Method for producing oligonucleotide

Abstract

Provided is a method for producing an oligonucleotide. A single-stranded oligonucleotide as a substrate is enzymatically linked using a complementary strand into which a site that reduces the stability of a hybrid formed between the complementary strand and the linking product is introduced to produce an oligonucleotide as a linking product.

Description

Technical Field

[0001] The present invention relates to a method for producing oligonucleic acid. Background Art

[0002] Oligonucleic acids such as siRNA or antisense nucleic acids show their usefulness as nucleic acid drugs, and their development has become active in recent years. Oligonucleic acids are mainly manufactured by chemical synthesis methods. Oligonucleic acids can be manufactured, for example, by utilizing synthetic methods such as the phosphoramidite method to extend the residues of nucleic acid residues one by one in series to synthesize. However, this method has problems such as the purity and yield of the product decreasing or the manufacturing efficiency being low as the chain length of the oligonucleotide becomes longer. Therefore, a short chain fragment of a synthetic oligonucleic acid is being sought and a parallel synthesis method of connecting them to obtain a long-chain oligonucleic acid is being obtained.

[0003] Oligonucleic acids can be produced, for example, by enzymatically ligating nucleic acid fragments using complementary chains.

[0004] In addition, regarding a method for amplifying a DNA sequence comprising enzymatically ligating nucleic acid fragments in the presence of complementary chains, there have been reports of techniques for introducing destabilizing moieties such as abasic moieties into complementary chains or nucleic acid fragments (Patent Document 1 and Non-Patent Document 1).

[0005] In addition, regarding a method for detecting single-stranded DNA comprising enzymatically ligating two complementary strands as probes in the presence of single-stranded DNA, a technique for introducing destabilizing elements such as abasic sites or mismatch sites into complementary strands has been reported (Non-Patent Document 2).

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent document 1: US 9,193,993 B1;

[0009] Non-patent literature

[0010] Non-patent document 1: Abu Kausar et al., Tuning DNA stability to achieve turnover in template for an enzymatic ligation reaction, Angew Chem Int Ed Engl. 2011 Sep 12; 50(38): 8922-6.

[0011] Non-patent document 2: BS Alladin-Mustan et al., Achieving room temperature DNA amplification by dialling in destabilization, Chem. Commun., 2015, 51, 9101-9104. Summary of the invention

[0012] Problems to be solved by the invention

[0013] An object of the present invention is to provide a method for producing an oligonucleic acid.

[0014] Means for solving problems

[0015] In order to solve the above-mentioned problems, the present inventors conducted intensive research and found that when a single-stranded oligonucleic acid as a substrate is subjected to enzymatic ligation to produce an oligonucleic acid as a ligation product, the ligation product can be efficiently produced by using a complementary chain into which a site that reduces the stability of the hybrid formed between the complementary chain and the ligation product is introduced, thereby completing the present invention.

[0016] That is, the present invention can be exemplified as follows.

[0017] [1] A method for producing a target oligonucleic acid, comprising the following steps:

[0018] A step of enzymatically ligating N (N is an integer greater than or equal to 2) single-stranded substrate oligonucleic acids in the presence of M (M is an integer greater than or equal to 1) single-stranded complementary oligonucleic acids to generate the target oligonucleic acid.

[0019] wherein the ligation between the substrate oligonucleotides at the 5' side and the 3' side of each linking portion is performed in the presence of one or more complementary oligonucleotides corresponding to the linking portion,

[0020] Each of the complementary oligonucleotides comprises a base sequence in which a destabilizing portion is introduced into the first base sequence,

[0021] The first base sequence is a base sequence consisting of a second base sequence and a third base sequence connected in the 5' to 3' direction,

[0022] The second base sequence is a complementary sequence to a partial sequence on the 5' side of the 3' side substrate oligonucleotide, or a complementary sequence to the full-length sequence of the 3' side substrate oligonucleotide,

[0023] The third base sequence is a complementary sequence to a partial sequence on the 3' side of the 5' side substrate oligonucleotide, or a complementary sequence to the full-length sequence of the 5' side substrate oligonucleotide.

[0024] The destabilizing portion is a site that reduces the stability of the hybrid formed between each of the complementary oligonucleic acids and the target oligonucleic acid.

[0025] [2] According to the above method, the following situations (1) to (5) are not included:

[0026] (1) The case where N is 2 and the destabilizing portion is composed of an abasic portion at the -1 position;

[0027] (2) The above N is 2, and the above destabilizing portion is composed of a linker between the -1 position and the +1 position substituted with a nucleic acid residue;

[0028] (3) The case where N is 2 and the destabilizing portion is composed of a substitution in which a nucleic acid residue at position -1 is substituted with a linker;

[0029] (4) The case where N is 2, and the destabilizing portion is composed of a combination of an abasic portion at the -1 position and an abasic portion at the +4 position;

[0030] (5) The case where N is 2, and the destabilizing portion is composed of a combination of an abasic portion at the -1 position and a mismatch portion at the +4 position.

[0031] [3] According to the above method, wherein the destabilizing portion includes an abasic portion, a mismatch portion, an insertion of a nucleic acid residue, a deletion of a nucleic acid residue, an insertion of a linker, a deletion of a linker, a substitution of a linker, or a combination thereof.

[0032] [4] According to the above method, wherein the above destabilizing portion includes the insertion of nucleic acid residues without the deletion of a linker and / or the deletion of nucleic acid residues without the insertion of a linker.

[0033] [5] According to the above method, the destabilization unit is composed of 1 to 20 groups of destabilization units.

[0034] [6] According to the above method, wherein the above destabilization unit is composed of 1 to 8 groups of destabilization units.

[0035] [7] According to the above method, wherein the destabilization unit is composed of 1 to 4 groups of destabilization units.

[0036] [8] According to the above method, wherein the destabilizing portion is composed of destabilizing portions whose number is less than 30% of the length of the first base sequence.

[0037] [9] According to the above method, wherein the destabilizing portion includes 1 to 8 abasic portions.

[0038]

[10] According to the above method, wherein the destabilizing portion includes 1 to 2 abasic portions.

[0039]

[11] According to the above method, wherein the destabilizing portion includes an abasic portion whose number is 30% or less relative to the length of the first base sequence.

[0040]

[12] According to the above method, wherein the destabilizing portion includes 1 to 8 mismatch portions.

[0041]

[13] According to the above method, wherein the destabilizing portion includes 1 to 3 mismatch portions.

[0042]

[14] According to the above method, wherein the destabilizing portion includes mismatch portions whose number is 30% or less in ratio to the length of the first base sequence.

[0043]

[15] According to the above method, wherein the destabilizing portion includes a deletion of nucleic acid residues whose number is less than 50% of the length of the first base sequence.

[0044]

[16] According to the above method, wherein the destabilizing portion includes the deletion of 1 to 8 groups of nucleic acid residues.

[0045]

[17] According to the above method, wherein the destabilizing portion includes the deletion of 1 to 3 groups of nucleic acid residues.

[0046]

[18] According to the above method, wherein the deletion of each group of nucleic acid residues consists of the deletion of 1 to 3 nucleic acid residues.

[0047]

[19] According to the above method, wherein the destabilizing portion includes the insertion of nucleic acid residues in a number that is 200% or less of the length of the first base sequence.

[0048]

[20] According to the above method, wherein the destabilizing portion includes the insertion of 1 to 8 groups of nucleic acid residues.

[0049]

[21] According to the above method, wherein the destabilizing portion includes the insertion of 1 to 4 groups of nucleic acid residues.

[0050]

[22] According to the above method, wherein the insertion of each group of nucleic acid residues consists of the insertion of 1 to 10 nucleic acid residues.

[0051]

[23] According to the above method, wherein, when the insertion of each group of nucleic acid residues is composed of insertion of two or more nucleic acid residues, a part or all of the nucleic acid residues have self-complementarity.

[0052]

[24] According to the above method, wherein the destabilizing portion includes the insertion of 1 to 8 connectors.

[0053]

[25] According to the above method, wherein the destabilizing portion includes the deletion of 1 to 8 linkers.

[0054]

[26] According to the above method, wherein the destabilizing portion comprises substitutions of 1 to 8 linkers.

[0055]

[27] According to the above method, wherein the destabilizing unit comprises the following (a), (b) or (c):

[0056] (a) a combination of deletion and / or insertion of the abasic portion at positions -1 to +1 and nucleic acid residues at positions other than -1 to +1;

[0057] (b) a combination of a mismatch at positions -1 to +1 and a deletion and / or insertion of a nucleic acid residue at a position other than positions -1 to +1;

[0058] (c) Mismatched portion at positions -1 to +1.

[0059]

[28] According to the above method, wherein the destabilizing parts (a), (b) and (c) are respectively the following (a1), (b1) and (c1):

[0060] (a1) a combination of one abasic part at positions -1 to +1 and one or two deletions of nucleic acid residues at positions other than -1 to +1 and / or one or two insertions of nucleic acid residues;

[0061] (b1) a combination of one mismatch at positions -1 to +1 and one or two deletions and / or one or two insertions of nucleic acid residues at positions other than -1 to +1;

[0062] (c1) One mismatched portion at positions -1 to +1.

[0063]

[29] According to the above method, wherein the destabilizing parts (a), (b) and (c) are respectively the following (a2), (b2) and (c2):

[0064] (a2) a combination of an abasic portion at positions -1 to +1, an insertion of a group of nucleic acid residues at a negative position other than position -1, and an insertion of a group of nucleic acid residues at a positive position other than position +1;

[0065] (b2) a combination of one mismatched portion at positions -1 to +1, one group of nucleic acid residues inserted at a negative position other than position -1, and one group of nucleic acid residues inserted at a positive position other than position +1;

[0066] (c2) One mismatched portion at positions -1 to +1.

[0067]

[30] According to the above method, the destabilizing portion reduces the melting temperature of the hybrid by 1 to 60°C.

[0068]

[31] According to the above method, the length of the above target oligonucleic acid is 10 to 200 residues.

[0069]

[32] According to the above method, wherein the length of each of the above substrate oligonucleotides is 5 to 50 residues.

[0070]

[33] According to the above method, the lengths of the second base sequence and the third base sequence are 5 to 50 residues respectively.

[0071]

[34] According to the above method, wherein the length of each of the complementary oligonucleotides is 5 to 300% based on the length of the target oligonucleotide as 100%.

[0072]

[35] The method as described above, wherein the length of each complementary oligonucleotide is 5 to 300% of the total length of the 5' substrate oligonucleotide and the 3' substrate oligonucleotide, with the total length of the 5' substrate oligonucleotide and the 3' substrate oligonucleotide being 100%.

[0073]

[36] According to the above method, in each of the above complementary oligonucleic acids, more than 5 residues of the nucleic acid residues constituting the above second base sequence remain, and more than 5 residues of the nucleic acid residues constituting the above third base sequence remain.

[0074]

[37] According to the above method, in each of the above complementary oligonucleotides, more than 50% of the nucleic acid residues constituting the above second base sequence remain, and more than 50% of the nucleic acid residues constituting the above third base sequence remain.

[0075]

[38] According to the above method, in each of the above complementary oligonucleic acids, more than three consecutive residues of the nucleic acid residues constituting the above second base sequence remain, and more than three consecutive residues of the nucleic acid residues constituting the above third base sequence remain.

[0076]

[39] According to the above method, the above target oligonucleic acid is composed of DNA residues, RNA residues, modified nucleic acid residues or a combination thereof.

[0077]

[40] According to the above method, wherein the above target oligonucleic acid is modified.

[0078]

[41] According to the above method, any one or more of the above complementary oligonucleotides are modified.

[0079]

[42] According to the above method, wherein the above modification includes modification of the phosphate part, modification of the sugar part, modification of the base part or a combination thereof.

[0080]

[43] According to the above method, wherein the modification of the phosphate moiety comprises phosphorothioate, boranophosphate, insertion of a linker or a combination thereof.

[0081]

[44] According to the above method, wherein the modification of the sugar part includes 2'-MOE, 2'-OMe, 2'-F, 4'-thio-2'-OMe, cross-linking formation between the 2' and 4' positions of the sugar part, modification of the 5' end of the oligonucleotide, modification of the 3' end of the oligonucleotide or a combination thereof.

[0082]

[45] According to the above method, wherein the above N is 2.

[0083]

[46] According to the above method, wherein the above N is greater than or equal to 3.

[0084]

[47] According to the above method, wherein the above N is less than 10.

[0085]

[48] ​​According to the above method, wherein the above M is less than N-1.

[0086]

[49] According to the above method, wherein the above M is N-1.

[0087]

[50] According to the above method, wherein the above M is greater than N-1.

[0088]

[51] According to the above method, the above step is carried out at 5 to 60°C.

[0089]

[52] According to the above method, the amount of each complementary oligonucleotide used is 50% or less of the smaller of the amount of the 5' substrate oligonucleotide used and the amount of the 3' substrate oligonucleotide used in terms of molar ratio.

[0090]

[53] According to the above method, wherein the concentration of each of the above substrate oligonucleotides in the reaction solution in the above step is 1 to 10000 μM.

[0091]

[54] According to the above method, the enzyme used for the above enzyme ligation is T3 DNA ligase. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] [ Figure 1A ] is a diagram showing the structure of the oligonucleotide (5' terminal substrate, 3' terminal substrate and complementary strand) used in the examples and the hybridization structure of the assumed oligonucleotide. In each hybridization structure, the upper side represents the substrate and the lower side represents the complementary strand. "Ab" represents the abasic part.

[0093] [ Figure 1B ] is a graph showing the amount of products when a ligation reaction of two substrates is carried out using a complementary chain into which an abasic portion is introduced or a complementary chain into which an abasic portion and a loop structure are introduced.

[0094] [ Figure 2] is a graph showing the product yield when a ligation reaction is carried out on two substrates whose sugar parts have been modified using a complementary chain into which an abasic part has been introduced or a complementary chain into which an abasic part and a ring structure have been introduced.

[0095] [ Figure 3 ] is a graph showing the product yield when a ligation reaction is carried out on two substrates whose phosphate parts have been modified using a complementary chain introduced with an abasic part, a complementary chain introduced with a loop structure, or a complementary chain introduced with an abasic part and a loop structure.

[0096] [ Figure 4 ] is a graph showing the product yield when a ligation reaction of two substrates is carried out using a complementary chain having an introduced loop structure.

[0097] [ Figure 5A ] is a diagram showing the structure of the oligonucleic acid used in the examples and the assumed hybridization structure of the substrate and the complementary chain. In each hybridization structure, the upper side represents the substrate and the lower side represents the complementary chain.

[0098] [ Figure 5B ] is a graph showing the product yield when a ligation reaction of two substrates is carried out using a complementary chain into which a deletion of one or more nucleic acid residues is introduced.

[0099] [ Figure 6 ] is a graph showing the product yield when a ligation reaction of two substrates is carried out using a complementary chain into which one or more ring structures are introduced.

[0100] [ Fig. 7A ] is a diagram showing the structure of the oligonucleotide (5' terminal substrate, 3' terminal substrate and complementary strand) used in the examples and the hybridization structure of the assumed oligonucleotide. In each hybridization structure, the upper side represents the substrate and the lower side represents the complementary strand. "Ab" represents the abasic part.

[0101] [ Figure 7B ] is a graph showing the product yield when a ligation reaction of two substrates is carried out using a complementary chain into which one or more abasic parts are introduced.

[0102] [ Fig. 8A ] is a diagram showing the structure of the oligonucleotide (5' terminal substrate, 3' terminal substrate and complementary strand) used in the examples and the hybridization structure of the assumed oligonucleotide. In each hybridization structure, the upper side represents the substrate and the lower side represents the complementary strand.

[0103] [ Figure 8B ] is a graph showing the product yield when a ligation reaction of two substrates is carried out using a complementary chain into which a mismatch portion is introduced.

[0104] [ Fig.9A] is a graph showing the product yield when a ligation reaction of two substrates is performed using a complementary chain with an abasic portion or a complementary chain with an abasic portion and a ring structure under various substrate concentrations and complementary chain concentrations. The concentration on the horizontal axis represents the substrate concentration (common to both the 5' terminal side substrate and the 3' terminal side substrate). The complementary chain concentration is 1 / 10 of the substrate concentration.

[0105] [ Fig. 9B ] is a graph showing the product generation rate per unit enzyme activity when a ligation reaction of two substrates is performed using a complementary chain with an abasic portion or a complementary chain with an abasic portion and a ring structure under various substrate concentrations and complementary chain concentrations. The concentration on the horizontal axis represents the substrate concentration (common to both the 5' terminal side substrate and the 3' terminal side substrate). The complementary chain concentration is 1 / 10 of the substrate concentration.

[0106] [ Fig.10 ] is a diagram showing the generation of products when a ligation reaction of two substrates of 50 residues is carried out using a complementary chain into which an abasic portion is introduced or a complementary chain into which an abasic portion and a loop structure are introduced.

[0107] [ Fig.11 ] is a diagram showing the generation of products when a ligation reaction of two substrates containing 50 residues of a modified nucleic acid is carried out using a complementary chain into which an abasic portion has been introduced or a complementary chain into which an abasic portion and a loop structure have been introduced.

[0108] [ Fig.12 ] is a diagram showing the generation of products when a three-substrate ligation reaction is carried out using a complementary chain into which an abasic portion is introduced or a complementary chain into which an abasic portion and a loop structure are introduced.

[0109] [ Fig.13 ] is a diagram showing the generation of products when a ligation reaction of three substrates containing a modified nucleic acid is carried out using a complementary chain into which an abasic portion has been introduced or a complementary chain into which an abasic portion and a loop structure have been introduced.

[0110] [ Fig.14 ] is a diagram showing the generation of products when a ligation reaction of two substrates comprising a modified nucleic acid and RNA is carried out using a complementary chain into which an abasic portion has been introduced or a complementary chain into which an abasic portion and a loop structure have been introduced. DETAILED DESCRIPTION

[0111] Hereinafter, the present invention will be described in detail.

[0112] The method of the present invention is a method for ligating single-stranded oligonucleotides. The ligated single-stranded oligonucleotides are also referred to as "single-stranded substrate oligonucleotides" or "substrate oligonucleotides", respectively.

[0113] By connecting the substrate oligonucleotides, an oligonucleotide formed by connecting the substrate oligonucleotides is generated. The generated oligonucleotide is also called a "target oligonucleotide". Therefore, the method of the present invention can be a method for producing a target oligonucleotide.

[0114] The method of the present invention includes a step of enzymatically ligating N (N is an integer greater than or equal to 2) substrate oligonucleotides. This step is also referred to as a "ligation step". The ligation step may be a step of enzymatically ligating N (N is an integer greater than or equal to 2) substrate oligonucleotides to generate a target oligonucleotide. The N (N is an integer greater than or equal to 2) substrate oligonucleotides ligated in the ligation step are also collectively referred to as a "substrate oligonucleotide set".

[0115] The connection process is implemented in the presence of a single-stranded oligonucleotide with a specific structure. The single-stranded oligonucleotide with a specific structure is also referred to as "single-stranded complementary oligonucleotide" or "complementary oligonucleotide". Specifically, the connection process can be implemented in the presence of M complementary oligonucleotides (M is an integer greater than 1). That is, the connection process can be a process in which N substrate oligonucleotides (N is an integer greater than 2) are enzymatically connected in the presence of M complementary oligonucleotides (M is an integer greater than 1). In addition, the connection process can also be a process in which N substrate oligonucleotides (N is an integer greater than 2) are enzymatically connected in the presence of M complementary oligonucleotides (M is an integer greater than 1) to generate a target oligonucleotide. More specifically, the connection process can be implemented in the presence of complementary oligonucleotides corresponding to each connecting portion. That is, each complementary oligonucleotide can be used to connect the substrate oligonucleotide of the connecting portion corresponding to the complementary oligonucleotide. More specifically, the connection between the substrate oligonucleotides on the 5' side and the 3' side of each connecting portion can be implemented in the presence of the complementary oligonucleotide corresponding to the connecting portion. That is, each complementary oligonucleotide can be used for ligation between the substrate oligonucleotides on the 5' side and the 3' side of the linking portion corresponding to the complementary oligonucleotide.

[0116] According to the method of the present invention, the ligation process can be effectively implemented, for example, by repeatedly using the complementary oligonucleotide in a catalytic manner in the ligation process. Specifically, for example, due to the effect of the destabilizing part, the complementary oligonucleotide is easily naturally dissociated from the ligation product (i.e., the target oligonucleotide) of the substrate oligonucleotide, and thus the complementary oligonucleotide can be reused in the ligation process. In other words, according to the method of the present invention, for example, the amount of complementary oligonucleotide used can be reduced.

[0117] <1> Target oligonucleotide

[0118] "Target oligonucleic acid" refers to an oligonucleic acid produced by the method of the present invention.

[0119] The target oligonucleic acid can exist in the form of a single-stranded oligonucleic acid, for example, or in the form of a double-stranded oligonucleic acid. That is, the target oligonucleic acid can be manufactured in the form of a single-stranded oligonucleic acid, for example, or in the form of a double-stranded oligonucleic acid. In addition, the target oligonucleic acid can be used in the form of a single-stranded oligonucleic acid, for example, or in the form of a double-stranded oligonucleic acid.

[0120] "Single-stranded oligonucleotide" refers to an oligonucleotide consisting of one nucleic acid chain that does not hybridize with other nucleic acid chains. That is, for example, "target oligonucleotide in the form of a single-stranded oligonucleotide" may refer to a target oligonucleotide in the form that does not hybridize with other nucleic acid chains. As long as it does not impair the purpose of the present invention, the single-stranded oligonucleotide may or may not form a double-stranded structure (e.g., a stem-loop structure) within the molecule.

[0121] "Double-stranded oligonucleotide" refers to an oligonucleotide composed of at least two nucleic acid chains that hybridize with each other. That is, for example, "a target oligonucleotide in the form of a double-stranded oligonucleotide" may refer to a target oligonucleotide in the form of hybridization with other nucleic acid chains. As other nucleic acid chains, complementary oligonucleotides may be listed. That is, the target oligonucleotide may exist, be manufactured and / or be used in the form of hybridization with one or more complementary oligonucleotides, for example.

[0122] The form of the target oligonucleotide (for example, existing, manufacturing and / or using in any form of a single-stranded oligonucleotide and a double-stranded oligonucleotide) can be appropriately set according to various conditions such as the purpose of the target oligonucleotide. There is no particular limitation on the purpose of the target oligonucleotide. As the purpose of the target oligonucleotide, it can be listed as: the use as an antisense nucleic acid, siRNA, miRNA mimics, decoy nucleic acid, aptamer or CpG oligonucleotide. In the case of using the target oligonucleotide as an antisense nucleic acid, aptamer or CpG oligonucleotide, the target oligonucleotide can, for example, exist, manufacture and / or use in the form of a single-stranded oligonucleotide. In the case of using the target oligonucleotide as an siRNA, miRNA mimics or decoy nucleic acid, the target oligonucleotide can, for example, exist, manufacture and / or use in the form of a double-stranded oligonucleotide or a single-stranded oligonucleotide with a double-stranded structure formed in the molecule.

[0123] There is no particular restriction on the type of nucleic acid residues constituting the target oligonucleotide. The type of nucleic acid residues constituting the target oligonucleotide can be appropriately set according to various conditions such as the purpose of the target oligonucleotide. As nucleic acid residues, there can be cited: DNA residues, RNA residues, modified nucleic acid residues. The target oligonucleotide may contain one or more nucleic acid residues. That is, the target oligonucleotide may contain, for example, DNA residues, RNA residues, modified nucleic acid residues or a combination thereof. The target oligonucleotide may be a nucleic acid chain consisting of one nucleic acid residue, or a nucleic acid chain consisting of a combination of two or more nucleic acid residues (i.e., a nucleic acid chain containing two or more nucleic acid residues in the molecule). That is, the target oligonucleotide may be, for example, a nucleic acid chain consisting of DNA residues, or a nucleic acid chain consisting of RNA residues, or a nucleic acid chain consisting of modified nucleic acid residues, or a nucleic acid chain consisting of a combination thereof (i.e., a nucleic acid chain containing two or more nucleic acid residues selected from DNA residues, RNA residues and modified nucleic acid residues in the molecule).

[0124] There is no particular restriction on the base sequence of the target oligonucleotide. The base sequence of the target oligonucleotide can be appropriately set according to various conditions such as the purpose of the target oligonucleotide. In the case of using the target oligonucleotide as an antisense nucleic acid, siRNA, miRNA mimics, decoy nucleic acid, aptamer or CpG oligonucleotide, the base sequence of the target oligonucleotide can be set to the target oligonucleotide as an antisense nucleic acid, siRNA, miRNA mimics, decoy nucleic acid, aptamer or CpG oligonucleotide to function. That is, for example, in the case of using the target oligonucleotide as an antisense nucleic acid, the base sequence of the target oligonucleotide can be set to the target oligonucleotide can form a double-stranded structure with the single-stranded RNA sequence of the object specifically. As the single-stranded RNA of the object of the antisense nucleic acid, it can be listed as: mRNA, pre-mRNA, miRNA. In addition, for example, in the case of using the target oligonucleotide as an siRNA or miRNA mimic, the base sequence of the target oligonucleotide can be set to the target oligonucleotide (specifically, the single-stranded structure generated by the dissociation of the target oligonucleotide) can form a double-stranded structure with the single-stranded RNA sequence of the object specifically. The single-stranded RNA as the object of siRNA or miRNA mimics can be exemplified as mRNA. In addition, for example, when the target oligonucleotide is used as a bait nucleic acid, an aptamer or a CpG oligonucleotide, the base sequence of the target oligonucleotide can be set so that the target oligonucleotide can specifically bind to the protein sequence of the object. The base sequence of the target oligonucleotide can be set, for example, so that the target oligonucleotide does not form a double-stranded structure in the molecule, or can be set so that the target oligonucleotide forms a double-stranded structure in the molecule.

[0125] There is no particular limitation on the length of the target oligonucleotide. The length of the target oligonucleotide can be appropriately set according to various conditions such as the purpose of the target oligonucleotide. The length of the target oligonucleotide can be, for example, 10 residues or more, 11 residues or more, 12 residues or more, 13 residues or more, 14 residues or more, 15 residues or more, 16 residues or more, 17 residues or more, 18 residues or more, 19 residues or more, 20 residues or more, 21 residues or more, 22 residues or more, 23 residues or more, 24 residues or more, 25 residues or more, 30 residues or more, 35 residues or more, 40 residues or more, 45 residues or more, 50 residues or more, 60 residues or more, 70 residues or more, 80 residues or more, 90 residues or more, 100 residues or more, 110 residues or more, 120 residues or more, 130 residues or more, 140 residues or more, 150 residues or more, 160 residues or more, 170 residues or more, 180 residues or more, or 190 residues or more. It is 200 residues or less, 190 residues or less, 180 residues or less, 170 residues or less, 160 residues or less, 150 residues or less, 140 residues or less, 130 residues or less, 120 residues or less, 110 residues or less, 100 residues or less, 90 residues or less, 80 residues or less, 70 residues or less, 60 residues or less, 50 residues or less, 45 residues or less, 40 residues or less, 35 residues or less, 30 residues or less, 25 residues or less, 24 residues or less, 23 residues or less, 22 residues or less, 21 residues or less, 20 residues or less, 19 residues or less, 18 residues or less, 17 residues or less, 16 residues or less, 15 residues or less, 14 residues or less, 13 residues or less, 12 residues or less, or 11 residues or less, and any non-contradictory combination thereof.Specifically, the length of the target oligonucleotide can be, for example, 10 to 11 residues, 11 to 12 residues, 12 to 13 residues, 13 to 14 residues, 14 to 15 residues, 15 to 16 residues, 16 to 17 residues, 17 to 18 residues, 18 to 19 residues, 19 to 20 residues, 20 to 21 residues, 21 to 22 residues, 22 to 23 residues, 23 to 24 residues, 24 to 25 residues, 25 to 30 residues, 30 to 35 residues, 35 to 40 residues. 170 residues, 170-180 residues, 180-190 residues or 190-200 residues. Specifically, the length of the target oligonucleotide can be, for example, 10 to 200 residues, 10 to 100 residues, 15 to 80 residues or 20 to 60 residues. Specifically, the length of the target oligonucleotide can be, for example, 10 to 200 residues, 10 to 150 residues, 15 to 120 residues, or 20 to 100 residues.

[0126] “DNA” without mentioning modification may refer to natural type DNA, and specifically, may refer to a nucleic acid whose nucleoside moiety is deoxyadenosine, deoxyguanosine, deoxycytidine, thymidine, or deoxyuridine.

[0127] “RNA” without mentioning modification may refer to natural RNA, and specifically, may refer to a nucleic acid whose nucleoside moiety is adenosine, guanosine, cytidine, 5-methyluridine, or uridine.

[0128] "Modified nucleic acid" may refer to nucleic acids other than natural DNA and natural RNA. Examples of modified nucleic acids include modified DNA and modified RNA. "Modified DNA" may refer to nucleic acids having the same structure as natural DNA except that they are modified. "Modified RNA" may refer to nucleic acids having the same structure as natural RNA except that they are modified.

[0129] The terms "the target oligonucleotide comprises a modified nucleic acid residue", "the target oligonucleotide is modified" and "the target oligonucleotide has a modification" are used interchangeably.

[0130] The terms "a nucleic acid residue is a modified nucleic acid residue", "a nucleic acid residue is modified" and "a nucleic acid residue has a modification" are used interchangeably.

[0131] The target oligonucleotide may or may not contain modified nucleic acid residues. That is, the target oligonucleotide may or may not be modified. The modification mode of the target oligonucleotide (for example, the type, position and amount of the modification) is not particularly limited as long as it does not damage the purpose of the present invention. The modification mode of the target oligonucleotide can be appropriately set according to various conditions such as the purpose of the target oligonucleotide. As modifications, there can be cited: modification of the phosphate portion, modification of the sugar portion, modification of the base portion. That is, the modification may, for example, include modification of the phosphate portion, modification of the sugar portion, modification of the base portion or a combination thereof. "Modification includes a certain modification A" means at least selecting modification A as a modification, including the situation where the modification is composed of modification A and the situation where the modification is composed of a combination of modification A and other modifications. For example, "modification includes modification of the phosphate portion" means at least selecting modification of the phosphate portion as a modification, including the situation where the modification is composed of modification of the phosphate portion and the situation where the modification is composed of a combination of modification of the phosphate portion and other modifications.

[0132] As the phosphate part, the following can be cited: the phosphate group present between the nucleic acid residues (i.e., the phosphate group that forms a phosphodiester bond between the nucleic acid residues). In the case where the phosphate group present between the nucleic acid residues is modified, the nucleic acid residue on the 3' side of the phosphate group (i.e., the nucleic acid residue having the phosphate group at the 5' position) is deemed to be modified. As modifications of the phosphate part, the following can be cited: phosphorothioate (i.e., the oxygen atom of the phosphate group is substituted with a sulfur atom; in other words, the phosphate group is substituted with a thiophosphate group) or boranophosphate (i.e., the oxygen atom of the phosphate group is substituted with borane (BH3); in other words, the phosphate group is substituted with a boranophosphate group). As modifications of the phosphate part, the following can also be cited: the insertion of a linker. "Linker" refers to a structure other than a nucleic acid residue. As a linker, the following can be cited: a hydrocarbon group. As a hydrocarbon group, the following can be cited: a non-aromatic hydrocarbon group or an aromatic hydrocarbon group. As a non-aromatic hydrocarbon group, the following can be cited: an alkylene group or an alkenylene group. The alkylene group can be straight chain or branched. Examples of the alkylene group include alkylene groups having 1 to 12, 1 to 8, 1 to 6 or 1 to 4 carbon atoms. Examples of the alkylene group having 1 to 4 carbon atoms include methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene and tert-butylene. The alkenylene group may be straight-chain or branched. Examples of the alkenylene group include alkenylene groups having 2 to 12, 2 to 8, 2 to 6 or 2 to 4 carbon atoms. Examples of the alkenylene group having 2 to 4 carbon atoms include vinylene, propenylene, isopropenylene, butenylene, isobutenylene and sec-butenylene. The alkenylene group may be, for example, cis or trans. Examples of the aromatic hydrocarbon group include aromatic hydrocarbon groups having 6 to 12 carbon atoms. Examples of the aromatic hydrocarbon group having 6 to 12 carbon atoms include benzylene. The linker may be bonded to a phosphate group at both ends, for example. That is, the linker can be bonded to the phosphate group at the 3' position of the nucleic acid residue on its 5' side and the phosphate group at the 5' position of the nucleic acid residue on its 3' side, for example. In other words, the insertion of the linker in the phosphate group between the nucleic acid residues can be to replace the phosphate group between the nucleic acid residues with a linker clamped by the phosphate group. The phosphate groups bonded by the linker can each be modified (for example, phosphorothioate or boranophosphate), or may not be modified. For example, in the case where the complementary oligonucleotide includes the insertion of a nucleic acid residue as a destabilizing portion, the linker can be inserted into the position corresponding to the insertion of the nucleic acid residue in the target oligonucleotide.

[0133] As the sugar part, deoxyribose as the sugar part of DNA or ribose as the sugar part of RNA can be listed. As the modification of the sugar part, 2'-MOE (i.e., O-methoxyethylation at the 2' position of the sugar part), 2'-OMe (i.e., O-methylation at the 2' position of the sugar part), 2'-F (i.e., fluorination at the 2' position of the sugar part), 4'-thio-2'-OMe (i.e., the oxygen atom at the 4' position of the sugar part is replaced by a sulfur atom and O-methylation at the 2' position of the sugar part), and cross-linking between the 2' and 4' positions of the sugar part can be listed. As the cross-linking between the 2' and 4' positions of the sugar part, LNA, BNANC 、BNA NC (Me), AmNA. As the modification of the sugar part, the modification of the 5' end of the oligonucleotide or the modification of the 3' end of the oligonucleotide can also be listed. As the modification of the 5' end of the oligonucleotide, the modification of 5'-amination, 5'-thiolation, 5'-dabsylation, 5'-fluoresceination, 5'-tetrafluorofluoresceination, 5'-phosphorylation, 5'-inverted thymidine, 5'-biotin addition, 5'-PEG addition, 5'-N-acetylgalactosamine (GalNAc) addition, 5'-peptide addition, 5'-cholesterol addition, 5'-tocopherol addition, 5'-fat chain addition, 5'-folic acid addition, 5'-polyamine addition, 5'-drug addition can be listed. Examples of modifications of the 3' end of the oligonucleotide include 3'-amination, 3'-thiolation, 3'-dabsylation, 3'-deoxidation, 3'-carboxylation, 3'-phosphorylation, 3'-inverted thymidine, 3'-biotin addition, 3'-PEG addition, 3'-N-acetylgalactosamine (GalNAc) addition, 3'-peptide addition, 3'-cholesterol addition, 3'-tocopherol addition, 3'-fat chain addition, 3'-folic acid addition, 3'-polyamine addition, and 3'-drug addition. Examples of drugs include anticancer drugs and other various drug components. Adducts such as drugs can be added to oligonucleic acids directly or via appropriate linkers (Do-Hun Kim et al., Design and clinical developments of aptamer-drug conjugates for targeted cancer therapy, Biomater Res. 2021 Nov 25; 25(1): 42.).

[0134] Examples of modifications of the phosphate moiety and sugar moiety include morpholino or peptidyl modification of the nucleic acid backbone. That is, examples of modified nucleic acids include morpholino nucleic acids and peptide nucleic acids (PNA).

[0135] Examples of the base part include adenine (A), thymine (T), guanine (G), cytosine (C), and uracil (U). Examples of modifications of the base part include alkylation (methylation, etc.), O-alkylation (O-methylation, etc.), N-alkylation (N-methylation, etc.), aminoalkylation (aminopropylation, etc.), alkoxylation (methoxylation, etc.), halogenation (bromination, fluorination, iodination, chlorination, etc.), acylation, amination, carboxylation, hydroxylation, oxidation, nitration, sulfation, aza-hydration, denitrification, and substitution with other various analogs. Specific examples of the modified base include: 8-haloadenine (halogen is bromine, fluorine, iodine or chlorine; the same applies hereinafter), 8-haloguanine, 5-halothymine, 5-halocytosine, 5-halouracil, 1-deazaadenine, 3-deazaadenine or 7-deazaadenine, 1-deazaguanine, 3-deazaguanine or 7-deazaguanine, 3-deazathymine, 3-deazacytosine, 3-deazauracil, 8-azaadenine, 8-azaguanine, 5-azathymine or 6-azathymine, 5-azacytosine or 6-azauracil. -azacytosine, 5-azauracil or 6-azauracil, 3-methyladenine, N6-methyladenine, O6-methylguanine, O4-methylthymine, 5-methylcytosine, 5-(2-amino)propyluracil, 2-aminoadenine, 8-oxoadenine, 8-oxoguanine, 2-thiothymine or 4-thiothymine, 2-thiocytosine or 4-thiocytosine, 2-thiouracil or 4-thiouracil, 2-aminopurine, 2,6-diaminopurine, hypoxanthine, 7-deaza-8-azahypoxanthine. In addition, as modified bases, specifically, artificial bases such as x, y, s, Ds, Pa, PxR, PICS, 5SICS, NaM, MMO2, TPT3, iG, iC, P, and Z can also be listed (Hirao et al., Artificial bases for the expansion of genetic information: Towards a new research field of Xenobiology, Chemistry and Biology Vol. 54, No. 11, 2016, pp. 835-840). As modifications of the base part, the deletion of the base part can be listed. In other words, the modified nucleic acid can be a nucleic acid lacking a base part (i.e., abasic nucleic acid).

[0136] The presence or absence and type of modification can be independently set for each nucleic acid residue constituting the target oligonucleotide. The target oligonucleotide may have one modification, or two or more modifications may be combined. The target oligonucleotide may, for example, include or not include the various modifications exemplified above. Each nucleic acid residue constituting the target oligonucleotide may have one modification, or two or more modifications may be combined. The nucleic acid residues constituting the target oligonucleotide may only be partially modified nucleic acid residues, or the nucleic acid residues constituting the target oligonucleotide may all be modified nucleic acid residues.

[0137] The ratio of the modified nucleic acid residues in the target oligonucleotide (i.e., the ratio of the number of modified nucleic acid residues to the number of nucleic acid residues constituting the target oligonucleotide) can be, for example, 0% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or less, 100% or less, 95% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, or 5% or less, and can also be a non-contradictory combination thereof. Specifically, the ratio of the modified nucleic acid residues in the target oligonucleotide can be, for example, 0-5%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95% or 95-100%. It should be noted that the ratio of the modified nucleic acid residues in the target oligonucleotides exemplified above can also be independently applied to the selected nucleic acid residues with any modification (for example, any of the modifications exemplified above). That is, for example, the ratio of the modified nucleic acid residues with a phosphate portion in the target oligonucleotide (that is, the ratio of the number of modified nucleic acid residues with a phosphate portion to the number of nucleic acid residues constituting the target oligonucleotide) can be set to the ratio of the modified nucleic acid residues in the target oligonucleotides exemplified above. In addition, for example, the ratio of the modified nucleic acid residues with a sugar portion in the target oligonucleotide (that is, the ratio of the number of modified nucleic acid residues with a sugar portion to the number of nucleic acid residues constituting the target oligonucleotide) can be set to the ratio of the modified nucleic acid residues in the target oligonucleotides exemplified above. In addition, for example, the ratio of the modified nucleic acid residues having a base portion in the target oligonucleotide (i.e., the ratio of the number of modified nucleic acid residues having a base portion to the number of nucleic acid residues constituting the target oligonucleotide) can be set to the ratio of the modified nucleic acid residues in the target oligonucleotide exemplified above.

[0138] The number of modified nucleic acid residues in the target oligonucleic acid may be, for example, 0 residues or more, 5 residues or more, 10 residues or more, 15 residues or more, 20 residues or more, 25 residues or more, 30 residues or more, 40 residues or more, 50 residues or more, 60 residues or more, 80 residues or more, 100 residues or more, 120 residues or more, 140 residues or more, 160 residues or more, or 180 residues or more; or 200 residues or less, 180 residues or less, 160 residues or less, 140 residues or less, 120 residues or less, 100 residues or less, 80 residues or less, 60 residues or less, 50 residues or less, 40 residues or less, 30 residues or less, 25 residues or less, 20 residues or less, 15 residues or less, 10 residues or less, or 5 residues or less; or any non-contradictory combination thereof. Specifically, the number of modified nucleic acid residues in the target oligonucleotide can be, for example, 0 to 5 residues, 5 to 10 residues, 10 to 15 residues, 15 to 20 residues, 20 to 25 residues, 25 to 30 residues, 30 to 40 residues, 40 to 50 residues, 50 to 60 residues, 60 to 80 residues, 80 to 100 residues, 100 to 120 residues, 120 to 140 residues, 140 to 160 residues, 160 to 180 residues or 180 to 200 residues. It should be noted that the number of modified nucleic acid residues in the target oligonucleotide exemplified above can also be independently applied to the selected nucleic acid residues with any modification (for example, any of the modifications exemplified above). That is, for example, the number of modified nucleic acid residues with a phosphate portion in the target oligonucleotide can be set to the number of modified nucleic acid residues in the target oligonucleotide exemplified above. In addition, for example, the number of modified nucleic acid residues with a sugar portion in the target oligonucleotide can be set to the number of modified nucleic acid residues in the target oligonucleotide exemplified above. Furthermore, for example, the number of nucleic acid residues having a modification of the base portion in the target oligonucleotide can be set to the number of modified nucleic acid residues in the target oligonucleotide exemplified above.

[0139] Unless otherwise specified, a modified nucleic acid residue having the same structure as deoxyribose at the 2' position of the sugar moiety is equivalent to a modified DNA residue. Unless otherwise specified, a modified nucleic acid residue having the same structure as ribose at the 2' position of the sugar moiety is equivalent to a modified RNA residue. Unless otherwise specified, a modified nucleic acid residue having a modification at the 2' position of the sugar moiety is equivalent to both a modified DNA residue and a modified RNA residue.

[0140] The target oligonucleic acid can be modified according to the modification method exemplified above when the substrate oligonucleic acid is connected, or it can be modified to the modification method exemplified above after the substrate oligonucleic acid is connected. That is, the modified target oligonucleic acid can be manufactured by connecting a substrate oligonucleic acid modified according to the modification method of the target oligonucleic acid, or it can be manufactured by connecting a substrate oligonucleic acid that is not modified according to the modification method of the target oligonucleic acid (for example, it can be unmodified) and modifying the connection product after connection.

[0141] <2>Substrate oligonucleotides and their compositions

[0142] The "substrate oligonucleic acid" refers to each single-stranded oligonucleic acid to be ligated in the ligation step, in other words, each nucleic acid fragment constituting the target oligonucleic acid.

[0143] "Single strand" about substrate oligonucleotide refers to the form of substrate oligonucleotide at the beginning of the method (specifically, the connection process) of the present invention. As the method (specifically, the connection process) of the present invention proceeds, the substrate oligonucleotide can form the form of double-stranded oligonucleotide. For example, as the method (specifically, the connection process) of the present invention proceeds, the substrate oligonucleotide can hybridize with the complementary oligonucleotide and form the form of double-stranded oligonucleotide.

[0144] The "substrate oligonucleotide group" is a collective term for the substrate oligonucleotides to be ligated in the ligation step. The substrate oligonucleotide group is composed of N (N is an integer greater than or equal to 2) substrate oligonucleotides.

[0145] The number (N) of substrate oligonucleotides is 2 or more. "The number of substrate oligonucleotides" refers to the number of substrate oligonucleotides connected in the connection process, in other words, it refers to the number of substrate oligonucleotides constituting the target oligonucleotide. In addition, in other words, "the number of substrate oligonucleotides" refers to the number of substrate oligonucleotides constituting the substrate oligonucleotide group. The number (N) of substrate oligonucleotides can be 2 or 3 or more. The number (N) of substrate oligonucleotides can be, for example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or 9 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, or 3 or less, or a combination thereof that is not contradictory. Specifically, the number (N) of substrate oligonucleotides can be, for example, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10. Specifically, the number (N) of substrate oligonucleotides can be, for example, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. The number (N) of substrate oligonucleotides may particularly be 2 to 3. The number (N) of substrate oligonucleotides may more particularly be 2.

[0146] The connecting portion between substrate oligonucleotides is also referred to as a "connecting portion". The two substrate oligonucleotides connected by each connecting portion are also referred to as "5' side substrate oligonucleotide" and "3' side substrate oligonucleotide". The term "5' side" and the term "5' terminal side" are used interchangeably. The term "3' side" and the term "3' terminal side" are used interchangeably. In each connecting portion, the 3' end of the 5' side substrate oligonucleotide is connected to the 5' end of the 3' side substrate oligonucleotide. It should be noted that the "5' side" and "3' side" of the substrate oligonucleotide respectively represent the same directions as the "5' side" and "3' side" of the target oligonucleotide. In addition, the "5' side" and "3' side" of the substrate oligonucleotide respectively represent the reverse directions of the "5' side" and "3' side" of the complementary oligonucleotide hybridized with the substrate oligonucleotide. In addition, the "5' side" and "3' side" of the substrate oligonucleotide represent the relative directions based on each connecting portion. That is, for example, in the case of connecting 3 substrate oligonucleotides A, B and C in the 5' to 3' direction, A and B are 5' side and 3' side substrate oligonucleotides, respectively, in the connection between A and B, B and C are 5' side and 3' side substrate oligonucleotides, respectively. The number of connectors is determined according to the number of substrate oligonucleotides. That is, if the number of substrate oligonucleotides is N (N is an integer greater than 2), the number of connectors is N-1, in other words, the connection step is implemented at the connector at N-1. In addition, in other words, the target oligonucleotide is composed of N substrate oligonucleotides connected at N-1. "Each connector" refers to the connector when the number of connectors is 1, and refers to each of these connectors when the number of connectors is 2 or more.

[0147] The structure of each substrate oligonucleotide (e.g., the type of nucleic acid residues, base sequence, length, and modification method) can be appropriately set according to the structure of the target oligonucleotide. That is, the structure of each substrate oligonucleotide can be consistent with the structure of the portion corresponding to each substrate oligonucleotide in the target oligonucleotide.

[0148] About the kind of nucleic acid residue, as described above.Each substrate oligonucleic acid can include one or more nucleic acid residues.That is, each substrate oligonucleic acid can include DNA residues, RNA residues, modified nucleic acid residues or their combination.Each substrate oligonucleic acid can be a nucleic acid chain consisting of one nucleic acid residue, or a nucleic acid chain consisting of a combination of two or more nucleic acid residues (that is, a nucleic acid chain containing two or more nucleic acid residues in the molecule).That is, each substrate oligonucleic acid can be a nucleic acid chain consisting of DNA residues, or a nucleic acid chain consisting of RNA residues, or a nucleic acid chain consisting of modified nucleic acid residues, or a nucleic acid chain consisting of a combination of them (that is, a nucleic acid chain containing two or more nucleic acid residues selected from DNA residues, RNA residues and modified nucleic acid residues in the molecule).

[0149] The base sequence of each substrate oligonucleotide is composed of a partial sequence of the base sequence of the target oligonucleotide. In addition, the base sequence of each substrate oligonucleotide is set to a connection sequence of the substrate oligonucleotide that is composed of the base sequence of the target oligonucleotide. For example, when the number of substrate oligonucleotides is 2, the base sequence of the 5' side substrate oligonucleotide is composed of the 5' side partial sequence of the target oligonucleotide, the base sequence of the 3' side substrate oligonucleotide is composed of the 3' side partial sequence of the target oligonucleotide, and the connection sequence of the 5' side substrate oligonucleotide and the 3' side substrate oligonucleotide is composed of the base sequence of the target oligonucleotide.

[0150] The length of each substrate oligonucleotide is not particularly limited as long as it does not impair the purpose of the present invention. That is, the length of each substrate oligonucleotide is set to be able to utilize the method of the present invention to connect substrate oligonucleotides. The length of each substrate oligonucleotide, for example, can be set to each substrate oligonucleotide can be hybridized with the corresponding complementary oligonucleotide." complementary oligonucleotide corresponding to substrate oligonucleotide" refers to the complementary oligonucleotide used in the junction between the substrate oligonucleotide connected thereto. In addition, the length of each substrate oligonucleotide can be set to: the length of the connection sequence formed by the substrate oligonucleotide connection is consistent with the length of the target oligonucleotide.

[0151] The length of each substrate oligonucleotide can be, for example, 5 residues or more, 6 residues or more, 7 residues or more, 8 residues or more, 9 residues or more, 10 residues or more, 11 residues or more, 12 residues or more, 13 residues or more, 14 residues or more, 15 residues or more, 17 residues or more, 20 residues or more, 25 residues or more, 30 residues or more, 35 residues or more, 40 residues or more, 45 residues or more, 50 residues or more, 55 residues or more, 60 residues or more, or 65 residues or more. The term "nucleotide sequence" may be 70 residues or less, 65 residues or less, 60 residues or less, 55 residues or less, 50 residues or less, 45 residues or less, 40 residues or less, 35 residues or less, 30 residues or less, 25 residues or less, 20 residues or less, 17 residues or less, 15 residues or less, 14 residues or less, 13 residues or less, 12 residues or less, 11 residues or less, 10 residues or less, 9 residues or less, 8 residues or less, 7 residues or less, or 6 residues or less, or any non-contradictory combination thereof. Specifically, the length of each substrate oligonucleotide can be, for example, 5 to 6 residues, 6 to 7 residues, 7 to 8 residues, 8 to 9 residues, 9 to 10 residues, 10 to 11 residues, 11 to 12 residues, 12 to 13 residues, 13 to 14 residues, 14 to 15 residues, 15 to 17 residues, 17 to 20 residues, 20 to 25 residues, 25 to 30 residues, 30 to 35 residues, 35 to 40 residues, 40 to 45 residues, 45 to 50 residues, 50 to 55 residues, 55 to 60 residues, 60 to 65 residues or 65 to 70 residues. Specifically, the length of each substrate oligonucleotide can be, for example, 5 to 70 residues, 5 to 50 residues, 5 to 30 residues, 5 to 20 residues or 5 to 15 residues.

[0152] For example, based on the length of the target oligonucleotide as 100%, the length of each substrate oligonucleotide can be more than 3%, more than 5%, more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95%, or it can be less than 97%, less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%, or it can be a non-contradictory combination thereof. Specifically, for example, the length of each substrate oligonucleotide may be 3-5%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95% or 95-97% based on the length of the target oligonucleotide as 100%. Specifically, for example, the length of each substrate oligonucleotide may be 3-97%, 10-90%, 20-80%, 30-70% or 40-60% based on the length of the target oligonucleotide as 100%.

[0153] The terms "the substrate oligonucleotide comprises a modified nucleic acid residue", "the substrate oligonucleotide is modified" and "the substrate oligonucleotide has a modification" are used interchangeably.

[0154] Each substrate oligonucleic acid may or may not include modified nucleic acid residues. That is, each substrate oligonucleic acid may or may not be modified. Each substrate oligonucleic acid may or may not be modified according to the modification mode of the target oligonucleic acid. For example, in the case of modifying the connection product after connecting the substrate oligonucleic acid, the modification mode of the substrate oligonucleic acid may be different from the modification mode of the target oligonucleic acid. About the modification of nucleic acid residues, as described above. Each substrate oligonucleic acid may have 1 modification, or may have 2 or more modifications in combination. Each substrate oligonucleic acid, for example, may or may not include various modifications of the above examples. Each nucleic acid residue constituting each substrate oligonucleic acid may have 1 modification, or may have 2 or more modifications in combination. The nucleic acid residues constituting each substrate oligonucleic acid may only be partially modified nucleic acid residues, and the nucleic acid residues constituting each substrate oligonucleic acid may also all be modified nucleic acid residues.

[0155] The ratio of the modified nucleic acid residues in each substrate oligonucleotide (i.e., the ratio of the number of modified nucleic acid residues to the number of nucleic acid residues constituting each substrate oligonucleotide) can be set so that the ratio of the modified nucleic acid residues in the connection sequence formed by connecting the substrate oligonucleotides is consistent with the ratio of the modified nucleic acid residues in the target oligonucleotide. In addition, the ratio of the modified nucleic acid residues in each substrate oligonucleotide can be, for example, 0% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or less, 100% or less, 95% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, or a non-contradictory combination thereof. Specifically, the ratio of the modified nucleic acid residues in each substrate oligonucleotide can be, for example, 0-5%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95% or 95-100%. It should be noted that the ratio of the modified nucleic acid residues in each substrate oligonucleotide of the above examples can also be independently applied to the selected nucleic acid residues with any modification (for example, any modification of the above examples). That is, for example, the ratio of the modified nucleic acid residues with a phosphate portion in each substrate oligonucleotide (that is, the ratio of the number of modified nucleic acid residues with a phosphate portion to the number of nucleic acid residues constituting each substrate oligonucleotide) can be set to the ratio of the modified nucleic acid residues in each substrate oligonucleotide of the above examples. In addition, for example, the ratio of the modified nucleic acid residues with sugar moieties in each substrate oligonucleic acid (i.e., the ratio of the number of modified nucleic acid residues with sugar moieties to the number of nucleic acid residues constituting each substrate oligonucleic acid) can be set to the ratio of the modified nucleic acid residues in each substrate oligonucleic acid of the above-mentioned examples. In addition, for example, the ratio of the modified nucleic acid residues with base moieties in each substrate oligonucleic acid (i.e., the ratio of the number of modified nucleic acid residues with base moieties to the number of nucleic acid residues constituting each substrate oligonucleic acid) can be set to the ratio of the modified nucleic acid residues in each substrate oligonucleic acid of the above-mentioned examples.

[0156] The number of modified nucleic acid residues in each substrate oligonucleotide can be set to be consistent with the number of modified nucleic acid residues in the connection sequence formed by the substrate oligonucleotide connection. In addition, the number of modified nucleic acid residues in each substrate oligonucleotide can be, for example, more than 0 residues, more than 5 residues, more than 10 residues, more than 15 residues, more than 20 residues, more than 25 residues, more than 30 residues, more than 35 residues, more than 40 residues, more than 45 residues, more than 50 residues, more than 55 residues, more than 60 residues or more than 65 residues, or less than 70 residues, less than 65 residues, less than 60 residues, less than 55 residues, less than 50 residues, less than 45 residues, less than 40 residues, less than 35 residues, less than 30 residues, less than 25 residues, less than 20 residues, less than 15 residues, less than 10 residues or less than 5 residues, or a combination that does not contradict them. Specifically, the length of each substrate oligonucleotide can be, for example, 0 to 5 residues, 5 to 10 residues, 10 to 15 residues, 15 to 20 residues, 20 to 25 residues, 25 to 30 residues, 30 to 35 residues, 35 to 40 residues, 40 to 45 residues, 45 to 50 residues, 50 to 55 residues, 55 to 60 residues, 60 to 65 residues or 65 to 70 residues. It should be noted that the number of modified nucleic acid residues in each substrate oligonucleotide exemplified above can also be independently applied to the selected nucleic acid residues with any modification (for example, any modification of the above examples). That is, for example, the number of modified nucleic acid residues with a phosphate portion in each substrate oligonucleotide can be set to the number of modified nucleic acid residues in each substrate oligonucleotide exemplified above. In addition, for example, the number of modified nucleic acid residues with a sugar portion in each substrate oligonucleotide can be set to the number of modified nucleic acid residues in each substrate oligonucleotide exemplified above. Furthermore, for example, the number of nucleic acid residues having a modification of the base portion in each substrate oligonucleotide can be set to the number of modified nucleic acid residues in each substrate oligonucleotide exemplified above.

[0157] Each substrate oligonucleotide, for example, may have a structure required or useful for the connection between substrate oligonucleotides. For example, the 5' end of the 3' side substrate oligonucleotide may be phosphorylated. That is, the connection between substrate oligonucleotides may be, for example, the connection between the 5' end of the 3' side substrate oligonucleotide and the 3' end of the hydroxyl group of the 5' side substrate oligonucleotide. In addition, for example, the 3' end of the 5' side substrate oligonucleotide may be phosphorylated. That is, the connection between substrate oligonucleotides may be, for example, the connection between the 5' end of the 3' side substrate oligonucleotide and the 3' end of the hydroxyl group of the 5' side substrate oligonucleotide. In addition, the phosphate portion at the 5' end or the 3' end may be modified or may not be modified. As a modification of the phosphate portion at the 5' end or the 3' end, it is possible to cite: thiophosphate (that is, the oxygen atom of the phosphate group is substituted with a sulfur atom) or borane phosphate (that is, the oxygen atom of the phosphate group is substituted with borane (BH3)). As a modification of the phosphate portion at the 5' end or the 3' end, it is also possible to cite: the insertion of a joint. About the insertion of a joint, as described above.

[0158] There is no particular restriction on the method for producing each substrate oligonucleic acid. Each substrate oligonucleic acid can be produced, for example, by sequentially connecting the nucleic acid residues constituting each substrate oligonucleic acid. The connection of nucleic acid residues can be implemented, for example, by the phosphoramidite method. In addition, each substrate oligonucleic acid can be produced, for example, by connecting the nucleic acid fragments constituting each substrate oligonucleic acid. The connection of nucleic acid fragments can be implemented, for example, by enzymatic means using complementary chains. The connection of nucleic acid fragments can be implemented, for example, by the method of the present invention.

[0159] <3>Complementary oligonucleotide

[0160] "Complementary oligonucleotide" refers to a single-stranded oligonucleotide having a specific structure used in the ligation step. In the ligation step, complementary oligonucleotides corresponding to each ligation part are used.

[0161] The "single strand" of complementary oligonucleotide refers to the form of complementary oligonucleotide at the beginning of the method (specifically the connection process) of the present invention. As the method (specifically the connection process) of the present invention proceeds, the complementary oligonucleotide can form the form of double-stranded oligonucleotide. For example, as the method (specifically the connection process) of the present invention proceeds, the complementary oligonucleotide can hybridize with the substrate oligonucleotide to form the form of double-stranded oligonucleotide.

[0162] The structure of the complementary oligonucleotide (eg, the type of nucleic acid residues, base sequence, length and modification pattern) can be appropriately set according to various conditions such as the structure of the 5' and 3' substrate oligonucleotides linked to the linker corresponding to the complementary oligonucleotide.

[0163] About the kind of nucleic acid residue, as described above.Each complementary oligonucleic acid can include one or more nucleic acid residues.That is, each complementary oligonucleic acid can include DNA residues, RNA residues, modified nucleic acid residues or their combination, for example.Each complementary oligonucleic acid can be a nucleic acid chain consisting of one nucleic acid residue, or a nucleic acid chain consisting of a combination of two or more nucleic acid residues (that is, a nucleic acid chain containing two or more nucleic acid residues in the molecule).That is, each complementary oligonucleic acid can be a nucleic acid chain consisting of DNA residues, or a nucleic acid chain consisting of RNA residues, or a nucleic acid chain consisting of modified nucleic acid residues, or a nucleic acid chain consisting of a combination of them (that is, a nucleic acid chain containing two or more nucleic acid residues selected from DNA residues, RNA residues and modified nucleic acid residues in the molecule).

[0164] The complementary oligonucleotide includes a base sequence in which a destabilizing portion is introduced into the first base sequence. The base sequence in which a destabilizing portion is introduced into the first base sequence is also referred to as a "destabilizing base sequence."

[0165] “The base sequence after the destabilizing portion is introduced into the first base sequence” refers to a modified sequence of the first base sequence, wherein the base sequence is the same as the first base sequence except for the introduction of the destabilizing portion (i.e., including the destabilizing portion). In other words, the first base sequence is the base sequence before the introduction of the destabilizing portion (i.e., excluding the destabilizing portion). It should be noted that the “introduction of the destabilizing portion” mentioned here is a record of the structure of a specific destabilizing base sequence, and does not specify the method for producing a specific destabilizing base sequence.

[0166] The first base sequence is a base sequence consisting of a second base sequence and a third base sequence connected in the 5' to 3' direction. "A base sequence consisting of a second base sequence and a third base sequence connected in the 5' to 3' direction" refers to a connection sequence of the second base sequence and the third base sequence, wherein the 3' end of the second base sequence is connected to the 5' end of the third base sequence. It should be noted that the "connection of the second base sequence and the third base sequence" mentioned here is a record for specifying the structure of the first base sequence, and does not specify the method for producing the first base sequence.

[0167] The second base sequence is a complementary sequence to the 5' side partial sequence of the 3' side substrate oligonucleotide, or a complementary sequence to the full-length sequence of the 3' side substrate oligonucleotide. "The 5' side partial sequence of the 3' side substrate oligonucleotide" refers to a partial sequence of the 3' side substrate oligonucleotide, which is composed of continuous nucleic acid residues including the 5' terminal nucleic acid residue (which is the nucleic acid residue connected to the 3' terminal of the 5' side substrate oligonucleotide).

[0168] The third base sequence is a complementary sequence to the 3' side partial sequence of the 5' side substrate oligonucleotide, or a complementary sequence to the full-length sequence of the 5' side substrate oligonucleotide. "3' side partial sequence of the 5' side substrate oligonucleotide" refers to a partial sequence of the 5' side substrate oligonucleotide, which is composed of continuous nucleic acid residues including the 3' terminal nucleic acid residue (which is the nucleic acid residue connected to the 5' terminal of the 3' side substrate oligonucleotide).

[0169] "Complementarity" between base sequences can mean that the base portion between base sequences can form a Watson-Crick type base pair or a base pair with an intensity equal to or stronger therewith. As a base pair considered as complementary, it can be enumerated: a Watson-Crick type base pair selected from the bases (they can be modified or not modified) of the above-mentioned examples or a base pair formed by a combination of base pairs with an intensity equal to or stronger therewith. Specifically, as a base pair considered as complementary, it can be enumerated: Watson-Crick type base pairs such as AT base pairs, AU base pairs, and GC base pairs. Specifically, as a base pair considered as complementary, it can also be enumerated: artificial base pairs such as xy base pairs, sy base pairs, Ds-Pa base pairs, Ds-PxR base pairs, PICS-PICS base pairs, 5SICS-NaM base pairs, 5SICS-MMO2 base pairs, TPT3-NaM base pairs, iG-iC base pairs, and PZ base pairs.

[0170] It should be noted that the first base sequence, the second base sequence, and the third base sequence can all be hypothetical base sequences for a specific destabilizing base sequence and do not actually need to exist. For example, in the case where the substrate oligonucleotide contains a nucleic acid residue A1 that cannot form a Watson-Crick type base pair or a base pair with the same or stronger strength with any base portion, the first base sequence, the second base sequence, or the third base sequence can be a hypothetical base sequence containing a nucleic acid residue A2, wherein the nucleic acid residue A2 has a hypothetical base portion that can form a Watson-Crick type base pair or a hypothetical base pair with the nucleic acid residue A1 with the same or stronger strength. Specifically, for example, when the substrate oligonucleotide contains an abasic nucleic acid residue B1, the abasic nucleic acid residue B1 cannot form a base pair with any base portion, but the first base sequence, the second base sequence, or the third base sequence may be a hypothetical base sequence containing a nucleic acid residue B2, wherein the nucleic acid residue B2 has a hypothetical base portion that can form a Watson-Crick type base pair or a hypothetical base pair with the abasic nucleic acid residue B1 that is equal to or stronger than the Watson-Crick type base pair. Unless otherwise specified, such a hypothetical base pair is regarded as a base pair with the same strength as the AT base pair in the calculation of the melting temperature (Tm).

[0171] The length of the first base sequence, the second base sequence and the third base sequence is not particularly limited, as long as it does not undermine the purpose of the present invention. That is, the length of the first base sequence, the second base sequence and the third base sequence is set to be able to connect substrate oligonucleotides by the method of the present invention. The length of the first base sequence is the total of the lengths of the second base sequence and the third base sequence. The length of the second base sequence can be set to, for example, a complementary oligonucleotide that can hybridize with a 3' side substrate oligonucleotide. The length of the third base sequence can be set to, for example, a complementary oligonucleotide that can hybridize with a 5' side substrate oligonucleotide.

[0172] The length of the first base sequence may be, for example, 10 residues or more, 11 residues or more, 12 residues or more, 13 residues or more, 14 residues or more, 15 residues or more, 16 residues or more, 17 residues or more, 20 residues or more, 25 residues or more, 30 residues or more, 35 residues or more, 40 residues or more, 45 residues or more, 50 residues or more, 60 residues or more, 70 residues or more, 80 residues or more, or 90 residues or more; or it may be 100 residues or less, 90 residues or less, 80 residues or less, 70 residues or less, 60 residues or less, 50 residues or less, 45 residues or less, 40 residues or less, 35 residues or less, 30 residues or less, 25 residues or less, 20 residues or less, 17 residues or less, 16 residues or less, 15 residues or less, 14 residues or less, 13 residues or less, 12 residues or less, or 11 residues or less; or it may be a non-contradictory combination thereof. Specifically, the length of the first base sequence may be, for example, 10 to 11 residues, 11 to 12 residues, 12 to 13 residues, 13 to 14 residues, 14 to 15 residues, 15 to 16 residues, 16 to 17 residues, 17 to 20 residues, 20 to 25 residues, 25 to 30 residues, 30 to 35 residues, 35 to 40 residues, 40 to 45 residues, 45 to 50 residues, 50 to 60 residues, 60 to 70 residues, 70 to 80 residues, 80 to 90 residues, or 90 to 100 residues. Specifically, the length of the first base sequence may be, for example, 10 to 100 residues, 10 to 50 residues, 10 to 40 residues, 12 to 30 residues, or 15 to 25 residues.

[0173] The length of the second base sequence and the third base sequence can be, for example, 4 residues or more, 5 residues or more, 6 residues or more, 7 residues or more, 8 residues or more, 9 residues or more, 10 residues or more, 11 residues or more, 12 residues or more, 13 residues or more, 14 residues or more, 15 residues or more, 17 residues or more, 20 residues or more, 25 residues or more, 30 residues or more, 35 residues or more, 40 residues or more, or 45 residues or less; or 50 residues or less, 45 residues or less, 40 residues or less, 35 residues or less, 30 residues or less, 25 residues or less, 20 residues or less, 17 residues or less, 15 residues or less, 14 residues or less, 13 residues or less, 12 residues or less, 11 residues or less, 10 residues or less, 9 residues or less, 8 residues or less, 7 residues or less, 6 residues or less, or 5 residues or less; or any non-contradictory combination thereof. Specifically, the lengths of the second base sequence and the third base sequence can be, for example, 4 to 5 residues, 5 to 6 residues, 6 to 7 residues, 7 to 8 residues, 8 to 9 residues, 9 to 10 residues, 10 to 11 residues, 11 to 12 residues, 12 to 13 residues, 13 to 14 residues, 14 to 15 residues, 15 to 17 residues, 17 to 20 residues, 20 to 25 residues, 25 to 30 residues, 30 to 35 residues, 35 to 40 residues, 40 to 45 residues, or 45 to 50 residues. Specifically, the lengths of the second base sequence and the third base sequence can be, for example, 4 to 50 residues, 4 to 25 residues, 4 to 20 residues, 5 to 15 residues, or 7 to 13 residues.

[0174] For example, the length of the first base sequence may be 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or less, 100% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less, or any combination thereof. Specifically, for example, the length of the first base sequence may be 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%, based on the total length of the 5' side and 3' side substrate oligonucleotides being 100%. Specifically, for example, the length of the first base sequence may be 5-100%, 5-50%, 5-20%, 20-100%, 20-50% or 50-100% based on the total length of the 5' and 3' substrate oligonucleotides being 100%.

[0175] For example, based on the length of the 3' side substrate oligonucleotide as 100%, the length of the second base sequence may be 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or less, 100% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less, or any combination thereof that does not contradict each other. Specifically, for example, based on the length of the 3' side substrate oligonucleotide as 100%, the length of the second base sequence may be 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%. Specifically, for example, based on the length of the 3' substrate oligonucleotide being 100%, the length of the second base sequence may be 5-100%, 5-50%, 5-20%, 20-100%, 20-50% or 50-100%.

[0176] For example, based on the length of the 5' side substrate oligonucleotide as 100%, the length of the third base sequence may be 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or less, 100% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less, or any combination thereof that does not contradict each other. Specifically, for example, based on the length of the 5' side substrate oligonucleotide as 100%, the length of the third base sequence may be 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%. Specifically, for example, based on the length of the 5' substrate oligonucleotide being 100%, the length of the third base sequence may be 5-100%, 5-50%, 5-20%, 20-100%, 20-50% or 50-100%.

[0177] When the substrate oligonucleotide includes a linker, the first base sequence includes a linker at the corresponding position. The "corresponding position" mentioned here, when the substrate oligonucleotide includes a linker between nucleic acid residues A and B, refers to between nucleic acid residues a and b in the first base sequence, where nucleic acid residues a and b are complementary nucleic acid residues of nucleic acid residues A and B, respectively. The linker contained in the substrate oligonucleotide may be the same as the linker contained in the corresponding position of the first base sequence.

[0178] "Destabilizing portion" refers to a site that reduces the stability of the hybrid formed between the complementary oligonucleotide and the target oligonucleotide. That is, by introducing the destabilizing portion into the first base sequence, the stability of the hybrid formed between the complementary oligonucleotide and the target oligonucleotide is reduced compared to before the destabilizing portion is introduced into the first base sequence. In other words, the stability of the hybrid formed between the complementary oligonucleotide and the target oligonucleotide is lower than the stability of the hybrid formed between the fully complementary oligonucleotide (here, referring to an oligonucleotide identical to the complementary oligonucleotide except that it contains the first base sequence instead of the destabilizing base sequence) and the target oligonucleotide. As a reduction in the stability of the hybrid, it can be cited as a reduction in the melting temperature (Tm) of the hybrid. The amount of Tm reduction due to the introduction of the destabilizing portion may be, for example, 1°C or more, 2°C or more, 3°C or more, 4°C or more, 5°C or more, 6°C or more, 7°C or more, 8°C or more, 9°C or more, 10°C or more, 11°C or more, 12°C or more, 13°C or more, 14°C or more, 15°C or more, 16°C or more, 17°C or more, 18°C ​​or more, 19°C or more, 20°C or more, 25°C or more, 30°C or more, 35°C or more, 40°C or more, 45°C or more, 50°C or more, or 55°C or more. It can also be below 60°C, below 55°C, below 50°C, below 45°C, below 40°C, below 35°C, below 30°C, below 25°C, below 20°C, below 19°C, below 18°C, below 17°C, below 16°C, below 15°C, below 14°C, below 13°C, below 12°C, below 11°C, below 10°C, below 9°C, below 8°C, below 7°C, below 6°C, below 5°C, below 4°C, below 3°C or below 2°C, and it can also be a non-contradictory combination of them. Specifically, the amount of reduction in Tm due to the introduction of the destabilizing portion can be, for example, 1-2°C, 2-3°C, 3-4°C, 4-5°C, 5-6°C, 6-7°C, 7-8°C, 8-9°C, 9-10°C, 10-11°C, 11-12°C, 12-13°C, 13-14°C, 14-15°C, 15-16°C, 16-17°C, 17-18°C, 18-19°C, 19-20°C, 20-25°C, 25-30°C, 30-35°C, 35-40°C, 40-45°C, 45-50°C, 50-55°C or 55-60°C. Specifically, the reduction in Tm due to the introduction of the destabilizing portion can be, for example, 1-60°C, 3-60°C, 5-60°C, 10-60°C, 1-40°C, 3-40°C, 5-40°C, 10-40°C, 1-20°C, 3-20°C, 5-20°C or 10-20°C.

[0179] The destabilizing moiety, for example, can reduce the stability of the hybrid formed between the complementary oligonucleotide and the target oligonucleotide, but does not completely prevent the hybridization of the complementary oligonucleotide with the 5' substrate oligonucleotide, and does not completely prevent the hybridization of the complementary oligonucleotide with the 3' substrate oligonucleotide.

[0180] The destabilizing moiety is not particularly limited as long as it improves the efficiency of producing the target oligonucleotide. Examples of improving the efficiency of producing the target oligonucleotide include increasing the molar ratio of the yield of the target oligonucleotide to the amount of complementary oligonucleotide used.

[0181] Examples of the destabilizing portion include an abasic portion, a mismatched portion, insertion of a nucleic acid residue, deletion of a nucleic acid residue, insertion of a linker, deletion of a linker, and substitution of a linker.

[0182] The "abasic moiety" refers to the following (1) or (2):

[0183] (1) Nucleic acid residues lacking a base moiety (i.e., abasic nucleic acid residues);

[0184] (2) A nucleic acid residue having an arbitrary base moiety, wherein the nucleic acid residue of the corresponding substrate oligonucleic acid is an abasic nucleic acid residue.

[0185] That is, in the abasic portion, the nucleic acid residues of either or both of the complementary oligonucleotide and the substrate oligonucleotide may be abasic nucleic acid residues. In the abasic portion, in particular, at least the nucleic acid residues of the complementary oligonucleotide may be abasic nucleic acid residues.

[0186] "Mismatching part" refers to the nucleic acid residue of the base part with base pair mismatch relative to the corresponding substrate oligonucleotide. "Base pair mismatching" refers to the base part that cannot form base pair between base sequences or the base part that forms base pair between base sequences, which is weaker than Watson-Crick type base pair. As the combination of bases regarded as mismatching, it can be enumerated: the combination of bases selected from the above-mentioned examples (these can be modified) that cannot form base pair or the combination of bases selected from the above-mentioned examples (these can be modified) that can form base pair with weaker strength than Watson-Crick type base pair. Specifically, as the combination of bases regarded as mismatching, it can be enumerated: the combination of A and A, the combination of A and G, the combination of A and C, the combination of T and T, the combination of T and G, the combination of T and C, the combination of T and U, the combination of U and U, the combination of U and G, the combination of U and C, the combination of G and G, the combination of C and C, the combination of I and U, the combination of I and A, the combination of I and C (I is hypoxanthine). As a combination of bases considered as mismatches, in particular, combinations of bases other than combinations of bases forming a wobble base pair can be mentioned. As a combination of bases forming a wobble base pair, combinations of U and G, combinations of I and U, combinations of I and A, and combinations of I and C can be mentioned. As a combination of bases other than combinations of bases forming a wobble base pair, combinations of A and A, combinations of A and G, combinations of A and C, combinations of T and T, combinations of T and G, combinations of T and C, combinations of T and U, combinations of U and U, combinations of U and C, combinations of G and G, and combinations of C and C can be mentioned.

[0187] The "insertion of nucleic acid residues" means that a nucleic acid residue is inserted between nucleic acid residues.

[0188] "Deletion of nucleic acid residues" refers to a deletion of nucleic acid residues.

[0189] "Insertion of a linker" refers to the insertion of a linker between nucleic acid residues. Regarding the linker, as described in the modification of the phosphate portion above. For example, the linker can be bonded to a phosphate group at both ends. That is, for example, the nucleic acid residue on the 5' side of the linker can be bonded to a phosphate group at the 3' position, and the nucleic acid residue on the 3' side can be bonded to a phosphate group at the 5' position. The insertion of a linker can be combined with the deletion of nucleic acid residues, for example. That is, for example, the nucleic acid residue can be substituted with a linker. That is, the nucleic acid residues at both ends of the deleted nucleic acid residue can be directly connected, for example, or can be connected via a linker. In other words, the deletion of nucleic acid residues may or may not be accompanied by the insertion of a linker. "Deletion of nucleic acid residues accompanied by the insertion of a linker" refers to the substitution of nucleic acid residues into a linker. "Deletion of nucleic acid residues without the insertion of a linker" refers to the deletion of nucleic acid residues other than the substitution of nucleic acid residues into a linker. In addition, the insertion of a linker can be combined with a destabilizing portion other than the deletion of nucleic acid residues, such as the insertion of nucleic acid residues. That is, for example, a linker and a nucleic acid residue connected thereto can be inserted together.

[0190] "Deletion of a linker" refers to the deletion of a linker. The deletion of a linker can be selected when the first base sequence contains a linker. The deletion of a linker can be combined with the insertion of a nucleic acid residue, for example. That is, for example, the linker can be substituted with a nucleic acid residue. In other words, the insertion of a nucleic acid residue may or may not be accompanied by the deletion of a linker. "Insertion of a nucleic acid residue accompanied by the deletion of a linker" refers to the substitution of a linker with a nucleic acid residue. "Insertion of a nucleic acid residue not accompanied by the deletion of a linker" refers to the insertion of a nucleic acid residue other than the substitution of a linker with a nucleic acid residue. In addition, the insertion of a linker can be combined with a destabilizing portion other than the insertion of a nucleic acid residue, such as the deletion of a nucleic acid residue. That is, for example, the linker and the nucleic acid residue connected thereto can be deleted together.

[0191] "Replacement of a linker" means that a linker is replaced with a different type of linker. The replacement of a linker can be selected when the first base sequence contains a linker. The length of the linker after replacement can be different from that of the linker before replacement (i.e., longer or shorter than the linker before replacement).

[0192] It should be noted that the destabilizers as exemplified above can function as destabilizers individually. However, when two or more destabilizers are used in combination, as long as the combination as a whole functions as a destabilizer, each destabilizer constituting the combination can function as a destabilizer individually or not.

[0193] As the destabilizing part, one destabilizing part can be used, or two or more destabilizing parts can be used in combination. The destabilizing part may, for example, include or not include each of the destabilizing parts exemplified above. The destabilizing part may, for example, include one or more destabilizing parts selected from the destabilizing parts exemplified above. Specifically, the destabilizing part may, for example, include an abasic part, a mismatch part, an insertion of a nucleic acid residue, a deletion of a nucleic acid residue, an insertion of a linker, a deletion of a linker, a replacement of a linker, or a combination thereof. "The destabilizing part includes a certain destabilizing part A" means that at least the destabilizing part A is selected as the destabilizing part, including the case where the destabilizing part is composed of the destabilizing part A and the case where the destabilizing part is composed of a combination of the destabilizing part A and other destabilizing parts. For example, "the destabilizing part includes an abasic part" means that at least the abasic part is selected as the destabilizing part, including the case where the destabilizing part is composed of the abasic part and the case where the destabilizing part is composed of a combination of the abasic part and other destabilizing parts. In addition, "the destabilizing portion includes a certain destabilizing portion A" means that, when the number or number of groups of destabilizing portions A is specified (here, indefinite articles (such as a and an) are not regarded as the specified number or number of groups), at least the destabilizing portion A is selected as the destabilizing portion, and the number or number of groups of destabilizing portions A contained in the destabilizing portion is limited to the specified number or number of groups. For example, "the destabilizing portion includes 1 abasic portion" means that at least the abasic portion is selected as the destabilizing portion, and the number of abasic portions contained in the destabilizing portion is limited to 1, including the case where the destabilizing portion is composed of 1 abasic portion and the case where the destabilizing portion is composed of a combination of 1 abasic portion and other destabilizing portions.

[0194] The destabilizing portion may, for example, include or not include the destabilizing portions exemplified above. The destabilizing portion may, for example, include or not include the abasic portion. The destabilizing portion may, for example, include or not include the mismatch portion. The destabilizing portion may, for example, include or not include the insertion of nucleic acid residues. The destabilizing portion may, for example, include or not include the deletion of nucleic acid residues. The destabilizing portion may, for example, include or not include the insertion of a linker. The destabilizing portion may, for example, include or not include the deletion of a linker. The destabilizing portion may, for example, include or not include the substitution of a linker.

[0195] There is no particular limitation on the scheme for introducing the destabilizing moiety (e.g., type, quantity, number of groups, and position of introduction) as long as the efficiency of generating the target oligonucleotide is improved. The scheme for introducing the destabilizing moiety can be appropriately set according to various conditions such as the structure of the substrate oligonucleotide.

[0196] The "number of destabilizing moieties" refers to the number of nucleic acid residues and linkers contained in the destabilizing base sequence that correspond to the destabilizing moieties. The number of any selected destabilizing moieties (e.g., any one or more of the destabilizing moieties exemplified above) can be, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% relative to the total number of destabilizing moieties.

[0197] The “number of groups of destabilizing parts” refers to the number of groups of destabilizing parts contained in the destabilizing base sequence. A “group of destabilizing parts” refers to one destabilizing part that is not continuous with a destabilizing part of the same kind or two or more continuous destabilizing parts of the same kind. Regarding the “group of destabilizing parts”, abasic parts are regarded as the same kind as each other, mismatch parts are regarded as the same kind as each other, insertions of nucleic acid residues are regarded as the same kind as each other, deletions of nucleic acid residues are regarded as the same kind as each other, insertions of linkers are regarded as the same kind as each other, deletions of linkers are regarded as the same kind as each other, and substitutions of linkers are regarded as the same kind as each other. “Insertion of two or more linkers, deletion of linkers or substitutions of linkers in succession” refers to the continuity of two or more nucleic acid residues with insertion of linkers, deletion of linkers or substitutions of linkers introduced on the 5' side. Relative to the total number of destabilization units, the number of groups of any selected destabilization units (for example, any one or more of the destabilization units in the above examples) can be, for example, more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95% or 100%.

[0198] The number of groups of destabilizing parts may be, for example, more than 1, more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 12, or more than 15, or less than 20, less than 15, less than 12, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, or less than 2, or any non-contradictory combination thereof. Specifically, the number of groups of destabilizing parts may be, for example, 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-12, 12-15, or 15-20. Specifically, the number of groups of destabilizing parts may be, for example, 1-20, 1-10, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1.

[0199] In addition, the number of groups of destabilizing parts (or, for example, the number of groups of destabilizing parts other than the insertion, deletion and replacement of linkers) may be, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or 7 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, or a non-contradictory combination thereof. Specifically, the number of groups of destabilizing parts (or, for example, the number of groups of destabilizing parts other than the insertion, deletion and replacement of linkers) may be, for example, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, or 7 to 8. Specifically, the number of groups of destabilizing parts (or, for example, the number of groups of destabilizing parts other than the insertion, deletion and replacement of linkers) may be, for example, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. The number of groups of destabilizing parts (or, for example, the number of groups of destabilizing parts other than the insertion, deletion and replacement of linkers) may be particularly 1, 2, or 3. In one embodiment, the number of destabilizing moieties (or, for example, the number of destabilizing moieties other than insertion of a linker, deletion of a linker, and substitution of a linker) may more particularly be 1 or 2. In one embodiment, the number of destabilizing moieties (or, for example, the number of destabilizing moieties other than insertion of a linker, deletion of a linker, and substitution of a linker) may more particularly be 3. The number of destabilizing moieties (or, for example, the number of destabilizing moieties other than insertion of a linker, deletion of a linker, and substitution of a linker) may be, for example, 5% or more, 10% or more, 15% or more, 20% or more, or 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less, in non-contradictory combinations thereof, in terms of the ratio to the length (number of residues) of the first base sequence. Specifically, the number of groups of destabilizing parts (or, for example, the number of groups of destabilizing parts other than the insertion of a linker, the deletion of a linker, and the substitution of a linker) can be, for example, 5 to 10%, 10 to 15%, 15 to 20%, 20 to 25%, or 25 to 30% in terms of the ratio to the length (number of residues) of the first base sequence. Specifically, the number of groups of destabilizing parts (or, for example, the number of groups of destabilizing parts other than the insertion of a linker, the deletion of a linker, and the substitution of a linker) can be, for example, 5 to 30%, 5 to 20%, or 5 to 15% in terms of the ratio to the length (number of residues) of the first base sequence.

[0200] For example, as a destabilizing portion, when the nucleic acid residue of 1 residue at position A of the first base sequence is substituted with a linker, the nucleic acid residue of 1 residue connected to a linker is inserted into position B of the first base sequence, and the nucleic acid residue of 4 consecutive residues not connected to a linker is inserted into position C of the first base sequence (here, positions A, B and C are not adjacent to each other), the number of destabilizing portions is 7 (specifically, the number of nucleic acid residue deletions is 1, the number of nucleic acid residue insertions is 5, and the number of linker insertions is 2), and the number of groups of destabilizing portions is 5 (specifically, the number of groups of nucleic acid residue deletions is 1, the number of groups of nucleic acid residue insertions is 2, and the number of linker insertions is 2).

[0201] It should be noted that, unless otherwise specified, the introduction scheme of the destabilization unit shall be interpreted according to the following standards.

[0202] (1) is interpreted as the number of destabilizing parts being the smallest (1 or more).

[0203] (2) When the number of destabilization sections is the same, it is interpreted that the number of groups of destabilization sections is the smallest (1 or more).

[0204] (3) When the number of destabilizing moieties and the number of groups of destabilizing moieties are the same, the position of introduction of the destabilizing moiety is -X or X (X is a positive integer; however, in the case of insertion of a nucleic acid residue, X may be 0), which is interpreted as minimizing the total number of Xs.

[0205] Unless otherwise specified, the position of introduction of the destabilizing portion is specifically the position in the first base sequence. Unless otherwise specified, the position in the first base sequence is based on the connection between the second base sequence and the third base sequence, with the 5' side (i.e., the second base sequence side) being negative and being represented by a serial number starting from -1, and the 3' side (i.e., the third base sequence side) being positive and being represented by a serial number starting from +1. The position on the 5' side (i.e., the second base sequence side) based on the connection between the second base sequence and the third base sequence is also referred to as a "negative position" or "position on the second base sequence side". The position on the 3' side (i.e., the third base sequence side) based on the connection between the second base sequence and the third base sequence is also referred to as a "positive position" or "position on the third base sequence side". That is, for example, when the second base sequence and the third base sequence are base sequences of 10 residues respectively, the nucleic acid residue at the 5' end of the second base sequence is at the -10 position, the nucleic acid residue at the 3' end of the second base sequence is at the -1 position, the nucleic acid residue at the 5' end of the third base sequence is at the +1 position, the nucleic acid residue at the 3' end of the third base sequence is at the +10 position, and the nucleic acid residue at the -1 position is connected to the nucleic acid residue at the +1 position. In the case of insertion of a nucleic acid residue, unless otherwise specified, "the insertion position of the nucleic acid residue is at the -X position" (X is a positive integer) means that a nucleic acid residue is inserted between the nucleic acid residue at the -X position and the nucleic acid residue at the -(X+1) position. In the case of insertion of a nucleic acid residue, unless otherwise specified, "the insertion position of the nucleic acid residue is at the +X position" (X is a positive integer) means that a nucleic acid residue is inserted between the nucleic acid residue at the +X position and the nucleic acid residue at the +(X+1) position. In the case of insertion of a nucleic acid residue, unless otherwise specified, "the insertion position of the nucleic acid residue is position 0" means that the nucleic acid residue is inserted between the nucleic acid residue at position -1 and the nucleic acid residue at position +1. In the case of insertion of a linker, deletion of a linker or substitution of a linker, unless otherwise specified, the introduction position of the destabilizing portion is specifically the position of the nucleic acid residue at the 5' side where the insertion of the linker, deletion of the linker or substitution of the linker is introduced. That is, for example, unless otherwise specified, "the insertion position of the linker is position +X" (X is a positive integer) means that the linker is inserted on the 5' side of the nucleic acid residue at position +X (i.e., between the nucleic acid residue at position +X and the nucleic acid residue at position +(X-1)).

[0206] For example, a destabilizing moiety may be introduced into one or both of the second base sequence and the third base sequence. When the number of destabilizing moieties is 2 or more, the position of introduction of the destabilizing moiety may be selected independently for each destabilizing moiety unless otherwise specified. The position of introduction of the destabilizing moiety may be, for example, position -1, position +1, the 5' terminal nucleic acid residue of the first base sequence, the 3' terminal nucleic acid residue of the first base sequence, or a position other than these. The introduction position of the destabilizing part can be, for example, -25 to +25, -20 to +20, -17 to +17, -15 to +15, -14 to +14, -13 to +13, -12 to +12, -11 to +11, -10 to +10, -9 to +9, -8 to +8, -7 to +7, -6 to +6, -5 to +5, -4 to +4, -3 to +3, -2 to +2 or -1 to +1. The introduction position of the destabilizing part can be, for example, -1 to -4, -2 to -5, -3 to -6, -4 to -7, -5 to -8, -6 to -9, -7 to -10, -8 to -11, -9 to -12, -10 to -13, -11 to -14, -12 to -15, -13 to -16, -14 to -17, -15 to -18, -16 to -19, -17 to -20, -18 to -21, -19 to -22, -20 to -23, -21 to -24 or -22 to -25. The introduction position of the destabilization part can be, for example, +1 to +4, +2 to +5, +3 to +6, +4 to +7, +5 to +8, +6 to +9, +7 to +10, +8 to +11, +9 to +12, +10 to +13, +11 to +14, +12 to +15, +13 to +16, +14 to +17, +15 to +18, +16 to +19, +17 to +20, +18 to +21, +19 to +22, +20 to +23, +21 to +24, +22 to +25, +23 to +26 or +24 to +27. The introduction position of the destabilizing portion can be, for example, -25, -24, -23, -22, -21, -20, -19, -18, -17, -16, -15, -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24 or +25.

[0207] In the destabilized base sequence, for example, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more nucleic acid residues may remain among the nucleic acid residues constituting the second base sequence. In the destabilized base sequence, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more nucleic acid residues may remain among the nucleic acid residues constituting the second base sequence. In the destabilized base sequence, for example, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more consecutive nucleic acid residues may remain among the nucleic acid residues constituting the second base sequence.

[0208] In the destabilized base sequence, for example, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or 9 or more nucleic acid residues may remain among the nucleic acid residues at positions -1 to -10 of the second base sequence. In the destabilized base sequence, for example, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more consecutive nucleic acid residues may remain among the nucleic acid residues at positions -1 to -10 of the second base sequence.

[0209] In the destabilized base sequence, for example, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more nucleic acid residues may remain among the nucleic acid residues constituting the third base sequence. In the destabilized base sequence, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more nucleic acid residues may remain among the nucleic acid residues constituting the third base sequence. In the destabilized base sequence, for example, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more consecutive nucleic acid residues may remain among the nucleic acid residues constituting the third base sequence.

[0210] In the destabilized base sequence, for example, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or 9 or more nucleic acid residues may remain among the nucleic acid residues at positions +1 to +10 of the third base sequence. In the destabilized base sequence, for example, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more consecutive nucleic acid residues may remain among the nucleic acid residues at positions +1 to +10 of the third base sequence.

[0211] "Nucleic acid residues remain in the destabilizing base sequence" means that the nucleic acid residues are not replaced by the destabilizing part. It should be noted that the insertion of nucleic acid residues, the insertion of linkers, the deletion of linkers and the replacement of linkers do not change the original nucleic acid residues, and therefore have no effect on the number of residual nucleic acid residues in the second base sequence or the third base sequence and the number of residual continuous nucleic acid residues in the second base sequence or the third base sequence.

[0212] The number of abasic parts may be, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more, or 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, or a non-contradictory combination thereof. Specifically, the number of abasic parts may be, for example, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, or 7 to 8. Specifically, the number of abasic parts may be, for example, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. The number of abasic parts may particularly be 1, 2, or 3. The number of abasic parts may more particularly be 1 or 2. The number of abasic parts may be, for example, 5% or more, 10% or more, 15% or more, 20% or more, or 25% or less, or 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less, or a non-contradictory combination thereof, as a ratio to the length (number of residues) of the first base sequence. Specifically, the number of the abasic portion may be, for example, 5 to 10%, 10 to 15%, 15 to 20%, 20 to 25%, or 25 to 30% in terms of the ratio to the length (number of residues) of the first base sequence. Specifically, the number of the abasic portion may be, for example, 5 to 30%, 5 to 20%, or 5 to 15% in terms of the ratio to the length (number of residues) of the first base sequence.

[0213] The number of groups in the abasic portion may be, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more, or 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, or a non-contradictory combination thereof. Specifically, the number of groups in the abasic portion may be, for example, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, or 7 to 8. Specifically, the number of groups in the abasic portion may be, for example, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. The number of groups in the abasic portion may particularly be 1, 2, or 3. The number of groups in the abasic portion may more particularly be 1 or 2. The number of groups in the abasic portion may be, for example, 5% or more, 10% or more, 15% or more, 20% or more, or 25% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less, or any combination thereof that does not conflict with each other. Specifically, the number of groups in the abasic portion may be, for example, 5 to 10%, 10 to 15%, 15 to 20%, 20 to 25%, or 25 to 30%, for example, 5 to 30%, 5 to 20%, or 5 to 15%, for example, 5 to 15%, for example, 5 to 20%, or 5 to 15%, for example, 5 to 15%, for example, 5 to 30%, 5 to 20%, or 5 to 15%, for example, ...15%, for example, 5 to 20%, or 5 to 15%, for example, 5 to 15%, for example, 5 to 10%, 25% or less, 20% or less, 15% or less, or any combination thereof that does not conflict with each other.

[0214] The number of abasic portions constituting one group of abasic portions may be, for example, 1 or more, 2 or more, 3 or more, or 4 or less, 5 or less, 4 or less, 3 or less, or 2 or less, or a non-contradictory combination thereof. Specifically, the number of abasic portions constituting one group of abasic portions may be, for example, 1 to 2, 2 to 3, 3 to 4, or 4 to 5. Specifically, the number of abasic portions constituting one group of abasic portions may be, for example, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. The number of abasic portions constituting one group of abasic portions may particularly be 1 or 2. The number of abasic portions constituting one group of abasic portions may more particularly be 1. When the number of groups of abasic portions is 2 or more, the number of abasic portions constituting one group of abasic portions may be selected independently for each group of abasic portions unless otherwise specified.

[0215] When the number of abasic parts is 2 or more, the introduction position of the abasic part can be selected independently for each abasic part unless otherwise specified. The introduction position of the abasic part can be, for example, the introduction position of the destabilizing part exemplified above. The introduction position of the abasic part can be particularly -1 to +1, or it can be a position other than this. In one embodiment, the first abasic part can be introduced at -1 or +1, and the second abasic part can be optionally further introduced at a position not adjacent to the first abasic nucleic acid (e.g., -5 to -7 or +5 to +7).

[0216] The number of mismatches may be, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more, or 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, or a non-contradictory combination thereof. Specifically, the number of mismatches may be, for example, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, or 7 to 8. Specifically, the number of mismatches may be, for example, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. The number of mismatches may particularly be 1, 2, or 3. The number of mismatches may more particularly be 1. The number of mismatches may be, for example, 5% or more, 10% or more, 15% or more, 20% or more, or 25% or less, or 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less, or a non-contradictory combination thereof, as a ratio to the length (number of residues) of the first base sequence. Specifically, the number of mismatches may be, for example, 5 to 10%, 10 to 15%, 15 to 20%, 20 to 25%, or 25 to 30% in terms of the ratio to the length (number of residues) of the first base sequence. Specifically, the number of mismatches may be, for example, 5 to 30%, 5 to 20%, or 5 to 15% in terms of the ratio to the length (number of residues) of the first base sequence.

[0217] The number of groups of mismatches may be, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more, or 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, or any combination thereof that does not contradict each other. Specifically, the number of groups of mismatches may be, for example, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, or 7 to 8. Specifically, the number of groups of mismatches may be, for example, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. The number of groups of mismatches may particularly be 1, 2, or 3. The number of groups of mismatches may more particularly be 1. The number of groups of mismatches may be, for example, 5% or more, 10% or more, 15% or more, 20% or more, or 25% or less, or 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less, or any combination thereof that does not contradict each other. Specifically, the number of groups of mismatches can be, for example, 5 to 10%, 10 to 15%, 15 to 20%, 20 to 25%, or 25 to 30% in terms of the ratio to the length (number of residues) of the first base sequence. Specifically, the number of groups of mismatches can be, for example, 5 to 30%, 5 to 20%, or 5 to 15% in terms of the ratio to the length (number of residues) of the first base sequence.

[0218] The number of mismatching parts constituting one group of mismatching parts may be, for example, more than 1, more than 2, more than 3 or more than 4, or less than 5, less than 4, less than 3 or less than 2, or a non-contradictory combination thereof. Specifically, the number of mismatching parts constituting one group of mismatching parts may be, for example, 1-2, 2-3, 3-4 or 4-5. Specifically, the number of mismatching parts constituting one group of mismatching parts may be, for example, 1-5, 1-4, 1-3, 1-2 or 1. The number of mismatching parts constituting one group of mismatching parts may particularly be 1 or 2. The number of mismatching parts constituting one group of mismatching parts may more particularly be 1. In a case where the number of groups of mismatching parts is more than 2, the number of mismatching parts constituting one group of mismatching parts may be selected independently for each group of mismatching parts unless otherwise specified.

[0219] When the number of mismatches is 2 or more, the position of introduction of the mismatches can be selected independently for each mismatch, unless otherwise specified. The position of introduction of the mismatches can be, for example, the position of introduction of the destabilizing portion in the above example. The position of introduction of the mismatches can be, in particular, positions -2 to +2. The position of introduction of the mismatches can be, more particularly, positions -1 to +1.

[0220] The number of inserted nucleic acid residues (i.e., the number of inserted nucleic acid residues) may be, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more, or 60 or less, 50 or less, 40 or less, 30 or less, 25 or less, 20 or less, 15 or less, 12 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, or a non-contradictory combination thereof. Specifically, the number of inserted nucleic acid residues may be, for example, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 12, 12 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 40, 40 to 50, or 50 to 60. Specifically, the number of nucleic acid residues inserted may be, for example, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. The number of nucleic acid residues inserted may particularly be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. The number of inserted nucleic acid residues may be, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 170% or more, 200% or more, or 300% or less, 250% or less, 200% or less, 170% or less, 150% or less, 140% or less, 130% or less, 120% or less, 110% or less, 100% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less, or any non-contradictory combination thereof. Specifically, the number of nucleic acid residues inserted may be, for example, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-110%, 110-120%, 120-130%, 130-140%, 140-150%, 150-170%, 170-200%, 200-250%, or 250-300% in terms of the ratio to the length (number of residues) of the first base sequence. Specifically, the number of nucleic acid residues inserted may be, for example, 5-300%, 5-200%, 5-150%, 5-100%, or 5-50% in terms of the ratio to the length (number of residues) of the first base sequence.

[0221] The number of groups of nucleic acid residues inserted may be, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more, or 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, or any combination thereof that does not contradict each other. Specifically, the number of groups of nucleic acid residues inserted may be, for example, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, or 7 to 8. Specifically, the number of groups of nucleic acid residues inserted may be, for example, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. The number of groups of nucleic acid residues inserted may particularly be 1, 2, or 3. The number of groups of nucleic acid residues inserted may more particularly be 2. The number of groups of nucleic acid residues inserted, for example, in terms of the ratio to the length (number of residues) of the first base sequence, may be 5% or more, 10% or more, 15% or more, 20% or more, or 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less, or any combination thereof that does not contradict each other. Specifically, the number of groups of nucleic acid residues inserted, for example, in terms of the ratio to the length (number of residues) of the first base sequence, may be 5 to 10%, 10 to 15%, 15 to 20%, 20 to 25%, or 25 to 30%. Specifically, the number of groups of nucleic acid residues inserted, for example, in terms of the ratio to the length (number of residues) of the first base sequence, may be 5 to 30%, 5 to 20%, or 5 to 15%.

[0222] The number of nucleic acid residue insertions constituting one set of nucleic acid residue insertions may be, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 12 or more, 15 or more, 20 or more, or 25 or more, or 30 or less, 25 or less, 20 or less, 15 or less, 12 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, or any combination thereof that is not contradictory. The number of nucleic acid residue insertions constituting one set of nucleic acid residue insertions may particularly be 2 or more. Specifically, the number of nucleic acid residue insertions constituting one set of nucleic acid residue insertions may be, for example, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 12, 12 to 15, 15 to 20, 20 to 25, or 25 to 30. Specifically, the number of nucleic acid residue insertions constituting one set of insertions of nucleic acid residues may be, for example, 1 to 30, 1 to 20, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. The number of nucleic acid residue insertions constituting one set of insertions of nucleic acid residues may particularly be 1, 2, 3, 4, 5, 6, 7, or 8. When the number of nucleic acid residue insertions constituting one set of insertions of nucleic acid residues is 2 or more, the number of nucleic acid residue insertions constituting one set of insertions of nucleic acid residues may be selected independently for each set of insertions of nucleic acid residues unless otherwise specified.

[0223] When the number of nucleic acid residues inserted is 2 or more, the position of introduction of the nucleic acid residues can be selected independently for each nucleic acid residue unless otherwise specified. The position of introduction of the nucleic acid residues can be, for example, the position of introduction of the destabilizing portion exemplified above. The position of introduction of the nucleic acid residues can be particularly -3 to -6 or +3 to +6. The position of introduction of the nucleic acid residues can be more particularly -5 or +5.

[0224] In the case where the number of nucleic acid residues inserted is 2 or more, unless otherwise specified, the type of base of the inserted nucleic acid residue can be selected independently for each nucleic acid residue. There is no particular restriction on the type of base of the inserted nucleic acid residue. The base of the inserted nucleic acid residue can be any base or no base. In the case of inserting 2 or more consecutive nucleic acid residues (hereinafter, also referred to as "continuous insertion residues"), part or all of the consecutive insertion residues may have self-complementarity. For example, more than 50%, more than 60%, more than 70%, more than 80% or more than 90% of the nucleic acid residues constituting the consecutive insertion residues may have self-complementarity. For example, at least the 5' terminal nucleic acid residue and the 3' terminal nucleic acid residue of the consecutive insertion residue may have or may not have self-complementarity. For example, 1 residue, 2 residues, 3 residues, 4 residues or more nucleic acid residues from the 5' end of the consecutive insertion residue and 1 residue, 2 residues, 3 residues, 4 residues or more nucleic acid residues from the 3' end may have self-complementarity. Specifically, for example, when the continuous insertion residue is TTGAA, 80% (= 4 residues / 5 residues) of the nucleic acid residues constituting the continuous insertion residue have self-complementarity, and the nucleic acid residues of 2 residues from the 5' end of the continuous insertion residue and the nucleic acid residues of 2 residues from the 3' end have self-complementarity. A part or all of the continuous insertion residues can form a double-stranded structure based on self-complementarity. The inserted nucleic acid residues (particularly the continuous insertion residues) are also referred to as "loops" or "loop structures".

[0225] The number of nucleic acid residue deletions (i.e., the number of nucleic acid residues deleted) may be, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more, or 60 or less, 50 or less, 40 or less, 30 or less, 25 or less, 20 or less, 15 or less, 12 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, or a non-contradictory combination thereof. Specifically, the number of nucleic acid residue deletions may be, for example, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 12, 12 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 40, 40 to 50, or 50 to 60. Specifically, the number of nucleic acid residue deletions may be, for example, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. The number of nucleic acid residue deletions may particularly be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. More particularly, the number of nucleic acid residue deletions may be 1, 2, or 3. The number of nucleic acid residue deletions may be, for example, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 50% or more, 60% or more, or 70% or more, or 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less, or any combination thereof that does not contradict each other. Specifically, the number of nucleic acid residue deletions may be, for example, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-40%, 40-50%, 50-60%, 60-70%, or 70-80% as a ratio to the length (number of residues) of the first base sequence. Specifically, the number of nucleic acid residues deleted may be, for example, 5 to 80%, 5 to 50%, 5 to 30%, or 5 to 20% in terms of the ratio to the length (number of residues) of the first base sequence.

[0226] The number of groups of nucleic acid residue deletions may be, for example, 1 or more, 2 or more, or 3 or more, or 4 or less, 3 or less, or 2 or less, or a non-contradictory combination thereof. Specifically, the number of groups of nucleic acid residue deletions may be, for example, 1 to 2, 2 to 3, or 3 to 4. Specifically, the number of groups of nucleic acid residue deletions may be, for example, 1 to 4, 1 to 3, 1 to 2, or 1. The number of groups of nucleic acid residue deletions may particularly be 1, 2, or 3. In one embodiment, the number of groups of nucleic acid residue deletions may more particularly be 1. In one embodiment, the number of groups of nucleic acid residue deletions may more particularly be 2. The number of groups of nucleic acid residue deletions may be, for example, 5% or more, 10% or more, 15% or more, 20% or more, or 25% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less, or a non-contradictory combination thereof, as a ratio to the length (number of residues) of the first base sequence. Specifically, the number of groups of nucleic acid residue deletions, for example, can be 5 to 10%, 10 to 15%, 15 to 20%, 20 to 25%, or 25 to 30% in terms of the ratio to the length (number of residues) of the first base sequence. Specifically, the number of groups of nucleic acid residue deletions, for example, can be 5 to 30%, 5 to 20%, or 5 to 15% in terms of the ratio to the length (number of residues) of the first base sequence.

[0227] The number of nucleic acid residue deletions constituting a deletion of one set of nucleic acid residues may be, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 12 or more, 15 or more, 20 or more, or 25 or more, or 30 or less, 25 or less, 20 or less, 15 or less, 12 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, or a non-contradictory combination thereof. Specifically, the number of nucleic acid residue deletions constituting a deletion of one set of nucleic acid residues may be, for example, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 12, 12 to 15, 15 to 20, 20 to 25, or 25 to 30. Specifically, the number of nucleic acid residue deletions constituting a deletion of one set of nucleic acid residues may be, for example, 1 to 30, 1 to 20, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. The number of nucleic acid residue deletions constituting a deletion of one set of nucleic acid residues may particularly be 1, 2, 3, 4, 5, 6, 7, or 8. The number of nucleic acid residue deletions constituting a deletion of one set of nucleic acid residues may more particularly be 1, 2, or 3. When the number of nucleic acid residue deletions is 2 or more, the number of nucleic acid residue deletions constituting one set of nucleic acid residue deletions may be selected independently for each set of nucleic acid residue deletions unless otherwise specified.

[0228] When the number of nucleic acid residue deletions is 2 or more, unless otherwise specified, the position of introduction of the nucleic acid residue deletion can be selected independently for each nucleic acid residue deletion. The position of introduction of the nucleic acid residue deletion can be, for example, the position of introduction of the destabilizing portion exemplified above. The position of introduction of the nucleic acid residue deletion can be particularly -3 to -6 or +3 to +6.

[0229] The number of insertions, deletions or substitutions of the linker (i.e., the number of inserted, deleted or substituted linkers) can be, for example, more than 1, more than 2, more than 3, more than 4, more than 5, more than 6, or more than 7, or less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, or less than 2, or a non-contradictory combination thereof. Specifically, the number of insertions, deletions or substitutions of the linker can be, for example, 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, or 7-8. Specifically, the number of insertions, deletions or substitutions of the linker can be, for example, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1. The number of insertions, deletions or substitutions of the linker can particularly be 1, 2, or 3. The number of insertions, deletions or substitutions of the linker can more particularly be 1. The number of insertions, deletions or substitutions of the linker, for example, in terms of the ratio to the length (number of residues) of the first base sequence, can be 5% or more, 10% or more, 15% or more, 20% or more, or 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less, or a combination thereof that does not contradict each other. Specifically, the number of insertions, deletions or substitutions of the linker, for example, in terms of the ratio to the length (number of residues) of the first base sequence, can be 5 to 10%, 10 to 15%, 15 to 20%, 20 to 25%, or 25 to 30%. Specifically, the number of insertions, deletions or substitutions of the linker, for example, in terms of the ratio to the length (number of residues) of the first base sequence, can be 5 to 30%, 5 to 20%, or 5 to 15%.

[0230] The number of insertions, deletions or substitutions of the linker may be, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more, or 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, or any combination thereof that does not contradict each other. Specifically, the number of insertions, deletions or substitutions of the linker may be, for example, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, or 7 to 8. Specifically, the number of insertions, deletions or substitutions of the linker may be, for example, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. The number of insertions, deletions or substitutions of the linker may particularly be 1, 2, or 3. The number of insertions, deletions or substitutions of the linker may more particularly be 1. The number of groups of insertion, deletion or substitution of the linker, for example, in terms of the ratio to the length (number of residues) of the first base sequence, can be 5% or more, 10% or more, 15% or more, 20% or more, or 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less, or a combination thereof that does not contradict each other. Specifically, the number of groups of insertion, deletion or substitution of the linker, for example, in terms of the ratio to the length (number of residues) of the first base sequence, can be 5 to 10%, 10 to 15%, 15 to 20%, 20 to 25%, or 25 to 30%. Specifically, the number of groups of insertion, deletion or substitution of the linker, for example, in terms of the ratio to the length (number of residues) of the first base sequence, can be 5 to 30%, 5 to 20%, or 5 to 15%.

[0231] The number of insertions, deletions or substitutions of the linkers constituting the insertion, deletion or substitution of one set of linkers may be, for example, 1 or more, 2 or more, 3 or more, or 4 or more, or 5 or less, 4 or less, 3 or less, or 2 or less, or a non-contradictory combination thereof. Specifically, the number of insertions, deletions or substitutions of the linkers constituting the insertion, deletion or substitution of one set of linkers may be, for example, 1 to 2, 2 to 3, 3 to 4, or 4 to 5. Specifically, the number of insertions, deletions or substitutions of the linkers constituting the insertion, deletion or substitution of one set of linkers may be, for example, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. The number of insertions, deletions or substitutions of the linkers constituting the insertion, deletion or substitution of one set of linkers may particularly be 1 or 2. The number of insertions, deletions or substitutions of the linkers constituting the insertion, deletion or substitution of one set of linkers may more particularly be 1. When the number of groups of insertions, deletions or substitutions of linkers is 2 or more, unless otherwise specified, the number of insertions, deletions or substitutions of the linkers constituting the insertion, deletion or substitution of one set of linkers may be selected independently for each group of insertions, deletions or substitutions of linkers.

[0232] When the number of insertions, deletions or substitutions of the linker is 2 or more, unless otherwise specified, the position of introduction of the insertion, deletion or substitution of the linker can be selected independently for the insertion, deletion or substitution of each linker. The position of introduction of the insertion, deletion or substitution of the linker can be, for example, the position of introduction of the destabilizing portion exemplified above.

[0233] The destabilizing portion may, for example, comprise an insertion of nucleic acid residues that is not accompanied by a linker deletion and / or a deletion of nucleic acid residues that is not accompanied by a linker insertion.

[0234] The destabilizing portion may, for example, include the insertion of more than two consecutive nucleic acid residues (i.e., a nucleic acid residue insertion group consisting of the insertion of more than two nucleic acid residues) and / or the deletion of more than two consecutive nucleic acid residues (i.e., a nucleic acid residue deletion group consisting of the deletion of more than two nucleic acid residues).

[0235] The destabilizing unit may include, for example, the following (A):

[0236] (A) Combination of deletion and / or insertion of the abasic portion and nucleic acid residues.

[0237] The abasic part in the destabilizing part (A) may be one abasic part or two or more abasic parts. The abasic part in the destabilizing part (A) may be, for example, one abasic part at positions -1 to +1, or a combination of one abasic part at positions -1 to +1 and one or more abasic parts at positions other than -1 to +1. The abasic part in the destabilizing part (A) may particularly be one abasic part at positions -1 to +1.

[0238] The destabilizing unit may include, for example, the following (B):

[0239] (B) Combination of mismatches with deletion and / or insertion of nucleic acid residues.

[0240] The mismatch part in the destabilizing part (B) may be one mismatch part or two or more mismatch parts. The mismatch part in the destabilizing part (B) may be, for example, one mismatch part at positions -1 to +1, or a combination of one mismatch part at positions -1 to +1 and one or more mismatch parts at positions other than positions -1 to +1. The mismatch part in the destabilizing part (B) may particularly be one mismatch part at positions -1 to +1.

[0241] The deletion of nucleic acid residues in the destabilizing portion (A) or (B) may be, for example, the deletion of one or more nucleic acid residues at positions other than -1 to +1. The deletion of nucleic acid residues in the destabilizing portion (A) or (B) may be, for example, the deletion of one or more groups of nucleic acid residues at positions other than -1 to +1. Specifically, the deletion of nucleic acid residues in the destabilizing portion (A) or (B) may be, for example, the deletion of one or two groups of nucleic acid residues at positions other than -1 to +1.

[0242] The insertion of nucleic acid residues in the destabilizing part (A) or (B) may be, for example, the insertion of one or more nucleic acid residues at positions other than -1 to +1. The insertion of nucleic acid residues in the destabilizing part (A) or (B) may be, for example, the insertion of one or more nucleic acid residues at positions other than -1 to +1. Specifically, the insertion of nucleic acid residues in the destabilizing part (A) or (B) may be, for example, the insertion of one or two groups of nucleic acid residues at positions other than -1 to +1. The insertion of nucleic acid residues in the destabilizing part (A) or (B) may particularly be the insertion of two groups of nucleic acid residues at positions other than -1 to +1. The insertion of nucleic acid residues in the destabilizing part (A) or (B) may more particularly be a combination of the insertion of one group of nucleic acid residues at negative positions other than -1 and the insertion of one group of nucleic acid residues at positive positions other than +1.

[0243] The destabilizing part may include, for example, the following (a), (b) or (c):

[0244] (a) a combination of deletion and / or insertion of the abasic portion at positions -1 to +1 and nucleic acid residues at positions other than -1 to +1;

[0245] (b) a combination of a mismatch at positions -1 to +1 and a deletion and / or insertion of a nucleic acid residue at a position other than positions -1 to +1;

[0246] (c) Mismatched portion at positions -1 to +1.

[0247] The destabilizing section (a) may be an example of the destabilizing section (A). The destabilizing section (b) may be an example of the destabilizing section (B).

[0248] The destabilizing part may specifically include: the destabilizing part (A) such as the destabilizing part (a) or the destabilizing part (B) such as the destabilizing part (b). The destabilizing part may more specifically include the destabilizing part (A) such as the destabilizing part (a).

[0249] The destabilizing portion (a) may be, for example, a combination of an abasic portion at positions -1 to +1 and a deletion of one or more nucleic acid residues and / or an insertion of one or more nucleic acid residues at positions other than -1 to +1. The destabilizing portion (a) may be, for example, a combination of an abasic portion at positions -1 to +1 and a deletion of one or more nucleic acid residues at positions other than -1 to +1 and / or an insertion of one or more nucleic acid residues. Specifically, the destabilizing portion (a) may be, for example, a combination of an abasic portion at positions -1 to +1 and a deletion of one or two nucleic acid residues at positions other than -1 to +1 and / or an insertion of one or two nucleic acid residues. The destabilizing portion (a) may particularly be a combination of an abasic portion at positions -1 to +1 and an insertion of one or two nucleic acid residues at positions other than -1 to +1. The destabilizing part (a) may more particularly be a combination of an abasic part at positions -1 to +1 and insertion of two groups of nucleic acid residues at positions other than -1 to +1. The destabilizing part (a) may more particularly be a combination of an abasic part at positions -1 to +1, insertion of a group of nucleic acid residues at negative positions other than -1, and insertion of a group of nucleic acid residues at positive positions other than +1.

[0250] The destabilizing portion (b) may be, for example, a combination of one mismatch at positions -1 to +1 and one or more deletions and / or one or more insertions of nucleic acid residues at positions other than -1 to +1. The destabilizing portion (b) may be, for example, a combination of one mismatch at positions -1 to +1 and one or more deletions and / or one or more insertions of nucleic acid residues at positions other than -1 to +1. Specifically, the destabilizing portion (b) may be, for example, a combination of one mismatch at positions -1 to +1 and one or two deletions and / or one or two insertions of nucleic acid residues at positions other than -1 to +1. The destabilizing portion (b) may particularly be a combination of one mismatch at positions -1 to +1 and one or two insertions of nucleic acid residues at positions other than -1 to +1. The destabilizing portion (b) may more particularly be a combination of one mismatch at positions -1 to +1 and two insertions of nucleic acid residues at positions other than -1 to +1. More specifically, the destabilizing portion (b) may be a combination of a mismatch portion at positions -1 to +1, an insertion of a group of nucleic acid residues at a negative position other than -1, and an insertion of a group of nucleic acid residues at a positive position other than +1.

[0251] As positions other than -1 to +1, there can be mentioned positions other than -1 to +1 selected from the above-exemplified introduction positions of the destabilizing portion. As positions other than -1 to +1, there can be mentioned -5 to -7, +5 to +7, -3 to -6, and +3 to +6. As negative positions other than -1, there can be mentioned negative positions other than -1 selected from the above-exemplified introduction positions of the destabilizing portion. As negative positions other than -1, there can be mentioned -5 to -7 or -3 to -6. As negative positions other than -1, there can be mentioned -3 to -6 more particularly. As positive positions other than +1, there can be mentioned positive positions other than +1 selected from the above-exemplified introduction positions of the destabilizing portion. As positive positions other than +1, there can be mentioned +5 to +7 or +3 to +6. As positive positions other than +1, there can be mentioned +3 to +6 more particularly.

[0252] In one embodiment, the situation that the destabilizing part is composed of 1 abasic part, 1 linker substituted with a nucleic acid residue, or 1 nucleic acid residue substituted with a linker can be excluded from the present invention. In one embodiment, the situation that the destabilizing part is composed of 1 abasic part at position -1, 1 linker between position -1 and position +1 substituted with a nucleic acid residue, or 1 nucleic acid residue at position -1 substituted with a linker can be excluded from the present invention. In one embodiment, the situation that the number (N) of substrate oligonucleotides is 2 and the destabilizing part is composed of 1 abasic part at position -1, 1 linker between position -1 and position +1 substituted with a nucleic acid residue, or 1 nucleic acid residue at position -1 substituted with a linker can be excluded from the present invention. In one embodiment, the present invention excludes the case where the number of substrate oligonucleotides (N) is 2, the number of complementary oligonucleotides (M) is 1, and the destabilizing portion is composed of a baseless portion at position -1, a linker between positions -1 and +1 replaced by a nucleic acid residue, or a nucleic acid residue at position -1 replaced by a linker.

[0253] In one embodiment, the destabilizing portion is composed of 1 abasic portion, 2 abasic portions, or a combination of 1 abasic portion and 1 mismatch portion, which can be excluded from the present invention. In one embodiment, the destabilizing portion is composed of 1 abasic portion at position -1, a combination of 1 abasic portion at position -1 and 1 abasic portion at a position other than -1 (e.g., position +4), or a combination of 1 abasic portion at position -1 and 1 mismatch portion at a position other than -1 (e.g., position +4), which can be excluded from the present invention. In one embodiment, the number (N) of substrate oligonucleotides is 2, and the destabilizing portion is composed of 1 abasic portion at position -1, a combination of 1 abasic portion at position -1 and 1 abasic portion at a position other than -1 (e.g., position +4), or a combination of 1 abasic portion at position -1 and 1 mismatch portion at a position other than -1 (e.g., position +4), which can be excluded from the present invention. In one embodiment, the situation in which the number of substrate oligonucleic acids (N) is 2, the number of complementary oligonucleic acids (M) is 1, and the destabilizing portion is composed of 1 abasic portion at position -1, a combination of 1 abasic portion at position -1 and 1 abasic portion at a position other than position -1 (e.g., position +4), or a combination of 1 abasic portion at position -1 and 1 mismatch portion at a position other than position -1 (e.g., position +4) can be excluded from the present invention.

[0254] The complementary oligonucleotide may or may not consist of a destabilizing base sequence. That is, the complementary oligonucleotide may further include a base sequence (hereinafter also referred to as an "addition sequence") on the 5' side and / or 3' side of the destabilizing base sequence. The complementary oligonucleotide may particularly consist of a destabilizing base sequence.

[0255] There is no particular limitation on the length of the addition sequence as long as the purpose of the present invention is not impaired.

[0256] The lengths of the 5'-side and 3'-side addition sequences may be, for example, both 0 residues, 1 residue, 2 residues, 3 residues, 4 residues, 5 residues, 7 residues, 10 residues, 15 residues, or 20 residues, or less than 25 residues, less than 20 residues, less than 15 residues, less than 10 residues, less than 7 residues, less than 5 residues, less than 4 residues, less than 3 residues, less than 2 residues, or less than 1 residue, or a non-contradictory combination thereof. Specifically, the lengths of the 5'-side and 3'-side addition sequences may be, for example, both 0 to 1 residue, 1 to 2 residues, 2 to 3 residues, 3 to 4 residues, 4 to 5 residues, 5 to 7 residues, 7 to 10 residues, 10 to 15 residues, 15 to 20 residues, or 20 to 25 residues. Specifically, the lengths of the 5'-side and 3'-side additional sequences may both be, for example, 0 to 25 residues, 0 to 15 residues, or 0 to 10 residues.

[0257] The lengths of the 5' and 3' additional sequences may be, for example, 0% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 100% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, or any non-contradictory combination thereof. The lengths of the 5' and 3' additional sequences may be, for example, 0 to 5%, 5 to 10%, 10 to 20%, 20 to 30%, 30 to 40%, 40 to 50%, 50 to 60%, 60 to 70%, 70 to 80%, 80 to 90%, or 90 to 100%, based on the total length of the 5' and 3' substrate oligonucleotides being 100%. Specifically, the lengths of the 5' and 3' additional sequences may be, for example, 0 to 100%, 0 to 50%, or 0 to 20%, based on the total length of the 5' and 3' substrate oligonucleotides being 100%.

[0258] The length of the complementary oligonucleotide can be, for example, 10 residues or more, 11 residues or more, 12 residues or more, 13 residues or more, 14 residues or more, 15 residues or more, 16 residues or more, 17 residues or more, 18 residues or more, 19 residues or more, 20 residues or more, 21 residues or more, 22 residues or more, 23 residues or more, 24 residues or more, 25 residues or more, 30 residues or more, 35 residues or more, 40 residues or more, 45 residues or more, 50 residues or more, 60 residues or more, 70 residues or more, 80 residues or more, 90 residues or more, 100 residues or more, 110 residues or more, 120 residues or more, 130 residues or more, 140 residues or more, 150 residues or more, 160 residues or more, 170 residues or more, 180 residues or more, or 190 residues or more. It is 200 residues or less, 190 residues or less, 180 residues or less, 170 residues or less, 160 residues or less, 150 residues or less, 140 residues or less, 130 residues or less, 120 residues or less, 110 residues or less, 100 residues or less, 90 residues or less, 80 residues or less, 70 residues or less, 60 residues or less, 50 residues or less, 45 residues or less, 40 residues or less, 35 residues or less, 30 residues or less, 25 residues or less, 24 residues or less, 23 residues or less, 22 residues or less, 21 residues or less, 20 residues or less, 19 residues or less, 18 residues or less, 17 residues or less, 16 residues or less, 15 residues or less, 14 residues or less, 13 residues or less, 12 residues or less, or 11 residues or less, and any non-contradictory combination thereof. Specifically, the length of the complementary oligonucleotide can be, for example, 10 to 11 residues, 11 to 12 residues, 12 to 13 residues, 13 to 14 residues, 14 to 15 residues, 15 to 16 residues, 16 to 17 residues, 17 to 18 residues, 18 to 19 residues, 19 to 20 residues, 20 to 21 residues, 21 to 22 residues, 22 to 23 residues, 23 to 24 residues, 24 to 25 residues, 25 to 30 residues, 30 to 35 residues, 35 to 40 residues. 140 residues, 140-150 residues, 150-160 residues, 160-170 residues, 170-180 residues, 180-190 residues or 190-200 residues.Specifically, the length of the complementary oligonucleotide can be, for example, 10 to 200 residues, 10 to 100 residues, 15 to 80 residues, or 20 to 60 residues.

[0259] The length of the complementary oligonucleotide may be, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 170% or more, 200% or more, 250% or more, 300% or more, 400% or more, 500% or more, 600% or more, 700% or more, 800% or more, 900% or more, 100% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 170% or more, 200% or more, 250% or more, 300% or more, 400% or more, 400% or more, 500% or more, 600% or more, 700% or more, 800% or more, 900% or more, 100% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 170% or more, 2 % or more or 350% or more; it may also be less than 400%, less than 350%, less than 300%, less than 250%, less than 200%, less than 170%, less than 150%, less than 140%, less than 130%, less than 120%, less than 110%, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20% or less than 10%; it may also be a non-contradictory combination of these. Specifically, the length of the complementary oligonucleotide can be, for example, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-110%, 110-120%, 120-130%, 130-140%, 140-150%, 150-170%, 170-200%, 200-250%, 250-300%, 300-350% or 350-400%, based on the total length of the 5' side and 3' side substrate oligonucleotides being 100%. Specifically, the length of the complementary oligonucleotide can be, for example, 5-400%, 5-300%, 5-200%, 5-150%, 5-100%, 5-50%, 5-20%, 20-150%, 20-100%, 20-50%, 50-150% or 50-100%, based on the total length of the 5' side and 3' side substrate oligonucleotides being 100%.

[0260] The length of the complementary oligonucleotide may be, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 170% or more, 200% or more, 250% or more, 300% or more, or 350% or more, based on the length of the target oligonucleotide as 100%. The amount of the content of the present invention may be more than 50%, or it may be less than 400%, less than 350%, less than 300%, less than 250%, less than 200%, less than 170%, less than 150%, less than 140%, less than 130%, less than 120%, less than 110%, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20% or less than 10%, and it may also be a non-contradictory combination of these. Specifically, the length of the complementary oligonucleotide can be, for example, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-110%, 110-120%, 120-130%, 130-140%, 140-150%, 150-170%, 170-200%, 200-250%, 250-300%, 300-350% or 350-400%, based on the length of the target oligonucleotide as 100%. Specifically, the length of the complementary oligonucleotide can be, for example, 5-400%, 5-300%, 5-200%, 5-150%, 5-100%, 5-50%, 5-20%, 20-150%, 20-100%, 20-50%, 50-150% or 50-100%, based on the length of the target oligonucleotide as 100%.

[0261] The terms "the complementary oligonucleotide comprises modified nucleic acid residues", "the complementary oligonucleotide is modified" and "the complementary oligonucleotide has a modification" are used interchangeably.

[0262] Each complementary oligonucleotide may or may not include modified nucleic acid residues. That is, each complementary oligonucleotide may or may not be modified. About the modification of nucleic acid residues, as described above. Each complementary oligonucleotide may have one modification, or may be combined with two or more modifications. Each complementary oligonucleotide, for example, may or may not include each modification of the above examples. Each nucleic acid residue constituting each complementary oligonucleotide may have one modification, or may be combined with two or more modifications. The nucleic acid residues constituting each complementary oligonucleotide may only be partially modified nucleic acid residues, and the nucleic acid residues constituting each complementary oligonucleotide may also all be modified nucleic acid residues. For example, in the case where the complementary oligonucleotide comprises a deletion of a nucleic acid residue as a destabilizing portion, a linker may be inserted into the position of the deletion of the nucleic acid residue.

[0263] The ratio of the modified nucleic acid residues in the complementary oligonucleotide (i.e., the ratio of the number of modified nucleic acid residues to the number of nucleic acid residues constituting the complementary oligonucleotide) can be, for example, 0% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or less, 100% or less, 95% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, or 5% or less, and can also be a non-contradictory combination thereof. Specifically, the ratio of the modified nucleic acid residues in the complementary oligonucleotide can be, for example, 0-5%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95% or 95-100%. It should be noted that the ratio of the modified nucleic acid residues in the complementary oligonucleotides exemplified above can also be independently applied to the selected nucleic acid residues with any modification (for example, any modification of the above examples). That is, for example, the ratio of the modified nucleic acid residues with a phosphate portion in the complementary oligonucleotide (that is, the ratio of the number of modified nucleic acid residues with a phosphate portion to the number of nucleic acid residues constituting the complementary oligonucleotide) can be set to the ratio of the modified nucleic acid residues in the complementary oligonucleotides exemplified above. In addition, for example, the ratio of the modified nucleic acid residues with a sugar portion in the complementary oligonucleotide (that is, the ratio of the number of modified nucleic acid residues with a sugar portion to the number of nucleic acid residues constituting the complementary oligonucleotide) can be set to the ratio of the modified nucleic acid residues in the complementary oligonucleotides exemplified above. In addition, for example, the ratio of modified nucleic acid residues having a base portion in a complementary oligonucleotide (i.e., the ratio of the number of modified nucleic acid residues having a base portion to the number of nucleic acid residues constituting the complementary oligonucleotide) can be set to the ratio of modified nucleic acid residues in the complementary oligonucleotide exemplified above.

[0264] The number of modified nucleic acid residues in the complementary oligonucleic acid may be, for example, 0 residues or more, 5 residues or more, 10 residues or more, 15 residues or more, 20 residues or more, 25 residues or more, 30 residues or more, 40 residues or more, 50 residues or more, 60 residues or more, 80 residues or more, 100 residues or more, 120 residues or more, 140 residues or more, 160 residues or more, or 180 residues or more; or 200 residues or less, 180 residues or less, 160 residues or less, 140 residues or less, 120 residues or less, 100 residues or less, 80 residues or less, 60 residues or less, 50 residues or less, 40 residues or less, 30 residues or less, 25 residues or less, 20 residues or less, 15 residues or less, 10 residues or less, or 5 residues or less; or any non-contradictory combination thereof. Specifically, the number of modified nucleic acid residues in the complementary oligonucleotide can be, for example, 0 to 5 residues, 5 to 10 residues, 10 to 15 residues, 15 to 20 residues, 20 to 25 residues, 25 to 30 residues, 30 to 40 residues, 40 to 50 residues, 50 to 60 residues, 60 to 80 residues, 80 to 100 residues, 100 to 120 residues, 120 to 140 residues, 140 to 160 residues, 160 to 180 residues or 180 to 200 residues. It should be noted that the number of modified nucleic acid residues in the complementary oligonucleotides exemplified above can also be independently applied to the selected nucleic acid residues with any modification (for example, any modification of the above examples). That is, for example, the number of modified nucleic acid residues with a phosphate portion in the complementary oligonucleotide can be set to the number of modified nucleic acid residues in the complementary oligonucleotides exemplified above. In addition, for example, the number of modified nucleic acid residues with a sugar portion in the complementary oligonucleotide can be set to the number of modified nucleic acid residues in the complementary oligonucleotides exemplified above. Furthermore, for example, the number of nucleic acid residues having a modification of the base portion in the complementary oligonucleotide can be set to the number of modified nucleic acid residues in the complementary oligonucleotide exemplified above.

[0265] In the connection process, M (M is an integer greater than or equal to 1) complementary oligonucleotides are used. The number of complementary oligonucleotides (M) is greater than or equal to 1. "The number of complementary oligonucleotides" refers to the number of complementary oligonucleotides used in the connection process. The number of complementary oligonucleotides (M) can be 1 or greater than or equal to 2.

[0266] When the number of connecting parts (N-1) is more than 2, complementary oligonucleotides can be selected independently for each of these connecting parts. In other words, when the number of connecting parts (N-1) is more than 2, destabilizing parts can be selected independently for each of these connecting parts (i.e., each complementary oligonucleotide corresponding to these connecting parts). For these connecting parts (i.e., the complementary oligonucleotides corresponding to these connecting parts), the destabilizing parts may be the same or different. For example, a destabilizing part can be selected independently from the scheme of the destabilizing part of the above-mentioned examples for each of these connecting parts (i.e., each complementary oligonucleotide corresponding to these connecting parts). Specifically, regarding the destabilizing part, for example, each of these connecting parts (i.e., each complementary oligonucleotide corresponding to these connecting parts) may independently include the above-mentioned destabilizing part (a) (i.e., a combination of abasic part at positions -1 to +1 and deletion and / or insertion of nucleic acid residues at positions other than -1 to +1), the above-mentioned destabilizing part (A) (i.e., a combination of abasic part and deletion and / or insertion of nucleic acid residues), the above-mentioned destabilizing part (b) (i.e., a combination of mismatch part at positions -1 to +1 and deletion and / or insertion of nucleic acid residues at positions other than -1 to +1), the above-mentioned destabilizing part (B) (i.e., a combination of mismatch part and deletion and / or insertion of nucleic acid residues), or the above-mentioned destabilizing part (c) (i.e., a mismatch part at positions -1 to +1). In addition, for example, any one of the above-mentioned exemplified destabilizing parts may be selected as the destabilizing part of the complementary oligonucleotide corresponding to any connecting part, and other embodiments of the above-mentioned destabilizing parts may be selected as the destabilizing part of the complementary oligonucleotide corresponding to other connecting parts. Specifically, for example, the destabilizing portion of the complementary oligonucleotide corresponding to any one of the connecting portions may include the destabilizing portion (a) or (b), and the destabilizing portion of the complementary oligonucleotide corresponding to the other connecting portion may include the destabilizing portion (c). In particular, the destabilizing portion of the complementary oligonucleotide corresponding to any one of the connecting portions may include the destabilizing portion (a), and the destabilizing portion of the complementary oligonucleotide corresponding to the other connecting portion may include the destabilizing portion (c).

[0267] In addition, in each connecting part, one complementary oligonucleotide can be used, and more than two complementary oligonucleotides can also be used. That is, the connection between the substrate oligonucleotides on the 5' side and the 3' side of each connecting part can be implemented in the presence of more than one complementary oligonucleotide corresponding to the connecting part. In addition, one complementary oligonucleotide can correspond to more than two connecting parts. In other words, one complementary oligonucleotide can be used in the connection between the substrate oligonucleotides on the 5' side and the 3' side of the connecting part more than two. In the case where one complementary oligonucleotide corresponds to more than two connecting parts, the complementary oligonucleotide can include the destabilizing base sequence corresponding to these connecting parts respectively. For example, in the case where three substrate oligonucleotides A, B and C are connected in the 5' to 3' direction in the presence of one complementary oligonucleotide, the complementary oligonucleotide can include the destabilizing base sequence for the connection of B and C and the destabilizing base sequence for the connection of A and B in the 5' to 3' direction. In this case, the third base sequence of the destabilizing base sequence for linking B and C and the second base sequence of the destabilizing base sequence for linking A and B may be partially or completely repeated, or may not be repeated.

[0268] The number of complementary oligonucleotides (M) may be less than the number of connectors (N-1), may be the same, or may be greater than the number of connectors. For example, when one complementary oligonucleotide is used independently in each connector, the number of complementary oligonucleotides (M) may be the same as the number of connectors (N-1). In addition, for example, when one complementary oligonucleotide corresponds to more than two connectors, the number of complementary oligonucleotides (M) may be less than the number of connectors (N-1). When the number of complementary oligonucleotides (M) is more than 2, destabilizing parts may be selected independently for these complementary oligonucleotides. For these complementary oligonucleotides, the destabilizing parts may be the same or different. For example, the destabilizing parts may be selected independently from the above-exemplified destabilizing parts schemes for these complementary oligonucleotides. Specifically, with respect to the destabilizing parts, for example, these complementary oligonucleotides may independently include the above-exemplified destabilizing parts (a), (b) or (c). In addition, for example, any of the above-exemplified destabilizing parts schemes may be selected as the destabilizing parts of any complementary oligonucleotide, and other schemes of the above-exemplified destabilizing parts may be selected as the destabilizing parts of other complementary oligonucleotides. Specifically, for example, the destabilizing portion of any complementary oligonucleotide may include the destabilizing portion (a) or (b), and the destabilizing portion of the other complementary oligonucleotide may include the destabilizing portion (c). In particular, the destabilizing portion of any complementary oligonucleotide may include the destabilizing portion (a), and the destabilizing portion of the other complementary oligonucleotide may include the destabilizing portion (c).

[0269] There is no particular limitation on the method for producing complementary oligonucleic acids. Complementary oligonucleic acids can be produced, for example, by sequentially connecting nucleic acid residues constituting complementary oligonucleic acids. The connection of nucleic acid residues can be carried out, for example, by the phosphoramidite method. In addition, complementary oligonucleic acids can be produced, for example, by connecting nucleic acid fragments constituting complementary oligonucleic acids. The connection of nucleic acid fragments can be carried out, for example, by enzymatic means using complementary chains. The connection of nucleic acid fragments can be carried out, for example, by the method of the present invention.

[0270] <4>Connection process

[0271] The ligation step is a step of enzymatically ligating the substrate oligonucleotide group. The term "ligation of the substrate oligonucleotide group" can be used interchangeably with the term "ligation of the substrate oligonucleotides constituting the group".

[0272] "Enzymatically ligating the substrate oligonucleotide group" means that the substrate oligonucleotide group is connected by a ligase. In the connection process, for example, the 5' side and 3' side substrate oligonucleotides are hybridized with the corresponding complementary oligonucleotides to form a double-stranded structure, and then the 5' side and 3' side substrate oligonucleotides can be connected by a ligase. The ligase is not particularly limited as long as it can connect the substrate oligonucleotides in the presence of complementary oligonucleotides. As ligases, DNA ligases or RNA ligases can be listed. As ligases, DNA ligases can be specifically listed. There is no particular restriction on the source of the ligase. Ligase can be derived from eukaryotic organisms such as mammals or yeast, from prokaryotes such as Escherichia coli (E. coli), and from viruses such as bacteriophages. As DNA ligases, T3 DNA ligases, T4 DNA ligases, T7 DNA ligases, Escherichia coli DNA ligases, and Taq DNA ligases can be listed. As RNA ligases, T4 RNA ligases such as T4 RNA ligase 2 or RtcB ligases can be listed. In one embodiment, the present invention excludes the case where the ligase is T4 DNA ligase. As a ligase, more particularly, T3 DNA ligase can be cited. DNA ligase can catalyze the connection between DNA or its modified nucleic acid. Therefore, DNA ligase can be used, for example, for the connection between DNA or its modified nucleic acid (i.e., the connection between the 5' side substrate oligonucleotide whose 3' end is DNA or its modified nucleic acid and the 3' side substrate oligonucleotide whose 5' end is DNA or its modified nucleic acid). In addition, certain DNA ligases such as T3 DNA ligase or T4 DNA ligase can also catalyze the connection between RNA or its modified nucleic acid in addition to catalyzing the connection between DNA or its modified nucleic acid. Therefore, certain DNA ligases such as T3 DNA ligase or T4 DNA ligase can be used, for example, for the connection between RNA or its modified nucleic acid (i.e., the connection between the 5' side substrate oligonucleotide whose 3' end is RNA or its modified nucleic acid and the 3' side substrate oligonucleotide whose 5' end is RNA or its modified nucleic acid). RNA ligase can catalyze the connection between RNA or its modified nucleic acid. Therefore, RNA ligase can be used, for example, to ligate RNAs or modified nucleic acids thereof (ie, to ligate a 5'-side substrate oligonucleotide whose 3' end is RNA or modified nucleic acid thereof with a 3'-side substrate oligonucleotide whose 5' end is RNA or modified nucleic acid thereof).

[0273] The connection process can be implemented in a liquid. The liquid for implementing the connection process is also referred to as a "reaction solution". Specifically, the connection process can be implemented by allowing a ligase, a substrate oligonucleotide and a complementary oligonucleotide to coexist in a reaction solution. The connection process can be implemented, for example, in a batch manner, or in a column manner. In the case of a batch manner, for example, the connection process can be implemented by mixing a ligase, a substrate oligonucleotide and a complementary oligonucleotide in the reaction solution in a container. The connection process can be implemented while standing, or while stirring or vibrating. In the case of a column, for example, the connection process can be implemented by allowing a reaction solution containing a substrate oligonucleotide and a complementary oligonucleotide to pass through a column filled with an immobilized ligase. As the reaction solution, an aqueous medium such as water or a buffer solution can be used. In addition to containing a ligase, a substrate oligonucleotide and a complementary oligonucleotide, the reaction solution can also contain components required for or useful in the connection process. As such components, pH buffers, Mg ions, ATP, and DTT can be cited.

[0274] There is no particular restriction on the implementation conditions of the connection process (for example, the pH of the reaction solution, the reaction temperature, the reaction time, and the amount or concentration of each component used), as long as the target oligonucleotide is generated. The implementation conditions of the connection process can be appropriately set according to various conditions such as the structure of the substrate oligonucleotide, the structure of the complementary oligonucleotide, and the type of ligase. It should be noted that, unless otherwise specified, "the concentration of a certain component in the reaction solution" may refer to the maximum concentration of the component in the reaction solution in the connection process. The maximum concentration of a certain component in the reaction solution in the connection process can be, for example, the concentration of the component in the reaction solution at the beginning of the connection process.

[0275] The pH of the reaction solution may be, for example, 5 to 10, 6 to 9, or 7 to 8.

[0276] The reaction temperature may be, for example, 5°C or more, 10°C or more, 15°C or more, 20°C or more, 25°C or more, 30°C or more, 35°C or more, 40°C or more, 45°C or more, 50°C or more, or 90°C or less, 80°C or less, 70°C or less, 60°C or less, 55°C or less, 50°C or less, 45°C or less, 40°C or less, 35°C or less, 30°C or less, 25°C or less, 20°C or less, 15°C or less, or 10°C or less, or a non-contradictory combination thereof. Specifically, the reaction temperature may be, for example, 5-10°C, 10-15°C, 15-20°C, 20-25°C, 25-30°C, 30-35°C, 35-40°C, 40-45°C, 45-50°C, 50-55°C, or 55-60°C. Specifically, the reaction temperature may be, for example, 5 to 60° C., 10 to 40° C., 15 to 35° C., or 20 to 30° C. The reaction temperature may particularly be room temperature (e.g., about 25° C.). In the connection step, heating may or may not be performed. The range of variation of the reaction temperature in the entire connection step may be, for example, below 30° C., below 25° C., below 20° C., below 15° C., below 10° C., or below 5° C.

[0277] The reaction time can be, for example, more than 0.5 hours, more than 1 hour, more than 2 hours, more than 4 hours, more than 6 hours, more than 9 hours, more than 12 hours, more than 15 hours, more than 18 hours, more than 24 hours or more than 30 hours, or less than 36 hours, less than 30 hours, less than 24 hours, less than 18 hours, less than 15 hours, less than 12 hours, less than 9 hours, less than 6 hours, less than 4 hours, less than 2 hours or less than 1 hour, or a non-contradictory combination thereof. Specifically, the reaction time can be, for example, 0.5 to 1 hour, 1 to 2 hours, 2 to 4 hours, 4 to 6 hours, 6 to 9 hours, 9 to 12 hours, 12 to 15 hours, 15 to 18 hours, 18 to 24 hours, 24 to 30 hours or 30 to 36 hours. Specifically, the reaction time can be, for example, 1 to 36 hours, 3 to 30 hours or 6 to 24 hours.

[0278] The usage of the 5' side and 3' side substrate oligonucleotides is, for example, not excessive or insufficient, and in order to connect these substrate oligonucleotides, they can be substantially the same in terms of molar ratio. "The usage of the 5' side and 3' side substrate oligonucleotides is substantially the same in terms of molar ratio" can mean that any one of the lesser of the usage of the 5' side and 3' side substrate oligonucleotides is more than 90%, more than 95%, more than 97% or more than 99% of the other in terms of molar ratio, and also includes the situation where the usage of the 5' side and 3' side substrate oligonucleotides is the same in terms of molar ratio. The molar concentrations of the 5' side and 3' side substrate oligonucleotides in the reaction solution are, for example, not excessive or insufficient, and in order to connect these substrate oligonucleotides, they can be substantially the same. "The molar concentrations of the 5' and 3' substrate oligonucleotides in the reaction solution are substantially the same" may mean that the smaller of the molar concentrations of the 5' and 3' substrate oligonucleotides is more than 90%, more than 95%, more than 97% or more than 99% of the other, and also includes the case where the molar concentrations of the 5' and 3' substrate oligonucleotides are the same.

[0279] The concentration of each substrate oligonucleic acid in the reaction solution can be, for example, more than 1 μM, more than 2 μM, more than 5 μM, more than 10 μM, more than 20 μM, more than 50 μM, more than 100 μM, more than 200 μM, more than 500 μM, more than 1000 μM, more than 2000 μM or more than 5000 μM; it can also be less than 10000 μM, less than 5000 μM, less than 2000 μM, less than 1000 μM, less than 500 μM, less than 200 μM, less than 100 μM, less than 50 μM, less than 20 μM, less than 10 μM, less than 5 μM or less, or a non-contradictory combination thereof. Specifically, the concentration of each substrate oligonucleotide in the reaction solution can be, for example, 1 to 2 μM, 2 to 5 μM, 5 to 10 μM, 10 to 20 μM, 20 to 50 μM, 50 to 100 μM, 100 to 200 μM, 200 to 500 μM, 500 to 1000 μM, 1000 to 2000 μM, 2000 to 5000 μM or 5000 to 10000 μM. Specifically, the concentration of each substrate oligonucleotide in the reaction solution can be, for example, 1 to 10000 μM, 10 to 10000 μM, 20 to 1000 μM or 100 to 500 μM.

[0280] The amount of complementary oligonucleotide used may be less than the lesser of the amount of the 5' substrate oligonucleotide used and the amount of the 3' substrate oligonucleotide used in terms of molar ratio. For example, the amount of complementary oligonucleotide used may be 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%, or 40% in terms of molar ratio, with the lesser of the amount of the 5' substrate oligonucleotide used and the amount of the 3' substrate oligonucleotide used being 100%, or may be 50%, 40%, 30%, 20%, 15%, 10%, 5%, 2%, 10%, 5%, 2%, 10%, 0.5%, 0.2%, 0.1%, 0.05%, or 0.02% in terms of molar ratio, or may be a non-contradictory combination thereof. Specifically, the amount of complementary oligonucleotide used, for example, based on the ratio of the amount of the 5' side substrate oligonucleotide used and the amount of the 3' side substrate oligonucleotide used, whichever is less, as 100%, can be 0.01-0.02%, 0.02-0.05%, 0.05-0.1%, 0.1-0.2%, 0.2-0.5%, 0.5-1%, 1-2%, 2-5%, 5-10%, 10-15%, 15-20%, 20-30%, 30-40%, or 40-50% in molar ratio. Specifically, the amount of complementary oligonucleotide used, for example, based on the ratio of the amount of the 5' side substrate oligonucleotide used and the amount of the 3' side substrate oligonucleotide used, whichever is less, as 100%, can be 0.01-50%, 0.1-50%, 1-50%, 2-30%, or 5-15% in molar ratio.

[0281] The molar concentration of the complementary oligonucleotide in the reaction solution may be smaller than the smaller of the molar concentration of the 5' substrate oligonucleotide in the reaction solution and the molar concentration of the 3' substrate oligonucleotide in the reaction solution. The molar concentration of the complementary oligonucleotide in the reaction solution may be, for example, 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%, or 40% or more, based on the smaller of the molar concentration of the 5' substrate oligonucleotide in the reaction solution and the molar concentration of the 3' substrate oligonucleotide in the reaction solution as 100%, or may be 50%, 40%, 30%, 20%, 15%, 10%, 5%, 2%, 10%, 5%, 2%, 10%, 0.5%, 0.2%, 0.1%, 0.05%, or 0.02% or less, or a non-contradictory combination thereof. Specifically, the molar concentration of the complementary oligonucleotide in the reaction solution can be 0.01-0.02%, 0.02-0.05%, 0.05-0.1%, 0.1-0.2%, 0.2-0.5%, 0.5-1%, 1-2%, 2-5%, 5-10%, 10-15%, 15-20%, 20-30%, 30-40% or 40-50%, for example, based on the smaller of the molar concentration of the 5' substrate oligonucleotide in the reaction solution and the molar concentration of the 3' substrate oligonucleotide in the reaction solution as 100%. Specifically, the molar concentration of the complementary oligonucleotide in the reaction solution can be 0.01 to 50%, 0.1 to 50%, 1 to 50%, 2 to 30% or 5 to 15%, for example, calculated as a ratio of the smaller of the molar concentration of the 5' substrate oligonucleotide in the reaction solution and the molar concentration of the 3' substrate oligonucleotide in the reaction solution as 100%.

[0282] The concentration of the complementary oligonucleic acid in the reaction solution can be, for example, 0.01 μM or more, 0.02 μM or more, 0.05 μM or more, 0.1 μM or more, 0.2 μM or more, 0.5 μM or more, 1 μM or more, 2 μM or more, 5 μM or more, 10 μM or more, 20 μM or more, 50 μM or more, 100 μM or more, 200 μM or more, or 500 μM or less, or 1000 μM or less, 500 μM or less, 200 μM or less, 100 μM or less, 50 μM or less, 20 μM or less, 10 μM or less, 5 μM or less, 2 μM or less, 1 μM or less, 0.5 μM or less, 0.2 μM or less, 0.1 μM or less, 0.05 μM or less, or 0.02 μM or less, and can also be a non-contradictory combination thereof. Specifically, the concentration of the complementary oligonucleotide in the reaction solution can be, for example, 0.01 to 0.02 μM, 0.02 to 0.05 μM, 0.05 to 0.1 μM, 0.1 to 0.2 μM, 0.2 to 0.5 μM, 0.5 to 1 μM, 1 to 2 μM, 2 to 5 μM, 5 to 10 μM, 10 to 20 μM, 20 to 50 μM, 50 to 100 μM, 100 to 200 μM, 200 to 500 μM or 500 to 1000 μM. Specifically, the concentration of the complementary oligonucleotide in the reaction solution can be, for example, 0.01 to 1000 μM, 0.1 to 1000 μM, 1 to 1000 μM, 2 to 100 μM or 10 to 50 μM.

[0283] The concentration of the ligase in the reaction solution may be, for example, 5 U / μL or more, 10 U / μL or more, 20 U / μL or more, 50 U / μL or more, 100 U / μL or more, 200 U / μL or more, or 500 U / μL or less, 1000 U / μL or less, 500 U / μL or less, 200 U / μL or less, 100 U / μL or less, 50 U / μL or less, 20 U / μL or less, or 10 U / μL or less, or any combination thereof. Specifically, the concentration of the ligase in the reaction solution may be, for example, 5 to 10 U / μL, 10 to 20 U / μL, 20 to 50 U / μL, 50 to 100 U / μL, 100 to 200 U / μL, 200 to 500 U / μL, or 500 to 1000 U / μL. Specifically, the concentration of the ligase in the reaction solution may be, for example, 5 to 1000 U / μL, 10 to 500 U / μL, or 20 to 200 U / μL.

[0284] The activity of the ligase can be defined, for example, as follows.

[0285] That is, for example, with respect to a ligase, the amount of enzyme that can ligate 50% of 100 ng of HindIII-digested λ DNA in 1 minute at 25°C in a reaction buffer of an appropriate composition (e.g., 66 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 7.5% polyethylene glycol (PEG 6000), pH 7.6 (25°C), total reaction volume 20 μl) can be defined as 1 unit [U].

[0286] Alternatively, for example, for ligase, 2×10 oligonucleotides can be ligated at 25° C. in a reaction buffer of an appropriate composition (e.g., 66 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 7.5% polyethylene glycol (PEG 6000), pH 7.6 (25° C.)) in the presence of a sufficient amount (e.g., 10-fold molar concentration of the substrate oligonucleotide) of complementary oligonucleotides for 1 minute. - 5 The amount of enzyme per nmol of substrate oligonucleotide is defined as 1 unit [U].

[0287] After the connection process begins, ligase, substrate oligonucleotide, complementary oligonucleotide and / or other components can be added to the reaction solution alone or in any combination. These components can be supplied once or multiple times, or they can be supplied continuously. In addition, the reaction conditions can be kept uniform from the beginning to the end of the connection process, or they can change during the implementation of the connection process. "Reaction conditions change during the implementation of the connection process" is not limited to reaction conditions changing over time, but also includes reaction conditions changing spatially. As the situation where reaction conditions change spatially, it can be listed as follows: when the connection process is implemented in a column format, reaction conditions such as reaction temperature or ligase concentration are different according to the position on the flow path.

[0288] In the case of connecting 3 or more substrate oligonucleotides (that is, in the case of the number of junctions being 2 or more), the order and timing of the connection of these substrate oligonucleotides are not particularly limited. In the case of connecting 3 or more substrate oligonucleotides, the connection of these substrate oligonucleotides can be implemented simultaneously, or can be implemented sequentially." Implementing the connection of 3 or more substrate oligonucleotides simultaneously" means that the connection in all junctions of these substrate oligonucleotides starts simultaneously." Implementing the connection of 3 or more substrate oligonucleotides sequentially" can mean starting the connection in a part of the junctions of these substrate oligonucleotides, and starting the connection in the remaining junctions during or after the connection in this part of the junctions is completed. That is, in the case of connecting 3 or more substrate oligonucleotides, the connection of these substrate oligonucleotides can be started simultaneously, or can be started sequentially. For example, in the case of connecting 3 substrate oligonucleotides A, B and C in the 5' to 3' direction, A, B and C can be connected simultaneously, A and B can be connected in advance, and then the junction of A and B and C can be connected, and B and C can be connected in advance, and then the junction of B and C and A can be connected. Specifically, for example, when three substrate oligonucleotides A, B, and C are linked in the 5' to 3' direction, the linking of A, B, and C may be started simultaneously, or the linking of A and B may be started in advance, and then the linking of B and C may be started during or after the linking of A and B is completed (depending on the progress of the linking of A and B, the linking of the linker of A and B and C may also be included), or the linking of B and C may be started in advance, and then the linking of B and A may be started during or after the linking of B and C is completed (depending on the progress of the linking of B and C, the linking of the linker of B and C and A may also be included). For example, by starting the linking step in the presence of complementary oligonucleotides for linking A, B, and C, A and B, and B and C, the linking of A, B, and C may be started simultaneously, and the linking of A, B, and C may be performed simultaneously. In addition, for example, by starting the ligation step in the absence of C and / or in the absence of an oligonucleotide complementary to ligation between B and C, and supplying C and / or an oligonucleotide complementary to ligation between B and C to the reaction solution after the start of the ligation step, the ligation between A and B can be started in advance, and then the ligation between B and C can be started during or after the ligation between A and B (depending on the degree of progress of the ligation between A and B, the ligation between A and B and C can also be included), so that the ligation between A, B, and C can be carried out in sequence. In addition, for example, by starting the ligation step in the absence of A and / or in the absence of an oligonucleotide complementary to ligation between A and B, and supplying A and / or an oligonucleotide complementary to ligation between A and B to the reaction solution after the start of the ligation step, the ligation between B and C can be started in advance, and then the ligation between B and A can be started during or after the ligation between B and C (depending on the degree of progress of the ligation between B and C, the ligation between B and C and A can also be included), so that the ligation between A, B, and C can be carried out in sequence.

[0289] In the case of connecting three or more substrate oligonucleotides, the connection of these substrate oligonucleotides can be generally consistent with the implementation of the method of the present invention. For example, in the case of connecting three substrate oligonucleotides A, B and C in the 5' to 3' direction, the connection of A, B and C (depending on the degree of the connection process, it also includes the connection of the connection of B and C to A and the connection of A and B to C) can be generally consistent with the implementation of the method of the present invention using A, B and C as substrate oligonucleotides.

[0290] In the case of connecting 3 or more substrate oligonucleotides, the connection of these substrate oligonucleotides can be consistent with the implementation of the method of the present invention for the connection in each connecting part independently. For example, in the case of connecting 3 substrate oligonucleotides A, B and C in the 5' to 3' direction, the connection of A and B (depending on the degree of the connection process, it also includes the connection of the connection of B and C and A) can be consistent with the implementation of the method of the present invention using A and B as substrate oligonucleotides, and the connection of B and C (depending on the degree of the connection process, it also includes the connection of A and B's connection and C) can be consistent with the implementation of the method of the present invention using B and C as substrate oligonucleotides.

[0291] By carrying out the ligation step in this manner, the target oligonucleotide is generated, thereby obtaining a reaction solution containing the target oligonucleotide.

[0292] The yield of the target oligonucleotide can be more than the amount of complementary oligonucleotide used in terms of molar ratio. That is, the target oligonucleotide with a molar ratio greater than the amount of complementary oligonucleotide used can be generated by repeatedly using complementary oligonucleotides in the connection process in a catalytic manner. The yield of the target oligonucleotide, for example, can be more than 100%, more than 110%, more than 120%, more than 130%, more than 150%, more than 170%, more than 200%, more than 250% or more than 300% in terms of molar ratio, based on the ratio of the amount of complementary oligonucleotide used as 100%. In the case where the number of connecting parts (N-1) is more than 2, the "usage amount of complementary oligonucleotide used" as a comparison object can refer to the usage of complementary oligonucleotides at any connection site. In the case where the number of connecting parts (N-1) is more than 2, the "usage amount of complementary oligonucleotide used" as a comparison object can be, for example, the smallest amount of complementary oligonucleotides used at these connection sites, the largest amount of complementary oligonucleotides used at these connection sites, or the average value of the amount of complementary oligonucleotides used at these connection sites. Regarding any ligation site, when the number of complementary oligonucleotides used at the ligation site is 2 or more, the “usage amount of complementary oligonucleotides” at the ligation site as a comparison object may mean the total usage amount of these complementary oligonucleotides.

[0293] With respect to any connection site, the yield of the connection product at the connection site may be more than the amount of complementary oligonucleotide used at the connection site in terms of molar ratio. That is, with respect to any connection site, by repeatedly using complementary oligonucleotides in the connection process in a catalytic manner, a connection product in an amount more than the amount of complementary oligonucleotide used in terms of molar ratio can be generated. With respect to any connection site, the yield of the connection product at the connection site, for example, with the amount of complementary oligonucleotide used at the connection site as 100%, may be more than 100%, more than 110%, more than 120%, more than 130%, more than 150%, more than 170%, more than 200%, more than 250% or more than 300% in terms of molar ratio. For any connection site, when the number of connection parts (N-1) is more than 2, the "yield of connection products" at the connection site refers to the total yield of all connection products generated via the connection at the connection site. For example, in the case where three substrate oligonucleotides A, B and C are ligated in the 5' to 3' direction, "the yield of the ligation product at the ligation site of A and B" may mean the total yield of the ligation product of A and B and the ligation product of A, B and C. For any ligation site, when the number of complementary oligonucleotides used in the ligation site is 2 or more, "the usage amount of complementary oligonucleotides" at the ligation site as a comparison object may mean the total usage amount of these complementary oligonucleotides.

[0294] The generation of the target oligonucleotide can be confirmed, for example, by a known method for detecting or identifying a compound. As such methods, HPLC, LC / MS, GC / MS, and NMR can be cited. One of these methods can be used alone, or two or more can be used in combination as appropriate. In the case where the complementary oligonucleotide is hybridized with the target oligonucleotide, they can be separated appropriately or not. Separation can be implemented, for example, by heating. The heating temperature can be, for example, above 40°C, above 50°C, or above 60°C, or below 100°C, below 90°C, or below 80°C, or a combination thereof. The target oligonucleotide can be appropriately recovered from the reaction solution. The recovery of the target oligonucleotide can be implemented, for example, by a known method for separating and purifying a compound. As such methods, ion exchange chromatography, reverse phase chromatography, affinity chromatography, chromatography such as gel filtration chromatography, electrophoresis such as polyacrylamide gel electrophoresis (PAGE), and membrane purification can be cited. One of these methods can be used alone, or two or more can be used in combination as appropriate. The target oligonucleotide can be purified to the desired degree. In addition, the target oligonucleotide can be modified, for example, to become the modification mode of the target oligonucleotide exemplified above.

[0295] Example

[0296] The present invention will be described in more detail below by way of non-limiting examples. It should be noted that, unless otherwise specified, the nucleic acid residues constituting the oligonucleotides used in the examples are unmodified DNA residues.

[0297] [Example 1] Ligation reaction using an oligonucleic acid having a complementary strand introduced with an abasic portion or a complementary strand having an abasic portion and a loop structure introduced

[0298] As complementary chains, the following five complementary chains were used: one complementary chain into which only the abasic part was introduced, three complementary chains into which the abasic part and the loop structure were introduced (three patterns in which the loop structure was inserted on the 5' side, the 3' side, or both sides), and one complete complementary chain into which no destabilizing part was introduced as a control ( Figure 1A and Table 1). An abasic moiety was introduced into the residue complementary to the linker, and a loop structure was introduced into a position away from the linker.

[0299] Using each complementary strand, a ligation reaction of oligonucleotides as substrates (5' terminal substrate and 3' terminal substrate) was carried out, and the generation of products (oligonucleotides formed by ligation of the 5' terminal substrate and the 3' terminal substrate) was compared.

[0300] The reaction solution composition was set to 20 μM of each substrate, 2 μM complementary strand, 60 units / μL T3 DNA ligase (New England Biolabs), 66 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 7.5% polyethylene glycol (PEG 6000), pH 7.6 (at 25° C.), and the reaction was carried out at 25° C. A portion of the reaction solution was recovered after 1 hour, 3 hours, and 16 hours, and diluted with 10 mM EDTA to stop the reaction.

[0301] The product was quantified by HPLC using an ACQUITY UPLC OST C18 column (1.7 μm 2.1×50 mm). HPLC was performed at a column temperature of 60°C, a detection wavelength of 254 nm, an injection volume of 10 μL, and a flow rate of 0.4 mL / min, using eluent A containing 100 mM hexafluoroisopropanol, 8 mM triethylamine, and 0.004% phosphoric acid and eluent B containing 10% methanol as mobile phases, and the analysis was performed by the linear gradient shown in Table 2. The product sample was analyzed in the same manner to confirm that the target product was obtained by the enzyme reaction. The amount of the product was calculated from the ratio of the peak area of ​​the product in the LC graph to the total peak area of ​​the substrate and the product.

[0302] Results Figure 1B. The amount of the product after 16 hours of reaction is as follows: relative to 2.0 μM in the case of a control fully complementary chain, it was increased to 7.8 μM by introducing the abasic part, and further increased to 9.9 to 12.3 μM by inserting a ring structure in addition to the introduction of the abasic part. That is, it was confirmed that the complementary chain introduced with a destabilizing part such as an abasic part or a ring structure has a catalytic effect (i.e., it is repeatedly used for the oligonucleotide connection reaction). In addition, it was confirmed that the combination of the abasic part and the ring structure is effective in improving the efficiency of the oligonucleotide connection reaction.

[0303] [Table 1]

[0304] Table 1 Oligonucleotides used

[0305]

[0306] pho: indicates modification of the 5' end with a phosphate group. (Ab): indicates an abasic moiety. Residues surrounded by rectangles: indicate inserted residues.

[0307] [Table 2]

[0308] Table 2 Gradient

[0309] Time (minutes) A％ B％ 0.00 95 5 8.00 75 25 8.10 10 90 10.50 10 90 10.60 95 5 14.00 95 5

[0310] [Example 2] Ligation reaction of oligonucleotides modified at the sugar moiety

[0311] The catalytic effect of the complementary chain introduced into the destabilizing part on the oligonucleotide modified at the 2' position of the sugar part was verified. As the 5' terminal side substrate and the 3' terminal side substrate, two oligonucleotides in which the sugar part of 1 residue or 5 residues was replaced with 2'-O-methoxyethyl were used respectively (Table 3). As complementary chains, a total of three complementary chains were used, namely, a control complete complementary chain, a complementary chain introduced into the abasic part, and a complementary chain introduced into the abasic part and a ring structure (Table 3).

[0312] The oligonucleotides (5' terminal substrate and 3' terminal substrate) were connected using each complementary chain, and the generation of products (oligonucleotides formed by connecting the 5' terminal substrate and the 3' terminal substrate) was compared. The reaction solution composition was set to 20 μM of each substrate, 2 μM complementary chain, 60 units / μL T3 DNA ligase (New England Biolabs), 66 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 7.5% polyethylene glycol (PEG 6000), pH 7.6 (at 25°C), and the reaction was carried out at 25°C. Under the HPLC conditions described in Example 1, the products after 0.25 hours, 1 hour, 4 hours and 24 hours were quantified, and the yield of the product was calculated. In this embodiment, the molar concentration of the complementary chain is 1 / 10 of the molar concentration of each substrate, so under the assumption that the complementary chain is used only once in the connection reaction, the maximum yield is 10%.

[0313] Results Figure 2 . In the case of using the control fully complementary chain, the two substrate patterns all had a yield of less than 10% after 24 hours of reaction. On the other hand, in the case of using the complementary chain with the introduction of the abasic part and the complementary chain with the introduction of the abasic part and the ring structure, a yield of 38 to 51% was obtained after 24 hours of reaction. That is, in the ligation reaction of the oligonucleotide with modified sugar part, the catalytic effect of the complementary chain with the introduction of the destabilizing part such as the abasic part or the ring structure was confirmed. The yield after 4 hours of reaction was 21% (when the sugar part of the substrate was modified at 2 places in total) and 13% (when the sugar part of the substrate was modified at 10 places in total) in the case of using the complementary chain with the introduction of the abasic part, in contrast, it was 30% (when the sugar part of the substrate was modified at 2 places in total) and 29% (when the sugar part of the substrate was modified at 10 places in total) in the case of using the complementary chain with the introduction of the abasic part and the ring structure. That is, in the ligation reaction of the oligonucleotide with modified sugar part, it was also confirmed that the combination of the abasic part and the ring structure was effective in improving the efficiency of the ligation reaction of the oligonucleotide.

[0314] [Table 3]

[0315] Table 3 Oligonucleotides used

[0316]

[0317] pho: indicates modification of the 5' end with a phosphate group. (m): indicates modification with a 2'-O-methoxyethyl group. (Ab): indicates an abasic moiety. Residues surrounded by rectangles: indicates inserted residues. [Example 3] Ligation reaction of oligonucleotides modified with a phosphate moiety

[0318] The catalytic effect of the complementary chain introduced into the destabilizing part on the oligonucleotide modified in the phosphate part was verified. As the 5' terminal side substrate and the 3' terminal side substrate, oligoDNA (PS-DNA) obtained by replacing all the phosphate bonds between nucleotides with thiophosphate bonds was used (Table 4). As complementary chains, a total of 4 complementary chains were used, including a control complete complementary chain, a complementary chain introduced into the abasic part, a complementary chain introduced into the ring structure, and a complementary chain introduced into the abasic part and the ring structure (Table 4).

[0319] The oligonucleotides (5' terminal substrate and 3' terminal substrate) were connected using each complementary chain, and the generation of products (oligonucleotides formed by connecting the 5' terminal substrate and the 3' terminal substrate) was compared. The reaction solution composition was set to 20 μM of each substrate, 2 μM complementary chain, 66 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 7.5% polyethylene glycol (PEG 6000), pH 7.6 (at 25°C), and the reaction was carried out at 25°C. Under the HPLC conditions described in Example 1, the products after 1 hour, 3 hours and 24 hours were quantified, and the yield of the products was calculated. In this example, the molar concentration of the complementary chain is 1 / 10 of the molar concentration of each substrate, so the maximum yield is 10% assuming that the complementary chain is used only once in the connection reaction.

[0320] Results Figure 3 . The yield after 24 hours was 13% when using the control fully complementary chain, but it increased to 43% when using the complementary chain introduced with the abasic part, and increased to 24% when using the complementary chain introduced with the loop structure. That is, in the ligation reaction of the oligonucleotide modified at the phosphate part, the catalytic effect of the complementary chain introduced with the destabilizing part such as the abasic part or the loop structure was also confirmed. The yield after 24 hours was further increased to 50% when using the complementary chain introduced with the abasic part and the loop structure. That is, in the ligation reaction of the oligonucleotide modified at the phosphate part, it was confirmed that the combination of the abasic part and the loop structure is effective in improving the efficiency of the ligation reaction of the oligonucleotide.

[0321] [Table 4]

[0322] Table 4 Oligonucleotides used

[0323]

[0324] pho: indicates modification of the 5' end with a phosphate group. (Ab): indicates an abasic moiety. ^: indicates substitution of a phosphate group with a phosphorothioate group. Residues surrounded by rectangles: indicate inserted residues.

[0325] [Example 4] Ligation reaction using oligonucleic acid with complementary chain inserted into loop structure

[0326] The catalytic effect of the complementary chain inserted into the loop structure was verified. As the position for inserting the loop structure, a position (-6 position or +6 position) 6 residues away from the connecting part to the 5' side or 3' side was selected (Table 5). The number of nucleic acid residues constituting the loop structure was set to 4 residues.

[0327] The oligonucleotides (5' terminal substrate and 3' terminal substrate) were connected using each complementary chain, and the generation of products (oligonucleotides formed by connecting the 5' terminal substrate and the 3' terminal substrate) was compared. The reaction solution composition was set to 20 μM of each substrate, 2 μM complementary chain, 60 units / μL T3 DNA ligase (New England Biolabs), 66 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 7.5% polyethylene glycol (PEG 6000), pH 7.6 (at 25°C), and the reaction was carried out at 25°C. Under the HPLC conditions described in Example 1, the products after 1 hour, 3 hours and 16 hours were quantified, and the yield of the product was calculated. In this embodiment, the molar concentration of the complementary chain is 1 / 10 of the molar concentration of each substrate, so under the assumption that the complementary chain is only used once in the connection reaction, the maximum yield is 10%.

[0328] Results Figure 4 . When using a complementary chain with a loop structure inserted at any position, the yield increases as the reaction time increases. The yield after 16 hours was 3.8% when using a control complete complementary chain, while it was 10.7% when using a complementary chain with a loop structure inserted at the 5' side and 6.7% when using a complementary chain with a loop structure inserted at the 3' side. That is, the catalytic effect of the complementary chain with a destabilizing part such as a loop structure introduced was confirmed.

[0329] [Table 5]

[0330] Table 5 Oligonucleotides used

[0331]

[0332] pho: indicates modification of the 5' end with a phosphate group. Residues surrounded by rectangles: indicates inserted residues. [Example 5] Ligation reaction using oligonucleotides of complementary chains into which deletions of nucleic acid residues are introduced

[0333] The catalytic effect of complementary chains with deletions of nucleic acid residues was verified. As complementary chains, a total of 7 complementary chains were used, including a control perfect complementary chain, three complementary chains (#3 to #1) with the 4th to 6th residues, the 5th to 6th residues, or the 6th residue deleted from the junction as the starting point, and three complementary chains (#6 to #4) with the 4th to 6th residues, the 5th to 6th residues, or the 6th residue deleted from the junction as the starting point (Table 6 and Figure 5A By deleting nucleic acid residues of the complementary chain, a loop structure can be formed in the substrate when the complementary chain hybridizes with the substrate ( Figure 5A ).

[0334] The oligonucleotides (5' terminal substrate and 3' terminal substrate) were connected using each complementary chain, and the generation of products (oligonucleotides formed by connecting the 5' terminal substrate and the 3' terminal substrate) was compared. The reaction solution composition was set to 20 μM of each substrate, 2 μM complementary chain, 60 units / μL T3 DNA ligase (New England Biolabs), 66 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 7.5% polyethylene glycol (PEG 6000), pH 7.6 (at 25°C), and the reaction was carried out at 25°C. Under the HPLC conditions described in Example 1, the products after 1 hour, 3 hours and 16 hours were quantified, and the yield of the product was calculated. In this embodiment, the molar concentration of the complementary chain is 1 / 10 of the molar concentration of each substrate, so under the assumption that the complementary chain is only used once in the connection reaction, the maximum yield is 10%.

[0335] Results Figure 5B . In the case of using any complementary chain into which a deletion of nucleic acid residues was introduced, the yield after 16 hours also exceeded the yield (5.1%) when the control fully complementary chain was used. That is, when the deletion of nucleic acid residues was selected as the destabilizing part introduced into the complementary chain, the catalytic effect of the complementary chain was also confirmed. In the case where the position of the deleted residue was set from the connecting part to the 5' side, the yield after 16 hours was the highest (10.5%) when the number of deleted residues was 1 residue, and the lowest (6.2%) when the number of deleted residues was 3 residues. In the case where the deleted residue was set from the connecting part to the 3' side, the yield after 16 hours was the highest (14.1%) when the number of deleted residues was 2 residues, and the lowest (9.2%) when the number of deleted residues was 1 residue.

[0336] Table 6 Oligonucleotides used

[0337]

[0338] pho: indicates modification of the 5' end with a phosphate group. - in #1 to #6: indicates a nucleic acid residue that is missing compared to the case of complete complementarity.

[0339] [Example 6] Study on the number of nucleic acid residues constituting the loop structure

[0340] The effect of the number of nucleic acid residues constituting the loop structure on the catalytic action of the complementary chain was evaluated. As the position for inserting the loop structure, a position 6 residues away from the linker to the 5' side or 3' side (-6 position or +6 position) was selected, and the number of nucleic acid residues constituting the loop structure was set to 1 to 8 residues (Table 7).

[0341] The oligonucleotides (5' terminal substrate and 3' terminal substrate) were connected using each complementary chain, and the generation of products (oligonucleotides formed by connecting the 5' terminal substrate and the 3' terminal substrate) was compared. The reaction solution composition was set to 20 μM of each substrate, 0.2 μM complementary chain, 60 units / μL T3 DNA ligase (New England Biolabs), 66 mMTris-HCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 7.5% polyethylene glycol (PEG 6000), pH 7.6 (at 25°C), and the reaction was carried out at 25°C. Under the HPLC conditions described in Example 1, the product after 16 hours was quantified and the yield of the product was calculated. In this embodiment, the molar concentration of the complementary chain is 1 / 100 of the molar concentration of each substrate, so under the assumption that the complementary chain is used only once in the connection reaction, the maximum yield is 1%.

[0342] The results are shown in Table 7. In the case of using a complementary chain with a loop structure of any number of residues inserted, a higher yield than the case of using a control complete complementary chain was obtained. That is, regardless of the number of nucleic acid residues constituting the loop structure introduced into the complementary chain, the catalytic effect of the complementary chain was confirmed. In particular, when the number of nucleic acid residues constituting the loop structure is 2 or more, a high effect was obtained.

[0343] [Table 7]

[0344] Table 7 Oligonucleotides used and yields

[0345]

[0346] pho: indicates modification of the 5' end with a phosphate group. Residues surrounded by rectangles: indicates inserted residues. [Example 7] Study on the insertion position of the loop structure

[0347] The effect of the insertion position of the loop structure on the catalytic action of the complementary chain was evaluated. As the position for inserting the loop structure, a position 3 to 6 residues away from the connecting part to the 3' side or the 5' side (-3 to -6 position or +3 to +6 position) was selected (Table 8). The number of nucleic acid residues constituting the loop structure was set to 4 residues.

[0348] The oligonucleotides (5' terminal substrate and 3' terminal substrate) were connected using each complementary chain, and the generation of products (oligonucleotides formed by connecting the 5' terminal substrate and the 3' terminal substrate) was compared. The reaction solution composition was set to 20 μM of each substrate, 0.2 μM complementary chain, 60 units / μL T3 DNA ligase (New England Biolabs), 66 mMTris-HCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 7.5% polyethylene glycol (PEG 6000), pH 7.6 (at 25°C), and the reaction was carried out at 25°C. Under the HPLC conditions described in Example 1, the product after 16 hours was quantified and the yield of the product was calculated. In this embodiment, the molar concentration of the complementary chain is 1 / 100 of the molar concentration of each substrate, so under the assumption that the complementary chain is used only once in the connection reaction, the maximum yield is 1%.

[0349] The results are shown in Table 8. In the case of using the complementary chain with the loop structure inserted at any insertion position, a higher yield was obtained than the case of using the control complete complementary chain. That is, regardless of the position of the loop structure introduced into the complementary chain, the catalytic effect of the complementary chain was confirmed. In particular, when the loop structure was inserted on either the 5' side or the 3' side of the connecting part, the reaction rate was the highest when the loop structure was inserted at a position 5 residues away from the connecting part (-5 position or +5 position).

[0350] [Table 8]

[0351] Table 8 Oligonucleotides used and yields

[0352]

[0353] pho: indicates modification of the 5' end with a phosphate group. Residues enclosed by a rectangle: indicates an inserted residue. [Example 8] Study of the base sequence of nucleic acid residues constituting a loop structure

[0354] The effect of the base sequence of nucleic acid residues constituting the loop structure on the catalytic action of the complementary chain was evaluated. The position of the loop structure insertion was set to a position 4 or 6 residues away from the connecting part to the 3' side (+4 position or +6 position), the number of nucleic acid residues constituting the loop structure was set to 4 residues, and 4 base sequences were set respectively (Table 9).

[0355] The oligonucleotides (5' terminal substrate and 3' terminal substrate) were connected using each complementary chain, and the generation of products (oligonucleotides formed by connecting the 5' terminal substrate and the 3' terminal substrate) was compared. The reaction solution composition was set to 20 μM of each substrate, 0.2 μM complementary chain, 60 units / μL T3 DNA ligase (New England Biolabs), 66 mMTris-HCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 7.5% polyethylene glycol (PEG 6000), pH 7.6 (at 25°C), and the reaction was carried out at 25°C. Under the HPLC conditions described in Example 1, the product after 16 hours was quantified and the yield of the product was calculated. In this embodiment, the molar concentration of the complementary chain is 1 / 100 of the molar concentration of each substrate, so under the assumption that the complementary chain is used only once in the connection reaction, the maximum yield is 1%.

[0356] The results are shown in Table 8. In the case of using the complementary chain into which the loop structure composed of any base sequence was inserted, a higher yield was obtained than when the control complete complementary chain was used. In other words, regardless of the base sequence of the nucleic acid residues constituting the loop structure introduced into the complementary chain, the catalytic effect of the complementary chain was confirmed.

[0357] [Table 9]

[0358] Table 9 Oligonucleotides used and yields

[0359]

[0360] pho: indicates modification of the 5' end with a phosphate group. (Ab): indicates an abasic moiety. Residues surrounded by rectangles: indicate inserted residues.

[0361] [Example 9] Study on the number of ring structures

[0362] The effect of the number of loop structures on the catalytic action of the complementary chain was evaluated. The number of nucleic acid residues constituting the loop structure was set to 4 residues, and complementary chains with loop structures inserted at 1 to 4 positions were designed (Table 10).

[0363] The oligonucleotides (5' terminal substrate and 3' terminal substrate) were connected using each complementary chain, and the generation of products (oligonucleotides formed by connecting the 5' terminal substrate and the 3' terminal substrate) was compared. The reaction solution composition was set to 20 μM of each substrate, 2 μM complementary chain, 60 units / μL T3 DNA ligase (New England Biolabs), 66 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 7.5% polyethylene glycol (PEG 6000), pH 7.6 (at 25°C), and the reaction was carried out at 25°C. Under the HPLC conditions described in Example 1, the products after 1 hour, 3 hours and 16 hours were quantified, and the yield of the product was calculated. In this embodiment, the molar concentration of the complementary chain is 1 / 10 of the molar concentration of each substrate, so under the assumption that the complementary chain is only used once in the connection reaction, the maximum yield is 10%.

[0364] Results Figure 6 . In the case of using a complementary chain with a loop structure inserted at 1 to 3 positions, a higher yield was obtained after 16 hours than when a control fully complementary chain was used, and the yield tended to increase with the increase in the number of inserted loop structures. That is, when the number of loop structures introduced into the complementary chain was 1 to 3, the catalytic effect of the complementary chain was confirmed. In addition, it was confirmed that the combination of 2 or 3 loop structures was effective in improving the efficiency of the oligonucleotide connection reaction. On the other hand, in the case of using a complementary chain with a loop structure inserted at 4 positions, a higher yield was obtained after 16 hours than when a control fully complementary chain was used only under 1 of the 4 conditions.

[0365] [Table 10]

[0366] Table 10 Oligonucleotides used

[0367]

[0368] pho: indicates modification of the 5' end with a phosphate group. Residues surrounded by rectangles: indicates inserted residues. [Example 10] Study on the position and number of introduced abasic sites

[0369] The effects of the position and number of abasic sites introduced on the catalytic activity of the complementary chain were evaluated. A complementary chain with 1 to 3 abasic sites introduced was designed. Fig. 7A and Table 11).

[0370] The oligonucleotides (5' terminal substrate and 3' terminal substrate) were connected using each complementary chain, and the generation of products (oligonucleotides formed by connecting the 5' terminal substrate and the 3' terminal substrate) was compared. The reaction solution composition was set to 20 μM of each substrate, 2 μM complementary chain, 60 units / μL T3 DNA ligase (New England Biolabs), 66 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 7.5% polyethylene glycol (PEG 6000), pH 7.6 (at 25°C), and the reaction was carried out at 25°C. Under the HPLC conditions described in Example 1, the products after 1 hour, 3 hours and 16 hours were quantified, and the yield of the product was calculated. In this embodiment, the molar concentration of the complementary chain is 1 / 10 of the molar concentration of each substrate, so under the assumption that the complementary chain is only used once in the connection reaction, the maximum yield is 10%.

[0371] Results Figure 7B . In the case of using a complementary chain with a baseless portion introduced at 1 or 2 positions, it was found that a higher yield was obtained after 1 hour than in the case of using a control completely complementary chain. In addition, a higher yield was obtained after 3 hours and 16 hours than in the case of using a control completely complementary chain. That is, in the case where the number of abasic portions introduced into the complementary chain was 1 to 2, the catalytic effect of the complementary chain was found. After 1 hour and 3 hours, in the case of using a complementary chain with a baseless portion introduced at two positions 9_14 of the complementary chain, a higher yield was obtained than in the case of using a complementary chain with a baseless portion introduced at one position 9 of the complementary chain, but after 16 hours, the maximum yield ( Figure 7B ) On the other hand, when a complementary chain into which three abasic sites were introduced was used, the yield decreased.

[0372] [Table 11]

[0373] Table 11 Oligonucleotides used

[0374]

[0375] pho: indicates modification of the 5' end with a phosphate group. (Ab): indicates an abasic moiety. The position of the abasic moiety is numbered with the 5' end of the complementary chain as 1.

[0376] [Example 11] Ligation reaction using complementary oligonucleotides with mismatches introduced

[0377] The catalytic effect of introducing a complementary strand into the mismatched part was verified. The introduction of mismatched base pairs into the linker was studied. The bases (T and G, respectively) of the complementary strand that form a pair with the base on the 3' side (A, -1 position) and the base on the 5' side (C, +1 position) of the linker were replaced with other bases and evaluated (Table 12, Fig. 8A ).

[0378] The oligonucleotides (5' terminal substrate and 3' terminal substrate) were connected using each complementary chain, and the generation of products (oligonucleotides formed by connecting the 5' terminal substrate and the 3' terminal substrate) was compared. The reaction solution composition was set to 20 μM of each substrate, 2 μM complementary chain, 60 units / μL T3 DNA ligase (New England Biolabs), 66 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 7.5% polyethylene glycol (PEG 6000), pH 7.6 (at 25°C), and the reaction was carried out at 25°C. Under the HPLC conditions described in Example 1, the products after 1 hour, 3 hours and 16 hours were quantified, and the yield of the product was calculated. In this embodiment, the molar concentration of the complementary chain is 1 / 10 of the molar concentration of each substrate, so under the assumption that the complementary chain is only used once in the connection reaction, the maximum yield is 10%.

[0379] Results Figure 8B The yield after 16 hours was 4.5% when using the control fully complementary chain, whereas it increased to 5.3 to 28.8% when using the complementary chain with the mismatch introduced. Figure 8B ). That is, even when the mismatched portion was selected as the destabilizing portion to be introduced into the complementary chain, the catalytic effect of the complementary chain was confirmed.

[0380] [Table 12]

[0381] Table 12 Oligonucleotides used

[0382]

[0383] pho: indicates modification of the 5' end with a phosphate group. Residues surrounded by rectangles: indicate mismatches.

[0384] [Example 12] Study on substrate concentration

[0385] The influence of substrate concentration and complementary strand concentration on the ligation reaction of substrate was evaluated. The substrate concentration was set to 20, 100, 300, 500 or 1000 μM. The complementary strand concentration was set to a molar concentration of 1 / 10 of the substrate concentration. As complementary strands, two complementary strands were used: a complementary strand with an abasic portion introduced and a complementary strand with an abasic portion and a ring structure introduced (Table 13).

[0386] At each substrate concentration and complementary chain concentration, each complementary chain was used to implement the oligonucleotide (5' terminal substrate and 3' terminal substrate) as a substrate. The generation of the product (oligonucleotide formed by connecting the 5' terminal substrate and the 3' terminal substrate) was compared. The reaction solution composition other than the substrate and the complementary chain was set to 60 units / μL T3 DNA ligase (New England Biolabs), 66mM Tris-HCl, 10mM MgCl2, 1mM ATP, 1mM DTT, 7.5% polyethylene glycol (PEG6000), pH7.6 (at 25°C), and the reaction was carried out at 25°C. Under the HPLC conditions described in Example 1, the product after 1, 3, 16 and 40 hours was quantified, and the yield of the product and the generation rate of the product per unit enzyme activity were calculated. In this embodiment, the molar concentration of the complementary chain is 1 / 10 of the molar concentration of each substrate, so under the assumption that the complementary chain is only used once in the connection reaction, the maximum yield is 10%.

[0387] Results Fig.9A and Fig. 9B . At any complementary chain concentration and substrate concentration, a yield exceeding the complementary chain to substrate addition ratio of 10% was obtained. In other words, the catalytic effect of the complementary chain was confirmed regardless of the complementary chain concentration and substrate concentration. When any complementary chain was used, the maximum yield ( Fig.9A The rate at which each enzyme's product is generated increases with increasing substrate concentration ( Fig. 9B ).

[0388] [Table 13]

[0389] Table 13 Oligonucleotides used

[0390]

[0391] pho: indicates modification of the 5' end with a phosphate group. (Ab): indicates an abasic moiety. Residues surrounded by rectangles: indicate inserted residues.

[0392] [Example 13] Generation of 100-residue oligonucleotides by ligation of 50-residue oligonucleotides

[0393] The catalytic effect of the complementary chain in the connection reaction using 2 substrates of 50 residues was verified. The complementary chain is designed to form a double strand in the region of 9 residues from the connecting portion to the 5' side or 3' side, and as a destabilizing portion, the baseless portion is introduced into the position of the complementary chain paired with the base (A, +1 position) of the connecting portion of the 3' terminal side substrate, the loop structure is introduced into the 5' terminal side (the position of the 3rd base from the upstream), the 3' terminal side (the position of the 3rd base from the downstream) of the complementary chain, or these destabilizing portions are combined for introduction. The oligonucleotides used are shown in Table 14.

[0394] The oligonucleotides (5' terminal substrate and 3' terminal substrate) were connected using each complementary chain, and the generation of products (oligonucleotides formed by connecting the 5' terminal substrate and the 3' terminal substrate) was compared. The reaction solution composition was set to 20 μM of each substrate, 2 μM complementary chain, 150 units / μL T3 DNA ligase (New England Biolabs), 66 mMTris-HCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 7.5% polyethylene glycol (PEG 6000), pH 7.6 (at 25°C), and the reaction was carried out at 25°C. Under the HPLC conditions described in Example 1, the product after 24 hours was quantified and the yield of the product was calculated. In this example, the molar concentration of the complementary chain is 1 / 10 of the molar concentration of each substrate, so the maximum yield is 10% assuming that the complementary chain is used only once in the connection reaction.

[0395] Results Fig.10 The yield after 24 hours was 9.7% when using the control complete complementary chain, 14.0% when using the complementary chain with the abasic part introduced, 57.8% when using the complementary chain with the abasic part and the 5' side loop structure introduced, 50.5% when using the complementary chain with the abasic part and the 3' side loop structure introduced, and 61.8% when using the complementary chain with the abasic part and the 5' side and 3' side loop structures introduced. That is, when generating an oligonucleotide of 100 residues in length by the ligation reaction of an oligonucleotide of 50 residues, the catalytic effect of the complementary chain was also confirmed, and the combination effect of the destabilizing part was confirmed.

[0396] [Table 14]

[0397] Table 14 Oligonucleotides used

[0398]

[0399] pho: indicates modification of the 5' end with a phosphate group. (Ab): indicates an abasic moiety. Residues surrounded by rectangles: indicate inserted residues.

[0400] [Example 14] Generation of 100-residue oligonucleotide by ligation reaction of 50-residue oligonucleotide containing modified nucleic acid

[0401] The catalytic effect of the complementary chain in the connection reaction using 2 substrates was verified, and the substrate was a substrate containing 2'-O-methoxyethyl, 2'-O-methyl, 2'-fluoro, 5-methylcytosine and thiophosphate as 50 residues of the modified nucleic acid. The complementary chain is designed to form a double strand in the region of 9 residues from the connecting portion to the 5' side or 3' side, and as a destabilizing portion, the baseless portion is introduced into the position of the complementary chain paired with the base (A, +1 position) of the connecting portion of the 3' terminal side substrate, and the loop structure is introduced into the 5' terminal side (the position of the third base from the upstream), the 3' terminal side (the position of the third base from the downstream) of the complementary chain or a combination of these destabilizing portions. The oligonucleotides used are shown in Table 15.

[0402] The oligonucleotides (5' terminal substrate and 3' terminal substrate) were connected using each complementary chain, and the generation of products (oligonucleotides formed by connecting the 5' terminal substrate and the 3' terminal substrate) was compared. The reaction solution composition was set to 20 μM of each substrate, 2 μM complementary chain, 150 units / μL T3 DNA ligase (New England Biolabs), 66 mMTris-HCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 7.5% polyethylene glycol (PEG 6000), pH 7.6 (at 25°C), and the reaction was carried out at 25°C. Under the HPLC conditions described in Example 1, the product after 24 hours was quantified and the yield of the product was calculated. In this example, the molar concentration of the complementary chain is 1 / 10 of the molar concentration of each substrate, so the maximum yield is 10% assuming that the complementary chain is used only once in the connection reaction.

[0403] Results Fig.11 The yield after 24 hours was 7.7% when using the control fully complementary chain, 8.3% when using the complementary chain with the abasic part introduced, 72.4% when using the complementary chain with the abasic part and the 5' side loop structure introduced, 27.3% when using the complementary chain with the abasic part and the 3' side loop structure introduced, and 88.3% when using the complementary chain with the abasic part and the 5' side and 3' side loop structures introduced. That is, when an oligonucleotide of 100 residues in length was generated by a ligation reaction of an oligonucleotide of 50 residues containing a modified nucleic acid, the catalytic effect of the complementary chain was confirmed, and the combined effect of the destabilizing part was confirmed.

[0404] [Table 15]

[0405] Table 15 Oligonucleotides used

[0406]

[0407] (m): indicates modification with 2'-O-methoxyethyl. (M): indicates modification with 2'-O-methyl. (F): indicates modification with 2'-fluoro. 5(m): indicates 5-methyldeoxycytidine modified with 2'-O-methoxyethyl. ^: indicates substitution of a phosphate group with a thiophosphate group. pho: indicates modification of the 5' end with a phosphate group. (Ab): indicates an abasic portion. Residues surrounded by rectangles: indicate inserted residues.

[0408] [Example 15] Ligation reaction of three oligonucleotides

[0409] The catalytic effect of the complementary chain in the ligation reaction using three substrates, namely, a 10-residue 5'-terminal substrate, a 20-residue central substrate, and a 10-residue 3'-terminal substrate, was verified. The complementary chain complementary to the 5'-terminal substrate and the central substrate was designed to form a double strand from the junction to the region of 9 residues each on the 5'-terminal side or the central substrate side, and as a destabilizing part, an abasic part was introduced to the position of the complementary chain paired with the base (A, +1 position) of the junction of the 3'-terminal substrate, and a loop structure was introduced to the 5'-terminal side (the position of the third base from the upstream), the 3'-terminal side (the position of the third base from the downstream) of the complementary chain, or these destabilizing parts were combined for introduction. The complementary chain complementary to the central substrate and the 3' terminal substrate was designed to form a double strand in the region of 8 residues from the junction to the central substrate side or the 3' terminal side, and as a destabilizing part, the residues of the complementary chain paired with the base (T, -1 position) of the junction of the 5' terminal substrate were mismatched (C). The oligonucleotides used are shown in Table 16.

[0410] The complementary chains corresponding to the two junctions were combined to carry out the ligation reaction of the oligonucleotides (5' terminal substrate, central substrate and 3' terminal substrate) as substrates, and the generation of products (oligonucleotides formed by ligating the 5' terminal substrate, central substrate and 3' terminal substrate) was compared. The combination of complementary chains used the case where completely complementary chains were used in both the ligation of the 5' terminal substrate and the central substrate and the ligation of the central substrate and the 3' terminal substrate as the control condition. As a study of complementary chains with destabilizing parts introduced, four complementary chains were used in the connection between the 5' terminal substrate and the central substrate, including the introduction of the abasic part to the complementary chain position paired with the base (A, +1 position) of the connection part of the 3' terminal substrate, the introduction of the loop structure to the 5' terminal side of the complementary chain (the position of the third base from the upstream), the introduction to the 3' terminal side (the position of the third base from the downstream), or the combination of these destabilizing parts. In the connection between the central substrate and the 3' terminal substrate, a complementary chain with mismatches introduced was used, and a total of four reaction conditions combining these were studied. The reaction solution composition was set to 20 μM of each substrate, 2 μM complementary chain, 150 units / μL T3 DNA ligase (New England Biolabs), 66 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 7.5% polyethylene glycol (PEG 6000), pH 7.6 (at 25°C), and the reaction was carried out at 25°C. The product was quantified after 72 hours and the yield of the product was calculated under the HPLC conditions described in Example 1. In this example, the molar concentration of the complementary chain was 1 / 10 of the molar concentration of each substrate, so the maximum yield was 10% assuming that the complementary chain was used only once in the ligation reaction.

[0411] Results Fig.12. In the ligation reaction using three substrates, the yield also increased with the extension of the reaction time. The yield after 72 hours was 1.6% under the control condition, 19.3% under the condition that the complementary chain with the abasic part was used in the ligation between the 5' terminal substrate and the central substrate, and the complementary chain with the mismatch was used in the ligation between the central substrate and the 3' terminal substrate, and 27.5% under the condition that the complementary chain with the abasic part and the loop structure (5' terminal side) was used in the ligation between the 5' terminal substrate and the central substrate, and the complementary chain with the mismatch was used in the ligation between the central substrate and the 3' terminal substrate. %, 24.4% under the condition that the complementary chain with the abasic part and the loop structure (3' end side) was used in the connection between the 5' end side substrate and the central substrate, and the complementary chain with the mismatch was used in the connection between the central substrate and the 3' end side substrate, and 33.9% under the condition that the complementary chain with the abasic part and the loop structure (5' end side and 3' end side) was used in the connection between the 5' end side substrate and the central substrate, and the complementary chain with the mismatch was used in the connection between the central substrate and the 3' end side substrate. That is, the catalytic effect of the complementary chain was also confirmed in the connection reaction of the three oligonucleotides.

[0412] [Table 16]

[0413] Table 16 Oligonucleotides used

[0414]

[0415] pho: indicates modification of the 5' end with a phosphate group. (Ab): indicates an abasic portion. Residues surrounded by rectangles: indicate mismatches. Residues underlined: indicate inserted residues.

[0416] [Example 16] Ligation reaction of three oligonucleotides containing modified nucleic acid

[0417] The catalytic effect of the complementary chain in the ligation reaction using three substrates, namely, a 10-residue 5'-terminal substrate containing 2'-O-methoxyethyl, 2'-O-methyl, 2'-fluoro, 5-methylcytosine and thiophosphate as modified nucleic acids, a 20-residue central substrate and a 10-residue 3'-terminal substrate, was verified. The complementary chain complementary to the 5'-terminal substrate and the central substrate was designed to form a double strand in a region of 9 residues from the connecting portion to the 5'-terminal side or the central substrate side, and as a destabilizing portion, an abasic portion was introduced to the position of the complementary chain that was paired with the base (A, +1 position) of the connecting portion of the 3'-terminal substrate, and a loop structure was introduced to the 5'-terminal side (the position of the third base from the upstream), the 3'-terminal side (the position of the third base from the downstream) of the complementary chain, or a combination of these destabilizing portions. The complementary chain complementary to the central substrate and the 3' terminal substrate was designed to form a double strand in the region of 8 residues from the junction to the central substrate side or the 3' terminal side, and as a destabilizing part, the residues of the complementary chain paired with the base (T, -1 position) of the junction of the 5' terminal substrate were mismatched (C). The oligonucleotides used are shown in Table 17.

[0418] The complementary chains corresponding to the two junctions were combined to carry out the ligation reaction of the oligonucleotides (5' terminal substrate, central substrate and 3' terminal substrate) as substrates, and the generation of products (oligonucleotides formed by ligating the 5' terminal substrate, central substrate and 3' terminal substrate) was compared. The combination of complementary chains used the case where completely complementary chains were used in both the ligation of the 5' terminal substrate and the central substrate and the ligation of the central substrate and the 3' terminal substrate as the control condition. As a study of complementary chains with destabilizing parts, four complementary chains obtained by introducing a baseless part into a complementary chain pairing with a base (A, +1 position) of a connecting part of a 3'-terminal substrate, introducing a loop structure into the 5'-terminal side of a complementary chain (position of the third base from the upstream), introducing into the 3'-terminal side (position of the third base from the downstream), or introducing these destabilizing parts in combination were used in the connection between the 5'-terminal substrate and the central substrate, and a complementary chain with mismatches introduced was used in the connection between the central substrate and the 3'-terminal substrate, and a total of four reaction conditions combining these were studied. The reaction solution composition was set to 20 μM of each substrate, 2 μM of complementary chain, 150 units / μL of T3 DNA ligase (New England Biolabs), 66 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 7.5% polyethylene glycol (PEG 6000), pH 7.6 (at 25°C), and the reaction was carried out at 25°C. The product was quantified 24 hours later and the yield of the product was calculated under the HPLC conditions described in Example 1. In this example, the molar concentration of the complementary chain was 1 / 10 of the molar concentration of each substrate, so the maximum yield was 10% assuming that the complementary chain was used only once in the ligation reaction.

[0419] Results Fig.13. In the ligation reaction using three substrates containing modified nucleic acids, the yield also increased with the extension of the reaction time. The yield after 24 hours was 1.6% under the control condition, 13.0% under the condition that the complementary chain with the abasic part was used in the ligation between the 5' terminal substrate and the central substrate, and the complementary chain with the mismatch was used in the ligation between the central substrate and the 3' terminal substrate, and 22.8% under the condition that the complementary chain with the abasic part and the ring structure (5' terminal side) was used in the ligation between the 5' terminal substrate and the central substrate, and the complementary chain with the mismatch was used in the ligation between the central substrate and the 3' terminal substrate. %, 21.1% under the condition that the complementary chain with the abasic part and the loop structure (3' end side) was used in the connection between the 5' end side substrate and the central substrate, and the complementary chain with the mismatch was used in the connection between the central substrate and the 3' end side substrate, and 22.5% under the condition that the complementary chain with the abasic part and the loop structure (5' end side and 3' end side) was used in the connection between the 5' end side substrate and the central substrate, and the complementary chain with the mismatch was used in the connection between the central substrate and the 3' end side substrate. That is, the catalytic effect of the complementary chain was also confirmed in the connection reaction of the three oligonucleotides containing the modified nucleic acid.

[0420] [Table 17]

[0421] Table 17 Oligonucleotides used

[0422]

[0423] (m): indicates modification with 2'-O-methoxyethyl. (M): indicates modification with 2'-O-methyl. (F): indicates modification with 2'-fluoro. 5(m): indicates 5-methyldeoxycytidine into which 2'-O-methoxyethyl has been introduced. ^: indicates substitution of a phosphate group with a thiophosphate group. pho: indicates modification of the 5' end with a phosphate group. (Ab): indicates an abasic portion. Residues surrounded by a rectangle: indicate a mismatch portion. Residues shown with an underline: indicate an inserted residue.

[0424] [Example 17] Ligation reaction of a 20-residue oligonucleotide composed of a modified nucleic acid and RNA

[0425] The catalytic effect of complementary chain in the ligation reaction of 10 residues of substrates consisting of 2 2'-O-methyl and RNA as modified nucleic acids was verified. The complementary chain is designed to form a double strand in the region of 9 residues from the connecting portion to the 5' side or 3' side, and as a destabilizing portion, the baseless portion is introduced into the position of the complementary chain paired with the base (A, +1 position) of the connecting portion of the 3' terminal side substrate, and the ring structure is introduced into the 5' terminal side (the position of the 3rd base from the upstream), the 3' terminal side (the position of the 3rd base from the downstream) of the complementary chain or the combination of these destabilizing portions is introduced. The oligonucleotides used are shown in Table 18.

[0426] The oligonucleotides (5' terminal substrate and 3' terminal substrate) were connected using each complementary chain as substrates, and the generation of products (oligonucleotides formed by connecting the 5' terminal substrate and the 3' terminal substrate) was compared. The reaction solution composition was set to 20 μM of each substrate, 2 μM complementary chain, 150 units / μL T3 DNA ligase (New England Biolabs), 66 mMTris-HCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 7.5% polyethylene glycol (PEG 6000), pH 7.6 (at 25°C), and the reaction was carried out at 25°C. Under the HPLC conditions described in Example 1, the product after 72 hours was quantified and the yield of the product was calculated. In this embodiment, the molar concentration of the complementary chain is 1 / 10 of the molar concentration of each substrate, so under the assumption that the complementary chain is used only once in the connection reaction, the maximum yield is 10%.

[0427] Results Fig.14 The yield after 72 hours was 17.5% when using the control fully complementary chain, 18.9% when using the complementary chain with the abasic part introduced, 33.0% when using the complementary chain with the abasic part and the 5' side loop structure introduced, 20.8% when using the complementary chain with the abasic part and the 3' side loop structure introduced, and 11.0% when using the complementary chain with the abasic part and the 5' side and 3' side loop structures introduced. That is, in the ligation reaction of the oligonucleic acid composed of the modified nucleic acid and RNA, the catalytic effect of the complementary chain was also confirmed, and the combined effect of the destabilizing part was confirmed.

[0428] Table 18 Oligonucleotides used

[0429]

[0430] Capital letters: DNA residues. Lowercase letters: RNA residues. (M): 2'-O-methyl modification. pho: 5'-terminal modification with a phosphate group. (Ab): Abasic residues. Residues surrounded by rectangles: Inserted residues.

[0431] Industrial Applicability

[0432] According to the present invention, oligonucleic acids can be efficiently ligated.

Claims

1. A method for producing a target oligonucleic acid, comprising the following steps: A step of enzymatically ligating N single-stranded substrate oligonucleic acids in the presence of M single-stranded complementary oligonucleic acids to generate the target oligonucleic acid, wherein N is an integer greater than 2, and M is an integer greater than 1, in, The ligation between the substrate oligonucleotides at the 5' side and the 3' side of each linking portion is carried out in the presence of one or more complementary oligonucleotides corresponding to the linking portion. Each of the complementary oligonucleotides comprises a base sequence in which a destabilizing portion is introduced into the first base sequence, The first base sequence is a base sequence consisting of a second base sequence and a third base sequence connected in the 5' to 3' direction, The second base sequence is a complementary sequence to a partial sequence on the 5' side of the 3' side substrate oligonucleotide, or a complementary sequence to the full-length sequence of the 3' side substrate oligonucleotide, The third base sequence is a complementary sequence to a partial sequence on the 3' side of the 5' side substrate oligonucleotide, or a complementary sequence to the full-length sequence of the 5' side substrate oligonucleotide. The destabilizing portion is a site that reduces the stability of the hybrid formed between each of the complementary oligonucleic acids and the target oligonucleic acid.

2. The method according to claim 1, wherein: The following situations (1) to (5) are not included: (1) The case where N is 2 and the destabilizing portion is composed of an abasic portion at the -1 position; (2) The above N is 2, and the above destabilizing portion is composed of a linker between the -1 position and the +1 position substituted with a nucleic acid residue; (3) The case where N is 2 and the destabilizing portion is composed of a substitution in which a nucleic acid residue at position -1 is substituted with a linker; (4) The case where N is 2, and the destabilizing portion is composed of a combination of an abasic portion at the -1 position and an abasic portion at the +4 position; (5) The case where N is 2, and the destabilizing portion is composed of a combination of an abasic portion at the -1 position and a mismatch portion at the +4 position.

3. The method according to claim 1 or 2, wherein: The destabilizing part includes an abasic part, a mismatch part, an insertion of a nucleic acid residue, a deletion of a nucleic acid residue, an insertion of a linker, a deletion of a linker, a substitution of a linker or a combination thereof.

4. The method according to any one of claims 1 to 3, wherein: The destabilizing portion includes insertion of a nucleic acid residue without deletion of a linker and / or deletion of a nucleic acid residue without insertion of a linker.

5. The method according to any one of claims 1 to 4, wherein The destabilization section is composed of 1 to 20 groups of destabilization sections.

6. The method according to any one of claims 1 to 5, wherein: The above-mentioned destabilization section is composed of 1 to 8 groups of destabilization sections.

7. The method according to any one of claims 1 to 6, wherein: The above-mentioned destabilization part is composed of 1 to 4 groups of destabilization parts.

8. The method according to any one of claims 1 to 7, wherein: The destabilizing portion is composed of a number of groups of the destabilizing portion that is 30% or less in ratio to the length of the first base sequence.

9. The method according to any one of claims 1 to 8, wherein: The destabilizing portion includes 1 to 8 abasic portions.

10. The method according to any one of claims 1 to 9, wherein: The destabilizing portion includes 1 to 2 abasic portions.

11. The method according to any one of claims 1 to 10, wherein: The destabilizing portion includes the abasic portion in an amount that is 30% or less of the length of the first base sequence.

12. The method according to any one of claims 1 to 11, wherein: The destabilizing portion includes 1 to 8 mismatching portions.

13. The method according to any one of claims 1 to 12, wherein: The destabilizing portion includes 1 to 3 mismatching portions.

14. The method according to any one of claims 1 to 13, wherein: The destabilizing portion includes mismatch portions whose number is 30% or less in terms of the ratio to the length of the first base sequence.

15. The method according to any one of claims 1 to 14, wherein: The destabilizing portion includes deletions of nucleic acid residues in an amount of 50% or less relative to the length of the first base sequence.

16. The method according to any one of claims 1 to 15, wherein: The destabilizing portion includes deletion of 1 to 8 groups of nucleic acid residues.

17. The method according to any one of claims 1 to 16, wherein: The destabilizing portion includes the deletion of 1 to 3 groups of nucleic acid residues.

18. The method according to claim 16 or 17, wherein: The deletion of each group of nucleic acid residues mentioned above consists of the deletion of 1 to 3 nucleic acid residues.

19. The method according to any one of claims 1 to 18, wherein: The destabilizing portion includes insertion of nucleic acid residues in a number that is 200% or less of the length of the first base sequence.

20. The method according to any one of claims 1 to 19, wherein: The destabilizing portion includes the insertion of 1 to 8 groups of nucleic acid residues.

21. The method according to any one of claims 1 to 20, wherein: The destabilizing portion includes the insertion of 1 to 4 groups of nucleic acid residues.

22. The method according to claim 20 or 21, wherein: The insertion of each group of nucleic acid residues mentioned above consists of the insertion of 1 to 10 nucleic acid residues.

23. The method according to any one of claims 20 to 22, wherein: When the insertion of each group of nucleic acid residues is composed of two or more nucleic acid residues, a part or all of the nucleic acid residues have self-complementarity.

24. The method according to any one of claims 1 to 23, wherein: The destabilizing portion includes the insertion of 1 to 8 connectors.

25. The method according to any one of claims 1 to 24, wherein: The destabilizing portion includes a deletion of 1 to 8 linkers.

26. The method according to any one of claims 1 to 25, wherein: The above-mentioned destabilizing part includes substitution of 1 to 8 linkers.

27. The method according to any one of claims 1 to 26, wherein: The destabilizing part includes the following (a), (b) or (c): (a) a combination of deletion and / or insertion of the abasic portion at positions -1 to +1 and nucleic acid residues at positions other than -1 to +1; (b) a combination of a mismatch at positions -1 to +1 and a deletion and / or insertion of a nucleic acid residue at a position other than positions -1 to +1; (c) Mismatched portion at positions -1 to +1.

28. The method according to claim 27, wherein: The destabilizing parts (a), (b) and (c) are respectively (a1), (b1) and (c1) below: (a1) a combination of one abasic part at positions -1 to +1 and one or two deletions of nucleic acid residues at positions other than -1 to +1 and / or one or two insertions of nucleic acid residues; (b1) a combination of one mismatch at positions -1 to +1 and one or two deletions and / or one or two insertions of nucleic acid residues at positions other than -1 to +1; (c1) One mismatched portion at positions -1 to +1.

29. The method according to claim 27, wherein: The destabilizing parts (a), (b) and (c) are respectively (a2), (b2) and (c2) below: (a2) a combination of an abasic portion at positions -1 to +1, an insertion of a group of nucleic acid residues at a negative position other than position -1, and an insertion of a group of nucleic acid residues at a positive position other than position +1; (b2) a combination of one mismatched portion at positions -1 to +1, one group of nucleic acid residues inserted at a negative position other than position -1, and one group of nucleic acid residues inserted at a positive position other than position +1; (c2) One mismatched portion at positions -1 to +1.

30. The method according to any one of claims 1 to 29, wherein: The destabilizing portion reduces the melting temperature of the hybrid by 1 to 60°C.

31. The method according to any one of claims 1 to 30, wherein: The length of the target oligonucleotide is 10 to 200 residues.

32. The method according to any one of claims 1 to 31, wherein: The length of each of the above substrate oligonucleotides is 5 to 50 residues.

33. The method according to any one of claims 1 to 32, wherein: The lengths of the second base sequence and the third base sequence are 5 to 50 residues, respectively.

34. The method according to any one of claims 1 to 33, wherein: The length of each complementary oligonucleotide is 5 to 300% based on the length of the target oligonucleotide being 100%.

35. The method according to any one of claims 1 to 34, wherein: The length of each complementary oligonucleotide is 5 to 300% based on the total length of the 5' substrate oligonucleotide and the 3' substrate oligonucleotide being 100%.

36. The method according to any one of claims 1 to 35, wherein: In each of the complementary oligonucleic acids, 5 or more of the nucleic acid residues constituting the second base sequence remain, and 5 or more of the nucleic acid residues constituting the third base sequence remain.

37. The method according to any one of claims 1 to 36, wherein: In each of the complementary oligonucleic acids, 50% or more of the nucleic acid residues constituting the second base sequence remain, and 50% or more of the nucleic acid residues constituting the third base sequence remain.

38. The method according to any one of claims 1 to 37, wherein: In each of the complementary oligonucleic acids, three or more consecutive residues among the nucleic acid residues constituting the second base sequence remain, and three or more consecutive residues among the nucleic acid residues constituting the third base sequence remain.

39. The method according to any one of claims 1 to 38, wherein: The above-mentioned target oligonucleotide is composed of DNA residues, RNA residues, modified nucleic acid residues or a combination thereof.

40. The method according to any one of claims 1 to 39, wherein: The above-mentioned target oligonucleotide is modified.

41. The method according to any one of claims 1 to 40, wherein: Any one or more of the complementary oligonucleotides are modified.

42. The method according to any one of claims 39 to 41, wherein: The above-mentioned modification includes modification of the phosphate moiety, modification of the sugar moiety, modification of the base moiety or a combination thereof.

43. The method of claim 42, wherein: The modification of the phosphate moiety includes phosphorothioation, boranophosphorylation, insertion of a linker or a combination thereof.

44. The method according to claim 42 or 43, wherein: The modification of the sugar moiety includes 2'-MOE, 2'-OMe, 2'-F, 4'-thio-2'-OMe, crosslinking between the 2' and 4' positions of the sugar moiety, modification of the 5' end of the oligonucleotide, modification of the 3' end of the oligonucleotide, or a combination thereof.

45. The method according to any one of claims 1 to 44, wherein: The above N is 2.

46. ​​The method according to any one of claims 1 to 45, wherein: The above-mentioned N is 3 or more.

47. The method according to any one of claims 1 to 46, wherein: The above-mentioned N is 10 or less.

48. The method according to any one of claims 1 to 47, wherein: The above M is smaller than N-1.

49. The method according to any one of claims 1 to 47, wherein: The above M is N-1.

50. The method according to any one of claims 1 to 47, wherein: The above M is greater than N-1.

51. The method according to any one of claims 1 to 50, wherein: The above steps are carried out at 5 to 60°C.

52. The method according to any one of claims 1 to 51, wherein: The amount of each complementary oligonucleotide used is 50% or less of the smaller amount of the 5' substrate oligonucleotide used and the 3' substrate oligonucleotide used in terms of molar ratio.

53. The method according to any one of claims 1 to 52, wherein: The concentration of each of the substrate oligonucleotides in the reaction solution in the above step is 1 to 10000 μM.

54. The method according to any one of claims 1 to 53, wherein: The enzyme used for the above enzymatic ligation is T3 DNA ligase.

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