Related product of inorganic pyrophosphatase in synthesis of nucleotide chain and application

Abstract

The invention discloses a related product of inorganic pyrophosphatase in synthesis of a nucleotide chain and application, and relates to the technical field of nucleic acid synthesis. The inorganic pyrophosphatase is added in the synthesis process of the nucleotide chain, so that by-products generated in the ligation reaction process can be greatly reduced or eliminated, the reaction speed is increased, the conversion rate and purity of the target product are further improved, and the effect of the target nucleotide chain can be fully exerted; and a new way is provided for high-quality large-scale synthesis of the nucleotide chain.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid synthesis, and in particular to products and applications of inorganic pyrophosphatase in the synthesis of nucleotide chains. Background Art

[0002] At present, the main method for synthesizing oligonucleotides in industry is solid phase synthesis. The synthesis of a large number of oligonucleotides requires multiple accumulations, and when synthesizing oligonucleotides, the yield decreases as the chain length increases. At the same time, as the chain length increases, the synthetic product contains a certain amount of non-target nucleotide chains whose length does not match the target nucleotide chain. After chemical synthesis, more non-target nucleotide chains cannot be effectively separated and removed from the synthetic product. The presence of non-target nucleotide chains in the synthetic product often affects the function and activity of the target nucleotide chain, especially for the target nucleotide chain antisense chain drugs, RNAi drugs, nucleic acid aptamer drugs and sgRNA drugs, etc. The presence of non-target nucleotide chains will seriously affect the target nucleotide chain to exert its efficacy.

[0003] For nucleotide chains with a length of less than or equal to 20mer, after chemical synthesis, the proportion of the final target nucleotide chain in the synthetic product can be increased through a purification process (for example, an ion column or a reverse column, etc.). Therefore, when synthesizing oligonucleotides, long fragments of 100-200mer are separated into short fragments, and RNA ligase is used to connect the short fragments into long fragments, thereby improving the purity of the oligonucleotides.

[0004] Existing enzyme ligation technology has at least the following problems: for high substrate concentrations, for example, the concentration of ligatable nicks exceeds 2.5mM, the reaction speed of the reaction solution containing only RNA ligase is slow and the conversion rate is low, especially when only one or part of the nicks complete the reaction first, while other nicks do not complete the ligation reaction.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide related products and applications of inorganic pyrophosphatase in the synthesis of nucleotide chains.

[0007] The present invention is achieved in that:

[0008] In a first aspect, embodiments of the present invention provide the use of inorganic pyrophosphatase in the synthesis of nucleotide chains or in the preparation of products for the synthesis of nucleotide chains.

[0009] In a second aspect, an embodiment of the present invention provides an enzyme ligation reagent, which includes: inorganic pyrophosphatase and nucleic acid ligase.

[0010] In a third aspect, an embodiment of the present invention provides a reaction solution, which includes: a double-stranded nucleic acid molecule with a nick and / or a nucleic acid substrate capable of forming the double-stranded nucleic acid molecule with a nick, and the enzyme-linked reagent described in the above embodiment.

[0011] In a fourth aspect, an embodiment of the present invention provides a composition, which includes: the components of the enzyme-linked reagent described in the above embodiments or the components of the reaction solution described in the above embodiments.

[0012] In a fifth aspect, an embodiment of the present invention provides a kit, which includes: the enzyme-linked reagent described in the preceding embodiment or the reaction solution described in the preceding embodiment or the composition described in the preceding embodiment.

[0013] In a sixth aspect, an embodiment of the present invention provides a method for synthesizing a nucleotide chain, comprising: synthesizing using the enzyme-linked reagent described in the preceding embodiment, or the reaction solution described in the preceding embodiment, or the composition described in the preceding embodiment, or the kit described in the preceding embodiment.

[0014] The present invention has the following beneficial effects:

[0015] By adding inorganic pyrophosphatase during the synthesis of nucleotide chains, the by-products produced in the ligation reaction process can be greatly reduced or eliminated, the reaction speed can be accelerated, and the conversion rate and purity of the target product can be improved, which is conducive to fully exerting the efficacy of the target nucleotide chain and providing a new way for high-quality and large-scale synthesis of nucleotide chains. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0017] Figure 1 The oligonucleotides and ligation products thereof used in Example 1 of the present invention;

[0018] Figure 2 The oligonucleotides and ligation products thereof used in Example 3 of the present invention;

[0019] Figure 3 The oligonucleotides and their ligation products used in Example 4 of the present invention. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0021] Explanation of terms

[0022] Oligonucleotide: generally refers to a linear polynucleotide fragment composed of 2 to 10 nucleotide residues connected by phosphodiester bonds. However, it should be noted that there is no strict regulation on the number of nucleotides in an oligonucleotide. In some literature, polynucleotide molecules containing 30 or more, up to 200, 300, 400, or 500 nucleotide residues can also be called oligonucleotides.

[0023] Natural ribonucleotides: composed of one molecule of phosphoric acid, one molecule of ribose (a five-carbon sugar), and one molecule of nitrogenous base. According to the type of nitrogenous base, ribonucleotides are divided into adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, and uracil ribonucleotides.

[0024] 2' deoxyribonucleotide: It is composed of one molecule of phosphoric acid, one molecule of 2' deoxyribose (the 2' of ribose is deoxygenated and becomes a hydrogen atom), and one molecule of nitrogenous base. According to the type of nitrogenous base, natural 2' deoxyribonucleotides are divided into adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymine deoxyribonucleotide.

[0025] Non-natural nucleotides: refers to nucleotides produced by modifying the phosphate group, nitrogenous base, sugar ring and glycosidic bond of natural nucleotides.

[0026] RNA: refers to a molecule formed by natural or non-natural ribonucleotides connected by phosphate bonds. Although typical RNA molecules are linked together by standard phosphodiester bonds, RNA as a drug can contain one or more non-standard bonds. RNA can be single-stranded or double-stranded, or can contain both single-stranded and double-stranded regions. In addition, ribonucleotides can also be divided into linear ribonucleotide chains and circular ribonucleotide chains according to their morphology. Circular ribonucleotide chains are composed of one or more linear ribonucleotide chains connected end to end by phosphate bonds, and have a closed ring structure.

[0027] DNA: refers to a molecule formed by 2' deoxyribonucleotides connected by phosphate bonds.

[0028] As used herein, "wild type" refers to a form found in nature, for example, a wild type protein sequence refers to a form found in nature, which can be isolated from a natural source and has not been intentionally modified or altered by man.

[0029] The term "nick" or "ligation nick" refers herein to the situation where a phosphodiester bond is missing between two adjacent nucleotide fragments in a double-stranded structure. A nick can be catalyzed by a double-stranded ligase to form a complete phosphodiester bond, i.e., the 3' terminal hydroxyl group of a nucleotide unit at the nick and the 5' monophosphate group of another nucleotide unit at the nick form a complete phosphodiester bond by double-stranded ligase catalysis. A "nick" can also be understood as a gap in a double-stranded nucleic acid molecule due to the breaking of a phosphodiester bond.

[0030] The term "gap" herein refers to a situation where a strand in a double-stranded structure is broken into two strands due to the loss of one or more consecutive nucleotides, and the two strands form a gap relationship.

[0031] The nucleic acid substrate described herein is an oligonucleotide capable of forming a double-stranded nucleic acid molecule with a nick, wherein the 5' end of the oligonucleotide contains a monophosphate group, and / or the 3' end contains a hydroxyl group.

[0032] The term "denaturation" herein refers to the process of breaking the hydrogen bonds between double-stranded nucleic acids such as double-stranded DNA, double-stranded RNA or DNA / RNA base pairs by incubation at high temperature, thereby converting double-stranded nucleic acids into single-stranded nucleic acids.

[0033] The term "annealing" herein refers to the process of slowly lowering the temperature of a nucleic acid solution that has been denatured at high temperature to a lower temperature so that the single-stranded nucleic acid reforms into a double-stranded nucleic acid.

[0034] The term "sequence identity percentage (%)" is used herein to refer to the comparison between polynucleotides and polypeptides, and is determined by comparing two optimally aligned sequences across a comparison window, wherein for optimal alignment of the two sequences, the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) compared to the reference sequence. The percentage can be calculated as follows: determine the number of positions where the same nucleic acid base or amino acid residue occurs in the two sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Alternatively, the percentage can be calculated as follows: determine the number of positions where the same nucleic acid base or amino acid residue occurs in the two sequences or the number of positions where the nucleic acid base or amino acid residue is aligned with a gap to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Those skilled in the art will appreciate that there are currently a variety of algorithms for sequence alignment, for example, the Smith-Waterman local homology algorithm (Smith and Waterman, Adv. Appl. Math., 2: 482

[1981] ), the Needleman-Wunsch global homology alignment algorithm (Needleman and Wunsch, J. Mol. Biol., 48: 443

[1970] ). These algorithms have been designed as related software, and researchers in the field can use these software to quickly align protein sequences or nucleotide sequences, such as the open software EMBOSS Water (https: / / www.ebi.ac.uk / jdispatcher / psa / emboss_water) based on the Smith-Waterman algorithm of the European Bioinformatics Institute (EMBL-EBI) and the EMBOSS needle software (https: / / www.ebi.ac.uk / jdispatcher / psa / emboss_needle) based on the Needleman-Wunsch algorithm. Protein sequence alignment and sequence alignment % identity can be performed using the open software EMBOSS Water software. The scoring matrix used in the alignment process is BLOSUM62, with the gap open score (GAP OPEN) set to 10 and the gap extension (GAP EXTEND) set to 1.

[0035] "Reference sequence" refers to a designated sequence used as the basis for sequence comparison. A reference sequence can be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Typically, a reference sequence is a length of at least 20 nucleotides or amino acid residues, a length of at least 25 residues, a length of at least 50 residues, a length of at least 100 residues, or the full length of a nucleic acid or polypeptide. Since two polynucleotides or polypeptides can each (1) contain a similar sequence (i.e., a portion of a complete sequence) between the two sequences, and (2) can also contain a different sequence between the two sequences, a sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides on a "comparison window" to identify and compare local regions of sequence similarity. In some embodiments, a "reference sequence" can be based on a primary amino acid sequence, wherein a reference sequence is a sequence that can have one or more changes in the primary sequence.

[0036] "Comparison window" refers to a conceptual segment of at least about 20 consecutive nucleotide positions or amino acid residues, wherein a sequence can be compared to a reference sequence of at least 20 consecutive nucleotides or amino acids, and wherein the portion of the sequence in the comparison window can contain 20% or less additions or deletions (i.e., gaps) for optimal alignment of the two sequences compared to the reference sequence (which does not contain additions or deletions). The comparison window can be longer than 20 consecutive residues, and optionally includes windows of 30, 40, 50, 100 or more.

[0037] In the case of numbering for a given amino acid or polynucleotide sequence, "corresponding to", "referenced to" or "relative to" refers to the numbering of the residues of the reference sequence specified when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is specified with respect to the reference sequence, rather than being specified by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as the amino acid sequence of a T4 RNA ligase 2 mutant, can be optimized for residue matching between the two sequences by introducing gaps to align with the reference sequence. In these cases, despite the presence of gaps, the numbering of the residues in the given amino acid or polynucleotide sequence is made according to the reference sequence compared thereto.

[0038] The present invention aims at the problems of the generation of by-products, slow enzyme-linked reactions and low conversion rates in the existing nucleotide chain synthesis process, especially when the concentration of the ligation nicks in the reaction solution is high (≥2.5mM) or the amount of the nucleic acid substrate in the reaction solution is ≥20g / L, the reaction rate and conversion rate are even lower. The inventors of the present application have discovered and verified for the first time that inorganic pyrophosphatase can effectively reduce or inhibit the generation of by-products, thereby accelerating the rate of the enzyme-linked reaction, and thereby improving the conversion rate and purity of the target nucleotide chain, especially when the concentration of the ligation nicks is high (≥2.5mM), the advantage of adding inorganic pyrophosphatase is more significant.

[0039] Specific technical solutions

[0040] In one aspect, embodiments of the present invention provide the use of inorganic pyrophosphatase in the synthesis of nucleotide chains or in the preparation of products for the synthesis of nucleotide chains.

[0041] In some embodiments, the product includes: a reagent, a kit, a chip, or a combination thereof.

[0042] On the other hand, an embodiment of the present invention provides an enzyme ligation reagent for use in the synthesis of nucleotide chains, which includes: inorganic pyrophosphatase and nucleic acid ligase.

[0043] The source of the inorganic pyrophosphatase includes any of Escherichia coli, yeast, and thermotolerant bacteria.

[0044] The inorganic pyrophosphatase may be a natural pyrophosphatase, or a mutant produced by enzyme mutation, modification and evolution, or an enzyme that can catalyze the elimination of by-products in the enzyme chain after fusion with enzymes in the same family or other families.

[0045] Nucleic acid ligase catalyzes the ligation nicks of nucleic acid substrates to form phosphodiester bonds to form the target product. Adding inorganic pyrophosphatase can greatly improve the conversion rate and purity of the synthetic target product, which is beneficial to fully exert the efficacy of the target product.

[0046] In some embodiments, the nucleic acid ligase includes a double-stranded ligase. A double-stranded ligase is a ligase that can seal a ligation nick in a double-stranded structure. The double-stranded ligase includes an RNA ligase, including a ligase that connects RNA double strands and / or RNA / DNA hybrid double strands. Inorganic pyrophosphatase is particularly suitable for the case where a double-stranded ligase is used to synthesize a nucleotide chain, as long as one of the double strands is RNA or the nick is RNA.

[0047] RNA ligase has three steps in the enzymatic ligation process: the first step is that the ligase reacts with ATP to generate a ligase-AMP intermediate and release pyrophosphate; the second step is that the ligase-AMP intermediate binds to the double-stranded substrate with a nick and transfers AMP to the 5'-phosphate end to form a double-stranded adenylated substrate with a nick; the third step is that the ligase catalyzes the 3'-OH of the nicked RNA to attack the 5' phosphate group, forming a new 3'-5' phosphodiester bond connecting the two nicks and releasing AMP. Inorganic pyrophosphatase can catalyze the elimination of byproducts in the first step reaction, thereby driving the chemical reaction to the right, thereby accelerating the rate of the enzyme ligation reaction and increasing the conversion rate.

[0048] The present application has no special restrictions on the specific selection of double-chain ligase, which can be selected according to actual conditions. A natural double-chain ligase can be used, or a mutant strain produced by enzyme mutation, transformation and evolution, or an enzyme that produces nick blocking activity after fusion with enzymes within the same enzyme family or with other families.

[0049] In some embodiments, the double-stranded ligase comprises any one or more of an Rnl2 family ligase and an Rnl5 family ligase.

[0050] In some embodiments, the double-stranded ligase comprises any one or more of T4 RNA ligase 2 and DraRn1 (from Naegleria gruberi).

[0051] In some embodiments, the enzyme-linked reagent further comprises: any one or more of a buffer, ATP, and a divalent ion.

[0052] In some embodiments, the divalent ions include: Mg 2+ , Mn 2+ 、Co 2+ and Zn 2+ Any one or more of .

[0053] In some embodiments, the buffer comprises any one of acetate buffer, phosphate buffer, Tris buffer and HEPES buffer.

[0054] On the other hand, an embodiment of the present invention provides a reaction solution, which includes: a double-stranded nucleic acid molecule with a nick and / or a nucleic acid substrate capable of forming the double-stranded nucleic acid molecule with a nick, and the enzyme-linked reagent described in any of the aforementioned embodiments.

[0055] In some embodiments, the final concentration of the inorganic pyrophosphatase in the enzyme-linked reagent in the reaction solution is 0.00001 mg / ml to 1 mg / ml, specifically, it can be any one of 0.00001, 0.00005, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.545, 0.6, 0.7, 0.8, 0.9 and 1 mg / mlL or a range between any two of them.

[0056] In some embodiments, the final concentration of the nucleic acid ligase in the enzyme ligation reagent in the reaction solution is 0.001 mg / ml to 20 mg / ml, specifically any one of 0.01, 0.05, 0.1, 0.5, 1, 2, 4, 6, 7, 8, 10, 12, 14, 16, 18 and 20 mg / ml or a range between any two of them.

[0057] It should be noted that the concentrations of the two enzymes can be adjusted according to actual conditions and there is no special limitation in this application.

[0058] In some embodiments, the double-stranded nucleic acid molecule with a nick includes any one or more of an RNA double-stranded molecule with a nick and an RNA / DNA hybrid double-stranded molecule with a nick.

[0059] In some embodiments, in the double-stranded nucleic acid molecule with a nick, the nick may refer to at least one strand in the double-stranded nucleic acid molecule having at least one nick, that is, there may be a nick on one strand or there may be nicks on both strands.

[0060] Specifically, the DNA / RNA hybrid double-stranded strand with a nick includes at least one deoxyribonucleic acid, and at least one end (3' terminal hydroxyl group and / or 5' terminal phosphate group) of at least one nick is RNA.

[0061] In some embodiments, the nicked double-stranded nucleic acid molecule comprises linear and / or circular shapes.

[0062] In some embodiments, the nicked double-stranded nucleic acid molecule comprises natural and / or modified.

[0063] In some embodiments, the double-stranded nucleic acid molecule with a nick includes a nucleic acid substrate or is formed by mixing or annealing nucleic acid substrates.

[0064] In some embodiments, the nucleic acid substrate comprises any one or more of a natural and / or modified RNA single strand, a natural and / or modified RNA double strand, a natural and / or modified DNA single strand, and a natural and / or modified DNA / RNA hybrid single strand and a natural and / or modified DNA / RNA hybrid double strand.

[0065] In some embodiments, in the nucleic acid substrate, the RNA single strand includes a linear RNA single strand and / or a circular RNA single strand, the RNA double strand includes a linear RNA double strand and / or a circular RNA double strand, the DNA single strand includes a linear DNA single strand and / or a circular DNA single strand, the DNA / RNA hybrid single strand includes a linear DNA / RNA hybrid single strand and / or a circular DNA / RNA hybrid single strand, and the DNA / RNA hybrid double strand includes a linear DNA / RNA hybrid double strand and / or a circular DNA / RNA hybrid double strand.

[0066] In some embodiments, the single-stranded RNA comprises any one or more of mRNA, antisense oligonucleotide, siRNAi, sgRNA, lncRNA, CircRNA and miRNA.

[0067] When the nucleic acid substrate is double-stranded and the double-stranded nucleic acid has a nick, the nucleic acid substrate itself is a double-stranded nucleic acid molecule with a nick. When the nucleic acid substrate is single-stranded, a double-stranded nucleic acid molecule with a nick can be formed by mixing the nucleic acid substrates or annealing after mixing the nucleic acid substrates.

[0068] In some embodiments, the annealing temperature of the nucleic acid substrate is 0-100°C, specifically any one of 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100°C or a range between any two of them.

[0069] In some embodiments, the process of forming a double-stranded nucleic acid molecule with a nick from a nucleic acid substrate can occur during the mixing of the nucleic acid substrate, without the need for a separate heating annealing process. The nucleic acid substrate, enzyme, ATP, Mg, etc. can be directly mixed into the mixture. 2+ The molecules necessary for the reaction are mixed with the solution and then connected. The nucleic acid substrates are specifically bound during the mixing process to form a double-stranded nucleic acid molecule with a nick.

[0070] In some embodiments, the fragment length of the nucleic acid substrate is ≥ 2 nt.

[0071] In some embodiments, the fragment length of the nucleic acid substrate is 2 to 200 nt, specifically, it can be any one of 2, 5, 7, 10, 13, 15, 17, 20, 23, 25, 27, 30, 33, 35, 37, 40, 43, 45, 47, 50, 53, 55, 57, 60, 63, 62, 67, 70, 73, 75, 77, 80, 85, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 nt or the range between any two.

[0072] In some embodiments, the number of the nucleic acid substrates is ≥ 1, for example, when used for circularization of circular RNA, the number of nucleic acid substrates is 1. For example, when used for circularization of circular RNA, the number of nucleic acid substrates is 1. The number can be any one of 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 or a range between any two of the above.

[0073] In some embodiments, the nucleic acid substrate includes a first fragment, a second fragment, a third fragment, and a fourth fragment, the 5' end of the first fragment contains a monophosphate group, the 3' end of the second fragment contains a hydroxyl group, and the first fragment and the second fragment can cooperate with each other and form a double-stranded structure together. The nucleic acid fragment includes a third fragment and a fourth fragment, the 3' end of the third fragment contains a hydroxyl group, the 5' end of the fourth fragment contains a monophosphate group, and the third fragment and the fourth fragment can cooperate with each other and form a double-stranded structure together. A notch is formed between the 5' end of the first fragment and the 3' end of the third fragment, and the first fragment and the third fragment are connected by a ligase to form a phosphodiester bond between the 3' end and the 5' end, and a target nucleotide chain can be formed after connection; a notch is formed between the 3' end of the second fragment and the 5' end of the fourth fragment, and the second fragment and the fourth fragment are connected by a ligase to form a phosphodiester bond between the 3' end and the 5' end, and a target nucleotide chain can be formed after connection.

[0074] In some embodiments, the molar ratio of any two nucleic acid fragments can be 0.1 to 1:1, specifically any one of 0.1:1, 0.2:1, 0.4:1, 0.6:1, 0.8:1, 1:1 or a range between any two of them.

[0075] The present application has no special restrictions on whether the nucleic acid substrate has a modification group. Compared with the scheme without adding inorganic pyrophosphatase, the scheme with added inorganic pyrophosphatase has fewer by-products, faster reaction speed, and higher conversion rate and purity of the target nucleotide chain.

[0076] The method provided in the embodiment of the present invention has no special limitation on the type of modification and is applicable to all types of modification.

[0077] In some embodiments, the modifications include modifications to enhance stability and / or reduce innate immune responses.

[0078] In some embodiments, the modification includes any one of modification of a phosphate group, modification of a base, modification of a sugar ring, and modification of a glycosidic bond.

[0079] In some embodiments, the modification of the phosphate group includes any one or more of the following: 5′-(E)-vinylphosphonate modification (5′-(E)-vinylphosphonate, 5′-VP), phosphorothioate modification, phosphotriester modification, 5′-methylphosphonate modification, 5′-morpholino modification, dithiophosphate modification, methoxypropylphosphonate modification, S-5′-C methyl analog modification, short-chain alkyl or cycloalkyl sugar bond modification, short-chain heteroatom or heterocyclic sugar bond modification, or the entire phosphate group is replaced by any one of amide, aminooxy, alkoxy and triazole.

[0080] In some embodiments, the modification of the base includes any one or more of the following: 2,4-difluorotoluidine ribonucleoside replacement, pseudouridine modification, 2-thiouridine modification, N1-methylpseudouridine modification, 5-methyluridine modification, 5-methoxyuridine modification, N6-methyladenosine modification, N6,N6-dimethyladenosine modification, 3-methyluridine modification, N7-methylguanosine modification, 2,7-dimethylguanosine modification, 2,2,7-trimethylguanosine modification, 5-methylcytidine modification, 5-hydroxymethylcytosine modification, 5-bromo-uracil modification, 5-iodo-uracil modification, propyne uridine nucleoside modification, N-ethylpiperidine-6-triazole-modified adenosine modification, 6'-phenylpyrrole cytosine modification, 2-aminopurine modification, inosine modification, 2,6-diaminopurine modification, 2-pyrimidone modification and 5-methylcytosine modification.

[0081] In some embodiments, the modification of the sugar ring includes any one or more of the following: 2'methoxy modification, 2'-deoxy-2'-fluoro modification, 2'O methoxyethyl modification, locked nucleic acid (LNA) modification, unlocked nucleic acid (UNA) modification, bridged nucleic acid (BNA) modification, tricyclo-DNA (tcDNA) modification, phosphodiamidate morpholino oligonucleotide (PMO) modification, 2'deoxy nucleic acid modification, (S)-constrained ethyl bicyclic nucleic acid modification, peptide nucleic acid modification and sugar mimetic modification.

[0082] In some embodiments, the sugar mimetic comprises any one or more of the following: a cyclobutyl group replacing a pentofuranosyl group in a cyclobutyl nucleoside, a morpholino group in a morpholino nucleic acid (MNA), a peptide backbone in a peptide nucleic acid (PNA), a polyethylene glycol backbone in a glycol nucleic acid (GNA), a threose backbone in a threose nucleic acid (TNA), and a butyl backbone in acyclic butyl nucleic acid (BuNA).

[0083] In some embodiments, the modification of the glycosidic bond includes, but is not limited to, replacing the CN bond of the glycosidic bond with any one of CC, CO and CS.

[0084] In some embodiments, the final concentration of the nucleic acid substrate in the reaction solution is 0.01 to 100 mM, specifically any one of or a range between any two of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 and 100 mM.

[0085] In some embodiments, the final concentration of the nucleic acid substrate is 1 to 200 g / L, specifically, it can be any one of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 g / L or a range between any two of them.

[0086] The reaction solution described in any of the aforementioned embodiments has no special restrictions on the amount of nucleic acid substrate used, and schemes with small or large amounts of nucleic acid substrate added are applicable. The advantages are more obvious when using high-concentration (≥20 g / L, for example, 40 g / L, 60 g / L) substrates, which can significantly promote the conversion rate of substrates to products and greatly reduce the content of non-target nucleotide chains in the synthetic products of nucleotide chains, so as to effectively improve the purity of target nucleotide chains in the synthetic products, thereby facilitating full utilization of the efficacy of the target products.

[0087] The double strands of the target product include a sense strand and an antisense strand. Usually, the sense strand will undergo an enzyme ligation reaction first under the action of RNA ligase, and the other strand will undergo an enzyme ligation reaction later. During the reaction, inorganic pyrophosphatase will eliminate the byproduct pyrophosphate in the reaction and promote the reaction to the direction of the target product. Preferably, the effect of inorganic pyrophosphate is more obvious in the later enzyme ligation process, which can promote the faster generation of the target product in the short fragment, and can greatly reduce the content of non-target nucleotide chains in the synthetic product of the nucleotide chain, so as to effectively improve the purity of the target nucleotide chain in the synthetic product.

[0088] In some embodiments, the nucleic acid substrate includes a fragment capable of synthesizing a sense strand, as a sense strand substrate, the sense strand substrates are connected by phosphodiester bonds to obtain the sense strand; the nucleic acid substrate includes a fragment capable of synthesizing an antisense strand, as an antisense strand substrate, the antisense strand substrates are connected by phosphodiester bonds to obtain the antisense strand. The number of sense strand substrates and antisense strand substrates is generally 1 to 10, specifically any one of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 or a range between any two.

[0089] The present application has no particular limitation on the length of the target nucleotide chain or target product. In some embodiments, the length of the target nucleotide chain or target product may be ≤100nt, 200nt or 500nt. In some embodiments, the length of the target nucleotide chain or target product may be ≥10nt, 20nt or 50nt.

[0090] In some embodiments, the sequence length of the target nucleotide chain can be 10 to 200 nt, specifically any one of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200 nt or a range between any two of them.

[0091] In some embodiments, the target nucleotide chain comprises linear and / or circular shapes.

[0092] In some embodiments, the target nucleotide chain is any one or more of a single-stranded RNA, a double-stranded RNA, a DNA / RNA hybrid single-stranded DNA / RNA hybrid double-stranded DNA / RNA.

[0093] In some embodiments, when the enzyme-linked reagent described in any of the preceding embodiments or the reaction solution described in any of the preceding embodiments is used to synthesize a nucleotide chain, the conversion rate of the target nucleotide chain or target product is ≥ 90%, 92%, 94%, 96%, 98%, or 99%.

[0094] In some embodiments, when the enzyme-linked reagent includes ATP, the final concentration of the ATP in the reaction solution is 0.01-50 mM, specifically any one of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 40, 50 mM or a range between any two of them.

[0095] In some embodiments, when the enzyme-linked reagent includes a divalent ion (e.g., Mg 2+ ), the final concentration of the divalent ion in the reaction solution is 0.01-100 mM, specifically, it can be any one of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 40, 60, 80 and 100 mM or a range between any two of them.

[0096] The present application has no special restrictions on the buffer. The buffer and its pH can be based on the pH corresponding to the optimal catalytic activity of the enzyme used as the reference value. For example, when the double-stranded ligase is T4 RNA ligase 2, the pH of the buffer can be 6.5-9.0.

[0097] In some embodiments, the concentration range of the buffer is 10-1000 mM, specifically any one of 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 mM or a range between any two of them, and a more preferred concentration is 100-500 mM.

[0098] In some embodiments, the pH of the buffer is 6.5-9.0, specifically any one of 6.5, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8 and 9.0 or a range between any two thereof.

[0099] In some embodiments, the reaction solution further comprises: any one or more of DTT, PEG and a surfactant. PEG can be used to increase the efficiency of the synthesis reaction, and the surfactant can increase the stability of the enzyme. The surfactant includes Trion X, TWEEN, etc.

[0100] On the other hand, an embodiment of the present invention provides a composition, which includes: each component of the enzyme-linked reagent described in any of the above embodiments or each component of the reaction solution described in any of the above embodiments.

[0101] In some embodiments, the composition further comprises a stop solution. The stop solution comprises EDTA. Specifically, the role of EDTA is to terminate the synthesis reaction by chelating divalent ions in the reaction solution. In some embodiments, the synthesis reaction of the nucleotide chain can also be terminated by heating to the denaturation temperature of the nucleotide chain (e.g., 80° C.) without adding EDTA.

[0102] In some embodiments, the final concentration of EDTA in the reaction solution can be 0.1-100 mM, specifically any one of 0.1, 1, 5, 6, 7, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mM or a range between any two of them.

[0103] On the other hand, an embodiment of the present invention provides a kit, which includes: the enzyme-linked reagent described in any embodiment or the reaction solution described in any embodiment or the composition described in any embodiment.

[0104] In addition, an embodiment of the present invention provides a method for synthesizing a target nucleotide chain, which comprises: synthesizing using the enzyme-linked reagent described in any of the preceding embodiments, or the reaction solution described in any of the preceding embodiments, or the composition described in any of the preceding embodiments, or the kit described in any of the preceding embodiments.

[0105] In some embodiments, the synthesis conditions include: a temperature of 15 to 55° C. and a time of 1 to 48 hours. The temperature can be any one of 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 37, 38, 40, 42, 44, 46, 48, 50, 52, 54, 55° C. or a range between any two thereof. The time can be any one of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 hours or a range between any two thereof. The specific reaction temperature and reaction time can be adjusted according to actual conditions and are not particularly limited in this application.

[0106] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0107] Example 1

[0108] 1. A reaction solution and a preparation method thereof, as follows.

[0109] (1) Obtaining a nucleic acid substrate.

[0110] The four nucleic acid fragments B1 to B4 (nucleic acid substrates) shown in Table 1 were synthesized on a solid phase. The fragments were dissolved and fixed to obtain the molar concentration of each fragment. The nucleic acid substrates and their ligation products were referred to Figure 1 .

[0111] Table 1: Non-natural nucleic acid substrates

[0112] Snippet name Sequence information (5'→3') SEQ ID NO: AS chain 5' B1 mUmUmUmGmCmUmCmAfAfCmUmGmUmU 1 AS strand 3' B2 pmCmGmGmAmAmUmGmGmA 2 SS strand 5' B3 mCmAfUmUfCmCmGfAfAmCmA 3 SS strand 3' B4 pmGmUmUmGmAmGmCmAmAmA 4

[0113] Note: The sequences shown in SEQ ID NO: 1 to 4 are the sequences before modification, m represents 2'OCH3 modification, and f represents 2'F modification. SEQ ID NO: 1 to 4 in the sequence table are the sequences before modification.

[0114] For convenience, the oligonucleotide generated by the ligation reaction of B1 and B2 is referred to as the sense strand (abbreviated as SS), and the oligonucleotide generated by B3 and B4 is referred to as the antisense strand (abbreviated as AS).

[0115] (2) Preparation of reaction solution.

[0116] Experimental group: 20 μL of mixed solution containing nucleic acid substrate (including B1-B4, the concentration of each fragment is 2.5 mM) was placed in a 200 μL PCR tube, and buffer, Mg 2+, ATP, RNA ligase and inorganic pyrophosphatase, and make up to 40 μL with sterile water for injection to obtain a reaction solution, see Table 2.

[0117] Table 2: Composition of the reaction solution

[0118]

[0119] Note: The formula of 10×reaction buffer is 500 mM Tris-Acetate, DTT 10 mM, pH=8.0; inorganic pyrophosphatase was purchased from Hongene Biotech, Pyrophosphatase Inorganic (yeast), catalog number ON-025; double-stranded ligase was purchased from Hongene Biotech, T4 RNA Ligase 2, catalog number ON-544-C010.

[0120] Control group: The preparation steps of the reaction solution were similar to those of the experimental group, except that inorganic pyrophosphatase was not added to the control group.

[0121] 2. Synthesis of nucleotide chains and analysis of results

[0122] The reaction solutions prepared in the experimental group and the control group were placed in a PCR instrument at 37°C, and 1 μL was taken at 1, 4, and 16 hours of reaction and 1 μL of 15 mM EDTA was added for HPLC-MS detection and analysis. The target product (target nucleotide chain) ratio was calculated by dividing the peak area of ​​the AS chain and the SS chain by the sum of the peak areas of the nucleotides on the entire spectrum as the product ratio. The experimental results of the experimental group and the control group are shown in Table 3.

[0123] Table 3: Product conversion rate of the experimental group and the control group at 1h, 4h and 16h in Example 1

[0124]

[0125] From the results, it can be seen that for the sequence in Table 1, the SS chain was first subjected to enzyme chain reaction, and the AS chain was then subjected to enzyme linkage reaction. After adding inorganic pyrophosphatase, the reaction rate of the AS chain was significantly higher than that of the reaction with only RNA polymerase added, proving that adding inorganic pyrophosphatase to the enzyme linkage reaction solution can accelerate the reaction rate of the enzyme linkage reaction and increase the product proportion of the target product. The promotion effect is particularly obvious for the AS chain with a slower reaction.

[0126] Example 2

[0127] 1. A reaction solution and a preparation method thereof

[0128] Three experimental groups were provided, experimental group 1 was the same as Example 1, experimental groups 2-3 were roughly the same as the reaction solution of Example 1, except that the final concentrations of each fragment of B1-B4 and ATP in the reaction solution were different, group 1: 2.5mM ligation notch, 20g / L (total amount of B1-B4); group 2: 5mM ligation notch, 40g / L; group 3: 7.5mM ligation notch, 60g / L. The concentration of ATP in the reaction system was consistent with the concentration of the ligation notch.

[0129] Experimental groups 1 to 3 were each provided with a control group, and the control group was not supplemented with inorganic pyrophosphatase.

[0130] 2. Synthesis of nucleotide chains and analysis of results

[0131] The synthesis of the nucleotide chain and the analysis of the results were roughly the same as in Example 1. The reaction product after 8 hours of reaction was tested and the results were as follows.

[0132] Table 4: Product conversion rate of the experimental group and the control group in Example 2 at 8 h

[0133]

[0134] It can be seen from the results of the experimental group that under the same enzyme amount, 20g / L, 40g / L and 60g / L can all achieve a conversion rate of about 87%. For the control group, the product conversion rate of the reaction concentration of 20g / L is about 87%, and the conversion rates of 40g / L and 60g / L are significantly reduced, especially the product conversion rate of the SS chain is significantly lower than that of the control group. Therefore, this embodiment proves that for high substrate concentrations, such as the concentration of connectable notches exceeding 2.5mM, the reaction speed of the reaction solution containing only RNA ligase is slow and the conversion rate is low, especially when only one or part of the notches complete the reaction first, while other notches do not complete the connection reaction. Adding inorganic pyrophosphatase to the reaction system can increase the reaction speed and conversion rate of high-concentration substrates, especially for two notches in the reaction process, inorganic pyrophosphatase has a significant advantage in improving the reaction rate of the corresponding notch.

[0135] Example 3

[0136] 1. A reaction solution and a preparation method thereof

[0137] Seven experimental groups were provided, which were substantially the same as the reaction solution of Example 1, except that: (1) the nucleic acid substrates were different; and (2) the final concentrations of the nucleic acid substrates and ATP in the reaction solution were different.

[0138] (1) Nucleic acid substrate

[0139] Information on nucleic acid substrates and their ligation products can be found in Figure 2 .

[0140] Table 5: Non-natural nucleic acid substrates

[0141] Snippet name Sequence information (5'→3') SEQ.ID.NO AS chain 5' B5 mCmCmUmCmUmCmUmUmGmAmUmAmUmG 5 AS strand 3' B6 pmAmGmAmUmCmUmGmCmA 6 SS strand 5' B7 mUmCmUmGmCmAmGmAmUmCmUmCmAmU 7 SS strand 3' B8 pmAmUmCmAmAmGmAmG 8

[0142] Note: The sequences shown in SEQ ID NO: 5 to 8 are the sequences before modification, m represents 2'OCH3 modification, and f represents 2'F modification. SEQ ID NO: 5 to 8 in the sequence table are the sequences before modification.

[0143] For convenience, the oligonucleotide generated by the ligation reaction of B5 and B6 is referred to as the sense strand (abbreviated as SS), and the oligonucleotide generated by B7 and B8 is referred to as the antisense strand (abbreviated as AS).

[0144] (2) Final concentration of nucleic acid substrate in the reaction solution.

[0145] Experimental group 1: 1.88mM connection notch, 15g / L (total amount of B5-B8); Experimental group 2: 3.76mM connection notch, 30g / L; Experimental group 3: 5.64mM connection notch, 45g / L; Experimental group 4: 7.52mM connection notch, 60g / L; Experimental group 5: 8.46mM connection notch, 67.5g / L; Experimental group 6: 9.4mM connection notch, 75g / L; Experimental group 7: 10.16mM connection notch, 81g / L. The concentration of ATP in the reaction system is consistent with the concentration of the connection notch.

[0146] Experimental groups 1 to 7 were each provided with a control group, and the control group was not supplemented with inorganic pyrophosphatase.

[0147] 2. Synthesis of nucleotide chains and analysis of results

[0148] The synthesis of nucleotide chains and the analysis of the results were roughly the same as in Example 1. The reaction products after 4h and 16h of reaction were tested, and the results were as follows.

[0149] Table 6: Product conversion rate of the experimental group and the control group at 4h and 16h in Example 3

[0150]

[0151] From the results of the experimental group, it can be seen that under the same enzyme amount, 15g / L~81g / L can achieve a conversion rate of about 90%. For the control group, the product conversion rate of the reaction concentration of 15g / L~45g / L is about 90%, and the conversion rate of 60g / L~81g / L is significantly reduced, especially the product conversion rate of the SS chain is lower than that of the control group. Therefore, this embodiment proves that for high substrate concentrations, such as the concentration of connectable notches exceeding 3.76mM, the reaction speed of the reaction solution containing only RNA ligase is slow and the conversion rate is low, especially when only one or part of the notches complete the reaction first, while other notches do not complete the connection reaction. Adding inorganic pyrophosphatase to the reaction system can increase the reaction speed and conversion rate of high-concentration substrates, especially for two notches in the reaction process, inorganic pyrophosphatase has a significant advantage in improving the reaction rate of the corresponding notch.

[0152] Example 4

[0153] 1. A reaction solution and a preparation method thereof, as follows.

[0154] Four experimental groups were provided, which were substantially the same as the reaction solution of Example 1, except that: (1) the nucleic acid substrates were different; and (2) the final concentrations of the nucleic acid substrates and ATP in the reaction solution were different.

[0155] (1) Obtaining a nucleic acid substrate.

[0156] The four nucleic acid fragments B9 to B12 (nucleic acid substrates) shown in Table 7 were synthesized on a solid phase, and the molar concentration of each fragment was obtained after the fragments were dissolved and fixed to volume.

[0157] Information on nucleic acid substrates and their ligation products can be found in Figure 3 .

[0158] Table 7: Non-natural nucleic acid substrates

[0159] Snippet name Sequence information (5'→3') SEQ.ID.NO AS chain 5' B9 mAmGmAmAmAmCmAmAmC 9 AS strand 3' B10 pmAmAfAmGmAmCfGmUmAmGmCmU 10 SS strand 5' B11 mCmUmAmCmGmUmCmUmUmUmGmUmU 11 SS strand 3' B12 pmGmUmUmUmCmUmU 12

[0160] Note: The sequences shown in SEQ ID NO:9-12 are the sequences before modification, m represents 2'OCH3 modification, and f represents 2'F modification. SEQ ID NO:9-12 in the sequence list are the sequences before modification.

[0161] For convenience, the oligonucleotide generated by the ligation reaction of B9 and B10 is referred to as the sense strand (abbreviated as SS), and the oligonucleotide generated by B11 and B12 is referred to as the antisense strand (abbreviated as AS).

[0162] (2) Final concentration of nucleic acid substrate in the reaction solution.

[0163] Experimental group 1: 2.5mM connection notch, 20g / L (total amount of B9-B12); Experimental group 2: 5mM connection notch, 40g / L; Experimental group 3: 10mM connection notch, 80g / L; Experimental group 4: 15mM connection notch, 120g / L;

[0164] The concentration of ATP in the reaction system is consistent with the concentration of the ligation nick.

[0165] Experimental groups 1 to 4 were each provided with a control group, and the control group was not supplemented with inorganic pyrophosphatase.

[0166] 2. Synthesis of nucleotide chains and analysis of results

[0167] The synthesis of nucleotide chains and the analysis of the results were roughly the same as in Example 1. The reaction products after 2h, 16h and 24h of reaction were tested, and the results were as follows.

[0168] Table 8: Product conversion rate of the experimental group and the control group at 2h, 16h and 24h in Example 4

[0169]

[0170] From the results of the experimental group, it can be seen that under the same enzyme amount, the conversion rate of 20g / L to 120g / L is about 90%. For the control group, the product conversion rate of the reaction concentration of 20g / L to 40g / L is about 95%, and the conversion rate of 80g / L to 120g / L is significantly reduced, especially the product conversion rate of the SS chain is lower than that of the control group. Therefore, this embodiment proves that for high substrate concentrations, such as the concentration of connectable notches exceeding 2.5mM, the reaction speed of the reaction solution containing only RNA ligase is slow and the conversion rate is low, especially when only one or part of the notches complete the reaction first, while other notches do not complete the connection reaction. Adding inorganic pyrophosphatase to the reaction system can increase the reaction speed and conversion rate of high-concentration substrates, especially for two notches in the reaction process, inorganic pyrophosphatase has a significant advantage in improving the reaction rate of the corresponding notch.

[0171] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of inorganic pyrophosphatase in the synthesis of nucleotide chains or in the preparation of products for the synthesis of nucleotide chains.

2. An enzyme-linked reagent, characterized in that It includes: Inorganic pyrophosphatase and nucleic acid ligase.

3. The enzyme-linked reagent according to claim 2, characterized in that The nucleic acid ligase includes a double-stranded ligase; Optionally, the double-stranded ligase includes a ligase that ligates RNA double strands and / or RNA / DNA hybrid double strands; Optionally, the double-stranded ligase comprises: any one or more of Rnl2 family ligase and Rnl5 family ligase; Optionally, the Rnl2 family ligase comprises T4 RNA ligase 2.

4. The enzyme-linked reagent according to claim 3, characterized in that The enzyme-linked reagent further comprises: any one or more of a buffer, ATP and a divalent ion; Optionally, the divalent ions include: Mg 2+ , Mn 2+ 、Co 2+ and Zn 2+ Any one or more of the following; Optionally, the buffer comprises any one of acetate buffer, phosphate buffer, Tris buffer and HEPES buffer.

5. A reaction solution, characterized in that It includes: A double-stranded nucleic acid molecule with a nick and / or a nucleic acid substrate capable of forming the double-stranded nucleic acid molecule with a nick, and the enzyme-linked reagent according to any one of claims 2 to 4.

6. The reaction solution according to claim 5, characterized in that The double-stranded nucleic acid molecule with a nick includes any one or more of an RNA double-stranded molecule with a nick and an RNA / DNA hybrid double-stranded molecule with a nick; Optionally, the RNA / DNA hybrid double strand includes: a hybrid double strand formed by a DNA single strand and an RNA single strand with complementary base pairing and / or a hybrid double strand formed by a DNA / RNA hybrid single strand and a DNA or RNA single strand with complementary base pairing; Optionally, the double-stranded nucleic acid molecule with a nick includes a nucleic acid substrate or is formed by mixing or annealing nucleic acid substrates; Optionally, the annealing temperature of the nucleic acid substrate is 0 to 100°C; Optionally, the nucleic acid substrate includes any one or more of a natural or modified RNA single strand, a natural or modified RNA double strand, a natural or modified DNA single strand, a natural or modified RNA / DNA hybrid single strand, and a natural or modified RNA / DNA hybrid double strand; Optionally, in the nucleic acid substrate, the RNA single strand includes a linear RNA single strand and / or a circular RNA single strand, the DNA single strand includes a linear DNA single strand and / or a circular DNA single strand, the DNA single strand includes a linear DNA single strand and / or a circular DNA single strand, the RNA / DNA hybrid single strand includes a linear RNA / DNA hybrid single strand and / or a circular RNA / DNA hybrid single strand, and the RNA / DNA hybrid double strand includes a linear RNA / DNA hybrid double strand and / or a circular RNA / DNA hybrid double strand; Optionally, the RNA includes: any one or more of mRNA, antisense oligonucleotide, siRNAi, sgRNA, lncRNA, CircRNA and miRNA; Optionally, the fragment length of the nucleic acid substrate is ≥2nt; Optionally, the fragment length of the nucleic acid substrate is 2 to 200 nt; Optionally, the number of the nucleic acid substrates is ≥1; Optionally, the final concentration of the nucleic acid substrate is 0.01 to 100 mM; Optionally, the final concentration of the nucleic acid substrate is 1-100 mM.

7. The reaction solution according to claim 5 or 6, characterized in that The final concentration of the inorganic pyrophosphatase in the enzyme-linked reagent in the reaction solution is 0.00001 mg / ml to 1 mg / ml; Optionally, the final concentration of the inorganic pyrophosphatase is 0.0001 mg / ml to 0.1 mg / ml; Optionally, the final concentration of the nucleic acid ligase in the enzyme ligation reagent in the reaction solution is 0.001 mg / ml to 20 mg / ml; Optionally, when the enzyme-linked reagent includes ATP, the final concentration of the ATP in the reaction solution is 0.01 to 50 mM; Optionally, the final concentration of ATP is 0.1-50 mM; Optionally, when the enzyme-linked reagent includes divalent ions, the final concentration of the divalent ions in the reaction solution is 0.01 to 100 mM; Optionally, the final concentration of the divalent ion is 0.1 to 100 mM; Optionally, when the enzyme-linked reagent includes a buffer, the pH of the buffer is 6.5-9.

0.

8. A composition, characterized in that It includes: The components of the enzyme-linked reagent according to any one of claims 2 to 4 or the components of the reaction solution according to any one of claims 5 to 7.

9. A kit, characterized in that: It includes: The enzyme-linked reagent according to any one of claims 2 to 4, the reaction solution according to any one of claims 5 to 7, or the composition according to claim 8.

10. A method for synthesizing a nucleotide chain, characterized in that: The method comprises: synthesizing using the enzyme-linked reagent according to any one of claims 2 to 4, the reaction solution according to any one of claims 5 to 7, the composition according to claim 8, or the kit according to claim 9; Optionally, the synthesis conditions include: temperature of 15 to 55° C. and time of 1 to 48 h.

Images