RNA ligase mutants, methods of making same, and use thereof in the production of nucleic acid products

Abstract

This application provides a class of RNA ligase mutants, their preparation methods, and their applications in the preparation of nucleic acid products, belonging to the field of biomedical technology. Compared with wild-type enzymes, the RNA ligase mutants with mutations of Y206A, V207A, I224A, or C226Y provided in this application significantly reduce non-specific ligation impurities generated in the ligation reaction, showing good prospects for industrial application.

Description

Technical Field

[0001] This application relates to the field of biomedicine, 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 various life-threatening diseases. siRNA (small interfering RNA) is considered one of the most promising applications in gene therapy. Since August 2018, Alnylam Pharmaceuticals has obtained approval for the siRNA therapy ONPATTRO. ® Since the approval of patisiran and GIVLAARI™ (givosiran), 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] Almac Sciences and Alnylam Pharmaceuticals jointly developed a biochemical co-process for the preparation of siRNA. This method involves splitting siRNA into multiple fragments, preparing small fragments using a solid-phase method, and then ligating them 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. However, small fragments may incorrectly form hairpin impurities, leading to incorrect ligation and impurities, which poses a challenge to downstream purification processes and the quality of the final siRNA product. Therefore, there is an urgent need for an economical RNA ligase that reduces the generation of hairpin impurities during non-specific ligation.

[0005] Summary of the Invention

[0006] In a first aspect, this application provides a class of RNA ligase mutants whose amino acid sequence contains at least one mutation at the following amino acid positions relative to SEQ ID NO: 1: Y206, V207, I224, C226.

[0007] 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).

[0008] Thirdly, this application provides a method for preparing the above-mentioned ligase mutant.

[0009] Fourthly, this application provides a composition comprising at least one ligase mutant as described in this application.

[0010] Fifthly, this application provides a kit containing at least one ligase mutant as described in this application.

[0011] Sixthly, this application also provides the use of the above-mentioned ligase mutants, compositions or kits in the preparation of nucleic acid products.

[0012] 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 at least one mutation selected from the following amino acid positions Y206, V207, I224, and C226 relative to SEQ ID NO: 1, wherein the mutation is selected from the substitution, deletion, or insertion of amino acid residues.

[0014] In some implementations, the variant includes a mutation at the Y206 position, the mutation being Y206A.

[0015] In some implementations, the variant includes a mutation at the V207 position, the mutation being V207A.

[0016] In some implementations, the variant includes a mutation at the I224 position, the mutation being I224A.

[0017] In some implementations, the variant includes a mutation at the C226 position, the mutation being C226Y.

[0018] In some embodiments, the amino acid sequence of the RNA ligase mutant has at least 97%, at least 98%, at least 99%, or higher sequence identity compared to the sequences shown in any of SEQ ID NO: 2-5. In some embodiments, the amino acid sequence of the RNA ligase mutant is as shown in any of SEQ ID NO: 2-5.

[0019] In some implementations, RNA ligase mutants may be provided in modified form, such as fusion proteins with amino acid tags that can be used in the separation, dissolution, and / or purification or identification processes of RNA ligases. Such amino acid tags include, but are not limited to, polyhistidine (His) tags, c-myc tags, FLAG tags, V5 tags, or hemagglutinin (HA) tags.

[0020] In some embodiments, the RNA ligase mutant possesses the activity of Rnl2 family ligases. In some embodiments, the RNA ligase mutant exhibits improved properties compared to a reference RNA ligase, with less non-specific ligation impurity production.

[0021] In some embodiments, the improved properties of the RNA ligase mutant are compared to those of an RNA ligase having the amino acid sequence SEQ ID NO: 1. In some embodiments, the reference RNA ligase is wild-type bacteriophage T4 RNA ligase 2.

[0022] Polynucleotide molecules This application provides a polynucleotide molecule encoding the RNA ligase mutant described in this application.

[0023] In some embodiments, the nucleotide sequence of the polynucleotide molecule is as shown in any of SEQ ID NO: 7-10. 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.

[0024] expression carrier This application provides an expression vector comprising a multinucleotide molecule encoding the RNA ligase mutant of this application.

[0025] 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.

[0026] 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(+).

[0027] 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).

[0028] 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.

[0029] 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.

[0030] 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).

[0031] Composition The composition provided in this application comprises at least one RNA ligase mutant described in this application.

[0032] 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.

[0033] Reagent test kit The kit provided in this application contains at least one RNA ligase mutant described in this application.

[0034] 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, and one or more polynucleotide substrates. In some embodiments, the kit further contains a linkage enhancer, 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).

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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).

[0044] 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.

[0045] Reaction conditions for connection The reaction system and incubation conditions suitable 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 reduction of hairpin impurities by RNA ligase ligation.

[0046] Other implementation plans: 1. An RNA ligase mutant, wherein the amino acid sequence relative to SEQ ID NO: 1 contains at least one mutation selected from the following amino acid positions: Y206, I207, I224, C226, wherein the mutation is selected from the substitution, deletion or insertion of amino acid residues.

[0047] 2. The mutant according to item 1, wherein the amino acid sequence relative to SEQ ID NO: 1 contains at least one of the following mutations: Y206A, V207A, I224A, or C226Y.

[0048] 3. The mutant described in item 1 has an amino acid sequence as shown in any of SEQ ID NO: 2-5.

[0049] 4. A biomaterial, characterized in that the biomaterial is selected from one or more of the following: 1) A polynucleotide molecule encoding any of the RNA ligase mutants described in items 1-3; 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).

[0050] 5. The method for preparing the mutant according to any one of items 1-3, comprising: (1) Culture host cells as described in section 4 under conditions suitable for mutant expression; and (2) Recover mutants.

[0051] 6. A composition comprising any of the mutants described in items 1-3.

[0052] 7. A kit containing any of the mutants described in items 1-3.

[0053] 8. The use of any of the mutants described in items 1-3, the composition described in item 6, or the kit described in item 7 in the preparation of nucleic acid products.

[0054] 9. A method for preparing a nucleic acid product, comprising contacting nucleic acid material with any of the RNA ligase mutants described in items 1-3 to obtain a target nucleic acid product.

[0055] 10. The nucleic acid material of the method according to item 9 comprises natural nucleic acid or modified nucleic acid, which is a single-stranded or double-stranded nucleic acid.

[0056] 11. According to the method described in item 9, the single-stranded nucleic acid or double-stranded nucleic acid is single-stranded RNA or double-stranded RNA.

[0057] 12. The nucleic acid product of the method according to item 9 contains a complementary portion of 12 to 27 bases in length.

[0058] 13. According to the method described in item 9, the nucleic acid product is siRNA.

[0059] 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.

[0060] Beneficial effects The RNA ligase mutant provided in this application, compared with the wild-type T4 RNA ligase 2 with an amino acid sequence as shown in SEQ ID NO: 1, significantly reduces the non-specific impurities generated in nucleic acid ligation reactions, and has good prospects for industrial application. Attached Figure Description

[0061] Figure 1 This is a schematic diagram illustrating the construction of recombinant plasmids; Figure 2 The results show the relative generation rate of Hairpin impurities. Detailed Implementation

[0062] 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.

[0063] Example 1: Preparation and expression level determination of T4 RNA ligase 2 mutant A His tag was added to the front end of the amino acid sequences SEQ ID NO: 1-5 of wild-type T4 RNA ligase 2 and its mutants. After DNA sequence synthesis, PCR amplification was performed (DNA sequence SEQ ID NO: 6-10). 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℃ in LB medium and then added to 200 mL of fermentation broth (LB medium). The culture was continued 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℃). The strain was then collected by centrifugation at 12000 rpm at 5℃. 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 T4 RNA ligase 2 enzyme solution. This enzyme solution was then dialyzed and transferred to 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 added, and the concentration was determined by HPLC. WT was wild-type T4 RNA ligase 2. The correspondence between the T4 RNA ligase 2 mutant and the amino acid sequence is shown in Table 1. Table 1: Correspondence between T4 RNA ligase 2 mutants and amino acid sequences

[0064] Example 2: Enzymatic preparation of double-stranded RNA products The above-mentioned T4 RNA ligase 2 wild-type and mutant were diluted to 12.5 ng / μL with storage buffer. The reaction components were prepared according to Tables 2 and 3. After adding the octet, the mixture was vortexed and incubated at 25°C for 2 h, followed by denaturation at 80°C for 5 min to terminate the reaction. The products were used for HPLC analysis. The substrate sequences are shown in Table 4. The relative yield of hairpin impurities = (yield of two substrate segments / yield of four substrate segments (AS+SS)) * 100% Table 2 Reaction System

[0065] Table 3 Reaction System

[0066] Table 4 Substrate Sequences

[0067] According to the test results, as shown in Table 5 and Figure 2 As shown, all of the above mutants can effectively reduce the production of hairpin impurities. Among them, I224A and Y206A can reduce the relative production rate of hairpin impurities from 12.19% in the wild type to 6.35% and 6.78%, respectively.

[0068] Table 5 Analysis of hairpin impurity generation results .

Claims

1. An RNA ligase mutant, characterized in that, The amino acid sequence of the mutant is shown in any of SEQ ID NO: 2-5.

2. A biomaterial, characterized in that, The biomaterial is selected from one or more of the following: 1) Encoding a polynucleotide molecule of the RNA ligase mutant 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 mutant according to claim 1, characterized in that, include: (1) Culture host cells in the biomaterial as described in claim 2 under conditions suitable for mutant expression; and (2) Recover mutants.

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

5. A reagent kit, characterized in that, The kit contains the mutant as described in claim 1.

6. The use of the mutant of claim 1, the composition of claim 4, or the kit of claim 5 in the preparation of nucleic acid products.

7. 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.

8. The method according to claim 7, characterized in that, The nucleic acid material is selected from single-stranded nucleic acid materials, double-stranded nucleic acid materials, and mixtures thereof.

9. The method according to claim 7, characterized in that, The nucleic acid material is RNA.

10. The method according to claim 7, characterized in that, The nucleic acid product is siRNA.

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