Method for producing oligonucleic acid

Abstract

Provided is a method for producing an oligonucleic acid. This method uses a complementary strand to which has been introduced a site for lowering the stability of a hybrid formed between the complementary strand and a linked product, so as to enzymatically link a single-strand oligonucleic acid, which is a substrate, and produce the oligonucleic acid which is a linked product.

Description

Oligonucleotide manufacturing method

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

[0002] Oligonucleotides such as siRNA and antisense nucleic acids have been shown to be useful as nucleic acid drugs, and their development has been intensified in recent years. Oligonucleotides are mainly produced by chemical synthesis. Oligonucleotides can be produced, for example, by serially extending nucleic acid residues one by one using a synthesis method such as the phosphoramidite method. However, this method has problems such as a decrease in product purity and yield as the chain length of the oligonucleotide increases, and low production efficiency. Therefore, a parallel synthesis method is needed in which short fragments of oligonucleotides are synthesized and then linked to obtain long-chain oligonucleotides.

[0003] Oligonucleic acids can be prepared, for example, by enzymatically ligating nucleic acid fragments with complementary strands.

[0004] Furthermore, in a method for amplifying a DNA sequence that involves enzymatically ligating nucleic acid fragments in the presence of a complementary strand, a technique has been reported in which a destabilizing moiety, such as an abasic site, is introduced into the complementary strand or the nucleic acid fragment (Patent Document 1 and Non-Patent Document 1).

[0005] Furthermore, in a method for detecting single-stranded DNA, which involves enzymatically ligating two complementary strands serving as probes in the presence of single-stranded DNA, a technique has been reported in which a destabilizing element such as an abasic site or a mismatch site is introduced into the complementary strand (Non-Patent Document 2).

[0006] US 9,193,993 B1

[0007] 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.BS Alladin-Mustan et al., Achieving room temperature DNA amplification by dialing in destabilization, Chem. Commun., 2015, 51, 9101-9104.

[0008] An objective of the present invention is to provide a method for producing an oligonucleic acid.

[0009] As a result of intensive research to solve the above problems, the present inventors have found that when single-stranded oligonucleic acids as substrates are enzymatically ligated to produce an oligonucleic acid as a ligation product, the ligation product can be efficiently produced by using a complementary strand into which a site that reduces the stability of a hybrid formed between the complementary strand and the ligation product has been introduced, thereby completing the present invention.

[0010] That is, the present invention can be exemplified as follows. [1] A method for producing a target oligonucleic acid, comprising the step of enzymatically ligating N (N is an integer of 2 or more) single-stranded substrate oligonucleic acids in the presence of M (M is an integer of 1 or more) single-stranded complementary oligonucleic acids to produce the target oligonucleic acid, wherein ligation between the 5'- and 3'-side substrate oligonucleic acids at each ligation site is carried out in the presence of one or more complementary oligonucleic acids corresponding to the ligation site, wherein each complementary oligonucleic acid comprises a base sequence in which a destabilizing portion has been introduced into a first base sequence, wherein the first base sequence is a base sequence consisting of a second base sequence and a third base sequence ligated in the 5'-to-3' direction, wherein the second base sequence is a complementary sequence to a 5'-side partial sequence of the 3'-side substrate oligonucleic acid or a complementary sequence to the full-length sequence of the 3'-side substrate oligonucleic acid, and wherein the third base sequence is a complementary sequence to a 3'-side partial sequence of the 5'-side substrate oligonucleic acid or a complementary sequence to the full-length sequence of the 5'-side substrate oligonucleic acid, The method, wherein the destabilizing portion is a portion that reduces the stability of a hybrid formed between each of the complementary oligonucleic acids and the target oligonucleic acid. [2] The method, except for the following cases (1) to (5): (1) when N is 2 and the destabilizing portion consists of one abasic portion at position −1; (2) when N is 2 and the destabilizing portion consists of a nucleic acid residue substituted with a linker between positions −1 and +1; (3) when N is 2 and the destabilizing portion consists of a nucleic acid residue substituted with a linker at position −1; (4) when N is 2 and the destabilizing portion consists of a combination of one abasic portion at position −1 and one abasic portion at position +4; or (5) when N is 2 and the destabilizing portion consists of a combination of one abasic portion at position −1 and one mismatch portion at position +4. [3] The method described above, wherein the destabilizing portion comprises 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. [4] The method described above, wherein the destabilizing portion comprises an insertion of a nucleic acid residue without deletion of a linker and / or a deletion of a nucleic acid residue without insertion of a linker. [5] The method described above, wherein the destabilizing portion consists of 1 to 20 sets of destabilizing portions.[6] The method, wherein the destabilizing portion consists of 1 to 8 sets of destabilizing portions. [7] The method, wherein the destabilizing portion consists of 1 to 4 sets of destabilizing portions. [8] The method, wherein the destabilizing portion consists of sets of destabilizing portions that account for 30% or less of the number of sets relative to the length of the first base sequence. [9] The method, wherein the destabilizing portion comprises 1 to 8 abasic portions.

[10] The method, wherein the destabilizing portion comprises 1 to 2 abasic portions.

[11] The method, wherein the destabilizing portion comprises a number of abasic portions that account for 30% or less of the number of sets relative to the length of the first base sequence.

[12] The method, wherein the destabilizing portion comprises 1 to 8 mismatch portions.

[13] The method, wherein the destabilizing portion comprises 1 to 3 mismatch portions.

[14] The method, wherein the destabilizing portion comprises a number of mismatch portions that account for 30% or less of the number of sets relative to the length of the first base sequence.

[15] The method described above, wherein the destabilizing portion comprises deletion of 50% or less of the number of nucleic acid residues relative to the length of the first base sequence.

[16] The method described above, wherein the destabilizing portion comprises deletion of 1 to 8 sets of nucleic acid residues.

[17] The method described above, wherein the destabilizing portion comprises deletion of 1 to 3 sets of nucleic acid residues.

[18] The method described above, wherein each set of deleted nucleic acid residues consists of deletion of 1 to 3 nucleic acid residues.

[19] The method described above, wherein the destabilizing portion comprises insertion of 200% or less of the number of nucleic acid residues relative to the length of the first base sequence.

[20] The method described above, wherein the destabilizing portion comprises insertion of 1 to 8 sets of nucleic acid residues.

[21] The method described above, wherein the destabilizing portion comprises insertion of 1 to 4 sets of nucleic acid residues.

[22] The method described above, wherein each set of inserted nucleic acid residues consists of insertion of 1 to 10 nucleic acid residues.

[23] The method, wherein when each set of insertions of nucleic acid residues consists of insertions of two or more nucleic acid residues, some or all of the nucleic acid residues are self-complementary.

[24] The method, wherein the destabilizing portion comprises insertion of 1 to 8 linkers.

[25] The method, wherein the destabilizing portion comprises deletion of 1 to 8 linkers.

[26] The method, wherein the destabilizing portion comprises substitution of 1 to 8 linkers.

[27] The method as described above, wherein the destabilizing portion comprises the following (a), (b), or (c): (a) a combination of an abasic portion 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; (b) a mismatch portion 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) a mismatch portion at positions −1 to +1.

[28] The method, wherein the destabilizing portions (a), (b), and (c) are the following (a1), (b1), and (c1), respectively: (a1) a combination of one abasic portion at positions -1 to +1 and one or two sets of deletions and / or insertions of nucleic acid residues at positions other than positions -1 to +1; (b1) a combination of one mismatch portion at positions -1 to +1 and one or two sets of deletions and / or insertions of nucleic acid residues at positions other than positions -1 to +1; (c1) one mismatch portion at positions -1 to +1.

[29] The method, wherein the destabilizing portions (a), (b), and (c) are (a2), (b2), and (c2) below, respectively: (a2) a combination of one abasic portion at positions −1 to +1, one set of nucleic acid residues inserted at minus positions other than −1, and one set of nucleic acid residues inserted at plus positions other than +1; (b2) a combination of one mismatch portion at positions −1 to +1, one set of nucleic acid residues inserted at minus positions other than −1, and one set of nucleic acid residues inserted at plus positions other than +1; (c2) one mismatch portion at positions −1 to +1.

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

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

[32] The method, wherein the length of each substrate oligonucleic acid is 5 to 50 residues.

[33] The method as described above, wherein the length of each of the second and third base sequences is 5 to 50 residues.

[34] The method as described above, wherein the length of each of the complementary oligonucleic acids is 5 to 300%, where the length of the target oligonucleic acid is taken as 100%.

[35] The method as described above, wherein the length of each of the complementary oligonucleic acids is 5 to 300%, where the sum of the lengths of the 5'- and 3'-substrate oligonucleic acids is taken as 100%.

[36] The method, wherein, in each of the complementary oligonucleic acids, 5 or more of the nucleic acid residues constituting the second base sequence and 5 or more of the nucleic acid residues constituting the third base sequence remain.

[37] The method, wherein, in each of the complementary oligonucleic acids, 50% or more of the nucleic acid residues constituting the second base sequence and 50% or more of the nucleic acid residues constituting the third base sequence remain.

[38] The method, wherein, in each of the complementary oligonucleic acids, 3 or more consecutive nucleic acid residues constituting the second base sequence and 3 or more consecutive nucleic acid residues constituting the third base sequence remain.

[39] The method, wherein the target oligonucleic acid consists of DNA residues, RNA residues, modified nucleic acid residues, or a combination thereof.

[40] The method, wherein the target oligonucleic acid is modified.

[41] The method, wherein one or more of the complementary oligonucleic acids are modified.

[42] The method described above, wherein the modification comprises a modification of the phosphate moiety, a modification of the sugar moiety, a modification of the base moiety, or a combination thereof.

[43] The method described above, wherein the modification of the phosphate moiety comprises phosphorothioation, boranophosphate, insertion of a linker, or a combination thereof.

[44] The method described above, wherein the modification of the sugar moiety comprises 2'-MOE, 2'-OMe, 2'-F, 4'-thio-2'-OMe, cross-linking between the 2' and 4' positions of the sugar moiety, modification of the 5' end of the oligonucleic acid, modification of the 3' end of the oligonucleic acid, or a combination thereof.

[45] The method described above, wherein N is 2.

[46] The method described above, wherein N is 3 or more.

[47] The method described above, wherein N is 10 or less.

[48] The method described above, wherein M is smaller than N-1.

[49] The method described above, wherein M is N-1.

[50] The method described above, wherein M is larger than N-1.

[51] The method as described above, wherein the step is carried out at 5 to 60° C.

[52] The method as described above, wherein the amount of each complementary oligonucleic acid used is 50% or less of the smaller of the amount of the 5'-side substrate oligonucleic acid used and the amount of the 3'-side substrate oligonucleic acid used, in terms of molar ratio.

[53] The method as described above, wherein the concentration of each substrate oligonucleic acid in the reaction solution in the step is 1 to 10,000 μM.

[54] The method as mentioned above, wherein the enzyme used for the enzymatic ligation is T3 DNA ligase.

[0011] 1 shows the structures of the oligonucleic acids (5'-end substrate, 3'-end substrate, and complementary strand) used in the examples, and the assumed hybridization structures of the oligonucleic acids. In each hybridization structure, the substrate is shown on the top and the complementary strand is shown on the bottom. "Ab" indicates an abasic moiety. This figure shows the amount of product obtained when a ligation reaction of two substrates is carried out using a complementary strand into which an abasic moiety has been introduced, or a complementary strand into which an abasic moiety and a loop structure have been introduced. This figure shows the yield of product obtained when a ligation reaction of two substrates whose sugar moieties have been modified using a complementary strand into which an abasic moiety has been introduced, or a complementary strand into which an abasic moiety and a loop structure have been introduced. This figure shows the yield of product obtained when a ligation reaction of two substrates whose phosphate moieties have been modified using a complementary strand into which an abasic moiety has been introduced, a complementary strand into which a loop structure ... is carried out using a complementary strand into which a loop structure has been introduced. 1 shows the structures of oligonucleic acids used in the examples and the expected hybridization structures of a substrate and a complementary strand. In each hybridization structure, the substrate is shown on the top and the complementary strand on the bottom. This figure shows the yield of products when a ligation reaction of two substrates is carried out using a complementary strand into which one or more nucleic acid residue deletions have been introduced. This figure shows the yield of products when a ligation reaction of two substrates is carried out using a complementary strand into which one or more loop structures have been introduced. This figure shows the structures of oligonucleic acids (5'-end substrate, 3'-end substrate, and complementary strand) used in the examples and the expected hybridization structures of oligonucleic acids. In each hybridization structure, the substrate is shown on the top and the complementary strand is shown on the bottom. "Ab" indicates an abasic moiety. This figure shows the yield of products when a ligation reaction of two substrates is carried out using a complementary strand into which one or more abasic moieties have been introduced.

[0023] Figures showing the structures of the oligonucleic acids (5'-end substrate, 3'-end substrate, and complementary strand) used in the examples and the assumed hybridization structures of the oligonucleic acids. In each hybridization structure, the substrate is shown on the top and the complementary strand on the bottom. Figures showing the yield of products when a ligation reaction of two substrates is carried out using a complementary strand into which a mismatch portion has been introduced. Figures showing the yield of products when a ligation reaction of two substrates is carried out using a complementary strand into which an abasic portion has been introduced or a complementary strand into which an abasic portion and a loop structure have been introduced, at various substrate and complementary strand concentrations.The horizontal axis indicates the substrate concentration (common to both the 5'-end substrate and the 3'-end substrate). The complementary strand concentration is 1 / 10 of the substrate concentration. This figure shows the rate of product formation per enzyme activity when ligation reactions of two substrates were performed using a complementary strand with an abasic portion introduced or a complementary strand with an abasic portion and a loop structure introduced at various substrate and complementary strand concentrations. The horizontal axis indicates the substrate concentration (common to both the 5'-end substrate and the 3'-end substrate). The complementary strand concentration is 1 / 10 of the substrate concentration. This figure shows the production of products when ligation reactions of two 50-residue substrates were performed using a complementary strand with an abasic portion introduced or a complementary strand with an abasic portion and a loop structure introduced. This figure shows the production of products when ligation reactions of two 50-residue substrates containing modified nucleic acids were performed using a complementary strand with an abasic portion introduced or a complementary strand with an abasic portion and a loop structure introduced. Figure 1 shows the production of products when a ligation reaction of three substrates is carried out using a complementary strand into which an abasic portion has been introduced or a complementary strand into which an abasic portion and a loop structure have been introduced. Figure 2 shows the production of products when a ligation reaction of three substrates containing modified nucleic acids is carried out using a complementary strand into which an abasic portion has been introduced or a complementary strand into which an abasic portion and a loop structure have been introduced. Figure 3 shows the production of products when a ligation reaction of two substrates containing modified nucleic acids and RNA is carried out using a complementary strand into which an abasic portion has been introduced or a complementary strand into which an abasic portion and a loop structure have been introduced.

[0012] The present invention will be described in detail below.

[0013] The method of the present invention is a method for ligating single-stranded oligonucleic acids. The single-stranded oligonucleic acids to be ligated are also referred to as "single-stranded substrate oligonucleic acid" or "substrate oligonucleic acid."

[0014] Ligation of the substrate oligonucleic acids results in the production of an oligonucleic acid to which the substrate oligonucleic acids are linked. The resulting oligonucleic acid is also referred to as a "target oligonucleic acid." Thus, the method of the present invention may be a method for producing a target oligonucleic acid.

[0015] The method of the present invention includes a step of enzymatically ligating N (N is an integer of 2 or more) substrate oligonucleic acids. 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 of 2 or more) substrate oligonucleic acids to produce a target oligonucleic acid. The N (N is an integer of 2 or more) substrate oligonucleic acids ligated in the ligation step are also collectively referred to as a "set of substrate oligonucleic acids."

[0016] The ligation step is carried out in the presence of single-stranded oligonucleic acids having a specific structure. Such single-stranded oligonucleic acids are also referred to as "single-stranded complementary oligonucleic acids" or "complementary oligonucleic acids." Specifically, the ligation step may be carried out in the presence of M (M is an integer of 1 or greater) complementary oligonucleic acids. That is, the ligation step may be a step of enzymatically ligating N (N is an integer of 2 or greater) substrate oligonucleic acids in the presence of M (M is an integer of 1 or greater) complementary oligonucleic acids. Alternatively, the ligation step may be a step of enzymatically ligating N (N is an integer of 2 or greater) substrate oligonucleic acids in the presence of M (M is an integer of 1 or greater) complementary oligonucleic acids to produce a target oligonucleic acid. More specifically, the ligation step may be carried out in the presence of complementary oligonucleic acids corresponding to each linking site. That is, each complementary oligonucleic acid may be used to link the substrate oligonucleic acid at the linking site corresponding to the complementary oligonucleic acid. More specifically, the linkage between the 5'- and 3'-side substrate oligonucleic acids at each linkage may be carried out in the presence of a complementary oligonucleic acid corresponding to the linkage, i.e., each complementary oligonucleic acid may be used for the linkage between the 5'- and 3'-side substrate oligonucleic acids at the linkage corresponding to the complementary oligonucleic acid.

[0017] According to the method of the present invention, for example, the complementary oligonucleic acid may be catalytically used in the repeated ligation step, thereby efficiently carrying out the ligation step. Specifically, for example, the action of the destabilizing moiety may make the complementary oligonucleic acid more likely to spontaneously dissociate from the ligation product of the substrate oligonucleic acid (i.e., the target oligonucleic acid), thereby allowing the complementary oligonucleic acid to be used in the repeated ligation step. In other words, according to the method of the present invention, for example, the amount of complementary oligonucleic acid used may be reduced.

[0018] <1> Target Oligonucleic Acid The term "target oligonucleic acid" refers to an oligonucleic acid produced by the method of the present invention.

[0019] The target oligonucleic acid may be present in the form of, for example, a single-stranded oligonucleic acid, or may be present in the form of a double-stranded oligonucleic acid.That is, the target oligonucleic acid may be produced in the form of, for example, a single-stranded oligonucleic acid, or may be produced in the form of a double-stranded oligonucleic acid.Furthermore, the target oligonucleic acid may be used in the form of, for example, a single-stranded oligonucleic acid, or may be used in the form of a double-stranded oligonucleic acid.

[0020] "Single-stranded oligonucleic acid" refers to an oligonucleic acid consisting of a single nucleic acid strand that is not hybridized with other nucleic acid strands. For example, "a target oligonucleic acid in the form of a single-stranded oligonucleic acid" may refer to a target oligonucleic acid in a form that is not hybridized with other nucleic acid strands. The single-stranded oligonucleic acid may or may not form a double-stranded structure (e.g., a stem-loop structure) within the molecule, as long as it does not impair the purpose of the present invention.

[0021] A "double-stranded oligonucleic acid" refers to an oligonucleic acid consisting of at least two nucleic acid strands hybridized to each other. That is, for example, a "target oligonucleic acid in the form of a double-stranded oligonucleic acid" may refer to a target oligonucleic acid in a form hybridized with another nucleic acid strand. The other nucleic acid strand may be a complementary oligonucleic acid. That is, the target oligonucleic acid may exist, be produced, and / or be used in a form hybridized with one or more complementary oligonucleic acids, for example.

[0022] The form of the target oligonucleic acid (e.g., whether it exists, is produced, and / or is used in the form of a single-stranded oligonucleic acid or a double-stranded oligonucleic acid) can be appropriately determined depending on various conditions, such as the intended use of the target oligonucleic acid. The intended use of the target oligonucleic acid is not particularly limited. Examples of intended uses of the target oligonucleic acid include use as an antisense nucleic acid, siRNA, miRNA mimic, decoy nucleic acid, aptamer, or CpG oligonucleic acid. When the target oligonucleic acid is used as an antisense nucleic acid, aptamer, or CpG oligonucleic acid, the target oligonucleic acid may exist, be produced, and / or be used, for example, as a single-stranded oligonucleic acid. When the target oligonucleic acid is used as an siRNA, miRNA mimic, or decoy nucleic acid, the target oligonucleic acid may exist, be produced, and / or be used, for example, as a double-stranded oligonucleic acid or as a single-stranded oligonucleic acid that forms an intramolecular duplex structure.

[0023] The type of nucleic acid residues constituting the target oligonucleic acid is not particularly limited. The type of nucleic acid residues constituting the target oligonucleic acid can be appropriately determined depending on various conditions, such as the intended use of the target oligonucleic acid. Examples of nucleic acid residues include DNA residues, RNA residues, and modified nucleic acid residues. The target oligonucleic acid may contain one or more types of nucleic acid residues. That is, the target oligonucleic acid may contain, for example, DNA residues, RNA residues, modified nucleic acid residues, or a combination thereof. The target oligonucleic acid may be a nucleic acid chain consisting of one type of nucleic acid residue, or a nucleic acid chain consisting of a combination of two or more types of nucleic acid residues (i.e., a nucleic acid chain containing two or more types of nucleic acid residues in the molecule). That is, the target oligonucleic acid may be, for example, a nucleic acid chain consisting of DNA residues, a nucleic acid chain consisting of RNA residues, 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 types of nucleic acid residues selected from DNA residues, RNA residues, and modified nucleic acid residues in the molecule).

[0024] The base sequence of the target oligonucleic acid is not particularly limited. The base sequence of the target oligonucleic acid can be appropriately set depending on various conditions, such as the intended use of the target oligonucleic acid. When the target oligonucleic acid is used as an antisense nucleic acid, siRNA, miRNA mimic, decoy nucleic acid, aptamer, or CpG oligonucleic acid, the base sequence of the target oligonucleic acid may be set so that the target oligonucleic acid functions as an antisense nucleic acid, siRNA, miRNA mimic, decoy nucleic acid, aptamer, or CpG oligonucleic acid, respectively. That is, for example, when the target oligonucleic acid is used as an antisense nucleic acid, the base sequence of the target oligonucleic acid may be set so that the target oligonucleic acid can form a sequence-specific duplex structure with the target single-stranded RNA. Examples of the target single-stranded RNA for antisense nucleic acids include mRNA, pre-mRNA, and miRNA. Furthermore, for example, when a target oligonucleic acid is used as an siRNA or miRNA mimic, the base sequence of the target oligonucleic acid may be designed so that the target oligonucleic acid (specifically, the single-stranded structure generated by dissociation of the target oligonucleic acid) can form a sequence-specific duplex structure with the target single-stranded RNA. Examples of target single-stranded RNA for siRNA or miRNA mimics include mRNA. Furthermore, for example, when a target oligonucleic acid is used as a decoy nucleic acid, aptamer, or CpG oligonucleic acid, the base sequence of the target oligonucleic acid may be designed so that the target oligonucleic acid can bind to a target protein in a sequence-specific manner. The base sequence of the target oligonucleic acid may be designed so that the target oligonucleic acid does not form a duplex structure within the molecule, or so that the target oligonucleic acid forms a duplex structure within the molecule.

[0025] The length of the target oligonucleic acid is not particularly limited. The length can be appropriately set depending on various conditions, such as the use of the target oligonucleic acid. The length of the target oligonucleic acid may be, for example, 10 or more residues, 11 or more residues, 12 or more residues, 13 or more residues, 14 or more residues, 15 or more residues, 16 or more residues, 17 or more residues, 18 or more residues, 19 or more residues, 20 or more residues, 21 or more residues, 22 or more residues, 23 or more residues, 24 or more residues, 25 or more residues, 30 or more residues, 35 or more residues, 40 or more residues, 45 or more residues, 50 or more residues, 60 or more residues, 70 or more residues, 80 or more residues, 90 or more residues, 100 or more residues, 110 or more residues, 120 or more residues, 130 or more residues, 140 or more residues, 150 or more residues, 160 or more residues, 170 or more residues, 180 or more residues, or 190 or more residues, and may be 200 or more. The number of residues may be 190 or less, 180 or less, 170 or less, 160 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, 45 or less, 40 or less, 35 or less, 30 or less, 25 or less, 24 or less, 23 or less, 22 or less, 21 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, or 11 or less, or any combination thereof that is compatible therewith. The length of the target oligonucleic acid is specifically, 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, It may be 40 to 45 residues, 45 to 50 residues, 50 to 60 residues, 60 to 70 residues, 70 to 80 residues, 80 to 90 residues, 90 to 100 residues, 100 to 110 residues, 110 to 120 residues, 120 to 130 residues, 130 to 140 residues, 140 to 150 residues, 150 to 160 residues, 160 to 170 residues, 170 to 180 residues, 180 to 190 residues, or 190 to 200 residues.The length of the target oligonucleic acid may be, for example, 10 to 200 residues, 10 to 100 residues, 15 to 80 residues, or 20 to 60 residues. The length of the target oligonucleic acid may be, for example, 10 to 200 residues, 10 to 150 residues, 15 to 120 residues, or 20 to 100 residues.

[0026] "DNA" without reference to modifications may refer to naturally occurring DNA, specifically nucleic acids in which the nucleoside moieties are deoxyadenosine, deoxyguanosine, deoxycytidine, thymidine, or deoxyuridine.

[0027] "RNA" without reference to modifications may refer to naturally occurring RNA, and specifically may refer to nucleic acids in which the nucleoside moiety is adenosine, guanosine, cytidine, 5-methyluridine, or uridine.

[0028] "Modified nucleic acid" may refer to a nucleic acid other than natural DNA and natural RNA. Modified nucleic acids include modified DNA and modified RNA. "Modified DNA" may refer to a nucleic acid that has the same structure as natural DNA except for the modification. "Modified RNA" may refer to a nucleic acid that has the same structure as natural RNA except for the modification.

[0029] The terms "the oligonucleic acid of interest comprises a modified nucleic acid residue," "the oligonucleic acid of interest is modified," and "the oligonucleic acid of interest has a modification" may be used interchangeably.

[0030] The terms "the nucleic acid residue is a modified nucleic acid residue," "the nucleic acid residue has been modified," and "the nucleic acid residue has a modification" may be used interchangeably.

[0031] The target oligonucleic acid may or may not contain modified nucleic acid residues. That is, the target oligonucleic acid may or may not be modified. The modification of the target oligonucleic acid (e.g., the type, position, and amount of modification) is not particularly limited as long as it does not impair the objectives of the present invention. The modification of the target oligonucleic acid can be appropriately determined depending on various conditions, such as the intended use of the target oligonucleic acid. Examples of modifications include modifications of the phosphate moiety, sugar moiety, and base moiety. That is, the modification may include, for example, modifications of the phosphate moiety, sugar moiety, base moiety, or a combination thereof. The phrase "the modification includes a certain modification A" means that at least modification A is selected as the modification, and includes cases where the modification consists of modification A and cases where the modification consists of a combination of modification A and another modification. For example, the phrase "the modification includes a modification of the phosphate moiety" means that at least a modification of the phosphate moiety is selected as the modification, and includes cases where the modification consists of a modification of the phosphate moiety and a combination of modification A and another modification.

[0032] Examples of phosphate moieties include phosphate groups present between nucleic acid residues (i.e., phosphate groups forming phosphodiester bonds between nucleic acid residues). When a phosphate group present between nucleic acid residues is modified, the nucleic acid residue 3' to the phosphate group (i.e., the nucleic acid residue having the phosphate group at the 5' position) is considered modified. Modifications of the phosphate moiety include phosphorothioation (i.e., substitution of the oxygen atom of the phosphate group with a sulfur atom; in other words, substitution of the phosphate group with a thiophosphate group) and boranophosphate (i.e., substitution of the oxygen atom of the phosphate group with borane (BH3); in other words, substitution of the phosphate group with a boranophosphate group). Modifications of the phosphate moiety also include the insertion of a linker. "Linker" refers to a structure other than a nucleic acid residue. Examples of linkers include hydrocarbon groups. Examples of hydrocarbon groups include non-aromatic hydrocarbon groups and aromatic hydrocarbon groups. Examples of non-aromatic hydrocarbon groups include alkylene groups and alkenylene groups. The alkylene group may be linear or branched. Examples of alkylene groups include alkylene groups having 1 to 12, 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Examples of alkylene groups having 1 to 4 carbon atoms include methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, and tert-butylene. The alkenylene group may be linear or branched. Examples of alkenylene groups include alkenylene groups having 2 to 12, 2 to 8, 2 to 6, or 2 to 4 carbon atoms. Examples of alkenylene groups having 2 to 4 carbon atoms include ethenylene, propenylene, isopropenylene, butenylene, isobutenylene, and sec-butenylene. The alkenylene group may be, for example, a cis or trans configuration. Examples of aromatic hydrocarbon groups include aromatic hydrocarbon groups having 6 to 12 carbon atoms. An example of an aromatic hydrocarbon group having 6 to 12 carbon atoms is a xylylene group. The linker may be bonded, for example, to a phosphate group at both ends. That is, the linker may be bonded, for example, to the phosphate group at the 3'-position of the nucleic acid residue on the 5'-side and to the phosphate group at the 5'-position of the nucleic acid residue on the 3'-side.In other words, the insertion of a linker at a phosphate group between nucleic acid residues may be achieved by replacing the phosphate group between nucleic acid residues with a linker sandwiched between phosphate groups. The phosphate groups to which the linker is attached may or may not be modified (e.g., phosphorothioated or boranophosphated). For example, when a complementary oligonucleic acid contains an inserted nucleic acid residue as a destabilizing moiety, the linker may be inserted at a position corresponding to the inserted nucleic acid residue in the target oligonucleic acid.

[0033] Examples of sugar moieties include deoxyribose, which is the sugar moiety of DNA, and ribose, which is the sugar moiety of RNA. Modifications of the sugar moiety include 2'-MOE (i.e., O-methoxyethylation of the 2' position of the sugar moiety), 2'-OMe (i.e., O-methylation of the 2' position of the sugar moiety), 2'-F (i.e., fluorination of the 2' position of the sugar moiety), 4'-thio-2'-OMe (i.e., substitution of the oxygen atom of the 4' position of the sugar moiety with a sulfur atom and O-methylation of the 2' position of the sugar moiety), and crosslink formation between the 2' and 4' positions of the sugar moiety. Examples of crosslink formation between the 2' and 4' positions of the sugar moiety include LNA and BNA. ＮＣ , BNA ＮＣ(Me), AmNA. Modifications of the sugar moiety include modifications of the 5' end of the oligonucleic acid and modifications of the 3' end of the oligonucleic acid. Modifications of the 5' end of the oligonucleic acid include 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'-aliphatic chain addition, 5'-folic acid addition, 5'-polyamine addition, and 5'-drug addition. Modifications of the 3' end of an oligonucleic acid include 3'-amination, 3'-thiolation, 3'-dabsylation, 3'-deoxylation, 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'-aliphatic chain addition, 3'-folic acid addition, 3'-polyamine addition, and 3'-drug addition. Drugs include anticancer drugs and various other pharmaceutical ingredients. An additive such as a drug may be attached to the oligonucleic acid, for example, directly or via a suitable linker (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.).

[0034] Modifications of the phosphate and sugar moieties include morpholino and peptidyl groups of the nucleic acid backbone, i.e., modified nucleic acids also include morpholino nucleic acids and peptide nucleic acids (PNAs).

[0035] Examples of the base moiety include adenine (A), thymine (T), guanine (G), cytosine (C), and uracil (U). Modifications of the base moiety 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, oxo-ation, nitration, thiolation, azation, deazation, and substitution with various other analogs. Specific examples of modified bases include 8-haloadenine (halo is bromo, fluoro, iodo, or chloro; the same applies below), 8-haloguanine, 5-halothymine, 5-halocytosine, 5-halouracil, 1-, 3-, or 7-deazaadenine, 1-, 3-, or 7-deazaguanine, 3-deazathymine, 3-deazacytosine, 3-deazauracil, 8-azaadenine, 8-azaguanine, 5- or 6-azathymine, 5- or 6-azacytosine, 5- or 6-azauracil, 3-methyladenine, N6-methyladenine, O6-methylguanine, O4-methylthymine, 5-methylcytosine, 5-(2-amino)propyluracil, 2-aminoadenine, 8-oxoadenine, 8-oxoguanine, 2- or 4-thiothymine, 2- or 4-thiocytosine, 2- or 4-thiouracil, 2-aminopurine, 2,6-diaminopurine, hypoxanthine, and 7-deaza-8-azahypoxanthine. Specific examples of modified bases include artificial bases such as x, y, s, Ds, Pa, PxR, PICS, 5SICS, NaM, MMO2, TPT3, iG, iC, P, and Z (Hirao et al., Toward a New Research Area, Xenobiology, from Artificial Base Pair Technology to Expand Genetic Information, Chemistry and Biology, Vol. 54, No. 11, 2016, pp. 835-840). Modifications of the base moiety also include deletion of the base moiety. In other words, modified nucleic acids may be nucleic acids lacking a base moiety (i.e., abasic nucleic acids).

[0036] The presence or absence and type of modification can be set independently for each nucleic acid residue constituting the target oligonucleic acid. The target oligonucleic acid may have one type of modification, or may have two or more types of modifications in combination. The target oligonucleic acid may or may not contain, for example, each of the modifications exemplified above. Each nucleic acid residue constituting the target oligonucleic acid may have one type of modification, or may have two or more types of modifications in combination. Only a portion of the nucleic acid residues constituting the target oligonucleic acid may be modified nucleic acid residues, or all of the nucleic acid residues constituting the target oligonucleic acid may be modified nucleic acid residues.

[0037] The ratio of modified nucleic acid residues in the target oligonucleic acid (i.e., the ratio of the number of modified nucleic acid residues to the number of nucleic acid residues constituting the target oligonucleic acid) 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, 90% or more, or 95% or more, or 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, or any compatible combination thereof. The ratio of modified nucleic acid residues in a target oligonucleic acid may 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%. The ratios of modified nucleic acid residues in a target oligonucleic acid exemplified above can also be applied independently to nucleic acid residues having a selected modification (e.g., any of the modifications exemplified above). That is, for example, the ratio of nucleic acid residues having a modification at the phosphate moiety in a target oligonucleic acid (i.e., the ratio of the number of nucleic acid residues having a modification at the phosphate moiety to the number of nucleic acid residues constituting the target oligonucleic acid) may be set to the ratios of modified nucleic acid residues in a target oligonucleic acid exemplified above. Furthermore, for example, the ratio of nucleic acid residues having a sugar moiety modification in a target oligonucleic acid (i.e., the ratio of the number of nucleic acid residues having a sugar moiety modification to the number of nucleic acid residues constituting the target oligonucleic acid) may be set to the ratio of modified nucleic acid residues in the target oligonucleic acid exemplified above.Furthermore, for example, the ratio of nucleic acid residues having a base moiety modification in a target oligonucleic acid (i.e., the ratio of the number of nucleic acid residues having a base moiety modification to the number of nucleic acid residues constituting the target oligonucleic acid) may be set to the ratio of modified nucleic acid residues in the target oligonucleic acid exemplified above.

[0038] The number of modified nucleic acid residues in the target oligonucleic acid may be, for example, 0 or more, 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, 80 or more, 100 or more, 120 or more, 140 or more, 160 or more, or 180 or more; or 200 or less, 180 or less, 160 or less, 140 or less, 120 or less, 100 or less, 80 or less, 60 or less, 50 or less, 40 or less, 30 or less, 25 or less, 20 or less, 15 or less, 10 or less, or 5 or less, or any compatible combination thereof. The number of modified nucleic acid residues in a target oligonucleic acid may 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. The above-exemplified numbers of modified nucleic acid residues in a target oligonucleic acid can also be applied independently to nucleic acid residues having any selected modification (e.g., any of the modifications exemplified above). That is, for example, the number of nucleic acid residues having a modification in the phosphate moiety in a target oligonucleic acid may be set to the number of modified nucleic acid residues in the above-exemplified target oligonucleic acid. Furthermore, for example, the number of nucleic acid residues having a modification in the sugar moiety in a target oligonucleic acid may be set to the number of modified nucleic acid residues in the above-exemplified target oligonucleic acid. Furthermore, for example, the number of nucleic acid residues having a modified base moiety in the target oligonucleic acid may be set to the number of modified nucleic acid residues in the target oligonucleic acid exemplified above.

[0039] Modified nucleic acid residues in which the structure of the 2' sugar moiety is the same as deoxyribose are considered to be modified DNA residues unless otherwise specified. Modified nucleic acid residues in which the structure of the 2' sugar moiety is the same as ribose are considered to be modified RNA residues unless otherwise specified. Modified nucleic acid residues with a modification at the 2' sugar moiety are considered to be both modified DNA residues and modified RNA residues unless otherwise specified.

[0040] The target oligonucleic acid may be modified in one of the modification modes exemplified above during ligation of the substrate oligonucleic acid, or may be modified to one of the modification modes exemplified above after ligation of the substrate oligonucleic acid. That is, the modified target oligonucleic acid may be produced, for example, by ligating substrate oligonucleic acids modified according to the modification mode of the target oligonucleic acid, or by ligating substrate oligonucleic acids that are not modified (for example, may be unmodified) according to the modification mode of the target oligonucleic acid and then modifying the ligation product.

[0041] <2> Substrate oligonucleic acid and set thereof The term "substrate oligonucleic acid" refers to each of the single-stranded oligonucleic acids to be ligated in the ligation step, in other words, each of the nucleic acid fragments constituting the target oligonucleic acid.

[0042] The term "single-stranded" with respect to the substrate oligonucleic acid refers to the form of the substrate oligonucleic acid at the start of the method of the present invention (specifically, the ligation step). The substrate oligonucleic acid can become a double-stranded oligonucleic acid as the method of the present invention (specifically, the ligation step) progresses. For example, the substrate oligonucleic acid can hybridize with a complementary oligonucleic acid to become a double-stranded oligonucleic acid as the method of the present invention (specifically, the ligation step) progresses.

[0043] The term "substrate oligonucleic acid set" is a general term for substrate oligonucleic acids to be ligated in the ligation step. The substrate oligonucleic acid set consists of N substrate oligonucleic acids (N is an integer of 2 or more).

[0044] The number (N) of substrate oligonucleic acids is 2 or more. "The number of substrate oligonucleic acids" refers to the number of substrate oligonucleic acids ligated in the ligation step, or in other words, the number of substrate oligonucleic acids constituting the target oligonucleic acid. Furthermore, "the number of substrate oligonucleic acids" refers to the number of substrate oligonucleic acids constituting a set of substrate oligonucleic acids. The number (N) of substrate oligonucleic acids may be 2 or 3 or more. The number (N) of substrate oligonucleic acids may 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 more, or 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 any compatible combination thereof. Specific examples of the number (N) of substrate oligonucleic acids may 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. The number (N) of substrate oligonucleic acids may be, for example, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. The number (N) of substrate oligonucleic acids may particularly be 2 to 3. The number (N) of substrate oligonucleic acids may more particularly be 2.

[0045] The linkage between substrate oligonucleic acids is also simply referred to as a "linkage." The two substrate oligonucleic acids linked at each linkage are also referred to as the "5'-side substrate oligonucleic acid" and the "3'-side substrate oligonucleic acid," respectively. The term "5'-side" may be used interchangeably with the term "5'-end side." The term "3'-side" may be used interchangeably with the term "3'-end side." At each linkage, the 3'-end of the 5'-side substrate oligonucleic acid is linked to the 5'-end of the 3'-side substrate oligonucleic acid. Note that the "5'-side" and "3'-side" of the substrate oligonucleic acid indicate the same direction as the "5'-side" and "3'-side" of the target oligonucleic acid, respectively. Furthermore, the "5'-side" and "3'-side" of the substrate oligonucleic acid indicate the opposite direction to the "5'-side" and "3'-side" of the complementary oligonucleic acid hybridized with the substrate oligonucleic acid, respectively. Furthermore, the terms "5' side" and "3' side" of the substrate oligonucleic acid indicate relative directions based on each linking portion. That is, for example, when three substrate oligonucleic acids A, B, and C are linked in the 5' to 3' direction, when A and B are linked, A and B are the 5' and 3' side substrate oligonucleic acids, respectively, whereas when B and C are linked, B and C are the 5' and 3' side substrate oligonucleic acids, respectively. The number of linking portions is determined according to the number of substrate oligonucleic acids. That is, when the number of substrate oligonucleic acids is N (N is an integer of 2 or more), the number of linking portions is N-1; in other words, the linking step is performed at N-1 linking portions. In other words, the target oligonucleic acid consists of N substrate oligonucleic acids linked at N-1 positions. When the number of linking portions is 1, "each linking portion" refers to that linking portion, and when the number of linking portions is 2 or more, it refers to each of those linking portions.

[0046] The structure of each substrate oligonucleic acid (e.g., type of nucleic acid residue, base sequence, length, and modification mode) can be appropriately set depending on the structure of the target oligonucleic acid. That is, the structure of each substrate oligonucleic acid may be the same as the structure of the portion of the target oligonucleic acid that corresponds to each substrate oligonucleic acid.

[0047] The types of nucleic acid residues are as described above. Each substrate oligonucleic acid may contain one or more types of nucleic acid residues. That is, each substrate oligonucleic acid may contain, for example, DNA residues, RNA residues, modified nucleic acid residues, or a combination thereof. Each substrate oligonucleic acid may be a nucleic acid chain consisting of one type of nucleic acid residue, or a nucleic acid chain consisting of a combination of two or more types of nucleic acid residues (i.e., a nucleic acid chain containing two or more types of nucleic acid residues in the molecule). That is, each substrate oligonucleic acid may be, for example, a nucleic acid chain consisting of DNA residues, a nucleic acid chain consisting of RNA residues, 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 types of nucleic acid residues selected from DNA residues, RNA residues, and modified nucleic acid residues in the molecule).

[0048] The base sequence of each substrate oligonucleic acid is a partial sequence of the base sequence of the target oligonucleic acid. The base sequence of each substrate oligonucleic acid is set so that the linking sequence of the substrate oligonucleic acid is the base sequence of the target oligonucleic acid. For example, when the number of substrate oligonucleic acids is two, the base sequence of the 5'-side substrate oligonucleic acid is the 5'-side partial sequence of the target oligonucleic acid, the base sequence of the 3'-side substrate oligonucleic acid is the 3'-side partial sequence of the target oligonucleic acid, and the linking sequence between the 5'-side substrate oligonucleic acid and the 3'-side substrate oligonucleic acid is the base sequence of the target oligonucleic acid.

[0049] The length of each substrate oligonucleic acid is not particularly limited as long as it does not impair the objectives of the present invention. That is, the length of each substrate oligonucleic acid is set so that the substrate oligonucleic acids can be linked by the method of the present invention. The length of each substrate oligonucleic acid may be set, for example, so that each substrate oligonucleic acid can hybridize with its corresponding complementary oligonucleic acid. A "complementary oligonucleic acid corresponding to a substrate oligonucleic acid" refers to a complementary oligonucleic acid used at the link between the substrate oligonucleic acid and the substrate oligonucleic acid linked to it. Furthermore, the length of each substrate oligonucleic acid can be set so that the length of the linking sequence to which the substrate oligonucleic acid is linked matches the length of the target oligonucleic acid.

[0050] The length of each substrate oligonucleic acid may be, for example, 5 or more residues, 6 or more residues, 7 or more residues, 8 or more residues, 9 or more residues, 10 or more residues, 11 or more residues, 12 or more residues, 13 or more residues, 14 or more residues, 15 or more residues, 17 or more residues, 20 or more residues, 25 or more residues, 30 or more residues, 35 or more residues, 40 or more residues, 45 or more residues, 50 or more residues, 55 or more residues, 60 or more residues, or 65 or more residues, or 70 or less, 65 or less residues, 60 or less residues, 55 or less residues, 50 or less, 45 or less residues, 40 or less residues, 35 or less residues, 30 or less, 25 or less residues, 20 or less, 17 or less, 15 or less residues, 14 or less, 13 or less, 12 or less, 11 or less residues, 10 or less, 9 or less residues, 8 or less residues, 7 or less residues, or 6 or less residues, or a compatible combination thereof. The length of each substrate oligonucleic acid may 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. The length of each substrate oligonucleic acid may be, for example, 5 to 70 residues, 5 to 50 residues, 5 to 30 residues, 5 to 20 residues, or 5 to 15 residues.

[0051] The length of each substrate oligonucleic acid may be, for example, 3% 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 more, or 97% 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, relative to the length of the target oligonucleic acid (100%), or any compatible combination thereof. The length of each substrate oligonucleic acid may be, for example, 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% of the length of the target oligonucleic acid, relative to the length of the target oligonucleic acid being 100%. The length of each substrate oligonucleic acid may be, for example, 3-97%, 10-90%, 20-80%, 30-70%, or 40-60% of the length of the target oligonucleic acid, relative to the length of the target oligonucleic acid being 100%.

[0052] The terms "substrate oligonucleic acid comprises a modified nucleic acid residue," "substrate oligonucleic acid is modified," and "substrate oligonucleic acid has a modification" may be used interchangeably.

[0053] Each substrate oligonucleic acid may or may not contain 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 depending on the modification mode of the target oligonucleic acid. For example, when the ligation product is modified after ligation of the substrate oligonucleic acids, the modification mode of the substrate oligonucleic acid may differ from the modification mode of the target oligonucleic acid. Modification of nucleic acid residues is as described above. Each substrate oligonucleic acid may have one type of modification, or may have two or more types of modifications in combination. Each substrate oligonucleic acid may or may not have, for example, each of the modifications exemplified above. Each nucleic acid residue constituting each substrate oligonucleic acid may have one type of modification, or may have two or more types of modifications in combination. Only a portion of the nucleic acid residues constituting each substrate oligonucleic acid may be modified nucleic acid residues, or all of the nucleic acid residues constituting each substrate oligonucleic acid may be modified nucleic acid residues.

[0054] The ratio of modified nucleic acid residues in each substrate oligonucleic acid (i.e., the ratio of the number of modified nucleic acid residues to the number of nucleic acid residues constituting each substrate oligonucleic acid) can be set so that the ratio of modified nucleic acid residues in the linked sequence to which the substrate oligonucleic acids are linked matches the ratio of modified nucleic acid residues in the target oligonucleic acid. Furthermore, the ratio of modified nucleic acid residues in each substrate oligonucleic acid 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, 90% or more, or 95% or more, or 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, or any compatible combination thereof. The ratio of modified nucleic acid residues in each substrate oligonucleic acid may 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%. The ratios of modified nucleic acid residues in each substrate oligonucleic acid exemplified above can also be applied independently to nucleic acid residues having a selected arbitrary modification (e.g., any of the modifications exemplified above). That is, for example, the ratio of nucleic acid residues having a modification at the phosphate moiety in each substrate oligonucleic acid (i.e., the ratio of the number of nucleic acid residues having a modification at the phosphate moiety to the number of nucleic acid residues constituting each substrate oligonucleic acid) may be set to the ratio of modified nucleic acid residues in each substrate oligonucleic acid exemplified above. Furthermore, for example, the ratio of nucleic acid residues having a sugar moiety modification in each substrate oligonucleic acid (i.e., the ratio of the number of nucleic acid residues having a sugar moiety modification to the number of nucleic acid residues constituting each substrate oligonucleic acid) may be set to the ratio of modified nucleic acid residues in each substrate oligonucleic acid exemplified above.Furthermore, for example, the ratio of nucleic acid residues having a base moiety modification in each substrate oligonucleic acid (i.e., the ratio of the number of nucleic acid residues having a base moiety modification to the number of nucleic acid residues constituting each substrate oligonucleic acid) may be set to the ratio of modified nucleic acid residues in each substrate oligonucleic acid exemplified above.

[0055] The number of modified nucleic acid residues in each substrate oligonucleic acid can be set so that the number of modified nucleic acid residues in the linked sequence to which the substrate oligonucleic acid is linked matches the number of modified nucleic acid residues in the target oligonucleic acid. Furthermore, the number of modified nucleic acid residues in each substrate oligonucleic acid can be, for example, 0 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, or 65 or more, or 70 or less, 65 or less, 60 or less, 55 or less, 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, 25 or less, 20 or less, 15 or less, 10 or less, or 5 or less, or any combination thereof that is consistent therewith. Specifically, the length of each substrate oligonucleic acid may 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. The number of modified nucleic acid residues in each of the substrate oligonucleic acids exemplified above can also be applied independently to nucleic acid residues having any selected modification (e.g., any of the modifications exemplified above). That is, for example, the number of nucleic acid residues having a modification in the phosphate moiety in each substrate oligonucleic acid may be set to the number of modified nucleic acid residues in each of the substrate oligonucleic acids exemplified above. Furthermore, for example, the number of nucleic acid residues having a modification in the sugar moiety in each substrate oligonucleic acid may be set to the number of modified nucleic acid residues in each of the substrate oligonucleic acids exemplified above. Furthermore, for example, the number of nucleic acid residues having a modified base moiety in each substrate oligonucleic acid may be set to the number of modified nucleic acid residues in each substrate oligonucleic acid exemplified above.

[0056] Each substrate oligonucleic acid may have, for example, a structure useful or necessary for linkage between substrate oligonucleic acids. For example, the 5'-end of the 3'-side substrate oligonucleic acid may be phosphorylated. That is, the linkage between substrate oligonucleic acids may be, for example, a linkage between the phosphate group at the 5'-end of the 3'-side substrate oligonucleic acid and the hydroxyl group at the 3'-end of the 5'-side substrate oligonucleic acid. Also, for example, the 3'-end of the 5'-side substrate oligonucleic acid may be phosphorylated. That is, the linkage between substrate oligonucleic acids may be, for example, a linkage between the hydroxyl group at the 5'-end of the 3'-side substrate oligonucleic acid and the phosphate group at the 3'-end of the 5'-side substrate oligonucleic acid. Furthermore, the phosphate moiety at the 5'-end or 3'-end may or may not be modified. Modifications of the phosphate moiety at the 5'-end or 3'-end include phosphorothioation (i.e., substitution of the oxygen atom of the phosphate group with a sulfur atom) and boranophosphate (i.e., substitution of the oxygen atom of the phosphate group with borane (BH3)). Modification of the phosphate moiety at the 5'- or 3'-end also includes insertion of a linker, which is described above.

[0057] The method for producing each substrate oligonucleic acid is not particularly limited. Each substrate oligonucleic acid can be produced, for example, by sequentially linking the nucleic acid residues that make up each substrate oligonucleic acid. Linking of nucleic acid residues can be carried out, for example, by the phosphoramidite method. Alternatively, each substrate oligonucleic acid can be produced, for example, by linking the nucleic acid fragments that make up each substrate oligonucleic acid. Linking of nucleic acid fragments can be carried out enzymatically, for example, using complementary strands. Linking of nucleic acid fragments may be carried out, for example, by the method of the present invention.

[0058] <3> Complementary oligonucleic acid The term "complementary oligonucleic acid" refers to a single-stranded oligonucleic acid having a specific structure that is used in the ligation step. In the ligation step, a complementary oligonucleic acid corresponding to each ligation site is used.

[0059] The term "single-stranded" with respect to a complementary oligonucleic acid refers to the form of the complementary oligonucleic acid at the start of the method of the present invention (specifically, the ligation step). The complementary oligonucleic acid can become a double-stranded oligonucleic acid as the method of the present invention (specifically, the ligation step) progresses. For example, the complementary oligonucleic acid can hybridize with a substrate oligonucleic acid to become a double-stranded oligonucleic acid as the method of the present invention (specifically, the ligation step) progresses.

[0060] The structure of the complementary oligonucleic acid (e.g., the type of nucleic acid residue, base sequence, length, and modification mode) can be appropriately set depending on various conditions such as the structures of the 5' and 3' substrate oligonucleic acids linked at the linking portions corresponding to the complementary oligonucleic acid.

[0061] The types of nucleic acid residues are as described above. Each complementary oligonucleic acid may contain one or more types of nucleic acid residues. That is, each complementary oligonucleic acid may contain, for example, DNA residues, RNA residues, modified nucleic acid residues, or a combination thereof. Each complementary oligonucleic acid may be a nucleic acid chain consisting of one type of nucleic acid residue, or a nucleic acid chain consisting of a combination of two or more types of nucleic acid residues (i.e., a nucleic acid chain containing two or more types of nucleic acid residues in the molecule). That is, each complementary oligonucleic acid may be, for example, a nucleic acid chain consisting of DNA residues, a nucleic acid chain consisting of RNA residues, 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 types of nucleic acid residues selected from DNA residues, RNA residues, and modified nucleic acid residues in the molecule).

[0062] The complementary oligonucleic acid includes a base sequence in which a destabilizing portion has been introduced into a first base sequence. The base sequence in which a destabilizing portion has been introduced into a first base sequence is also referred to as a "destabilizing base sequence."

[0063] "A base sequence in which a destabilizing part has been introduced into a first base sequence" means a modified sequence of the first base sequence, which is identical to the first base sequence except for the introduction of a destabilizing part (i.e., the inclusion of a destabilizing part). In other words, the first base sequence is the base sequence before the destabilizing part is introduced (i.e., the destabilizing part is not included). Note that "introduction of a destabilizing part" as used herein is a description for specifying the structure of the destabilizing base sequence, and does not specify the method for producing the destabilizing base sequence.

[0064] The first base sequence is a base sequence consisting of a second base sequence and a third base sequence linked in the 5' to 3' direction. "A base sequence consisting of a second base sequence and a third base sequence linked in the 5' to 3' direction" means a linked sequence of the second base sequence and the third base sequence, in which the 3' end of the second base sequence is linked to the 5' end of the third base sequence. Note that the "linkage of the second base sequence and the third base sequence" used here is a description for specifying the structure of the first base sequence, and does not specify the method for producing the first base sequence.

[0065] The second base sequence is a complementary sequence of the 5'-side partial sequence of the 3'-side substrate oligonucleic acid, or a complementary sequence of the full-length sequence of the 3'-side substrate oligonucleic acid. The "5'-side partial sequence of the 3'-side substrate oligonucleic acid" means a partial sequence of the 3'-side substrate oligonucleic acid, consisting of consecutive nucleic acid residues including the 5'-terminal nucleic acid residue (this is the nucleic acid residue linked to the 3'-end of the 5'-side substrate oligonucleic acid).

[0066] The third base sequence is a complementary sequence of the 3'-side partial sequence of the 5'-side substrate oligonucleic acid, or a complementary sequence of the full-length sequence of the 5'-side substrate oligonucleic acid. The "3'-side partial sequence of the 5'-side substrate oligonucleic acid" means a partial sequence of the 5'-side substrate oligonucleic acid, consisting of consecutive nucleic acid residues including the 3'-terminal nucleic acid residue (this is the nucleic acid residue linked to the 5'-end of the 3'-side substrate oligonucleic acid).

[0067] "Complementary" between base sequences may mean that the base moieties between the base sequences can form a Watson-Crick base pair or a base pair of equal or greater strength. Examples of base pairs that are considered complementary include Watson-Crick base pairs selected from the bases exemplified above (which may or may not be modified), or base pairs of combinations that can form base pairs of equal or greater strength. Specific examples of base pairs that are considered complementary include Watson-Crick base pairs such as A-T base pairs, A-U base pairs, and G-C base pairs. Specific examples of base pairs that are considered complementary include unnatural base pairs such as x-y base pairs, s-y base pairs, Ds-Pa base pairs, Ds-PxR base pairs, PICS-PICS base pairs, 5SICS-NaM base pairs, 5SICS-MM02 base pairs, TPT3-NaM base pairs, iG-iC base pairs, and P-Z base pairs.

[0068] It should be noted that the first, second, and third base sequences may all be hypothetical base sequences for identifying destabilizing base sequences, and do not necessarily exist.For example, when the substrate oligonucleic acid contains a nucleic acid residue A1 that cannot form a Watson-Crick base pair or a base pair with any base moiety with a strength equal to or greater than that, the first, second, or third base sequence may be a hypothetical base sequence containing a nucleic acid residue A2 having a hypothetical base moiety that can form a Watson-Crick base pair or a hypothetical base pair with the nucleic acid residue A1 with a strength equal to or greater than that.Specifically, for example, when the substrate oligonucleic acid contains an abasic nucleic acid residue B1, the abasic nucleic acid residue B1 cannot form a base pair with any base moiety, but the first, second, or third base sequence may be a hypothetical base sequence containing a nucleic acid residue B2 having a hypothetical base moiety that can form a Watson-Crick base pair or a hypothetical base pair with the abasic nucleic acid residue B1 with a strength equal to or greater than that. In calculating the melting temperature (Tm), such hypothetical base pairs are considered to be base pairs of the same strength as AT base pairs, unless otherwise specified.

[0069] The lengths of the first, second, and third base sequences are not particularly limited as long as they do not impair the objectives of the present invention. That is, the lengths of the first, second, and third base sequences are set so that substrate oligonucleic acids can be ligated by the method of the present invention. The length of the first base sequence is the sum of the lengths of the second and third base sequences. The length of the second base sequence may be set, for example, so that a complementary oligonucleic acid can hybridize with the 3' substrate oligonucleic acid. The length of the third base sequence may be set, for example, so that a complementary oligonucleic acid can hybridize with the 5' substrate oligonucleic acid.

[0070] The length of the first base sequence may be, for example, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 60 or more, 70 or more, 80 or more, or 90 or more residues, or 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, 25 or less, 20 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, or 11 or less residues, or any compatible combination thereof. 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. 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.

[0071] The length of each of the second and third base sequences may be, 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, 17 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, or 45 or more residues, or 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, 25 or less, 20 or less, 17 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, or 5 or less, or any compatible combination thereof. The length of each of the second and third base sequences may 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. The length of each of the second and third base sequences may be, for example, 4 to 50 residues, 4 to 25 residues, 4 to 20 residues, 5 to 15 residues, or 7 to 13 residues.

[0072] The length of the first base sequence 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, or 90% 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, or 10% or less, or any compatible combination thereof, when the total length of the 5'-side and 3'-side substrate oligonucleic acid is taken as 100%. The length of the first base sequence may be, for example, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% relative to the total length of the 5'- and 3'-substrate oligonucleic acids, respectively. The length of the first base sequence may be, for example, 5-100%, 5-50%, 5-20%, 20-100%, 20-50%, or 50-100% relative to the total length of the 5'- and 3'-substrate oligonucleic acids, respectively.

[0073] The length of the second base sequence 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, or 90% 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, or 10% or less, or a compatible combination thereof, relative to the length of the 3'-side substrate oligonucleic acid as 100%. Specifically, the length of the second base sequence may be, for example, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%. Specifically, the length of the second base sequence may be, for example, 5 to 100%, 5 to 50%, 5 to 20%, 20 to 100%, 20 to 50%, or 50 to 100%, relative to the length of the 3'-side substrate oligonucleic acid, which is 100%.

[0074] The length of the third base sequence 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, or 90% 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, or 10% or less, or a compatible combination thereof, relative to the length of the 5'-side substrate oligonucleic acid as 100%. Specifically, the length of the third base sequence may be, for example, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% relative to the length of the 5'-side substrate oligonucleic acid as 100%. Specifically, the length of the third base sequence may be, for example, 5 to 100%, 5 to 50%, 5 to 20%, 20 to 100%, 20 to 50%, or 50 to 100%, relative to the length of the 5'-side substrate oligonucleic acid, which is 100%.

[0075] When the substrate oligonucleic acid contains a linker, the first base sequence contains the linker at a corresponding position. Here, "corresponding position" means between nucleic acid residues a and b in the first base sequence when the substrate oligonucleic acid contains a linker between nucleic acid residues A and B, 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 oligonucleic acid may be the same as the linker contained at the corresponding position in the first base sequence.

[0076] The term "destabilizing portion" refers to a portion that reduces the stability of a hybrid formed between a complementary oligonucleic acid and a target oligonucleic acid. That is, introduction of a destabilizing portion into the first base sequence reduces the stability of a hybrid formed between the complementary oligonucleic acid and the target oligonucleic acid compared to before introduction of the destabilizing portion into the first base sequence. In other words, the stability of a hybrid formed between a complementary oligonucleic acid and a target oligonucleic acid is lower than the stability of a hybrid formed between a perfectly complementary oligonucleic acid (here, this refers to an oligonucleic acid that is identical to the complementary oligonucleic acid except that it contains the first base sequence instead of the destabilizing base sequence) and the target oligonucleic acid. Examples of reduced hybrid stability include a decrease in the melting temperature (Tm) of the hybrid. The amount of decrease in Tm due to the introduction of a destabilizing moiety 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. The temperature may be 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, 19°C or less, 18°C ​​or less, 17°C or less, 16°C or less, 15°C or less, 14°C or less, 13°C or less, 12°C or less, 11°C or less, 10°C or less, 9°C or less, 8°C or less, 7°C or less, 6°C or less, 5°C or less, 4°C or less, 3°C or less, or 2°C or less, or any compatible combination thereof. The amount of decrease in Tm due to the introduction of a destabilizing moiety may be, specifically, for example, 1 to 2°C, 2 to 3°C, 3 to 4°C, 4 to 5°C, 5 to 6°C, 6 to 7°C, 7 to 8°C, 8 to 9°C, 9 to 10°C, 10 to 11°C, 11 to 12°C, 12 to 13°C, 13 to 14°C, 14 to 15°C, 15 to 16°C, 16 to 17°C, 17 to 18°C, 18 to 19°C, 19 to 20°C, 20 to 25°C, 25 to 30°C, 30 to 35°C, 35 to 40°C, 40 to 45°C, 45 to 50°C, 50 to 55°C, or 55 to 60°C.The amount of decrease in Tm due to the introduction of the destabilizing moiety may be, specifically, for example, 1 to 60°C, 3 to 60°C, 5 to 60°C, 10 to 60°C, 1 to 40°C, 3 to 40°C, 5 to 40°C, 10 to 40°C, 1 to 20°C, 3 to 20°C, 5 to 20°C, or 10 to 20°C.

[0077] The destabilizing portion may, for example, reduce the stability of the hybrid formed between the complementary oligonucleic acid and the target oligonucleic acid, but may not completely inhibit hybridization between the complementary oligonucleic acid and the 5'-side substrate oligonucleic acid, and may not completely inhibit hybridization between the complementary oligonucleic acid and the 3'-side substrate oligonucleic acid.

[0078] The destabilizing moiety is not particularly limited as long as it improves the efficiency of production of the target oligonucleic acid. Improvement of the efficiency of production of the target oligonucleic acid can be achieved by increasing the molar ratio of the amount of target oligonucleic acid produced to the amount of complementary oligonucleic acid used.

[0079] Destabilizing moieties include abasic moieties, mismatched moieties, insertions of nucleic acid residues, deletions of nucleic acid residues, insertions of linkers, deletions of linkers, and substitutions of linkers.

[0080] "Abasic portion" means either (1) or (2) below: (1) a nucleic acid residue lacking a base portion (i.e., an abasic nucleic acid residue); (2) a nucleic acid residue having any base portion, the nucleic acid residue of which in the corresponding substrate oligonucleic acid is an abasic nucleic acid residue.

[0081] That is, in the abasic portion, the nucleic acid residues of either or both of the complementary oligonucleic acid and the substrate oligonucleic acid may be abasic nucleic acid residues. In particular, in the abasic portion, at least the nucleic acid residues of the complementary oligonucleic acid may be abasic nucleic acid residues.

[0082] "Mismatched portion" refers to a nucleic acid residue having a base portion that is mismatched with the corresponding substrate oligonucleic acid. "Mismatched base pair" may mean that the base portion cannot form a base pair between the base sequences, or that the strength of the base pair formed between the base portions of the base sequences is less than that of a Watson-Crick base pair. Examples of base combinations that are considered to be mismatched include combinations of bases that cannot form base pairs selected from the above-exemplified bases (which may be modified), and combinations of bases that can form base pairs with a strength less than that of a Watson-Crick base pair selected from the above-exemplified bases (which may be modified). Specific examples of base combinations that are considered to be mismatched include 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, and the combination of I and C (I is hypoxanthine). The combination of bases that are considered to be mismatches includes, in particular, combinations of bases other than those that form wobble base pairs. The combinations of bases that form wobble base pairs include the combination of U and G, the combination of I and U, the combination of I and A, and the combination of I and C. The combinations of bases other than those that form wobble base pairs include 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 C, the combination of G and G, and the combination of C and C.

[0083] "Insertion of a nucleic acid residue" means that a nucleic acid residue is inserted between nucleic acid residues.

[0084] "Deletion of a nucleic acid residue" means that a nucleic acid residue is deleted.

[0085] "Insertion of a linker" means that a linker is inserted between nucleic acid residues. Linkers are as described above in the modification of the phosphate moiety. The linker may be bound to phosphate groups at both ends, for example. That is, the linker may be bound to the phosphate group at the 3'-position of the nucleic acid residue on the 5'-side and the phosphate group at the 5'-position of the nucleic acid residue on the 3'-side. Insertion of a linker may be combined with deletion of a nucleic acid residue, for example. That is, for example, a nucleic acid residue may be replaced with a linker. That is, the nucleic acid residues on both ends of the deleted nucleic acid residue may be linked, for example, directly or via a linker. In other words, deletion of a nucleic acid residue may or may not be accompanied by insertion of a linker. "Deletion of a nucleic acid residue accompanied by insertion of a linker" means replacement of a nucleic acid residue with a linker. "Deletion of a nucleic acid residue without insertion of a linker" means deletion of a nucleic acid residue other than replacement of the nucleic acid residue with a linker. The insertion of a linker may also be combined with a destabilizing moiety other than the deletion of a nucleic acid residue, such as the insertion of a nucleic acid residue. That is, for example, the linker and the nucleic acid residue linked thereto may be inserted together.

[0086] "Linker deletion" means that the linker is deleted. Linker deletion can be selected when the first base sequence contains a linker. Linker deletion may be combined with, for example, the insertion of a nucleic acid residue. That is, for example, the linker may be replaced 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 linker deletion" means the substitution of a linker with a nucleic acid residue. "Insertion of a nucleic acid residue without linker deletion" means the insertion of a nucleic acid residue other than the substitution of a linker with a nucleic acid residue. Furthermore, linker insertion may 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 linked thereto may be deleted together.

[0087] "Linker substitution" means that a linker is replaced with a different type of linker. Linker substitution can be selected when the first base sequence contains a linker. The substituted linker may, for example, be of a different length from the linker before substitution (i.e., longer or shorter than the linker before substitution).

[0088] Any of the destabilizing moieties exemplified above may function as a destabilizing moiety on its own. However, when two or more destabilizing moieties are used in combination, each of the destabilizing moieties constituting the combination may or may not function as a destabilizing moiety on its own, as long as the combination functions as a destabilizing moiety as a whole.

[0089] The destabilizing portion may be a single destabilizing portion, or a combination of two or more destabilizing portions. The destabilizing portion may, for example, include each of the destabilizing portions exemplified above, or may not include any of the destabilizing portions exemplified above. The destabilizing portion may, for example, include one or more destabilizing portions selected from the destabilizing portions exemplified above. Specific examples of the destabilizing portion include 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 linker substitution, or a combination thereof. The phrase "the destabilizing portion includes a certain destabilizing portion A" means that at least a destabilizing portion A is selected as the destabilizing portion, and includes cases where the destabilizing portion consists of a destabilizing portion A and a combination of a destabilizing portion A and another destabilizing portion. For example, the phrase "the destabilizing portion includes an abasic portion" means that at least an abasic portion is selected as the destabilizing portion, and includes cases where the destabilizing portion consists of an abasic portion and a combination of abasic portion and another destabilizing portion. Furthermore, "the destabilizing portion includes a certain destabilizing portion A" means that, when the number or number of sets of destabilizing portions A is specified (here, indefinite articles (e.g., a and an) are not considered to specify the number or number of sets), at least a destabilizing portion A is selected as a destabilizing portion, and the number or number of sets of destabilizing portions A included in the destabilizing portion is limited to the specified number or number of sets. For example, "the destabilizing portion includes one abasic portion" means that at least an abasic portion is selected as a destabilizing portion, and the number of abasic portions included in the destabilizing portion is limited to one, and includes cases where the destabilizing portion consists of one abasic portion and cases where the destabilizing portion consists of a combination of one abasic portion and another destabilizing portion.

[0090] The destabilizing portion may or may not include, for example, any of the destabilizing portions exemplified above. The destabilizing portion may or may not include, for example, an abasic portion. The destabilizing portion may or may not include, for example, a mismatch portion. The destabilizing portion may or may not include, for example, an insertion of a nucleic acid residue. The destabilizing portion may or may not include, for example, a deletion of a nucleic acid residue. The destabilizing portion may or may not include, for example, an insertion of a linker. The destabilizing portion may or may not include, for example, a deletion of a linker. The destabilizing portion may or may not include, for example, a substitution of a linker.

[0091] The manner in which the destabilizing moiety is introduced (e.g., type, number, number of sets, and introduction position) is not particularly limited as long as it improves the efficiency of producing the target oligonucleic acid. The manner in which the destabilizing moiety is introduced can be appropriately determined depending on various conditions, such as the structure of the substrate oligonucleic acid.

[0092] The "number of destabilizing portions" refers to the number of nucleic acid residues and linkers corresponding to the destabilizing portions contained in the destabilizing base sequence. The number of selected destabilizing portions (e.g., any one or more of the destabilizing portions exemplified above) relative to the total number of destabilizing portions may 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%.

[0093] "The number of sets of destabilizing portions" refers to the number of sets of destabilizing portions contained in a destabilizing base sequence. "A set of destabilizing portions" refers to one destabilizing portion that is not contiguous with the same type of destabilizing portion, or two or more contiguous destabilizing portions of the same type. With regard to a "set of destabilizing portions," abasic portions are considered to be the same type, mismatch portions are considered to be the same type, nucleic acid residue insertions are considered to be the same type, nucleic acid residue deletions are considered to be the same type, linker insertions are considered to be the same type, linker deletions are considered to be the same type, and linker substitutions are considered to be the same type. "Two or more consecutive linker insertions, linker deletions, or linker substitutions" refers to two or more consecutive nucleic acid residues into which linker insertions, linker deletions, or linker substitutions have been introduced on the 5' side. The number of sets of any selected destabilizing portion (e.g., any one or more of the destabilizing portions exemplified above) relative to the total number of sets of destabilizing portions may 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%.

[0094] The number of sets of destabilizing portions 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, or 15 or more, or 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 compatible combination thereof. The number of sets of destabilizing portions may specifically 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, or 15 to 20. The number of sets of destabilizing portions may specifically be, for example, 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.

[0095] Furthermore, the number of sets of destabilizing moieties (or, for example, the number of sets of destabilizing moieties other than linker insertion, linker deletion, and linker substitution) 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 is not contradictory. The number of sets of destabilizing moieties (or, for example, the number of sets of destabilizing moieties other than linker insertion, linker deletion, and linker substitution) may specifically 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. The number of sets of destabilizing moieties (or, for example, the number of sets of destabilizing moieties other than linker insertion, linker deletion, and linker substitution) may specifically 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 sets of destabilizing moieties (or, e.g., the number of sets of destabilizing moieties other than linker insertions, linker deletions, and linker substitutions) may particularly be 1, 2, or 3. In one embodiment, the number of sets of destabilizing moieties (or, e.g., the number of sets of destabilizing moieties other than linker insertions, linker deletions, and linker substitutions) may, more particularly, be 1 or 2. In one embodiment, the number of sets of destabilizing moieties (or, e.g., the number of sets of destabilizing moieties other than linker insertions, linker deletions, and linker substitutions) may, more particularly, be 3. The number of sets of destabilizing portions (or, for example, the number of sets of destabilizing portions other than linker insertions, linker deletions, and linker substitutions) may be, for example, 5% or more, 10% 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, or 10% or less, or any compatible combination thereof, as a ratio to the length (number of residues) of the first base sequence. Specifically, the number of sets of destabilizing portions (or, for example, the number of sets of destabilizing portions other than linker insertions, linker deletions, and linker substitutions) may be, for example, 5-10%, 10-15%, 15-20%, 20-25%, or 25-30% as a ratio to the length (number of residues) of the first base sequence.The number of sets of destabilizing portions (or, for example, the number of sets of destabilizing portions other than linker insertions, linker deletions, and linker substitutions) may specifically be, for example, 5 to 30%, 5 to 20%, or 5 to 15% as a ratio to the length (number of residues) of the first base sequence.

[0096] For example, if, as a destabilizing portion, one nucleic acid residue at position A of the first base sequence is replaced with a linker, one nucleic acid residue linked to the linker is inserted at position B of the first base sequence, and four consecutive nucleic acid residues not linked to a linker are inserted at position C of the first base sequence (where positions A, B, and C are not adjacent to each other), the number of destabilizing portions is 7 (specifically, the number of deleted nucleic acid residues is 1, the number of inserted nucleic acid residues is 5, and the number of inserted linkers is 2), and the number of sets of destabilizing portions is 5 (specifically, the number of deleted nucleic acid residue sets is 1, the number of inserted nucleic acid residue sets is 2, and the number of inserted linkers sets is 2).

[0097] Unless otherwise specified, the manner in which destabilizing moieties are introduced shall be interpreted according to the following criteria: (1) The number of destabilizing moieties shall be interpreted as being the smallest (but not less than 1); (2) When the number of destabilizing moieties is the same, the number of sets of destabilizing moieties shall be interpreted as being the smallest (but not less than 1); (3) When the number of destabilizing moieties and the number of sets of destabilizing moieties are the same, the position at which the destabilizing moiety is introduced shall be the −X position or the X position (X is a positive integer; however, in the case of insertion of a nucleic acid residue, X may be 0), and the sum of X shall be interpreted as being the smallest.

[0098] Unless otherwise specified, the position at which the destabilizing portion is introduced is specified as a position in the first base sequence. Unless otherwise specified, the position in the first base sequence is indicated by a consecutive number starting from -1, with the 5' side (i.e., the second base sequence side) being negative, and a consecutive number starting from +1, with the 3' side (i.e., the third base sequence side) being positive, based on the junction between the second and third base sequences. The position on the 5' side (i.e., the second base sequence side) based on the junction between the second and third base sequences is also referred to as the "minus 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 junction between the second and third base sequences is also referred to as the "plus position" or "position on the third base sequence side." That is, for example, if the second and third base sequences each have 10 residues, the nucleic acid residue at the 5' end of the second base sequence is at position -10, the nucleic acid residue at the 3' end of the second base sequence is at position -1, the nucleic acid residue at the 5' end of the third base sequence is at position +1, and the nucleic acid residue at the 3' end of the third base sequence is at position +10, and the nucleic acid residues at positions -1 and +1 are linked. In the case of an insertion of a nucleic acid residue, the phrase "the insertion position of the nucleic acid residue is at position -X" (X is a positive integer) means, unless otherwise specified, that the nucleic acid residue is inserted between the nucleic acid residue at position -X and the nucleic acid residue at position -(X+1). In the case of an insertion of a nucleic acid residue, the phrase "the insertion position of the nucleic acid residue is at position +X" (X is a positive integer) means, unless otherwise specified, that the nucleic acid residue is inserted between the nucleic acid residue at position +X and the nucleic acid residue at position +(X+1). In the case of inserting a nucleic acid residue, "the insertion position of the nucleic acid residue is position 0" means, unless otherwise specified, 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 inserting, deleting, or substituting a linker, the position at which the destabilizing moiety is introduced is specified as the position of the nucleic acid residue at which the linker is inserted, deleted, or substituted on the 5' side, unless otherwise specified. That is, for example, "the insertion position of the linker is position +X" (X is a positive integer) means, unless otherwise specified, 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)).

[0099] The destabilizing portion may be introduced, for example, into one or both of the second and third base sequences. When the number of destabilizing portions is two or more, the position at which the destabilizing portion is introduced may be selected independently for each destabilizing portion, unless otherwise specified. The position at which the destabilizing portion is introduced 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 another position. The position at which the destabilizing moiety is introduced may be, for example, positions −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 position at which the destabilizing moiety is introduced may be, for example, positions -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 position at which the destabilizing moiety is introduced may 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 positions of the destabilizing parts are, for example, -25th position, -24th position, -23rd position, -22nd position, -21st position, -20th position, -19th position, -18th position, -17th position, -16th position. -15th, -14th, -13th, -12th, -11th, -10th, -9th, -8th, -7th, -6th, -5th, -4th, -3rd, -2nd, - 1st place, +1st place, +2nd place, +3rd place, +4th place, +5th place, +6th place, +7th place, +8th place, +9th place, +10th place, +11th place, +12th place, +13th place, +14th place, It may be +15th, +16th, +17th, +18th, +19th, +20th, +21st, +22nd, +23rd, +24th, or +25th.

[0100] In the destabilizing 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 destabilizing 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 destabilizing 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.

[0101] In the destabilizing 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 destabilizing 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.

[0102] In the destabilizing 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 destabilizing base sequence, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the nucleic acid residues constituting the third base sequence may remain. In the destabilizing 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.

[0103] In the destabilizing 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 destabilizing 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.

[0104] "Nucleic acid residues remain in the destabilizing base sequence" means that the nucleic acid residues are not substituted with destabilizing moieties. Note that insertion of nucleic acid residues, insertion of linkers, deletion of linkers, and substitution of linkers do not change the original nucleic acid residues, and therefore do not affect the number of remaining nucleic acid residues in the second or third base sequence or the number of remaining consecutive nucleic acid residues in the second or third base sequence.

[0105] The number of basic moieties 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 compatible combination thereof. Specific examples of the number of basic moieties 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. Specific examples of the number of basic moieties 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 basic moieties may particularly be 1, 2, or 3. The number of basic moieties may, more particularly, be 1 or 2. The number of basal sequences may be, for example, 5% or more, 10% 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, or 10% or less, as a ratio to the length (number of residues) of the first base sequence, or a compatible combination thereof. Specific examples of the number of basal sequences may be 5-10%, 10-15%, 15-20%, 20-25%, or 25-30% as a ratio to the length (number of residues) of the first base sequence. Specific examples of the number of basal sequences may be 5-30%, 5-20%, or 5-15% as a ratio to the length (number of residues) of the first base sequence.

[0106] The number of sets of abasic portions 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 compatible combination thereof. Specific examples of the number of sets of abasic portions 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. Specific examples of the number of sets of abasic portions 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 sets of abasic portions may particularly be 1, 2, or 3. The number of sets of abasic portions may, more particularly, be 1 or 2. The number of sets of basal moieties may be, for example, 5% or more, 10% or more, 15% or more, 20% or more, or 25% or more as a ratio to the length (number of residues) of the first base sequence, or 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less, or a compatible combination thereof. Specific examples of the number of sets of basal moieties may be, for example, 5-10%, 10-15%, 15-20%, 20-25%, or 25-30% as a ratio to the length (number of residues) of the first base sequence. Specific examples of the number of sets of basal moieties may be, for example, 5-30%, 5-20%, or 5-15% as a ratio to the length (number of residues) of the first base sequence.

[0107] The number of abasic sites constituting one set of abasic sites 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 any compatible combination thereof. The number of abasic sites constituting one set of abasic sites may specifically be, for example, 1 to 2, 2 to 3, 3 to 4, or 4 to 5. The number of abasic sites constituting one set of abasic sites may specifically be, for example, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. The number of abasic sites constituting one set of abasic sites may particularly be 1 or 2. The number of abasic sites constituting one set of abasic sites may more particularly be 1. When the number of sets of abasic sites is 2 or more, the number of abasic sites constituting one set of abasic sites may be selected independently for each set of abasic sites, unless otherwise specified.

[0108] When the number of abasic portions is two or more, the position at which each abasic portion is introduced may be selected independently for each abasic portion unless otherwise specified. The position at which the abasic portion is introduced may be, for example, the position at which the destabilizing portion is introduced, as exemplified above. The position at which the abasic portion is introduced may be, in particular, between positions −1 and +1, or may be any other position. In one embodiment, a first abasic portion may be introduced at position −1 or +1, and optionally, a second abasic portion may be introduced at a position not adjacent to the first abasic nucleic acid (e.g., positions −5 to −7 or +5 to +7).

[0109] The number of mismatching portions 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 compatible combination thereof. The number of mismatching portions may specifically 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. The number of mismatching portions may specifically 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 mismatching portions may particularly be 1, 2, or 3. The number of mismatching portions may, more particularly, be 1. The number of mismatches, as a ratio to the length (number of residues) of the first base sequence, may be, for example, 5% or more, 10% 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, or 10% or less, or any compatible combination thereof. Specifically, the number of mismatches, as a ratio to the length (number of residues) of the first base sequence, may be, for example, 5 to 10%, 10 to 15%, 15 to 20%, 20 to 25%, or 25 to 30%. Specifically, the number of mismatches, as a ratio to the length (number of residues) of the first base sequence, may be, for example, 5 to 30%, 5 to 20%, or 5 to 15%.

[0110] The number of sets of mismatching portions 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 compatible combination thereof. The number of sets of mismatching portions may specifically 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. The number of sets of mismatching portions may specifically 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 sets of mismatching portions may particularly be 1, 2, or 3. The number of sets of mismatching portions may, more particularly, be 1. The number of sets of mismatch portions may be, for example, 5% or more, 10% or more, 15% or more, 20% or more, or 25% or more, as a ratio to the length (number of residues) of the first base sequence, or 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less, or any compatible combination thereof. Specifically, the number of sets of mismatch portions may be, for example, 5-10%, 10-15%, 15-20%, 20-25%, or 25-30% as a ratio to the length (number of residues) of the first base sequence. Specifically, the number of sets of mismatch portions may be, for example, 5-30%, 5-20%, or 5-15% as a ratio to the length (number of residues) of the first base sequence.

[0111] The number of mismatching portions constituting one set of mismatching portions 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 any combination thereof that is not contradictory. The number of mismatching portions constituting one set of mismatching portions may specifically be, for example, 1 to 2, 2 to 3, 3 to 4, or 4 to 5. The number of mismatching portions constituting one set of mismatching portions may specifically be, for example, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. The number of mismatching portions constituting one set of mismatching portions may particularly be 1 or 2. The number of mismatching portions constituting one set of mismatching portions may, more particularly, be 1. When the number of sets of mismatching portions is 2 or more, the number of mismatching portions constituting one set of mismatching portions may be selected independently for each set of mismatching portions, unless otherwise specified.

[0112] When the number of mismatching sites is two or more, the position at which the mismatching site is introduced may be selected independently for each mismatching site, unless otherwise specified. The position at which the mismatching site is introduced may be, for example, the position at which the destabilizing site is introduced, as exemplified above. The position at which the mismatching site is introduced may particularly be between positions -2 and +2. The position at which the mismatching site is introduced may more particularly be between positions -1 and +1.

[0113] 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 any compatible combination thereof. The number of inserted nucleic acid residues may specifically 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. The number of inserted nucleic acid residues may specifically 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 inserted nucleic acid residues may specifically 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, as a ratio to the length (number of residues) of the first base sequence, 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 250% 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 compatible combination thereof.The number of inserted nucleic acid residues may be, for example, 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%, 90 to 100%, 100 to 110%, 110 to 120%, 120 to 130%, 130 to 140%, 140 to 150%, 150 to 170%, 170 to 200%, 200 to 250%, or 250 to 300% as a ratio to the length (number of residues) of the first base sequence. The number of inserted nucleic acid residues may be, for example, 5 to 300%, 5 to 200%, 5 to 150%, 5 to 100%, or 5 to 50% as a ratio to the length (number of residues) of the first base sequence.

[0114] The number of sets of insertions 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, 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 compatible combination thereof. The number of sets of insertions of nucleic acid residues may specifically 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. The number of sets of insertions of nucleic acid residues may specifically 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 sets of insertions of nucleic acid residues may particularly be 1, 2, or 3. The number of sets of insertions of nucleic acid residues may, more particularly, be 2. The set number of inserted nucleic acid residues may be, for example, 5% or more, 10% 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, or 10% or less, as a ratio to the length (number of residues) of the first base sequence, or a compatible combination thereof. Specific examples of the set number of inserted nucleic acid residues may be, for example, 5-10%, 10-15%, 15-20%, 20-25%, or 25-30% as a ratio to the length (number of residues) of the first base sequence. Specific examples of the set number of inserted nucleic acid residues may be, for example, 5-30%, 5-20%, or 5-15% as a ratio to the length (number of residues) of the first base sequence.

[0115] 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 compatible combination thereof. The number of nucleic acid residue insertions constituting one set of nucleic acid residue insertions may particularly be 2 or more. The number of nucleic acid residue insertions constituting one set of nucleic acid residue insertions may specifically 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. The number of nucleic acid residue insertions constituting one set of nucleic acid residue insertions may specifically 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 nucleic acid residue insertions may particularly be 1, 2, 3, 4, 5, 6, 7, or 8. When the number of sets of nucleic acid residue insertions is two or more, the number of nucleic acid residue insertions constituting one set of nucleic acid residue insertions may be selected independently for each set of nucleic acid residue insertions, unless otherwise specified.

[0116] When the number of nucleic acid residue insertions is two or more, the insertion position of each nucleic acid residue may be independently selected unless otherwise specified. The insertion position of the nucleic acid residue may be, for example, the insertion position of the destabilizing moiety exemplified above. The insertion position of the nucleic acid residue may particularly be positions -3 to -6 or +3 to +6. The insertion position of the nucleic acid residue may more particularly be positions -5 or +5.

[0117] When the number of inserted nucleic acid residues is two or more, the type of base of the inserted nucleic acid residue may be selected independently for each nucleic acid residue unless otherwise specified. The type of base of the inserted nucleic acid residue is not particularly limited. The base of the inserted nucleic acid residue may be any base, or may be abasic. When inserting two or more consecutive nucleic acid residues (hereinafter also referred to as "consecutively inserted residues"), some or all of the consecutively inserted residues may be self-complementary. For example, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the nucleic acid residues constituting the consecutively inserted residues may be self-complementary. For example, at least the 5'-terminal nucleic acid residue and the 3'-terminal nucleic acid residue of the consecutively inserted residues may or may not be self-complementary. For example, one, two, three, four, or more nucleic acid residues from the 5'-end of the consecutively inserted residues and one, two, three, four, or more nucleic acid residues from the 3'-end may be self-complementary. Specifically, for example, when the consecutively inserted residues are TTGAA, 80% (= 4 residues / 5 residues) of the nucleic acid residues constituting the consecutively inserted residues are self-complementary, and the two nucleic acid residues from the 5'-end and the two nucleic acid residues from the 3'-end of the consecutively inserted residues are self-complementary. Some or all of the consecutively inserted residues may form a double-stranded structure based on self-complementarity, for example. The inserted nucleic acid residues (particularly the consecutively inserted residues) are also referred to as a "loop" or "loop structure."

[0118] The number of deleted nucleic acid residues (i.e., the number of deleted 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 any compatible combination thereof. The number of deleted nucleic acid residues may specifically 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. The number of deleted nucleic acid residues may specifically 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 deleted nucleic acid residues may particularly be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. The number of deleted nucleic acid residues may, more particularly, be 1, 2, or 3. The number of deleted nucleic acid residues 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, 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 a compatible combination thereof, as a ratio to the length (number of residues) of the first base sequence. Specifically, the number of deleted nucleic acid residues 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%. Specifically, the number of deleted nucleic acid residues may be, for example, 5 to 80%, 5 to 50%, 5 to 30%, or 5 to 20% as a ratio to the length (number of residues) of the first base sequence.

[0119] The number of sets of deleted nucleic acid residues 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 any combination thereof that is consistent. The number of sets of deleted nucleic acid residues may specifically be, for example, 1 to 2, 2 to 3, or 3 to 4. The number of sets of deleted nucleic acid residues may specifically be, for example, 1 to 4, 1 to 3, 1 to 2, or 1. The number of sets of deleted nucleic acid residues may particularly be 1, 2, or 3. In one embodiment, the number of sets of deleted nucleic acid residues may more particularly be 1. In one embodiment, the number of sets of deleted nucleic acid residues may more particularly be 2. The number of sets of deleted nucleic acid residues may be, for example, 5% or more, 10% 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, or 10% or less, as a ratio to the length (number of residues) of the first base sequence, or any combination thereof that is consistent. The set number of deleted nucleic acid residues may be, for example, 5 to 10%, 10 to 15%, 15 to 20%, 20 to 25%, or 25 to 30% as a ratio to the length (number of residues) of the first base sequence. The set number of deleted nucleic acid residues may be, for example, 5 to 30%, 5 to 20%, or 5 to 15% as a ratio to the length (number of residues) of the first base sequence.

[0120] The number of deleted nucleic acid residues constituting one set of deleted 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 compatible combination thereof. Specifically, the number of deleted nucleic acid residues constituting one set of deleted 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. The number of deleted nucleic acid residues constituting one set of deleted nucleic acid residues may specifically 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 deleted nucleic acid residues constituting one set of deleted nucleic acid residues may particularly be 1, 2, 3, 4, 5, 6, 7, or 8. The number of deleted nucleic acid residues constituting one set of deleted nucleic acid residues may, more particularly, be 1, 2, or 3. When the number of deleted nucleic acid residue sets is two or more, the number of deleted nucleic acid residues constituting one set of deleted nucleic acid residues may be selected independently for each set of deleted nucleic acid residues, unless otherwise specified.

[0121] When the number of nucleic acid residue deletions is two or more, the position for introducing the nucleic acid residue deletions may be selected independently for each nucleic acid residue deletion, unless otherwise specified. The position for introducing the nucleic acid residue deletions may be, for example, the position for introducing the destabilizing moiety exemplified above. The position for introducing the nucleic acid residue deletions may be, in particular, positions −3 to −6 or +3 to +6.

[0122] The number of linker insertions, deletions, or substitutions (i.e., the number of inserted, deleted, or substituted 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 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 compatible combination thereof. The number of linker insertions, deletions, or substitutions may specifically 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. The number of linker insertions, deletions, or substitutions may specifically 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 linker insertions, deletions, or substitutions may particularly be 1, 2, or 3. The number of linker insertions, deletions, or substitutions may, more particularly, be 1. The number of linker insertions, deletions, or substitutions may be, for example, 5% or more, 10% 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, or 10% or less, as a ratio to the length (number of residues) of the first base sequence, or any compatible combination thereof. Specific examples of the number of linker insertions, deletions, or substitutions may be, for example, 5-10%, 10-15%, 15-20%, 20-25%, or 25-30% as a ratio to the length (number of residues) of the first base sequence. Specific examples of the number of linker insertions, deletions, or substitutions may be, for example, 5-30%, 5-20%, or 5-15% as a ratio to the length (number of residues) of the first base sequence.

[0123] The number of sets of linker insertions, deletions, or substitutions 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 compatible combination thereof. The number of sets of linker insertions, deletions, or substitutions may specifically 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. The number of sets of linker insertions, deletions, or substitutions may specifically 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 sets of linker insertions, deletions, or substitutions may particularly be 1, 2, or 3. The number of sets of linker insertions, deletions, or substitutions may, more particularly, be 1. The set number of linker insertions, deletions, or substitutions may be, for example, 5% or more, 10% 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, or 10% or less, as a ratio to the length (number of residues) of the first base sequence, or any compatible combination thereof. Specific examples of the set number of linker insertions, deletions, or substitutions may be, for example, 5-10%, 10-15%, 15-20%, 20-25%, or 25-30% as a ratio to the length (number of residues) of the first base sequence. Specific examples of the set number of linker insertions, deletions, or substitutions may be, for example, 5-30%, 5-20%, or 5-15% as a ratio to the length (number of residues) of the first base sequence.

[0124] The number of linker insertions, deletions, or substitutions constituting one set of linker insertions, deletions, or substitutions 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 any compatible combination thereof. The number of linker insertions, deletions, or substitutions constituting one set of linker insertions, deletions, or substitutions may specifically be, for example, 1 to 2, 2 to 3, 3 to 4, or 4 to 5. The number of linker insertions, deletions, or substitutions constituting one set of linker insertions, deletions, or substitutions may specifically be, for example, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. The number of linker insertions, deletions, or substitutions constituting one set of linker insertions, deletions, or substitutions may particularly be 1 or 2. The number of linker insertions, deletions, or substitutions constituting one set of linker insertions, deletions, or substitutions may, more particularly, be 1. When the number of sets of linker insertions, deletions, or substitutions is 2 or more, the number of linker insertions, deletions, or substitutions constituting one set of linker insertions, deletions, or substitutions may be selected independently for each set of linker insertions, deletions, or substitutions, unless otherwise specified.

[0125] When the number of linker insertions, deletions, or substitutions is two or more, the position for introducing the linker insertions, deletions, or substitutions may be selected independently for each linker insertion, deletion, or substitution unless otherwise specified. The position for introducing the linker insertions, deletions, or substitutions may be, for example, the position for introducing the destabilizing portion exemplified above.

[0126] The destabilizing moiety may, for example, comprise an insertion of a nucleic acid residue without the deletion of a linker and / or a deletion of a nucleic acid residue without the insertion of a linker.

[0127] The destabilizing portion may comprise, for example, an insertion of two or more consecutive nucleic acid residues (i.e., a set of nucleic acid residue insertions consisting of two or more nucleic acid residue insertions) and / or a deletion of two or more consecutive nucleic acid residues (i.e., a set of nucleic acid residue deletions consisting of two or more nucleic acid residue deletions).

[0128] The destabilizing portion may include, for example, (A) the following: (A) A combination of an abasic portion and deletion and / or insertion of nucleic acid residues.

[0129] The abasic moiety in the destabilizing portion (A) may be one abasic moiety or two or more abasic moieties. The abasic moiety in the destabilizing portion (A) may be, for example, one abasic moiety at positions −1 to +1, or a combination of one abasic moiety at positions −1 to +1 and one or more abasic moieties at positions other than positions −1 to +1. The abasic moiety in the destabilizing portion (A) may particularly be one abasic moiety at positions −1 to +1.

[0130] The destabilizing portion may include, for example, the following (B): (B) A combination of a mismatch portion and a deletion and / or insertion of a nucleic acid residue.

[0131] The mismatch portion in the destabilizing portion (B) may be one mismatch portion or two or more mismatch portions. The mismatch portion in the destabilizing portion (B) may be, for example, one mismatch portion at positions −1 to +1, or a combination of one mismatch portion at positions −1 to +1 and one or more mismatch portions at positions other than positions −1 to +1. The mismatch portion in the destabilizing portion (B) may particularly be one mismatch portion at positions −1 to +1.

[0132] 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 positions -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 sets of nucleic acid residues at positions other than positions -1 to +1. The deletion of nucleic acid residues in the destabilizing portion (A) or (B) may specifically be, for example, the deletion of one or two sets of nucleic acid residues at positions other than positions -1 to +1.

[0133] The insertion of nucleic acid residues in the destabilizing portion (A) or (B) may be, for example, the insertion of one or more nucleic acid residues at positions other than the -1 to +1 positions. The insertion of nucleic acid residues in the destabilizing portion (A) or (B) may be, for example, the insertion of one or more sets of nucleic acid residues at positions other than the -1 to +1 positions. The insertion of nucleic acid residues in the destabilizing portion (A) or (B) may specifically be, for example, the insertion of one or two sets of nucleic acid residues at positions other than the -1 to +1 positions. The insertion of nucleic acid residues in the destabilizing portion (A) or (B) may particularly be the insertion of two sets of nucleic acid residues at positions other than the -1 to +1 positions. The insertion of nucleic acid residues in the destabilizing portion (A) or (B) may more particularly be a combination of the insertion of one set of nucleic acid residues at minus positions other than the -1 position and the insertion of one set of nucleic acid residues at plus positions other than the +1 position.

[0134] The destabilizing portion may comprise, for example, (a), (b), or (c) of the following: (a) an abasic portion at positions −1 to +1 in combination with deletions and / or insertions of nucleic acid residues at positions other than positions −1 to +1; (b) a mismatch portion at positions −1 to +1 in combination with deletions and / or insertions of nucleic acid residues at positions other than positions −1 to +1; (c) a mismatch portion at positions −1 to +1.

[0135] The destabilizing portion (a) may be an example of the destabilizing portion (A), and the destabilizing portion (b) may be an example of the destabilizing portion (B).

[0136] The destabilizing portion may in particular comprise the destabilizing portion (A), such as the destabilizing portion (a), or the destabilizing portion (B), such as the destabilizing portion (b). The destabilizing portion may more in particular comprise the destabilizing portion (A), such as the destabilizing portion (a).

[0137] The destabilizing portion (a) may be, for example, a combination of one abasic portion at positions -1 to +1 and one or more deletions and / or insertions of one or more nucleic acid residues at positions other than positions -1 to +1. The destabilizing portion (a) may be, for example, a combination of one abasic portion at positions -1 to +1 and one or more deletions and / or insertions of one or more sets of nucleic acid residues at positions other than positions -1 to +1. Specifically, the destabilizing portion (a) may be, for example, a combination of one abasic portion at positions -1 to +1 and one or two deletions and / or insertions of one or two sets of nucleic acid residues at positions other than positions -1 to +1. The destabilizing portion (a) may particularly be a combination of one abasic portion at positions -1 to +1 and one or two insertions of nucleic acid residues at positions other than positions -1 to +1. The destabilizing portion (a) may more particularly be a combination of one abasic portion at positions −1 to +1 and two sets of nucleic acid residue insertions at positions other than positions −1 to +1. The destabilizing portion (a) may more particularly be a combination of one abasic portion at positions −1 to +1 and one set of nucleic acid residue insertions at minus positions other than position −1 and one set of nucleic acid residue insertions at plus positions other than position +1.

[0138] The destabilizing portion (b) may be, for example, a combination of one mismatch portion at positions -1 to +1 and one or more deletions and / or one or more insertions of nucleic acid residues at positions other than positions -1 to +1. The destabilizing portion (b) may be, for example, a combination of one mismatch portion at positions -1 to +1 and one or more deletions and / or one or more insertions of nucleic acid residues at positions other than positions -1 to +1. Specifically, the destabilizing portion (b) may be, for example, a combination of one mismatch portion at positions -1 to +1 and one or two deletions and / or one or two insertions of nucleic acid residues at positions other than positions -1 to +1. The destabilizing portion (b) may particularly be a combination of one mismatch portion at positions -1 to +1 and one or two insertions of nucleic acid residues at positions other than positions -1 to +1. The destabilizing portion (b) may more particularly be a combination of one mismatch at positions −1 to +1 and two sets of nucleic acid residue insertions at positions other than positions −1 to +1. The destabilizing portion (b) may more particularly be a combination of one mismatch at positions −1 to +1 and one set of nucleic acid residue insertions at minus positions other than position −1 and one set of nucleic acid residue insertions at plus positions other than position +1.

[0139] Examples of positions other than positions -1 to +1 include positions other than positions -1 to +1 selected from the above-exemplified positions for introducing a destabilizing moiety. Particular examples of positions other than positions -1 to +1 include positions -5 to -7, +5 to +7, -3 to -6, and +3 to +6. Examples of negative positions other than position -1 include positions other than position -1 selected from the above-exemplified positions for introducing a destabilizing moiety. Particular examples of negative positions other than position -1 include positions -5 to -7 and -3 to -6. More particular examples of negative positions other than position -1 include positions -3 to -6. Examples of positive positions other than position +1 include positions other than position +1 selected from the above-exemplified positions for introducing a destabilizing moiety. Particular examples of positive positions other than position +1 include positions +5 to +7 and +3 to +6. More particular examples of positive positions other than position +1 include positions +3 to +6.

[0140] In one embodiment, the present invention may exclude cases where the destabilizing portion consists of one abasic portion, a substitution of one linker with a nucleic acid residue, or a substitution of one nucleic acid residue with a linker. In one embodiment, the present invention may exclude cases where the destabilizing portion consists of one abasic portion at position -1, a substitution of one linker between positions -1 and +1 with a nucleic acid residue, or a substitution of one nucleic acid residue at position -1 with a linker. In one embodiment, the present invention may exclude cases where the number of substrate oligonucleic acids (N) is 2 and the destabilizing portion consists of one abasic portion at position -1, a substitution of one linker between positions -1 and +1 with a nucleic acid residue, or a substitution of one nucleic acid residue at position -1 with a linker. In one aspect, the present invention may exclude cases where the number of substrate oligonucleic acids (N) is 2, the number of complementary oligonucleic acids (M) is 1, and the destabilizing moiety consists of one abasic moiety at −1, one linker between positions −1 and +1 to a nucleic acid residue, or one nucleic acid residue at position −1 to a linker.

[0141] In one embodiment, the present invention may exclude cases in which the destabilizing portion consists of one abasic portion, two abasic portions, or a combination of one abasic portion and one mismatch portion. In one embodiment, the present invention may exclude cases in which the destabilizing portion consists of one abasic portion at position −1, a combination of one abasic portion at position −1 and one abasic portion at a position other than position −1 (e.g., position +4), or a combination of one abasic portion at position −1 and one mismatch portion at a position other than position −1 (e.g., position +4). In one embodiment, the present invention may exclude cases in which the number of substrate oligonucleic acids (N) is 2 and the destabilizing portion consists of one abasic portion at position −1, a combination of one abasic portion at position −1 and one abasic portion at a position other than position −1 (e.g., position +4), or a combination of one abasic portion at position −1 and one mismatch portion at a position other than position −1 (e.g., position +4). In one aspect, the present invention may exclude cases where 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 one abasic portion at position −1, a combination of one abasic portion at position −1 and one abasic portion at a position other than position −1 (e.g., position +4), or a combination of one abasic portion at position −1 and one mismatch portion at a position other than position −1 (e.g., position +4).

[0142] The complementary oligonucleic acid may or may not consist of a destabilizing base sequence. That is, the complementary oligonucleic acid may contain an additional base sequence (hereinafter also referred to as an "additional sequence") on the 5' and / or 3' side of the destabilizing base sequence. The complementary oligonucleic acid may particularly consist of a destabilizing base sequence.

[0143] The length of the additional sequence is not particularly limited as long as it does not impair the object of the present invention.

[0144] The lengths of the 5'- and 3'-additional sequences may both be, for example, 0 or more, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 7 or more, 10 or more, 15 or more, or 20 or more residues, or 25 or less, 20 or less, 15 or less, 10 or less, 7 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less residue, or any compatible combination thereof. The lengths of the 5'- and 3'-additional sequences may both specifically be, for example, 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. The lengths of the 5'- and 3'-additional sequences may both specifically be, for example, 0 to 25 residues, 0 to 15 residues, or 0 to 10 residues.

[0145] The lengths of the 5'- and 3'-additional sequences may each 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 90% 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 5% or less, relative to the total length of the 5'- and 3'-substrate oligonucleic acid, which is taken as 100%, or may be any compatible combination thereof. The lengths of the 5'- and 3'-additional sequences may each be, for example, 0-5%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%, relative to the total length of the 5'- and 3'-substrate oligonucleic acid being 100%. Specifically, the lengths of the 5'- and 3'-additional sequences may each be, for example, 0-100%, 0-50%, or 0-20%, relative to the total length of the 5'- and 3'-substrate oligonucleic acid being 100%.

[0146] The length of the complementary oligonucleic acid may be, for example, 10 or more residues, 11 or more residues, 12 or more residues, 13 or more residues, 14 or more residues, 15 or more residues, 16 or more residues, 17 or more residues, 18 or more residues, 19 or more residues, 20 or more residues, 21 or more residues, 22 or more residues, 23 or more residues, 24 or more residues, 25 or more residues, 30 or more residues, 35 or more residues, 40 or more residues, 45 or more residues, 50 or more residues, 60 or more residues, 70 or more residues, 80 or more residues, 90 or more residues, 100 or more residues, 110 or more residues, 120 or more residues, 130 or more residues, 140 or more residues, 150 or more residues, 160 or more residues, 170 or more residues, 180 or more residues, or 190 or more residues, and may be 200 or more. The number of residues may be 190 or less, 180 or less, 170 or less, 160 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, 45 or less, 40 or less, 35 or less, 30 or less, 25 or less, 24 or less, 23 or less, 22 or less, 21 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, or 11 or less, or any combination thereof that is compatible therewith. The length of the complementary oligonucleic acid is specifically, 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, The length may be 40 to 45 residues, 45 to 50 residues, 50 to 60 residues, 60 to 70 residues, 70 to 80 residues, 80 to 90 residues, 90 to 100 residues, 100 to 110 residues, 110 to 120 residues, 120 to 130 residues, 130 to 140 residues, 140 to 150 residues, 150 to 160 residues, 160 to 170 residues, 170 to 180 residues, 180 to 190 residues, or 190 to 200 residues. The length of the complementary oligonucleic acid may specifically be, for example, 10 to 200 residues, 10 to 100 residues, 15 to 80 residues, or 20 to 60 residues.

[0147] The length of the complementary oligonucleic acid 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 It may be 350% or more, 400% or less, 350% or less, 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 compatible combination thereof. Specifically, the length of the complementary oligonucleic acid may be, for example, 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%, 90 to 100%, 100 to 110%, 110 to 120%, 120 to 130%, 130 to 140%, 140 to 150%, 150 to 170%, 170 to 200%, 200 to 250%, 250 to 300%, 300 to 350%, or 350 to 400% of the total length of the 5'- and 3'-substrate oligonucleic acid, taken as 100%. Specifically, the length of the complementary oligonucleic acid may be, for example, 5 to 400%, 5 to 300%, 5 to 200%, 5 to 150%, 5 to 100%, 5 to 50%, 5 to 20%, 20 to 150%, 20 to 100%, 20 to 50%, 50 to 150%, or 50 to 100%, relative to the sum of the lengths of the 5'- and 3'-substrate oligonucleic acids being 100%.

[0148] The length of the complementary oligonucleic acid 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, relative to the length of the target oligonucleic acid, which is taken as 100%. or 400% or less, 350% or less, 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 compatible combination thereof. Specifically, the length of the complementary oligonucleic acid may be, for example, 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%, 90 to 100%, 100 to 110%, 110 to 120%, 120 to 130%, 130 to 140%, 140 to 150%, 150 to 170%, 170 to 200%, 200 to 250%, 250 to 300%, 300 to 350%, or 350 to 400%, relative to the length of the target oligonucleic acid being 100%. Specifically, the length of the complementary oligonucleic acid may be, for example, 5 to 400%, 5 to 300%, 5 to 200%, 5 to 150%, 5 to 100%, 5 to 50%, 5 to 20%, 20 to 150%, 20 to 100%, 20 to 50%, 50 to 150%, or 50 to 100%, relative to the length of the target oligonucleic acid being 100%.

[0149] The terms "the complementary oligonucleic acid comprises a modified nucleic acid residue," "the complementary oligonucleic acid is modified," and "the complementary oligonucleic acid has a modification" may be used interchangeably.

[0150] Each complementary oligonucleic acid may or may not contain modified nucleic acid residues. That is, each complementary oligonucleic acid may or may not be modified. Modification of nucleic acid residues is as described above. Each complementary oligonucleic acid may have one type of modification, or two or more types of modifications in combination. Each complementary oligonucleic acid may or may not contain, for example, the modifications exemplified above. Each nucleic acid residue constituting each complementary oligonucleic acid may have one type of modification, or two or more types of modifications in combination. Only a portion of the nucleic acid residues constituting each complementary oligonucleic acid may be modified nucleic acid residues, or all of the nucleic acid residues constituting each complementary oligonucleic acid may be modified nucleic acid residues. For example, when a complementary oligonucleic acid contains a deletion of a nucleic acid residue as a destabilizing moiety, a linker may be inserted at the position of the deletion of the nucleic acid residue.

[0151] The ratio of modified nucleic acid residues in a complementary oligonucleic acid (i.e., the ratio of the number of modified nucleic acid residues to the number of nucleic acid residues constituting the complementary oligonucleic acid) 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, 90% or more, or 95% or more, or 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, or any compatible combination thereof. The ratio of modified nucleic acid residues in a complementary oligonucleic acid may 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%. The ratios of modified nucleic acid residues in the complementary oligonucleic acid exemplified above can also be applied independently to nucleic acid residues having any selected modification (e.g., any of the modifications exemplified above). That is, for example, the ratio of nucleic acid residues having a modification at the phosphate moiety in a complementary oligonucleic acid (i.e., the ratio of the number of nucleic acid residues having a modification at the phosphate moiety to the number of nucleic acid residues constituting the complementary oligonucleic acid) may be set to the ratios of modified nucleic acid residues in the complementary oligonucleic acid exemplified above. Furthermore, for example, the ratio of nucleic acid residues having a sugar moiety modification in a complementary oligonucleic acid (i.e., the ratio of the number of nucleic acid residues having a sugar moiety modification to the number of nucleic acid residues constituting the complementary oligonucleic acid) may be set to the ratio of modified nucleic acid residues in the complementary oligonucleic acid exemplified above.Furthermore, for example, the ratio of nucleic acid residues having a base moiety modification in a complementary oligonucleic acid (i.e., the ratio of the number of nucleic acid residues having a base moiety modification to the number of nucleic acid residues constituting the complementary oligonucleic acid) may be set to the ratio of modified nucleic acid residues in the complementary oligonucleic acid exemplified above.

[0152] The number of modified nucleic acid residues in the complementary oligonucleic acid may be, for example, 0 or more, 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, 80 or more, 100 or more, 120 or more, 140 or more, 160 or more, or 180 or more; or 200 or less, 180 or less, 160 or less, 140 or less, 120 or less, 100 or less, 80 or less, 60 or less, 50 or less, 40 or less, 30 or less, 25 or less, 20 or less, 15 or less, 10 or less, or 5 or less, or any compatible combination thereof. The number of modified nucleic acid residues in a complementary oligonucleic acid may 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. The number of modified nucleic acid residues in the complementary oligonucleic acid exemplified above can also be applied independently to nucleic acid residues having any selected modification (e.g., any of the modifications exemplified above). That is, for example, the number of nucleic acid residues having a modification at the phosphate moiety in a complementary oligonucleic acid may be set to the number of modified nucleic acid residues in the complementary oligonucleic acid exemplified above. Furthermore, for example, the number of nucleic acid residues having modifications in the sugar moiety in the complementary oligonucleic acid may be set to the number of modified nucleic acid residues in the complementary oligonucleic acid exemplified above, and the number of nucleic acid residues having modifications in the base moiety in the complementary oligonucleic acid may be set to the number of modified nucleic acid residues in the complementary oligonucleic acid exemplified above.

[0153] In the ligation step, M (M is an integer of 1 or more) complementary oligonucleic acids are used. The number (M) of complementary oligonucleic acids is 1 or more. "The number of complementary oligonucleic acids" means the number of complementary oligonucleic acids used in the ligation step. The number (M) of complementary oligonucleic acids may be 1 or 2 or more.

[0154] When the number of linking portions (N-1) is 2 or more, the complementary oligonucleic acid can be selected independently for each of those linking portions. In other words, when the number of linking portions (N-1) is 2 or more, the destabilizing portion can be selected independently for each of those linking portions (i.e., for each complementary oligonucleic acid corresponding to those linking portions). The destabilizing portion may or may not be the same for each of those linking portions (i.e., for each complementary oligonucleic acid corresponding to those linking portions). For example, the destabilizing portion may be selected independently for each of those linking portions (i.e., for each complementary oligonucleic acid corresponding to those linking portions) from the destabilizing portion embodiments exemplified above. Specifically, the destabilizing portion may include, for example, the destabilizing portion (A) (i.e., a combination of an abasic portion and deletion and / or insertion of nucleic acid residues) such as the destabilizing portion (a) (i.e., a combination of an abasic portion at positions −1 to +1 and deletion and / or insertion of nucleic acid residues at positions other than positions −1 to +1), the destabilizing portion (B) (i.e., a combination of a mismatch portion and deletion and / or insertion of nucleic acid residues) such as the destabilizing portion (b) (i.e., a combination of a mismatch portion at positions −1 to +1 and deletion and / or insertion of nucleic acid residues at positions other than positions −1 to +1), or the destabilizing portion (c) (i.e., a mismatch portion at positions −1 to +1). Alternatively, for example, any of the destabilizing portions exemplified above may be selected as the destabilizing portion of a complementary oligonucleic acid corresponding to one of the linking portions, and another of the destabilizing portions exemplified above may be selected as the destabilizing portion of a complementary oligonucleic acid corresponding to another linking portion. Specifically, for example, the destabilizing portion of a complementary oligonucleic acid corresponding to one of the linking portions may contain the destabilizing portion (a) or (b), and the destabilizing portion of a complementary oligonucleic acid corresponding to another linking portion may contain the destabilizing portion (c). In particular, the destabilizing portion of a complementary oligonucleic acid corresponding to one of the linking portions may contain the destabilizing portion (a), and the destabilizing portion of a complementary oligonucleic acid corresponding to another linking portion may contain the destabilizing portion (c).

[0155] Furthermore, one complementary oligonucleic acid may be used at each linking site, or two or more complementary oligonucleic acids may be used. That is, the linkage between the substrate oligonucleic acids on the 5' and 3' sides at each linking site may be carried out in the presence of one or more complementary oligonucleic acids corresponding to the linking site. Furthermore, one complementary oligonucleic acid may correspond to two or more linking sites. In other words, one complementary oligonucleic acid may be used commonly for linking the substrate oligonucleic acids on the 5' and 3' sides at two or more linking sites. When one complementary oligonucleic acid corresponds to two or more linking sites, the complementary oligonucleic acid may contain a destabilizing base sequence corresponding to each of the linking sites. For example, when three substrate oligonucleic acids A, B, and C are linked from 5' to 3' in the presence of one complementary oligonucleic acid, the complementary oligonucleic acid may contain, from 5' to 3', a destabilizing base sequence for linking B and C and a destabilizing base sequence for linking A and B. In this case, the third base sequence for the destabilizing base sequence for linking B and C and the second base sequence for the destabilizing base sequence for linking A and B may or may not overlap, partially or completely.

[0156] The number of complementary oligonucleic acids (M) may be smaller than, the same as, or larger than the number of linking portions (N-1). For example, when one complementary oligonucleic acid is used independently at each linking portion, the number of complementary oligonucleic acids (M) may be the same as the number of linking portions (N-1). Furthermore, for example, when one complementary oligonucleic acid corresponds to two or more linking portions, the number of complementary oligonucleic acids (M) may be smaller than the number of linking portions (N-1). When the number of complementary oligonucleic acids (M) is two or more, the destabilizing portion can be independently selected for each of the complementary oligonucleic acids. The destabilizing portion may or may not be the same for each of the complementary oligonucleic acids. For example, the destabilizing portion may be independently selected from the destabilizing portion embodiments exemplified above for each of the complementary oligonucleic acids. Specifically, for example, the destabilizing portion may independently include the destabilizing portion (a), (b), or (c) for each of the complementary oligonucleic acids. Furthermore, for example, any of the above-exemplified destabilizing portions may be selected as the destabilizing portion of one of the complementary oligonucleic acids, and another of the above-exemplified destabilizing portions may be selected as the destabilizing portion of the other complementary oligonucleic acid. Specifically, for example, the destabilizing portion of one of the complementary oligonucleic acids may contain the destabilizing portion (a) or (b), and the destabilizing portion of the other complementary oligonucleic acid may contain the destabilizing portion (c). In particular, the destabilizing portion of one of the complementary oligonucleic acids may contain the destabilizing portion (a), and the destabilizing portion of the other complementary oligonucleic acid may contain the destabilizing portion (c).

[0157] The method for producing a complementary oligonucleic acid is not particularly limited. A complementary oligonucleic acid can be produced, for example, by sequentially linking nucleic acid residues constituting the complementary oligonucleic acid. Linking of nucleic acid residues can be carried out, for example, by the phosphoramidite method. Alternatively, a complementary oligonucleic acid can be produced, for example, by linking nucleic acid fragments constituting the complementary oligonucleic acid. Linking of nucleic acid fragments can be carried out, for example, enzymatically using complementary chains. Linking of nucleic acid fragments may be carried out, for example, by the method of the present invention.

[0158] <4> Ligation Step The ligation step is a step of enzymatically ligating a set of substrate oligonucleic acids. The term "ligation of a set of substrate oligonucleic acids" may be used interchangeably with the term "ligation of substrate oligonucleic acids constituting a set."

[0159] "Enzymatically ligating a set of substrate oligonucleic acids" means that the set of substrate oligonucleic acids is ligated by a ligase. In the ligation step, for example, the 5' and 3' substrate oligonucleic acids may hybridize with their corresponding complementary oligonucleic acids to form a double-stranded structure, and then the 5' and 3' substrate oligonucleic acids may be ligated by a ligase. The ligase is not particularly limited as long as it can ligate substrate oligonucleic acids in the presence of complementary oligonucleic acids. Examples of the ligase include DNA ligase and RNA ligase. Examples of the ligase include DNA ligase. The origin of the ligase is not particularly limited. The ligase may be derived from eukaryotes such as mammals or yeast, prokaryotes such as E. coli, or viruses such as phages. Examples of DNA ligases include T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, and Taq DNA ligase. Examples of RNA ligases include T4 RNA ligases such as T4 RNA ligase 2 and RtcB ligase. In one embodiment, the present invention may exclude cases where the ligation enzyme is T4 DNA ligase. More specifically, examples of ligation enzymes include T3 DNA ligase. DNA ligases may catalyze the ligation between DNAs or modified nucleic acids thereof. Thus, DNA ligases may be used, for example, to ligate between DNAs or modified nucleic acids thereof (i.e., ligation between a 5'-side substrate oligonucleic acid whose 3'-terminus is DNA or a modified nucleic acid and a 3'-side substrate oligonucleic acid whose 5'-terminus is DNA or a modified nucleic acid). Furthermore, certain DNA ligases, such as T3 DNA ligase and T4 DNA ligase, may catalyze the ligation between RNAs or modified nucleic acids in addition to the ligation between DNAs or modified nucleic acids. Therefore, certain DNA ligases such as T3 DNA ligase and T4 DNA ligase may be used, for example, to ligate RNA or modified nucleic acids thereof (i.e., ligation of a 5'-end substrate oligonucleic acid whose 3'-end is RNA or modified nucleic acid to a 3'-end substrate oligonucleic acid whose 5'-end is RNA or modified nucleic acid). RNA ligase may catalyze ligation between RNA or modified nucleic acids.Therefore, RNA ligase may be used, for example, to ligate RNA or modified nucleic acids thereof (i.e., to ligate a 5'-end substrate oligonucleic acid whose 3'-end is RNA or modified nucleic acid to a 3'-end substrate oligonucleic acid whose 5'-end is RNA or modified nucleic acid).

[0160] The ligation step can be carried out in a liquid. The liquid in which the ligation step is carried out is also referred to as a "reaction solution." Specifically, the ligation step can be carried out by allowing a ligation enzyme, a substrate oligonucleic acid, and a complementary oligonucleic acid to coexist in a reaction solution. The ligation step can be carried out, for example, in a batch system or a column system. In the batch system, the ligation step can be carried out, for example, by mixing the ligation enzyme, the substrate oligonucleic acid, and the complementary oligonucleic acid in a reaction solution in a container. The ligation step can be carried out stationary or with stirring or shaking. In the column system, the ligation step can be carried out, for example, by passing a reaction solution containing the substrate oligonucleic acid and the complementary oligonucleic acid through a column filled with an immobilized ligation enzyme. An aqueous medium such as water or a buffer solution can be used as the reaction solution. In addition to the ligation enzyme, the substrate oligonucleic acid, and the complementary oligonucleic acid, the reaction solution may contain components useful or necessary for the ligation step. Such components include a pH buffer, Mg ions, ATP, and DTT.

[0161] The conditions for carrying out the ligation step (e.g., pH of the reaction solution, reaction temperature, reaction time, and amounts and concentrations of various components used) are not particularly limited as long as the target oligonucleic acid is produced. The conditions for carrying out the ligation step can be appropriately set depending on various conditions such as the structure of the substrate oligonucleic acid, the structure of the complementary oligonucleic acid, and the type of ligation enzyme. Unless otherwise specified, the "concentration of a certain component in the reaction solution" may refer to the maximum concentration of that component in the reaction solution during the ligation step. The maximum concentration of a certain component in the reaction solution during the ligation step may be, for example, the concentration of that component in the reaction solution at the start of the ligation step.

[0162] The pH of the reaction solution may be, for example, 5 to 10, 6 to 9, or 7 to 8.

[0163] The reaction temperature may be, for example, 5°C or higher, 10°C or higher, 15°C or higher, 20°C or higher, 25°C or higher, 30°C or higher, 35°C or higher, 40°C or higher, 45°C or higher, 50°C or higher, or 55°C or higher, or 90°C or lower, 80°C or lower, 70°C or lower, 60°C or lower, 55°C or lower, 50°C or lower, 45°C or lower, 40°C or lower, 35°C or lower, 30°C or lower, 25°C or lower, 20°C or lower, 15°C or lower, or 10°C or lower, or a compatible combination thereof.Specific examples of the reaction temperature may be, for example, 5 to 10°C, 10 to 15°C, 15 to 20°C, 20 to 25°C, 25 to 30°C, 30 to 35°C, 35 to 40°C, 40 to 45°C, 45 to 50°C, 50 to 55°C, or 55 to 60°C. 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). Heating may or may not be performed in the linking step. The fluctuation range of the reaction temperature throughout the linking step may be, for example, 30°C or less, 25°C or less, 20°C or less, 15°C or less, 10°C or less, or 5°C or less.

[0164] The reaction time may be, for example, 0.5 hours or more, 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, 9 hours or more, 12 hours or more, 15 hours or more, 18 hours or more, 24 hours or more, or 30 hours or more, or 36 hours or less, 30 hours or less, 24 hours or less, 18 hours or less, 15 hours or less, 12 hours or less, 9 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, or 1 hour or less, or a compatible combination thereof. Specific examples of the reaction time include 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. Specific examples of the reaction time include 1 to 36 hours, 3 to 30 hours, or 6 to 24 hours.

[0165] The amounts of the 5'- and 3'-substrate oligonucleic acids used may be substantially the same in terms of molar ratio, for example, so that the substrate oligonucleic acids are linked without excess or deficiency. "The amounts of the 5'- and 3'-substrate oligonucleic acids used are substantially the same in terms of molar ratio" may mean that the smaller of the amounts of the 5'- and 3'-substrate oligonucleic acids used is 90% or more, 95% or more, 97% or more, or 99% or more of the other in terms of molar ratio, and also includes cases where the amounts of the 5'- and 3'-substrate oligonucleic acids used are the same in terms of molar ratio. The molar concentrations of the 5'- and 3'-substrate oligonucleic acids in the reaction solution may be substantially the same, for example, so that the substrate oligonucleic acids are linked without excess or deficiency. The phrase "the molar concentrations of the 5'- and 3'-substrate oligonucleic acids in the reaction solution are substantially the same" may mean that the smaller of the molar concentrations of the 5'- and 3'-substrate oligonucleic acids is 90% or more, 95% or more, 97% or more, or 99% or more of the other, and also includes cases where the molar concentrations of the 5'- and 3'-substrate oligonucleic acids are the same.

[0166] The concentration of each substrate oligonucleic acid in the reaction solution may be, for example, 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, 500 μM or more, 1000 μM or more, 2000 μM or more, or 5000 μM or more, or 10,000 μM or less, 5000 μM or less, 2000 μM or less, 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, or 2 μM or less, or any compatible combination thereof. The concentration of each substrate oligonucleic acid in the reaction solution may 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. The concentration of each substrate oligonucleic acid in the reaction solution may be, for example, 1 to 10,000 μM, 10 to 10,000 μM, 20 to 1,000 μM, or 100 to 500 μM.

[0167] The amount of complementary oligonucleic acid used may be less than the smaller of the amount of the 5'-side substrate oligonucleic acid used and the amount of the 3'-side substrate oligonucleic acid used, in molar ratio. The amount of complementary oligonucleic acid used may be, for example, 0.01% or more, 0.02% or more, 0.05% or more, 0.1% or more, 0.2% or more, 0.5% or more, 1% or more, 2% or more, 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, or 40% or more in molar ratio, or 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, 5% or less, 2% or less, 1% or less, 0.5% or less, 0.2% or less, 0.1% or less, 0.05% or less, or 0.02% or less, or any compatible combination thereof, where the smaller of the amount of 5'-side substrate oligonucleic acid used and the amount of 3'-side substrate oligonucleic acid used is taken as 100%. The amount of complementary oligonucleic acid used may be, for example, 0.01 to 0.02%, 0.02 to 0.05%, 0.05 to 0.1%, 0.1 to 0.2%, 0.2 to 0.5%, 0.5 to 1%, 1 to 2%, 2 to 5%, 5 to 10%, 10 to 15%, 15 to 20%, 20 to 30%, 30 to 40%, or 40 to 50% by molar ratio, where the smaller of the amount of 5' substrate oligonucleic acid used and the amount of 3' substrate oligonucleic acid used is taken as 100%. The amount of complementary oligonucleic acid used may be, for example, 0.01 to 50%, 0.1 to 50%, 1 to 50%, 2 to 30%, or 5 to 15% by molar ratio, where the smaller of the amount of 5' substrate oligonucleic acid used and the amount of 3' substrate oligonucleic acid used is taken as 100%.

[0168] The molar concentration of the complementary oligonucleic acid in the reaction solution may be lower than the smaller of the molar concentration of the 5' substrate oligonucleic acid in the reaction solution and the molar concentration of the 3' substrate oligonucleic acid in the reaction solution. The molar concentration of the complementary oligonucleic acid in the reaction solution may be, for example, 0.01% or more, 0.02% or more, 0.05% or more, 0.1% or more, 0.2% or more, 0.5% or more, 1% or more, 2% or more, 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, or 40% or more, or 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, 5% or less, 2% or less, 1% or less, 0.5% or less, 0.2% or less, 0.1% or less, 0.05% or less, or 0.02% or less, or any compatible combination thereof, where the smaller of the molar concentration of the 5'-side substrate oligonucleic acid in the reaction solution and the molar concentration of the 3'-side substrate oligonucleic acid in the reaction solution is taken as 100%. Specifically, the molar concentration of the complementary oligonucleic acid in the reaction solution may be, for example, 0.01 to 0.02%, 0.02 to 0.05%, 0.05 to 0.1%, 0.1 to 0.2%, 0.2 to 0.5%, 0.5 to 1%, 1 to 2%, 2 to 5%, 5 to 10%, 10 to 15%, 15 to 20%, 20 to 30%, 30 to 40%, or 40 to 50%, where the smaller of the molar concentration of the 5' substrate oligonucleic acid in the reaction solution and the molar concentration of the 3' substrate oligonucleic acid in the reaction solution is taken as 100%. Specifically, the molar concentration of the complementary oligonucleic acid in the reaction solution may be, for example, 0.01 to 50%, 0.1 to 50%, 1 to 50%, 2 to 30%, or 5 to 15%, where the smaller of the molar concentration of the 5' substrate oligonucleic acid in the reaction solution and the molar concentration of the 3' substrate oligonucleic acid in the reaction solution is taken as 100%.

[0169] The concentration of the complementary oligonucleic acid in the reaction solution may 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 more, 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, or any compatible combination thereof. The concentration of the complementary oligonucleic acid in the reaction solution may 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. The concentration of the complementary oligonucleic acid in the reaction solution may 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.

[0170] The concentration of the ligating enzyme 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 more, or 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 a compatible combination thereof.Specific examples of the concentration of the ligating enzyme 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. The concentration of the ligating enzyme in the reaction solution may be, specifically, for example, 5 to 1000 U / μL, 10 to 500 U / μL, or 20 to 200 U / μL.

[0171] The activity of a ligase may be defined, for example, as follows:

[0172] That is, for example, with regard to a ligase, the amount of enzyme that ligates 50% of 100 ng of HindIII-digested λ DNA in a reaction buffer of appropriate composition (e.g., 66 mM Tris-HCl, 10 mM MgCl, 1 mM ATP, 1 mM DTT, 7.5% Polyethylene glycol (PEG 6000), pH 7.6 (at 25°C), total reaction volume 20 μl) at 25°C for 1 minute may be defined as 1 unit [U].

[0173] For example, ligation enzymes are used in a reaction buffer of an appropriate composition (e.g., 66 mM Tris-HCl, 10 mM MgCl, 1 mM ATP, 1 mM DTT, 7.5% Polyethylene glycol (PEG 6000), pH 7.6 (at 25°C)) at 25°C for 1 minute, with 2 × 10 -5 The amount of enzyme that ligates nmol of substrate oligonucleic acid in the presence of a sufficient amount (for example, a molar concentration 10 times that of the substrate oligonucleic acid) of complementary oligonucleic acid may be defined as 1 unit [U].

[0174] After the start of the ligation step, a ligation enzyme, substrate oligonucleic acid, complementary oligonucleic acid, and / or other components may be additionally supplied to the reaction solution, either alone or in any combination. These components may be supplied once, multiple times, or continuously. Furthermore, the reaction conditions may be uniform from the start to the end of the ligation step, or may change during the ligation step. "Reaction conditions change during the ligation step" does not necessarily mean that the reaction conditions change over time, but also includes that the reaction conditions change spatially. An example of a case where the reaction conditions change spatially is when the ligation step is performed in a column system, and the reaction conditions, such as the reaction temperature and ligation enzyme concentration, vary depending on the position in the flow path.

[0175] When three or more substrate oligonucleic acids are ligated (i.e., when the number of linkages is two or more), the order and timing of ligation of the substrate oligonucleic acids are not particularly limited. When three or more substrate oligonucleic acids are ligated, the ligation of the substrate oligonucleic acids may be performed simultaneously or sequentially, for example. "Performing the ligation of three or more substrate oligonucleic acids simultaneously" may mean starting the ligation of all linkages of the substrate oligonucleic acids simultaneously. "Performing the ligation of three or more substrate oligonucleic acids sequentially" may mean starting the ligation of some linkages of the substrate oligonucleic acids, and starting the ligation of the remaining linkages while the ligation of those linkages is in progress or after completion. That is, when three or more substrate oligonucleic acids are ligated, the ligation of the substrate oligonucleic acids may be started simultaneously or sequentially, for example. For example, when three substrate oligonucleic acids A, B, and C are ligated in the 5' to 3' direction, A, B, and C may be ligated simultaneously, A and B may be ligated in advance and then the conjugate of A and B may be ligated to C, or B and C may be ligated in advance and then the conjugate of B and C may be ligated to A. Specifically, for example, when three substrate oligonucleic acids A, B, and C are ligated in the 5' to 3' direction, ligation of A, B, and C may be initiated simultaneously, ligation of A and B may be initiated in advance and then ligation of B and C may be initiated while ligation of A and B is in progress or after completion (including ligation of the conjugate of A and B to C depending on the degree of progress of ligation of A and B), or ligation of B and C may be initiated in advance and then ligation of B and A (including ligation of the conjugate of B and C to A depending on the degree of progress of ligation of B and C) may be initiated while ligation of B and C is in progress or after completion. For example, by initiating the ligation step in the presence of complementary oligonucleic acids for ligating A, B, and C, A and B, and B and C, ligation of A, B, and C can be initiated simultaneously, and ligation of A, B, and C can be carried out simultaneously.Alternatively, for example, by initiating the ligation step in the absence of C and / or the absence of a complementary oligonucleic acid for linking B and C, and then supplying C and / or a complementary oligonucleic acid for linking B and C to the reaction solution after the initiation of the ligation step, it is possible to initiate the ligation of A and B in advance, and then initiate the ligation of B and C (including the ligation of the conjugate of A and B to C, depending on the degree of progress of the ligation of A and B) after the ligation of A and B is underway or is completed, thereby allowing the ligation of A, B, and C to be carried out sequentially. Alternatively, for example, by initiating the ligation step in the absence of A and / or the absence of a complementary oligonucleic acid for linking A and B, and then supplying A and / or a complementary oligonucleic acid for linking A and B to the reaction solution after the initiation of the ligation step, it is possible to initiate the ligation of B and C in advance, and then initiate the ligation of B and A (including the ligation of the conjugate of B and C to A, depending on the degree of progress of the ligation of B and C) after the ligation of B and C is underway or is completed, thereby allowing the ligation of A, B, and C to be carried out sequentially.

[0176] When three or more substrate oligonucleic acids are ligated, the ligation of the substrate oligonucleic acids as a whole may be considered to be an implementation of the method of the present invention. For example, when three substrate oligonucleic acids A, B, and C are ligated in the 5' to 3' direction, the ligation of A, B, and C (including the ligation of the ligated product of B and C to A and the ligation of the ligated product of A and B to C, depending on the degree of progress of the ligation step) may be considered to be an implementation of the method of the present invention in which A, B, and C are used as substrate oligonucleic acids.

[0177] When three or more substrate oligonucleic acids are ligated, the ligation of those substrate oligonucleic acids may independently correspond to the implementation of the method of the present invention for each ligation at each ligation site. For example, when three substrate oligonucleic acids A, B, and C are ligated in the 5' to 3' direction, the ligation of A and B (including the ligation of A and the conjugate of B and C, depending on the progress of the ligation step) may correspond to the implementation of the method of the present invention using A and B as substrate oligonucleic acids, and the ligation of B and C (including the ligation of C and the conjugate of A and B, depending on the progress of the ligation step) may correspond to the implementation of the method of the present invention using B and C as substrate oligonucleic acids.

[0178] By carrying out the ligation step in this manner, the target oligonucleic acid is produced, and thus a reaction solution containing the target oligonucleic acid is obtained.

[0179] The amount of target oligonucleic acid produced may be greater in molar ratio than the amount of complementary oligonucleic acid used. That is, by repeatedly using the complementary oligonucleic acid catalytically in the ligation step, a target oligonucleic acid may be produced in a molar ratio greater than the amount of complementary oligonucleic acid used. For example, the amount of target oligonucleic acid produced may be greater than 100%, 110% or more, 120% or more, 130% or more, 150% or more, 170% or more, 200% or more, 250% or more, or 300% or more, assuming that the amount of complementary oligonucleic acid used is 100%. The "amount of complementary oligonucleic acid used" to be compared may refer to the amount of complementary oligonucleic acid used at any ligation site when the number of ligation sites (N-1) is 2 or more. When the number of linkages (N-1) is 2 or more, the "amount of complementary oligonucleic acids used" to be compared may be, for example, the minimum amount of complementary oligonucleic acids used at those linkage sites, the maximum amount of complementary oligonucleic acids used at those linkage sites, or the average amount of complementary oligonucleic acids used at those linkage sites. For any linkage site, the "amount of complementary oligonucleic acids used" at that linkage site to be compared may mean the total amount of complementary oligonucleic acids used when the number of complementary oligonucleic acids used at that linkage site is 2 or more.

[0180] For any ligation site, the amount of ligation product produced at that ligation site may be greater in molar ratio than the amount of complementary oligonucleic acid used at that ligation site. That is, for any ligation site, the complementary oligonucleic acid may be catalytically repeatedly used in the ligation step, thereby producing a ligation product in a molar ratio greater than the amount of complementary oligonucleic acid used. For any ligation site, the amount of ligation product produced at that ligation site may be, for example, greater than 100%, 110% or more, 120% or more, 130% or more, 150% or more, 170% or more, 200% or more, 250% or more, or 300% or more, based on the amount of complementary oligonucleic acid used at that ligation site being 100%. For any ligation site, the "amount of ligation product produced" at that ligation site may refer to the total amount of all ligation products produced through ligation at that ligation site when the number of linkages (N-1) is 2 or more. For example, when three substrate oligonucleic acids A, B, and C are ligated in the 5' to 3' direction, the "amount of ligation product produced at the ligation site between A and B" may mean the total amount of the ligation product between A and B and the ligation product between A, B, and C. For any ligation site, the "amount of complementary oligonucleic acid used" at the ligation site to be compared may mean the total amount of complementary oligonucleic acids used when two or more complementary oligonucleic acids are used at the ligation site.

[0181] The production of the target oligonucleic acid can be confirmed, for example, by known techniques used for detecting or identifying compounds. Such techniques include HPLC, LC / MS, GC / MS, and NMR. These techniques may be used alone, or two or more may be used in appropriate combination. When a complementary oligonucleic acid hybridizes to the target oligonucleic acid, they may or may not be separated as appropriate. Separation can be performed, for example, by heating. The heating temperature may be, for example, 40°C or higher, 50°C or higher, or 60°C or higher, or 100°C or lower, 90°C or lower, or 80°C or lower, or a combination thereof. The target oligonucleic acid can be recovered from the reaction solution as appropriate. Recovery of the target oligonucleic acid can be performed, for example, by known techniques used for separating and purifying compounds. Such techniques include chromatography such as ion exchange chromatography, reverse-phase chromatography, affinity chromatography, and gel filtration chromatography; electrophoresis such as polyacrylamide gel electrophoresis (PAGE); and membrane purification. These techniques may be used alone or in combination of two or more. The target oligonucleic acid may be purified to a desired degree. Furthermore, the target oligonucleic acid may be modified, for example, to obtain the modification form of the target oligonucleic acid exemplified above.

[0182] The present invention will be explained in more detail below with reference to non-limiting examples. Unless otherwise specified, the nucleic acid residues constituting the oligonucleic acids used in the examples are unmodified DNA residues.

[0183] Example 1 Ligation Reaction of Oligonucleotides Using Complementary Strands with an Abasic Region or with an Abasic Region and a Loop Structure Five complementary strands were used: one with only an abasic region introduced, three with an abasic region and a loop structure (three patterns in which the loop structure was inserted at the 5' end, the 3' end, or both), and one control, a completely complementary strand without any destabilizing region ( FIG. 1A and Table 1 ). The abasic region was introduced at the residue complementary to the linking region, and the loop structure was introduced at a position distant from the linking region.

[0184] A ligation reaction of substrate oligonucleic acids (5'-end substrate and 3'-end substrate) was carried out using each complementary strand, and the production of products (oligonucleic acids in which the 5'-end substrate and the 3'-end substrate are ligated) was compared.

[0185] The reaction mixture consisted of 20 μM of each substrate, 2 μM of 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 was incubated at 25°C. Aliquots of the reaction mixture were collected after 1, 3, and 16 hours and diluted with 10 mM EDTA to terminate the reaction.

[0186] The product was quantified by HPLC using an ACQUITY UPLC OST C18 Column (1.7 μm, 2.1 × 50 mm). The HPLC conditions were: column temperature 60°C, detection wavelength 254 nm, injection volume 10 μL, flow rate 0.4 mL / min, mobile phase A containing 100 mM hexafluoroisopropanol, 8 mM triethylamine, and 0.004% phosphoric acid, and eluent B containing 10% methanol, with a linear gradient as shown in Table 2. A product sample was similarly analyzed to confirm the enzymatic reaction yielded the desired product. The amount of product was calculated from the ratio of the product peak area to the sum of the substrate and product peak areas in the LC chart.

[0187] The results are shown in Figure 1B. The amount of product after 16 hours of reaction was 2.0 μM when the control perfectly complementary strand was used. The introduction of an abasic segment increased the amount to 7.8 μM, and the insertion of a loop structure in addition to the introduction of an abasic segment further increased the amount to 9.9-12.3 μM. This confirms that complementary strands incorporating destabilizing segments such as abasic segments and loop structures possess catalytic activity (i.e., can be repeatedly utilized in oligonucleotide ligation reactions). Furthermore, it was confirmed that the combination of an abasic segment and a loop structure is effective in improving the efficiency of oligonucleotide ligation reactions.

[0188]

[0189]

[0190] Example 2: Ligation reaction of oligonucleotides with modified sugar moieties The catalytic activity of complementary strands with destabilizing moieties introduced into oligonucleotides modified at the 2' position of the sugar moiety was examined. Two types of oligonucleotides were used as the 5'-end substrate and the 3'-end substrate, respectively, in which one or five sugar residues were substituted with 2'-O-methoxyethyl (Table 3). Three types of complementary strands were used: a control perfectly complementary strand, a complementary strand with an abasic moiety introduced, and a complementary strand with an abasic moiety and a loop structure introduced (Table 3).

[0191] Ligation reactions of substrate oligonucleotides (5'- and 3'-terminal substrates) were carried out using each complementary strand, and the production of products (oligonucleotides in which the 5'- and 3'-terminal substrates were ligated) was compared. The reaction mixture consisted of 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. The product was quantified after 0.25, 1, 4, and 24 hours using the HPLC conditions described in Example 1, and the product yield was calculated. In this example, the molar concentration of the complementary strand was 1 / 10 of the molar concentration of each substrate, so the maximum yield was 10% assuming that each complementary strand was used in a single ligation reaction.

[0192] The results are shown in Figure 2. When the control perfectly complementary strand was used, the yield was less than 10% after 24 hours of reaction for both substrate patterns. In contrast, when complementary strands with an abasic moiety or with an abasic and loop structure were used, yields of 38-51% were obtained after 24 hours of reaction. This confirms the catalytic activity of complementary strands with destabilizing moieties, such as abasic and loop structures, even in the ligation reaction of sugar-modified oligonucleotides. After 4 hours of reaction, the yields were 21% (when the substrate had two sugar modifications) and 13% (when the substrate had 10 sugar modifications) when complementary strands with an abasic moiety were used, whereas the yields were 30% (when the substrate had two sugar modifications) and 29% (when the substrate had 10 sugar modifications) when complementary strands with an abasic and loop structure were used. That is, it was confirmed that the combination of an abasic portion and a loop structure is effective in improving the efficiency of the ligation reaction of oligonucleic acids, even in the ligation reaction of oligonucleic acids with modified sugar portions.

[0193]

[0194] Example 3: Ligation Reaction of Phosphate-Modified Oligonucleotides The catalytic activity of complementary strands containing destabilizing moieties was examined for oligonucleotides containing modified phosphate moieties. OligoDNAs in which all internucleotide phosphate ester bonds were replaced with thiophosphate ester bonds (PS-DNA) were used as the 5'-end and 3'-end substrates, respectively (Table 4). Four types of complementary strands were used: a control perfectly complementary strand, a complementary strand containing an abasic portion, a complementary strand containing a loop structure, and a complementary strand containing both an abasic portion and a loop structure (Table 4).

[0195] Ligation reactions of substrate oligonucleotides (5'- and 3'-terminal substrates) were carried out using each complementary strand, and the production of products (oligonucleotides in which the 5'- and 3'-terminal substrates were ligated) was compared. The reaction mixture consisted of 20 μM of each substrate, 2 μM complementary strand, 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 1 hour, 3 hours, and 24 hours using the HPLC conditions described in Example 1, and the product yield was calculated. In this example, the molar concentration of the complementary strand was 1 / 10 of the molar concentration of each substrate, so the maximum yield was 10% assuming that each complementary strand was used in a single ligation reaction.

[0196] The results are shown in Figure 3. The yield after 24 hours was 13% when the control perfect complementary chain was used, but improved to 43% when the complementary chain with an abasic moiety was used and to 24% when the complementary chain with a loop structure was used. In other words, the catalytic action of complementary chains with destabilizing moieties such as abasic moieties and loop structures was confirmed even in the ligation reaction of oligonucleotides with modified phosphate moieties. The yield after 24 hours was further improved to 50% when the complementary chain with an abasic moiety and loop structure was used. In other words, it was confirmed that the combination of an abasic moiety and a loop structure is effective in improving the efficiency of the ligation reaction of oligonucleotides, even in the ligation reaction of oligonucleotides with modified phosphate moieties.

[0197]

[0198] Example 4: Ligation reaction of oligonucleotides using a complementary strand with a loop structure inserted The catalytic activity of a complementary strand with a loop structure inserted was examined. The position for inserting the loop structure was selected to be six residues away from the ligation site on the 5' or 3' side (position -6 or +6) (Table 5). The loop structure consisted of four nucleic acid residues.

[0199] Ligation reactions of substrate oligonucleotides (5'- and 3'-terminal substrates) were carried out using each complementary strand, and the production of products (oligonucleotides in which the 5'- and 3'-terminal substrates were ligated) was compared. The reaction mixture consisted of 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. The product was quantified after 1, 3, and 16 hours using the HPLC conditions described in Example 1, and the product yield was calculated. In this example, the molar concentration of the complementary strand was 1 / 10 of the molar concentration of each substrate, so the maximum yield was 10% assuming that each complementary strand was used in a single ligation reaction.

[0200] The results are shown in Figure 4. Regardless of the position of the loop structure inserted into the complementary strand, the yield increased with increasing reaction time. The yield after 16 hours was 3.8% when the control perfectly complementary strand was used, 10.7% when the complementary strand with the loop structure inserted at the 5' end was used, and 6.7% when the complementary strand with the loop structure inserted at the 3' end was used. This confirms the catalytic activity of the complementary strand with a destabilizing portion such as a loop structure introduced.

[0201]

[0202] Example 5: Ligation Reaction of Oligonucleotides Using Complementary Strands with Nucleic Acid Residue Deletions The catalytic activity of complementary strands with nucleic acid residue deletions was examined. Seven complementary strands were used: a control perfectly complementary strand; three complementary strands (#3 to #1) with deletions of residues 4 to 6, 5 to 6, or 6 on the 5' side of the junction; and three complementary strands (#6 to #4) with deletions of residues 4 to 6, 5 to 6, or 6 on the 3' side of the junction (Table 6 and Figure 5A). Deletion of nucleic acid residues in the complementary strand can result in the formation of a loop structure in the substrate when the complementary strand hybridizes with the substrate (Figure 5A).

[0203] Ligation reactions of substrate oligonucleotides (5'- and 3'-terminal substrates) were carried out using each complementary strand, and the production of products (oligonucleotides in which the 5'- and 3'-terminal substrates were ligated) was compared. The reaction mixture consisted of 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. The product was quantified after 1, 3, and 16 hours using the HPLC conditions described in Example 1, and the product yield was calculated. In this example, the molar concentration of the complementary strand was 1 / 10 of the molar concentration of each substrate, so the maximum yield was 10% assuming that each complementary strand was used in a single ligation reaction.

[0204] The results are shown in Figure 5B. Regardless of the complementary strand containing the deleted nucleic acid residues, the yield after 16 hours was higher than the yield (5.1%) obtained using the control, fully complementary strand. This confirms the catalytic activity of the complementary strand, even when the deletion of nucleic acid residues is used as a destabilizing moiety. When the deletion residue was located 5' from the junction, the yield after 16 hours was highest when one residue was deleted (10.5%) and lowest when three residues were deleted (6.2%). When the deletion residue was located 3' from the junction, the yield after 16 hours was highest when two residues were deleted (14.1%) and lowest when one residue was deleted (9.2%).

[0205]

[0206] Example 6: Examination of the number of nucleic acid residues constituting the loop structure The effect of the number of nucleic acid residues constituting the loop structure on the catalytic activity of the complementary strand was evaluated. The position for inserting the loop structure was selected to be 6 residues away from the 5' or 3' side of the linking site (position -6 or +6), and the number of nucleic acid residues constituting the loop structure was set to 1 to 8 residues (Table 7).

[0207] Ligation reactions of substrate oligonucleotides (5'-end substrate and 3'-end substrate) were carried out using each complementary strand, and the production of products (oligonucleotides in which the 5'-end substrate and 3'-end substrate were ligated) was compared. The reaction mixture consisted of 20 μM of each substrate, 0.2 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. The product was quantified after 16 hours using the HPLC conditions described in Example 1, and the product yield was calculated. In this example, the molar concentration of the complementary strand was 1 / 100 of the molar concentration of each substrate, so the maximum yield was 1% assuming that the complementary strand was used in only one ligation reaction.

[0208] The results are shown in Table 7. When using complementary strands with loop structures of any number of residues, higher yields were obtained than when using the control perfectly complementary strand. In other words, the catalytic action of complementary strands was confirmed regardless of the number of nucleic acid residues constituting the loop structure introduced into the complementary strand. In particular, a high effect was obtained when the number of nucleic acid residues constituting the loop structure was two or more.

[0209]

[0210] Example 7: Examination of the insertion position of a loop structure The effect of the insertion position of a loop structure on the catalytic activity of the complementary strand was evaluated. The positions selected for the insertion of the loop structure were 3 to 6 residues away from the 3' or 5' side of the linking site (positions -3 to -6 or +3 to +6) (Table 8). The number of nucleic acid residues constituting the loop structure was set to 4.

[0211] Ligation reactions of substrate oligonucleotides (5'-end substrate and 3'-end substrate) were carried out using each complementary strand, and the production of products (oligonucleotides in which the 5'-end substrate and 3'-end substrate were ligated) was compared. The reaction mixture consisted of 20 μM of each substrate, 0.2 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. The product was quantified after 16 hours using the HPLC conditions described in Example 1, and the product yield was calculated. In this example, the molar concentration of the complementary strand was 1 / 100 of the molar concentration of each substrate, so the maximum yield was 1% assuming that the complementary strand was used in only one ligation reaction.

[0212] The results are shown in Table 8. Regardless of the insertion position of the loop structure, a higher yield was obtained when using a complementary strand than when using the control perfectly complementary strand. This confirms the catalytic activity of the complementary strand, regardless of the position of the loop structure introduced into the complementary strand. In particular, regardless of whether the loop structure was inserted at the 5' or 3' side of the linker, the highest reaction rate was obtained when the loop structure was inserted at a position five residues away from the linker (position -5 or +5).

[0213]

[0214] Example 8: Examination of the base sequence of nucleic acid residues constituting the loop structure The effect of the base sequence of nucleic acid residues constituting the loop structure on the catalytic activity of the complementary strand was evaluated. The position at which the loop structure was inserted was 4 or 6 residues away from the linkage on the 3' side (+4 or +6 position), and the number of nucleic acid residues constituting the loop structure was 4, with four different base sequences designed for each position (Table 9).

[0215] Ligation reactions of substrate oligonucleotides (5'-end substrate and 3'-end substrate) were carried out using each complementary strand, and the production of products (oligonucleotides in which the 5'-end substrate and 3'-end substrate were ligated) was compared. The reaction mixture consisted of 20 μM of each substrate, 0.2 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. The product was quantified after 16 hours using the HPLC conditions described in Example 1, and the product yield was calculated. In this example, the molar concentration of the complementary strand was 1 / 100 of the molar concentration of each substrate, so the maximum yield was 1% assuming that the complementary strand was used in only one ligation reaction.

[0216] The results are shown in Table 8. When a complementary strand into which a loop structure consisting of any of the base sequences was inserted was used, a higher yield was obtained than when the control perfectly complementary strand was used. In other words, the catalytic action of the complementary strand was confirmed regardless of the base sequence of the nucleic acid residues constituting the loop structure introduced into the complementary strand.

[0217]

[0218] Example 9: Investigation of the number of loop structures The effect of the number of loop structures on the catalytic activity of the complementary strand was evaluated. The number of nucleic acid residues constituting the loop structure was set to four, and complementary strands were designed with one to four loop structures inserted (Table 10).

[0219] Ligation reactions of substrate oligonucleotides (5'- and 3'-terminal substrates) were carried out using each complementary strand, and the production of products (oligonucleotides in which the 5'- and 3'-terminal substrates were ligated) was compared. The reaction mixture consisted of 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. The product was quantified after 1, 3, and 16 hours using the HPLC conditions described in Example 1, and the product yield was calculated. In this example, the molar concentration of the complementary strand was 1 / 10 of the molar concentration of each substrate, so the maximum yield was 10% assuming that each complementary strand was used in a single ligation reaction.

[0220] The results are shown in Figure 6. When complementary strands with one to three loop structures inserted were used, higher yields were obtained after 16 hours than when the control perfectly complementary strand was used, and the amount of product tended to increase as the number of loop structures inserted increased. This confirms the catalytic activity of complementary strands when the number of loop structures introduced into the complementary strand was one to three. It was also confirmed that a combination of two or three loop structures is effective in improving the efficiency of the oligonucleotide ligation reaction. On the other hand, when complementary strands with four loop structures inserted were used, a higher yield was obtained after 16 hours than when the control perfectly complementary strand was used under only one of the four conditions.

[0221]

[0222] Example 10: Examination of the position and number of abasic sites introduced The influence of the position and number of abasic sites introduced on the catalytic activity of the complementary strand was evaluated. Complementary strands were designed with one to three abasic sites introduced (Figure 7A and Table 11).

[0223] Ligation reactions of substrate oligonucleotides (5'- and 3'-terminal substrates) were carried out using each complementary strand, and the production of products (oligonucleotides in which the 5'- and 3'-terminal substrates were ligated) was compared. The reaction mixture consisted of 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. Using the HPLC conditions described in Example 1, the product was quantified after 1, 3, and 16 hours, and the product yield was calculated. In this example, the molar concentration of the complementary strand was 1 / 10 of the molar concentration of each substrate, so the maximum yield was 10% assuming that each complementary strand was used in a single ligation reaction.

[0224] The results are shown in Figure 7B. Complementary strands containing one or two abasic sites tended to produce higher yields than the control perfect complementary strand after 1 hour, and also produced higher yields than the control perfect complementary strand after 3 and 16 hours. This confirms the catalytic activity of complementary strands containing one or two abasic sites. After 1 and 3 hours, complementary strands containing two abasic sites (positions 9-14) produced higher yields than complementary strands containing a single abasic site (position 9). However, after 16 hours, the highest yield was achieved with complementary strands containing a single abasic site (position 9) (Figure 7B). However, the yield decreased when complementary strands containing three abasic sites were used.

[0225]

[0226] Example 11: Ligation reaction of oligonucleotides using complementary strands with mismatched sites introduced The catalytic activity of complementary strands with mismatched sites introduced was examined. The introduction of mismatched base pairs into the ligation site was investigated. The bases (T and G, respectively) in the complementary strand that pair with the base on the 3' side (A, position -1) and the base on the 5' side (C, position +1) of the ligation site were substituted with other bases and evaluated (Table 12, Figure 8A).

[0227] Ligation reactions of substrate oligonucleotides (5'- and 3'-terminal substrates) were carried out using each complementary strand, and the production of products (oligonucleotides in which the 5'- and 3'-terminal substrates were ligated) was compared. The reaction mixture consisted of 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. The product was quantified after 1, 3, and 16 hours using the HPLC conditions described in Example 1, and the product yield was calculated. In this example, the molar concentration of the complementary strand was 1 / 10 of the molar concentration of each substrate, so the maximum yield was 10% assuming that each complementary strand was used in a single ligation reaction.

[0228] The results are shown in Figure 8B. The yield after 16 hours was 4.5% when the control perfectly complementary strand was used, but increased to 5.3-28.8% when the mismatched complementary strand was used (Figure 8B). This indicates that the catalytic activity of the complementary strand was confirmed even when the mismatched complementary strand was selected as the destabilizing site.

[0229]

[0230] Example 12: Study of Substrate Concentration The effects of substrate concentration and complementary strand concentration on the substrate ligation reaction were evaluated. The substrate concentration was set to 20, 100, 300, 500, or 1000 μM. The complementary strand concentration was set to 1 / 10 the molar concentration of the substrate. Two types of complementary strands were used: one with an abasic portion introduced and one with an abasic portion and a loop structure introduced (Table 13).

[0231] Ligation reactions of substrate oligonucleotides (5'-end substrate and 3'-end substrate) were carried out using each complementary strand at various substrate and complementary strand concentrations, and the production of products (oligonucleotides in which the 5'-end substrate and the 3'-end substrate were ligated) was compared. The reaction mixture, excluding the substrate and complementary strand, consisted of 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. The product was quantified after 1, 3, 16, and 40 hours using the HPLC conditions described in Example 1, and the product yield and the product production rate per enzyme activity were calculated. In this example, the molar concentration of the complementary strand was 1 / 10 of the molar concentration of each substrate, so the maximum yield was 10% assuming that the complementary strand was used in only one ligation reaction.

[0232] The results are shown in Figures 9A and 9B. At all complementary strand and substrate concentrations, yields exceeding 10%, the ratio of complementary strand to substrate, were obtained. This confirms the catalytic action of the complementary strand, regardless of complementary strand or substrate concentration. Regardless of the complementary strand used, the maximum yield was obtained at a substrate concentration of 300 μM (Figure 9A). The rate of product production per enzyme increased with increasing substrate concentration (Figure 9B).

[0233]

[0234] Example 13: Production of a 100-residue oligonucleotide by ligation of 50-residue oligonucleotides. The catalytic activity of complementary strands in ligation reactions using two 50-residue substrates was examined. The complementary strands were designed to form double strands in regions of 9 residues on either the 5' or 3' side of the ligation site. An abasic moiety was introduced as a destabilizing moiety into the complementary strand at a position that pairs with the base (A, +1) at the ligation site of the 3'-terminal substrate. A loop structure was introduced into the complementary strand at the 5' end (3 bases upstream) or 3' end (3 bases downstream), or a combination of these destabilizing moieties was introduced. The oligonucleotides used are shown in Table 14.

[0235] Ligation reactions of substrate oligonucleotides (5'-end substrate and 3'-end substrate) were carried out using each complementary strand, and the production of products (oligonucleotides in which the 5'-end substrate and 3'-end substrate were ligated) was compared. The reaction mixture consisted of 20 μM of each substrate, 2 μM complementary strand, 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. After 24 hours, the product was quantified using the HPLC conditions described in Example 1, and the product yield was calculated. In this example, the molar concentration of the complementary strand was 1 / 10 of the molar concentration of each substrate, so the maximum yield was 10% assuming that each complementary strand was used in a single ligation reaction.

[0236] The results are shown in Figure 10. The yield after 24 hours was 9.7% when the control perfectly complementary strand was used and 14.0% when the complementary strand containing an abasic portion was used, whereas it was 57.8% when the complementary strand containing an abasic portion and a 5' loop structure was used, 50.5% when the complementary strand containing an abasic portion and a 3' loop structure was used, and 61.8% when the complementary strand containing an abasic portion and both 5' and 3' loop structures was used. Thus, the catalytic action of the complementary strand and the combined effect of the destabilizing portion were confirmed even in the production of a 100-residue oligonucleotide by ligation of 50-residue oligonucleotides.

[0237]

[0238] Example 14: Production of a 100-residue oligonucleotide by ligation of 50-residue oligonucleotides containing modified nucleic acids. The catalytic activity of complementary strands in ligation reactions was examined using two 50-residue substrates containing 2'-O-methoxyethyl, 2'-O-methyl, 2'-fluoro, 5-methylcytosine, and thiophosphate groups as modified nucleic acids. The complementary strands were designed to form double strands in a region of 9 residues on either the 5' or 3' side of the ligation site. An abasic site was introduced as a destabilizing moiety into the complementary strand at a position paired with the base (A, +1) at the ligation site of the 3'-terminal substrate. A loop structure was introduced into the complementary strand at the 5' end (3 bases upstream) or 3' end (3 bases downstream), or a combination of these destabilizing moieties was introduced. The oligonucleotides used are listed in Table 15.

[0239] Ligation reactions of substrate oligonucleotides (5'-end substrate and 3'-end substrate) were carried out using each complementary strand, and the production of products (oligonucleotides in which the 5'-end substrate and 3'-end substrate were ligated) was compared. The reaction mixture consisted of 20 μM of each substrate, 2 μM complementary strand, 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. After 24 hours, the product was quantified using the HPLC conditions described in Example 1, and the product yield was calculated. In this example, the molar concentration of the complementary strand was 1 / 10 of the molar concentration of each substrate, so the maximum yield was 10% assuming that each complementary strand was used in a single ligation reaction.

[0240] The results are shown in Figure 11. The yield after 24 hours was 7.7% when the control perfectly complementary strand was used and 8.3% when the complementary strand containing an abasic portion was used, whereas it was 72.4% when the complementary strand containing an abasic portion and a 5' loop structure was used, 27.3% when the complementary strand containing an abasic portion and a 3' loop structure was used, and 88.3% when the complementary strand containing an abasic portion and both 5' and 3' loop structures was used. In other words, the catalytic action of the complementary strand was confirmed in the production of a 100-residue oligonucleotide by the ligation reaction of 50-residue oligonucleotides containing modified nucleic acids, and the combined effect of the destabilizing portion was also confirmed.

[0241]

[0242] Example 15: Ligation Reaction of Three Oligonucleotides The catalytic activity of complementary strands in a ligation reaction using three substrates: a 10-residue 5'-end substrate, a 20-residue central substrate, and a 10-residue 3'-end substrate was examined. The complementary strands complementary to the 5'-end substrate and the central substrate were designed to form double strands in a region of 9 residues from the ligation site toward the 5'-end or the central substrate, respectively. An abasic moiety was introduced as a destabilizing moiety into the complementary strand at a position paired with the base (A, +1) at the ligation site of the 3'-end substrate. A loop structure was introduced into the complementary strand at the 5'-end (3 bases upstream) or 3'-end (3 bases downstream), or a combination of these destabilizing moieties was introduced. The complementary strands to the central substrate and the 3'-end substrate were designed to form double strands in a region of 8 residues from the linkage toward the central substrate or 3'-end, respectively, and the residue of the complementary strand that pairs with the base at the linkage of the 5'-end substrate (T, position -1) was mismatched (C) to form a destabilizing portion. The oligonucleotides used are shown in Table 16.

[0243] The ligation reaction of the substrate oligonucleotides (5'-end substrate, central substrate, and 3'-end substrate) was carried out by combining complementary strands corresponding to the two ligation sites, and the production of products (oligonucleotides in which the 5'-end substrate, central substrate, and 3'-end substrate are ligated) was compared. As a control condition, the combination of complementary strands used for both the ligation of the 5'-end substrate and central substrate and the ligation of the central substrate and 3'-end substrate were completely complementary. Four types of destabilizing sites were investigated: an abasic site at the junction between the 5' and central substrates, paired with the base at the junction of the 3' substrate (A, +1 position), a loop site at the 5' end (three bases upstream) of the complementary strand, a mismatch site at the junction between the central and 3' substrates, and a combination of these destabilizing sites. Four different reaction conditions were also investigated. The reaction mixture consisted of 20 μM of each substrate, 2 μM complementary strand, 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). The product yield was calculated by quantifying the product after 72 hours under the HPLC conditions described in Example 1. In this example, the molar concentration of the complementary strand was 1 / 10 of the molar concentration of each substrate, so the maximum yield was 10% assuming that the complementary strand was used in only one ligation reaction.

[0244] The results are shown in Figure 12. In the ligation reaction using the three substrates, the yield also increased as the reaction time was extended. The yield after 72 hours was 1.6% under the control conditions, 19.3% under the conditions where a complementary strand with an abasic site introduced at the ligation between the 5'-end substrate and the central substrate and a mismatch introduced at the ligation between the central substrate and the 3'-end substrate were used, 27.5% under the conditions where a complementary strand with an abasic site and a loop structure (5'-end) introduced at the ligation between the 5'-end substrate and the central substrate and a mismatch introduced at the ligation between the central substrate and the 3'-end substrate were used, and 28.5% under the conditions where a complementary strand with an abasic site and a loop structure (5'-end) introduced at the ligation between the 5'-end substrate and the central substrate and a mismatch introduced at the ligation between the central substrate and the 3'-end substrate were used. The yield was 24.4% when a complementary strand with an abasic region and a loop structure (3'-end) was used to link the terminal substrate and the central substrate, and a complementary strand with a mismatch introduced to link the central substrate and the 3'-end substrate, and the yield was 33.9% when a complementary strand with an abasic region and a loop structure (5'-end and 3'-end) was used to link the 5'-end substrate and the central substrate, and a complementary strand with a mismatch introduced to link the central substrate and the 3'-end substrate. In other words, the catalytic action of the complementary strand was confirmed in the ligation reactions of the three oligonucleotides.

[0245]

[0246] Example 16: Ligation Reaction of Three Oligonucleotides Containing Modified Nucleic Acids The catalytic activity of complementary strands in ligation reactions was examined using three modified nucleic acids: a 10-residue 5'-end substrate containing a 2'-O-methoxyethyl group, a 2'-O-methyl group, a 2'-fluoro group, a 5-methylcytosine group, and a thiophosphate group; a 20-residue central substrate; and a 10-residue 3'-end substrate. The complementary strands complementary to the 5'-end substrate and the central substrate were designed to form double strands in a region of 9 residues from the junction toward the 5'-end or the central substrate, respectively. An abasic moiety was introduced as a destabilizing moiety into the complementary strand at a position paired with the base (A, +1) at the junction of the 3'-end substrate. A loop structure was introduced into the complementary strand at the 5'-end (3 bases upstream) or the 3'-end (3 bases downstream), or a combination of these destabilizing moieties was introduced. The complementary strands to the central substrate and the 3'-end substrate were designed to form double strands in a region of 8 residues from the linkage toward the central substrate or 3'-end, respectively, and the residue of the complementary strand that pairs with the base at the linkage of the 5'-end substrate (T, position -1) was made a mismatch (C) to serve as a destabilizing portion. The oligonucleotides used are shown in Table 17.

[0247] The ligation reaction of the substrate oligonucleotides (5'-end substrate, central substrate, and 3'-end substrate) was carried out by combining complementary strands corresponding to the two ligation sites, and the production of products (oligonucleotides in which the 5'-end substrate, central substrate, and 3'-end substrate are ligated) was compared. As a control condition, the combination of complementary strands used for both the ligation of the 5'-end substrate and central substrate and the ligation of the central substrate and 3'-end substrate were completely complementary. Four types of destabilizing sites were investigated: an abasic site at the junction between the 5' and central substrates, paired with the base at the junction of the 3' substrate (A, +1 position), a loop site at the 5' end (three bases upstream) of the complementary strand, a mismatch site at the junction between the central and 3' substrates, and a combination of these destabilizing sites. Four different reaction conditions were also investigated. The reaction mixture consisted of 20 μM of each substrate, 2 μM complementary strand, 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). The product yield was calculated by quantifying the product after 24 hours under the HPLC conditions described in Example 1. In this example, the molar concentration of the complementary strand was 1 / 10 of the molar concentration of each substrate, so the maximum yield was 10% assuming that the complementary strand was used in only one ligation reaction.

[0248] The results are shown in Figure 13. In the ligation reactions using the three substrates containing modified nucleic acids, the yield also increased as the reaction time was extended. The yield after 24 hours was 1.6% under the control conditions, 13.0% under the conditions in which a complementary strand with an abasic site introduced into the ligation between the 5'-end substrate and the central substrate and a mismatch introduced into the ligation between the central substrate and the 3'-end substrate were used, 22.8% under the conditions in which a complementary strand with an abasic site and a loop structure (5'-end) introduced into the ligation between the 5'-end substrate and the central substrate and a mismatch introduced into the ligation between the central substrate and the 3'-end substrate were used, and 23.8% under the conditions in which a complementary strand with an abasic site and a loop structure (5'-end) introduced into the ligation between the 5'-end substrate and the central substrate and a mismatch introduced into the ligation between the central substrate and the 3'-end substrate were used. The yield was 21.1% when a complementary strand with an abasic region and a loop structure (3'-end) was used to link the terminal substrate and the central substrate, and a complementary strand with a mismatch introduced to link the central substrate and the 3'-end substrate, and 22.5% when a complementary strand with an abasic region and a loop structure (5'-end and 3'-end) was used to link the 5'-end substrate and the central substrate, and a complementary strand with a mismatch introduced to link the central substrate and the 3'-end substrate. In other words, the catalytic action of the complementary strand was confirmed in the ligation reactions of three oligonucleotides, including modified nucleic acids.

[0249]

[0250] Example 17: Ligation Reaction of 20-Residue Oligonucleotides Composed of Modified Nucleic Acids and RNA The catalytic activity of complementary strands in ligation reactions using two 10-residue substrates composed of 2'-O-methyl groups and RNA as modified nucleic acids was examined. The complementary strands were designed to form double strands in a region of 9 residues on either the 5' or 3' side of the linking site. An abasic site was introduced as a destabilizing moiety into the complementary strand at a position paired with the base (A, +1) at the linking site of the 3'-terminal substrate. A loop structure was introduced into the complementary strand at the 5'-terminal (3 bases upstream) or 3'-terminal (3 bases downstream), or a combination of these destabilizing moieties was introduced. The oligonucleotides used are listed in Table 18.

[0251] Ligation reactions of substrate oligonucleotides (5'-end substrate and 3'-end substrate) were carried out using each complementary strand, and the production of products (oligonucleotides in which the 5'-end substrate and 3'-end substrate were ligated) was compared. The reaction mixture consisted of 20 μM of each substrate, 2 μM complementary strand, 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 using the HPLC conditions described in Example 1, and the product yield was calculated. In this example, the molar concentration of the complementary strand was 1 / 10 of the molar concentration of each substrate, so the maximum yield was 10% assuming that the complementary strand was used in only one ligation reaction.

[0252] The results are shown in Figure 14. The yield after 72 hours was 17.5% when the control perfectly complementary strand was used and 18.9% when the complementary strand containing an abasic portion was used, whereas it was 33.0% when the complementary strand containing an abasic portion and a 5' loop structure was used, 20.8% when the complementary strand containing an abasic portion and a 3' loop structure was used, and 11.0% when the complementary strand containing an abasic portion and both 5' and 3' loop structures was used. In other words, the catalytic action of the complementary strand was confirmed in the ligation reaction of oligonucleic acids composed of modified nucleic acids and RNA, and the combined effect of the destabilizing portion was also confirmed.

[0253]

[0254] According to the present invention, oligonucleic acids can be efficiently ligated.

Claims

1. A method for producing target oligonucleotides, The process includes a step of enzymatically linking N strands (where N is an integer of 2 or more) of single-stranded substrate oligonucleotides in the presence of M strands (where M is an integer of 1 or more) of single-stranded complementary oligonucleotides to produce the target oligonucleotide, The linkage between the 5' and 3' sides of the substrate oligonucleotide at each linkage is performed in the presence of one or more complementary oligonucleotides corresponding to the linkage. Each of the complementary oligonucleotides includes a base sequence in which an unstable region 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 linked in the 5' to 3' direction, The second base sequence is either a complementary sequence to the 5' portion of the 3' substrate oligonucleotide, or a complementary sequence to the full-length sequence of the 3' substrate oligonucleotide. The third base sequence is either a complementary sequence to the 3' portion of the 5' substrate oligonucleotide, or a complementary sequence to the full-length sequence of the 5' substrate oligonucleotide. A method wherein the destabilizing portion is a site that reduces the stability of the hybrid formed between each complementary oligonucleotide and the target oligonucleotide.

2. The method according to claim 1, except in the cases (1) to (5) below: (1) When N is 2 and the destabilizing portion consists of one base-free portion at position -1; (2) When N is 2 and the destabilization portion consists of a substitution of a nucleic acid residue of one linker between the -1 and +1 positions; (3) When N is 2 and the destabilization portion consists of the substitution of a single nucleic acid residue at the -1 position into a linker; (4) When N is 2, and the destabilization portion consists of a combination of one base-free portion at position -1 and one base-free portion at position +4; (5) When N is 2, and the destabilization portion consists of a combination of one base-free portion at position -1 and one mismatch portion at position +4.

3. The method according to claim 1 or 2, wherein the destabilized portion includes a base-free portion, a mismatched portion, insertion of a nucleic acid residue, deletion of a nucleic acid residue, insertion of a linker, deletion of a linker, substitution of a linker, or a combination thereof.

4. The method according to claim 1 or 2, wherein the destabilized portion includes the insertion of a nucleic acid residue without linker deletion and / or the deletion of a nucleic acid residue without linker insertion.

5. The method according to claim 1 or 2, wherein the destabilizing portion consists of 1 to 20 sets of destabilizing portions.

6. The method according to claim 1 or 2, wherein the destabilizing portion consists of 1 to 8 sets of destabilizing portions.

7. The method according to claim 1 or 2, wherein the destabilizing portion consists of 1 to 4 sets of destabilizing portions.

8. The method according to claim 1 or 2, wherein the destabilization portion comprises a number of destabilization portions equal to 30% or less of the length of the first base sequence.

9. The method according to claim 1 or 2, wherein the destabilizing portion comprises 1 to 8 base-free portions.

10. The method according to claim 1 or 2, wherein the destabilizing portion comprises one or two base-free portions.

11. The method according to claim 1 or 2, wherein the destabilized portion includes a number of base-free portions that are 30% or less in ratio to the length of the first base sequence.

12. The method according to claim 1 or 2, wherein the destabilizing portion includes 1 to 8 mismatch portions.

13. The method according to claim 1 or 2, wherein the destabilizing portion includes one to three mismatch portions.

14. The method according to claim 1 or 2, wherein the destabilizing portion includes a number of mismatch portions that are 30% or less in ratio to the length of the first base sequence.

15. The method according to claim 1 or 2, wherein the destabilized portion includes the deletion of a number of nucleic acid residues that is 50% or less in ratio to the length of the first base sequence.

16. The method according to claim 1 or 2, wherein the destabilized portion includes the deletion of 1 to 8 sets of nucleic acid residues.

17. The method according to claim 1 or 2, wherein the destabilized portion includes the deletion of 1 to 3 sets of nucleic acid residues.

18. The method according to claim 16, wherein each set of nucleic acid residue deletions consists of the deletion of one to three nucleic acid residues.

19. The method according to claim 1 or 2, wherein the destabilization portion includes the insertion of a number of nucleic acid residues that is 200% or less in ratio to the length of the first base sequence.

20. The method according to claim 1 or 2, wherein the destabilization portion includes the insertion of 1 to 8 sets of nucleic acid residues.

21. The method according to claim 1 or 2, wherein the destabilization portion includes the insertion of 1 to 4 sets of nucleic acid residues.

22. The method according to claim 20, wherein each set of nucleic acid residue insertions consists of the insertion of 1 to 10 nucleic acid residues.

23. The method according to claim 20, wherein, if each set of nucleic acid residue insertions consists of the insertion of two or more nucleic acid residues, some or all of the nucleic acid residues are self-complementary.

24. The method according to claim 1 or 2, wherein the destabilizing portion includes the insertion of 1 to 8 linkers.

25. The method according to claim 1 or 2, wherein the destabilized portion includes the absence of 1 to 8 linkers.

26. The method according to claim 1 or 2, wherein the destabilizing portion includes the substitution of 1 to 8 linkers.

27. The method according to claim 1 or 2, wherein the destabilizing portion includes (a), (b), or (c) below: (a) A combination of a base-free region at positions -1 to +1 and deletion and / or insertion of nucleic acid residues at positions other than -1 to +1; (b) A combination of a mismatch at positions -1 to +1 and deletion and / or insertion of nucleic acid residues at positions other than -1 to +1; (c) Mismatch in ranks -1 to +1.

28. The method according to claim 27, wherein the destabilizing parts (a), (b), and (c) are (a1), (b1), and (c1), respectively: (a1) A combination of a single base-free region at position -1 to +1 and the deletion of one or two sets of nucleic acid residues and / or insertion of one or two sets of nucleic acid residues at positions other than -1 to +1; (b1) A combination of one mismatch at position -1 to +1 and one or two sets of deletions and / or insertions of nucleic acid residues at positions other than -1 to +1; (c1) One mismatch at position -1 to +1.

29. The method according to claim 27, wherein the destabilizing parts (a), (b), and (c) are, respectively, (a2), (b2), and (c2): (a2) A combination of one base-free region at positions -1 to +1, one set of nucleic acid residues inserted at a negative position other than -1, and one set of nucleic acid residues inserted at a positive position other than +1; (b2) A combination of one mismatch site between position -1 and +1, one set of nucleic acid residues inserted at a negative position other than -1, and one set of nucleic acid residues inserted at a positive position other than +1; (c2) One mismatch in ranks -1 to +1.

30. The method according to claim 1 or 2, wherein the destabilizing portion lowers the melting temperature of the hybrid by 1 to 60°C.

31. The method according to claim 1 or 2, wherein the length of the target oligonucleotide is 10 to 200 residues.

32. The method according to claim 1 or 2, wherein the length of each substrate oligonucleotide is 5 to 50 residues. 。

33. The method according to claim 1 or 2, wherein the lengths of the second and third base sequences are each 5 to 50 residues.

34. The method according to claim 1 or 2, wherein the length of each complementary oligonucleotide is 5 to 300% of the length of the target oligonucleotide, with the length of the target oligonucleotide being 100%.

35. The method according to claim 1 or 2, wherein the length of each complementary oligonucleotide is 5 to 300% of the sum of the lengths of the 5' and 3' substrate oligonucleotides, with the sum being 100%.

36. The method according to claim 1 or 2, wherein in each complementary oligonucleotide, five or more nucleic acid residues constituting the second base sequence remain, and five or more nucleic acid residues constituting the third base sequence remain.

37. The method according to claim 1 or 2, wherein in each complementary oligonucleotide, 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 claim 1 or 2, wherein in each complementary oligonucleotide, three or more consecutive nucleic acid residues constituting the second base sequence remain, and three or more consecutive nucleic acid residues constituting the third base sequence remain.

39. The method according to claim 1 or 2, wherein the target oligonucleotide comprises a DNA residue, an RNA residue, a modified nucleic acid residue, or a combination thereof.

40. The method according to claim 1 or 2, wherein the target oligonucleotide is modified.

41. The method according to claim 1 or 2, wherein one or more of the complementary oligonucleotides are modified.

42. The method according to claim 39, wherein the modification includes modification of the phosphate group, modification of the sugar group, modification of the base group, or a combination thereof.

43. The method according to claim 42, wherein the modification of the phosphate portion includes phosphorothioate, boranophosphate, linker insertion, or a combination thereof.

44. The method according to claim 42, wherein the modification of the sugar portion includes 2'-MOE, 2'-OMe, 2'-F, 4'-thio-2'-OMe, crosslinking between the 2' and 4' positions of the sugar portion, 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 claim 1 or 2, wherein N is 2.

46. The method according to claim 1 or 2, wherein N is 3 or more.

47. The method according to claim 1 or 2, wherein N is 10 or less.

48. The method according to claim 1 or 2, wherein M is less than N-1.

49. The method according to claim 1 or 2, wherein M is N-1.

50. The method according to claim 1 or 2, wherein M is greater than N-1.

51. The method according to claim 1 or 2, wherein the above step is carried out at 5 to 60°C.

52. The method according to claim 1 or 2, wherein the amount of each complementary oligonucleotide used is 50% or less of the smaller of the amount of the 5'-side substrate oligonucleotide used and the amount of the 3'-side substrate oligonucleotide used, in molar ratio.

53. The method according to claim 1 or 2, wherein the concentration of each substrate oligonucleotide in the reaction solution in the step is 1 to 10,000 μM.

54. The method according to claim 1 or 2, wherein the enzyme used for the enzymatic linkage is a T3 DNA ligase.