RNA polymerase variants and their use in the preparation of rna by in vitro transcription
By introducing I543, E600, or N601 mutations into RNA polymerase variants, the problem of incomplete RNA products in mRNA vaccine production has been solved, enabling the preparation of high-yield and high-integrity RNA products under low magnesium ion conditions, which is suitable for the production of various RNA drugs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NANJING VAZYME BIOTECH CO LTD
- Filing Date
- 2026-05-26
- Publication Date
- 2026-06-30
Smart Images
Figure CN122303188A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of biotechnology, and in particular to RNA polymerase variants and their applications. Background Technology
[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 an information carrier to guide the production of functional proteins or peptides in the human body. Since the beginning of the 21st century, mRNA technology has matured and made remarkable progress in the research of cancer, rare diseases, genetic diseases, and infectious diseases.
[0003] Current methods for preparing mRNA vaccines mainly involve in vitro transcription (IVT) synthesis using T7 RNA polymerase (T7 RNAP). Compared to traditional vaccines, mRNA vaccines have advantages such as shorter development cycles, stronger immunogenicity, and simpler production processes. However, the production of mRNA vaccines involves multiple biological processes and raw material processing, requiring quality assessments of various aspects such as mRNA integrity, residual immunogenic impurities (dsRNA), and capping rate. Among these, the assessment of mRNA integrity is a crucial step.
[0004] The formation of incomplete RNA fragments is mainly caused by two factors: 1) degradation of RNA products, T7 RNAP cometallic ions Mg 2+ The addition of Mg2+ is an important factor promoting the hydrolysis of mRNA and the generation of incomplete RNA fragments; 2) premature termination of transcription. In the complex process of T7 RNAP catalyzing the transcription of mRNA, transcription may stop before reaching the end due to some factors, thus producing incomplete fragments (Camperi et al., 2024; Lu et al., 2020; He et al., 2024). Therefore, in the process of in vitro RNA synthesis, it is necessary to improve the integrity of RNA products, reduce the generation of incomplete fragments, and reduce Mg2+. 2+ The addition of concentration and reduction of truncated forms caused by premature transcription termination are crucial. Summary of the Invention
[0005] This application provides an RNA polymerase variant that not only significantly reduces the production of incomplete fragments and improves the integrity of transcribed RNA products in low-magnesium in vitro transcription, but also increases the yield and capping rate of RNA products in low-magnesium in vitro transcription reaction systems. This application also provides methods for preparing these variants and their applications in in vitro RNA synthesis.
[0006] 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: I543, E600 or N601.
[0007] Secondly, this application provides a class of biological materials selected from one or more of the following: 1) The polynucleotide molecule encoding the above variants; 2) Expression vectors containing polynucleotide molecules as described in 1); 3) Host cells containing the polynucleotide molecules described in 1), or host cells containing the expression vectors described in 2).
[0008] Thirdly, this application provides a method for preparing the aforementioned RNA polymerase variant.
[0009] Fourthly, this application provides a composition comprising at least one RNA polymerase variant as described in this application.
[0010] Fifthly, this application provides a kit comprising at least one RNA polymerase variant as described in this application.
[0011] Sixthly, this application provides the application of the above-mentioned RNA polymerase variant in the in vitro transcription preparation of RNA.
[0012] Seventhly, this application also provides a method for preparing RNA. Invention Details RNA polymerase variants The RNA polymerase variant provided in this application contains at least one mutation selected from the following amino acid sites compared to SEQ ID NO: 1: I543, E600, or N601; the mutation type can be selected from substitution or deletion.
[0014] In some embodiments, the amino acid sequence of the variant contains any mutation selected from the following relative to SEQ ID NO: 1: I543L, E600T, E600R, or N601K.
[0015] In some embodiments, the amino acid sequence of the variant is as shown in any of SEQ ID NO: 2-5.
[0016] biomaterials 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: 7-10.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] 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).
[0021] Preparation methods of RNA polymerase variants 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.
[0022] 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.
[0023] 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.
[0024] Composition The composition provided in this application comprises at least one RNA polymerase variant described in this application.
[0025] 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.
[0026] 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.
[0027] Reagent test kit The kit provided in this application contains at least one RNA polymerase variant as described in this application.
[0028] 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.
[0029] application 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. In some embodiments, the in vitro transcription is carried out under low magnesium conditions, wherein the concentration of magnesium ions is 10-25 mM, preferably 15-20 mM, and most preferably 17.5 mM.
[0030] This application also provides the use of at least one RNA polymerase variant as described in this application in the synthesis of RNA drugs.
[0031] Preparation method 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.
[0032] 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 at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% higher integrity compared to the product prepared 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).
[0033] 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).
[0034] 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, 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.
[0035] Other implementation plans 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: I543, E600, or N601.
[0036] 2. The variants described in item 1, having an amino acid sequence as shown in any of SEQ ID NO: 2-5.
[0037] 3. Biological materials, selected from one or more of the following: 1) A polynucleotide molecule encoding an RNA polymerase variant as described in either item 1 or item 2; 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).
[0038] 4. A method for preparing a variant as described in either item 1 or item 2, characterized in that it comprises: (1) culturing a host cell as described in item 3; and (2) recovering the variant.
[0039] 5. A composition comprising a variant as described in either item 1 or item 2.
[0040] 6. A kit containing a variant as described in either item 1 or item 2.
[0041] 7. Use of any variant described in item 1 or 2, the composition described in item 5, or the kit described in item 6 in in vitro transcription.
[0042] 8. 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: 2-5.
[0043] 9. Application of RNA polymerase variants as described in items 1-2 in RNA drug synthesis.
[0044] 10. A method for preparing RNA, characterized in that the method comprises contacting a DNA template, a modified or unmodified nucleoside triphosphate, and any RNA polymerase variant described in item 1 or 2, incubating in an in vitro transcription reaction system to obtain a target RNA product.
[0045] Beneficial effects 1. This application provides multiple RNA polymerase variants that not only significantly reduce the generation of incomplete fragments in in vitro transcription under low magnesium ion conditions and improve the integrity of transcribed RNA products, but also increase the yield and capping rate of RNA products in in vitro transcription reaction systems under low magnesium ion conditions, and have broad template and application scenario adaptability.
[0046] 2. The RNA polymerase mutant provided in this application can reduce the purification cost in the RNA drug production process, rapidly prepare RNA drugs that meet the requirements for use, and provide an efficient tool enzyme for the large-scale production of RNA drugs. Attached Figure Description
[0047] Figure 1 This is a schematic diagram illustrating the construction of recombinant plasmids; Figure 2 The effect of different RNA polymerase variants on RNA capping rate. Detailed Implementation
[0048] 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.
[0049] Example 1: Preparation of RNA polymerase variants DNA fragments were synthesized based on the DNA sequences shown in SEQ ID NO: 6-10 (encoding amino acid sequences corresponding to SEQ ID NO: 1-5). 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 antibiotic (ampicillin) resistance plate plating, cloned strains were obtained. After successful recombination was confirmed, the obtained strains were incubated overnight at 37°C. The resulting single colonies 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 continued for 4-6 h. The strain was collected by centrifugation at 12000 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, affinity chromatography was performed to purify 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, 1-2, and 1-3. Table 1-1
[0050] Table 1-2
[0051] Table 1-3
[0052] Example 2: Detection of In vitro transcriptional reaction integrity and yield (1) Dilute the enzyme stock solution to 0.875 μg / μL 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). Prepare the MIX solution according to the reaction system (20 μL) in Table 2 (Note: In control group 1, the amount of MgCl2 added is replaced with 2 μL of 300 mM (final concentration 30 mM)). Aliquot the MIX solution into octet arrays, mix and centrifuge. Place the octet arrays on a PCR instrument and react at 37℃ for 2 h. Add 36 μL of magnetic beads (Vazyme, catalog number: N412) and mix well. 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. Use One Drop to detect the content. Yield (μg) = concentration (ng / μL) * volume (μL) / 1000. (2) Take 200 ng RNA and use the Qsep400 fully automated nucleic acid analyzer for capillary electrophoresis detection. Use the R1 clip (injection and separation 4KV, 20 nt Marker) to detect the integrity of mRNA (peak area of intact RNA product / peak area of total RNA product).
[0053] Table 2: Reaction System Proportions
[0054] Table 3-1 Increased yield of RNA polymerase mutant in low magnesium reaction system
[0055] Table 3-2 Integrity of mRNA Products
[0056] The test results are shown in Tables 3-1 and 3-2. Compared with wild-type T7 RNAP (control group), the mutants significantly increased mRNA production in the low magnesium ion system; compared with wild-type (control group) in the normal system, the low magnesium ion system significantly increased mRNA production. 2+ The T7 RNAPWT and its mutants under the system can effectively improve the integrity of RNA products, especially the E600R mutant, which can effectively improve the integrity to 90%.
[0057] Example 3: Capping Rate Detection After pretreatment using the mRNA Capping Rate Detection Kit (Vazyme, catalog number: DD3510-01), the capping rate of the mRNA product was detected by MS. (1) Capped RNA was prepared according to the system in Example 2 (the DNA template was replaced with a co-transcription template (SEQ ID NO. 12), and a final concentration of 1 mM CleanCap AG cap (Trilink, catalog number N-7113) was added). The reaction was carried out under the same conditions on a PCR instrument at 37°C for 2 hours. After purification of the evolution product, the mRNA was bound to the probe. The reaction system is shown in Table 4, and the reaction conditions are shown in Table 5. Table 4: Reaction System Proportions
[0058] Table 5: Reaction Conditions
[0059] (2) RNase H digestion: Prepare the digestion reaction system according to Table 6, vortex thoroughly to mix evenly, and place in a PCR instrument. React at 25°C for 20 min. Table 6: Composition of the enzyme digestion reaction system
[0060] (3) SA magnetic bead bonding ① Magnetic bead cleaning: Take 9 μl of SA magnetic beads (Cat.No.:SM017005) into a centrifuge tube, place it on a magnetic rack, and wait for the solution to become clear. Then, use a pipette to remove the supernatant. Remove the centrifuge tube from the magnetic rack, add 200 μL of RNase-free H2O to rinse, place it on the magnetic rack, and wait for the solution to become clear. Then, use a pipette to remove the supernatant, and add another 200 μL of RNase-free H2O to rinse once more.
[0061] ②Reaction conditions: Remove the centrifuge tube from the magnetic rack, add the enzyme digestion product to the SA magnetic beads (solid), pipette and mix thoroughly 20-30 times, place on a tumbler and incubate at room temperature for 30 minutes to allow the magnetic beads to fully combine with the enzyme digestion product.
[0062] (4) Rinsing and elution ① Place the product from the previous step on a magnetic rack for 2-3 minutes until the solution becomes clear, then use a pipette to remove the supernatant; ② Add 200 μL of rinsing solution to rinse, being careful not to blow away the magnetic beads, let stand for 0.5~1 min, and then use a pipette to remove the supernatant; ③ Repeat step ②; ④ Remove the centrifuge tube from the magnetic rack, add 30 μL of elution buffer, and mix thoroughly by pipetting 10-20 times to ensure even dispersion of the magnetic beads and complete elution; ⑤ Place it in a PCR instrument and react at 85℃ for 3 min. Immediately after that, place it on a magnetic rack. After the solution becomes clear (0.5~1 min), aspirate the supernatant into a new centrifuge tube. The supernatant is the desired product. ⑥ The above product was sent to a Thermo Scientific Vanquish Flex-Qrbitrap Exploris 120 chromatographic instrument for capping rate determination (mobile phase: Phase A: 2% hexafluoroisopropanol-1% N'N-diisopropylethylamine aqueous solution; Phase B: 2% hexafluoroisopropanol-1% N'N-diisopropylethylamine methanol solution; column: Nano ChromCore C18 3μm, 4.6*100mm; ion mode: negative ion; scan mode: full scan; scan range: 600-3000). Capping rate calculation formula: mRNA capping rate (%) = (capped mRNA / (capped mRNA + uncapped mRNA)) × 100%. Detection results are as follows: Figure 2 The results show that, compared to the conventional 30mM Mg 2+ The capping rate in the WT group was 82.08%. Both the WT group and the mutant were at low Mg levels of 17.5 mM. 2+ The RNA products prepared by catalysis in the addition system can be effectively improved, and all mutants can increase the capping rate to >95%.
Claims
1. An RNA polymerase variant, characterized in that, The amino acid sequence of the variant contains a mutation selected from any of the following sites relative to SEQ ID NO: 1: I543, E600, or N601.
2. The variant as claimed in claim 1, characterized in that, The amino acid sequences of the variants are shown in any of SEQ ID NO: 2-5.
3. A biomaterial, characterized in that, The biomaterial is selected from one or more of the following: 1) A polynucleotide molecule encoding an RNA polymerase variant as described in any one of claims 1 or 2; 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).
4. The method for preparing the variant as described in any one of claims 1 or 2, characterized in that, include: (1) Culturing the host cells as described in claim 3; and (2) recycling variants.
5. The composition, characterized in that, Includes the variants as described in any of claims 1 or 2.
6. A reagent kit, characterized in that, Includes the variants as described in any of claims 1 or 2.
7. The use of any variant of claim 1 or 2, the composition of claim 5, or the kit of claim 6 in in vitro transcription.
8. 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 any of SEQ ID NO: 2-5.
9. A method for preparing RNA, characterized in that, The method comprises contacting a DNA template, a modified or unmodified nucleoside triphosphate, with any of the RNA polymerase variants described in claim 1 or 2, incubating in an in vitro transcription reaction system, and obtaining the target RNA product.
10. The use of any of the RNA polymerase variants described in claims 1-2 in the synthesis of RNA drugs.