A mutant RNA ligase and its use in the preparation of nucleic acid products
By preparing RNA ligase mutants, the problems of complexity, low yield, and environmental pollution associated with traditional siRNA preparation methods have been solved, achieving the preparation of highly active and pure siRNA suitable for large-scale production.
Patent Information
- Application Number
- CN202611485445.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-09-23
- Publication Date
- 2026-11-03
AI Technical Summary
Traditional methods for preparing siRNA are complex to operate, have low yields, are difficult to purify, are expensive, and cause environmental pollution. Chemical synthesis methods for preparing siRNA also face challenges in large-scale production.
An RNA ligase mutant is provided, which enhances ligation activity by mutating the amino acid sequence at position 59, and siRNA is prepared by a biochemical method and by a composition synthesis method.
It significantly improves the activity of RNA ligase, simplifies the preparation process, increases yield and purity, reduces environmental impact, and is suitable for large-scale production.
Smart Images
Figure CN123027192A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of biotechnology, and in particular to RNA ligase mutants, their preparation methods, and their applications in the preparation of nucleic acid products. Background Technology
[0002] Gene therapy, by targeting disease-causing genes in a sequence-specific manner, enables more precise and personalized treatment of a variety of life-threatening diseases. siRNA (small interfering RNA) is considered one of the most promising applications in gene therapy. Since the FDA approval of the siRNA drugs Patisiran and Givosiiran, siRNA therapy has entered a phase of rapid development.
[0003] Traditional methods for preparing siRNA mainly involve chemical synthesis, including solid-phase synthesis methods such as the phosphoramidite method and liquid-phase synthesis. These methods are complex, have low yields, are difficult to purify, and are expensive, making them unsuitable for large-scale production. Furthermore, chemical methods for preparing siRNA require large amounts of organic solvents, causing environmental pollution and contradicting the trend of green manufacturing.
[0004] A combined biochemical method for preparing siRNA (Stephanie Paul, Darren Gray, Jill Caswell, Joshua Brooks, Wenjie Ye, Thomas S. Moody, Roumen Radinov, Lubomir Nechev; Convergent Biocatalytic Mediated Synthesis of siRNA. ACS Chem. Biol. 20 October 2023; 18 (10): 2183–2187.) involves splitting siRNA into multiple fragments. Small fragments are first prepared using a solid-phase method, and then ligated using a ligase to prepare complete siRNA. This method significantly increases yield and product purity, while the mild enzymatic ligation reduces the environmental stress associated with chemical methods. Therefore, there is a need to provide more RNA ligases with high ligation activity to offer options for industrial use. Summary of the Invention
[0005] In a first aspect, this application provides a novel RNA ligase mutant whose amino acid sequence contains a site mutation at position 59 relative to SEQ ID NO:1, wherein the mutation is selected from substitution, deletion or insertion of amino acid residues, and wherein the mutant has at least 85% but less than 100% sequence identity with SEQ ID NO:1.
[0006] Secondly, this application provides one or more biological materials selected from the following: 1) The polynucleotide molecule encoding the ligase mutant; 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).
[0007] Thirdly, this application provides a method for preparing the above-mentioned ligase mutant.
[0008] Fourthly, this application provides a composition comprising at least one ligase mutant as described in this application.
[0009] Fifthly, this application provides a kit containing at least one ligase mutant as described in this application.
[0010] Sixthly, this application also provides the use of the above-mentioned ligase mutants, compositions or kits in the preparation of nucleic acid products.
[0011] Seventhly, this application also provides a method for preparing nucleic acid products. Invention Details RNA ligase mutant This application provides an RNA ligase mutant, wherein the amino acid sequence of the RNA ligase mutant contains a site mutation at position 59 relative to SEQ ID NO:1, wherein the mutation is selected from substitution, deletion or insertion of amino acid residues, and wherein the mutant has at least 85% but less than 100% sequence identity with SEQ ID NO:1.
[0013] In some implementations, the 59th position is mutated to K59R.
[0014] In some embodiments, the amino acid sequence of the mutant is shown in SEQ ID NO:2.
[0015] Polynucleotide molecules This application provides a polynucleotide molecule encoding the RNA ligase mutant described in this application.
[0016] In some embodiments, the nucleotide sequence of the polynucleotide molecule is shown in SEQ ID NO: 4. The polynucleotide molecule described in this application may have various modifications in its coding region, as long as the mutant amino acid sequence of this application does not change with codon degeneracy or with the preferred codon in the organism expressing the mutant.
[0017] expression carrier This application provides an expression vector comprising a multinucleotide molecule encoding the RNA ligase mutant of this application.
[0018] In some implementations, the expression vector typically contains elements such as a multiple cloning site, an antibiotic resistance gene, and a replication initiation site. In some implementations, the expression vector also contains one or more regulatory sequences (such as enhancer, promoter, and terminator sequences) that are operatively linked to a polynucleotide molecule encoding the mutant.
[0019] In some implementations, the expression vector can be a plasmid, viral vector, bacteriophage, or artificial chromosome. The expression vector can also be an integrative or non-integrative vector. An integrative vector can be one in which the entire vector is integrated into the host cell's genome. Alternatively, an integrative vector can be one in which only a portion (e.g., an expression unit) is integrated into the host cell's genome. The expression vector can also be a DNA vector or an RNA vector (e.g., a retrovirus). The expression vector can also be a widely used expression vector. Examples of such expression vectors include pUC (e.g., pUC19, pUC18), pSTV, pBR (e.g., pBR322), pHSG (e.g., pHSG299, pHSG298, pHSG399, pHSG398), RSF (e.g., RSF1010), pACYC (e.g., pACYC177, pACYC184), pMW (e.g., pMW119, pMW118, pMW219, pMW218), pQE (e.g., pQE30), and derivatives thereof. In some embodiments, the expression vector described in this application is pET-30a(+).
[0020] host cells This application provides a host cell comprising the aforementioned polynucleotide molecule or the aforementioned expression vector. The host cell can be any cell favorable for expression of the mutant of this application, i.e., any cell susceptible to transformation, transfection, or transduction using the expression vector of this application, encompassing any cell progeny that differs from the parent cell due to mutations occurring during replication. In some embodiments, the host cell is a prokaryotic cell, selected 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] Methods for preparing mutants This application provides a method for preparing the above-mentioned RNA ligase mutant, comprising: (1) culturing the host cell described in this application under conditions suitable for mutant expression; and (2) recovering the mutant.
[0022] In some implementations, the method for recovering mutants 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 step of purifying the mutant, 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).
[0024] Composition The composition provided in this application comprises at least one RNA ligase mutant described in this application.
[0025] The composition described in this application can be a composition for storing RNA ligase mutants. In some embodiments, the composition described in this application may optionally contain, in addition to the aforementioned RNA ligase mutant, components such as buffering agents (e.g., Tris base, Tris-HCl, HEPES, MOPS), salts (e.g., NaCl, KCl, (NH4)2SO4), enzyme inhibitors (e.g., EDTA, EDTA-2Na), reducing agents (e.g., DTT), and stabilizers (e.g., glycerol). In some embodiments, the composition described in this application for storing RNA ligase mutants comprises: RNA ligase mutant, Tris-HCl, KCl, (NH4)2SO4, EDTA, DTT, and glycerol.
[0026] Reagent test kit The kit provided in this application contains at least one RNA ligase mutant described in this application.
[0027] The kit described in this application may also contain one or more reaction reagents (such as buffers, nucleotide substrates, such as ATP or dATP, RNase inhibitors, one or more polynucleotide substrates). In some embodiments, the kit may also contain a linker, including polyethylene glycol (e.g., PEG 6000, PEG 8000, etc.). 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).
[0028] Applications or uses This application provides the use of the above-mentioned RNA ligase mutants, compositions, or kits in the preparation of nucleic acid products.
[0029] This application also provides the use of the above-mentioned RNA ligase mutants, compositions, or kits in various methods, including but not limited to polynucleotide synthesis, preparation of RNA drugs, etc.
[0030] Methods for preparing nucleic acid products This application also provides a method for manufacturing nucleic acid products, including ligating nucleic acid materials in the presence of the RNA ligase mutant of this application to generate nucleic acid products. The nucleic acid materials may be selected from single-stranded nucleic acid materials, double-stranded nucleic acid materials, and mixtures thereof.
[0031] Nucleic acid materials Nucleic acids in nucleic acid materials and products can be classified into natural nucleic acids and modified nucleic acids. Natural nucleic acids are composed of natural nucleotides, which refer to naturally occurring nucleotides such as ATP, GTP, CTP, UTP, dATP, dGTP, dCTP, and dTTP. Modified nucleic acids refer to nucleic acids that contain non-natural nucleotides.
[0032] The nucleic acid materials can be single or multiple. When using multiple nucleic acid materials, the method of this application can be used for the following ligation methods: ligation of multiple double-stranded nucleic acids with protruding ends, ligation of single or multiple double-stranded nucleic acids with protruding ends to single or multiple single-stranded nucleic acids, ligation of multiple single-stranded nucleic acids, etc. The number of nucleic acid materials used when using multiple nucleic acid materials is not particularly limited as long as it is two or more; it can be a small number, such as 2-10, 2-8, 3-7, 4-6, etc., but it can also be more than 10.
[0033] There is no particular limitation on the length of the nucleic acid material. For example, longer nucleic acid materials exceeding 1000 bases in length can be used. Alternatively, shorter nucleic acid materials can be used when it is desired to produce shorter nucleic acid products. For shorter nucleic acid materials, for example, the length can be 5 bases or more, preferably 6 bases or more, more preferably 7 bases or more, further preferably 8 bases or more, and particularly preferably 9 bases or more. In addition, for shorter nucleic acid materials, the length can also be 19 bases or less, preferably 18 bases or less, more preferably 17 bases or less, further preferably 16 bases or less, and particularly preferably 15 bases or less.
[0034] Nucleic acid materials can be manufactured through chemical synthesis (e.g., solid-phase synthesis, liquid-phase synthesis) or enzymatic synthesis. Nucleic acid materials can be one or more of DNA and RNA.
[0035] nucleic acid products Nucleic acid products contain complementary base pairs. Examples of such nucleic acid products include double-stranded nucleic acids and single-stranded nucleic acids containing double-stranded-like structure portions (e.g., circular nucleic acids such as hairpin-shaped and dumbbell-shaped nucleic acids). Double-stranded nucleic acids can be double-stranded nucleic acids in which each strand is one of the aforementioned nucleic acids, and examples include: double-stranded RNA, double-stranded DNA, heteroduplex nucleic acids formed from RNA and DNA, double-stranded nucleic acids formed from RNA and RNA-DNA hybrid nucleic acids, double-stranded nucleic acids formed from DNA and RNA-DNA hybrid nucleic acids, and double-stranded nucleic acids formed from RNA-DNA hybrid nucleic acids. Examples of double-stranded nucleic acids include siRNA and heteroduplex nucleic acids.
[0036] In some embodiments, the nucleic acid product may contain the aforementioned modified residues in its complementary portion. For example, double-stranded nucleic acids or circular nucleic acids containing modified nucleotide residues (e.g., double-stranded nucleic acids or circular nucleic acids containing modified nucleotide residues in their complementary portions).
[0037] Nucleic acid products can be nucleic acids formed solely by complementary base pairs, or they can be nucleic acids containing both complementary and unpaired non-complementary bases. The length of the complementary and / or non-complementary bases is not particularly limited, and they can be relatively short. For example, a shorter complementary base can be 11–27 bases, 12–27 bases, 15–27 bases, or 18–27 bases in length. Similarly, a shorter non-complementary base can be 1–16 bases, 1–10 bases, 1–5 bases, or 1, 2, or 3 bases in length. When a nucleic acid product contains both complementary and non-complementary bases, the complementary bases can be continuous or discontinuous, separated by non-complementary bases. The length of the nucleic acid product is not particularly limited, and it can be relatively short. For example, a shorter nucleic acid product can be 20–80 bases or 24–74 bases in length.
[0038] Reaction conditions for connection The appropriate reaction system and incubation conditions for generating the target nucleic acid product are well known in the art. Those skilled in the art can determine suitable reaction conditions, such as the pH value of the reaction system, reaction temperature, reaction time, salt concentration, or whether to add exogenous cofactors, taking into account the optimal activity of RNA ligase.
[0039] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. If any definition of a specific term disclosed in the prior art differs from any definition provided herein, the definition provided herein shall prevail.
[0040] In this application, the "identity" value with a certain amino acid sequence refers to the percentage of identity calculated using NCBI-BLAST software (BLASTP) with default settings.
[0041] Beneficial effects
[0042] The RNA ligase mutant provided in this application has significantly improved activity compared with the wild-type RNA ligase shown in SEQ ID NO: 1, and has good prospects for industrial application. Attached Figure Description
[0043] Figure 1 This is a schematic diagram illustrating the construction of recombinant plasmids; Figure 2 Results of preparation of double-stranded RNA products for wild-type and mutant RNA ligases. Detailed Implementation
[0044] 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.
[0045] Example 1: Preparation and expression level determination of RNA ligase A His tag was added to the front end of the amino acid sequence SEQ ID NO: 1-2 of the RNA ligase. After DNA sequence synthesis, PCR amplification was performed (DNA sequence SEQ ID NO: 3-4). The DNA was then introduced into the NdeI and XhoI restriction sites of the expression vector pET-30a(+) to obtain a recombinant expression vector, which was transformed into E. coli BL21(DE3). After screening by antibiotic resistance plate plating, cloned strains were obtained. After successful recombination, the obtained strain was activated overnight at 37°C in LB medium, then added to 200 mL of fermentation broth (LB medium) and cultured until the OD600 value reached 0.6–0.8. IPTG was added to a final concentration of 0.5 mM, and the strain was cultured for another 18 h (16°C). The strain was then collected by centrifugation at 12000 rpm at 5°C. The collected strain was washed with 0.2 M PBS buffer (pH 7.0), and 15 mL of buffer was added for ultrasonic disruption to obtain crude enzyme solution. The crude enzyme solution was purified by nickel column affinity chromatography (His trap HP (GE Healthcare Corp., Chicago, IL, USA), 17524801) and Q-column purification (Bestarose HP, BorgLyn (Shanghai)) to obtain ligase solution. This ligase solution was then dialyzed to a storage buffer (10 mM Tris-HCl, 50 mM KCl, 35 mM (NH4)2SO4, 0.1 mM DTT, 0.1 mM... EDTA (50% Glycerol, pH 7.5) was used, and its concentration was determined by HPLC. WT was a wild-type ligase derived from Acinetobacter phage ZZ1, and the correspondence between the ligase and the amino acid sequence is shown in Table 1.
[0046] Table 1
[0047] Example 2: Enzymatic preparation of double-stranded RNA products The above wild-type ligase and its mutant were diluted to 0.5 ng / μL with storage buffer. The reaction components were prepared according to Table 2. After adding the octet, the mixture was shaken and incubated at 25°C for 2 h. The substrate sequences are shown in Table 3.
[0048] Table 2: Reaction System
[0049] Table 3: Substrate Sequences
[0050] After the above reaction is completed, the product is sent for HPLC analysis.
[0051] The HPLC analysis methods are as follows: Instrument: Thermo Fisher Scientific VF Mobile phase A: 100 mM TEAA Mobile phase B: Acetonitrile Chromatographic column: Waters ACQUITY UPLC Peptide BEH C18 Column 300A, 1.7 μm, 2.1 x 50 mm Column temperature: 55℃; Flow rate: 0.5 ml / min; Wavelength: 260 nm Test results are shown Figure 2 As shown in Table 4, the mutant effectively improves the product conversion rate compared to the wild type.
[0052] Table 4 .
Claims
1. An RNA ligase mutant, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO:
2.
2. A polynucleotide molecule, characterized in that, It encodes the RNA ligase mutant of claim 1.
3. An expression carrier, characterized in that, It comprises the polynucleotide molecule of claim 2.
4. A host cell, characterized in that... It comprises the polynucleotide molecule of claim 2, or the expression vector of claim 3.
5. The method for preparing the mutant according to claim 1, characterized in that, include: (1) Culturing the host cells as described in claim 4; and (2) Recover RNA ligase mutants.
6. A composition or kit, characterized in that, The composition or kit contains the mutant as described in claim 1.
7. The use of the mutant of claim 1, the composition of claim 6, or the kit in the preparation of nucleic acid products.
8. A method for preparing nucleic acid products, characterized in that, The method includes contacting nucleic acid material with the RNA ligase mutant of claim 1 to obtain the target nucleic acid product; The nucleic acid material is selected from single-stranded nucleic acid materials and / or double-stranded nucleic acid materials.
9. The method according to claim 8, characterized in that, The nucleic acid material is RNA, and the nucleic acid product is siRNA.
10. A fusion protein, characterized in that, The fusion protein is a tag that assists in expression or purification by linking the N-terminus and / or C-terminus of the RNA ligase mutant of claim 1.