Composition for synthesizing nucleotide chain and synthesis method for nucleotide chain

Abstract

A composition for synthesizing a nucleotide chain and a synthesis method for a nucleotide chain, which belong to the technical field of nucleotides. The composition for synthesizing a nucleotide chain comprises a nucleotide fragment and a double-stranded ligase. The nucleotide fragment is used for forming a double-stranded structure; the double-stranded ligase is a ligase capable of sealing a nick in the double-stranded structure; and at least one strand of the double-stranded structure is a target nucleotide chain. The nucleotide fragment comprises a first fragment and a second fragment; the 5' end of the first fragment contains a monophosphate group, and the first fragment is synthesized in the 3' to 5' direction; and the 3' end of the second fragment contains a hydroxyl group, and the second fragment is synthesized in the 5' to 3' direction.

Description

Composition for synthesizing nucleotide chains and method for synthesizing nucleotide chains

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application number 202311627360.X filed with the Chinese Patent Office on November 30, 2023, entitled “Composition for synthesizing nucleotide chains and method for synthesizing nucleotide chains”, the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present disclosure relates to the technical field of nucleotide synthesis, and in particular, to a composition for synthesizing nucleotide chains and a method for synthesizing nucleotide chains. Background Art

[0004] Currently, nucleotide chain synthesis typically involves sequentially extending nucleotide residues one base at a time to produce the target nucleotide chain. Because chemical nucleotide chain synthesis reactions cannot achieve 100% efficiency, the purity and yield of the synthesized target nucleotide chain decrease as the length of the target nucleotide chain increases, resulting in the inclusion of a certain amount of non-target nucleotide chains whose lengths do not match the target nucleotide chain.

[0005] For nucleotide chains less than or equal to 20mer in length, after chemical synthesis, the proportion of the final target nucleotide chain in the synthetic product can be increased through purification processes (e.g., ion columns or reverse columns, etc.); however, since the length of some non-target nucleotide chains is relatively close to that of the target nucleotide chain (e.g., the length of the non-target nucleotide chain is only 1mer or 2mer different from that of the target nucleotide chain), the properties of these non-target nucleotide chains are very similar to those of the target nucleotide chain, resulting in the inability to effectively separate and remove these non-target nucleotide chains from the synthetic product. For nucleotide chains longer than 20mer, especially those longer than 50mer, and even those longer than 100mer, after chemical synthesis, more non-target nucleotide chains cannot be effectively separated and removed from the synthetic product.

[0006] The presence of non-target nucleotide chains in the synthetic product will affect the function and activity of the target nucleotide chain, especially for antisense chain drugs, RNAi drugs, nucleic acid aptamer drugs and sgRNA drugs, the presence of non-target nucleotide chains will seriously affect the efficacy of the target nucleotide chain. Summary of the Invention

[0007] The purpose of the present disclosure is to provide a composition for synthesizing nucleotide chains and a method for synthesizing nucleotide chains, which can reduce the content of non-target nucleotide chains in the synthesis product of the nucleotide chain to improve the purity of the target nucleotide chain in the synthesis product.

[0008] In a first aspect, the present disclosure provides a composition for synthesizing nucleotide chains, comprising: nucleotide fragments and a double-stranded ligase; wherein the nucleotide fragments are used to form a double-stranded structure; the double-stranded ligase is a ligase that can close the gap in the double-stranded structure; and at least one chain in the double-stranded structure is a target nucleotide chain; the nucleotide fragments include a first fragment and a second fragment; the 5' end of the first fragment contains a monophosphate group, and the first fragment is synthesized in a 3' to 5' direction; the 3' end of the second fragment contains a hydroxyl group, and the second fragment is synthesized in a 5' to 3' direction.

[0009] In the composition for synthesizing nucleotide chains provided by the present disclosure, the nucleotide fragments include 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 fragment and the second fragment can cooperate with each other and jointly form a double-stranded structure, which can significantly reduce the content of non-target nucleotide chains in the synthetic product of the nucleotide chain, thereby effectively improving the purity of the target nucleotide chain in the synthetic product, and thus facilitating full utilization of the efficacy of the target nucleotide chain.

[0010] In combination with the first aspect, in an optional embodiment of the present disclosure, the nucleotide fragment further includes a third fragment, the 3' end of the third fragment contains a hydroxyl group, and the 5' end of the third fragment contains a monophosphate group.

[0011] Alternatively, the third segment is synthesized in a 3' to 5' direction, or the third segment is synthesized in a 5' to 3' direction.

[0012] In combination with the first aspect, in an optional embodiment of the present disclosure, the composition for synthesizing a nucleotide chain further includes a template chain, and the template chain and the nucleotide fragments are used to form a double-stranded structure together; and the nucleotide fragments are used to form a target nucleotide chain.

[0013] In combination with the first aspect, in an optional embodiment of the present disclosure, the template chain is a linear structure; the template chain can be reverse complementary to the sequence at the 5' end of the first fragment to form a first complementary region; the template chain can be reverse complementary to the sequence at the 3' end of the second fragment to form a second complementary region; a gap can be formed between the 5' end of the first fragment and the 3' end of the second fragment.

[0014] In the above technical solution, the template chain and the nucleotide fragments can form a double-stranded structure, and the nucleotide fragments can form the target nucleotide chain.

[0015] In conjunction with the first aspect, in an optional embodiment of the present disclosure, the template strand has a hairpin structure; the template strand can be reverse complementary to the sequence at the 5' end of the first fragment, forming a first complementary region; the template strand can be reverse complementary to the sequence at the 3' end of the second fragment, forming a second complementary region. The 5' end of the template strand contains a monophosphate group, and a gap can be formed between the 5' end of the template strand and the 3' end of the first fragment; or the 3' end of the template strand contains a hydroxyl group, and a gap can be formed between the 3' end of the template strand and the 5' end of the second fragment.

[0016] In the above technical solution, the template chain and the nucleotide fragments can form a double-stranded structure, and the nucleotide fragments can form the target nucleotide chain.

[0017] In conjunction with the first aspect, in an optional embodiment of the present disclosure, the first fragment is a hairpin structure; the first fragment can be reverse complementary to the sequence at the 3' end 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; the nucleotide chain after the first and second fragments are connected is the target nucleotide chain. Alternatively, the second fragment is a hairpin structure; the second fragment can be reverse complementary to the sequence at the 5' end 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; the nucleotide chain after the first and second fragments are connected is the target nucleotide chain.

[0018] In the above technical solution, the first fragment and the second fragment can form a double-stranded structure together, and the first fragment and the second fragment can form a target nucleotide chain after being connected.

[0019] In combination with 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 structures, and a gap can be formed between at least two template fragments; the template chain can be reverse complementary to the sequence at the 5' end of the first fragment to form a first complementary region; the template chain can be reverse complementary to the sequence at the 3' end of the second fragment to form a second complementary region; 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 used to form a target nucleotide chain.

[0020] In the above technical solution, the template chain and the nucleotide fragments can form a double-stranded structure together, and the nucleotide fragments can form the target nucleotide chain.

[0021] Optionally, the template fragment includes a fourth fragment and a fifth fragment; the 5' end of the fourth fragment contains a monophosphate group, and the fourth fragment is synthesized in a 3' to 5' direction; the 3' end of the fifth fragment contains a hydroxyl group, 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.

[0022] In combination with the first aspect, in an optional embodiment of the present disclosure, the nucleotide fragment further includes a sixth fragment and a seventh fragment; the 5' end of the sixth fragment contains a monophosphate group, and the sixth fragment is synthesized in a 3' to 5' direction; the 3' end of the seventh fragment contains a hydroxyl group, and the seventh fragment 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 fragment and the seventh fragment 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 fragment and the second fragment together form a second target nucleotide chain. The first target nucleotide chain and the second target nucleotide chain are used to jointly form a double-stranded structure.

[0023] In the above technical solution, the sixth fragment and the seventh fragment together form the first target nucleotide chain, and the first fragment and the second fragment can together form the second target nucleotide chain, which can further effectively reduce the content of non-target nucleotide chains in the synthetic product of the nucleotide chain, so as to further effectively improve the purity of the target nucleotide chain in the synthetic product, thereby facilitating full utilization of the efficacy of the target nucleotide chain.

[0024] In combination with the first aspect, in an optional embodiment of the present disclosure, the nucleotide fragment further includes a sixth fragment and a seventh fragment; the 5' end of the sixth fragment contains a monophosphate group, and the sixth fragment is synthesized in a 3' to 5' direction; the 3' end of the seventh fragment contains a hydroxyl group, and the seventh fragment 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 fragment and the seventh fragment 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, and a 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 used to jointly form a second target nucleotide chain. The first target nucleotide chain and the second target nucleotide chain are used to jointly form a double-stranded structure.

[0025] In the above technical solution, the sixth fragment and the seventh fragment together form the first target nucleotide chain, and the first fragment, the third fragment and the second fragment can together form the second target nucleotide chain, which can further effectively reduce the content of non-target nucleotide chains in the synthetic product of the nucleotide chain, so as to further effectively improve the purity of the target nucleotide chain in the synthetic product, thereby facilitating full utilization of the efficacy of the target nucleotide chain.

[0026] In conjunction with the first aspect, in an optional embodiment of the present disclosure, when the nucleotide fragment is DNA, the double-strand ligase is a DNA double-strand ligase; the DNA double-strand ligase includes T4 DNA ligase; when the nucleotide fragment is RNA, the double-strand ligase is an RNA double-strand ligase; the RNA double-strand ligase includes Rnl2 family ligases and Rnl5 family ligases. Alternatively, or alternatively, the nucleotide fragment contains 4 to 200 bases. Alternatively, or alternatively, the composition for synthesizing a nucleotide chain further includes a buffer containing magnesium ions.

[0027] Optionally, the nucleotide fragment contains 4 to 120 bases.

[0028] In combination with the first aspect, in an optional embodiment of the present disclosure, the nucleotide fragment contains or does not contain a modification group; or / and, the template chain contains or does not contain a modification group.

[0029] In combination with the first aspect, in an optional embodiment of the present disclosure, the method for preparing a template chain includes: performing a reaction to synthesize the template chain according to the target sequence of the template chain, and then not purifying the synthesized product; or / and, the method for preparing a nucleotide fragment includes: performing a reaction to synthesize the nucleotide fragment according to the target sequence of the nucleotide fragment, and then not purifying the synthesized product.

[0030] In the above technical solution, even if the template chain and nucleotide fragments are not purified after synthesis, the content of non-target nucleotide chains in the synthetic product of the nucleotide chain can be greatly reduced, thereby effectively improving the purity of the target nucleotide chain in the synthetic product, which is conducive to fully exerting the efficacy of the target nucleotide chain.

[0031] In a second aspect, the present disclosure provides a method for synthesizing a nucleotide chain, which comprises: performing a nucleotide chain synthesis reaction using the composition for synthesizing a nucleotide chain provided in any one of the first aspects above.

[0032] The method for synthesizing a nucleotide chain provided by the present disclosure comprises a first fragment synthesized in a 3' to 5' direction and containing a monophosphate group at the 5' end, and a second fragment synthesized in a 5' to 3' direction and containing a hydroxyl group at the 3' end, in the synthetic raw materials. The first fragment and the second fragment can cooperate with each other to form a double-stranded structure, which can significantly reduce the content of non-target nucleotide chains in the synthetic product of the nucleotide chain, thereby effectively improving the purity of the target nucleotide chain in the synthetic product, thereby facilitating full utilization of the efficacy of the target nucleotide chain.

[0033] In a third aspect, the present disclosure provides a reagent or kit comprising: a composition for synthesizing a nucleotide chain provided in any one of the first aspects above.

[0034] The form of the reagent includes but is not limited to liquid, solid or semi-solid. The reagent also includes buffer, ATP, MgCl2, DTT, ligase, etc.

[0035] Ligase includes but is not limited to T4 RNA Ligase2 and double-stranded ligase.

[0036] When the nucleotide fragment is DNA, the double-strand ligase is DNA double-strand ligase; illustratively, the DNA double-strand ligase includes T4 DNA ligase (T4 DNA Ligase) or Taq DNA Ligase.

[0037] As described above, when the nucleotide fragment is RNA, the double-stranded ligase is an RNA double-stranded ligase; illustratively, the RNA double-stranded ligase includes Rnl2 family ligases and Rnl5 family ligases, for example, T4 RNA ligase2 and DraRnl (from Naegleria gruberi).

[0038] In the above technical solution, the kit also includes a solid phase support for the synthesis of nucleotide chains (such as 5'-Dimethoxytrityl-N4-acetyl-Cytidine, 2'-TBDMS-3'-succinoyl-long chain alkylamino-CPG), a synthesis column, a diluent, a detergent, an activator, a deprotection reagent, an oxidizing reagent, a sulfurizing reagent, a blocking reagent and a termination reaction solution, etc.

[0039] The deprotection reagent is trichloroacetic acid solution, and the activating agent is 5-ethylthiotetrazole (ETT) solution.

[0040] The oxidizing agent is iodine (I2) solution; the sulfiding agent is phenylacetyl disulfide; the blocking 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.

[0041] The reaction termination solution is selected from EDTA.

[0042] In a fourth aspect, the present disclosure provides use of the composition for synthesizing a nucleotide chain provided in any one of the first aspects in synthesizing a nucleotide chain. Beneficial effects

[0043] In the composition for synthesizing nucleotide chains provided by the present disclosure, the nucleotide fragments include 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 fragment and the second fragment can cooperate with each other and jointly form a double-stranded structure, which can significantly reduce the content of non-target nucleotide chains in the synthetic product of the nucleotide chain, thereby effectively improving the purity of the target nucleotide chain in the synthetic product, and thus facilitating full utilization of the efficacy of the target nucleotide chain. DETAILED DESCRIPTION

[0044] The embodiments of the present disclosure will be described in detail below with reference to the examples. However, those skilled in the art will appreciate that the following examples are intended only to illustrate the present disclosure and should not be construed as limiting the scope of the present disclosure. Where specific conditions are not specified in the examples, the experiments were performed under conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, all are commercially available conventional products.

[0045] In this disclosure, a nucleotide chain refers to a polymer formed by the condensation of two or more nucleotide monomers through a phosphodiester bond; a phosphodiester bond is typically formed by the hydroxyl group on the 3' carbon of the sugar ring of a nucleoside and the 5' phosphate of another nucleoside monomer. In this disclosure, a nucleotide chain refers to a nucleotide chain having 100 or fewer bases, 200 or fewer bases, or 500 or fewer bases. This disclosure does not limit the specific number of bases in a nucleotide chain.

[0046] In the present disclosure, "synthesis from 3' to 5' direction" means that the synthesis direction of the nucleotide fragment is from the 3' end of the nucleotide fragment to the 5' end of the nucleotide fragment; "synthesis from 5' to 3' direction" means that the synthesis direction of the nucleotide fragment is from the 5' end of the nucleotide fragment to the 3' end of the nucleotide fragment.

[0047] In the present disclosure, for the synthesis method using the 3' to 5' direction, n-1 impurity refers to: a nucleotide product with one base missing relative to the 5' end of the target synthesis product, n-2 impurity refers to: a nucleotide product with two bases missing relative to the 5' end of the target synthesis product, n-3 impurity refers to: a nucleotide product with three bases missing relative to the 5' end of the target synthesis product, nx impurity refers to: a nucleotide product with x bases missing relative to the 5' end of the target synthesis product, n+1 impurity refers to: a nucleotide product with one base extra relative to the 5' end of the target synthesis product, and n+2 impurity refers to: a nucleotide product with two bases extra relative to the 5' end of the target synthesis product.

[0048] In the present disclosure, for the synthesis method using the 5' to 3' direction, n-1 impurity refers to: a nucleotide product with one base missing relative to the 3' end of the target synthesis product, n-2 impurity refers to: a nucleotide product with two bases missing relative to the 3' end of the target synthesis product, n-3 impurity refers to: a nucleotide product with three bases missing relative to the 3' end of the target synthesis product, nx impurity refers to: a nucleotide product with x bases missing relative to the 3' end of the target synthesis product; n+1 impurity refers to: a nucleotide product with one base extra relative to the 3' end of the target synthesis product, and n+2 impurity refers to: a nucleotide product with two bases extra relative to the 3' end of the target synthesis product.

[0049] In the present disclosure, the template chain refers to a nucleotide chain that is partially or completely reverse complementary to a nucleotide fragment; the template chain can be a DNA chain or an RNA chain, and can contain or not contain a modification group.

[0050] In the present disclosure, a double-stranded structure refers to: double-stranded DNA with reverse complementary sequences, double-stranded RNA with reverse complementary sequences, or a double-stranded structure formed by single-stranded RNA and single-stranded DNA with reverse complementary sequences.

[0051] In the present disclosure, at least one of the double-stranded chains is a target nucleotide chain, which means that both of the two nucleotide chains with reverse complementary sequences may be target nucleotide chains, or only one of them may be a target nucleotide chain.

[0052] In the present disclosure, a gap refers to a position where a phosphodiester bond is missing between two adjacent nucleotide units (which can be a nucleotide fragment or a template chain) in a double-stranded structure; a complete phosphodiester bond can be formed at the gap by catalysis of a double-stranded ligase, that is, the 3' terminal hydroxyl group of one nucleotide unit at the gap and the 5' monophosphate group of another nucleotide unit at the gap are catalyzed by the double-stranded ligase to form a complete phosphodiester bond.

[0053] In the present disclosure, gap sealing 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 the present disclosure, a phosphate donor refers to a nucleotide unit (which can be a nucleotide fragment or a template strand) with a monophosphate group at its 5' end; a hydroxyl acceptor refers to a nucleotide unit (which can be a nucleotide fragment or a template strand) with a hydroxyl group at its 3' end. Under the catalysis of a double-stranded ligase, the monophosphate group at the 5' end of the phosphate donor and the hydroxyl group at the 3' end of the hydroxyl acceptor can form a complete phosphodiester bond. The phosphate donor can be a modified phosphate donor, for example, a thioated or halogenated phosphate donor.

[0055] In the present disclosure, double-stranded ligase refers to an enzyme that has a sealing effect on the gap in the double-stranded structure, that is, an enzyme that can form a complete phosphodiester bond at the gap in the double-stranded structure.

[0056] The synthesis of nucleotide chains is generally carried out by sequentially extending nucleotide residues one base at a time to prepare the target nucleotide chain, but the efficiency of the chemical synthesis reaction of nucleotide chains cannot reach 100%.

[0057] For the synthesis of DNA chains, the efficiency of synthesizing each base of the DNA chain is roughly 99.0% to 99.6% based on the phosphoramidite principle. Calculated based on the median of 99.3%, when synthesizing a 10mer DNA chain, the target DNA chain accounts for 93% of the synthesized product; when synthesizing a 20mer DNA chain, the target DNA chain accounts for 87% of the synthesized product; when synthesizing a 40mer DNA chain, the target DNA chain accounts for 76% of the synthesized product; and when synthesizing an 80mer DNA chain, the target DNA chain accounts for 57% of the synthesized product. For the synthesis of RNA chains, the phosphoramidite principle is used for synthesis, and the efficiency of synthesizing each base of the RNA chain is approximately 98.0%. When synthesizing an RNA chain with a length of 10mer, the target RNA chain accounts for 82% of the synthetic product; when synthesizing an RNA chain with a length of 20mer, the target RNA chain accounts for 67% of the synthetic product; when synthesizing an RNA chain with a length of 40mer, the target RNA chain accounts for 45% of the synthetic product; and when synthesizing an RNA chain with a length of 80mer, the target RNA chain accounts for 20% of the synthetic product.

[0058] Among the synthetic products of the nucleotide chain, in addition to the target nucleotide chain (i.e., full-length product), non-target nucleotide chains (i.e., non-full-length products, i.e., non-target nucleotide chains whose length does not match the target nucleotide chain) are usually non-full-length truncated nucleotide fragments such as n-1 impurities, n-2 impurities, n-3 impurities, etc., and there are also small amounts of n+1 impurities and n+2 impurities.

[0059] For nucleotide chains less than or equal to 20mers, after chemical synthesis, the proportion of the final target nucleotide chain in the synthetic product can be increased through purification processes (e.g., ion columns or reverse columns, etc.); however, the n-1 impurities and n-2 impurities in the synthetic product cannot be effectively separated and removed from the synthetic product because their properties are too similar to those of the target nucleotide chain. For nucleotide chains longer than 20mers, especially those longer than 50mers, or even those longer than 100mers, after chemical synthesis, during the purification process of the synthetic product, in addition to the n-1 impurities, n-2 impurities, n+1 impurities, and n+2 impurities, n-3 impurities, n-4 impurities, n-5 impurities, and even n-6 impurities, n-7 impurities, n-8 impurities, n-9 impurities, n-10 impurities, or longer truncated nucleotide fragments cannot be effectively separated and removed from the synthetic product. The presence of non-target nucleotide chains in the synthetic products often affects the function and activity of the target nucleotide chains, especially for antisense chain drugs, RNAi drugs, nucleic acid aptamer drugs and sgRNA drugs, the presence of non-target nucleotide chains will seriously affect the efficacy of the target nucleotide chains.

[0060] To this end, the present disclosure provides a composition for synthesizing nucleotide chains, which composition includes: nucleotide fragments and double-stranded ligase; wherein the nucleotide fragments are used to form a double-stranded structure; the double-stranded ligase is a ligase that can close the gap in the double-stranded structure; and at least one chain in the double-stranded structure is a target nucleotide chain; the nucleotide fragments include a first fragment and a second fragment; the 5' end of the first fragment contains a monophosphate group, and the first fragment is synthesized in a 3' to 5' direction; the 3' end of the second fragment contains a hydroxyl group, and the second fragment is synthesized in a 5' to 3' direction.

[0061] In the composition for synthesizing nucleotide chains provided above, the nucleotide fragments include "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"; in the process of carrying out the synthesis reaction of the nucleotide chain using the composition for synthesizing nucleotide chains provided above, the first fragment acts as a phosphate donor and the second fragment acts as a hydroxyl acceptor, and the nucleotide fragment containing the first fragment and the second fragment can form a double-stranded structure, a gap can be generated in the double-stranded structure, and the gap can be closed under the catalysis of a double-stranded ligase to complete the synthesis reaction of the target nucleotide chain; the above-mentioned synthesis reaction can significantly reduce the content of non-target nucleotide chains (especially n-1 impurities, n-2 impurities, n+1 impurities and n+2 impurities) in the synthesis product of the nucleotide chain, so as to effectively improve the purity of the target nucleotide chain in the synthesis product, thereby facilitating full utilization of the efficacy of the target nucleotide chain.

[0062] It is understood that when the nucleotide fragment is DNA, the double-stranded ligase that performs gap sealing is DNA double-stranded ligase; when the nucleotide fragment is RNA, the double-stranded ligase that performs gap sealing is RNA double-stranded ligase.

[0063] It can be understood that the first fragment and the second fragment can be DNA at the same time, the first fragment and the second fragment can be RNA at the same time, the first fragment can be DNA and the second fragment can be RNA, or the first fragment can be RNA and the second fragment can be DNA, as long as the first fragment and the second fragment form a double-stranded structure together.

[0064] In some optional embodiments of the present disclosure, the nucleotide fragment may include, in addition to the aforementioned first and second fragments, a third fragment, wherein the 3' end of the third fragment contains a hydroxyl group and the 5' end of the third fragment contains a monophosphate group.

[0065] When the nucleotide fragments include the first fragment, the second fragment and the third fragment at the same time, the first fragment, the second fragment and the third fragment are used to jointly form a double-stranded structure, which can also significantly reduce the content of non-target nucleotide chains (especially n-1 impurities, n-2 impurities, n+1 impurities and n+2 impurities) in the synthetic product of the nucleotide chain, thereby effectively improving the purity of the target nucleotide chain in the synthetic product, which is conducive to fully exerting the efficacy of the target nucleotide chain.

[0066] In some optional embodiments of the present disclosure, the third segment is synthesized in a 3' to 5' direction; or, the third segment is synthesized in a 5' to 3' direction. When the nucleotide segment includes the first segment, the second segment, and the third segment, the third segment is synthesized in a 3' to 5' direction or a 5' to 3' direction. Both of these can significantly reduce the content of non-target nucleotide chains (particularly n-1 impurities, n-2 impurities, n+1 impurities, and n+2 impurities) in the synthetic product of the nucleotide chain, thereby effectively improving the purity of the target nucleotide chain in the synthetic product, thereby facilitating full utilization of the efficacy of the target nucleotide chain.

[0067] In some optional embodiments of the present disclosure, the nucleotide fragment may also have other fragments. As long as the nucleotide fragment has at least one first fragment and at least one second fragment, and all the fragments in the nucleotide fragment can together form a double-stranded structure, it is possible to significantly reduce the content of non-target nucleotide chains (especially n-1 impurities, n-2 impurities, n+1 impurities and n+2 impurities) in the synthetic product of the nucleotide chain, thereby effectively improving the purity of the target nucleotide chain in the synthetic product, which is conducive to fully exerting the efficacy of the target nucleotide chain.

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

[0069] In some optional embodiments of the present disclosure, the template strand has a linear structure; the template strand can be reverse complementary to the sequence at the 5' end of the first fragment, forming a first complementary region; and the template strand can be reverse complementary to the sequence at the 3' end of the second fragment, forming a second complementary region; and a gap can be formed between the 5' end of the first fragment and the 3' end of the second fragment. This approach allows the template strand and the nucleotide fragments to form a double-stranded structure, and the nucleotide fragments can form the target nucleotide strand.

[0070] As an example, when "the nucleotide fragment only includes the aforementioned first fragment and second fragment, and the template chain is a straight-chain structure", the 5' end of the template chain can be reverse complementary to the sequence of the 5' end of the first fragment to form a first complementary region; the 3' end of the template chain can be reverse complementary to the sequence of the 3' end of the second fragment to form a second complementary region; 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 template chain together form a double-stranded structure, the first fragment and the second fragment together form a target nucleotide chain, and the gap between the first fragment and the second fragment can be closed under the catalysis of the double-stranded ligase to form a complete phosphodiester bond, thereby completing the synthesis reaction of the target nucleotide chain.

[0071] As an example, when "the nucleotide fragments simultaneously include the aforementioned first fragment, second fragment and third fragment, and the template chain is a linear structure", the 5' end of the template chain can be reverse complementary to the sequence of the 5' end of the first fragment to form a first complementary region; the 3' end of the template chain can be reverse complementary to the sequence of the 3' end of the second fragment to form a second complementary region; the middle region of the template chain (i.e., the region between the 3' end and the 5' end) can be reverse complementary to the third fragment to form a third complementary region; 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 third fragment and the hydroxyl group at the 3' end of the second fragment. The first fragment, the second fragment and the third fragment together form a double-stranded structure with the template chain. The first fragment, the second fragment and the third fragment together form a target nucleotide chain. The gap between the first fragment and the third fragment and the gap between the second fragment and the third fragment can both be closed under the catalysis of the double-stranded ligase to form a complete phosphodiester bond, thereby completing the synthesis reaction of the target nucleotide chain.

[0072] In some optional embodiments of the present disclosure, the template chain is a hairpin structure; the template chain can be reverse complementary to the sequence at the 5' end of the first fragment to form a first complementary region; the template chain can be reverse complementary to the sequence at the 3' end of the second fragment to form a second complementary region. 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. The above method can achieve that the template chain and the nucleotide fragment can form a double-stranded structure together, and the nucleotide fragment can form a target nucleotide chain.

[0073] As an example, when "the nucleotide fragment only includes the aforementioned first fragment and second fragment, and the template chain is a hairpin structure", there are the following two examples:

[0074] Example 1: The template chain can be reverse complementary to the sequence at the 5' end of the first fragment to form a first complementary region, and the template chain can be reverse complementary to the sequence at the 3' end of the second fragment to form a second complementary region, 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 second fragment; the 5' end of the template chain contains a monophosphate group, and the monophosphate group at the 5' end of the template chain can form a gap with the hydroxyl group at the 3' end of the first fragment; the first fragment, the second fragment and the template chain together form a double-stranded structure, and the first fragment and the second fragment together form a target nucleotide chain, and the gap between the first fragment and the second fragment and the gap between the first fragment and the template chain can be closed under the catalysis of the 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 be reverse complementary to the sequence at the 5' end of the first fragment to form a first complementary region, and the template chain can be reverse complementary to the sequence at the 3' end of the second fragment to form a second complementary region, 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 second fragment; the 3' end of the template chain contains a hydroxyl group, and the hydroxyl group at the 3' end of the template chain can form a gap with the monophosphate group at the 5' end of the second fragment; the first fragment, the second fragment and the template chain together form a double-stranded structure, and the first fragment and the second fragment together form a target nucleotide chain, and the gap between the first fragment and the second fragment and the gap between the second fragment and the template chain can be closed under the catalysis of the double-stranded ligase to form a complete phosphodiester bond, thereby completing the synthesis reaction of the target nucleotide chain.

[0076] As an example, when "the nucleotide fragment includes the aforementioned first fragment, second fragment, and third fragment at the same time, and the template chain is a hairpin structure", there are the following two examples:

[0077] Example 1: The template strand can be reverse complementary to the sequence at the 5' end of the first fragment, forming a first complementary region. The template strand can be reverse complementary to the sequence at the 3' end of the second fragment, forming a second complementary region. 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, and 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. The third fragment is reverse complementary to a portion of the template strand, forming a third complementary region. The 5' end of the template strand contains a monophosphate group, and the monophosphate group at the 5' end of the template strand can form a gap with the hydroxyl group at the 3' end of the first fragment. The first, second, and third fragments together with the template strand form a double-stranded structure. The first, second, and third fragments together form a target nucleotide chain. The gaps between the first and third fragments, the gap between the second and third fragments, and the gap between the first fragment and the template strand can all be sealed under the catalysis of a double-stranded ligase to form complete phosphodiester bonds, thereby completing the synthesis reaction of the target nucleotide chain.

[0078] Example 2: The template strand can be reverse complementary to the sequence at the 5' end of the first fragment, forming a first complementary region. The template strand can be reverse complementary to the sequence at the 3' end of the second fragment, forming a second complementary region. 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 also 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. The third fragment is reverse complementary to a portion of the template strand, forming a third complementary region. The 3' end of the template strand contains a hydroxyl group, and the hydroxyl group at the 3' end of the template strand can form a gap with the monophosphate group at the 5' end of the second fragment. The first, second, and third fragments together with the template strand form a double-stranded structure. The first, second, and third fragments together form a target nucleotide chain. The gaps between the first and third fragments, the gap between the second and third fragments, and the gap between the second fragment and the template strand can all be sealed under the catalysis of a double-stranded ligase to form complete phosphodiester bonds, thereby completing the synthesis reaction of the target nucleotide chain.

[0079] In some optional embodiments of the present disclosure, when the nucleotide unit in the composition for synthesizing a nucleotide chain has only nucleotide fragments (i.e., the first fragment and the second fragment), the first fragment or the second fragment can be a hairpin structure, and the first fragment and the second fragment together form a double-stranded structure. For example, the first fragment is a hairpin structure; the first fragment can be reverse complementary to the sequence of the 3' end of the second fragment, and the 5' end of the first fragment can form a gap with the 3' end of the second fragment; the nucleotide chain after the first fragment and the second fragment are connected is the target nucleotide chain. Or, the second fragment is a hairpin structure; the second fragment can be reverse complementary to the sequence of the 5' end of the first fragment, and the 3' end of the second fragment can form a gap with the 5' end of the first fragment; the nucleotide chain after the first fragment and the second fragment are connected is the target nucleotide chain. The above method can achieve that the first fragment and the second fragment can form a double-stranded structure together, and the first fragment and the second fragment can form a target nucleotide chain after connection.

[0080] As an example, when "the nucleotide unit in the composition for synthesizing a nucleotide chain has only a first fragment and a second fragment, and the first fragment is a hairpin structure", the 3' end of the second fragment is reverse complementary to the first 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 second fragment. The first fragment and the second fragment together form a double-stranded structure. The gap between the first fragment and the second fragment can be closed under the catalysis of a double-stranded ligase to form a complete phosphodiester bond, thereby completing the synthesis reaction of the target nucleotide chain. The nucleotide chain after the first fragment and the second fragment are connected is the target nucleotide chain.

[0081] As an example, when "the nucleotide unit in the composition for synthesizing a nucleotide chain has only a first fragment and a second fragment, and the second fragment is a hairpin structure", the 5' end of the first fragment is reverse complementary to the second fragment, and 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 fragment and the second fragment together form a double-stranded structure. The gap between the first fragment and the second fragment can be closed under the catalysis of a double-stranded ligase to form a complete phosphodiester bond, thereby completing the synthesis reaction of the target nucleotide chain. The nucleotide chain after the first fragment and the second fragment are connected is the target nucleotide chain.

[0082] In some optional embodiments of the present disclosure, the template strand comprises at least two template fragments, the template fragments are linear structures, and a gap may be formed between at least two template fragments; the template strand may be reverse complementary to the sequence at the 5' end of the first fragment, forming a first complementary region; the template strand may be reverse complementary to the sequence at the 3' end of the second fragment, forming a second complementary region; a gap may be formed between the 5' end of the first fragment and the 3' end of the second fragment; and the nucleotide fragments are used to form a target nucleotide strand. The above approach enables the template strand and the nucleotide fragments to form a double-stranded structure, and the nucleotide fragments can form the target nucleotide strand.

[0083] As an example, when "the nucleotide fragment only includes the aforementioned first fragment and second fragment, the template fragment is a straight-chain structure, and the template chain has two template fragments", a gap is formed between the two template fragments, and the two template fragments together form a template chain; the template chain can be reverse complementary to the sequence at the 5' end of the first fragment to form a first complementary region, and the template chain can be reverse complementary to the sequence at the 3' end of the second fragment to form a second complementary region; 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 together form a double-stranded structure, and the first fragment and the second fragment together form a target nucleotide chain, and the gap between the first fragment and the second fragment and the gap between the two template fragments can be closed under the catalysis of double-stranded ligase to form a complete phosphodiester bond, thereby completing the synthesis reaction of the target nucleotide chain.

[0084] As an example, when "the nucleotide fragment includes the aforementioned first fragment, second fragment and third fragment at the same time, the template fragment is a linear structure, and the template chain has two template fragments", a gap is formed between the two template fragments, and the two template fragments together form a template chain; the 5' end of the template chain can be reverse complementary to the sequence of the 5' end of the first fragment to form a first complementary region, and the 3' end of the template chain can be reverse complementary to the sequence of the 3' end of the second fragment to form a second complementary region; the middle region of the template chain (i.e., the region between the 3' end and the 5' end) can be reverse complementary to the third fragment to form a third complementary region; the 5' end of the first fragment can be reverse complementary to the sequence of the 3' end of the second fragment to form a third complementary region. A gap is formed between the monophosphate group at the 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, the first fragment, the second fragment, the third fragment and the two template fragments together form a double-stranded structure, the first fragment, the second fragment and the third fragment together form a target nucleotide chain, the gap between the first fragment and the third fragment, the gap between the second fragment and the third fragment, and the gap between the two template fragments can all be closed under the catalysis of the double-stranded ligase to form a complete phosphodiester bond, thereby completing the synthesis reaction of the target nucleotide chain.

[0085] In some optional embodiments of the present disclosure, the aforementioned template fragment includes a fourth fragment and a fifth fragment; the 5' end of the fourth fragment contains a monophosphate group, and the fourth fragment is synthesized in a 3' to 5' direction; the 3' end of the fifth fragment contains a hydroxyl group, 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 with the situation where the group at the 3' end, the group at the 5' end and the synthesis direction of the template fragment are not limited, the above-mentioned scheme of limiting "the template fragment includes a fourth fragment and a fifth fragment" is beneficial to further reduce the content of non-target nucleotide chains in the synthetic product of the nucleotide chain, so as to further effectively improve the purity of the target nucleotide chain in the synthetic product, and thus help to fully exert the efficacy of the target nucleotide chain.

[0087] As an example, when "the nucleotide fragment only includes the aforementioned first fragment and second fragment, the template fragment is a linear structure, the template chain has two template fragments, and the template fragment has a fourth fragment and a fifth fragment", a gap is formed between the 5' end of the fourth fragment and the 3' end of the fifth fragment, and the fourth fragment and the fifth fragment together form the template chain; the template chain can be reverse complementary to the sequence at the 5' end of the first fragment to form a first complementary region, and the template chain can be reverse complementary to the sequence at the 3' end of the second fragment to form a second complementary region; 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 and the second fragment are used to jointly form a target nucleotide chain; the sequence of the target nucleotide chain is reverse complementary to the sequence of the template chain to form a double-stranded structure, and the gap between the first fragment and the second fragment and the gap between the fourth fragment and the fifth fragment can be closed under the catalysis of the double-stranded ligase to form a complete phosphodiester bond, thereby completing the synthesis reaction of the target nucleotide chain.

[0088] As an example, when "the nucleotide fragment includes the aforementioned first fragment, second fragment and third fragment at the same time, the template fragment is a linear structure, the template chain has two template fragments, and the template fragment has a fourth fragment and a fifth fragment", a gap is formed between the 5' end of the fourth fragment and the 3' end of the fifth fragment, and the fourth fragment and the fifth fragment together form a template chain; the 5' end of the template chain can be reverse complementary to the sequence of the 5' end of the first fragment to form a first complementary region, and the 3' end of the template chain can be reverse complementary to the sequence of the 3' end of the second fragment to form a second complementary region; the middle region of the template chain (i.e., the region between the 3' end and the 5' end) can be reverse complementary to the third fragment, A third complementary region is formed; 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 third fragment and the hydroxyl group at the 3' end of the second fragment, and the first fragment, the third fragment and the second fragment are used to jointly form a target nucleotide chain; the sequence of the target nucleotide chain and the sequence of the template chain are reversely complementary to form a double-stranded structure, and the gap between the first fragment and the third fragment, the gap between the second fragment and the third fragment, and the gap between the fourth fragment and the fifth fragment can all be closed under the catalysis of the double-stranded ligase to form a complete phosphodiester bond, thereby completing the synthesis reaction of the target nucleotide chain.

[0089] In some optional embodiments of the present disclosure, the nucleotide fragment further includes a sixth fragment and a seventh fragment; the 5' end of the sixth fragment contains a monophosphate group, and the sixth fragment is synthesized in a 3' to 5' direction; the 3' end of the seventh fragment contains a hydroxyl group, and the seventh fragment 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 fragment and the seventh fragment 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 fragment and the second fragment together form a second target nucleotide chain. The first target nucleotide chain and the second target nucleotide chain are used to form a double-stranded structure together.

[0090] In the above scheme, the sixth fragment and the seventh fragment together form the first target nucleotide chain, and the first fragment and the second fragment can together form the second target nucleotide chain, which can further effectively reduce the content of non-target nucleotide chains in the synthetic product of the nucleotide chain, so as to further effectively improve the purity of the target nucleotide chain in the synthetic product, thereby facilitating the full utilization of the efficacy of the target nucleotide chain.

[0091] As an example, when "the nucleotide fragments simultaneously include the aforementioned first fragment, second fragment, sixth fragment and seventh fragment, and the two complementary nucleotide chains in the double-stranded structure are each 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 fragment and the seventh fragment 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 fragment and the second fragment together form the second target nucleotide chain; the first target nucleotide chain and the second target nucleotide chain serve as templates for each other, and the sequence of the first target nucleotide chain and the sequence of the second target nucleotide chain are reversely complementary to form a double-stranded structure, and the gap between the first fragment and the second fragment and the gap between the sixth fragment and the seventh fragment can both be closed under the catalysis of the double-stranded ligase to form a complete phosphodiester bond.

[0092] In one embodiment, when the nucleotide fragments further include a sixth fragment and a seventh fragment, and also include the aforementioned 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. The first, third, and second fragments are used to together form the second target nucleotide chain. The first and second target nucleotide chains are used to together form a double-stranded structure.

[0093] In the above scheme, the sixth fragment and the seventh fragment together form the first target nucleotide chain, and the first fragment, the third fragment and the second fragment can together form the second target nucleotide chain, which can further effectively reduce the content of non-target nucleotide chains in the synthetic product of the nucleotide chain, so as to further effectively improve the purity of the target nucleotide chain in the synthetic product, thereby facilitating the full utilization of the efficacy of the target nucleotide chain.

[0094] As an example, when "the nucleotide fragments include the aforementioned first fragment, second fragment, third fragment, sixth fragment and seventh fragment at the same time, and the two complementary nucleotide chains in the double-stranded structure are each 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 fragment and the seventh fragment together form the first target nucleotide chain; the 5' end of the first target nucleotide chain can be reverse complementary to the sequence of the 5' end of the first fragment to form a first complementary region, and the 3' end of the first target nucleotide chain can be reverse complementary to the sequence of the 3' end of the second fragment to form a second complementary region; the middle region of the first target nucleotide chain (i.e., the region between the 3' end and the 5' end) can be reverse complementary to the third fragment to form a The third complementary region; 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 third fragment and the hydroxyl group at the 3' end of the second fragment, and the first fragment, the third fragment, and the second fragment are used to jointly form a second target nucleotide chain; the first target nucleotide chain and the second target nucleotide chain serve as templates for each other, and the sequence of the first target nucleotide chain and the sequence of the second target nucleotide chain are reversely complementary to form a double-stranded structure, and the gap between the first and third fragments, the gap between the second and third fragments, and the gap between the sixth and seventh fragments can all be gap-sealed under the catalysis of a double-stranded ligase to form a complete phosphodiester bond. As mentioned above, when the nucleotide fragments are DNA, the double-stranded ligase is a DNA double-stranded ligase; as an example, the DNA double-stranded ligase includes T4 DNA ligase (T4 DNA Ligase) or Taq DNA Ligase, etc.

[0095] As described above, when the nucleotide fragment is RNA, the double-stranded ligase is an RNA double-stranded ligase; illustratively, the RNA double-stranded ligase includes Rnl2 family ligases and Rnl5 family ligases, for example, T4 RNA ligase2 and DraRnl (from Naegleria gruberi).

[0096] It should be noted that the present disclosure does not limit the specific selection of double-stranded ligase, which can be selected according to actual conditions. It can be a natural double-stranded ligase, a mutant strain produced by enzyme mutation, transformation and evolution, or an enzyme with gap-sealing activity after fusion with enzymes within the same family or with enzymes in other families.

[0097] In some optional embodiments of the present disclosure, the nucleotide fragment contains 4 to 200 bases, which can effectively reduce the content of non-target nucleotide chains in the synthetic product of the nucleotide chain, thereby effectively improving the purity of the target nucleotide chain in the synthetic product, and further facilitating the full utilization of the efficacy of the target nucleotide chain.

[0098] In one embodiment, the nucleotide fragment contains 4 to 120 bases, which is beneficial to further reduce the content of non-target nucleotide chains in the synthetic product of the nucleotide chain, thereby effectively improving the purity of the target nucleotide chain in the synthetic product, and further facilitating full utilization of the efficacy of the target nucleotide chain.

[0099] In some optional embodiments of the present disclosure, the template chain contains a modification group.

[0100] As an example, the modification group of the template chain can be a modification group on the base, a modification group on the sugar ring, a modification group on the phosphate group, etc., which is not limited in the present disclosure.

[0101] It should be noted that in other optional embodiments of the present disclosure, the template chain may not contain a modifying group, and the present disclosure does not limit whether the template chain contains a modifying group.

[0102] In some optional embodiments of the present disclosure, the nucleotide fragment contains a modification group.

[0103] It should be noted that in other optional embodiments of the present disclosure, the nucleotide fragment may not contain a modification group, and the present disclosure does not limit whether the nucleotide fragment contains a modification group.

[0104] As an example, the modification group of the nucleotide fragment can be a modification group on the base, a modification group on the sugar ring, a modification group on the phosphate group, etc., which is not limited in the present disclosure.

[0105] In some optional embodiments of the present disclosure, the method for preparing a template chain includes: performing a reaction to synthesize the template chain according to the target sequence of the template chain, and then not purifying the synthesized product. Since the composition for synthesizing a nucleotide chain provided by the present disclosure comprises "a first fragment synthesized in a 3' to 5' direction and containing a monophosphate group at the 5' end" and "a second fragment synthesized in a 5' to 3' direction and containing a hydroxyl group at the 3' end", even if no purification treatment is performed after the template chain is synthesized, the content of non-target nucleotide chains in the synthetic product of the nucleotide chain can be effectively reduced, thereby effectively improving the purity of the target nucleotide chain in the synthetic product, thereby facilitating full utilization of the efficacy of the target nucleotide chain.

[0106] It is understood that if the synthesis of the template chain involves the protection of a protecting group, the protecting group can also be removed during the synthesis process.

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

[0108] It should be noted that in other optional embodiments of the present disclosure, the method for preparing the template chain may also include: performing a reaction to synthesize the template chain according to the target sequence of the template chain, and then purifying the synthesized product to obtain a template chain with higher purity.

[0109] It should be noted that the present disclosure does not limit the synthesis path of the template chain, and the H-phosphonate principle, the phosphoramidite principle, or the enzymatic synthesis principle may be adopted; solid-phase synthesis or liquid-phase synthesis may be adopted.

[0110] In some optional embodiments of the present disclosure, the method for preparing a nucleotide fragment includes: performing a reaction to synthesize the nucleotide fragment according to the target sequence of the nucleotide fragment, and then not purifying the synthesized product. Since the composition for synthesizing a nucleotide chain provided by the present disclosure comprises "a first fragment synthesized in a 3' to 5' direction and containing a monophosphate group at the 5' end" and "a second fragment synthesized in a 5' to 3' direction and containing a hydroxyl group at the 3' end", even if the nucleotide fragment is not purified after synthesis, the content of non-target nucleotide chains in the synthetic product of the nucleotide chain can be effectively reduced, thereby effectively improving the purity of the target nucleotide chain in the synthetic product, thereby facilitating full utilization of the efficacy of the target nucleotide chain.

[0111] It is understood that if the synthesis of the nucleotide fragment involves the protection of a protecting group, the protecting group can also be removed during the synthesis process.

[0112] In some optional embodiments of the present disclosure, the method for preparing nucleotide fragments includes: performing a reaction to synthesize the nucleotide fragments according to the target sequence of the nucleotide fragments, and then only performing a desalting treatment on the synthesized products.

[0113] It should be noted that, in other optional embodiments of the present disclosure, the method for preparing nucleotide fragments may also include: performing a reaction to synthesize nucleotide fragments according to the target sequence of the nucleotide fragments, and then purifying the synthesized products to obtain nucleotide fragments with higher purity.

[0114] It should be noted that the present disclosure does not limit the synthesis path of the nucleotide fragments, and the H-phosphonate principle, the phosphoramidite principle, or the enzymatic synthesis principle may be adopted; solid-phase synthesis or liquid-phase synthesis may be adopted.

[0115] As an example, in the composition for synthesizing a nucleotide chain, the concentration of the nucleotide fragments can be 1 μM to 100 mM; and the molar ratio between any two nucleotide fragments can be (0.1:1) to (1:0.1). It should be noted that the concentration of the nucleotide fragments and the concentration of the template chain can be adjusted according to actual conditions and are not limited in this disclosure.

[0116] As an example, the concentration of double-stranded ligase in the composition for synthesizing nucleotide chains can be 0.01 U / μL to 10 U / μL. It should be noted that the concentration of double-stranded ligase can be adjusted according to actual conditions and is not limited in this disclosure.

[0117] In some optional embodiments of the present disclosure, the composition for synthesizing a nucleotide chain further comprises a buffer containing divalent ions.

[0118] As an example, the divalent ions can be magnesium ions or manganese ions, etc.; the buffer can be a phosphate buffer, a Tris buffer or a HEPES buffer, etc.; the pH range of the buffer is selected based on the pH corresponding to the optimal catalytic activity of the enzyme used in the composition. For example, when the dual ligase is T4 RNA Ligase2, the pH value of the buffer can be 6.5 to 9.0.

[0119] In some optional embodiments of the present disclosure, the composition for synthesizing a nucleotide chain further comprises DTT.

[0120] In some optional embodiments of the present disclosure, the composition for synthesizing nucleotide chains further comprises EDTA; the function of EDTA is to terminate the synthesis reaction by chelating divalent ions in the buffer.

[0121] As an example, PEG can be added to the composition for synthesizing nucleotide chains to increase the efficiency of the synthesis reaction, a surfactant can be added to increase the stability of the enzyme, and auxiliary factors such as ATP can be added.

[0122] It is understandable that the composition for synthesizing nucleotide chains may only have a template chain, nucleotide fragments and double-stranded ligase. As for other substances required for realizing the nucleotide synthesis reaction, such as buffer, ATP, DTT and EDTA, they can be added accordingly during the reaction process.

[0123] The present disclosure also provides a method for synthesizing a nucleotide chain, which comprises: performing a nucleotide chain synthesis reaction using the composition for synthesizing a nucleotide chain as provided above.

[0124] The method for synthesizing a nucleotide chain provided by the present disclosure comprises a "first fragment synthesized in a 3' to 5' direction and containing a monophosphate group at the 5' end" and a "second fragment synthesized in a 5' to 3' direction and containing a hydroxyl group at the 3' end" in the synthetic raw materials. The first fragment and the second fragment can cooperate with each other, which can significantly reduce the content of non-target nucleotide chains (especially n-1 impurities, n-2 impurities, n+1 impurities and n+2 impurities) in the synthetic product of the nucleotide chain, thereby effectively improving the purity of the target nucleotide chain in the synthetic product, thereby facilitating full utilization of the efficacy of the target nucleotide chain.

[0125] It can be understood that in the method for synthesizing a nucleotide chain provided in the present disclosure, the composition used to synthesize the nucleotide chain includes a template chain, a nucleotide fragment and a double-chain ligase; for the relevant selection of the template chain, the nucleotide fragment and the double-chain ligase, please refer to the above content and will not be repeated here.

[0126] As an example, the synthesis reaction of the nucleotide chain is carried out at 37° C. for about 4 hours. The specific reaction temperature and reaction time can be adjusted according to actual conditions and are not limited in this disclosure.

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

[0128] The present disclosure provides a reagent or kit, which includes: the composition for synthesizing nucleotide chains provided above.

[0129] The form of the reagent includes but is not limited to liquid, solid or semi-solid. The reagent also includes buffer, ATP, MgCl2, DTT, ligase, etc.

[0130] Ligase includes but is not limited to T4 RNA Ligase2 and double-stranded ligase.

[0131] When the nucleotide fragment is DNA, the double-strand ligase is DNA double-strand ligase; illustratively, the DNA double-strand ligase includes T4 DNA ligase (T4 DNA Ligase) or Taq DNA Ligase.

[0132] As described above, when the nucleotide fragment is RNA, the double-stranded ligase is an RNA double-stranded ligase; illustratively, the RNA double-stranded ligase includes Rnl2 family ligases and Rnl5 family ligases, for example, T4 RNA ligase2 and DraRnl (from Naegleria gruberi).

[0133] In the above technical solution, the kit also includes a solid phase support for the synthesis of nucleotide chains (such as 5'-Dimethoxytrityl-N4-acetyl-Cytidine, 2'-TBDMS-3'-succinoyl-long chain alkylamino-CPG), a synthesis column, a diluent, a detergent, an activator, a deprotection reagent, an oxidizing reagent, a sulfurizing reagent, a blocking reagent and a termination reaction solution, etc.

[0134] The deprotection reagent is trichloroacetic acid solution, and the activating agent is 5-ethylthiotetrazole (ETT) solution.

[0135] The oxidizing agent is iodine (I2) solution; the sulfurizing agent is phenylacetyl disulfide; the blocking 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 terminating solution is EDTA.

[0136] The present disclosure also provides use of the above composition for synthesizing nucleotide chains in synthesizing nucleotide chains.

[0137] To make the purpose, technical solutions, and advantages of the embodiments of the present disclosure more clear, the technical solutions in the embodiments of the present disclosure are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer were followed. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0138] Example 1

[0139] This embodiment provides a method for synthesizing a nucleotide chain, comprising the following steps:

[0140] (1) Synthesis of nucleotide fragment 1 using the 3' to 5' direction:

[0141] The synthesis equipment used was Mermade 12 synthesizer, and the synthesis scale was 10 μM.

[0142] 5'-Dimethoxytrityl-N4-acetyl-Cytidine,2'-TBDMS-3'-succinoyl-long chain alkylamino-CPG from Glen Research was selected as the solid phase carrier.

[0143] 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) 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) phosphoramidite (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 As 3'-phosphoramidite monomers, 5'-O-(4,4-dimethoxytrityl)-2'-O-methyl-N2-isobutyrylguanosine-3'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (i.e., 5'-DMT-2'-OMe-G(iBu)-3'-CE-Phosphoramidite) were dissolved in anhydrous acetonitrile to obtain corresponding 0.1 M monomer solutions.

[0144] A 3 wt % trichloroacetic acid (TCA) solution (solvent: dichloromethane) was selected as a deprotection agent for dimethoxytrityl (DMT).

[0145] A 0.25 M 5-ethylthiotetrazolium (ETT) solution (solvent: acetonitrile) was selected as the activating agent.

[0146] A 0.05 M iodine (I2) solution (the solvent is a mixture of pyridine and water with a volume ratio of 9:1) is selected as the oxidizing agent.

[0147] Phenylacetylisulfide (PADS) was selected as the sulfurization reagent.

[0148] A mixture of acetic anhydride (Ac2O) and acetonitrile in a volume ratio of 1:4 was selected as blocking reagent A.

[0149] A mixture of N-methylimidazole, pyridine and acetonitrile in a volume ratio of 2:3:5 was selected as blocking reagent B.

[0150] 20 μmol of the aforementioned solid phase support was loaded into the synthesis column, and the target product sequence (5'-mA*mG*mG*AUGCGCUAAGUAGCGUGCGUUUUAGUACUCUGGAAACAGAAUCUAC-3', SEQ ID NO. 1) was synthesized from the 3' end to the 5' end. Each base synthesis underwent one cycle, and each cycle had the following steps: 1. 2 mL of the aforementioned deprotection reagent was added to the synthesis column for deprotection for 40 s, and then the system in the synthesis column was washed with acetonitrile; 2. 470 μL of the aforementioned activator and 630 μL of the corresponding monomer solution were added to the synthesis column for coupling for 6 min, and then the system in the synthesis column was washed with acetonitrile; 3. 1.6 mL of the oxygenation reagent or 1.6 mL of the thiolation reagent was added to the synthesis column, treated for 3 min, and then the system in the synthesis column was washed with acetonitrile; 4. The aforementioned blocking reagent A (1 mL) and blocking reagent B (1 mL) were added to the synthesis column, and after 30 s, the next cycle was entered.

[0151] The solid support in the synthesis column was washed with 90 mL of acetonitrile and transferred to a 15 mL centrifuge tube. A mixture of 10 mL of aqueous ammonia and a 40 wt% aqueous methylamine solution (V:V, 1:1) was added to the tube and incubated at 65°C for 30 minutes. The mixture was centrifuged at 13,000 rpm for 3 minutes, and the supernatant was collected and dried to obtain the first system. Next, 4 mL of a 12 wt% tetraethylammonium chloride solution (in dimethyl sulfoxide) was added to the first system at 25°C and sonicated for 1 hour to remove the tert-butyldimethylsilyl groups. The precipitate was then washed with 12 mL of n-butanol and then with 4 mL of ethanol. The mixture was then centrifuged at 13,000 rpm for 3 minutes, the supernatant removed, and the precipitate dissolved in 1 mL of ultrapure water. The resulting synthetic products are shown in Table 1.

[0152] Table 1

[0153] In Table 1, m represents 2'OMe modification, that is, the 2' position of the nucleoside sugar is modified by a methoxy group; * represents a phosphorus-sulfur bond.

[0154] (2) Synthesis of nucleotide fragment 1 using the 5' to 3' direction:

[0155] The synthesis equipment used was Mermade 12 synthesizer, and the synthesis scale was 10 μM.

[0156] 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 selected as the solid phase carrier.

[0157] 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]adenosine [silyl]-N2-isobutyrylguanosine-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 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 e) 3'-O-(4,4-dimethoxytrityl)-2'-O-methyl-N2-isobutyrylguanosine-3'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (i.e., 3'-DMT-2'-OMe-iBu-G-5'-CE-Phosphoramidite) as a 5'phosphoramidite monomer, and the above 5'phosphoramidite monomers were respectively dissolved in anhydrous acetonitrile to obtain corresponding 0.1 M monomer solutions.

[0158] A 3 wt % trichloroacetic acid (TCA) solution (solvent: dichloromethane) was selected as a deprotection agent for dimethoxytrityl (DMT).

[0159] A 0.25 M 5-ethylthiotetrazolium (ETT) solution (solvent: acetonitrile) was selected as the activating agent.

[0160] A 0.05 M iodine (I2) solution (the solvent is a mixture of pyridine and water with a volume ratio of 9:1) was selected as the oxidant.

[0161] Phenylacetylisulfide (PADS) was selected as the vulcanizing agent.

[0162] A mixture of acetic anhydride (Ac2O) and acetonitrile in a volume ratio of 1:4 was selected as blocking reagent A.

[0163] A mixture of N-methylimidazole, pyridine and acetonitrile in a volume ratio of 2:3:5 was selected as blocking reagent B.

[0164] 20 μmol of the aforementioned solid phase support was loaded into the synthesis column, and the target product sequence (5'-mA*mG*mG*AUGCGCUAAGUAGCGUGCGUUUUAGUACUCUGGAAACAGAAUCUAC-3', SEQ ID NO. 1) was synthesized from the 5' end to the 3' end. Each base synthesis underwent one cycle, and each cycle had the following steps: 1. 2 mL of the aforementioned deprotection reagent was added to the synthesis column for deprotection for 40 s, and then the system in the synthesis column was washed with acetonitrile; 2. 470 μL of the aforementioned activator and 630 μL of the corresponding monomer solution were added to the synthesis column for coupling for 6 min, and then the system in the synthesis column was washed with acetonitrile; 3. 1.6 mL of the oxygenation reagent or 1.6 mL of the thiolation reagent was added to the synthesis column, treated for 3 min, and then the system in the synthesis column was washed with acetonitrile; 4. The aforementioned blocking reagent A (1 mL) and blocking reagent B (1 mL) were added to the synthesis column, and after 30 s, the next cycle was entered.

[0165] The solid support in the synthesis column was washed with 90 mL of acetonitrile and transferred to a 15 mL centrifuge tube. A mixture of 10 mL of aqueous ammonia and a 40 wt% aqueous methylamine solution (V:V, 1:1) was added to the tube and incubated at 65°C for 30 minutes. The mixture was centrifuged at 13,000 rpm for 3 minutes, and the supernatant was collected and dried to obtain the first system. Next, 4 mL of a 12 wt% tetraethylammonium chloride solution (in dimethyl sulfoxide) was added to the first system at 25°C and sonicated for 1 hour to remove the tert-butyldimethylsilyl groups. The precipitate was washed with 12 mL of n-butanol and then with 4 mL of ethanol. The mixture was then centrifuged at 13,000 rpm for 3 minutes, the supernatant removed, and the precipitate dissolved in 1 mL of ultrapure water. The resulting synthetic products are shown in Table 2.

[0166] Table 2

[0167] In Table 2, m represents 2'-ome modification, that is, the 2' position of the nucleoside sugar is modified by a methoxy group; * represents a phosphorus-sulfur bond.

[0168] (3) Synthesis of nucleotide fragment 2 using the 3' to 5' direction:

[0169] The synthesis of nucleotide fragment 2 synthesized in the 3' to 5' direction is basically the same as step (1), except that only [3'-O-(4,4-dimethoxytrityloxy)-2,2'-dicarboxyethyl]propyl-(2-cyanoethyl)-(N,N-isopropyl)phosphoramidite (i.e., [3-(4,4'-Dimethoxytrityloxy)-2,2-dicarboxyethyl]propyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite) is used as the 3' phosphoramidite monomer.

[0170] The obtained synthetic products are shown in Table 3.

[0171] Table 3

[0172] In Table 3, m represents 2'OMe modification, that is, the 2' position of the nucleoside sugar is modified by a methoxy group; * represents a phosphorus-sulfur bond; and p represents a monophosphate group.

[0173] (4) Synthesis of nucleotide fragment 2 using the 5' to 3' direction:

[0174] The synthesis of nucleotide fragment 2 synthesized in the 5' to 3' direction is basically the same as step (2), except that 3-(4,4'-Dimethoxytrityloxy)-2,2-(dicarboxymethylamido)propyl-1-O-succinoyl-long chain alkylamino-CPG from Glen Research was used as the solid phase support.

[0175] The obtained synthetic products are shown in Table 4.

[0176] Table 4

[0177] In Table 4, m represents 2'OMe modification, that is, the 2' position of the nucleoside sugar is modified by a methoxy group; * represents a phosphorus-sulfur bond; and p represents a monophosphate group.

[0178] (5) Synthesis of template strand 1 using 3' to 5' direction:

[0179] The synthesis equipment used was Mermade 12 synthesizer, and the synthesis scale was 10 μM.

[0180] 5'-Dimethoxytrityl-N-benzoyl-2'-deoxyThymidine-3'-succinoyl-long chain alkylamino-CPG from Glen Research was selected as the solid phase support.

[0181] The phosphoamidite was selected from 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'-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) were used as 3'-phosphoramidite monomers. These 3'-phosphoramidite monomers were respectively dissolved in anhydrous acetonitrile to obtain the corresponding 0.1 M monomer solutions.

[0182] A 3 wt % trichloroacetic acid (TCA) solution (solvent: dichloromethane) was selected as a deprotection agent for dimethoxytrityl (DMT).

[0183] A 0.25 M 5-ethylthiotetrazolium (ETT) solution (solvent: acetonitrile) was selected as the activating agent.

[0184] A 0.05 M iodine (I2) solution (the solvent is a mixture of pyridine and water with a volume ratio of 9:1) is selected as the oxidizing agent.

[0185] Phenylacetylisulfide (PADS) was selected as the sulfurization reagent.

[0186] A mixture of acetic anhydride (Ac2O) and acetonitrile in a volume ratio of 1:4 was selected as blocking reagent A.

[0187] A mixture of N-methylimidazole, pyridine and acetonitrile in a volume ratio of 2:3:5 was selected as blocking reagent B.

[0188] 20 μmol of the aforementioned solid phase support was loaded into the synthesis column, and synthesis was started from the 3' end to the 5' end according to the target product sequence (3'-TGAGACCTTTGTCTTAGATGATTTTGTTCCGTTTTACGGA-5', SEQ ID NO.15). Each base synthesis underwent one cycle, and each cycle had the following steps: 1. 2 mL of the aforementioned deprotection reagent was added to the synthesis column for deprotection for 40 seconds, and then the system in the synthesis column was cleaned with acetonitrile; 2. 470 μL of the aforementioned activator and 630 μL of the corresponding monomer solution were added to the synthesis column for coupling for 6 minutes, and then the system in the synthesis column was cleaned with acetonitrile; 3. 1.6 mL of the oxygenation reagent or 1.6 mL of the thiolation reagent was added to the synthesis column, and after treatment for 3 minutes, the system in the synthesis column was cleaned with acetonitrile; 4. The aforementioned blocking reagent A (1 mL) and blocking reagent B (1 mL) were added to the synthesis column, and after 30 seconds, the next cycle was entered.

[0189] The sequence of template strand 1 is as follows:

[0190] 3'-TGAGACCTTTGTCTCTAGATGATTTTGTTCCGTTTTACGGA-5', SEQ ID NO. 15.

[0191] (6) Synthesis reaction of nucleotide chain:

[0192] In a tris buffer with a pH of 8.0, containing 400 μM ATP, 2 mM MgCl2, 1 mM DTT, 200 μM template chain 1, 200 μM nucleotide fragment 1 (synthesized in the 3' to 5' direction or the 5' to 3' direction), and 200 μM nucleotide fragment 2 (synthesized in the 3' to 5' direction or the 5' to 3' direction), T4 RNA Ligase2 was added (and its final concentration in the reaction solution was 0.3 U / μL), and after reacting at 37°C for 4 hours, EDTA was added (and its final concentration in the reaction solution was 3 mM) to terminate the reaction to obtain a synthetic product.

[0193] Among them, the specific reactions are divided into 4 reactions, as shown in Table 5.

[0194] Table 5

[0195] Example 2

[0196] This embodiment provides a method for synthesizing a nucleotide chain, comprising the following steps:

[0197] (1) Synthesis of nucleotide fragment 5 using the 3' to 5' direction:

[0198] The synthesis steps were the same as step (1) in Example 1, and the obtained synthetic products are shown in Table 6.

[0199] Table 6

[0200] In Table 6, m represents 2'OMe modification, that is, the 2' position of the nucleoside sugar is modified with a methoxy group; * represents a phosphorus-sulfur bond; and f represents 2'F modification, that is, the 2' position of the nucleoside sugar is modified with a fluorine group.

[0201] (2) Synthesis of nucleotide fragment 5 using the 5' to 3' direction:

[0202] The synthesis steps were the same as step (2) in Example 1, and the obtained synthetic products are shown in Table 7.

[0203] Table 7

[0204] In Table 7, m represents 2'OMe modification, that is, the 2' position of the nucleoside sugar is modified with a methoxy group; * represents a phosphorus-sulfur bond; and f represents 2'F modification, that is, the 2' position of the nucleoside sugar is modified with a fluorine group.

[0205] (3) Synthesis of nucleotide fragment 6 using the 3' to 5' direction:

[0206] The synthesis steps were the same as step (1) in Example 1, and the obtained synthetic products are shown in Table 8.

[0207] Table 8

[0208] In Table 8, m represents 2'OMe modification, ie, the 2' position of the nucleoside sugar is modified with a methoxy group; p represents a monophosphate group; and f represents 2'F modification, ie, the 2' position of the nucleoside sugar is modified with a fluorine group.

[0209] (4) Synthesis of nucleotide fragment 6 using the 5' to 3' direction:

[0210] The synthesis steps were the same as step (2) in Example 1, and the obtained synthetic products are shown in Table 9.

[0211] Table 9

[0212] In Table 9, m represents 2'OMe modification, ie, the 2' position of the nucleoside sugar is modified with a methoxy group; p represents a monophosphate group; and f represents 2'F modification, ie, the 2' position of the nucleoside sugar is modified with a fluorine group.

[0213] (5) Synthesis of nucleotide fragment 7 using 3' to 5' direction synthesis:

[0214] The synthesis steps were the same as step (1) in Example 1, and the obtained synthetic products are shown in Table 10.

[0215] Table 10

[0216] In Table 10, m represents 2'OMe modification, i.e., the 2' position on 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 on the nucleoside sugar is modified with a fluorine group.

[0217] (6) Synthesis of nucleotide fragment 7 using the 5' to 3' direction:

[0218] The synthesis steps were the same as step (2) in Example 1, and the obtained synthetic products are shown in Table 11.

[0219] Table 11

[0220] In Table 11, m represents 2'OMe modification, i.e., the 2' position on 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 on the nucleoside sugar is modified with a fluorine group.

[0221] (7) Synthesis of template strand 2 using a 3' to 5' direction:

[0222] The synthesis equipment used was Mermade 12 synthesizer, and the synthesis scale was 10 μM.

[0223] 5'-Dimethoxytrityl-2'-deoxyThymidine-3'-succinoyl-long chain alkylamino-CPG from Glen Research was selected as the solid phase carrier.

[0224] The phosphoamidite was selected from 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'-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) were used as 3'-phosphoramidite monomers. These 3'-phosphoramidite monomers were respectively dissolved in anhydrous acetonitrile to obtain the corresponding 0.1 M monomer solutions.

[0225] A 3 wt % trichloroacetic acid (TCA) solution (solvent: dichloromethane) was selected as a deprotection agent for dimethoxytrityl (DMT).

[0226] A 0.25 M 5-ethylthiotetrazolium (ETT) solution (solvent: acetonitrile) was selected as the activating agent.

[0227] A 0.05 M iodine (I2) solution (the solvent is a mixture of pyridine and water with a volume ratio of 9:1) is selected as the oxidizing agent.

[0228] Phenylacetylisulfide (PADS) was selected as the sulfurization reagent.

[0229] A mixture of acetic anhydride (Ac2O) and acetonitrile in a volume ratio of 1:4 was selected as blocking reagent A.

[0230] A mixture of N-methylimidazole, pyridine and acetonitrile in a volume ratio of 2:3:5 was selected as blocking reagent B.

[0231] 20 μmol of the aforementioned solid phase carrier was loaded into the synthesis column and the target product sequence (3'-TGTTTTCGTTTTGTCCAGATC-5', 5'-CTAGACCTGTTTTGCTTTTGT-3' SEQ ID NO.16) Starting from the 3' end, synthesis is carried out toward the 5' end, and each base synthesis undergoes one cycle. Each base synthesis undergoes one cycle, and each cycle has the following steps: 1. Add 2 mL of the aforementioned deprotection reagent to the synthesis column for deprotection for 40 seconds, and then use acetonitrile to clean the system in the synthesis column; 2. Add 470 μL of the aforementioned activator and 630 μL of the corresponding monomer solution to the synthesis column for coupling for 6 minutes, and then use acetonitrile to clean the system in the synthesis column; 3. Add 1.6 mL of the oxygenation reagent or 1.6 mL of the thiolation reagent to the synthesis column, treat for 3 minutes, and then use acetonitrile to clean the system in the synthesis column; 4. Add the aforementioned blocking reagent A (1 mL) and blocking reagent B (1 mL) to the synthesis column, and after 30 seconds, enter the next cycle.

[0232] The sequence of template strand 2 is as follows:

[0233] 3'-TGTTTTCGTTTTGTCCAGATC-5', that is, 5'-CTAGACCTGTTTTGCTTTTGT-3'SEQ ID NO. 16.

[0234] (8) Synthesis reaction of nucleotide chain:

[0235] In a tris buffer with a pH of 8.0, containing 400 μM ATP, 2 mM MgCl2, 1 mM DTT, 200 μM template chain 2, 200 μM nucleotide fragment 5 (synthesized in the 3' to 5' direction or the 5' to 3' direction), 200 μM nucleotide fragment 6 (synthesized in the 3' to 5' direction or the 5' to 3' direction), and 200 μM nucleotide fragment 7 (synthesized in the 3' to 5' direction or the 5' to 3' direction), T4 RNA Ligase2 was added (and its final concentration in the reaction solution was 0.5 U / μL), and after reacting at 37°C for 5 h, EDTA was added (and its final concentration in the reaction solution was 3 mM) to terminate the reaction to obtain a synthetic product.

[0236] Among them, the specific reactions are divided into 8 reactions, as shown in Table 12.

[0237] Table 12

[0238] Example 3

[0239] This embodiment provides a method for synthesizing a nucleotide chain, comprising the following steps:

[0240] (1) Synthesis of nucleotide fragment 8 using the 3' to 5' direction:

[0241] The synthesis steps were the same as step (1) in Example 1, and the obtained synthetic products are shown in Table 13.

[0242] Table 13

[0243] In Table 13, m represents 2'OMe modification, ie, the 2' position on the nucleoside sugar is modified with a methoxy group; p represents a monophosphate group; and GalNAc represents N-acetylgalactosamine.

[0244] (2) Synthesis of nucleotide fragment 8 using the 5' to 3' direction:

[0245] The synthesis steps were the same as step (2) in Example 1, and the obtained synthetic products are shown in Table 14.

[0246] Table 14

[0247] In Table 14, m represents 2'OMe modification, ie, the 2' position on the nucleoside sugar is modified with a methoxy group; p represents a monophosphate group; and GalNAc represents N-acetylgalactosamine.

[0248] (3) Synthesis of nucleotide fragment 9 using the 3' to 5' direction:

[0249] The synthesis steps were the same as step (1) in Example 1, and the obtained synthetic products are shown in Table 15.

[0250] Table 15

[0251] In Table 15, m represents 2'OMe modification, i.e., the 2' position on the nucleoside sugar is modified with a methoxy group; * represents a phosphorus-sulfur bond; d represents deoxynucleoside, i.e., the 2' position on the nucleoside sugar is hydrogen; and f represents 2'F modification, i.e., the 2' position on the nucleoside sugar is modified with a fluorine group.

[0252] (4) Synthesis of nucleotide fragment 9 using the 5' to 3' direction:

[0253] The synthesis steps were the same as step (2) in Example 1, and the obtained synthetic products are shown in Table 16.

[0254] Table 16

[0255] In Table 16, m represents 2'OMe modification, i.e., the 2' position on the nucleoside sugar is modified with a methoxy group; * represents a phosphorus-sulfur bond; d represents deoxynucleoside, i.e., the 2' position on the nucleoside sugar is hydrogen; and f represents 2'F modification, i.e., the 2' position on the nucleoside sugar is modified with a fluorine group.

[0256] (5) Synthesis reaction of nucleotide chain:

[0257] In a tris buffer at pH 8.0, containing 400 μM ATP, 2 mM MgCl2, 1 mM DTT, 100 μM of nucleotide fragment 5 prepared in Example 2 (synthesized in the 3' to 5' direction or the 5' to 3' direction), 100 μM of nucleotide fragment 6 prepared in Example 2 (synthesized in the 3' to 5' direction or the 5' to 3' direction), 100 μM of nucleotide fragment 7 prepared in Example 2 (synthesized in the 3' to 5' direction or the 5' to 3' direction), 100 μM of nucleotide fragment 8 (synthesized in the 3' to 5' direction or the 5' to 3' direction), and 100 μM of nucleotide fragment 9 (synthesized in the 3' to 5' direction or the 5' to 3' direction), T4 RNA was added. Ligase2 (and its final concentration in the reaction solution is 0.4 U / μL) was added, and after reacting at 37° C. for 4 hours, EDTA (and its final concentration in the reaction solution is 3 mM) was added to terminate the reaction to obtain the synthetic product.

[0258] Among them, the specific reactions are divided into 8 reactions, as shown in Table 17.

[0259] Table 17

[0260] Experimental Example 1

[0261] Liquid chromatography-mass spectrometry (LC-MS) analysis was performed on the synthetic products corresponding to the four reactions performed in Table 5 of Example 1. The analysis results are shown in Table 18.

[0262] Table 18

[0263] As can be seen from Table 18, the contents of n-1 impurities, n-2 impurities, and n-3 impurities in the synthetic product corresponding to Reaction 3 are the lowest, indicating that when the nucleotide fragment 1 containing a hydroxyl group at the 3' end is synthesized in the 5' to 3' direction and the nucleotide fragment 2 containing a monophosphate group at the 5' end is synthesized in the 3' to 5' direction, the impurity content in the synthetic product of the nucleotide chain can be effectively reduced.

[0264] Experimental Example 2

[0265] Liquid chromatography-mass spectrometry (LC-MS) analysis was performed on the synthetic products corresponding to the 8 reactions performed in Table 12 of Example 2. The analysis results are shown in Table 19.

[0266] Table 19

[0267] As can be seen from Table 19, the contents of n-1 impurities, n-2 impurities, and n-3 impurities in the synthetic products corresponding to reactions 9 and 11 are the lowest, indicating that when the nucleotide fragment 5 containing a hydroxyl group at the 3' end is synthesized in the 5' to 3' direction and the nucleotide fragment 7 containing a monophosphate group at the 5' end is synthesized in the 3' to 5' direction, the impurity content in the synthetic product of the nucleotide chain can be effectively reduced.

[0268] Experimental Example 3

[0269] The synthetic products corresponding to the 8 reactions performed in Table 17 of Example 3 were analyzed by liquid chromatography-mass spectrometry (LC-MS). The analysis results are shown in Table 20.

[0270] Table 20

[0271] As can be seen from Table 20, the contents of n-1 impurities, n-2 impurities, and n-3 impurities in the synthesis product corresponding to reaction 14 (i.e., the two target nucleotide chains, one of which is the nucleotide chain formed by linking nucleotide fragments 8 and nucleotide fragment 9, and the other is the nucleotide chain formed by linking nucleotide fragments 5, nucleotide fragment 6, and nucleotide fragment 7) are the lowest. This indicates that when "nucleotide fragment 8 containing a monophosphate group at the 5' end is synthesized in the 3' to 5' direction and nucleotide fragment 9 containing a hydroxyl group at the 3' end is synthesized in the 5' to 3' direction," and when "nucleotide fragment 5 containing a hydroxyl group at the 3' end is synthesized in the 5' to 3' direction and nucleotide fragment 7 containing a monophosphate group at the 5' end is synthesized in the 3' to 5' direction," the impurity content in the synthesis product of the two complementary target nucleotide chains can be effectively reduced.

[0272] In summary, the composition for synthesizing nucleotide chains provided by the present disclosure can significantly reduce the content of non-target nucleotide chains in the synthesis product of the nucleotide chain, thereby effectively improving the purity of the target nucleotide chain in the synthesis product, thereby facilitating full utilization of the efficacy of the target nucleotide chain.

[0273] The embodiments described above are some of the embodiments of the present disclosure, rather than all of them. The detailed description of the embodiments of the present disclosure is not intended to limit the scope of the present disclosure for which protection is sought, but rather merely represents selected embodiments of the present disclosure. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present disclosure without creative effort are within the scope of protection of the present disclosure. Industrial Applicability:

[0274] The composition for synthesizing nucleotide chains provided by the present disclosure can significantly reduce the content of non-target nucleotide chains in the synthesis product of the nucleotide chain, thereby effectively improving the purity of the target nucleotide chain in the synthesis product, thereby facilitating full utilization of the efficacy of the target nucleotide chain.

Claims

1. A composition for synthesizing nucleotide chains, characterized in that Includes: nucleotide fragments and double-stranded ligase; Wherein, the nucleotide fragments are used to form a double-stranded structure; the double-stranded ligase is a ligase that can close the gap in the double-stranded structure; and at least one chain in the double-stranded structure is a target nucleotide chain; The nucleotide fragments include a first fragment and a second fragment; the 5' end of the first fragment contains a monophosphate group, and the first fragment is synthesized in a 3' to 5' direction; the 3' end of the second fragment contains a hydroxyl group, and the second fragment is synthesized in a 5' to 3' direction.

2. The composition for synthesizing nucleotide chains according to claim 1, characterized in that The nucleotide fragments further include a third fragment, the 3' end of the third fragment contains a hydroxyl group, and the 5' end of the third fragment contains a monophosphate group; Optionally, the third fragment is synthesized in a 3' to 5' direction, or the third fragment is synthesized in a 5' to 3' direction.

3. The composition for synthesizing nucleotide chains according to claim 1 or 2, characterized in that The composition for synthesizing a nucleotide chain also includes a template chain, wherein the template chain and the nucleotide fragment are used to form the double-stranded structure together; and the nucleotide fragment is used to form the target nucleotide chain.

4. The composition for synthesizing nucleotide chains according to claim 3, characterized in that The template chain is a linear structure; the template chain can be reverse complementary to the sequence at the 5' end of the first fragment to form a first complementary region; the template chain can be reverse complementary to the sequence at the 3' end of the second fragment to form a second complementary region; the gap can be formed between the 5' end of the first fragment and the 3' end of the second fragment.

5. The composition for synthesizing nucleotide chains according to claim 3, characterized in that The template strand is a hairpin structure; the template strand can be reverse complementary to the sequence at the 5' end of the first fragment to form a first complementary region; the template strand can be reverse complementary to the sequence at the 3' end of the second fragment to form a second complementary region; The 5' end of the template chain contains a monophosphate group, and the 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 the gap can be formed between the 3' end of the template chain and the 5' end of the second fragment.

6. The composition for synthesizing nucleotide chains according to claim 1, characterized in that The first fragment is a hairpin structure; the first fragment can be reverse complementary to the sequence at the 3' end of the second fragment, and the 5' end of the first fragment can form the gap with the 3' end of the second fragment; the nucleotide chain after the first fragment and the second fragment are connected is the target nucleotide chain; Alternatively, the second fragment is a hairpin structure; the second fragment can be reverse complementary to the sequence at the 5' end of the first fragment, and the 3' end of the second fragment can form the gap with the 5' end of the first fragment; the nucleotide chain after the first fragment and the second fragment are connected is the target nucleotide chain.

7. The composition for synthesizing nucleotide chains according to claim 3, characterized in that The template chain has at least two template fragments, the template fragments are linear structures, and the gap can be formed between at least the two template fragments; The template strand can be reverse complementary to the sequence at the 5' end of the first fragment to form a first complementary region; the template strand can be reverse complementary to the sequence at the 3' end of the second fragment to form a second complementary region; the 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 used to form the target nucleotide strand; Optionally, the template fragment includes a fourth fragment and a fifth fragment; the 5' end of the fourth fragment contains a monophosphate group, and the fourth fragment is synthesized in the 3' to 5' direction; the 3' end of the fifth fragment contains a hydroxyl group, and the fifth fragment is synthesized in the 5' to 3' direction; the gap is formed between the 5' end of the fourth fragment and the 3' end of the fifth fragment; the gap is formed between the 5' end of the first fragment and the 3' end of the second fragment.

8. The composition for synthesizing nucleotide chains according to any one of claims 1 to 7, characterized in that: The nucleotide fragments further include a sixth fragment and a seventh fragment; the 5' end of the sixth fragment contains a monophosphate group, and the sixth fragment is synthesized in a 3' to 5' direction; the 3' end of the seventh fragment contains a hydroxyl group, and the seventh fragment 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 fragment and the seventh fragment together form a 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 fragment and the second fragment together form a second target nucleotide chain; The first target nucleotide chain and the second target nucleotide chain are used to form the double-stranded structure together.

9. The composition for synthesizing a nucleotide chain according to any one of claims 2 to 7, characterized in that: The nucleotide fragments further include a sixth fragment and a seventh fragment; the 5' end of the sixth fragment contains a monophosphate group, and the sixth fragment is synthesized in a 3' to 5' direction; the 3' end of the seventh fragment contains a hydroxyl group, and the seventh fragment 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 fragment and the seventh fragment together form a 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 used to form a second target nucleotide chain together; The first target nucleotide chain and the second target nucleotide chain are used to form the double-stranded structure together.

10. The composition for synthesizing nucleotide chains according to any one of claims 1 to 9, characterized in that: When the nucleotide fragments are DNA, the double-stranded ligase is a DNA double-stranded ligase; the DNA double-stranded ligase includes T4 DNA ligase; when the nucleotide fragments are RNA, the double-stranded ligase is an RNA double-stranded ligase; the RNA double-stranded ligase includes Rnl2 family ligase and Rnl5 family ligase; Or / and, the nucleotide fragment contains 4 to 200 bases; Or / and, the composition for synthesizing a nucleotide chain further comprises a buffer containing magnesium ions; optionally, the nucleotide fragment contains 4 to 120 bases.

11. The composition for synthesizing nucleotide chains according to any one of claims 3 to 5 and claim 7, characterized in that: The nucleotide fragment may or may not contain a modification group; Or / and, the template chain contains or does not contain a modification group.

12. The composition for synthesizing nucleotide chains according to any one of claims 3 to 5 and claim 7, characterized in that: The method for preparing the template chain comprises: performing a reaction for synthesizing the template chain according to the target sequence of the template chain, and then not purifying the synthesized product; Or / and, the method for preparing the nucleotide fragment comprises: performing a reaction for synthesizing the nucleotide fragment according to the target sequence of the nucleotide fragment, and then not purifying the synthesized product.

13. A method for synthesizing a nucleotide chain, characterized in that: include: A nucleotide chain synthesis reaction is carried out using the composition for synthesizing a nucleotide chain according to any one of claims 1 to 12.

14. A reagent or a kit, characterized in that: It includes: The composition for synthesizing a nucleotide chain according to any one of claims 1 to 12.

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

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