymerase variants, compositions or kits in in vitro transcription. Invention Details RNA polymerase variants The RNA polymerase variant provided in this application is a phage T7 RNA polymerase (T7 RNAP) variant, whose amino acid sequence contains a mutation at the following amino acid site relative to SEQ ID NO: 1: V634, wherein the mutation type is selected from substitution or deletion.

[0012] In some implementations, the variant is replaced by T at the V634 position.

[0013] In some embodiments, the amino acid sequence of the variant contains a mutation at the following amino acid site relative to SEQ ID NO: 1: V634+P508, wherein the mutation type is selected from substitution or deletion.

[0014] In some implementations, the variant is replaced by T at the V634 position.

[0015] In some implementations, the variant is replaced by K at position P508.

[0016] In some embodiments, the variant, relative to SEQ ID NO: 1, comprises any mutation selected from the following sites: V634T+P508K+C347V, V634T, V634T+H346N, V634T+P508K, V634T+P508K+P657K, V634T+P508K+A881C, V634T+P508K+P657K+A881C, V634T+P 508K+Y623H+A881C, V634T+H346S+V349I+A881C, V634T+P508K+V473K, V634T+P5 08K+N560G, V634T+P508K+F814Y, V634T+P508K+G572Q, V634T+P508K+T630V, V63 4T+P508K+P657R, V634T+P508K+S661D, V634T+P508K+A503T, V634T+P508K+E56 5P, V634T+P508K+H346E, V634T+P508K+C347P, V634T+P508K+E350D, V634T+P508 K+V559T, V634T+P508K+D879K, V634T+P508K+G469S, V634T+P508K+V567P, V634T +P508K+P348D, V634T+P508K+I352L, V634T+P508K+T566K or V634T+P508K+D879P.

[0017] In some embodiments, the amino acid sequence of the RNA polymerase variant has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity compared to any of the sequences described in SEQ ID NO: 2-30. In some embodiments, the amino acid sequence of the variant is as shown in any of SEQ ID NO: 2-30.

[0018] In some embodiments, the amino acid sequence of the RNA polymerase variant has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity compared to any of the sequences described in SEQ ID NO: 2-22, 25-29. In some embodiments, the amino acid sequence of the RNA polymerase is as shown in any of SEQ ID NO: 2-22, 25-29.

[0019] Polynucleotide molecules The polynucleotide molecule provided in this application encodes any of the RNA polymerase variants described in this application. In some embodiments, the nucleotide sequence of the polynucleotide molecule comprises any of the nucleotide sequences shown in SEQ ID NO: 32-60. The polynucleotide molecule described in this application may have various modifications in its coding region, as long as the amino acid sequence of the variant does not change with codon degeneracy or with the preferred codon in the organism expressing the variant.

[0020] expression carrier The expression vectors provided in this application refer to linear or circular DNA molecules, typically containing elements such as multiple cloning sites, resistance genes, and replication origin sites. In some embodiments, the expression vector described in this application is pQE-80L.

[0021] In some embodiments, the vector described in this application comprises a multinucleotide molecule encoding a variant of the RNA polymerase described in this application. In some further embodiments, the vector also comprises one or more regulatory sequences (such as enhancer, promoter, and terminator sequences) operatively linked to the multinucleotide molecule encoding the variant.

[0022] host cells The host cell provided in this application can be 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 of this application, and includes any cell progeny that is different from the parent cell due to mutations that occur during replication.

[0023] In some embodiments, the host cell described in this application comprises the above-described polynucleotide molecule or the expression vector.

[0024] 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).

[0025] Methods for preparing variants This application provides a method for preparing the above-mentioned RNA polymerase variant, comprising: (1) culturing the host cell described in this application under conditions suitable for the expression of the variant; and (2) recovering the variant.

[0026] 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.

[0027] 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, and ammonium sulfate precipitation).

[0028] Composition The composition provided in this application comprises at least one RNA polymerase variant described in this application.

[0029] The composition described in this application can be a composition for storing RNA polymerase variants. In some embodiments, in addition to the aforementioned RNA polymerase variants, the composition described in this 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 this application comprises: RNA polymerase variant, Tris-HCl, NaCl, EDTA, DTT, Triton X-100, and glycerol.

[0030] The compositions of this application may also be in vitro transcription reaction compositions. In some embodiments, the compositions, in addition to the RNA polymerase variants described above, further comprise one or more in vitro transcription reaction reagents (e.g., buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, etc.). In some embodiments, the compositions further comprise a DNA template. In some embodiments, the compositions further comprise a cap analogue.

[0031] In some embodiments, the in vitro transcription reaction composition of this application comprises: an RNA polymerase variant, a buffer component, a modified or unmodified nucleoside triphosphate, an RNase inhibitor, a pyrophosphatase, magnesium ions, water, and a cap analog. In some embodiments, the in vitro transcription reaction composition of this application comprises: an RNA polymerase variant, a buffer component, a modified or unmodified nucleoside triphosphate, an RNase inhibitor, a pyrophosphatase, magnesium ions, water, a cap analog, and a DNA template.

[0032] Reagent test kit The kit provided in this application contains at least one RNA polymerase variant described in this application.

[0033] In some embodiments, the kit may also contain one or more in vitro transcription reaction reagents (e.g., buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, etc.). In some embodiments, the kit also contains a cap analogue. In some embodiments, each component in the kit (if applicable) may be provided in liquid form (e.g., in solution) or in solid form (e.g., dry powder).

[0034] The kit described in this application may include one or more containers containing one or more components described in this application and optional instructions for use.

[0035] Applications or uses This application provides the use of the above-mentioned RNA polymerase variants, compositions, or kits in in vitro transcription.

[0036] This application also provides the use of the above-mentioned RNA polymerase variants, compositions, or kits in various methods, including but not limited to RNA preparation, RNA probe preparation, RNA vaccine preparation, and protein preparation.

[0037] Methods for preparing RNA This application provides a method for preparing RNA. In some embodiments, the method includes contacting a DNA template, modified or unmodified nucleoside triphosphates, with at least one RNA polymerase variant described in this application, incubating in an in vitro transcription reaction system to obtain a target RNA product. In some embodiments, the RNA product may be dsRNA, ssRNA, mRNA, siRNA, miRNA, piRNA, shRNA, or gRNA.

[0038] Suitable in vitro transcription reaction systems and incubation conditions for generating RNA products are well known in the art. Those skilled in the art can determine appropriate reaction system pH, reaction temperature, reaction time, salt concentration, or whether to add exogenous cofactors, taking into account the optimal activity of RNA polymerase. In some embodiments, the in vitro transcription reaction system described in this application includes in vitro transcription reaction reagents: one or more buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, etc. In some embodiments, the concentration of modified or unmodified nucleoside triphosphates is selected from 10 mM to 20 mM, specifically from 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, and 20 mM. In some embodiments, the incubation temperature in the incubation step described in this application is 30°C to 50°C, preferably 37°C. In some embodiments, the incubation time in the incubation step described in this application is 20 min to 240 min, preferably 60 min.

[0039] In some embodiments, the RNA product or capped mRNA product prepared using the method described in this application exhibits higher yield compared to that prepared using wild-type RNA polymerase. In some embodiments, the mRNA product prepared using the method described in this application effectively reduces high-concentration substrate inhibition and achieves higher yield compared to that prepared using wild-type RNA polymerase.

[0040] In vitro transcription reaction reagents The in vitro transcription reaction reagent described in this application includes buffer components, nucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatase, magnesium ions, and water (such as DEPC-water, RNase-free water, DNase-free water, sterile purified water, deionized water, distilled water, etc.).

[0041] In some embodiments, the buffering component may be selected from one or more of the following: phosphate buffer, Tris buffer, MOPS buffer, HEPES buffer, citrate buffer, acetate buffer, malate buffer, MES buffer, histidine buffer, PIPES buffer, bis-tris buffer, and ethanolamine buffer.

[0042] In some embodiments, the nucleoside triphosphate may be selected from modified or unmodified nucleoside triphosphates (including analogues thereof). In some embodiments, the nucleoside triphosphate may be selected from unmodified ATP, GTP, CTP, or UTP. In some embodiments, the nucleoside triphosphate may be selected from modified nucleoside triphosphates, and the modification types on the nucleoside include, but are not limited to, m1A (N1-methyladenosine), m6A (N6-methyladenosine), m5C (5-methylcytidine), 5moU (5-methoxyuridine), ψ (pseudouridine), m1ψ (N1-methyl-pseudouridine), and labeled nucleoside triphosphates (the label may be biotin, fluorescent substances, digoxigenin, radioactive elements, etc.).

[0043] In some embodiments, the in vitro transcription reaction reagent described in this application may be selected from any commercially available RNA in vitro transcription reagent.

[0044] Other implementation plans: 1. An RNA polymerase variant whose amino acid sequence contains the following amino acid site substitution relative to SEQ ID NO: 1: V634.

[0045] 2. A variant as described in item 1, wherein the variant is replaced by T at position V634.

[0046] 3. The variant described in item 1, wherein the amino acid sequence relative to SEQ ID NO: 1 contains any mutation selected from the following sites: V634T, V634T+H346N, V634T+P508K, V634T+P508K+P657K, V634T+P508K+A881C, V634T+P508K+P657K+A881C, V634T+P508K+Y623H+A881C, V 634T+H346S+V349I+A881C, V634T+P508K+C347V, V634T+P508K+V473K, V634T+P5 08K+N560G, V634T+P508K+F814Y, V634T+P508K+G572Q, V634T+P508K+T630V, V63 4T+P508K+P657R, V634T+P508K+S661D, V634T+P508K+A503T, V634T+P508K+E56 5P, V634T+P508K+H346E, V634T+P508K+C347P, V634T+P508K+E350D, V634T+P508 K+V559T, V634T+P508K+D879K, V634T+P508K+G469S, V634T+P508K+V567P, V634T +P508K+P348D, V634T+P508K+I352L, V634T+P508K+T566K or V634T+P508K+D879P.

[0047] 4. The variants described in item 1, having an amino acid sequence as shown in any of SEQ ID NO: 2-30.

[0048] 5. A polynucleotide molecule that encodes a variant as described in any of items 1-4.

[0049] 6. An expression vector comprising a polynucleotide molecule as described in item 5.

[0050] 7. A host cell containing a polynucleotide molecule as described in item 5, or an expression vector as described in item 6.

[0051] 8. A method for preparing any of the variants described in items 1-4, comprising: (1) Culture host cells as described in section 7 under conditions suitable for variant expression; and (2) Recycle variants.

[0052] 9. A composition comprising any of the variants described in items 1-4.

[0053] 10. The composition as described in item 9, further comprising a hat analogue.

[0054] 11. The composition as described in item 9 or 10, further comprising a DNA template.

[0055] 12. A kit containing any of the variants described in items 1-4.

[0056] 13. The use of any variant described in items 1-4, any composition described in items 9-11, or the kit described in item 12 in in vitro transcription.

[0057] 14. A method for preparing RNA, comprising contacting a DNA template, a modified or unmodified nucleoside triphosphate, with any of the RNA polymerase variants described in items 1-4, incubating in an in vitro transcription reaction system to obtain a target RNA product.

[0058] Beneficial effects This application provides a class of RNA polymerase variants and their preparation methods. By modifying wild-type T7 RNA polymerase, RNA polymerase variants with high catalytic efficiency are obtained. Using such variants, the inhibitory effect of high substrate concentration can be reduced, significantly increasing the yield of mRNA products during in vitro transcription, avoiding resource waste, and saving production costs. This is of great significance for the economical production of RNA. Attached Figure Description

[0059] Figure 1 This is a schematic diagram illustrating the construction of recombinant plasmids; Figure 2 Results of RNA polymerase yield assays for wild-type and variant RNA polymerases; Figure 3 Results of RNA polymerase yield detection for wild-type and mutant V634T+P508K+C347V. Detailed Implementation

[0060] 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.

[0061] In this application embodiment, unit enzyme activity (U) is defined as the amount of enzyme that can be activated by 1 nmol of enzyme 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.

[0062] Example 1: Preparation of RNA polymerase variants The RNA polymerases shown in Table 1 were synthesized using DNA sequences (SEQ ID NO: 31-60) and then amplified by PCR. The resulting DNA was then introduced into the BseRI and HindIII restriction sites of the expression vector pQE-80L to obtain a recombinant expression vector. The constructed vector was then introduced into the DNA via chemical transformation. E. coli In BL21(DE3), the bacteria were plated on LB agar plates containing ampicillin and incubated overnight at 37°C. Single colonies that grew were then subjected to plasmid extraction and sequencing to obtain recombinant engineered bacteria containing the target gene. Successfully sequenced colonies were then... E.coli After overnight activation culture in LB medium, the recombinant strain was inoculated into 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 continued for 4–6 h. The strain was collected by centrifugation at 12000 rpm and 4°C. The collected strain was washed with 0.2 M PBS buffer (pH 7.0) to obtain the bacterial cells. After sonication, affinity chromatography was performed to purify the RNA polymerase stock solution (His trap HP, 29-0510-21, Cytiva). The correspondence between RNA polymerase variants and amino acid sequences is shown in Table 1. Table 1: Correspondence between RNA polymerase variants and amino acid sequences

[0063] Example 2: Preparation of mRNA by in vitro transcription reaction The enzyme stock solution was diluted with storage buffer (50 mM Tris-HCl (25℃, pH 7.9), 100 mM NaCl, 0.1 mM EDTA, 2 mM DTT, 0.1% Triton X-100, 50% Glycerol) to an enzyme activity of 300 U / μL. The MIX solution was prepared according to the reaction system (20 μL) in Table 2 and transferred to an EP tube. The MIX solution was then transferred to an octet, mixed, and centrifuged. The octet was placed on a PCR instrument and reacted at 37℃ for 3 hours. 36 μL of magnetic beads (Vazyme, catalog number: N412) were added, mixed, and incubated at room temperature for 2–5 minutes. The mixture was placed on a magnetic rack to purify the mRNA. After purification, the mRNA was transferred to an RNase-free centrifuge tube. The purified mRNA was then analyzed using a one-drop assay (RNA yield (μg) = concentration (ng / μL) * volume (μL) / 1000).

[0064] Table 2: Reaction System Proportions

[0065] Table 3

[0066] like Figure 2 As shown in Table 3, in an in vitro transcription reaction system containing 15 mM NTPs, WT production was significantly inhibited. All mutants could reduce the inhibitory effect of high concentrations of NTPs. Among them, the mutant V634T+P508K+C347V could significantly increase the production from 13.92 μg catalyzed by WT to 328.7 μg.

[0067] Example 3: Preparation of mRNA by in vitro transcription reaction The enzyme stock solution was diluted with storage buffer (50 mM Tris-HCl (25℃, pH 7.9), 100 mM NaCl, 0.1 mM EDTA, 2 mM DTT, 0.1% Triton X-100, 50% Glycerol) to an enzyme activity of 300 U / μL. The MIX solution was prepared according to the reaction system (20 μL) in Table 2 and transferred to an EP tube. The MIX solution was then transferred to an octet, mixed, and centrifuged. The octet was placed on a PCR instrument and reacted at 37℃ for 5 h. 36 μL of magnetic beads (Vazyme, catalog number: N412) were added, mixed, and incubated at room temperature for 2–5 min. The mixture was placed on a magnetic rack to purify the mRNA. After purification, the mRNA was transferred to an RNase-free centrifuge tube. The purified mRNA was then analyzed using a one-drop assay (RNA yield (μg) = concentration (ng / μL) * volume (μL) / 1000).

[0068] like Figure 3 As shown, the addition of 15 mM NTPs significantly inhibited the yield of wild-type NTPs. Extending the reaction time to 5 h did not significantly increase the yield of wild-type NTPs. However, the mutant V634T+P508K+C347V significantly increased the yield from 17.63 μg catalyzed by wild-type NTPs to 332.55 μg.

Claims

1. An RNA polymerase variant, characterized in that, The amino acid sequences of the variants are shown in any of SEQ ID NO: 2-30.

2. A biomaterial, characterized in that, The biomaterial is selected from one or more of the following: 1) Encoding a polynucleotide molecule that is a variant of the RNA polymerase as described in claim 1; 2) An expression vector containing the polynucleotide molecule 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 host cells in the biomaterial as described in claim 2; and (2) Recycle variants.

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

5. A reagent kit, characterized in that, The kit comprises 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. 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.

Images