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

Abstract

Provided are a related product and use of inorganic pyrophosphatase in the synthesis of a nucleotide chain, relating to the technical field of nucleic acid synthesis. By adding inorganic pyrophosphatase in a synthesis process of a nucleotide chain, by-products generated in a ligation reaction process can be greatly reduced or eliminated, the rate of reaction is increased, and thus the conversion rate and purity of a target product are improved, thereby facilitating the full exertion of the effect of the target nucleotide chain and providing a new approach to high-quality large-scale nucleotide chain synthesis.

Description

Inorganic pyrophosphatases: related products and applications in nucleotide chain synthesis Cross-references to related applications This disclosure claims priority to Chinese Patent Application No. 2025101109508, filed on January 23, 2025, entitled “Related Products and Applications of Inorganic Pyrophosphatase in the Synthesis of Nucleotide Chains,” the entire contents of which are incorporated herein by reference. Technical Field This disclosure relates to the field of nucleic acid synthesis technology, and more specifically, to products and applications of inorganic pyrophosphatases in the synthesis of nucleotide chains. Background Technology Currently, the main method for industrial synthesis of oligonucleotides is solid-phase synthesis. Synthesizing large quantities of oligonucleotides requires multiple accumulations, and the yield decreases with increasing chain length. Furthermore, increasing chain length leads to the presence of a certain amount of non-target nucleotide chains in the synthetic product, whose lengths do not match the target nucleotide chain. After chemical synthesis, even 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 drugs targeting antisense strands, RNAi drugs, nucleic acid aptamer drugs, and sgRNA drugs, where the presence of non-target nucleotide chains can severely impair the efficacy of the target nucleotide chain. For nucleotide chains with a length of 20 mere or less, after chemical synthesis, the proportion of the final target nucleotide chain in the synthetic product can be increased through purification processes (e.g., ion column or reverse column). Therefore, when synthesizing oligonucleotides, long fragments of 100-200 mere 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. Existing enzyme ligation technologies have at least the following problems: for high substrate concentrations, for example, when the concentration of ligable nicks exceeds 2.5 mM, the reaction rate of reaction solutions containing only RNA ligase is slow and the conversion rate is low. In particular, only one or a portion of the nicks complete the reaction first, while other nicks do not complete the ligation reaction. In view of this, this disclosure is hereby made. Summary of the Invention The purpose of this disclosure is to provide products and applications related to inorganic pyrophosphatases in the synthesis of nucleotide chains. This disclosure is implemented as follows: In a first aspect, embodiments of this disclosure provide the application of inorganic pyrophosphatases in the synthesis of nucleotide chains or in the preparation of products containing synthesized nucleotide chains. Secondly, embodiments of this disclosure provide an enzyme ligation reagent comprising: inorganic pyrophosphatase and nucleic acid ligase. Thirdly, embodiments of this disclosure provide a reaction solution comprising: a notched double-stranded nucleic acid molecule and / or a nucleic acid substrate capable of forming the notched double-stranded nucleic acid molecule, and the enzyme-linked reagent described in the foregoing embodiments. Fourthly, embodiments of this disclosure provide a composition comprising: each component of the enzyme-linked reagent described in the foregoing embodiments or each component of the reaction solution described in the foregoing embodiments. Fifthly, this disclosure provides a kit comprising: the enzyme-linked reagent described in the foregoing embodiments, the reaction solution described in the foregoing embodiments, or the composition described in the foregoing embodiments. In a sixth aspect, embodiments of this disclosure provide a method for synthesizing nucleotide chains, comprising: synthesizing using the enzyme ligation reagent, reaction solution, composition, or kit described in the foregoing embodiments. In a seventh aspect, embodiments of this disclosure provide products synthesized by the synthesis method described in the foregoing embodiments. This disclosure has the following beneficial effects: By adding inorganic pyrophosphatase during the synthesis of nucleotide chains, the byproducts generated during the ligation reaction can be significantly reduced or eliminated, the reaction rate can be accelerated, and the conversion rate and purity of the target product can be improved. This is beneficial for fully utilizing the effectiveness of the target nucleotide chain and provides a new approach for the high-quality, large-scale synthesis of nucleotide chains. Attached Figure Description To more clearly illustrate the technical solutions of the embodiments of this disclosure, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of this disclosure and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort. Figure 1 shows the oligonucleotides and their ligation products used in Example 1 of this disclosure; Figure 2 shows the oligonucleotides and their ligation products used in Example 3 of this disclosure; Figure 3 shows the oligonucleotides and their ligation products used in Example 4 of this disclosure. Detailed Implementation To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions in the embodiments of this disclosure will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. Terminology Explanation Oligonucleotides: Generally, they refer to linear polynucleotide fragments consisting of 2 to 10 nucleotide residues linked by phosphodiester bonds. However, it should be noted that there is no strict rule regarding the number of nucleotides in oligonucleotides. In some literature, polynucleotide molecules containing 30 or even more, up to 200, 300, 400, or 500 nucleotide residues, can also be called oligonucleotides. Natural ribonucleotides are composed of one molecule of phosphate, one molecule of ribose (a pentose sugar), and one nitrogenous base. Based on the type of nitrogenous base, ribonucleotides are classified into adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, and uracil ribonucleotides. 2'-Deoxyribonucleotides: Composed of one molecule of phosphate, one molecule of 2'-deoxyribose (the 2' of the ribose undergoes deoxygenation, becoming a hydrogen atom), and one nitrogenous base. Based on the type of nitrogenous base, naturally occurring 2'-deoxyribonucleotides are classified into adenine deoxyribonucleotides, guanine deoxyribonucleotides, cytosine deoxyribonucleotides, and thymine deoxyribonucleotides. Non-natural nucleotides: Nucleotides produced by modifying the phosphate groups, nitrogenous bases, sugar rings, and glycosidic bonds of natural nucleotides. RNA: A molecule formed by natural or non-natural ribonucleotides linked by phosphodiester bonds. While typical RNA molecules are linked together by standard phosphodiester bonds, RNA used as a drug may contain one or more non-standard bonds. RNA can be single-stranded or double-stranded, or contain both single-stranded and double-stranded regions. Furthermore, ribonucleotides can be classified according to their morphology into linear ribonucleotide chains and cyclic ribonucleotide chains. Cyclic ribonucleotide chains consist of one or more linear ribonucleotide chains linked end-to-end by phosphodiester bonds, forming a closed ring structure. DNA: refers to molecules formed by 2' deoxyribonucleotides linked together by phosphate ester bonds. As used in this article, "wild type" refers to the form found in nature. For example, wild type protein sequences are forms found in nature that can be isolated from natural sources and have not been intentionally modified or altered by humans. The term "nick" or "linkage nick" in this article refers to the absence of a phosphodiester bond between two adjacent nucleotide segments in a double-stranded structure. A nick can be catalyzed by double-strand ligase to form a complete phosphodiester bond; that is, the 3' hydroxyl group of one nucleotide unit at the nick site forms a complete phosphodiester bond with the 5' monophosphate group of the other nucleotide unit at the same nick site through double-strand ligase catalysis. A "nick" can also be understood as a gap in a double-stranded nucleic acid molecule caused by the breakage of a phosphodiester bond. In this article, the term "gap" refers to a situation in which one strand of a double-stranded structure breaks into two strands due to the absence of one or more consecutive nucleotides, and these two strands form a gap. The nucleic acid substrates described in this article are oligonucleotides capable of forming nicked double-stranded nucleic acid molecules, wherein the 5' end of the oligonucleotide contains a monophosphate group and / or the 3' end contains a hydroxyl group. The term "denaturation" in this article 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, through high-temperature incubation, thereby converting double-stranded nucleic acids into single-stranded nucleic acids. The term "annealing" in this article refers to the process of slowly cooling a nucleic acid solution that has been denatured at high temperatures to a lower temperature, allowing single-stranded nucleic acids to reform into double-stranded nucleic acids. This article uses the term "sequence identity percentage (%)" to refer to comparisons between polynucleotides and peptides, and it is determined by comparing two optimally aligned sequences across a comparison window. For optimal alignment of the two sequences, the portion of the polynucleotide or peptide sequence within the comparison window may contain additions or deletions (i.e., vacancies) compared to the reference sequence. The percentage can be calculated as follows: determine the number of positions in both sequences where the same nucleic acid base or amino acid residue appears 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 in both sequences where the same nucleic acid base or amino acid residue appears, or the number of positions where the nucleic acid base or amino acid residue is aligned with a vacancy 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 understand that there are currently various algorithms available for sequence alignment, such as the Smith-Waterman local homology algorithm (Smith and Waterman, Adv. Appl. Math., 2:482).

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

[1970] These algorithms have been designed into related software, allowing researchers in the field to quickly align protein or nucleotide sequences. Examples include EMBOSS Water (https: / / www.ebi.ac.uk / jdispatcher / psa / emboss_water), an open-source software based on the Smith-Waterman algorithm, and EMBOSS needle (https: / / www.ebi.ac.uk / jdispatcher / psa / emboss_needle), based on the Needleman-Wunsch algorithm, both developed by the European Institute for Bioinformatics (EMBL-EBI). Protein sequence alignment and sequence alignment consistency percentages can be performed using the open-source EMBOSS Water software. The scoring matrix used during alignment is BLOSUM62, with the gap open score set to 10 and the gap extension score set to 1. A “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, such as a segment of a full-length gene or polypeptide sequence. Typically, a reference sequence is at least 20 nucleotides or amino acid residues long, at least 25 residues long, at least 50 residues long, at least 100 residues long, or the full length of a nucleic acid or polypeptide. Since two polynucleotides or polypeptides can each (1) contain sequences similar to each other (i.e., a portion of the complete sequence) and (2) also contain sequences different from each other, sequence comparisons between two (or more) polynucleotides or polypeptides are 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 the reference sequence is a sequence that may have one or more variations in the primary sequence. A “comparison window” refers to a conceptual segment of at least about 20 consecutive nucleotide positions or amino acid residues, wherein the sequence can be compared with a reference sequence of at least 20 consecutive nucleotides or amino acids, and wherein, for optimal alignment of the two sequences, the portion of the sequence within the comparison window may contain 20% or less of additions or deletions (i.e., vacancies). The comparison window may be longer than 20 consecutive residues and optionally includes windows of 30, 40, 50, 100, or longer. In the context of numbering a given amino acid or polynucleotide sequence, "corresponding to," "referring to," or "relative to" refers to the number of reference sequence residues specified when the given amino acid or polynucleotide sequence is compared to a reference sequence. In other words, the number or position of residues in a given polymer is specified with respect to a reference sequence, rather than by the actual numerical position of residues 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 have its residue matching optimized by introducing vacancies to align with a reference sequence. In these cases, the numbering of residues in the given amino acid or polynucleotide sequence is made according to the reference sequence with which it is aligned, despite the presence of vacancies. This disclosure addresses the problems of byproduct generation, slow enzyme ligation reaction rate, and low conversion rate in existing nucleotide chain synthesis processes. The reaction rate and conversion rate are particularly low when the concentration of ligation defects in the reaction solution is high (≥2.5 mM) or the amount of nucleic acid substrate in the reaction solution is ≥20 g / L. The inventors of this disclosure have for the first time discovered and verified that inorganic pyrophosphatase can effectively reduce or inhibit the generation of byproducts, thereby accelerating the enzyme ligation reaction rate and improving the conversion rate and purity of the target nucleotide chain. The advantages of adding inorganic pyrophosphatase are even more significant when the concentration of ligation defects is high (≥2.5 mM). Specific technical solutions On the one hand, embodiments of this disclosure provide the application of inorganic pyrophosphatases in the synthesis of nucleotide chains or in the preparation of products synthesized from nucleotide chains. In some embodiments, the product includes: reagents, kits, chips, or combinations thereof. On the other hand, embodiments of this disclosure provide an enzyme ligation reagent configured for the synthesis of nucleotide chains, comprising: an inorganic pyrophosphatase and a nucleic acid ligase. Inorganic pyrophosphatases can be derived from any of Escherichia coli, yeast, and thermostable bacteria. Inorganic pyrophosphatases can be natural pyrophosphatases, mutant strains resulting from enzyme mutations or evolution, or enzymes that can catalyze the elimination of byproducts in the enzyme chaining process after fusion with enzymes within the same family or with other families. Nucleic acid ligases catalyze the formation of phosphodiester bonds from the ligation notches of nucleic acid substrates to form the target product. The addition of inorganic pyrophosphatase can significantly improve the conversion rate and purity of the synthesized target product, thereby facilitating the full utilization of the target product's efficacy. In some embodiments, the nucleic acid ligase includes a double-stranded ligase. A double-stranded ligase is a ligase capable of blocking nicks in a double-stranded structure. The double-stranded ligase includes RNA ligases, including ligases that connect RNA double strands and / or RNA / DNA hybrid double strands. Inorganic pyrophosphatases are particularly suitable for nucleotide chain synthesis using double-stranded ligases, provided that one strand of the double strand is RNA or the nick is RNA. RNA ligase operates in three steps during ligation: First, the ligase reacts with ATP to generate the ligase-AMP intermediate, releasing pyrophosphate. Second, the ligase-AMP intermediate binds to the nicked double-stranded substrate, transferring AMP to the 5'-phosphate terminus, forming a nicked adenylated double strand. Third, the ligase catalyzes the attack of the 3'-OH group of the nicked RNA on the 5' phosphate group, forming a new 3'-5' phosphodiester bond connecting the two nicks and releasing AMP. Inorganic pyrophosphatases can catalyze the elimination of byproducts from the first step, thus driving the chemical reaction to the right and accelerating the ligation rate, thereby increasing the conversion rate. This disclosure does not impose any special restrictions on the specific selection of double-stranded ligases. They can be selected according to the actual situation. Natural double-stranded ligases can be used, as well as mutant strains produced by enzyme mutation, modification and evolution, and enzymes that produce nick-blocking activity after fusion with enzymes in the same family or other families. In some embodiments, the double-stranded ligase includes any one or more of the Rnl2 family ligases and the Rnl5 family ligases. In some embodiments, the double-stranded ligase includes any one or more of T4 RNA ligase 2 and DraRnl (from Naegleria gruberi). In some embodiments, the enzyme ligation reagent further includes any one or more of buffer solution, ATP, and divalent ions. In some embodiments, the divalent ion includes: Mg 2+ Mn 2+ Co 2+ and Zn 2+ Any one or more of the following. In some embodiments, the buffer solution includes any one of acetate buffer, phosphate buffer, Tris buffer, and HEPES buffer. On the other hand, embodiments of this disclosure provide a reaction solution comprising: a notched double-stranded nucleic acid molecule and / or a nucleic acid substrate capable of forming the notched double-stranded nucleic acid molecule, and the enzyme-linked reagent described in any of the foregoing embodiments. In some embodiments, the final concentration of inorganic pyrophosphatase in the enzyme-linked reagent in the reaction solution is 0.00001 mg / ml to 1 mg / ml, specifically within any one or any two of the following: 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 / ml. 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 or any two of 0.01, 0.05, 0.1, 0.5, 1, 2, 4, 6, 7, 8, 10, 12, 14, 16, 18 and 20 mg / ml. It should be noted that the concentrations of the two enzymes can be adjusted according to the actual situation, and there are no special restrictions in this disclosure. In some embodiments, the notched double-stranded nucleic acid molecule includes any one or more of notched RNA double strands and notched RNA / DNA hybrid double strands. In some embodiments, in the notched double-stranded nucleic acid molecule, the notch can refer to at least one notch on at least one strand of the double-stranded nucleic acid molecule, that is, there can be a notch on one strand or there can be a notch on both strands. Specifically, the notched DNA / RNA hybrid double strand includes at least one deoxyribonucleic acid, and at least one notched end (3' hydroxyl group and / or 5' phosphate group) is RNA. In some embodiments, the notched double-stranded nucleic acid molecule comprises linear and / or circular molecules. In some embodiments, the notched double-stranded nucleic acid molecule comprises natural and / or modified varieties. In some embodiments, the notched double-stranded nucleic acid molecule comprises a nucleic acid substrate or is formed by mixing or annealing nucleic acid substrates. In some embodiments, the nucleic acid substrate includes any one or more of the following: natural and / or modified RNA single strand, natural and / or modified RNA double strand, natural and / or modified DNA single strand, and natural and / or modified DNA / RNA hybrid single strand and natural and / or modified DNA / RNA hybrid double strand. In some embodiments, in the nucleic acid substrate, the RNA single strand includes linear RNA single strand and / or circular RNA single strand, the RNA double strand includes linear RNA double strand and / or circular RNA double strand, the DNA single strand includes linear DNA single strand and / or circular DNA single strand, the DNA / RNA hybrid single strand includes linear DNA / RNA hybrid single strand and / or circular DNA / RNA hybrid single strand, and the DNA / RNA hybrid double strand includes linear DNA / RNA hybrid double strand and / or circular DNA / RNA hybrid double strand. In some embodiments, the RNA single strand includes any one or more of mRNA, antisense oligonucleotide, siRNA, sgRNA, lncRNA, circRNA, and miRNA. When the nucleic acid substrate is double-stranded with notches, the nucleic acid substrate itself is a notched double-stranded nucleic acid molecule. When the nucleic acid substrate is single-stranded, a notched double-stranded nucleic acid molecule can be formed by mixing the nucleic acid substrate or by annealing the mixed nucleic acid substrate. In some embodiments, the annealing temperature of the nucleic acid substrate is 0 to 100°C, specifically any one or any two of the following: 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 and 100°C. In some embodiments, the process of forming notched double-stranded nucleic acid molecules from nucleic acid substrates can occur during the mixing of nucleic acid substrates without a separate heating and annealing process. The nucleic acid substrates, enzymes, ATP, and Mg can be directly mixed. 2+ After the necessary molecules and solutions for the reaction are mixed, they are linked together. The nucleic acid substrate binds specifically during the mixing process to form a notched double-stranded nucleic acid molecule. In some embodiments, the fragment length of the nucleic acid substrate is ≥2 nt. In some embodiments, the fragment length of the nucleic acid substrate is 2 to 200 nt, specifically any one or any two of the following: 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, and 200 nt. In some embodiments, the number of nucleic acid substrates is ≥1. For example, when configured for the circularization of circular RNA, the number of nucleic acid substrates is 1. Specifically, this number can be any one or a range between any two of the following: 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, and 50. In some embodiments, the nucleic acid substrate includes a first fragment, a second fragment, a third fragment, and a fourth fragment. The first fragment has a monophosphate group at its 5' end, and the second fragment has a hydroxyl group at its 3' end. The first and second fragments can cooperate to form a double-stranded structure. The nucleic acid fragments include a third fragment and a fourth fragment. The third fragment has a hydroxyl group at its 3' end, and the fourth fragment has a monophosphate group at its 5' end. The third and fourth fragments can cooperate to form a double-stranded structure. A notch is formed between the 5' end of the first fragment and the 3' end of the third fragment. The first and third fragments are linked by a ligase to form a phosphodiester bond between their 3' and 5' ends, resulting in a target nucleotide chain. Similarly, a notch is formed between the 3' end of the second fragment and the 5' end of the fourth fragment. The second and fourth fragments are linked by a ligase to form a phosphodiester bond between their 3' and 5' ends, resulting in a target nucleotide chain. In some embodiments, the molar ratio of any two nucleic acid fragments can be 0.1 to 1:1, specifically any one or any two of 0.1:1, 0.2:1, 0.4:1, 0.6:1, 0.8:1 and 1:1. This disclosure does not impose any particular restrictions on whether the nucleic acid substrate has a modifying group. Compared with the method without the addition of inorganic pyrophosphatase, the scheme with added inorganic pyrophosphatase produces fewer byproducts, has a faster reaction rate, and has a higher conversion rate and purity of the target nucleotide chain. The method provided in this disclosure has no special restrictions on the type of modification and is applicable to all types of modifications. In some embodiments, the modification includes modifications that enhance stability and / or reduce innate immune responses. In some embodiments, the modification includes any one of the following: modification of phosphate groups, modification of bases, modification of sugar rings, and modification of glycosidic bonds. In some embodiments, the modification of the phosphate group includes any one or more of the following: 5′-(E)-vinylphosphonate (5′-VP), thiophosphate, triphosphate, 5′-methylphosphonate, 5′-morpholino, dithiophosphate, methoxypropylphosphonate, S-5′-C methyl analog, short-chain alkyl or cycloalkyl sugar inter-bond, short-chain heteroatom or heterocyclic sugar inter-bond, or the entire phosphate group is substituted by any one of amide, aminooxy, alkoxy and triazole. In some embodiments, the base modification includes any one or more of the following: 2,4-difluorotoluylribonucleoside substitution, pseudouridine modification, 2-thiouridine modification, N1-methylpseudouridine modification, 5-methyluridine modification, 5-methoxyuridine modification, N6-methyladenosine modification, N6,N6-dimethyladenosine modification, 3-methylureaside modification, N7-methylguanosine modification, 2,7-dimethylguanosine modification, 2,2,7-trimethylguanosine modification, 5-methylcytidine modification, 5-hydroxymethylcytosine modification, 5-bromo-uridine modification, 5-iodo-uridine modification, propynyluridine nucleoside modification, adenosine modification with N-ethylpiperidine-6-triazole modification, 6'-phenylpyrrolecytosine modification, 2-aminopurine modification, inosine modification, 2,6-diaminopurine modification, 2-pyrimidinone modification, and 5-methylcytosine modification. In some embodiments, the modification of the sugar ring includes any one or more of the following: 2'-methoxy modification, 2'-deoxy-2'-fluorine 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)-restricted ethyl bicyclic nucleic acid modification, peptide nucleic acid modification, and glycomimetic modification. In some embodiments, the glycomimetic includes any one or more of the following: a cyclobutyl group replacing the pentafuranose group in cyclobutyl nucleotides, a morpholino group in morpholino nucleic acid (MNA), a peptide backbone in peptide nucleic acid (PNA), a polyethylene glycol backbone in glycol nucleic acid (GNA), a threose backbone in threose nucleic acid (TNA), and a butyl backbone in acyclic butyl nucleic acid (BuNA). In some embodiments, the modification of the glycosidic bond includes, but is not limited to, replacing the CN bond connecting the glycosidic bond with any one of CC, CO, and CS. In some embodiments, the final concentration of the nucleic acid substrate in the reaction solution is 0.01–100 mM, specifically any one or any two of the following: 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. In some embodiments, the final concentration of the nucleic acid substrate is 1–200 g / L, specifically any one or any two of the following: 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, and 200 g / L. The reaction solution described in any of the foregoing embodiments has no special limitation on the amount of nucleic acid substrate used. Schemes with small or large amounts of nucleic acid substrate can be applied. The advantages are more obvious when using high concentrations (≥20g / L, for example, 40g / L, 60g / L) of substrate, which can significantly promote the conversion rate of substrate to product and greatly reduce the content of non-target nucleotide chains in the synthesized nucleotide chain, thereby effectively improving the purity of the target nucleotide chain in the synthesized product and thus helping to fully exert the efficacy of the target product. The target product consists of a sense strand and an antisense strand. Typically, the sense strand undergoes ligation first under the action of RNA ligase, while the other strand undergoes ligation subsequently. During the reaction, inorganic pyrophosphatase eliminates the byproduct pyrophosphate, promoting the reaction towards the formation of the target product. The effect of inorganic pyrophosphate is more pronounced during the subsequent ligation process, accelerating the formation of the target product from the shorter fragment. This significantly reduces the content of non-target nucleotide chains in the synthesized product, effectively improving the purity of the target nucleotide chain. In some embodiments, the nucleic acid substrate includes a fragment capable of synthesizing the sense strand as a sense strand substrate, and the sense strand substrates are linked by phosphodiester bonds to obtain the sense strand; the nucleic acid substrate includes a fragment capable of synthesizing the antisense strand as an antisense strand substrate, and the antisense strand substrates are linked by phosphodiester bonds to obtain the antisense strand. The number of sense and antisense strand substrates is typically 1 to 10, specifically any one or any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. This disclosure does not impose any particular limitation on the length of the target nucleotide chain or the target product. In some embodiments, the length of the target nucleotide chain or the target product may be ≤100nt, 200nt, or 500nt. In some embodiments, the length of the target nucleotide chain or the target product may be ≥10nt, 20nt, or 50nt. In some embodiments, the sequence length of the target nucleotide chain can be 10 to 200 nt, specifically any one or any two of the following: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, and 200 nt. In some embodiments, the target nucleotide chain comprises linear and / or cyclic strands. In some embodiments, the target nucleotide chain is any one or more of RNA single-stranded, RNA double-stranded, DNA / RNA hybrid single-stranded, and DNA / RNA hybrid double-stranded. In some embodiments, when synthesizing nucleotide chains using the enzyme-linked reagent or reaction solution described in any of the foregoing embodiments, the conversion rate of the target nucleotide chain or target product is ≥90%, 92%, 94%, 96%, 98%, or 99%. In some embodiments, when the enzyme-linked reagent includes ATP, the final concentration of ATP in the reaction solution is 0.01 to 50 mM, specifically any one or any two of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 40 and 50 mM. In some embodiments, when the enzyme-linked reagent comprises a divalent ion (e.g., Mg²⁺) 2+ When the divalent ion is in the reaction solution, the final concentration of the divalent ion is 0.01 to 100 mM, specifically any one or any two of the following: 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 40, 60, 80 and 100 mM. This disclosure does not impose any special restrictions on the buffer solution. The buffer solution and its pH can be based on the pH corresponding to the optimal catalytic activity of the enzyme used. For example, when the double-stranded ligase is T4 RNA ligase 2, the pH of the buffer solution can be 6.5 to 9.0. In some embodiments, the concentration range of the buffer solution is 10-1000 mM, specifically any one or any two of the following: 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 and 1000 mM, with a preferred concentration of 100-500 mM. In some embodiments, the pH of the buffer solution is 6.5 to 9.0, specifically any one or any two 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. In some embodiments, the reaction solution further includes any one or more of DTT, PEG, and surfactants. PEG can be used to increase the efficiency of the synthesis reaction, and surfactants can increase the stability of the enzyme. Surfactants include Trion X, TWEEN, etc. On the other hand, embodiments of this disclosure provide a composition comprising: each component of the enzyme-linked reagent described in any of the foregoing embodiments or each component of the reaction solution described in any of the foregoing embodiments. In some embodiments, the composition further includes a termination solution. The termination solution includes EDTA. Specifically, EDTA terminates 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. In some embodiments, the final concentration of EDTA in the reaction solution can be 0.1 to 100 mM, specifically any one or any two of 0.1, 1, 5, 6, 7, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 mM. On the other hand, this disclosure provides a kit comprising: the enzyme-linked reagent described in any of the preceding embodiments, the reaction solution described in any of the preceding embodiments, or the composition described in any of the preceding embodiments. On the other hand, embodiments of this disclosure provide a method for synthesizing a target nucleotide chain, comprising: synthesizing using the enzyme ligation reagent, reaction solution, composition, or kit described in any of the foregoing embodiments. In some embodiments, the synthesis conditions include: a temperature of 15–55°C and a time of 1–48 h. Specifically, the temperature can be any one or a range between any two of the following: 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 37, 38, 40, 42, 44, 46, 48, 50, 52, 54, and 55°C. Specifically, the time can be any one or a range between any two of the following: 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, and 48 h. The specific reaction temperature and reaction time can be adjusted according to the actual situation, and there are no special restrictions in this disclosure. Furthermore, embodiments of this disclosure provide products synthesized by the synthesis methods described in any of the foregoing embodiments. The features and performance of this disclosure will be further described in detail below with reference to embodiments. Example 1 1. A reaction solution and its preparation method, as detailed below. (1) Obtain nucleic acid substrate. The four nucleic acid fragments B1 to B4 (nucleic acid substrates) shown in Table 1 were synthesized in a solid phase. After dissolving and adjusting the volume, the molar concentration of each fragment was obtained. The nucleic acid substrates and their ligation products are shown in Figure 1. Table 1: Non-natural nucleic acid substrates Note: The sequences shown in SEQ ID NO:1 to 4 in the computer-readable sequence listing are the unmodified sequences, m indicates 2'-OCH3 modification, and f indicates 2'-F modification. For convenience, the oligonucleotide generated by the linkage 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). (2) Preparation of reaction solution. Experimental group: 20 μL of a mixed solution containing nucleic acid substrates (B1-B4, each fragment at a concentration of 2.5 mM) was placed in a 200 μL PCR tube, and buffer and Mg were added. 2+ ATP, RNA ligase, and inorganic pyrophosphatase were added to a final volume of 40 μL with sterile water for injection to obtain the reaction solution, as shown in Table 2. Table 2: Components of the reaction solution Note: The 10× reaction buffer formula is 500mM Tris-Acetate, 10mM DTT, pH=8.0; the inorganic pyrophosphatase was purchased from Hongene Biotech, Pyrophosphatase Inorganic (yeast), catalog number ON-025; the double-stranded ligase was purchased from Hongene Biotech, T4 RNA Ligase 2, catalog number ON-544-C010. Control group: The preparation steps of the reaction solution are roughly the same as those of the experimental group. The difference is that inorganic pyrophosphatase is not added to the control group. 2. Synthesis and Result Analysis of Nucleotide Chains The reaction solutions prepared for the experimental and control groups were placed in a PCR instrument at 37℃. At 1, 4, and 16 h of reaction, 1 μL of the solution was added to 1 μL of 15 mM EDTA for HPLC-MS analysis. The percentage of the target product (target nucleotide chain) was calculated by dividing the peak areas of the AS and SS chains by the sum of the peak areas of all nucleotides in the entire spectrum. The experimental results for the experimental and control groups are shown in Table 3. Table 3: Product conversion rates of the experimental and control groups at 1h, 4h, and 16h in Example 1 The results show that for the sequences in Table 1, the SS chain was subjected to enzyme chain reaction first, followed by the AS chain. The reaction rate of the AS chain after adding inorganic pyrophosphatase was significantly higher than that after adding RNA polymerase alone. This demonstrates that adding inorganic pyrophosphatase to the enzyme ligation reaction solution can accelerate the reaction rate and increase the proportion of the target product. The promoting effect is particularly pronounced for the slower-reacting AS chain. Example 2 1. A reaction solution and its preparation method Three experimental groups were provided. Experimental group 1 was the same as in Example 1. Experimental groups 2 and 3 had roughly the same reaction solution as in Example 1, except that the final concentrations of each fragment (B1-B4) and ATP in the reaction solution were different. Group 1: 2.5 mM ligation defect, 20 g / L (total amount of B1-B4); Group 2: 5 mM ligation defect, 40 g / L; Group 3: 7.5 mM ligation defect, 60 g / L. The concentration of ATP in the reaction system was consistent with the concentration of the ligation defect. Experimental groups 1-3 each had a control group, in which no inorganic pyrophosphatase was added. 2. Synthesis and Result Analysis of Nucleotide Chains The synthesis and analysis of the nucleotide chains were largely the same as in Example 1. The reaction product after 8 hours of reaction was tested, and the results are as follows. Table 4: Product conversion rates of the experimental and control groups after 8 hours in Example 2 The experimental results show that, at the same enzyme concentration, 20 g / L, 40 g / L, and 60 g / L all achieved a conversion rate of approximately 87%. However, for the control group, the product conversion rate at a reaction concentration of 20 g / L was approximately 87%, while the conversion rates at 40 g / L and 60 g / L were significantly lower, especially for the SS chain, which had a significantly lower conversion rate than the control group. Therefore, this example demonstrates that for high substrate concentrations, such as nick concentrations exceeding 2.5 mM, reaction solutions containing only RNA ligase exhibit slow reaction rates and low conversion rates, particularly when only one or a portion of the nicks completes the reaction first, while other nicks fail to complete the ligation reaction. Adding inorganic pyrophosphatase to the reaction system can improve the reaction rate and conversion rate of high-concentration substrates, especially for nicks with two nicks in the reaction process; inorganic pyrophosphatase has a significant advantage in increasing the reaction rate of the subsequent nicks. Example 3 1. A reaction solution and its preparation method Seven experimental groups were provided, which were roughly the same as the reaction solution in Example 1, except that: (1) the nucleic acid substrate was different; and (2) the final concentrations of the nucleic acid substrate and ATP in the reaction solution were different. (1) Nucleic acid substrate Information on nucleic acid substrates and their ligation products is shown in Figure 2. Table 5: Non-natural nucleic acid substrates Note: The sequences shown in SEQ ID NO:5 to 8 in the computer-readable sequence listing are the unmodified sequences, m indicates 2'-OCH3 modification, and f indicates 2'-F modification. 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). (2) The final concentration of the nucleic acid substrate in the reaction solution. Experimental Group 1: 1.88 mM ligation defect, 15 g / L (total B5-B8); Experimental Group 2: 3.76 mM ligation defect, 30 g / L; Experimental Group 3: 5.64 mM ligation defect, 45 g / L; Experimental Group 4: 7.52 mM ligation defect, 60 g / L; Experimental Group 5: 8.46 mM ligation defect, 67.5 g / L; Experimental Group 6: 9.4 mM ligation defect, 75 g / L; Experimental Group 7: 10.16 mM ligation defect, 81 g / L. The concentration of ATP in the reaction system was consistent with the concentration at the ligation defect. Experimental groups 1 through 7 each had a control group, in which no inorganic pyrophosphatase was added. 2. Synthesis and Result Analysis of Nucleotide Chains The synthesis and analysis of the nucleotide chains were largely the same as in Example 1. The reaction products after 4 hours and 16 hours of reaction were tested, and the results are as follows. Table 6: Product conversion rates of the experimental and control groups at 4h and 16h in Example 3 The experimental results show that, under the same enzyme concentration, concentrations from 15 g / L to 81 g / L all achieved a conversion rate of approximately 90%. However, the conversion rate of the control group (60 g / L to 81 g / L) was significantly lower, particularly for the SS chain. Therefore, this example demonstrates that for high substrate concentrations, such as nick concentrations exceeding 3.76 mM, reaction solutions containing only RNA ligase exhibit slow reaction rates and low conversion rates. Specifically, only one or a portion of the nicks completes the reaction first, while other nicks fail to complete the ligation reaction. Adding inorganic pyrophosphatase to the reaction system can improve the reaction rate and conversion rate of high-concentration substrates, especially for nicks with two nicks in the reaction process; inorganic pyrophosphatase has a significant advantage in increasing the reaction rate of the subsequent nicks. Example 4 1. A reaction solution and its preparation method, as detailed below. Four experimental groups were provided, which were roughly the same as the reaction solution in Example 1, except that: (1) the nucleic acid substrate was different; and (2) the final concentrations of the nucleic acid substrate and ATP in the reaction solution were different. (1) Obtain nucleic acid substrate. The four nucleic acid fragments B9 to B12 (nucleic acid substrates) shown in Table 7 were synthesized in a solid phase. After dissolving and adjusting the volume, the molar concentration of each fragment was obtained. Information on nucleic acid substrates and their ligation products is shown in Figure 3. Table 7: Non-natural nucleic acid substrates Note: The sequences shown in SEQ ID NO:9 to 12 in the computer-readable sequence listing are the unmodified sequences, m indicates 2'-OCH3 modification, and f indicates 2'-F modification. 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). (2) The final concentration of the nucleic acid substrate in the reaction solution. Experimental group 1: 2.5mM linker defect, 20g / L (total amount of B9-B12); Experimental group 2: 5mM linker defect, 40g / L; Experimental group 3: 10mM linker defect, 80g / L; Experimental group 4: 15mM linker defect, 120g / L. The concentration of ATP in the reaction system is the same as the concentration at the junction notch. Experimental groups 1 through 4 each had a control group, in which no inorganic pyrophosphatase was added. 2. Synthesis and Result Analysis of Nucleotide Chains The synthesis and analysis of nucleotide chains were largely the same as in Example 1. The reaction products were taken at 2h, 16h and 24h for detection, and the results are as follows. Table 8: Product conversion rates of the experimental and control groups at 2h, 16h, and 24h in Example 4 The experimental results show that, under the same enzyme concentration, the conversion rate was around 90% at concentrations ranging from 20 g / L to 80 g / L. In contrast, the conversion rate of the control group at a reaction concentration of 20 g / L was around 95%, while the conversion rates at concentrations of 40 g / L to 120 g / L were significantly lower, especially for the AS chain, where the conversion rate was lower than in the experimental group. Therefore, this example demonstrates that for high substrate concentrations, such as nick concentrations exceeding 2.5 mM, the reaction solution containing only RNA ligase exhibits slow reaction speed and low conversion rate, particularly when only one or a portion of the nicks completes the reaction first, while other nicks fail to complete the ligation reaction. Adding inorganic pyrophosphatase to the reaction system can improve the reaction rate and conversion rate of high-concentration substrates, especially for nicks with two nicks in the reaction process; inorganic pyrophosphatase has a significant advantage in increasing the reaction rate of the subsequent nicks. The above description is merely an optional embodiment of this disclosure and is not intended to limit this disclosure. Various modifications and variations can be made to this disclosure by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the scope of protection of this disclosure. Industrial applicability By adding inorganic pyrophosphatase during the synthesis of nucleotide chains, the byproducts generated during the ligation reaction can be significantly reduced or eliminated, the reaction rate can be accelerated, and the conversion rate and purity of the target product can be improved. This is beneficial for fully utilizing the effectiveness of the target nucleotide chain and provides a new approach for the high-quality, large-scale synthesis of nucleotide chains.

Claims

1. Use of inorganic pyrophosphatase in the synthesis of nucleotide chain or the product of the synthesis of nucleotide chain.

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

3. The enzyme-linked reagent of claim 2, wherein, The nucleic acid ligase comprises double-stranded ligase.

4. The enzyme-linked reagent of claim 3, wherein, The double-stranded ligase comprises ligase connecting RNA double-stranded and / or RNA / DNA hybrid double-stranded.

5. The enzyme-linked reagent of claim 3, wherein, The double-stranded ligase comprises any one or more of Rnl2 family ligase and Rnl5 family ligase.

6. The enzyme-linked reagent of claim 5, wherein, The Rnl2 family ligase comprises T4 RNA ligase 2.

7. The enzyme-linked reagent according to any one of claims 2 to 6, characterized in that, The enzyme reagent further comprises any one or more of buffer, ATP and divalent ion; Optionally, the divalent ions include any one or more of: Mg 2+ , Mn 2+ , Co 2+ , and Zn 2+ . Optionally, the buffer comprises any one of acetic acid buffer, phosphate buffer, Tris buffer and HEPES buffer.

8. A reaction solution, characterized by, It comprises: double-stranded nucleic acid molecule with nick and / or nucleic acid substrate capable of forming the double-stranded nucleic acid molecule with nick, and the enzyme reagent of any one of claims 2-7.

9. The reaction solution according to claim 8, wherein, The double-stranded nucleic acid molecule with nick comprises any one or more of RNA double-stranded with nick and RNA / DNA hybrid double-stranded with nick; Optionally, the RNA / DNA hybrid double-stranded comprises hybrid double-stranded formed by one DNA single strand and one RNA single strand in base complementary pairing and / or hybrid double-stranded formed by one DNA / RNA hybrid single strand and one DNA or RNA single strand in base complementary pairing; Optionally, the double-stranded nucleic acid molecule with nick comprises nucleic acid substrate or is formed after mixing or mixed annealing of nucleic acid substrate; Optionally, the annealing temperature of the nucleic acid substrate is 0-100℃; Optionally, the nucleic acid substrate comprises any one or more of natural or modified RNA single strand, natural or modified RNA double strand, natural or modified DNA single strand and natural or modified RNA / DNA hybrid single strand and natural or modified RNA / DNA hybrid double strand; Optionally, in the nucleic acid substrate, the RNA single strand comprises linear RNA single strand and / or circular RNA single strand, the DNA single strand comprises linear DNA single strand and / or circular DNA single strand, the DNA single strand comprises linear DNA single strand and / or circular DNA single strand, and the RNA / DNA hybrid single strand comprises linear RNA / DNA hybrid single strand and / or circular RNA / DNA hybrid single strand, and the RNA / DNA hybrid double strand comprises linear RNA / DNA hybrid double strand and / or circular RNA / DNA hybrid double strand; Optionally, the RNA comprises any one or more of mRNA, antisense oligonucleotide, siRNA, 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 2-200nt; Optionally, the number of the nucleic acid substrate is ≥1; Optionally, the final concentration of the nucleic acid substrate is 0.01-100mM; Optionally, the final concentration of the nucleic acid substrate is 1-100mM.

10. The reaction solution according to claim 8 or 9, characterized by 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-linked 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 the ATP is 0.1 to 50 mM. Optionally, when the enzyme-linked reagent includes a divalent ion, the final concentration of the divalent ion 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 to 9.

0.

11. A composition characterized in that, It comprises: each component of the enzyme-linked reagent according to any one of claims 2 to 7 or each component of the reaction solution according to any one of claims 8 to 10.

12. A kit characterized in that, It comprises: the enzyme-linked reagent according to any one of claims 2 to 7 or the reaction solution according to any one of claims 8 to 10 or the composition according to claim 8.

13. A method of synthesizing a nucleotide chain, characterized by, It comprises: performing synthesis using the enzyme-linked reagent according to any one of claims 2 to 7 or the reaction solution of any one of claims 8 to 10 or the composition according to claim 11 or the kit according to claim 12.

14. The method of synthesis of claim 13, wherein, The conditions of the synthesis include: temperature is 15 to 55°C, and time is 1 to 48 hours.

15. The product synthesized by the synthesis method according to claim 13 or 14.

15. The product synthesized by the synthesis method according to claim 13 or 14.

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