Method for synthesizing nucleotide chain

Abstract

Provided is a method for synthesizing a nucleotide chain. The synthesis method comprises adjusting a synthesis temperature of the nucleotide chain to ≥42°C or performing, during the synthesis, at least one procedure of high-temperature denaturation and low-temperature annealing, so that the efficient ligation of natural nucleic acid fragments and / or non-natural nucleic acid fragments is achieved. The method can reduce by-products generated during the synthesis of the nucleotide chain, improve the synthesis efficiency of a target nucleotide chain, and facilitate large-scale and high-quality production of nucleotide chains.

Description

A method for synthesizing nucleotide chains Cross-references to related applications This disclosure claims priority to Chinese Patent Application No. 2025101109531, filed on January 23, 2025, entitled "A Method for Synthesizing a Nucleotide Chain", the entire contents of which are incorporated herein by reference. Technical Field This disclosure relates to the biological field, and more specifically, to a method for synthesizing nucleotide chains. Background Technology RNA drugs are a novel class of drugs that have attracted much attention in recent years, and have been widely studied in the treatment of various diseases, including cancer, rare diseases, gastrointestinal diseases, cardiovascular diseases, and metabolic disorders. Compared with the development of traditional small molecule drugs and protein drugs, RNA drugs have many advantages, such as rapid synthesis, high targeting, and high personalization. RNA drugs can be broadly classified into four categories: RNA aptamers, antisense oligonucleotides (ASO), RNA interference drugs (miRNA and siRNA), and messenger RNA (mRNA). Besides being used as RNA drugs, RNA also plays an important role in gene editing technology. In the widely used CRISPR-Cas gene editing technology, single-stranded guide RNA (gRNA) can form a complex with the DNA endonuclease Cas and locate the gene site to be edited through complementary pairing, initiating the Cas-mediated gene editing process. With the rise of RNA products in the pharmaceutical field, the demand for RNA synthesis is increasing year by year. Currently, the chemical synthesis of nucleotide chains generally involves preparing the target nucleotide chain by sequentially extending nucleotide residues one base at a time in series. Since the efficiency of the chemical synthesis reaction of nucleotide chains cannot reach 100%, the purity and yield of the synthesized target nucleotide chain both decrease with increasing target nucleotide chain length, resulting in the presence of a certain amount of non-target nucleotide chains in the synthesized product that do not match the length of the target nucleotide chain. For nucleotide chains with a length of 20 nt or less, purification processes (e.g., ion exchange column or reverse-phase column) can increase the proportion of the final target nucleotide chain in the synthetic product. However, because some non-target nucleotide chains are very close in length to the target nucleotide chain (e.g., the length of the non-target nucleotide chain differs from the target nucleotide chain by only 1 nt or 2 nt), their properties are extremely similar to the target nucleotide chain, making it impossible to effectively separate and remove these non-target nucleotide chains from the synthetic product. For nucleotide chains longer than 20 nt, especially those longer than 50 nt, or even longer than 100 nt, chemical synthesis results in even more non-target nucleotide chains failing to be effectively separated and removed from the synthetic product. The presence of non-target nucleotide chains in the synthesized product often affects the function and activity of the target nucleotide chain, especially for drugs whose target nucleotide chain is an antisense strand, RNAi drug, nucleic acid aptamer drug, or sgRNA drug. The presence of non-target nucleotide chains can seriously affect the efficacy of the target nucleotide chain. In view of this, this disclosure is hereby made. Summary of the Invention The purpose of this disclosure is to provide a method for synthesizing nucleotide chains. This disclosure is implemented as follows: In a first aspect, embodiments of this disclosure provide a method for synthesizing a target nucleotide chain, comprising: performing a ligation reaction on a nicked double-stranded nucleic acid molecule and / or a nucleic acid substrate capable of forming the nicked double-stranded nucleic acid molecule at a first set temperature; wherein the first set temperature is ≥42°C. Secondly, embodiments of this disclosure provide a method for synthesizing a target nucleotide chain, comprising: performing a ligation reaction on a nicked double-stranded nucleic acid molecule and / or a nucleic acid substrate capable of forming the nicked double-stranded nucleic acid molecule; and, during the ligation reaction, performing at least one high-temperature denaturation-low-temperature annealing procedure as described in the foregoing embodiments. Thirdly, embodiments of this disclosure provide products synthesized by the synthesis method described in any of the foregoing embodiments. This disclosure has the following beneficial effects: By controlling the synthesis temperature of the target nucleotide chain to ≥42℃ or performing at least one high-temperature denaturation-low-temperature annealing procedure during synthesis, efficient ligation of natural and / or non-natural nucleic acid fragments is achieved, effectively reducing byproducts generated during nucleotide chain synthesis, improving the synthesis efficiency of the target nucleotide chain, and facilitating large-scale, high-quality production 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 is a schematic diagram of the nucleic acid substrates used in Examples 1-5 after ligation; Figure 2 is a schematic diagram of the nucleic acid substrate used in Example 6 after ligation; Figure 3 is a schematic diagram of the nucleic acid substrate used in Example 7 after ligation. 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. The following description uses a number of technical terms. To ensure a clearer and more consistent understanding of the specification and claims, including the scope of these terms, the following definitions are provided. 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: Ribonucleotides that exist in nature and consist of one molecule of phosphate, one molecule of ribose (a pentose sugar), and one nitrogenous base. Based on the type of nitrogenous base, natural 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 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 into linear and circular ribonucleotide chains based on their morphology. Circular RNA (circRNA) is RNA with a closed circular structure, formed by one or more linear ribonucleotide chains linked end-to-end by phosphodiester 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. In this article, the term "nick" 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 paper uses the term "sequence identity" (%) to refer to comparisons between polynucleotides and peptides, and determines it by comparing two optimally aligned sequences across a comparison window. For optimal alignment of two sequences, the portion of the polynucleotide or peptide sequence in the comparison window compared to the reference sequence may contain additions or deletions (i.e., vacancies). 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. Specific technical solutions On one hand, embodiments of this disclosure provide a method for synthesizing nucleotide chains, comprising: performing a ligation reaction on a notched double-stranded nucleic acid molecule and / or a nucleic acid substrate capable of forming the notched double-stranded nucleic acid molecule at a first set temperature; wherein the first set temperature is ≥42°C. In some embodiments, the first set temperature can be any one or a range between any two of 42, 45, 48, 50, 52, 55, 57 and 60°C. In some embodiments, the reaction time under the first set temperature condition is ≥30s. In some embodiments, the reaction time under the first set temperature condition is 5 min to 16 h, specifically it can be any one or any two of the following: 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, and 16 h. In some embodiments, before, during, and / or after the reaction at the first set temperature, the synthesis method further includes performing at least one high-temperature denaturation-low-temperature annealing procedure; wherein the high-temperature denaturation includes a first incubation at a second set temperature, the second set temperature being ≥ the first set temperature; and the low-temperature annealing includes a second incubation at a third set temperature, the third set temperature being < the second set temperature. In some embodiments, the second set temperature is 42 to 60°C, specifically any one or any two of 42, 44, 45, 46, 48, 50, 52, 54, 56, 58 and 60°C. In some embodiments, the third set temperature is ≥4°C. In some embodiments, the third set temperature is any one of 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40°C. In some embodiments, the synthesis method further includes performing the high-temperature denaturation-low-temperature annealing process two or more times. "Two or more times" includes two or more instances. In some embodiments, the first incubation time is ≥30s. In some embodiments, the first incubation time is 5 min to 16 h, specifically any one or any two of the following: 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, and 16 h. In some embodiments, the second incubation time is ≥30s. In some embodiments, the second incubation time is 5 min to 16 h, specifically any one or any two of the following: 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, and 16 h. On the other hand, embodiments of this disclosure provide a method for synthesizing a target nucleotide chain, comprising: performing a ligation reaction on a notched double-stranded nucleic acid molecule and / or a nucleic acid substrate capable of forming the notched double-stranded nucleic acid molecule; and, during the ligation reaction, performing at least once the high-temperature denaturation-low-temperature annealing procedure described in any of the foregoing embodiments. In some embodiments, the synthesis method includes performing the high-temperature denaturation-low-temperature annealing procedure two or more times. In some embodiments, the term "two or more times" specifically includes ≥2, 3, 4, 5, 6, 7, 8, 9, and 10 times. The high-temperature denaturation-low-temperature annealing process can eliminate non-target nicked double-stranded nucleic acid molecules or double-stranded nucleic acid molecules with "gaps" formed due to mismatches, thereby improving ligation efficiency and reducing the formation of ligation byproducts. Furthermore, since high temperatures can damage the structure of double strands, direct reaction at high temperatures may lead to reduced ligation efficiency. Therefore, by first raising the temperature to open up non-specific double-stranded nucleic acids, and then annealing to a slightly lower temperature, more target double-stranded nicked molecules can be formed and ligated, improving ligation efficiency and reducing byproduct formation. In some embodiments, the notched double-stranded nucleic acid molecule includes any one or more of notched RNA double strands and notched DNA / RNA 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 0–100°C. Specifically, it can be any one or any two of the following temperatures: 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100°C or higher. 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. When the nucleic acid substrate is a circular, notched double-stranded nucleic acid molecule, the number of nucleic acid substrates can be 1. In some embodiments, the number of nucleic acid substrates is 1 to 50, specifically any one or 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. The method of this application has no special restrictions on the type of modification and is applicable to all types of modifications. In some embodiments, the modifications include modifications that enhance stability and / or reduce innate immune response. 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 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 replacing the CN bond connecting the glycosidic bond with any one of CC, CO, and CS. In some embodiments, the target nucleotide chain has a sequence length of ≥2nt, 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, the synthesis method further includes separating and purifying the ligated product to obtain the target nucleotide chain. In some embodiments, the synthesis method further includes adding a ligase during the ligation reaction, the ligase linking the notches with phosphodiester bonds. In some embodiments, the ligase comprises an RNA ligase that ligates double strands. In some embodiments, the ligase includes RNA ligases from the Rnl2 and Rnl5 families. In some embodiments, the ligase comprises a thermostable RNA ligase that ligates double strands. A thermostable ligase is one that exhibits high stability at a set temperature during the ligation process. In some embodiments, the ligase comprises wild-type T4 RNA ligase 2 or a mutant thereof. In some embodiments, the amino acid sequence of the wild-type T4 RNA ligase 2 has at least 80% identity with the sequence shown in SEQ ID NO: 1 or 2. In some embodiments, at least 80% identity specifically refers to sequence identity within the range of any one or any two of 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, and 100%. In some embodiments, the mutant has a mutation at any one or more of the following positions in the amino acid sequence of the wild-type T4 RNA ligase 2: positions 62, 103, 166, 168, 193, 217, 260, 297, 298, 303, 306, 311, 313, and 318. A mutation is defined as a substitution or deletion of an amino acid residue at the corresponding position. In some embodiments, the mutation at position 62 includes A62W, the mutation at position 103 includes P103G, the mutation at position 166 includes N166P, the mutation at position 168 includes L168F, and the mutation at position 193 includes N193K, where "N193K" refers to the substitution of N with K at position 193 of the amino acid sequence; the mutation at position 217 includes any one of R217A, R217Q, R217N, and R217P; the mutation at position 2... The mutation at position 60 includes C260D; the mutation at position 297 includes T297A; the mutation at position 298 includes S298E; the mutation at position 303 includes T303D; the mutation at position 306 includes any one of Q306A, Q306D, and Q306E; the mutation at position 311 includes any one of S311E, S311D, S311A, and S311V; and the mutation at position 313 includes I313V. In some embodiments, the mutant has any one or a combination of the following mutations relative to the wild-type T4 RNA ligase 2: A62W, P103G, N166P, L168F, R217P, R217A, R217N, R217Q, and C260D. In some embodiments, the mutant has any of the following mutation combinations and combinations thereof relative to the wild-type T4 RNA ligase 2: N193K-S311D, N193K-R217P, N193K-T303D, N193K-T297A, T297A-Q306D, N193K-I313V, R217P-S298E, R217P-T297A, R217P-I313V, and S311E-I313V. In some embodiments, the mutant has any of the following mutation combinations or combinations thereof relative to the wild-type T4 RNA ligase 2: R217P-T297A-S311E, R217P-S298E-S311D, R217P-T297A-I313V, N193K-R217P-C260D, R217P-S311E-I313V, R217P-T303D-I313V, N193K-R217P-T303D-S311E, N193K-R217P-T303D-I313V, N193K-R217P-T303D-V318T, N1 93K-R217P-S311E-I313V, N193K-R217P-T303D-S311E-I313V, A62W-N193K-R217P-T303D-S311E-I313V, P103G-N193K-R217P-T303D-S311 E-I313V, N166P-N193K-R217P-T303D-S311E-I313V, L168F-N193K-R217P-T303D-S311E-I313V, N193K-R217P-C260D-T303D-S311E-I313V -V318T, L168F-N193K-R217P-C260D-S311E-T303D-I313V-V318T, A62W-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T, P103G-L 168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T, A62W-P103G-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T, A62W-P103 G-N166P-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T, A62W-N166P-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T, P103G-N166P-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T and N166P-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T. In some embodiments, the reaction time of the synthesis method is ≥5 min. In some embodiments, it can be 5 min to 16 h, specifically any one or any two of the following: 5 min, 30 min, 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, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, and 16 h. In some embodiments, the system of the synthetic reaction comprises: nucleic acid substrate, enzyme, ATP, and Mg. 2+ . On the other hand, this disclosure also provides a kit comprising: reagents configured to carry out the synthesis method described in any of the foregoing embodiments. On the other hand, embodiments of this disclosure also provide the use of the target reagent in the preparation of a product configured to synthesize a nucleotide chain, wherein the target reagent is a reagent configured to carry out the synthesis method described in any of the foregoing embodiments. In some embodiments, the reagents configured to carry out the synthesis method described in any of the foregoing embodiments include: the thermostable ligase and the reaction solution for synthesizing nucleotide chains described in any of the foregoing embodiments. In some embodiments, the reaction solution for synthesizing nucleotide chains includes any one or more of the following: 40–60 mM Tris HCl (pH 7.8–8.2), 10–14 mM MgCl2, 0.1–5 mM DTT, and 1–10 mM ATP. Each concentration represents the effective concentration of each component in the reaction system. Specifically, the effective concentration of TrisHCl can be any one or any two of the following: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60 mM. The effective concentration of MgCl2 can be any one or any two of the following: 10, 11, 11.5, 12, 12.5, 13, and 14 mM. The effective concentration of DTT can be any one or any two of the following: 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 mM. The effective concentration of ATP can be any one or any two of the following: 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, and 10 mM. In some embodiments, for every 0.5–30 mM of nucleic acid substrate, a ligase with a final concentration of 0.03 μM–30 μM is added. Specifically, the 0.5–30 mM concentration can be any one or any two of the following: 0.5, 1, 1.5, 1.6, 1.8, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 15, 20, 25, and 30 mM. Similarly, the 0.03–30 μM concentration can be any one or any two of the following: 0.03, 0.04, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, and 30 μM. Furthermore, embodiments of this disclosure also 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 A method for synthesizing a nucleotide chain, comprising: A 20 μL reaction solution containing each oligonucleotide (nucleic acid substrate) at a final concentration of 1.6 mM, 50 mM TrisHCl (pH 8.0), 12 mM MgCl2, 1 mM DTT, 4 mM ATP, and 0.005 mg / mL ligase was added to a 200 μL microtube and the reaction was incubated at the set temperature using a PCR instrument. Two hours after the start of the reaction, 1 μL of the reaction solution was sampled and 49 μL of 10 mM EDTA solution was added to terminate the reaction. The set temperature is 45°C. Example 2 A method for synthesizing a nucleotide chain is substantially the same as the method provided in Example 1, except that the temperature is set to 50°C. Example 3 A method for synthesizing a nucleotide chain is substantially the same as the method provided in Example 1, except that the temperature is set at 42°C. Example 4 A method for synthesizing a nucleotide chain is substantially the same as the method provided in Example 1, except that the temperature is set at 37°C. Example 5: The synthesis of nucleotide chains at high temperatures reduces the generation of byproducts. Using the four oligonucleotide fragments in Table 1 as nucleic acid substrates, and based on the synthesis methods provided in Examples 1-4, wild-type T4 Rnl2 (amino acid sequence as shown in SEQ ID NO:2) and its mutants Mut3, Mut6 and Mut9 were used as thermostable ligases to ligate the four oligonucleotide fragments as shown in Figure 1. Mut3 has an N193K-R217P-C260D mutation based on SEQ ID NO:2. Mut6 has the N193K-R217P-S311E-I313V mutation based on SEQ ID NO:2. Mut9 has the L168F-N193K-R217P-T303D-S311E-I313V mutation based on SEQ ID NO:2. For convenience, the oligonucleotides generated by the ligation reaction of F1 and F2 are referred to as sense strands (abbreviated as SS), and the oligonucleotides generated by F3 and F4 are referred to as antisense strands (abbreviated as AS). Table 1. Oligonucleotides used in the ligation reactions of Examples 1-5 Note: "Pho" indicates a 5' phosphate group; "-" indicates a phosphate diester bond. After the reaction, the products were separated using HPLC, and the components of each peak were analyzed using mass spectrometry. The proportion of each product was calculated by dividing the peak area of ​​each product on the HPLC chromatogram by the sum of the peak areas of all nucleotides on the entire chromatogram. The results of the examples are shown in Table 2. Table 2. Product formation in the ligation reaction. Note: "ND" indicates that the corresponding product was not detected during liquid chromatography and mass spectrometry analysis. In this embodiment, the reaction at 37°C for 2 hours yielded a 62.4% yield of the target product from wild-type T4Rnl2, along with 13.6% of non-target byproducts. When the reaction temperature was increased to 42°C, the yield of the target product decreased slightly, but the byproducts decreased by approximately 50%, indicating that increasing the reaction temperature does indeed reduce byproduct formation. Further increases in reaction temperature to 45°C and 50°C further reduced byproduct formation. At 50°C, no byproducts were produced, but because wild-type T4Rnl2 is essentially inactivated at high temperatures, the target product yield was only 10.8%. However, using Mut3, Mut6, and Mut9 at 45°C for 2 hours yielded 88.9%, 88.2%, and 89.5% of the target product, respectively, with byproduct yields of 1.9%, 2.6%, and 2.3%, respectively. When Mut3, Mut6, and Mut9 were reacted at 50°C for 2 hours, the yields of the target product were 90.2%, 90.0%, and 91.6%, respectively, and no byproducts were detected by liquid chromatography-mass spectrometry. The results of this example demonstrate that using the thermostable mutants of this disclosure at high temperatures can significantly reduce byproducts caused by base mismatches. Furthermore, in this example, the yield of the target product under high-temperature conditions was significantly higher than that under 37°C conditions. Example 6: Releasing non-natural nucleic acid substrates at high temperatures reduces the generation of byproducts. This embodiment is based on the nucleotide chain synthesis method provided in Examples 1-4. It uses the four oligonucleotide fragments from Table 3 as nucleic acid substrates and Mut3, Mut6, and Mut9 (same as in Example 5) as ligases for ligation. A schematic diagram of the nucleic acid substrate ligation is shown in Figure 2. Wild-type T4 Rnl2 (SEQ ID NO:2) purified under the same conditions was used as a control group for the reaction. In this embodiment, the ribonucleotides used as nucleic acid substrates are all non-natural ribonucleotides, including several non-natural ribonucleotides commonly used in RNA drug design: 2'-methoxy modification at the 2' position of the pentose ring (2'-OCH3), 2'-deoxy-2'-fluorine (2'-F), 5'-(E)-vinylphosphonate modification at the 5' position of the pentose ring, thiomodification at the α-phosphate position, and deoxyribonucleotides incorporated at certain sites. For ease of description, the oligonucleotides generated by the ligation reactions of F1 and F2 are referred to as sense strands (abbreviated as SS), and the oligonucleotides generated by F3 and F4 are referred to as antisense strands (abbreviated as AS). Table 3 shows the oligonucleotides used in the ligation reaction of Example 6. Notes: "Pho" indicates a 5' phosphate group; "-" indicates a phosphodiester bond; m indicates 2' methoxy modification (2'-OCH3); "f" indicates 2' deoxy-2' fluorine modification (2'-F); "s" indicates α-phosphate thiomodification; "d" indicates that the nucleotide used is a deoxyribonucleotide; "VP" indicates 5′-(E)-vinylphosphonate modification (5′-VP). The detection and analysis methods are the same as those described in Example 5, and the results are shown in Table 4. Table 4. Product formation in the ligation reaction of Example 6 Note: "ND" indicates that the corresponding product was not detected during liquid chromatography and mass spectrometry analysis. In this embodiment, the reaction at 37°C for 2 hours yielded a 65.4% yield of the target product from wild-type T4Rnl2, along with 14.9% of non-target byproducts. When the reaction temperature was increased to 42°C, the yield of the target product decreased slightly, but the byproducts decreased by approximately 60%. Further increases in reaction temperature to 45°C and 50°C further reduced the production of byproducts. At 50°C, no byproducts were observed, but because wild-type T4Rnl2 is essentially inactivated at high temperatures, the yield of the target product was only 11%. However, using Mut3, Mut6, and Mut9 at 45°C for 2 hours yielded 89.5%, 90.2%, and 88.2% of the target product, respectively, with byproduct yields of 1.7%, 2.1%, and 2.0%, respectively. When Mut3, Mut6, and Mut9 were reacted at 50°C for 2 hours, the yields were 89.2%, 88.6%, and 90.1%, respectively, and no byproducts were detected by liquid chromatography-mass spectrometry. The results of this example demonstrate that reacting the thermostable mutants of this disclosure at high temperatures can significantly reduce byproducts caused by base mismatches. This effect is not limited to ribonucleotide chains composed of natural ribonucleotides but also applies to ribonucleotide chains composed of non-natural ribonucleotides. Example 7: Improving the efficiency of nucleotide synthesis and reducing byproduct generation by adding a "high-temperature denaturation-low-temperature annealing" procedure. 20 μL of reaction solution containing each oligonucleotide (nucleic acid substrate) shown in Table 5 at a final concentration of 1.6 mM, 50 mM Tris HCl (pH 8.0), 12 mM MgCl2, 1 mM DTT, 4 mM ATP, and 0.005 mg / mL ligase was added to a 200 μL microtube, and the reaction shown in Figure 3 was performed using a PCR instrument with temperature controlled. The ligases used were wild-type T4 Rnl2, thermostable ligases L168F-N193K-R217P-T303D-S311E-I313V (Mut9), and A62W-P103G-N166P-L168F-N193K-R217P-T303D-S311E-I313V (Mut23). Mut23 is based on SEQ ID NO:2 and has the A62W-P103G-N166P-L168F-N193K-R217P-T303D-S311E-I313V mutation. This embodiment includes the following experimental groups: Experimental groups 1, 6, and 11 were directly incubated at 37°C for 4 hours; experimental groups 2, 7, and 12 were directly incubated at 45°C for 4 hours; experimental groups 3, 8, and 13 were directly incubated at 55°C for 4 hours; experimental groups 4, 9, and 14 were incubated at 45°C for 1 hour, then heated to 55°C for 1 hour, and then annealed to 45°C for 2 hours; experimental groups 5, 10, and 15 were first incubated at 55°C for 1 hour, then annealed to 45°C for 1 hour, then heated to 55°C for 1 hour, and then annealed to 45°C for 1 hour. After the reaction was completed, 1 μL of the reaction solution was sampled and 49 μL of 10 mM EDTA solution was added to terminate the reaction, and liquid chromatography-mass spectrometry was performed to analyze the reaction products. Table 5. Oligonucleotides used in Example 7 Notes: "Pho" indicates a 5' phosphate group; "-" indicates a phosphodiester bond; m indicates 2' methoxy modification (2'-OCH3); "f" indicates 2'-deoxy-2' fluorine (2'-F) modification; "s" indicates α-phosphate thiomodification; "d" indicates that the nucleotide used is a deoxyribonucleotide; "VP" indicates 5′-(E)-vinylphosphonate modification (5′-VP). The detection and analysis methods are the same as those described in Example 5, and the results are shown in Table 6. Table 6. Formation of the linkage reaction products in Example 7 Note: "ND" indicates that the corresponding product was not detected during liquid chromatography and mass spectrometry analysis. The results of the examples show that reacting Mut9 at 55°C for 4 hours (Experimental Group 8) yielded a 46.8% yield with no detectable byproducts, while reacting wild-type T4 Rnl2 at 55°C for 4 hours (Experimental Group 4) produced almost no detectable target product. This result verifies that Mut9 has better thermal stability and that reacting with Mut9 at 55°C effectively reduces byproduct formation. However, according to the results of the examples, the byproducts generated by reacting Mut9 at 55°C for 4 hours were significantly lower than those generated by reacting Mut9 at 45°C for 4 hours, but the yield of Mut9 at 55°C for 4 hours (Experimental Group 8) was 46.8%, lower than the 68.2% yield of Mut9 at 45°C for 4 hours. Using Mut9, incubation was first performed at 45°C for 1 hour, then increased to 55°C for 1 hour, and finally annealed to 45°C for 2 hours (Experimental Group 9). The yield of the target product was 89.7%, higher than the 68.2% yield of Mut9 directly incubated at 45°C for 4 hours (Experimental Group 7), while the byproduct ratio was even lower, at only 2%. Using Mut9, incubation was first performed at 55°C for 1 hour, then annealed to 45°C for 1 hour, followed by increasing to 55°C for 1 hour, and finally annealed to 45°C for 1 hour, resulting in a yield of 91.1% (Experimental Group 10), with no byproducts detected. The experimental results of Mut23 in this example were basically consistent with those of Mut9. These results indicate that when using the thermally stable T4 Rnl2 mutant for ligation, adding a "high-temperature denaturation-low-temperature annealing" step can significantly improve the yield of the target product while reducing byproduct generation. The sequence information involved in this application is shown in the table below. The sequences shown in SEQ ID NO:3 to 14 in the computer-readable sequence list are the unmodified sequences. 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 controlling the synthesis temperature of the target nucleotide chain to ≥42℃ or performing at least one high-temperature denaturation-low-temperature annealing procedure during synthesis, efficient ligation of natural and / or non-natural nucleic acid fragments is achieved, effectively reducing byproducts generated during nucleotide chain synthesis, improving the synthesis efficiency of the target nucleotide chain, and facilitating large-scale, high-quality production of nucleotide chains.

Claims

1. A method for synthesizing a target nucleotide chain, characterized in that, It includes: The notched double-stranded nucleic acid molecule and / or the nucleic acid substrate capable of forming the notched double-stranded nucleic acid molecule are subjected to a ligation reaction at a first set temperature; wherein the first set temperature is ≥42°C.

2. The synthesis method according to claim 1, characterized in that, The first set temperature is 42–60°C; Optionally, the reaction time under the first set temperature condition is ≥30s; Optionally, the reaction time under the first set temperature condition is 5 min to 16 h; Optionally, before, during, and / or after the reaction at the first set temperature, the synthesis method further includes performing at least one high-temperature denaturation-low-temperature annealing procedure; The high-temperature denaturation includes a first incubation at a second set temperature, wherein the second set temperature is greater than or equal to the first set temperature; the low-temperature annealing includes a second incubation at a third set temperature, wherein the third set temperature is less than the second set temperature. Optionally, the second set temperature is 42–60°C; Optionally, the third set temperature is ≥4℃; Optionally, the synthesis method includes performing the high-temperature denaturation-low-temperature annealing procedure more than twice; Optionally, the first incubation time is ≥30s; Optionally, the first incubation period is 5 min to 16 h; Optionally, the second incubation time is ≥30s; Optionally, the second incubation period is 5 min to 16 h.

3. A method for synthesizing a target nucleotide chain, characterized in that, It includes: The notched double-stranded nucleic acid molecule and / or a nucleic acid substrate capable of forming the notched double-stranded nucleic acid molecule are coupled together. During the connection reaction, the high-temperature denaturation-low-temperature annealing procedure as described in claim 2 is performed at least once; Optionally, the synthesis method includes performing the high-temperature denaturation-low-temperature annealing procedure more than twice.

4. The synthesis method according to any one of claims 1 to 3, characterized in that, The synthesis method further includes adding a ligase during the ligation reaction; Optionally, the ligase includes an RNA ligase that ligates double strands; Optionally, the ligase includes RNA ligases of the Rnl2 family and the Rnl5 family; Optionally, the ligase comprises: a thermostable RNA ligase for ligating double strands; Optionally, the ligase includes T4 RNA ligase 2 wild-type or a mutant thereof; Optionally, the amino acid sequence of the wild-type T4 RNA ligase 2 has at least 80% identity with the sequence shown in SEQ ID NO:1 or 2; Optionally, the mutant has mutations at any one or more of the following positions in the amino acid sequence of the wild-type T4 RNA ligase 2: position 62, position 103, position 166, position 168, position 193, position 217, position 260, position 297, position 298, position 303, position 306, position 311, position 313, and position 318. Optionally, the mutant has any one or a combination of the following mutations relative to the wild-type T4 RNA ligase 2: A62W, P103G, N166P, L168F, R217P, R217A, R217N, R217Q, and C260D; Optionally, the mutant has any of the following mutation combinations and combinations thereof relative to the wild-type T4 RNA ligase 2: N193K-S311D, N193K-R217P, N193K-T303D, N193K-T297A, T297A-Q306D, N193K-I313V, R217P-S298E, R217P-T297A, R217P-I313V, and S311E-I313V; Optionally, the mutant has any of the following mutation combinations or combinations thereof relative to the wild-type T4 RNA ligase 2: R217P-T297A-S311E, R217P-S298E-S311D, R217P-T297A-I313V, N193K-R217P-C260D, R217P-S311E-I313V, R217P-T303D-I313V, N193K-R217P-T303D-S311E, N193K-R217P-T303D-I313V, N193K-R217P-T303D-V318T, N193K-R217P-S311E-I 313V, N193K-R217P-T303D-S311E-I313V, A62W-N193K-R217P-T303D-S311E-I313V, P103G-N193K-R217P-T303D-S311E-I313V, N166P- N193K-R217P-T303D-S311E-I313V, L168F-N193K-R217P-T303D-S311E-I313V, N193K-R217P-C260D-T303D-S311E-I313V-V318T, L168 F-N193K-R217P-C260D-S311E-T303D-I313V-V318T, A62W-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T, P103G-L168F-N19 3K-R217P-C260D-T303D-S311E-I313V-V318T, A62W-P103G-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T, A62W-P103G-N16 6P-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T, A62W-N166P-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T, P103G-N166P-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T, and N166P-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T.

5. The synthesis method according to claims 1 to 4, characterized in that, The notched double-stranded nucleic acid molecule includes any one or more of notched RNA double strands and notched DNA / RNA hybrid double strands; Optionally, the notched double-stranded nucleic acid molecule may be linear and / or circular; Optionally, the notched double-stranded nucleic acid molecule includes a nucleic acid substrate and / or a double-stranded nucleic acid molecule formed by mixing the nucleic acid substrate; Optionally, 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, natural and / or modified DNA / RNA hybrid single strand, and natural and / or modified DNA / RNA hybrid double strand; Optionally, 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; Optionally, the RNA single strand includes any one or more of the following: mRNA, antisense oligonucleotide, siRNA, sgRNA, lncRNA, circRNA, and miRNA; Optionally, the fragment length of the nucleic acid substrate is ≥2 nt; Optionally, the fragment length of the nucleic acid substrate is 2 to 200 nt.

6. The synthesis method according to claim 5, characterized in that, The modifications include: modifications that enhance stability and / or reduce innate immune responses; Optionally, the modification includes any one of the following: modification of phosphate groups, modification of bases, modification of sugar rings, and modification of glycosidic bonds.

7. The synthesis method according to claim 6, characterized in that, The modification of the phosphate group includes any one or more of the following: 5′-(E)-vinylphosphonic acid modification, thiophosphate modification, phosphate triester modification, 5′-methylphosphonate modification, 5′-morpholino modification, dithiophosphate modification, methoxypropylphosphonate modification, S-5′-C methyl analog modification, short-chain alkyl or cycloalkyl sugar inter-bond modification, short-chain heteroatom or heterocyclic sugar inter-bond modification, or the entire phosphate group is substituted by any one of amide, aminooxy, alkoxy and triazole groups; Optionally, the base modification includes any one or more of the following: 2,4-difluorotolyl ribonucleoside 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; Optionally, 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 modification, unlocked nucleic acid modification, bridged nucleic acid modification, tricyclic DNA modification, phosphodiamid morpholino oligonucleotide modification, 2'-deoxy nucleic acid modification, (S)-restricted ethyl bicyclic nucleic acid modification, peptide nucleic acid modification, and glycomimetic modification; Optionally, the sugar mimic includes any one or more of the following: cyclobutyl in cyclobutyl nucleotides replacing the pentafuranose group, morpholino in morpholino nucleic acid, peptide backbone in peptide nucleic acid, polyethylene glycol backbone in ethylene glycol nucleic acid, threonose backbone in threonose nucleic acid, and butyl backbone in acyclic butyl nucleic acid. Optionally, the modification of the glycosidic bond includes replacing the CN bond connecting the glycosidic bond with any one of CC, CO, and CS.

8. The synthesis method according to any one of claims 1 to 7, characterized in that, The target nucleotide chain has a sequence length of ≥2 nt; Optionally, the target nucleotide chain has a sequence length ≥20 nt; Optionally, the target nucleotide chain has a sequence length ≥50 nt.

9. The synthesis method according to any one of claims 1 to 8, characterized in that, The reaction time of the synthesis method is ≥5 min.

10. The synthesis method according to claim 9, characterized in that, The reaction time of the synthesis method is 5 min to 16 h.

11. The synthesis method according to claim 10, characterized in that, The reaction time of the synthesis method is 1 h to 16 h.

12. The synthesis method according to any one of claims 1 to 11, characterized in that, The target nucleotide chain includes linear and / or cyclic strands.

13. The synthesis method according to any one of claims 1 to 12, characterized in that, The target nucleotide chain is any one or more of the following: RNA single-strand, RNA double-strand, DNA / RNA hybrid single-strand, and DNA / RNA hybrid double-strand.

14. The synthesis method according to any one of claims 1 to 13, characterized in that, The synthesis method further includes separating and purifying the ligated product to obtain the target nucleotide chain.

15. The product synthesized by the synthetic method according to any one of claims 1 to 14.

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