Composition for the synthesis of nucleotide chains and method for the synthesis of nucleotide chains

Abstract

A composition for the synthesis of a nucleotide chain and a method for the synthesis of a nucleotide chain, belonging to the art of nucleotides. The composition for the synthesis of a nucleotide chain comprises a nucleotide fragment and a double-stranded ligase, wherein the nucleotide fragment is for forming a double-stranded structure, the double-stranded ligase is a ligase capable of closing gaps in the double-stranded structure, and at least one of the strands of the double-stranded structure is a target nucleotide chain, the nucleotide fragment comprises a first fragment and a second fragment, the first fragment contains a monophosphate group at its 5' end and the first fragment is synthesized in the direction from 3' to 5', the second fragment contains a hydroxyl group at its 3' end and the second fragment is synthesized in the direction from 5' to 3'.

Description

Technical Field

[0001] The present disclosure belongs to the technical field of nucleotide synthesis, and specifically relates to a composition for synthesizing a nucleotide chain and a method for synthesizing a nucleotide chain.

[0002] Cross-reference to Related Applications This application claims priority based on a Chinese application filed with the Chinese Patent Office on November 30, 2023, with application number 202311627360.X and titled "Composition for Synthesizing Nucleotide Chain and Method for Synthesizing Nucleotide Chain", and all of its content is incorporated herein by reference.

Background Art

[0003] Currently, the synthesis of a nucleotide chain generally prepares a target nucleotide chain by sequentially extending and synthesizing nucleotide residues one base at a time in series. Since the efficiency of the chemical synthesis reaction of the nucleotide chain cannot reach 100%, both the purity of the synthesized target nucleotide chain product and the yield of the target product decrease as the length of the target nucleotide chain increases, and the synthetic product contains a certain amount of non-target nucleotide chains with lengths that do not match the target nucleotide chain.

[0004] For nucleotide chains with a length of 20mer or less, after chemical synthesis, a purification process (e.g., ion-exchange chromatography column or reverse-phase chromatography column) is used to increase the proportion of the final target nucleotide chain in the synthetic product. However, some non-target nucleotide chains are relatively 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 1mer or 2mer), and these non-target nucleotide chains cannot be effectively separated and removed from the synthetic product because their properties are very similar to those of the target nucleotide chain. For nucleotide chains longer than 20mer, especially those longer than 50mer, and even longer than 100mer, a larger proportion of non-target nucleotide chains cannot be separated and removed from the synthetic product after chemical synthesis.

[0005] The presence of non-target nucleotide chains in synthetic products impairs the function and activity of the target nucleotide chain. In particular, when the target nucleotide chain is an antisense chain drug, RNAi drug, nucleic acid aptamer drug, or sgRNA drug, the presence of non-target nucleotide chains significantly impairs the efficacy of the target nucleotide chain. [Overview of the project]

[0006] The present disclosure aims to provide a composition for synthesizing nucleotide chains and a method for synthesizing nucleotide chains that can reduce the content of non-target nucleotide chains in the synthesized nucleotide chain product and increase the purity of target nucleotide chains in the synthesized product.

[0007] In a first embodiment, the present disclosure provides a composition for the synthesis of a nucleotide chain. The composition comprises a nucleotide fragment and a double-stranded ligase, wherein the nucleotide fragment is for forming a double-stranded structure, the double-stranded ligase is a ligase capable of closing gaps in the double-stranded structure, and at least one of the strands of the double-stranded structure is a target nucleotide chain, and the nucleotide fragment comprises a first fragment and a second fragment, wherein the first fragment contains a monophosphate group at its 5' end and the first fragment is synthesized in a 3'-to-5' direction, and the second fragment contains a hydroxyl group at its 3' end and the second fragment is synthesized in a 5'-to-3' direction.

[0008] In the composition for the synthesis of nucleotide chains according to this disclosure, the nucleotide fragment comprises a first fragment synthesized in the 3' to 5' direction and containing a monophosphate group at the 5' end, and a second fragment synthesized in the 5' to 3' direction and containing a hydroxyl group at the 3' end, wherein the first and second fragments combine to form a double-stranded structure, thereby significantly reducing the content of non-target nucleotide chains in the synthesized nucleotide chain product, effectively increasing the purity of the target nucleotide chain in the synthesized product, and contributing to the full expression of the efficacy of the target nucleotide chain.

[0009] In an optional embodiment of the present disclosure based on the first aspect, the nucleotide fragment further comprises a third fragment, the third fragment comprising a hydroxyl group at its 3' end and a monophosphate group at its 5' end.

[0010] The third fragment is either synthesized in the direction from 3' to 5', or synthesized in the direction from 5' to 3'.

[0011] Based on the first aspect, in an optional embodiment of the present disclosure, the composition for the synthesis of a nucleotide chain further comprises a template chain, wherein the template chain and nucleotide fragments are for jointly forming a double-stranded structure, and the nucleotide fragments are for forming a target nucleotide chain.

[0012] Based on the first aspect, in an optional embodiment of the present disclosure, the template chain has a linear structure and can form a first complementary region in inverse complement to the arrangement of the 5' ends of the first fragment, and can form a second complementary region in inverse complement to the arrangement of the 3' ends of the second fragment, and can form a gap between the 5' ends of the first fragment and the 3' ends of the second fragment.

[0013] In the above proposed technology, the template chain and the nucleotide fragment can work together to form a double-stranded structure, and the nucleotide fragment can form a target nucleotide chain.

[0014] Based on the first aspect, in an optional embodiment of the present disclosure, the template chain has a hairpin structure, and the template chain can form a first complementary region in reverse complement to the 5' end arrangement of the first fragment, and the template chain can form a second complementary region in reverse complement to the 3' end arrangement of the second fragment, and the 5' end of the template chain contains a monophosphate group and a gap can be formed between the 5' end of the template chain and the 3' end of the first fragment, or the 3' end of the template chain contains a hydroxyl group and a gap can be formed between the 3' end of the template chain and the 5' end of the second fragment.

[0015] In the above proposed technology, the template chain and the nucleotide fragment can work together to form a double-stranded structure, and the nucleotide fragment can form a target nucleotide chain.

[0016] Based on the first aspect, in an optional embodiment of the present disclosure, the first fragment has a hairpin structure, the first fragment is inversely complementary to the 3' end sequence of the second fragment, and a gap can be formed between the 5' end of the first fragment and the 3' end of the second fragment, and the nucleotide chain formed by the joining of the first and second fragments is the target nucleotide chain, or the second fragment has a hairpin structure, the second fragment is inversely complementary to the 5' end sequence of the first fragment, and a gap can be formed between the 3' end of the second fragment and the 5' end of the first fragment, and the nucleotide chain formed by the joining of the first and second fragments is the target nucleotide chain.

[0017] In the above proposed technology, the first and second fragments can work together to form a double-stranded structure, and the first and second fragments can bind together to form a target nucleotide chain.

[0018] Based on the first aspect, in an optional embodiment of the present disclosure, the template chain has at least two template fragments, the template fragments are linear in structure, a gap can be formed between the at least two template fragments, the template chain can form a first complementary region in reverse complement to the 5' end sequence of the first fragment, the template chain can form a second complementary region in reverse complement to the 3' end sequence of the second fragment, a gap can be formed between the 5' end of the first fragment and the 3' end of the second fragment, and the nucleotide fragments are for forming a target nucleotide chain.

[0019] In the above proposed technology, the template chain and nucleotide fragments can work together to form a double-stranded structure, and the nucleotide fragments can form a target nucleotide chain.

[0020] Optionally, the template fragment comprises one fourth fragment and one fifth fragment, wherein the fourth fragment contains a monophosphate group at its 5' end and is synthesized in a 3'-to-5' direction, the fifth fragment contains a hydroxyl group at its 3' end and is synthesized in a 5'-to-3' direction, a gap is formed between the 5' end of the fourth fragment and the 3' end of the fifth fragment, and a gap is formed between the 5' end of the first fragment and the 3' end of the second fragment.

[0021] In an optional embodiment of the present disclosure based on the first aspect, the nucleotide fragment further comprises a sixth fragment and a seventh fragment, wherein the sixth fragment contains a monophosphate group at its 5' end and is synthesized in a 3'-to-5' direction, the seventh fragment contains a hydroxyl group at its 3' end and is synthesized in a 5'-to-3' direction, a gap is formed between the 5' end of the sixth fragment and the 3' end of the seventh fragment, and the sixth and seventh fragments together form a first target nucleotide chain, a gap is formed between the 5' end of the first fragment and the 3' end of the second fragment, and the first and second fragments together form a second target nucleotide chain, and the first and second target nucleotide chains together form a double-stranded structure.

[0022] In the above proposed technology, the sixth and seventh fragments jointly form the first target nucleotide chain, and the first and second fragments jointly form the second target nucleotide chain. This further effectively reduces the content of non-target nucleotide chains in the synthesized nucleotide chain product, further effectively increases the purity of the target nucleotide chain in the synthesized product, and contributes to the full exertion of the efficacy of the target nucleotide chain.

[0023] Based on the first aspect, in an optional embodiment of the present disclosure, the nucleotide fragment further comprises a sixth fragment and a seventh fragment, wherein the sixth fragment contains a monophosphate group at its 5' end and is synthesized in a 3'-to-5' direction, the seventh fragment contains a hydroxyl group at its 3' end and is synthesized in a 5'-to-3' direction, a gap is formed between the 5' end of the sixth fragment and the 3' end of the seventh fragment, and the sixth and seventh fragments together form a first target nucleotide chain, a gap is formed between the 3' end of the third fragment and the 5' end of the first fragment, a gap is formed between the 5' end of the third fragment and the 3' end of the second fragment, and the first, third and second fragments together form a second target nucleotide chain, and the first and second target nucleotide chains together form a double-stranded structure.

[0024] In the above technical solution, the sixth fragment and the seventh fragment jointly form the first target nucleotide strand, and the first fragment, the third fragment and the second fragment jointly form the second target nucleotide strand, so as to further effectively reduce the content of non-target nucleotide strands in the synthesis product of the nucleotide strand, further effectively increase the purity of the target nucleotide strand in the synthesis product, and contribute to the full exertion of the efficacy of the target nucleotide strand.

[0025] Based on the first aspect, in an alternative embodiment of the present disclosure, when the nucleotide fragment is DNA, the double-stranded ligase is a DNA double-stranded ligase, and the DNA double-stranded ligase includes T4 DNA ligase. When the nucleotide fragment is RNA, the double-stranded ligase is an RNA double-stranded ligase, and the RNA double-stranded ligase includes Rnl2 family ligase and Rnl5 family ligase, and / or the nucleotide fragment includes 4 to 200 bases, and / or the composition for the synthesis of the nucleotide strand further includes a buffer solution containing magnesium ions.

[0026] Optionally, the nucleotide fragment includes 4 to 120 bases.

[0027] Based on the first aspect, in an alternative embodiment of the present disclosure, the nucleotide fragment may or may not include a modification group, and / or the template strand may or may not include a modification group.

[0028] Based on the first aspect, in an alternative embodiment of the present disclosure, the method for preparing the template strand includes performing a synthesis reaction of the template strand according to the target sequence of the template strand, and then not performing a purification treatment on the synthesis product, and / or the method for preparing the nucleotide fragment includes performing a synthesis reaction of the nucleotide fragment according to the target sequence of the nucleotide fragment, and then not performing a purification treatment on the synthesis product.

[0029] In the above technical solution, after synthesizing the template strand and the nucleotide fragment, even without performing purification treatment, it is possible to effectively reduce the content of non-target nucleotide strands in the synthesis product of the nucleotide strand, effectively increase the purity of the target nucleotide strand in the synthesis product, and contribute to the full exertion of the efficacy of the target nucleotide strand.

[0030] In a second aspect, the present disclosure provides a method for synthesizing a nucleotide strand. The synthesis method includes performing a nucleotide strand synthesis reaction using a composition for synthesizing a nucleotide strand according to any one of the above first aspects.

[0031] The method for synthesizing a nucleotide strand according to the present disclosure has a first fragment synthesized in the 3' to 5' direction and containing a monophosphate group at the 5' end, and a second fragment synthesized in the 5' to 3' direction and containing a hydroxyl group at the 3' end as raw materials for synthesis. Therefore, the first fragment and the second fragment combine and jointly form a double-stranded structure, thereby significantly reducing the content of non-target nucleotide strands in the synthesis product of the nucleotide strand, effectively increasing the purity of the target nucleotide strand in the synthesis product, and contributing to the full exertion of the efficacy of the target nucleotide strand.

[0032] In a third aspect, the present disclosure provides a reagent or a kit. The reagent or kit includes a composition for synthesizing a nucleotide strand according to any one of the above first aspects.

[0033] The form of the reagent includes, but is not limited to, liquid, solid or semi-solid. The reagent further includes a buffer solution, ATP, MgCl2, DTT, ligase, etc.

[0034] The ligase includes, but is not limited to, T4 RNA Ligase2 and double-stranded ligase.

[0035] When the nucleotide fragment is DNA, the double-stranded ligase is a DNA double-stranded ligase, and exemplary examples of DNA double-stranded ligases include T4 DNA ligase (T4 DNA Ligase) or Taq DNA Ligase, etc.

[0036] As described above, when the nucleotide fragment is RNA, the double-stranded ligase is an RNA double-stranded ligase. Exemplary examples of RNA double-stranded ligases include Rnl2 family ligases and Rnl5 family ligases, such as T4 RNA ligase2 and DraRnl (derived from Naegleria gruberi).

[0037] In the above proposed technology, the kit further comprises a solid support for the synthesis of nucleotide chains (e.g., 5'-Dimethoxytrityl-N4-acetyl-Cytidine, 2'-TBDMS-3'-succinoyl-long chain alkylamino-CPG), a synthesis column, a diluent, a washing agent, an activator, a deprotection agent, an oxidizing agent, a sulfidating agent, a closure agent, and a reaction termination solution.

[0038] The deprotection agent is a solution of trichloroacetic acid, and the activator is a solution of 5-ethylthiotetrazole (i.e., ETT).

[0039] The oxidizing agent is iodine (I2) solution, the sulfiding agent is phenylacetyl disulfide, and the occluding agent is a mixture of acetic anhydride (Ac2O) and acetonitrile in a volume ratio of 1:4, and / or a mixture of N-methylimidazole, pyridine, and acetonitrile in a volume ratio of 2:3:5.

[0040] The reaction termination solution is selected from EDTA.

[0041] In a fourth embodiment, the disclosure provides the use of a composition for the synthesis of nucleotide chains according to any one of the first embodiments described above in the synthesis of nucleotide chains.

[0042] Beneficial effects In the composition for the synthesis of nucleotide chains according to this disclosure, the nucleotide fragment comprises a first fragment synthesized in the 3' to 5' direction and containing a monophosphate group at the 5' end, and a second fragment synthesized in the 5' to 3' direction and containing a hydroxyl group at the 3' end. The first and second fragments combine to form a double-stranded structure, thereby significantly reducing the content of non-target nucleotide chains in the synthesized nucleotide chain product, effectively increasing the purity of the target nucleotide chain in the synthesized product, and contributing to the full expression of the efficacy of the target nucleotide chain. [Modes for carrying out the invention]

[0043] The embodiments of this disclosure will be described in detail below using examples. As those skilled in the art will see, the following examples are for illustrative purposes only and do not limit the scope of this disclosure. Where specific conditions are not specified in the examples, it is possible to perform the procedures under conventional conditions or conditions recommended by the manufacturer. Where the manufacturer is not specified for reagents or instruments, commercially available conventional products can be used.

[0044] As used herein, the terms "or / and" describe a relationship between related objects, indicating that three types of relationships exist. For example, A or / and B represents three types of relationships: A alone exists, both A and B exist, and B alone exists.

[0045] In this disclosure, a nucleotide chain refers to a polymer obtained by condensation polymerization of two or more nucleotide monomers via phosphodiester bonds. A phosphodiester bond is typically formed by esterification of a hydroxyl group attached to the 3' carbon of the sugar ring of one nucleotide with the 5' phosphate of another nucleotide monomer. In this disclosure, a nucleotide chain may have 100 or fewer bases, 200 or fewer bases, or 500 or fewer bases, and the specific number of bases in a nucleotide chain is not limited in this disclosure.

[0046] In this disclosure, “synthesized in the 3' to 5' direction” means that a nucleotide fragment is synthesized from its 3' end to its 5' end. “Synthesized in the 5' to 3' direction” means that a nucleotide fragment is synthesized from its 5' end to its 3' end.

[0047] In this disclosure, for a synthesis method performed in the 3' to 5' direction, n-1 impurities refer to nucleotide products in which one base is deleted at the 5' end compared to the target synthesis product, n-2 impurities refer to nucleotide products in which two bases are deleted at the 5' end compared to the target synthesis product, n-3 impurities refer to nucleotide products in which three bases are deleted at the 5' end compared to the target synthesis product, nx impurities refer to nucleotide products in which x bases are deleted at the 5' end compared to the target synthesis product, n+1 impurities refer to nucleotide products in which one base is added at the 5' end compared to the target synthesis product, and n+2 impurities refer to nucleotide products in which two bases are added at the 5' end compared to the target synthesis product.

[0048] In this disclosure, for a synthesis method performed in the 5' to 3' direction, n-1 impurities refer to nucleotide products in which one base is deleted at the 3' end compared to the target synthetic product, n-2 impurities refer to nucleotide products in which two bases are deleted at the 3' end compared to the target synthetic product, n-3 impurities refer to nucleotide products in which three bases are deleted at the 3' end compared to the target synthetic product, nx impurities refer to nucleotide products in which x bases are deleted at the 3' end compared to the target synthetic product, n+1 impurities refer to nucleotide products in which one base is added at the 3' end compared to the target synthetic product, and n+2 impurities refer to nucleotide products in which two bases are added at the 3' end compared to the target synthetic product.

[0049] In this disclosure, the template chain refers to a nucleotide chain that is inversely complementary to some or all of a nucleotide fragment. The template chain may be a DNA chain, an RNA chain, and may or may not contain modifying groups.

[0050] In this disclosure, a double-stranded structure refers to a double-stranded structure consisting of reverse-complementary double-stranded DNA, reverse-complementary double-stranded RNA, or reverse-complementary single-stranded RNA and single-stranded DNA.

[0051] In this disclosure, "at least one strand of a double-stranded structure is a target nucleotide" means that both of the two nucleotide strands, whose sequences are inversely complementary, may be target nucleotide strands, or only one may be a target nucleotide strand.

[0052] In this disclosure, a gap refers to a site in a double-stranded structure where a phosphodiester bond is lacking between two adjacent nucleotide units (which may be nucleotide fragments or a template chain). By catalytic action of a double-stranded ligase, a complete phosphodiester bond can be formed at the gap site; that is, the 3' hydroxyl group of one nucleotide unit at the gap site and the 5' monophosphate group of the other nucleotide unit at the gap site form a complete phosphodiester bond by catalytic action of the double-stranded ligase.

[0053] In this disclosure, gap closure refers to the process of forming a complete phosphodiester bond at the gap between two adjacent nucleotide units in a double-stranded structure.

[0054] In this disclosure, a phosphate group donor refers to a nucleotide unit (which may be a nucleotide fragment or a template chain) containing a monophosphate group at its 5' end. A hydroxyl group acceptor refers to a nucleotide unit (which may be a nucleotide fragment or a template chain) containing a hydroxyl group at its 3' end. Under the catalytic action of a double-stranded ligase, the monophosphate group at the 5' end of the phosphate group donor and the hydroxyl group at the 3' end of the hydroxyl group acceptor can form a complete phosphodiester bond. The phosphate group donor may be a modified phosphate group donor, such as one modified by sulfur or halogen.

[0055] In this disclosure, double-stranded ligase refers to an enzyme that plays a role in closing gaps in a double-stranded structure, that is, an enzyme that causes a complete phosphodiester bond to form at the gap site in the double-stranded structure.

[0056] Nucleotide chain synthesis generally involves preparing the target nucleotide chain by sequentially extending nucleotide residues one base at a time in series. However, the efficiency of the chemical synthesis reaction of nucleotide chains cannot reach 100%.

[0057] When synthesizing DNA strands, the phosphoramidite method is used, and the synthesis efficiency of each base in the DNA strand is approximately 99.0% to 99.6%. Calculating with the median value of 99.3%, when synthesizing a 10-mer DNA strand, the target DNA strand content in the synthesized product is 93%; when synthesizing a 20-mer DNA strand, the target DNA strand content is 87%; when synthesizing a 40-mer DNA strand, the target DNA strand content is 76%; and when synthesizing an 80-mer DNA strand, the target DNA strand content is 57%. In the case of RNA chain synthesis, the phosphoramidite method is used, and the synthesis efficiency of each base of the RNA chain is approximately 98.0%. When synthesizing an RNA chain of 10mer length, the target RNA chain content in the synthesized product is 82%. When synthesizing an RNA chain of 20mer length, the target RNA chain content in the synthesized product is 67%. When synthesizing an RNA chain of 40mer length, the target RNA chain content in the synthesized product is 45%. When synthesizing an RNA chain of 80mer length, the target RNA chain content in the synthesized product is 20%.

[0058] In the synthetic product of a nucleotide chain, in addition to the target nucleotide chain (i.e., the full-length product), non-target nucleotide chains (i.e., non-full-length products whose length does not match that of the target nucleotide chain) are also included, and these non-target nucleotide chains are generally non-full-length cleaved nucleotide fragments such as n-1 impurities, n-2 impurities, and n-3 impurities, with small amounts of n+1 and n+2 impurities.

[0059] For nucleotide chains of 20mer or less, after chemical synthesis, the proportion of the final target nucleotide chain in the synthesized product can be increased through purification processes (e.g., ion-exchange chromatography columns or reverse-phase chromatography columns). However, n-1 and n-2 impurities in the synthesized product cannot be effectively separated and removed because their properties are similar to those of the target nucleotide chain. For nucleotide chains longer than 20mer, especially those longer than 50mer, and even longer than 100mer, after chemical synthesis, the purification process of the synthesized product cannot effectively separate and remove not only n-1, n-2, n+1, and n+2 impurities, but also n-3, n-4, n-5, and even n-6, n-7, n-8, n-9, n-10 impurities or longer cleaved nucleotide fragments. The presence of non-target nucleotide chains in the synthesized product often impairs the function and activity of the target nucleotide chain. In particular, when the target nucleotide chain is an antisense chain drug, RNAi drug, nucleic acid aptamer drug, or sgRNA drug, the efficacy of the target nucleotide chain is significantly impaired due to the presence of non-target nucleotide chains.

[0060] In view of this, the present disclosure provides a composition for the synthesis of a nucleotide chain. The composition comprises a nucleotide fragment and a double-stranded ligase, wherein the nucleotide fragment is for forming a double-stranded structure, the double-stranded ligase is a ligase capable of closing gaps in the double-stranded structure, and at least one of the strands of the double-stranded structure is a target nucleotide chain, the nucleotide fragment comprises a first fragment and a second fragment, the first fragment having a monophosphate group at its 5' end and being synthesized in a 3'-to-5' direction, and the second fragment having a hydroxyl group at its 3' end and being synthesized in a 5'-to-3' direction.

[0061] In the above-described composition for the synthesis of nucleotide chains, the nucleotide fragment comprises a "first fragment synthesized in the direction from 3' to 5' and containing a monophosphate group at the 5' end" and a "second fragment synthesized in the direction from 5' to 3' and containing a hydroxyl group at the 3' end." In the nucleotide chain synthesis reaction using the above-described composition, the first fragment functions as a phosphate group donor, and the second fragment functions as a hydroxyl group acceptor. The nucleotide fragments, including the first and second fragments, can form a double-stranded structure, a gap can be formed in the double-stranded structure, and the gap can be closed under the catalytic action of a double-stranded ligase, thereby completing the synthesis reaction of the target nucleotide chain. According to the above synthesis reaction, the content of non-target nucleotide chains (especially n-1 impurities, n-2 impurities, n+1 impurities, and n+2 impurities) in the nucleotide chain synthesis product can be significantly reduced, effectively increasing the purity of the target nucleotide chain in the synthesis product and contributing to the full expression of the efficacy of the target nucleotide chain.

[0062] When the nucleotide fragment is DNA, the double-stranded ligase that closes the gap is a DNA double-stranded ligase; when the nucleotide fragment is RNA, the double-stranded ligase that closes the gap is an RNA double-stranded ligase.

[0063] The first and second fragments may both be DNA, or both be RNA, or the first fragment may be DNA and the second fragment may be RNA, or the first fragment may be RNA and the second fragment may be DNA, as long as the first and second fragments can form a double-stranded structure.

[0064] In some optional embodiments of the present disclosure, the nucleotide fragment further comprises a third fragment in addition to the first and second fragments described above, the third fragment having a hydroxyl group at its 3' end and a monophosphate group at its 5' end.

[0065] When a nucleotide fragment contains a first, second, and third fragment, these fragments form a double-stranded structure, which significantly reduces the content of non-target nucleotide chains (especially n-1, n-2, n+1, and n+2 impurities) in the synthetic product of the nucleotide chain. This effectively increases the purity of the target nucleotide chain in the synthetic product and contributes to the full expression of the efficacy of the target nucleotide chain.

[0066] In some select embodiments of this disclosure, the third fragment is synthesized in a 3'-to-5' direction, or in a 5'-to-3' direction. When the nucleotide fragment comprises the first, second, and third fragments, whether the third fragment is synthesized in a 3'-to-5' direction or a 5'-to-3' direction, it can significantly reduce the content of non-target nucleotide chains (particularly n-1, n-2, n+1, and n+2 impurities) in the nucleotide chain synthesis product, effectively increasing the purity of the target nucleotide chain in the synthesis product and contributing to the full efficacy of the target nucleotide chain.

[0067] In some select embodiments of this disclosure, the nucleotide fragment may further comprise other fragments, and if the nucleotide fragment has at least one first fragment and at least one second fragment, and all fragments in the nucleotide fragment can collectively form a double-stranded structure, then the content of non-target nucleotide chains (particularly n-1, n-2, n+1, and n+2 impurities) in the synthetic product of the nucleotide chain can be significantly reduced, effectively increasing the purity of the target nucleotide chain in the synthetic product and contributing to the full exertion of the efficacy of the target nucleotide chain.

[0068] In some select embodiments of the present disclosure, the composition for the synthesis of a nucleotide chain further comprises a template chain, wherein the template chain and nucleotide fragments (i.e., a first fragment and a second fragment) are to work together to form a double-stranded structure, and the nucleotide fragments are to form a target nucleotide chain.

[0069] In some select embodiments of the present disclosure, the template chain is linear in structure, and the template chain can form a first complementary region in reverse complement to the 5' end sequence of the first fragment, and the template chain can form a second complementary region in reverse complement to the 3' end sequence of the second fragment, and a gap can be formed between the 5' end of the first fragment and the 3' end of the second fragment. According to the above configuration, the template chain and the nucleotide fragments can work together to form a double-stranded structure, and the nucleotide fragments can form a target nucleotide chain.

[0070] For example, if the nucleotide fragments consist only of the first and second fragments described above, and the template chain is linear, the 5' end of the template chain can form a first complementary region in reverse complement to the sequence of the 5' end of the first fragment, the 3' end of the template chain can form a second complementary region in reverse complement to the sequence of the 3' end of the second fragment, a gap is formed between the monophosphate group at the 5' end of the first fragment and the hydroxyl group at the 3' end of the second fragment, the first fragment, the second fragment and the template chain together form a double-stranded structure, the first and second fragments together form the target nucleotide chain, the gap between the first and second fragments can be closed under the catalytic action of a double-stranded ligase to form a complete phosphodiester bond, thereby completing the synthesis reaction of the target nucleotide chain.

[0071] Exemplary, if the nucleotide fragment comprises the first, second, and third fragments described above, and the template chain has a linear structure, then the 5' end of the template chain can form a first complementary region in reverse complement to the sequence of the 5' end of the first fragment, the 3' end of the template chain can form a second complementary region in reverse complement to the sequence of the 3' end of the second fragment, the middle region of the template chain (i.e., the region between the 3' and 5' ends) can form a third complementary region in reverse complement to the third fragment, a gap can be formed between the monophosphate group at the 5' end of the first fragment and the hydroxyl group at the 3' end of the third fragment, and a gap can be formed between the monophosphate group at the 5' end of the first fragment and the hydroxyl group at the 3' end of the third fragment. The first, second, and third fragments work together with the template chain to form a double-stranded structure, and the first, second, and third fragments work together to form the target nucleotide chain. The gaps between the first and third fragments, as well as the gaps between the second and third fragments, are closed under the catalytic action of a double-stranded ligase to form a complete phosphodiester bond, thereby completing the synthesis reaction of the target nucleotide chain.

[0072] In some select embodiments of the present disclosure, the template chain has a hairpin structure, and the template chain can form a first complementary region in reverse complement to the 5' end sequence of the first fragment, and the template chain can form a second complementary region in reverse complement to the 3' end sequence of the second fragment. The 5' end of the template chain may contain a monophosphate group and a gap may be formed between the 5' end of the template chain and the 3' end of the first fragment, or the 3' end of the template chain may contain a hydroxyl group and a gap may be formed between the 3' end of the template chain and the 5' end of the second fragment. According to the above configuration, the template chain and the nucleotide fragments can work together to form a double-stranded structure, and the nucleotide fragments can form a target nucleotide chain.

[0073] For example, if "the nucleotide fragment contains only the first and second fragments mentioned above, and the template chain has a hairpin structure," there are two examples as follows:

[0074] Example 1: The template chain can form a first complementary region in reverse complement to the 5' end sequence of the first fragment, and the template chain can form a second complementary region in reverse complement to the 3' end sequence of the second fragment, a gap is formed between the monophosphate group at the 5' end of the first fragment and the hydroxyl group at the 3' end of the second fragment, a monophosphate group is included at the 5' end of the template chain, and a gap is formed between the monophosphate group at the 5' end of the template chain and the hydroxyl group at the 3' end of the first fragment. The first fragment, the second fragment and the template chain work together to form a double-stranded structure, the first and second fragments work together to form the target nucleotide chain, and the gap between the first and second fragments, as well as the gap between the first fragment and the template chain, can be closed under the catalytic action of a double-stranded ligase to form a complete phosphodiester bond, thereby completing the synthesis reaction of the target nucleotide chain.

[0075] Example 2: The template chain can form a first complementary region in reverse complement to the 5' end sequence of the first fragment, and the template chain can form a second complementary region in reverse complement to the 3' end sequence of the second fragment, a gap is formed between the monophosphate group at the 5' end of the first fragment and the hydroxyl group at the 3' end of the second fragment, a hydroxyl group is included at the 3' end of the template chain, and a gap is formed between the hydroxyl group at the 3' end of the template chain and the monophosphate group at the 5' end of the second fragment. The first fragment, the second fragment and the template chain work together to form a double-stranded structure, the first and second fragments work together to form the target nucleotide chain, and the gaps between the first and second fragments, as well as the gap between the second fragment and the template chain, can be closed under the catalytic action of a double-stranded ligase to form a complete phosphodiester bond, thereby completing the synthesis reaction of the target nucleotide chain.

[0076] For example, if "the nucleotide fragment includes the first, second, and third fragments mentioned above, and the template chain has a hairpin structure," there are two examples as follows:

[0077] Example 1: The template chain can form a first complementary region in reverse complement to the 5' end arrangement of the first fragment, the template chain can form a second complementary region in reverse complement to the 3' end arrangement of the second fragment, the third fragment is located between the first and second fragments, a gap is formed between the monophosphate group at the 5' end of the first fragment and the hydroxyl group at the 3' end of the third fragment, a gap is formed between the hydroxyl group at the 3' end of the second fragment and the monophosphate group at the 5' end of the third fragment, and the third fragment and a portion of the template chain form a third complementary region in reverse complement, the template chain contains a monophosphate group at the 5' end, and a gap can be formed between the monophosphate group at the 5' end of the template chain and the hydroxyl group at the 3' end of the first fragment. The first, second, and third fragments work together with the template chain to form a double-stranded structure, and the first, second, and third fragments work together to form the target nucleotide chain. The gaps between the first and third fragments, the second and third fragments, and the first fragment and the template chain are closed under the catalytic action of a double-stranded ligase to form a complete phosphodiester bond, thereby completing the synthesis reaction of the target nucleotide chain.

[0078] Example 2: The template chain can form a first complementary region in reverse complement to the 5' end arrangement of the first fragment, the template chain can form a second complementary region in reverse complement to the 3' end arrangement of the second fragment, the third fragment is located between the first and second fragments, a gap is formed between the monophosphate group at the 5' end of the first fragment and the hydroxyl group at the 3' end of the third fragment, a gap is formed between the hydroxyl group at the 3' end of the second fragment and the monophosphate group at the 5' end of the third fragment, and the third fragment and a portion of the template chain form a third complementary region in reverse complement, the template chain contains a hydroxyl group at the 3' end, and a gap can be formed between the hydroxyl group at the 3' end of the template chain and the monophosphate group at the 5' end of the second fragment. The first, second, and third fragments work together with the template chain to form a double-stranded structure, and the first, second, and third fragments work together to form the target nucleotide chain. The gaps between the first and third fragments, the second and third fragments, and the second fragment and the template chain are also closed under the catalytic action of a double-stranded ligase to form a complete phosphodiester bond, thereby completing the synthesis reaction of the target nucleotide chain.

[0079] In some select embodiments of the present disclosure, when the nucleotide units in a composition for the synthesis of a nucleotide chain consist only of nucleotide fragments (i.e., a first fragment and a second fragment), the first or second fragment has a hairpin structure, and the first and second fragments together form a double-stranded structure. For example, the first fragment has a hairpin structure, the first fragment is inversely complementary to the 3' end sequence of the second fragment, and a gap can be formed between the 5' end of the first fragment and the 3' end of the second fragment, and the nucleotide chain formed by the joining of the first and second fragments is the target nucleotide chain. Alternatively, the second fragment has a hairpin structure, the second fragment is inversely complementary to the 5' end sequence of the first fragment, and a gap can be formed between the 3' end of the second fragment and the 5' end of the first fragment, and the nucleotide chain formed by the joining of the first and second fragments is the target nucleotide chain. According to the above method, the first and second fragments can work together to form a double-stranded structure, and the first and second fragments can bind together to form a target nucleotide chain.

[0080] For example, if the nucleotide unit in the composition for the synthesis of a nucleotide chain consists only of a first and a second fragment, and the first fragment has a hairpin structure, then the 3' end of the second fragment is inversely complementary to the first fragment, a gap is formed between the monophosphate group at the 5' end of the first fragment and the hydroxyl group at the 3' end of the second fragment, and the first and second fragments work together to form a double-stranded structure. The gap between the first and second fragments can be closed under the catalytic action of a double-stranded ligase to form a complete phosphodiester bond, thereby completing the synthesis reaction of the target nucleotide chain, and the nucleotide chain formed by the joining of the first and second fragments is the target nucleotide chain.

[0081] For example, if the nucleotide unit in the composition for the synthesis of a nucleotide chain consists only of a first and a second fragment, and the second fragment has a hairpin structure, then the 5' end of the first fragment is inversely complementary to the second fragment, a gap is formed between the hydroxyl group at the 3' end of the second fragment and the monophosphate group at the 5' end of the first fragment, the first and second fragments work together to form a double-stranded structure, and the gap between the first and second fragments can be closed under the catalytic action of a double-stranded ligase to form a complete phosphodiester bond, thereby completing the synthesis reaction of the target nucleotide chain, and the nucleotide chain formed by the joining of the first and second fragments is the target nucleotide chain.

[0082] In some select embodiments of the present disclosure, the template chain has at least two template fragments, the template fragments are linear in structure, a gap can be formed between at least two template fragments, the template chain can form a first complementary region in reverse complement to the 5' end sequence of the first fragment, the template chain can form a second complementary region in reverse complement to the 3' end sequence of the second fragment, a gap is formed between the 5' end of the first fragment and the 3' end of the second fragment, and the nucleotide fragments form a target nucleotide chain. According to the above configuration, the template chain and the nucleotide fragments can work together to form a double-stranded structure, and the nucleotide fragments can form a target nucleotide chain.

[0083] Exemplary, if the nucleotide fragment comprises the first and second fragments described above, the template fragment is linear in structure, and the template chain has two template fragments, a gap is formed between the two template fragments, the two template fragments can jointly form a template chain, the template chain can form a first complementary region in reverse complement to the 5' end sequence of the first fragment, the template chain can form a second complementary region in reverse complement to the 3' end sequence of the second fragment, a gap is formed between the monophosphate group at the 5' end of the first fragment and the hydroxyl group at the 3' end of the second fragment, and the first fragment, the second fragment and the two template fragments jointly form a double-stranded structure, the first and second fragments can jointly form a target nucleotide chain, and the gap between the first and second fragments, as well as the gap between the two template fragments, can be closed under the catalytic action of a double-stranded ligase to form a complete phosphodiester bond, thereby completing the synthesis reaction of the target nucleotide chain.

[0084] Exemplary, if the nucleotide fragment comprises the first, second, and third fragments described above, the template fragment is linear in structure, and the template chain has two template fragments, then a gap is formed between the two template fragments, the two template fragments together form a template chain, the 5' end of the template chain can form a first complementary region in reverse complement to the sequence of the 5' end of the first fragment, the 3' end of the template chain can form a second complementary region in reverse complement to the sequence of the 3' end of the second fragment, the middle region of the template chain (i.e., the region between the 3' and 5' ends) can form a third complementary region in reverse complement to the third fragment, a gap is formed between the monophosphate group at the 5' end of the first fragment and the hydroxyl group at the 3' end of the third fragment, and a gap is formed between the monophosphate group at the 5' end of the first fragment and the hydroxyl group at the 3' end of the third fragment. The first, second, and third fragments and the two template fragments work together to form a double-stranded structure, and the first, second, and third fragments work together to form the target nucleotide chain. The gaps between the first and third fragments, the second and third fragments, and the two template fragments are also closed under the catalytic action of a double-stranded ligase to form a complete phosphodiester bond, thereby completing the synthesis reaction of the target nucleotide chain.

[0085] In some select embodiments of the present disclosure, the above template fragment comprises one fourth fragment and one fifth fragment, wherein the fourth fragment contains a monophosphate group at its 5' end and the fourth fragment is synthesized in a 3'-to-5' direction, the fifth fragment contains a hydroxyl group at its 3' end and the fifth fragment is synthesized in a 5'-to-3' direction, a gap is formed between the 5' end of the fourth fragment and the 3' end of the fifth fragment, and a gap is formed between the 5' end of the first fragment and the 3' end of the second fragment.

[0086] Compared to not specifying the 3' end group of the template fragment, the 5' end group of the template fragment, and the synthesis direction of the template fragment, the above proposal, which specifies that "the template fragment contains one fourth fragment and one fifth fragment," can further reduce the content of non-target nucleotide chains in the nucleotide chain synthesis product, more effectively increase the purity of the target nucleotide chain in the synthesis product, and contribute to the full exertion of the efficacy of the target nucleotide chain.

[0087] Exemplary, if the nucleotide fragment comprises only the first and second fragments described above, the template fragment has a linear structure, the template chain has two template fragments, and the template fragment has one fourth fragment and one fifth fragment, then a gap is formed between the 5' end of the fourth fragment and the 3' end of the fifth fragment, the fourth and fifth fragments together form a template chain, the template chain can form a first complementary region in reverse complement to the sequence of the 5' end of the first fragment, the template chain can form a second complementary region in reverse complement to the sequence of the 3' end of the second fragment, a gap is formed between the monophosphate group at the 5' end of the first fragment and the hydroxyl group at the 3' end of the second fragment, and the first and second fragments together form a target nucleotide chain. The sequence of the target nucleotide chain and the sequence of the template chain form a double-stranded structure in an inverse complementary manner, and the gaps between the first and second fragments, as well as the gaps between the fourth and fifth fragments, can be closed under the catalytic action of the double-stranded ligase to form a complete phosphodiester bond, thereby completing the synthesis reaction of the target nucleotide chain.

[0088] For example, if "the nucleotide fragment comprises the above-mentioned first, second, and third fragments, the template fragment has a linear structure, the template chain has two template fragments, and the template fragment has one fourth fragment and one fifth fragment," then a gap is formed between the 5' end of the fourth fragment and the 3' end of the fifth fragment, the fourth and fifth fragments together form a template chain, the 5' end of the template chain can form a first complementary region in reverse complement to the sequence of the 5' end of the first fragment, and the 3' end of the template chain is the 3' end of the second fragment The sequence can form a second complementary region in reverse complement to the template chain, the middle region of the template chain (i.e., the region between the 3' and 5' ends) can form a third complementary region in reverse complement to the third fragment, a gap is formed between the monophosphate group at the 5' end of the first fragment and the hydroxyl group at the 3' end of the third fragment, a gap is formed between the monophosphate group at the 5' end of the third fragment and the hydroxyl group at the 3' end of the second fragment, and the first, third, and second fragments work together to form the target nucleotide chain. The sequence of the target nucleotide chain and the sequence of the template chain form a double-stranded structure in reverse complement to each other, and the gaps between the first and third fragments, the second and third fragments, and the fourth and fifth fragments can be closed under the catalytic action of a double-stranded ligase to form a complete phosphodiester bond, thereby completing the synthesis reaction of the target nucleotide chain.

[0089] In some select embodiments of the present disclosure, the nucleotide fragment further comprises a sixth fragment and a seventh fragment, wherein the sixth fragment contains a monophosphate group at its 5' end and is synthesized in a 3'-to-5' direction, and the seventh fragment contains a hydroxyl group at its 3' end and is synthesized in a 5'-to-3' direction. A gap is formed between the 5' end of the sixth fragment and the 3' end of the seventh fragment, and the sixth and seventh fragments together form a first target nucleotide chain. A gap is formed between the 5' end of the first fragment and the 3' end of the second fragment, and the first and second fragments together form a second target nucleotide chain. The first and second target nucleotide chains together form a double-stranded structure.

[0090] In the above proposal, the sixth and seventh fragments jointly form the first target nucleotide chain, and the first and second fragments jointly form the second target nucleotide chain. This further effectively reduces the content of non-target nucleotide chains in the nucleotide chain synthesis product, further effectively increases the purity of the target nucleotide chain in the synthesis product, and contributes to the full exertion of the efficacy of the target nucleotide chain.

[0091] For example, if the nucleotide fragment comprises the first, second, sixth, and seventh fragments, and both complementary nucleotide chains in the double-stranded structure are target nucleotide chains, a gap is formed between the 5' end of the sixth fragment and the 3' end of the seventh fragment, and the sixth and seventh fragments together form the first target nucleotide chain; a gap is formed between the 5' end of the first fragment and the 3' end of the second fragment, and the first and second fragments together form the second target nucleotide chain. The first and second target nucleotide chains serve as templates for each other, and the sequences of the first and second target nucleotide chains form a double-stranded structure in reverse complementarity. The gaps between the first and second fragments, as well as the gaps between the sixth and seventh fragments, can be closed under the catalytic action of a double-stranded ligase to form a complete phosphodiester bond.

[0092] In one embodiment, if "the nucleotide fragment further comprises the sixth and seventh fragments, and also comprises the third fragment," a gap is formed between the 5' end of the sixth fragment and the 3' end of the seventh fragment, and the sixth and seventh fragments together form the first target nucleotide chain. A gap is formed between the 3' end of the third fragment and the 5' end of the first fragment, and a gap is formed between the 5' end of the third fragment and the 3' end of the second fragment, and the first, third, and second fragments together form the second target nucleotide chain. The first and second target nucleotide chains together form a double-stranded structure.

[0093] In the above proposal, the sixth and seventh fragments work together to form the first target nucleotide chain, and the first, third, and second fragments work together to form the second target nucleotide chain. This further effectively reduces the content of non-target nucleotide chains in the synthesized nucleotide chain product, further effectively increases the purity of the target nucleotide chain in the synthesized product, and contributes to the full exertion of the efficacy of the target nucleotide chain.

[0094] For example, if the nucleotide fragment comprises the above-mentioned first, second, third, sixth, and seventh fragments, and both complementary nucleotide chains in the double-stranded structure are target nucleotide chains, then a gap is formed between the 5' end of the sixth fragment and the 3' end of the seventh fragment, and the sixth and seventh fragments together form the first target nucleotide chain, and the 5' end of the first target nucleotide chain can form the first complementary region in reverse complement to the sequence of the 5' end of the first fragment, and the 3' end of the first target nucleotide chain is the second fragment The 3' end sequence of the first fragment can be reverse-complementary to form a second complementary region, the middle region of the first target nucleotide chain (i.e., the region between the 3' and 5' ends) can be reverse-complementary to form a third complementary region with the third fragment, a gap is formed between the monophosphate group at the 5' end of the first fragment and the hydroxyl group at the 3' end of the third fragment, a gap is formed between the monophosphate group at the 5' end of the third fragment and the hydroxyl group at the 3' end of the second fragment, and the first, third, and second fragments together form the second target nucleotide chain. The first and second target nucleotide chains serve as templates for each other, the sequences of the first and second target nucleotide chains form a double-stranded structure in reverse complementarity, and the gaps between the first and third fragments, the gaps between the second and third fragments, and the gaps between the sixth and seventh fragments are closed under the catalytic action of a double-stranded ligase to form a complete phosphodiester bond. As described above, when the nucleotide fragment is DNA, the double-stranded ligase is a DNA double-stranded ligase, and exemplary examples of DNA double-stranded ligases include T4 DNA ligase or Taq DNA ligase.

[0095] As described above, when the nucleotide fragment is RNA, the double-stranded ligase is an RNA double-stranded ligase. Exemplary examples of RNA double-stranded ligases include Rnl2 family ligases and Rnl5 family ligases, such as T4 RNA ligase2 and DraRnl (derived from Naegleria gruberi).

[0096] This disclosure does not limit the specific selection of double-stranded ligases, and they can be selected according to the actual situation. The double-stranded ligase may be a natural double-stranded ligase, a mutant strain produced by enzyme mutation or genetic evolution, or an enzyme having gap-closing activity produced by fusing a congeneral enzyme or / or an enzyme within another family.

[0097] In some select embodiments of this disclosure, the nucleotide fragment comprises 4 to 200 bases, which can relatively effectively reduce the content of non-target nucleotide chains in the synthetic product of the nucleotide chain, thereby effectively increasing the purity of the target nucleotide chain in the synthetic product and contributing to the full efficacy of the target nucleotide chain.

[0098] In one embodiment, the nucleotide fragment contains 4 to 120 bases, which further reduces the content of non-target nucleotide chains in the nucleotide chain synthesis product, effectively increasing the purity of the target nucleotide chain in the synthesis product and contributing to the full exertion of the efficacy of the target nucleotide chain.

[0099] In some optional embodiments of this disclosure, the template chain includes a modifying group.

[0100] Exemplary examples of template chain modifying groups include, but are not limited to, modifying groups on bases, modifying groups on sugar rings, and modifying groups on phosphate groups.

[0101] In other optional embodiments of the present disclosure, the template chain may not contain modifying groups, and the present disclosure is not limited to whether or not the template chain contains modifying groups.

[0102] In some optional embodiments of this disclosure, the nucleotide fragment comprises a modifying group.

[0103] In other optional embodiments of the present disclosure, the nucleotide fragment may not contain a modifying group, and the present disclosure is not limited to whether or not the nucleotide fragment contains a modifying group.

[0104] Exemplary examples of modifying groups on nucleotide fragments include, but are not limited to, modifying groups on bases, modifying groups on sugar rings, and modifying groups on phosphate groups.

[0105] In some select embodiments of the present disclosure, a method for preparing a template chain includes carrying out a synthesis reaction of the template chain according to the target sequence of the template chain, and then not purifying the synthesized product. Because the composition for the synthesis of nucleotide chains according to the present disclosure has a "first fragment synthesized in the direction from 3' to 5' and containing a monophosphate group at the 5' end" and a "second fragment synthesized in the direction from 5' to 3' and containing a hydroxyl group at the 3' end," it is possible to effectively reduce the content of non-target nucleotide chains in the synthesized nucleotide chain product and effectively increase the purity of the target nucleotide chain in the synthesized product without performing a purification treatment after the synthesis of the template chain, thereby contributing to the full exertion of the efficacy of the target nucleotide chain.

[0106] If the protection of a protecting group is involved in the synthesis process of the template chain, a deprotection operation of the protecting group may be performed during the synthesis process.

[0107] In some select embodiments of the present disclosure, a method for preparing a template chain includes carrying out a synthesis reaction of the template chain according to a target sequence of the template chain, and then performing only a desalting treatment on the synthesized product.

[0108] In other optional embodiments of the present disclosure, the method for preparing the template chain may include carrying out a synthesis reaction of the template chain according to the target sequence of the template chain, and then purifying the synthesized product to obtain a template chain of higher purity.

[0109] In this disclosure, the synthesis route of the template chain is not limited, and synthesis may be performed using the H-phosphonate method, the phosphoramidite method, or the enzymatic method, and may be performed using solid-phase synthesis or liquid-phase synthesis.

[0110] In some select embodiments of the present disclosure, a method for preparing nucleotide fragments includes performing a synthesis reaction of nucleotide fragments according to a target sequence of nucleotide fragments, and then not purifying the synthesized product. Because the composition for synthesizing nucleotide chains according to the present disclosure has a "first fragment synthesized in the 3' to 5' direction and containing a monophosphate group at the 5' end" and a "second fragment synthesized in the 5' to 3' direction and containing a hydroxyl group at the 3' end," it is possible to effectively reduce the content of non-target nucleotide chains in the synthesized nucleotide chain product and effectively increase the purity of the target nucleotide chain in the synthesized product without performing a purification treatment after the synthesis of the nucleotide fragments, thereby contributing to the full expression of the efficacy of the target nucleotide chain.

[0111] If the protection of a protecting group is involved in the synthesis process of a nucleotide fragment, a deprotection operation of the protecting group may be performed during the synthesis process.

[0112] In some select embodiments of the present disclosure, a method for preparing nucleotide fragments includes carrying out a synthesis reaction of nucleotide fragments according to a target sequence of nucleotide fragments, and then performing only a desalting treatment on the synthesized product.

[0113] In other optional embodiments of the present disclosure, a method for preparing nucleotide fragments may include performing a synthesis reaction of nucleotide fragments according to a target sequence of nucleotide fragments, and then purifying the synthesized product to obtain a nucleotide fragment of higher purity.

[0114] In this disclosure, the synthesis pathway for nucleotide fragments is not limited, and synthesis may be performed using the H-phosphonate method, the phosphoramidite method, the enzymatic method, and it may be solid-phase synthesis or liquid-phase synthesis.

[0115] Exemplary, in a composition for the synthesis of a nucleotide chain, the concentration of the nucleotide fragment is 1 μM to 100 mM. In the nucleotide fragment, the molar ratio of any two fragments is (0.1:1) to (1:0.1). The concentrations of the nucleotide fragment and the template chain can be adjusted according to the actual circumstances and are not limited in this disclosure.

[0116] Exemplary, in compositions for the synthesis of nucleotide chains, the concentration of double-stranded ligase is 0.01 U / μL to 10 U / μL. The concentration of double-stranded ligase can be adjusted according to the actual circumstances and is not limited to this disclosure.

[0117] In some optional embodiments of the present disclosure, the composition for the synthesis of nucleotide chains further comprises a buffer containing a divalent ion.

[0118] For example, divalent ions include magnesium ions or manganese ions. Buffers include phosphate buffer, Tris buffer, or HEPES buffer. The pH of the buffer is selected based on the pH at which the enzyme used in the composition achieves optimal catalysis. For example, if the double-stranded ligase is T4 RNA Ligase 2, the pH of the buffer should be between 6.5 and 9.0.

[0119] In some optional embodiments of this disclosure, the composition for the synthesis of nucleotide chains further comprises DTT.

[0120] In some select embodiments of the present disclosure, the composition for the synthesis of nucleotide chains further comprises EDTA, the action of which EDTA terminates the synthesis reaction by chelating divalent ions in the buffer.

[0121] For example, PEG may be added to a composition for the synthesis of nucleotide chains to increase the reaction efficiency of the synthesis, a surfactant may be added to improve the stability of the enzyme, or a cofactor such as ATP may be added.

[0122] The composition for synthesizing nucleotide chains may consist only of a template chain, a nucleotide fragment, and a double-stranded ligase. Other substances necessary for the nucleotide synthesis reaction, such as buffers, ATP, DTT, and EDTA, can be added as appropriate during the reaction process.

[0123] This disclosure further provides a method for synthesizing nucleotide chains. The synthesis method includes carrying out a nucleotide chain synthesis reaction using the above-described composition for nucleotide chain synthesis.

[0124] The method for synthesizing nucleotide chains according to this disclosure has as raw materials a "first fragment synthesized in the direction from 3' to 5' and containing a monophosphate group at the 5' end" and a "second fragment synthesized in the direction from 5' to 3' and containing a hydroxyl group at the 3' end." Therefore, by combining the first and second fragments, the content of non-target nucleotide chains (especially n-1 impurities, n-2 impurities, n+1 impurities, and n+2 impurities) in the synthesized nucleotide chain product can be significantly reduced, effectively increasing the purity of the target nucleotide chain in the synthesized product and contributing to the full expression of the efficacy of the target nucleotide chain.

[0125] In the method for synthesizing nucleotide chains according to this disclosure, the composition for synthesizing nucleotide chains includes a template chain, a nucleotide fragment, and a double-stranded ligase. The selection of the template chain, nucleotide fragment, and double-stranded ligase can be found in the above description and is omitted here.

[0126] Exemplary, the nucleotide chain synthesis reaction is carried out at 37°C for approximately 4 hours, and the specific reaction temperature and reaction time can be adjusted according to the actual circumstances and are not limited to those described herein.

[0127] As described above, the addition of EDTA chelates the divalent ions in the buffer, thereby terminating the nucleotide chain synthesis reaction. In other feasible embodiments, the nucleotide chain synthesis reaction may be terminated by heating to the denaturation temperature of the nucleotide chain (e.g., 80°C).

[0128] This disclosure provides a reagent or kit comprising a composition for the synthesis of the nucleotide chain described above.

[0129] The reagents may be in liquid, solid, or semi-solid form, but are not limited to these. The reagents may further include buffers, ATP, MgCl2, DTT, ligases, and the like.

[0130] Ligases include, but are not limited to, T4 RNA Ligase 2 and double-stranded ligases.

[0131] When the nucleotide fragment is DNA, the double-stranded ligase is a DNA double-stranded ligase, and exemplary examples of DNA double-stranded ligases include T4 DNA ligase (T4 DNA Ligase) or Taq DNA Ligase, etc.

[0132] As described above, when the nucleotide fragment is RNA, the double-stranded ligase is an RNA double-stranded ligase. Exemplary examples of RNA double-stranded ligases include Rnl2 family ligases and Rnl5 family ligases, such as T4 RNA ligase2 and DraRnl (derived from Naegleria gruberi).

[0133] In the above proposed technology, the kit further comprises a solid support for the synthesis of nucleotide chains (e.g., 5'-Dimethoxytrityl-N4-acetyl-Cytidine, 2'-TBDMS-3'-succinoyl-long chain alkylamino-CPG), a synthesis column, a diluent, a washing agent, an activator, a deprotection agent, an oxidizing agent, a sulfidating agent, a closure agent, and a reaction termination solution.

[0134] The deprotection agent is a solution of trichloroacetic acid, and the activator is a solution of 5-ethylthiotetrazole (i.e., ETT).

[0135] The oxidizing agent is iodine (I2) solution, the sulfiding agent is phenylacetyl disulfide, and the clotting agent is a mixture of acetic anhydride (Ac2O) and acetonitrile in a volume ratio of 1:4, and / or a mixture of N-methylimidazole, pyridine, and acetonitrile in a volume ratio of 2:3:5. The reaction termination solution is selected from EDTA.

[0136] This disclosure further provides the use of the above-mentioned compositions for the synthesis of nucleotide chains in the synthesis of nucleotide chains.

[0137] To more clearly explain the purpose, technical proposals, and advantages of the examples of this disclosure, the technical proposals in the examples of this disclosure will be described clearly and completely below. Where specific conditions are not specified in the examples, it is possible to perform the procedures under conventional conditions or conditions recommended by the manufacturer. For reagents or instruments where the manufacturer is not specified, commercially available conventional products can be used.

[0138] Example 1 This embodiment provides a method for synthesizing nucleotide chains. The synthesis method includes the following steps.

[0139] (1) Synthesis of nucleotide fragment 1 synthesized in the direction from 3' to 5' A Mermade12 synthesis system was used as the synthesis instrument, and the synthesis scale was 10 μM.

[0140] 5'-Dimethoxytrityl-N4-acetyl-Cytidine and 2'-TBDMS-3'-succinoyl-long-chain alkylamino-CPG, both manufactured by Glen Research, were used as solid phase supports.

[0141] N6-Benzoyl-5'-O-(4,4-dimethoxytrityl)-2'-O-[(tert-butyl)dimethylsilyl]adenosine-3'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (i.e., 5'-DMT-2'-O-TBDMS-A(Bz)-3'-CE-Phosphoramidite), 5'-O-(4,4-dimethoxytrityl)-2'-O-[(tert-butyl)dimethylsilyl]-N2-isobutyrylguanosine-3'-(2-cyanoethyl-N,N-diisopropyl) Pill) phosphoramidite (i.e., 5'-DMT-2'-O-TBDMS-G(iBu)-3'-CE-Phosphoramidite), N4-acetyl-5'-O-(4,4-dimethoxytrityl)-2'-O-[(tert-butyl)dimethylsilyl]cytidine-3'-(2-cyanoethyl-N,N-diisopropyl)phosphoamidite (i.e., 5'-DMT-2'-O-TBDMS-C(Ac)-3'-CE-Phosphoramidite), 5'-O-(4,4-dimethoxytrityl) -2-O-[(tert-butyl)dimethylsilyl]uridine-3-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (i.e., 5'-DMT-2'-O-TBDMS-U-3'-CE-Phosphoramidite), N6-benzoyl-5'-O-(4,4-dimethoxytrityl)-2'-O-methyladenosine-3'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (i.e., 5'-DMT-2'-OMe-A(Bz)-3'-CE-Phosphoram Using 5'-DMT-2'-OMe-G(iBu)-3'-CE-Phosphoramidite (i.e., 5'-DMT-2'-OMe-G(iBu)-3'-CE-Phosphoramidite) and 5'-O-(4,4-dimethoxytrityl)-2'-O-methyl-N2-isobutyrylguanosine-3'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite as 3'-phosphoramidite monomers, each of the above 3'-phosphoramidite monomers was dissolved in anhydrous acetonitrile to obtain corresponding 0.1 M (mol / L) monomer solutions.

[0142] A 3 wt% trichloroacetic acid (i.e., TCA) solution (with dichloromethane as the solvent) was used as a deprotection reagent for dimethoxytrityl (i.e., DMT).

[0143] A 0.25 M solution of 5-ethylthiotetrazole (i.e., ETT) (with acetonitrile as the solvent) was used as an activator.

[0144] A 0.05 M iodine(I2) solution (with a solvent of pyridine and water in a volume ratio of 9:1) was used as the oxidizing agent.

[0145] Phenylacetylisulfide (i.e., PADS) was used as the sulfidation agent.

[0146] A mixture of acetic anhydride (Ac2O) and acetonitrile in a volume ratio of 1:4 was used as closure reagent A.

[0147] A mixture of N-methylimidazole, pyridine, and acetonitrile in a volume ratio of 2:3:5 was used as closure reagent B.

[0148] 20 μmol of the above solid support was placed in a synthesis column, and synthesis was carried out from the 3' end to the 5' end according to the target product sequence (5'-mA*mG*mG*AUGCGCUAAGUAGCGUGCGUUUUAGUACUCUGGAAACAGAAUCUAC-3', SEQ ID NO.1), with each base synthesis completing one cycle. Each cycle included the following steps: 1. 2 mL of the above deprotection reagent was added to the synthesis column for 40 seconds of deprotection, and the system in the synthesis column was washed with acetonitrile. 2. 470 μL of the above activator and 630 μL of the corresponding monomer solution were added to the synthesis column for coupling for 6 minutes, and the system in the synthesis column was washed with acetonitrile. 3. 1.6 mL of oxidizing reagent or 1.6 mL of sulfiding reagent was added to the synthesis column for 3 minutes, and the system in the synthesis column was washed with acetonitrile. 4. Add closure reagent A (1 mL) and closure reagent B (1 mL) to the synthesis column, wait 30 seconds, and then proceed to the next cycle.

[0149] The solid support in the synthesis column was washed with 90 mL of acetonitrile and transferred to a 15 mL centrifuge tube. At 65°C, 10 mL of a mixed solution of aqueous ammonia / 40 wt% aqueous methylamine (V:V=1:1) was added to the centrifuge tube and incubated for 30 minutes. The mixture was centrifuged at 13000 rpm for 3 minutes, the supernatant was collected and dried to obtain the first system. Then, at 25°C, 4 mL of a 12 wt% tetraethylammonium chloride solution (solvent was dimethyl sulfoxide) was added to the first system, and the mixture was sonicated for 1 hour to remove the tert-butyldimethylsilyl group. The precipitate was then washed with 12 mL of n-butanol, further washed with 4 mL of ethanol, and centrifuged at 13000 rpm for 3 minutes to remove the supernatant. The precipitate was dissolved with 1 mL of ultrapure water, and the resulting synthesis product is shown in Table 1.

[0150] [Table 1]

[0151] In Table 1, m represents 2'OMe modification, meaning the 2' position in the nucleoside sugar is modified with a methoxy group, and * represents a phosphorus-sulfur bond.

[0152] (2) Synthesis of nucleotide fragment 1 synthesized in the direction from 5' to 3' A Mermade12 synthesis system was used as the synthesis instrument, and the synthesis scale was 10 μM.

[0153] 3'-O-(4,4-dimethoxytrityl)-N6-benzoyl-2'-O-methyladenosine 5'-long-chain alkylamino CPG (i.e., 3'-Dimethoxytrityl-2'-OMe-A(Bz)-5'-succinoyl-long-chain alkylamino-CPG) was used as the solid phase support.

[0154] N6-Benzoyl-3'-O-(4,4-dimethoxytrityl)-2'-O-[(tert-butyl)dimethylsilyl]adenosine-5'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (i.e., 3'-DMT-2'-O-TBDMS-A(Bz)-5'-CE-Phosphoramidite), 3'-O-(4,4-dimethoxytrityl)-2'-O-[(tert-butyl)dimethylsilyl]-N2-I Sobutyrylguanosine-5'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (i.e., 3'-DMT-2'-O-TBDMS-G(iBu)-5'-CE-Phosphoramidite), N4-acetyl-3'-O-(4,4-dimethoxytrityl)-2'-O-[(tert-butyl)dimethylsilyl]cytidine-5'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (i.e., 3'-DMT-2'-O-TBDMS-C(Ac)-5'-CE-Phosphoramidite), 3'-O-(4,4-dimethoxytrityl)-2'-O-[(tert-butyl)dimethylsilyl]uridine-5'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (i.e., 3'-DMT-2'-O-TBDMS-U-5'-CE-Phosphoramidite), 3'-O-(4,4-dimethoxytri (Cyl)-2'-O-methyl-N2-isobutyrylguanosine-3'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (i.e., 3'-DMT-2'-OMe-iBu-G-5'-CE-Phosphoramidite) was selected as the 5'-phosphoramidite monomer, and each of the above 5'-phosphoramidite monomers was dissolved in anhydrous acetonitrile to obtain the corresponding 0.1 M monomer solution.

[0155] A 3 wt% trichloroacetic acid (i.e., TCA) solution (with dichloromethane as the solvent) was used as a deprotection reagent for dimethoxytrityl (i.e., DMT).

[0156] A 0.25 M solution of 5-ethylthiotetrazole (i.e., ETT) (with acetonitrile as the solvent) was used as an activator.

[0157] A 0.05 M iodine (I2) solution (the solvent being a mixture of pyridine and water in a volume ratio of 9:1) was used as the oxidizing agent.

[0158] Phenylacetylisulfide (i.e., PADS) was used as the sulfurizing agent.

[0159] A mixture of acetic anhydride (Ac2O) and acetonitrile in a volume ratio of 1:4 was used as closure reagent A.

[0160] A mixture of N-methylimidazole, pyridine, and acetonitrile in a volume ratio of 2:3:5 was used as closure reagent B.

[0161] 20 μmol of the above solid support was placed in a synthesis column, and synthesis was carried out from the 5' end to the 3' end according to the target product sequence (5'-mA*mG*mG*AUGCGCUAAGUAGCGUGCGUUUUAGUACUCUGGAAACAGAAUCUAC-3', SEQ ID NO.1), with each base synthesis completing one cycle. Each cycle included the following steps: 1. 2 mL of the above deprotection reagent was added to the synthesis column for 40 seconds to deprotect it, and the system in the synthesis column was washed with acetonitrile. 2. 470 μL of the above activator and 630 μL of the corresponding monomer solution were added to the synthesis column for 6 minutes to perform coupling, and the system in the synthesis column was washed with acetonitrile. 3. 1.6 mL of oxidizing reagent or 1.6 mL of sulfiding reagent was added to the synthesis column and treated for 3 minutes, and the system in the synthesis column was washed with acetonitrile. 4. Add closure reagent A (1 mL) and closure reagent B (1 mL) to the synthesis column, wait 30 seconds, and then proceed to the next cycle.

[0162] The solid support in the synthesis column was washed with 90 mL of acetonitrile and transferred to a 15 mL centrifuge tube. At 65°C, 10 mL of a mixed solution of aqueous ammonia / 40 wt% aqueous methylamine (V:V, 1:1) was added to the centrifuge tube and incubated for 30 minutes. The mixture was centrifuged at 13000 rpm for 3 minutes, the supernatant was collected and dried to obtain the first system. At 25°C, 4 mL of a 12 wt% tetraethylammonium chloride solution (solvent was dimethyl sulfoxide) was added to the first system, and the mixture was sonicated for 1 hour to remove the tert-butyldimethylsilyl group. The precipitate was then washed with 12 mL of n-butanol, further washed with 4 mL of ethanol, and centrifuged at 13000 rpm for 3 minutes to remove the supernatant. The precipitate was dissolved with 1 mL of ultrapure water, and the resulting synthesis product is shown in Table 2.

[0163] [Table 2]

[0164] In Table 2, m represents 2'-OMe modification, meaning the 2' position in the nucleoside sugar is modified with a methoxy group, and * represents a phosphorus-sulfur bond.

[0165] (3) Synthesis of nucleotide fragment 2 synthesized in the direction from 3' to 5' The synthesis of nucleotide fragment 2, synthesized in the 3' to 5' direction, is essentially the same as in step (1), with the following differences: Only [3'-O-(4,4-dimethoxytrityl)-4,4'-dicarboxyethyl]propyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (i.e., [3-(4,4'-Dimethoxytrityloxy)-4,4-dicarboxyethyl]propyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite) was selected as the 3'-phosphoramidite monomer.

[0166] The resulting synthetic products are shown in Table 3.

[0167] [Table 3]

[0168] In Table 3, m represents a 2'OMe modification, i.e., the 2' position in the nucleoside sugar is modified with a methoxy group, * represents a phosphorus-sulfur bond, and p represents a monophosphate group.

[0169] (4) Synthesis of nucleotide fragment 2 synthesized in the direction from 5' to 3' The synthesis of nucleotide fragment 2, synthesized in the 5' to 3' direction, is essentially the same as in step (2), with the following differences: 3'-O-(4,4'-Dimethoxytrityloxy)-4,4'(dicarboxymethylamido)propyl-1-O-succinoyl-long chain alkylamino-CPG, manufactured by Glen Research, was used as the solid support.

[0170] The resulting synthetic products are shown in Table 4.

[0171] [Table 4]

[0172] In Table 4, m represents a 2'OMe modification, i.e., the 2' position in the nucleoside sugar is modified with a methoxy group, * represents a phosphorus-sulfur bond, and p represents a monophosphate group.

[0173] (5) Synthesis of template chain 1 synthesized in the direction from 3' to 5' A Mermade12 synthesis system was used as the synthesis instrument, and the synthesis scale was 10 μM.

[0174] (5'-Dimethoxytrityl-N-benzoyl-2'-deoxythymidine-3'-succinoyl-long-chain alkylamino-CPG), manufactured by Glen Research, was used as the solid phase support.

[0175] N6-benzoyl-5'-O-(4,4-dimethoxytrityl)-2'-deoxyadenosine-3'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (i.e., 5'-DMT-dA(Bz)-3'-CE-Phosphoramidite), 5'-O-(4,4-dimethoxytrityl)-N2-isobutyryl-2'-deoxyguanosine-3'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (i.e., 5'-DMT-dG(iBu)- Using 3'-CE-Phosphoramidite), N4-acetyl-5'-O-(4,4-dimethoxytrityl)-2'-deoxycytidine-3'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (i.e., 5'-DMT-dC(Ac)-3'-CE-Phosphoramidite), and 5'-O-(4,4-dimethoxytrityl)-2'-deoxythymidine-3-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (i.e., 5'-DMT-dT-3'-CE-Phosphoramidite) as 3'-phosphoramidite monomers, each of the above 3'-phosphoramidite monomers was dissolved in anhydrous acetonitrile to obtain the corresponding 0.1 M monomer solutions.

[0176] A 3 wt% trichloroacetic acid (i.e., TCA) solution (with dichloromethane as the solvent) was used as a deprotection reagent for dimethoxytrityl (i.e., DMT).

[0177] A 0.25 M solution of 5-ethylthiotetrazole (i.e., ETT) (with acetonitrile as the solvent) was used as an activator.

[0178] A 0.05 M iodine(I2) solution (with a solvent of pyridine and water in a volume ratio of 9:1) was used as the oxidizing agent.

[0179] Phenylacetylisulfide (i.e., PADS) was used as the sulfidation agent.

[0180] A mixture of acetic anhydride (Ac2O) and acetonitrile in a volume ratio of 1:4 was used as closure reagent A.

[0181] A mixture of N-methylimidazole, pyridine, and acetonitrile in a volume ratio of 2:3:5 was used as closure reagent B.

[0182] 20 μmol of the above solid support was placed in a synthesis column, and synthesis was carried out from the 3' end to the 5' end according to the target product sequence (3'-TGAGACCTTTGTCTTAGATGATTTTGTTCCGTTTTACGGA-5', SEQ ID NO. 15), with each base synthesis completing one cycle. Each cycle included the following steps: 1. 2 mL of the above deprotection reagent was added to the synthesis column for 40 seconds of deprotection, and the system in the synthesis column was washed with acetonitrile. 2. 470 μL of the above activator and 630 μL of the corresponding monomer solution were added to the synthesis column for 6 minutes of coupling, and the system in the synthesis column was washed with acetonitrile. 3. 1.6 mL of oxidizing reagent or 1.6 mL of sulfiding reagent was added to the synthesis column and treated for 3 minutes, and the system in the synthesis column was washed with acetonitrile. 4. 1 mL of the above closure reagent A and 1 mL of closure reagent B were added to the synthesis column, and after 30 seconds, the next cycle began.

[0183] The arrangement of template chain 1 is as follows:

[0184] 3'-TGAGACCTTTGTCTTAGATGATTTTGTTCCGTTTTACGGA-5', SEQ ID NO.15.

[0185] (6) Synthesis reaction of nucleotide chains In a tris buffer at pH 8.0, 400 μM ATP, 2 mM MgCl2, 1 mM DTT, 200 μM template strand 1, 200 μM nucleotide fragment 1 (synthesized in the 3'-5' direction or the 5'-3' direction), and 200 μM nucleotide fragment 2 (synthesized in the 3'-5' direction or the 5'-3' direction) were added. T4 RNA Ligase 2 (final concentration in the reaction mixture was 0.3 U / μL) was added, and the reaction was carried out at 37°C for 4 hours. After that, EDTA (final concentration in the reaction mixture was 3 mM) was added to terminate the reaction and obtain the synthesized product.

[0186] As shown in Table 5, there are four specific reactions.

[0187] [Table 5]

[0188] Example 2 This embodiment provides a method for synthesizing nucleotide chains. The synthesis method includes the following steps.

[0189] (1) Synthesis of nucleotide fragment 5 synthesized in the direction from 3' to 5' The synthesis steps are the same as in step (1) of Example 1, and the resulting synthetic products are shown in Table 6.

[0190] [Table 6] In Table 6, m represents 2'OMe modification, i.e., the 2' position in the nucleoside sugar is modified with a methoxy group; * represents a phosphorus-sulfur bond; and f represents 2'F modification, i.e., the 2' position in the nucleoside sugar is modified with fluorine.

[0191] (2) Synthesis of nucleotide fragment 5 synthesized in the direction from 5' to 3' The synthesis steps are the same as in step (2) of Example 1, and the resulting synthetic products are shown in Table 7.

[0192] [Table 7]

[0193] In Table 7, m represents 2'OMe modification, i.e., the 2' position in the nucleoside sugar is modified with a methoxy group; * represents a phosphorus-sulfur bond; and f represents 2'F modification, i.e., the 2' position in the nucleoside sugar is modified with fluorine.

[0194] (3) Synthesis of nucleotide fragment 6 synthesized in the direction from 3' to 5' The synthesis steps are the same as in step (1) of Example 1, and the resulting synthetic products are shown in Table 8.

[0195] [Table 8]

[0196] In Table 8, m represents 2'OMe modification, i.e., the 2' position in the nucleoside sugar is modified with a methoxy group; p represents a monophosphate group; and f represents 2'F modification, i.e., the 2' position in the nucleoside sugar is modified with fluorine.

[0197] (4) Synthesis of nucleotide fragment 6 synthesized in the direction from 5' to 3' The synthesis steps are the same as in step (2) of Example 1, and the resulting synthetic products are shown in Table 9.

[0198] [Table 9]

[0199] In Table 9, m represents 2'OMe modification, i.e., the 2' position of the nucleoside sugar is modified with a methoxy group, p represents a monophosphate group, and f represents 2'F modification, i.e., the 2' position of the nucleoside sugar is modified with fluorine.

[0200] (5) Synthesis of nucleotide fragment 7 synthesized in the direction from 3' to 5' The synthesis steps are the same as in step (1) of Example 1, and the resulting synthetic products are shown in Table 10.

[0201] [Table 10]

[0202] In Table 10, m represents 2'OMe modification, i.e., the 2' position in the nucleoside sugar is modified with a methoxy group; * represents a phosphorus-sulfur bond; p represents a monophosphate group; and f represents 2'F modification, i.e., the 2' position in the nucleoside sugar is modified with fluorine.

[0203] (6) Synthesis of nucleotide fragment 7 synthesized in the direction from 5' to 3' The synthesis steps are the same as in step (2) of Example 1, and the resulting synthetic products are shown in Table 11.

[0204] [Table 11]

[0205] In Table 11, m represents 2'OMe modification, i.e., the 2' position in the nucleoside sugar is modified with a methoxy group; * represents a phosphorus-sulfur bond; p represents a monophosphate group; and f represents 2'F modification, i.e., the 2' position in the nucleoside sugar is modified with fluorine.

[0206] (7) Synthesis of template chain 2 synthesized in the direction from 3' to 5' A Mermade12 synthesis system was used as the synthesis instrument, and the synthesis scale was 10 μM.

[0207] 5'-Dimethoxytrityl-2'-deoxythymidine-3'-succinoyl-long-chain alkylamino-CPG, manufactured by Glen Research, was used as the solid phase support.

[0208] N6-Benzoyl-5'-O-(4,4-dimethoxytrityl)-2'-deoxyadenosine-3'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (i.e., 5'-DMT-dA(Bz)-3'-CE-Phosphoramidite), 5'-O-(4,4-dimethoxytrityl)-N2-isobutyryl-2'-deoxyguanosine-3'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (i.e., 5'-DMT-dG(iBu)-3'-CE-Phosphoramidite), N4-Acetyl-5'-O-(4,4-dimethoxytrityl)-2'-de Oxycytidine-3'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (i.e., 5'-DMT-dC(Ac)-3'-CE-Phosphoramidite) and 5'-O-(4,4-dimethoxytrityl)-2'-deoxythymidine-3-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (i.e., 5'-DMT-dT-3'-CE-Phosphoramidite) were used as 3'phosphoramidite monomers. Each of the above 3'phosphoramidite monomers was dissolved in anhydrous acetonitrile to obtain the corresponding 0.1 M monomer solutions.

[0209] A 3 wt% trichloroacetic acid (i.e., TCA) solution (with dichloromethane as the solvent) was used as a deprotection reagent for dimethoxytrityl (i.e., DMT).

[0210] A 0.25 M solution of 5-ethylthiotetrazole (i.e., ETT) (with acetonitrile as the solvent) was used as an activator.

[0211] A 0.05 M iodine(I2) solution (with a solvent of pyridine and water in a volume ratio of 9:1) was used as the oxidizing agent.

[0212] Phenylacetylisulfide (i.e., PADS) was used as the sulfidation agent.

[0213] A mixture of acetic anhydride (Ac2O) and acetonitrile in a volume ratio of 1:4 was used as closure reagent A.

[0214] A mixture of N-methylimidazole, pyridine, and acetonitrile in a volume ratio of 2:3:5 was used as closure reagent B.

[0215] 20 μmol of the above solid support was placed in a synthesis column, and synthesis was carried out from the 3' end to the 5' end according to the target product sequence (3'- TGTTTTCGTTTTGTCCAGATC-5', i.e., 5'-CTAGACCTGTTTTGCTTTTGT-3' SEQ ID NO. 16), with each base synthesis completing one cycle. Each cycle included the following steps: 1. 2 mL of the above deprotection reagent was added to the synthesis column for 40 seconds of deprotection, and the system in the synthesis column was washed with acetonitrile. 2. 470 μL of the above activator and 630 μL of the corresponding monomer solution were added to the synthesis column for coupling for 6 minutes, and the system in the synthesis column was washed with acetonitrile. 3. 1.6 mL of oxidizing reagent or 1.6 mL of sulfiding reagent was added to the synthesis column and treated for 3 minutes, and the system in the synthesis column was washed with acetonitrile. 4. 1 mL of the above closure reagent A and 1 mL of closure reagent B were added to the synthesis column, and after 30 seconds, the next cycle began.

[0216] The arrangement of template chain 2 is as follows:

[0217] 3'-TGTTTTCGTTTTGTCCAGATC-5', i.e., 5'-CTAGACCTGTTTTGCTTTTGT-3', SEQ ID NO.16.

[0218] (8) Synthesis reactions of nucleotide chains In tris buffer at pH 8.0, 400 μM ATP, 2 mM MgCl2, 1 mM DTT, 200 μM template strand 2, 200 μM nucleotide fragment 5 (synthesized in the 3'-to-5' direction or 5'-to-3' direction), 200 μM nucleotide fragment 6 (synthesized in the 3'-to-5' direction or 5'-to-3' direction), and 200 μM nucleotide fragment 7 (synthesized in the 3'-to-5' direction or 5'-to-3' direction) were added. T4 RNA Ligase 2 (final concentration in the reaction mixture was 0.5 U / μL) was added, and the reaction was carried out at 37°C for 5 hours. After that, EDTA (final concentration in the reaction mixture was 3 mM) was added to terminate the reaction and obtain the synthesized product.

[0219] As shown in Table 12, there are eight specific reactions.

[0220] [Table 12]

[0221] Example 3 This embodiment provides a method for synthesizing nucleotide chains. The synthesis method includes the following steps.

[0222] (1) Synthesis of nucleotide fragment 8 synthesized in the direction from 3' to 5' The synthesis steps are the same as in step (1) of Example 1, and the resulting synthetic products are shown in Table 13.

[0223] [Table 13]

[0224] In Table 13, m represents 2'OMe modification, i.e., the 2' position in the nucleoside sugar is modified with a methoxy group, p represents a monophosphate group, and GalNAc represents N-acetylgalactosamine.

[0225] (2) Synthesis of nucleotide fragment 8 synthesized in the direction from 5' to 3' The synthesis steps are the same as in step (2) of Example 1, and the resulting synthetic products are shown in Table 14.

[0226] [Table 14]

[0227] In Table 14, m represents 2'OMe modification, i.e., the 2' position in the nucleoside sugar is modified with a methoxy group, p represents a monophosphate group, and GalNAc represents N-acetylgalactosamine.

[0228] (3) Synthesis of nucleotide fragment 9 synthesized in the direction from 3' to 5' The synthesis steps are the same as in step (1) of Example 1, and the resulting synthetic products are shown in Table 15.

[0229] [Table 15]

[0230] In Table 15, m represents a 2'OMe modification, i.e., the 2' position in the nucleoside sugar is modified with a methoxy group; * represents a phosphorus-sulfur bond; d represents a deoxyribonucleotide, i.e., the 2' position in the nucleoside sugar is hydrogen; and f represents a 2'F modification, i.e., the 2' position in the nucleoside sugar is modified with fluorine.

[0231] (4) Synthesis of nucleotide fragment 9 synthesized in the direction from 5' to 3' The synthesis steps are the same as in step (2) of Example 1, and the resulting synthetic products are shown in Table 16.

[0232] [Table 16]

[0233] In Table 16, m represents a 2'OMe modification, i.e., the 2' position in the nucleoside sugar is modified with a methoxy group; * represents a phosphorus-sulfur bond; d represents a deoxyribonucleotide, i.e., the 2' position in the nucleoside sugar is hydrogen; and f represents a 2'F modification, i.e., the 2' position in the nucleoside sugar is modified with fluorine.

[0234] (5) Synthesis reaction of nucleotide chains In tris buffer at pH 8.0, there is a mixture containing 400 μM ATP, 2 mM MgCl2, 1 mM DTT, 100 μM nucleotide fragment 5 prepared in Example 2 (synthesized in the 3'-5' direction or 5'-3' direction), 100 μM nucleotide fragment 6 prepared in Example 2 (synthesized in the 3'-5' direction or 5'-3' direction), 100 μM nucleotide fragment 7 prepared in Example 2 (synthesized in the 3'-5' direction or 5'-3' direction), 100 μM nucleotide fragment 8 (synthesized in the 3'-5' direction or 5'-3' direction), and 100 μM nucleotide fragment 9 (synthesized in the 3'-5' direction or 5'-3' direction), along with T4 RNA. Ligase 2 (final concentration in the reaction solution was 0.4 U / μL) was added, and the reaction was carried out at 37°C for 4 hours. Then, EDTA (final concentration in the reaction solution was 3 mM) was added to terminate the reaction and obtain the synthetic product.

[0235] As shown in Table 17, there are eight specific reactions.

[0236] [Table 17]

[0237] Experimental Example 1 Liquid chromatography-mass spectrometry (LC-MS) was used to analyze each of the synthetic products corresponding to the four reactions shown in Table 5 of Example 1, and the analysis results are shown in Table 18.

[0238] [Table 18]

[0239] As can be seen from Table 18, the content of n-1, n-2, and n-3 impurities is lowest in the synthetic product corresponding to reaction 3. In other words, when nucleotide fragment 1, which has a hydroxyl group at the 3' end, is synthesized in the direction from 5' to 3' and nucleotide fragment 2, which has a monophosphate group at the 5' end, is synthesized in the direction from 3' to 5', the content of impurities in the synthetic product of the nucleotide chain can be effectively reduced.

[0240] Experimental Example 2 Liquid chromatography-mass spectrometry (LC-MS) was used to analyze each of the eight synthetic products corresponding to the reactions shown in Table 12 of Example 2, and the analysis results are shown in Table 19.

[0241] [Table 19]

[0242] As can be seen from Table 19, the content of n-1, n-2, and n-3 impurities is lowest in the synthetic products corresponding to reactions 9 and 11. In other words, when nucleotide fragment 5, which has a hydroxyl group at the 3' end, is synthesized in the direction from 5' to 3', and nucleotide fragment 7, which has a monophosphate group at the 5' end, is synthesized in the direction from 3' to 5', the content of impurities in the synthetic product of the nucleotide chain can be effectively reduced.

[0243] Experimental Example 3 Liquid chromatography-mass spectrometry (LC-MS) was used to analyze each of the eight synthetic products corresponding to the reactions shown in Table 17 of Example 3, and the analysis results are shown in Table 20.

[0244] [Table 20]

[0245] As can be seen from Table 20, the content of n-1, n-2, and n-3 impurities is lowest in the synthetic product corresponding to reaction 14 (i.e., one of the two target nucleotide chains was a nucleotide chain formed by the linkage of nucleotide fragment 8 and nucleotide fragment 9, and the other was a nucleotide chain formed by the linkage of nucleotide fragment 5, nucleotide fragment 6, and nucleotide fragment 7). In other words, when "nucleotide fragment 8, which has a monophosphate group at the 5' end, is synthesized in the direction from 3' to 5' and nucleotide fragment 9, which has a hydroxyl group at the 3' end, is synthesized in the direction from 5' to 3'", and "nucleotide fragment 5, which has a hydroxyl group at the 3' end, is synthesized in the direction from 5' to 3' and nucleotide fragment 7, which has a monophosphate group at the 5' end, is synthesized in the direction from 3' to 5'", the content of impurities in the synthetic product of the two complementary target nucleotide chains can be effectively reduced.

[0246] As described above, the composition for the synthesis of nucleotide chains according to this disclosure can significantly reduce the content of non-target nucleotide chains in the synthesized nucleotide chain product, effectively increase the purity of the target nucleotide chain in the synthesized product, and contribute to the full exertion of the efficacy of the target nucleotide chain.

[0247] The embodiments described above are only a selection of the embodiments of this disclosure, not all embodiments. The detailed description of the embodiments of this disclosure is only a selection of embodiments and does not limit the scope of the scope of protection of this disclosure. All other embodiments that a person skilled in the art could obtain without using their inventive ability based on the embodiments of this disclosure are also within the scope of protection of this disclosure. [Industrial applicability]

[0248] The composition for the synthesis of nucleotide chains according to this disclosure can significantly reduce the content of non-target nucleotide chains in the synthesized nucleotide chain product, effectively increase the purity of the target nucleotide chain in the synthesized product, and contribute to the full exertion of the efficacy of the target nucleotide chain.

Claims

1. A composition for the synthesis of nucleotide chains, It contains nucleotide fragments and double-stranded ligases, The nucleotide fragment is for forming a double-stranded structure, the double-stranded ligase is a ligase capable of closing the gap in the double-stranded structure, and at least one of the strands of the double-stranded structure is a target nucleotide strand. The nucleotide fragment comprises a first fragment and a second fragment, wherein the first fragment contains a monophosphate group at its 5' end and the first fragment is synthesized in a 3' to 5' direction, and the second fragment contains a hydroxyl group at its 3' end and the second fragment is synthesized in a 5' to 3' direction. A composition for the synthesis of nucleotide chains characterized by the following.

2. The nucleotide fragment further comprises a third fragment, the third fragment having a hydroxyl group at its 3' end and a monophosphate group at its 5' end. The third fragment is optionally synthesized in the direction from 3' to 5', or the third fragment is synthesized in the direction from 5' to 3'. The composition for the synthesis of nucleotide chains according to feature 1.

3. The composition for the synthesis of the nucleotide chain further comprises a template chain, wherein the template chain and the nucleotide fragments are for jointly forming the double-stranded structure, and the nucleotide fragments are for forming the target nucleotide chain. A composition for the synthesis of nucleotide chains according to claim 1 or 2.

4. The template chain has a linear structure, and the template chain can form a first complementary region in the opposite complementary relationship to the arrangement of the 5' end of the first fragment, and the template chain can form a second complementary region in the opposite complementary relationship to the arrangement of the 3' end of the second fragment, and the gap can be formed between the 5' end of the first fragment and the 3' end of the second fragment. The composition for the synthesis of nucleotide chains according to feature 3.

5. The template chain has a hairpin structure, and the template chain can form a first complementary region in inverse complement to the arrangement of the 5' end of the first fragment, and the template chain can form a second complementary region in inverse complement to the arrangement of the 3' end of the second fragment. The template chain contains a monophosphate group at its 5' end, and the gap can be formed between the 5' end of the template chain and the 3' end of the first fragment, or the template chain contains a hydroxyl group at its 3' end, and the gap can be formed between the 3' end of the template chain and the 5' end of the second fragment. The composition for the synthesis of nucleotide chains according to feature 3.

6. The first fragment has a hairpin structure, the first fragment is inversely complementary to the 3' end sequence of the second fragment, the gap can be formed between the 5' end of the first fragment and the 3' end of the second fragment, and the nucleotide chain formed by the joining of the first and second fragments is the target nucleotide chain. Alternatively, the second fragment may have a hairpin structure, be inversely complementary to the 5' end sequence of the first fragment, and be able to form the gap between the 3' end of the second fragment and the 5' end of the first fragment, and the nucleotide chain formed by the joining of the first and second fragments may be the target nucleotide chain. The composition for the synthesis of nucleotide chains according to feature 1.

7. The template chain has at least two template fragments, the template fragments have a linear structure, and the gap can be formed between at least two of the template fragments. The template chain is capable of forming a first complementary region in reverse complement to the 5' end sequence of the first fragment, the template chain is capable of forming a second complementary region in reverse complement to the 3' end sequence of the second fragment, the gap is capable of forming the gap between the 5' end of the first fragment and the 3' end of the second fragment, and the nucleotide fragment is for forming the target nucleotide chain. Optionally, the template fragment comprises one fourth fragment and one fifth fragment, wherein the fourth fragment contains a monophosphate group at its 5' end and is synthesized in a 3'-to-5' direction, the fifth fragment contains a hydroxyl group at its 3' end and is synthesized in a 5'-to-3' direction, the gap is formed between the 5' end of the fourth fragment and the 3' end of the fifth fragment, and the gap is formed between the 5' end of the first fragment and the 3' end of the second fragment. The composition for the synthesis of nucleotide chains according to feature 3.

8. The nucleotide fragment further comprises a sixth fragment and a seventh fragment, wherein the sixth fragment contains a monophosphate group at its 5' end and is synthesized in a 3'-to-5' direction, and the seventh fragment contains a hydroxyl group at its 3' end and is synthesized in a 5'-to-3' direction. The gap is formed between the 5' end of the sixth fragment and the 3' end of the seventh fragment, and the sixth and seventh fragments together form the first target nucleotide chain. The gap is formed between the 5' end of the first fragment and the 3' end of the second fragment, and the first and second fragments together form a second target nucleotide chain. The first target nucleotide chain and the second target nucleotide chain are for the purpose of jointly forming the double-stranded structure. A composition for the synthesis of a nucleotide chain according to any one of claims 1 to 7.

9. The nucleotide fragment further comprises a sixth fragment and a seventh fragment, wherein the sixth fragment contains a monophosphate group at its 5' end and is synthesized in a 3'-to-5' direction, and the seventh fragment contains a hydroxyl group at its 3' end and is synthesized in a 5'-to-3' direction. The gap is formed between the 5' end of the sixth fragment and the 3' end of the seventh fragment, and the sixth and seventh fragments together form the first target nucleotide chain. The gap is formed between the 3' end of the third fragment and the 5' end of the first fragment, and the gap is formed between the 5' end of the third fragment and the 3' end of the second fragment, and the first fragment, the third fragment and the second fragment are to work together to form a second target nucleotide chain. The first target nucleotide chain and the second target nucleotide chain are for the joint formation of the double-stranded structure. A composition for the synthesis of nucleotide chains according to any one of claims 2 to 7.

10. If the nucleotide fragment is DNA, the double-stranded ligase is a DNA double-stranded ligase, and the DNA double-stranded ligase includes T4 DNA ligase; if the nucleotide fragment is RNA, the double-stranded ligase is an RNA double-stranded ligase, and the RNA double-stranded ligase includes Rnl2 family ligase and Rnl5 family ligase. or / and the nucleotide fragment comprises 4 to 200 bases, or / and the composition for the synthesis of the nucleotide chain further comprises a buffer containing magnesium ions, and optionally the nucleotide fragment comprises 4 to 120 bases. A composition for the synthesis of a nucleotide chain according to any one of claims 1 to 9.

11. The nucleotide fragment may or may not contain a modifying group. or / and the template chain may or may not contain modifying groups. A composition for the synthesis of a nucleotide chain according to any one of claims 3 to 5 or 7.

12. The method for preparing the template chain includes carrying out a synthesis reaction of the template chain according to the target sequence of the template chain, and then not performing any purification treatment on the synthesized product. OR / AND, the method for preparing the nucleotide fragment includes carrying out a synthesis reaction of the nucleotide fragment according to the target sequence of the nucleotide fragment, and then not performing any purification treatment on the synthesized product. A composition for the synthesis of a nucleotide chain according to any one of claims 3 to 5 or 7.

13. The method includes carrying out a nucleotide chain synthesis reaction using the composition for nucleotide chain synthesis described in any one of claims 1 to 12. A method for synthesizing nucleotide chains characterized by the following:

14. The composition for the synthesis of nucleotide chains according to any one of claims 1 to 12 includes A reagent or kit characterized by the following features.

15. Use of the composition for the synthesis of nucleotide chains according to any one of claims 1 to 12 in the synthesis of nucleotide chains.

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