Method for producing modified oligonucleotides containing complementary moieties

Abstract

This invention provides an efficient method for producing oligonucleotides containing complementary regions, such as siRNA and heterodouble-stranded oligonucleotides. [Solution] More specifically, a method for producing a modified oligonucleotide containing a complementary portion of 11 to 27 base lengths, The method includes treating a total of four or more oligonucleotide raw material fragments in the presence of an oligonucleotide ligase to produce the modified oligonucleotide, The total of four or more oligonucleotide raw material fragments correspond to oligonucleotide raw material fragments obtained when the modified oligonucleotide is separated at fragment linkages that satisfy the following conditions (i) to (v): (i) There is one or more fragment linkers in each chain side of the complementary portion, and there are a total of two or more fragment linkers in the modified oligonucleotide; (ii) When the modified oligonucleotide is separated at the fragment linkage, the protruding end is formed in the complementary portion, and the protruding end is 1 to 10 bases long; (iii) At least one oligonucleotide raw material fragment contains a modified nucleotide, (iv) Of the total of four or more oligonucleotide raw material fragments, four oligonucleotide raw material fragments include a complementary portion of 5 to 25 bases in length, (v) A method is provided in which the sum of the base lengths corresponding to each strand of the complementary portion of the oligonucleotide raw material fragment is 11 to 27 base lengths.

Description

[Technical Field]

[0001] This invention relates to a method for producing modified oligonucleotides containing complementary moieties. [Background technology]

[0002] Oligonucleotides such as siRNA and antisense drugs have demonstrated their usefulness as nucleic acid drugs, and their development has become increasingly active in recent years. Oligonucleotides are mainly manufactured by synthetic methods, for example, by sequentially extending nucleotide residues one base at a time in a series using solid-phase synthesis such as the phosphoramidite method. However, this method has drawbacks, such as a decrease in product purity and yield as the oligonucleotide chain length increases, and low manufacturing efficiency. Therefore, there is a need for a parallel synthesis method that synthesizes oligonucleotides as individual short-chain fragments and then condenses them to obtain the desired oligonucleotide.

[0003] Patent Document 1 describes a method for producing a single-stranded oligonucleotide by annealing multiple oligonucleotide raw material fragments, which correspond to fragments obtained by dividing the target oligonucleotide, with a template oligonucleotide complementary to the target oligonucleotide, enzymatically condensing the annealed oligonucleotide raw material fragments together, and separating the resulting target oligonucleotide chain from the template oligonucleotide. Non-patent document 1 describes the PEGylation of oligoDNA by linking an oligoDNA fragment and a PEGylated oligoDNA fragment at their adhesive ends with DNA ligase. However, since Non-patent document 1 only uses natural oligoDNA fragments, it is unclear whether enzymatic condensation of oligonucleotides containing complementary regions is possible using oligonucleotides containing short chains and modified bases, which are suggested to have reduced annealing ability, as raw materials. Non-patent documents 2 and 3 describe ligating a nick formed by annealing one oligonucleotide chain with two complementary oligonucleotide raw material fragments. Non-Patent Document 4 describes the formation of double-stranded RNA having a size of 48 mer or more by ligating a 24-mer double-stranded oligo RNA having an adhesive end with RNA ligase. However, in Non-Patent Document 4, since both the substrate and the product are as long as 24 bases and 48 bases, respectively, the reaction is carried out only under conditions where the annealing ability of the substrate is high. Therefore, when using an oligonucleotide substrate in which the ligation activity of the enzyme is expected to decrease due to the introduction of short-chain and modified bases, which is suggested to significantly reduce the annealing ability, it was not known whether an enzymatic reaction having two or more condensation points would proceed. Non-Patent Document 5 describes the synthetic preparation of siRNA. Non-Patent Document 6 describes that RNA ligase DraRnl is included in the Rnl5 family.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

[0006] The objective of the present invention is to provide an efficient method for producing oligonucleotides containing complementary parts, such as siRNA and heterodouble-stranded oligonucleotides. [Means for solving the problem]

[0007] As a result of diligent research, the inventors of the present invention have discovered that by treating four or more oligonucleotide raw material fragments, which correspond to fragments obtained by separating both complementary parts of an oligonucleotide containing the target complementary part, with oligonucleotide ligase, oligonucleotides containing complementary parts such as double hemispheres can be directly constructed. They have also found that this method can be produced with higher manufacturing efficiency and higher purity compared to serial synthesis methods such as solid-phase synthesis, and have completed the present invention.

[0008] Traditionally, relatively short double-stranded oligonucleotides, such as siRNA, have been produced by chemical synthesis because it is easy and convenient to synthesize. Specifically, the two constituent oligonucleotide chains were chemically synthesized separately (e.g., solid-phase synthesis), purified, and then annealed. Therefore, methods using enzymatic condensation have not been widely reported, and among those, only methods for producing relatively long double-stranded oligonucleotides of 28 nucleotides or more from four or more oligonucleotide starting fragments have been reported. Shorter oligonucleotides naturally require shorter oligonucleotide starting fragments, but it is thought that oligonucleotide starting fragments with shorter base lengths also have reduced annealing ability. It was also thought that the annealing ability would be further reduced if the constituent nucleotides were modified. Therefore, since the annealing ability of the oligonucleotide starting fragments was considered important in enzymatic oligonucleotide synthesis methods using ligases, methods for producing even shorter oligonucleotides, such as those with a length of less than 28 nucleotides, using four or more oligonucleotide starting fragments with ligases had not been attempted. However, after diligent research, the inventors unexpectedly discovered that, when using ligase and four or more oligonucleotide raw material fragments, double-stranded oligonucleotides with a length of less than 28 bases can be successfully produced without being affected by annealing ability. Furthermore, they found that the purity of the oligonucleotides produced also improved in the case of short-chain oligonucleotides, leading to the completion of the present invention.

[0009] In other words, the present invention is as follows: [1] A method for producing a modified oligonucleotide containing a complementary portion of 11 to 27 bases in length, The method comprises treating a total of four or more oligonucleotide raw material fragments in the presence of an oligonucleotide ligase. The total of four or more oligonucleotide raw material fragments correspond to oligonucleotide raw material fragments obtained when the modified oligonucleotide is separated at fragment linkages that satisfy the following conditions (i) to (v): (i) There is one or more fragment linkers in each chain side of the complementary portion, and there are a total of two or more fragment linkers in the modified oligonucleotide; (ii) When the modified oligonucleotide is separated at the fragment linkage, the protruding end is formed in the complementary portion, and the protruding end is 1 to 10 bases long; (iii) At least one oligonucleotide raw material fragment contains a modified nucleotide, (iv) Of the total of four or more oligonucleotide raw material fragments, four oligonucleotide raw material fragments include a complementary portion of 5 to 25 bases in length, (v) The sum of the base lengths corresponding to each strand of the complementary portion of the oligonucleotide raw material fragment is 11 to 27 base lengths in all cases. The method of [1] in [2](ii) wherein the overhang is 2 to 6 bases long. The method of [1] or [2], wherein the portions of the complementary parts of the four oligonucleotide raw material fragments specified in [3](iv) that are not the protruding ends are 4 to 16 base pairs long. [4] Any of the methods [1] to [3], wherein the oligonucleotide ligase is an RNA ligase. [5] The method of [4], wherein the oligonucleotide ligase is a double-stranded RNA ligase. [6] The method of [5], wherein the double-stranded RNA ligase is an RNA ligase of the Rnl2 family or the Rnl5 family. [7] Any method [1] to [6] wherein the modified oligonucleotide contains a modified nucleotide residue. The method of [7], wherein the modified nucleotide residue is a 1′, 2′, 3′, or 4′ chemically modified nucleotide residue, a 5′- or 3′-phosphate group modified nucleotide residue, a cross-linked modified nucleotide residue, a carrier-added modified nucleotide residue, or a sugar backbone-substituted nucleotide residue. [9] The modified nucleotide residue is i) a 1′, 2′, 3′, or 4′ chemically modified nucleotide residue in which the 1′, 2′, 3′, or 4′ position is substituted with C , 1~6 alkyloxy C 1~6 alkylene, -O-C 1~6 alkyl, -O-C 6~14 aryl, -C-aryl, a halogen atom, -O-C 1~6 alkyl N-amido C 1~6 alkylene, -O-C 1~6 alkyl-(C 1~6 alkyl-)amino-C 1~6 alkylene, or -O-amino C 1~6 alkyl (e.g., -O-aminopropyl, -O-AP); ii) a 5′- or 3′-phosphate group modified nucleotide residue in which the hydroxyl group is optionally substituted with a protecting group and is substituted with -O-P(S)(OH)2, -NH-P(O)(OH)2, or -NH-P(S)(OH)2; [ iii) a cross-linked modified nucleotide residue in which the 2′ position and the 4′ position are substituted with 2′-O-C 1~6 alkylene-4′, 2′-O-ethylene-4′, 2′-O-methyl-substituted methylene-4′, 2′-O-C 1~6 alkylene-O-C 1~6 alkylene-4′, 2′-O-N(R)-C 1~6 alkylene-4′ (where R represents methyl, a hydrogen atom, or benzyl), 2′-N(R)-C(O)-4′, 2′-NH-C 1~6 alkylene-4′, or 2′-C 1~6 alkylene-4′, or in which the 3′ position and the 5′ position are substituted with 3′-C 1~6 alkylene-5′; or iv) Hexitol nucleic acid (HNA) residues, cyclohexenyl nucleic acid (CeNA) residues, or morpholino nucleic acid (PMO) residues The method described in [8].

[10] Any method [1] to [9] wherein the total molar ratio of any two oligonucleotide raw material fragments selected from a total of four or more oligonucleotide raw material fragments is 0.5 to 2.

[11] Any of the methods [1] to

[10] , wherein the oligonucleotide raw material fragment is treated under a monovalent cation salt concentration of 10 mM or less.

[12] Any method of [1] to

[11] , wherein the oligonucleotide raw material fragment mixed solution is not placed at a high temperature and then cooled down before the above treatment.

[13] Any method [1] to

[12] that suppresses the generation of impurities in the modified oligonucleotide.

[14] Any method of [1] to

[13] , further comprising purifying the modified oligonucleotide. [Effects of the Invention]

[0010] According to the method of the present invention, modified oligonucleotides such as siRNA and heterodouble-stranded oligonucleotides can be efficiently produced with high purity. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 is a schematic diagram showing an example of the mechanism of the present invention. [Figure 2-1] Figures 2-1 to 2-6 are graphs showing the amount of siRNA produced in each case of the T4 RNA ligase 2 concentration when six combinations of four short native RNA fragments (combination numbers 1 to 6) for generating the same siRNA in Example 1 were reacted with T4 RNA ligase 2. [Figure 2-2] Figures 2-1 to 2-6 are as described above. [Figure 2-3] Figures 2-1 to 2-6 are as described above. [Figure 2-4] Figures 2-1 to 2-6 are as described above. [Figure 2-5] Figures 2-1 to 2-6 are as described above. [Figure 2-6] Figures 2-1 to 2-6 are as described above. [Figure 3-1] Figures 3-1 to 3-4 are graphs showing the time course of siRNA production at various reaction temperatures when a combination of four short-chain native RNA fragments was reacted with T4 RNA ligase 2 in Example 2. [Figure 3-2] Figures 3-1 to 3-4 are as described above. [Figure 3-3] Figures 3-1 to 3-4 are as described above. [Figure 3-4] Figures 3-1 to 3-4 are as described above. [Figure 4] Figure 4 shows the confirmation of siRNA and oligonucleotide preparations generated from modified RNA at each T4 RNA ligase concentration in Example 3 by HPLC analysis. [Figure 5] Figure 5 shows the HPLC analysis charts of the reaction products in Example 5, both without enzyme and in the presence of Deinococcus radiodurans-derived RNA ligase (DraRnl), as well as the RNA preparations (sense strand and antisense strand). [Figure 6] Figure 6 shows a schematic diagram of the reaction product in Example 6, as well as HPLC analysis charts of the reaction product in the absence of enzyme and in the presence of T4 RNA ligase 2. [Figure 7] Figure 7 shows the relationship between four oligonucleotide starting material fragments (including oligonucleotide starting material fragments containing mismatched base pairs) and the double-stranded modified oligonucleotides generated from them. [Figure 8] Figure 8 shows the relationship between five or six oligonucleotide raw material fragments and the double-stranded modified oligonucleotides generated therefrom. [Figure 9]Figure 9 shows the confirmation of double-stranded modified oligonucleotides in reactions using five or six oligonucleotide starting material fragments, as determined by HPLC analysis. [Figure 10] Figure 10 shows the relationship between four oligonucleotide raw material fragments, each containing an oligonucleotide raw material fragment with a DMTr group attached to its 5' end, and the double-stranded modified oligonucleotides generated from them. [Figure 11] Figure 11 shows the relationship between four oligonucleotide raw material fragments, each containing an oligonucleotide raw material fragment with a carrier attached to its 5' end, and the double-stranded modified oligonucleotides generated from them. [Figure 12] Figure 12 shows the relationship between four oligonucleotide raw material fragments in which the phosphate group at the linkage of nucleotide residues was replaced with a thiophosphate group, and the double-stranded modified oligonucleotides generated from them. [Figure 13] Figure 13 shows the relationship between four oligonucleotide raw material fragments and the hairpin-shaped modified oligonucleotides generated from them. [Figure 14] Figure 14 shows the relationship between four oligonucleotide starting fragments and the double-stranded modified oligonucleotides generated from them, used to compare the effect of the base length of the protruding ends of oligonucleotide starting fragments on reactivity. [Figure 15] Figure 15 shows the relationship between four oligonucleotide starting material fragments and the double-stranded modified oligonucleotides generated from them, used to compare the effect of product base length on reactivity. [Figure 16] Figure 16 shows the relationship between four oligonucleotide starting material fragments used to investigate the initial reaction rate at high substrate concentrations and the double-stranded modified oligonucleotides generated from them. [Figure 17] Figure 17 shows the relationship between four oligonucleotide starting material fragments used for comparing the base lengths of the products and the double-stranded modified oligonucleotides generated from them. [Modes for carrying out the invention]

[0012] (Summary of the present invention) The present invention will now be described. To facilitate the explanation of the present invention, an example of the mechanism of the present invention is shown in a schematic diagram in Figure 1. However, this schematic diagram is merely an example for explaining the present invention and does not limit the present invention.

[0013] The present invention provides a method for producing a modified oligonucleotide containing a complementary portion of 11 to 27 base pairs (hereinafter sometimes referred to as the "target modified oligonucleotide"). The method of the present invention includes treating a total of four or more oligonucleotide raw material fragments in the presence of an oligonucleotide ligase to produce the target modified oligonucleotide. The following describes in detail the target modified oligonucleotide produced by the method of the present invention, the oligonucleotide raw material fragments and oligonucleotide ligase used in the method of the present invention, and the conditions for each treatment for carrying out the method of the present invention.

[0014] (Target modified oligonucleotide) The modified oligonucleotide of the present invention produced by the present invention is a modified oligonucleotide containing a complementary moiety of 11 to 27 bases in length.

[0015] An "oligonucleotide" is an oligomer that contains nucleotide residues as monomer units. Examples of oligonucleotides include oligoRNA, oligoDNA, and RNA-DNA hybrid oligonucleotides.

[0016] Oligonucleotides can be classified into "natural oligonucleotides" and "modified oligonucleotides." "Natural oligonucleotides" are oligonucleotides composed of nucleotide residues that make up polynucleotides (RNA and DNA) contained in cells (adenosine (A), guanosine (G), cytidine (C), uridine (U), deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC), thymidine (dT). Hereinafter referred to as "natural nucleotide residues"). "Modified oligonucleotides" are oligonucleotides other than "natural oligonucleotides," and are oligonucleotides that contain components other than natural nucleotide residues (hereinafter referred to as "modified residues"). Examples of modified residues include modified nucleotide residues, amino acid residues, and linkers. Examples of modified nucleotide residues include nucleotide residues containing modifications described later. Amino acids include amino acid derivatives. Examples of amino acids include glycine, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, tryptophan, serine, threonine, asparagine, glutamine, tyrosine, cysteine, aspartic acid, glutamic acid, histidine, lysine, arginine, and their derivatives. An amino acid derivative is an amino acid in which any atom or group in the amino acid is substituted with another atom or group. Examples include amino acids in which a hydrogen atom in the amino group, a hydrogen atom in the carboxyl group, an oxygen atom, a hydroxyl group, any atom or group in the side chain, or a hydrogen atom bonded to a skeletal carbon atom (e.g., α-, β-, γ-, δ-carbon atoms) is substituted with another atom (e.g., a halogen atom such as a fluorine atom, chlorine atom, bromine atom, or iodine atom) or group (e.g., a substituent after substitution in the chemical modification described later).

[0017] In the context of "modified nucleotide residues," "modification" includes substitution of substituents on the sugar portion (ribose or deoxyribose) of a nucleotide residue, substitution of the sugar portion (sugar backbone) itself, and modification of the nucleic acid base portion of a nucleotide residue (e.g., substitution of substituents on the nucleic acid base portion).

[0018] Examples of "substitution of substituents in the sugar portion of nucleotide residues" include substitutions of 1'-H, 2'-OH (ribose only), 2'-H, 3'-OH, 3'-NH2, 3'-H, 3'-phosphate group, 4'-H, 5'-phosphate group, or combinations thereof. Here, "phosphate group" includes not only -OP(O)(OH)2 but also groups in which the oxygen atom is replaced by a sulfur atom or NH (e.g., -O-P(S)(OH)2, -NH-P(O)(OH)2, -NH-P(S)(OH)2). Also, the hydroxyl group (-OH) in the phosphate group may be OR * (In the formula, R * The term "phosphate group" also includes groups that have been replaced by organic groups such as protecting groups for phosphate groups (e.g., protected phosphate groups). Examples of such substitutions include 1', 2', 3', or 4'-chemical modifications (substitution of other substituents at the 1', 2', 3', or 4' site), 5'- or 3'-phosphate group modifications (substitution of other substituents for the 5'- or 3'-phosphate group), crosslinking modifications (substitution of two 1', 2', 3', or 4' sites to crosslink each other), and carrier addition modifications (substitution of carriers at the 1', 2', 3', 4', or 5' site).

[0019] Chemical modifications may be introduced, for example, to improve the degradation resistance of oligonucleotides. Examples of substituents to be substituted in chemical modifications include C 1~6 Alkyloxy C 1~6 Alkylene (e.g., methoxyethyl: MOE), -OC 1~6 Alkyl (e.g., -O-Me), -OC 6~14 Aryl (e.g., -O-phenyl), -C-aryl (e.g., -C-phenyl), halogen atom (e.g., fluorine atom), -OC 1~6 Alkyl N-amide C 1~6 Alkylenes (e.g., -ON-methylacetamide, -O-NMA), -OC 1~6 Alkyl-(C 1~6 Alkyl-)amino-C 1~6 Alkylenes (e.g., -O-dimethylaminoethoxyethyl, -O-DMAEOE), and -O-aminoC 1~6Examples include alkyl groups (e.g., -O-aminopropyl, -O-AP). Chemical modifications are preferably 2'-modification (substitution of the 2' position) or 3'-modification (substitution of the 3' position), with 2'-modification (substitution of the 2' position) being more preferred. Examples of substituents after substitution in 2'-modification include 2'-C 1~6 Alkyloxy C 1~6 Alkylenes (e.g., 2'-methoxyethyl), 2'-OC 1~6 Alkyl (e.g., 2'-O-Me), 2'-OC 6~14 Aryl (e.g., 2'-O-phenyl), 2'-C-aryl (e.g., 2'-C-phenyl), 2'-halogen atoms (e.g., 2'-F), 2'-OC 1~6 Alkyl N-amide C 1~6 Alkylenes (e.g., 2'-ON-methylacetamide, 2'-O-NMA), 2'-OC 1~6 Alkyl-(C 1~6 Alkyl-)amino-C 1~6 Alkylenes (e.g., 2'-O-dimethylaminoethoxyethyl, 2'-O-DMAEOE), and 2'-O-amino C 1~6 Examples include alkyl groups (e.g., 2'-O-aminopropyl, 2'-O-AP). Examples of substituents after 3'-chemical modification include 3'-OP(O)(OH) 2、 3'-O-Ρ(S)(OH)2, 3'-NH-Ρ(O)(OH)2, 3'-NH-Ρ(S)(OH)2), and OR when the hydroxyl group (-OH) in the phosphate group is OR * (In the formula, R * Examples include groups that have been replaced by organic groups (such as phosphate protecting groups, which will be discussed later).

[0020] 5'- or 3'-phosphate group modifications may be introduced, for example, to improve the degradation resistance of oligonucleotides. Examples of 5'- or 3'-phosphate group modifications include substitution of a phosphate group (-OP(O)(OH)2) with a group in which the oxygen atom in the phosphate group is replaced by a sulfur atom or NH. Examples of such groups include -O-Ρ(S)(OH)2 (thiophosphate group: phosphorothioate type modification), -NH-Ρ(O)(OH)2, and -NH-Ρ(S)(OH)2. In addition, in 5'- or 3'-phosphate group modifications, the hydroxyl group (-OH) in the phosphate group is replaced with OR * (In the formula, R * This also includes groups that have been replaced by organic groups such as phosphate protecting groups (e.g., protected phosphate groups). Examples of phosphate protecting groups include the trityl (Tr) group, the p-methoxyphenyldiphenylmethyl (MMTr) group, the di(p-methoxyphenyl)phenylmethyl (DMTr) group, and the cyanoethyl (CN-C2H4-) group.

[0021] Cross-linking modifications may be introduced, for example, to improve the three-dimensional stability of nucleotide residues. Examples of cross-linking modifications include 2'4'-cross-linking modifications (substitutions that cross-link 2'-OH and 4'-H) and 3'5'-cross-linking modifications (substitutions that cross-link 3'-H and 5'-H). An example of a 2'4'-cross-linking modification is the 2'-OC of 2'-OH and 4'-H. 1~6 Substitution to alkylene-4' (e.g., 2'-O-methylene-4' (Loc nucleic acid: LNA), 2'-O-ethylene-4' (ethylene-crosslinked nucleic acid: ENA), substitution to 2'-O-methyl-substituted methylene-4' (constructed-ethyl crosslinked nucleic acid: a type of BNA (cEt-BNA)), 2'-OC of 2'-OH and 4'-H) 1~6 Alkylene-OC 1~6 Substitution to alkylene-4' (e.g., 2'-O-methylene-O-methylene-4' (crosslinked nucleic acid: a type of BNA (BNA) COC )), 2'-ON(R)-C of 2'-OH and 4'-H 1~6 Substitution to alkylene-4' (e.g., 2'-ON(R)-methylene-4' (crosslinked nucleic acid: a type of BNA (BNA) NC), where R represents a methyl, hydrogen atom or benzyl atom), substitution of 2'-NH2 and 4'-H to 2'-N(R)-C(O)-4' (e.g., 2'-N(methyl)-C(O)-4' (amide-bridged nucleic acid: AmNA)), 2'-NH2 and 4'-H to 2'-NH-C 1~6 Substitution to alkylene-4' (e.g., 2'-NH-methylene-4'), 2'-C of 2'-H and 4'-H 1~6 Substitution to alkylene-4' (e.g., 2'-methyl-substituted ethylene-4') is one example. Furthermore, 3'5'-crosslinking modifications include, for example, the 3'-C of 3'-H and 5'-H. 1~6 Substitution to alkylene-5' is one example (e.g., 3'-ethylene-5' (bicyclonucleotide: Bc nucleic acid), a type of Bc nucleic acid: tc nucleic acid, etc.)

[0022] The carrier used in carrier modification may be a carrier that improves or imparts properties such as stability, targeting, or pharmacokinetics to the target modified oligonucleotide. Such a carrier can be appropriately selected from known carriers depending on the intended use. Examples of carriers include N-acetylgalactosamine (GalNAc), peptides, phosphate, cholesterol, tocopherol, fatty acid chains, and folic acid. The attachment site in carrier modification is preferably the 3' or 5' site corresponding to the end of the target modified oligonucleotide.

[0023] Modified nucleotide residues (sugar backbone-substituted nucleotide residues) that involve "substitution of the sugar portion of the nucleotide residue itself" include, for example, nucleotide residues in oligonucleotides such as hexitol nucleic acid (HNA) and cyclohexenyl nucleic acid (CeNA) that involve substitution of a 5-membered ring sugar to a 6-membered ring pseudosugar. Furthermore, morpholino nucleic acid (PMO) residues, which are nucleotide-like artificial compounds having a morpholino ring structure that is not degraded by enzymes in the body (e.g., nucleases such as RNase) and does not induce an immune response, can also be considered as modified nucleotide residues that involve "substitution of the sugar portion of the nucleotide residue itself."

[0024] Examples of "modification of the nucleic acid base portion of a nucleotide residue" include alkyl substitution of the nucleic acid base portion of a nucleotide residue (for example, substitution of a methyl group at the 5th position of a cytosyl group).

[0025] An oligonucleotide containing a complementary region refers to an oligonucleotide that contains a structure in which complementary nucleotide sequences are paired together. Examples of oligonucleotides containing a complementary region include double-stranded oligonucleotides and single-stranded oligonucleotides containing a double-strand-like structure (e.g., hairpin oligonucleotides, dumbbell oligonucleotides, and other loop-type oligonucleotides). A double-stranded oligonucleotide may be a double-stranded oligonucleotide in which each strand is one of the oligonucleotides described above. Examples include double-stranded oligoRNA, double-stranded oligoDNA, hetero-double-stranded oligonucleotides consisting of oligoRNA and oligoDNA, double-stranded oligonucleotides consisting of oligoRNA and RNA-DNA hybrid oligonucleotides, and double-stranded oligonucleotides consisting of RNA-DNA hybrid oligonucleotides. Examples of double-stranded oligonucleotides include siRNA and hetero-double-stranded oligonucleotides. In an oligonucleotide containing a complementary region, the portion in which complementary nucleotide sequences are paired together is called the "complementary region." The term "complementary region" refers not only to the complementary region within an oligonucleotide containing the complementary region, but also to the portion of the oligonucleotide raw material fragment corresponding to the complementary region when the oligonucleotide containing the complementary region is separated into oligonucleotide raw material fragments. For convenience, one of the complementary nucleotide sequences in the complementary region is sometimes called the "sense strand," and the other complementary nucleotide sequence is sometimes called the "antisense strand." In this invention, the terms "sense" and "antisense" are merely convenient designations referring to either one or the other of the complementary region, and are not intended to have any biological significance (especially significance in RNAi). Oligonucleotides containing the complementary region may or may not contain a loop region. A "loop region" refers to a linker that connects the sense side and the antisense side of the complementary region at the same end (for example, the 5' end and the 3' end). Oligonucleotides containing the complementary region are particularly used for post-transcriptional gene silencing (e.g., RNA interference (RNAi)).

[0026] The target modified oligonucleotide contains the modified residues described above in its complementary portion. Examples of the target modified oligonucleotide include double-stranded oligonucleotides or loop-type oligonucleotides containing modified nucleotide residues (e.g., double-stranded oligonucleotides or loop-type oligonucleotides containing modified nucleotide residues in their complementary portion), and loop-type oligonucleotides containing modified nucleotide residues or residues other than nucleotide residues (e.g., amino acid residues or linkers) in the loop portion (e.g., International Publication No. 2012 / 005368). In the target modified oligonucleotide, some nucleotide residues may be modified nucleotide residues, or all nucleotide residues may be modified nucleotide residues, but if the "modified nucleotide residues" are morpholino nucleic acid (PMO) residues, it is preferable that some nucleotide residues in the target modified oligonucleotide are morpholino nucleic acid (PMO) residues. Furthermore, the target modified oligonucleotides include gapmers, which are oligonucleotides that have modified nucleotide residues at both ends of their sequence and a gap region in the middle of their sequence that is recognized by RNase; and mixedmers, which are oligonucleotides in which modified nucleotide residues are mixed within their sequence; and fully modified oligonucleotides, which are oligonucleotides in which all nucleotide residues in their sequence are modified nucleotide residues, and other oligonucleotides that do not induce RNase activity.

[0027] In the present invention, the complementary portion in the target modified oligonucleotide is 11 to 27 bases long (e.g., 12 to 27 bases, 15 to 27 bases, or 18 to 27 bases). For example, if the target modified oligonucleotide is a double-stranded modified oligonucleotide consisting only of the complementary portion, it may be 11 to 27 bases long. Alternatively, the target modified oligonucleotide may have a non-complementary portion in addition to the complementary portion. In this case, the non-complementary portion may be 1 to 16 bases long, for example, 1 to 10 bases long, preferably 1 to 5 bases long, and more preferably 1, 2, or 3 bases long. In the target modified oligonucleotide having a non-complementary portion in addition to a complementary portion of 11 to 27 bases long, the complementary portion of 11 to 27 bases long may be in a continuous form, or it may be in a discontinuous form separated by mismatched base pairs as the non-complementary portion.

[0028] The total number of residues of the target modified oligonucleotide may be appropriately selected based on the function of the target modified oligonucleotide and the conditions in the method of the present invention. For example, the total number of residues of the target modified oligonucleotide may be 24 to 74.

[0029] (Oligocynonucleotide raw material fragments) The total of four or more oligonucleotide raw material fragments used as raw materials in the method of the present invention can be designed to correspond to oligonucleotide raw material fragments obtained when the target modified oligonucleotide is separated at fragment linkage sites (also called "cleavage sites") that satisfy the following conditions (i) to (v): (i) There is one or more fragment linkers in each chain side of the complementary portion, and there are a total of two or more fragment linkers in the modified oligonucleotide; (ii) When the modified oligonucleotide is separated at the fragment linkage, an overhang (also called an "adhesive end") is formed in the complementary portion, and the overhang is 1 to 10 bases long; (iii) At least one oligonucleotide raw material fragment contains a modified nucleotide, (iv) Of the total of four or more oligonucleotide raw material fragments, four oligonucleotide raw material fragments include a complementary portion of 5 to 25 bases in length, (v) The sum of the base lengths corresponding to each strand of the complementary portion of the oligonucleotide raw material fragment is 11 to 27 base lengths in all cases.

[0030] The number of oligonucleotide raw material fragments is four or more, preferably four to six (four, five, or six). The number of oligonucleotide raw material fragments can also be characterized in terms of the number of fragments that mainly correspond to the sense strand and antisense strand that constitute the target modified oligonucleotide (mainly double-stranded nucleic acid). From the above condition (i), it is understood that the number of oligonucleotide raw material fragments that mainly correspond to such sense strands and antisense strands is two or more, each. The number of oligonucleotide raw material fragments that correspond to such sense strands and antisense strands may be three or four fragments, each, with two or three fragments being preferred. The overhanging end in the above condition (ii) may be either a 5' overhanging end or a 3' overhanging end. The "complementary portion" in the above condition (iv) refers to the portion of the oligonucleotide raw material fragment that corresponds to the complementary portion in the target modified oligonucleotide. In the present invention, the terms "fragment linkage" and "cleavage site" have the same meaning. "Fragment linkage" ("cleavage site") means a site that is set for convenience in designing the combination of oligonucleotide raw material fragments, and does not mean a site that is actually cleaved in the method of the present invention. The four oligonucleotide raw material fragments in (iv) above may be designed such that the desired modified oligonucleotide contains a complementary portion of preferably 5 to 25 bases in length, more preferably 5 to 20 bases in length, and even more preferably 5 to 17 bases in length.

[0031] The base length of the "complementary portion" in the above condition (iv) is sufficient to be a base length that allows for pairing, and is only required to be 1 base length or longer. Furthermore, since the purity, yield, and production efficiency of the product may decrease as the base length increases in oligonucleotide synthesis, it is preferable that four of the oligonucleotide raw material fragments out of the total oligonucleotide raw material fragments be designed so that the complementary portion is 17 base lengths or less. It is preferable that the two strands constituting the complementary portion are 5 to 25 base lengths (e.g., 5 to 22 base lengths, 5 to 20 base lengths, 5 to 17 base lengths, 8 to 25 base lengths, 8 to 22 base lengths, 8 to 20 base lengths, 8 to 17 base lengths).

[0032] The protruding end is, for example, 1 to 10 bases long, preferably 1 to 8 bases long, more preferably 1 to 6 bases long, and even more preferably 2 to 6 bases long, 3 to 6 bases long, or 4 to 6 bases long.

[0033] The numerical values ​​of the base lengths of the "complementary portion" and the base lengths of the overhanging end in condition (iv) above are set to values ​​that satisfy the range described above and are consistent with each other. For example, if the overhanging end is 5 base lengths, the complementary portion may be 6 to 25 base lengths in order to form the overhanging end, and for example, if the overhanging end is 6 base lengths, the complementary portion may be 7 to 25 base lengths in order to form the overhanging end.

[0034] The portions of the "complementary portion" of the four oligonucleotide raw material fragments defined in condition (iv) above, excluding the protruding ends, are preferably 4 to 24 nucleotides long, 4 to 21 nucleotides long, 4 to 19 nucleotides long, or 4 to 16 nucleotides long.

[0035] In certain embodiments, the four oligonucleotide raw material fragments defined in condition (iv) above may each be 5 nucleotides or longer, preferably 6 nucleotides or longer, more preferably 7 nucleotides or longer, even more preferably 8 nucleotides or longer, and particularly preferably 9 nucleotides or longer. Such four oligonucleotide raw material fragments may also be 19 nucleotides or shorter, preferably 18 nucleotides or shorter, more preferably 17 nucleotides or shorter, even more preferably 16 nucleotides or shorter, and particularly preferably 15 nucleotides or shorter. Such four oligonucleotide raw material fragments may also be 5 to 19 nucleotides in length, preferably 6 to 18 nucleotides, more preferably 7 to 17 nucleotides, even more preferably 8 to 16 nucleotides, and particularly preferably 9 to 15 nucleotides.

[0036] The 5' end of the oligonucleotide raw material fragment corresponding to the 5' end of the target modified oligonucleotide may remain a 5'-phosphate group, be substituted with a 5'-OH group, have a 5'-phosphate group modification introduced, or have the same structure as the 5' end of the target modified oligonucleotide. Examples of 5'-phosphate group modifications include those mentioned above. From the viewpoint of the linking reaction by oligonucleotide ligase, it is preferable that the 5' end of the other oligonucleotide raw material fragments remain a 5'-phosphate group. The 3' end of the oligonucleotide raw material fragment corresponding to the 3' end of the target modified oligonucleotide may remain a 3'-OH group, have a 3'-phosphate group modification introduced, or have the same structure as the 3' end of the target modified oligonucleotide. Examples of 3'-phosphate group modifications include those mentioned above. From the viewpoint of the linking reaction by oligonucleotide ligase, it is preferable that the 3' end of the other oligonucleotide raw material fragments remain a 3'-OH group.

[0037] Oligonucleotide raw material fragments may be in a free form, complexed, or immobilized form.

[0038] Oligonucleotide raw material fragments may be produced by known chemical or enzymatic synthesis methods. Known chemical synthesis methods include solid-phase and liquid-phase synthesis methods, such as those described in International Publication No. 2012 / 157723 and International Publication No. 2005 / 070859.

[0039] If the addition of a functional portion to a target modified oligonucleotide is desired, the oligonucleotide raw material fragment may have the functional portion added to the corresponding portion.

[0040] (Ligae treatment) Oligonucleotide ligases are enzymes that link oligonucleotide raw material fragments together. In the method of the present invention, oligonucleotide raw material fragments are linked together at "cleavage sites" (also called "linking sites") by the catalytic action of oligonucleotide ligases to produce the desired modified oligonucleotide. Examples of oligonucleotide ligases include RNA ligases and DNA ligases. RNA ligases may be either single-stranded RNA ligases or double-stranded RNA ligases, with double-stranded RNA ligases being preferred. Examples of double-stranded RNA ligases include RNA ligases of the Rnl2 family (sometimes called "RNA ligase 2") and RNA ligases of the Rnl5 family. As long as the objective of the present invention is achieved, RNA ligases derived from any biological species or virus species may be used as RNA ligases, for example, T4 phage-derived RNA ligases (T4RNA ligase 1, T4RNA ligase 2) may be used. As the DNA ligase, any DNA ligase derived from any biological species or virus species may be used, insofar as the objectives of the present invention are achieved; for example, a DNA ligase derived from the T4 phage may be used.

[0041] Treatment in the presence of oligonucleotide ligase (hereinafter referred to as "ligase treatment") is a reaction in which oligonucleotide starting material fragments are linked by the catalytic action of oligonucleotide ligase. The operation of ligase treatment involves mixing the oligonucleotide starting material fragments with oligonucleotide ligase. In ligase treatment, all oligonucleotide starting material fragments and oligonucleotide ligase may be mixed and the linking reaction carried out in one step. Alternatively, in ligase treatment, as a multi-step linking reaction, some oligonucleotide starting material fragments and oligonucleotide ligase may be mixed and the linking reaction carried out, and then the remaining oligonucleotide starting material fragments and reactants may be mixed and the next linking reaction carried out. Mixing may be done by adding oligonucleotide ligase to oligonucleotide starting material fragments, adding oligonucleotide starting material fragments to a system containing oligonucleotide ligase, or adding oligonucleotide starting material fragments and oligonucleotide ligase to a system for reaction.

[0042] An aqueous solution can be used as the system for ligase treatment. A buffer solution is preferred as the aqueous solution. Examples of buffer solutions include phosphate buffer, Tris buffer, carbonate buffer, acetate buffer, and citrate buffer. The pH may be, for example, about 5 to 9. For example, if the concentration of oligonucleotide raw material fragments in the ligase treatment is high, the pH may be 7.5 to 9.0 (for example, 8.0 to 8.5).

[0043] The concentration of each oligonucleotide raw material fragment in the ligase treatment should be sufficient to dissolve the oligonucleotide raw material fragment and produce the desired modified oligonucleotide. The concentration of each oligonucleotide raw material fragment may be, for example, 1 μM or more, 10 μM or more, 50 μM or more, 100 μM or more, 300 μM or more, 500 μM or more, or 1000 μM or more. The concentration of each oligonucleotide raw material fragment may also be, for example, 1 M, 100 mM, or 10 mM or less. When efficient mass production of the desired modified oligonucleotide is particularly desired, it is preferable to use each oligonucleotide raw material fragment at a concentration of 100 μM or more among the above concentrations and at a pH range of 7.5 to 9.0 among the above pH ranges.

[0044] In the ligase treatment, the total number of moles of oligonucleotide raw material fragments is preferably approximately equal, from the viewpoint of improving production efficiency by reducing the amount of unreacted oligonucleotide raw material fragments. In order for the total number of moles of oligonucleotide raw material fragments to be approximately equal, the total molar ratio of any two oligonucleotide raw material fragments selected from a total of four or more oligonucleotide raw material fragments may be, for example, within the range of 0.5 to 2, preferably 1 / 1.8 to 1.8, more preferably 1 / 1.5 to 1.5, even more preferably 1 / 1.2 to 1.2, and particularly preferably 1 / 1.1 to 1.1.

[0045] The concentration of oligonucleotide ligase in the ligase treatment should be sufficient to produce the desired modified oligonucleotide. The concentration of oligonucleotide ligase may be, for example, 0.01 U / μL or higher, preferably 0.02 U / μL or higher, more preferably 0.03 U / μL or higher, and even more preferably 0.04 U / μL or higher. The concentration of oligonucleotide ligase may be, for example, 1 U / μL or less, preferably 0.5 U / μL or less, more preferably 0.2 U / μL or less, and even more preferably 0.1 U / μL or less. More specifically, the concentration of oligonucleotide ligase may be, for example, 0.01 to 1 U / μL, preferably 0.02 to 0.5 U / μL, more preferably 0.03 to 0.2 U / μL, and even more preferably 0.04 to 0.1 U / μL.

[0046] The ligase treatment system may contain cofactors for oligonucleotide ligase. Examples of cofactors for oligonucleotide ligase include ATP and divalent metal salts (e.g., magnesium salts such as magnesium chloride). The treatment system may also contain stabilizers for oligonucleotide ligase. Examples of stabilizers for oligonucleotide ligase include antioxidants (e.g., reducing agents such as dithiothreitol and mercaptoethanol). The ligase treatment system may contain surfactants to maintain enzyme stability and improve reaction rate. Examples of surfactants include nonionic surfactants (e.g., surfactants in the Triton series such as Triton X-100) and ionic surfactants. Examples of ionic surfactants include cationic surfactants, anionic surfactants, and amphoteric surfactants. The ligase treatment system may also contain polyethylene glycol to improve reaction rate.

[0047] The system for ligase treatment may have a low concentration of monovalent cation salts or may not contain monovalent cation salts at all. The concentration of monovalent cation salts in the treatment system may be, for example, 10 mM or less, preferably 1 mM or less, more preferably 0.1 mM or less, and even more preferably 0.01 mM or less. Particularly preferably, the treatment system may not contain monovalent cation salts at all. Examples of monovalent cation salts include salts of monovalent cations such as lithium ions, sodium ions, potassium ions, rubidium ions, cesium ions, and ammonium ions with anions such as fluoride ions, chloride ions, bromide ions, and iodide ions.

[0048] The temperature during ligase treatment should be sufficient to activate the oligonucleotide ligase. Such a temperature may be, for example, 2 to 50°C, preferably 16 to 50°C, and more preferably 25 to 50°C.

[0049] The duration of the ligase treatment should be sufficient to generate the desired modified oligonucleotide. This duration may be, for example, 1 to 72 hours.

[0050] According to the present invention, the generation and contamination of the target modified oligonucleotide with base lengths other than the target base length, such as N-1mer and N+1mer, can be suppressed. For example, in the present invention, the target modified oligonucleotide is manufactured as a single double-stranded nucleic acid (e.g., siRNA, hetero-double-stranded oligonucleotide). The sense strand and antisense strand constituting such a single double-stranded nucleic acid have base lengths of N and M, respectively. The base lengths of the N and M strands are independently 11 to 30 base lengths (e.g., 18 to 30 base lengths). The base lengths of the N and M strands may also independently be 11 to 27 base lengths (e.g., 18 to 27 base lengths). In the present invention, the impurities of the target modified oligonucleotide are intended to be nucleic acid contaminants other than the target modified oligonucleotide. The sense strand and antisense strand constituting such nucleic acid impurities consist of (N±α) and M base lengths, N and (M±β) base lengths, or (N±α) and (M±β) base lengths, respectively, rather than N and M base lengths. Here, N and M are as described above, and α and β are, for example, 1, 2, or 3.

[0051] (Other optional steps) The method of the present invention may include a step of synthesizing oligonucleotide raw material fragments (e.g., chemical synthesis such as solid-phase synthesis). Since the method of the present invention can suppress the generation of nucleic acid contaminants other than the target modified oligonucleotide, the purification of the target modified oligonucleotide from the synthesized oligonucleotide raw material fragment sample can be omitted. However, even if the target modified oligonucleotide is purified, the method of the present invention can suppress the generation of double-stranded nucleic acid contaminants (impurities) caused by small amounts of unintended oligonucleotide raw material fragments that may remain after such purification, so the purification of the target modified oligonucleotide may be performed. Such purification can be performed, for example, by methods such as chromatography (e.g., HPLC, IEX) or gel filtration.

[0052] The method of the present invention may include a reaction termination step after the ligase treatment step. Examples of reaction termination steps include high-temperature treatment (e.g., 80°C), deactivation of oligonucleotide ligase by adding acid, alkali, or organic solvent, and removal of cofactor metal ions by adding a chelating agent such as EDTA. Another method involves immobilizing the enzyme on a support and carrying out the reaction, then removing the enzyme from the reaction solution by membrane separation.

[0053] The method of the present invention may include a step of purifying the desired modified oligonucleotide after the ligase treatment step. For example, this step can be carried out by any suitable method such as chromatography (e.g., HPLC) or gel filtration.

[0054] In annealing oligonucleotide raw material fragments, it is common practice to heat the oligonucleotide raw material fragment mixture to a high temperature to denature the fragments (make them unpaired), and then gradually cool the high-temperature oligonucleotide raw material fragment mixture by air cooling or the like to form pairs between complementary nucleotide sequences. However, in the method of the present invention, the desired modified oligonucleotide can be produced in a simplified operation without performing such heating for denaturation and cooling for pairing (heat-cooling treatment) before the ligase treatment step. Examples of placing the mixture at a high temperature include holding the oligonucleotide raw material fragment mixture at 65°C or higher, 70°C or higher, 75°C or higher, 80°C or higher, 85°C or higher, 90°C or higher, 95°C or higher, or 100°C or higher (e.g., for 5 minutes or more, or 10 minutes or more). Cooling methods include, for example, leaving the oligonucleotide raw material fragment mixture solution standing at room temperature (e.g., 15-25°C or 20-25°C) or a predetermined temperature (e.g., 37°C) for (e.g., 5 hours or more), or maintaining the oligonucleotide raw material fragment mixture solution at a predetermined temperature (e.g., 37°C) for (e.g., 15 minutes or more).

[0055] To omit the heating-cooling process, the time the oligonucleotide raw material fragment mixture solution is exposed to high temperatures before the ligase treatment step may be controlled to, for example, less than 5 minutes, 4.5 minutes or less, 4 minutes or less, 3.5 minutes or less, 3 minutes or less, 2.5 minutes or less, 2 minutes or less, 1.5 minutes or less, 1 minute or less, or 0.5 minutes or less.

[0056] To omit the heating-cooling process, in the method of the present invention, the solution containing the oligonucleotide raw material fragments may be kept at 2 to 50°C from the time the oligonucleotide raw material fragments are mixed in the solution until the ligase treatment step is performed. That is, in the method of the present invention, any mixing of all the oligonucleotide raw material fragments and oligonucleotide ligases included in the above combination, any intervals between mixing, and the ligase reaction may be carried out under conditions of 2 to 50°C. In such embodiments, the method of the present invention includes the following: (1) Mixing all oligonucleotide raw material fragments and oligonucleotide ligases contained in the above combination, which are present in separate systems, under conditions that all mixing is carried out at 2 to 50°C and the mixture is maintained at 2 to 50°C during the intervals between mixing, to obtain a mixed solution; and (2) React the mixed solution while maintaining it at 2-50°C to obtain a solution containing the desired modified oligonucleotide.

[0057] In this embodiment, all oligonucleotide raw material fragments included in the above combination are obtained in separate systems. In this embodiment, the present invention may be carried out, for example, by mixing the oligonucleotide raw material fragments at 2 to 50°C to obtain an oligonucleotide raw material fragment mixture, and then mixing the oligonucleotide raw material fragment mixture with oligonucleotide ligase at 2 to 50°C. In this embodiment, the present invention may be carried out, for example, by sequentially adding the oligonucleotide raw material fragments to a solution containing oligonucleotide ligase at 2 to 50°C.

[0058] The method of the present invention can be used, for example, in the industrial production of desired modified oligonucleotides on a large scale. [Examples]

[0059] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0060] [Example 1] Comparison of fragment combination patterns using native RNA 1) Synthesis of substrates and product preparations The effect of the base length of the short-chain native RNA fragments on the enzymatic synthesis of siRNA from four short-chain native RNA fragments was evaluated. The target siRNA was a double-stranded RNA1-S (21 mer) and RNA1-A (23 mer) as shown in Table 1 (hereinafter referred to as the sense strand and antisense strand, respectively). Eighteen RNA fragments shown in Table 1 were synthesized, and these were used to evaluate the six patterns of fragment combinations shown in Table 2.

[0061] [Table 1]

[0062] [Table 2]

[0063] 2) Ligation reaction by T4 RNA ligase 2 Four oligonucleotide fragments were reacted with T4 RNA ligase 2 (New England Biolabs). The reaction mixture consisted of 50 mM Tris-HCl, 2 mM MgCl2, 1 mM dithiothreitol, 400 μM ATP, pH 7.5, and 10 μM RNA fragments, with a reaction volume of 10 μL. The product concentration was compared at enzyme addition concentrations of 0.025, 0.05, 0.1, and 0.2 U / μL. The reaction was stopped by heating at 80 °C for 5 minutes after reacting at 25 °C using a thermal cycler.

[0064] 3) Analysis by HPLC The reaction solution was analyzed by HPLC using an Xbridge Oligonucleotide BEH C18 column (Waters, 2.5 μm, 4.6 mm × 50 mm). The analytical conditions were: column temperature 60°C, detection wavelength 254 nm, injection volume 10 μL, and flow rate 0.4 mL / min. The mobile phase was analyzed using a linear gradient with eluent A (hexafluoroisopropanol-triethylamine) and eluent B (methanol). The concentration of the ligation product was quantified by similar analysis of the sense and antisense strand standards.

[0065] 4) Results Figure 2 shows the accumulation of sense and antisense strands for each combination of base lengths of the fragments. In combination 3, there was almost no accumulation of ligation products, but in the other combinations, it was observed that the accumulation of ligation products increased with increasing enzyme concentration.

[0066] [Example 2] Evaluation of the effect of reaction temperature on the ligation reaction of native RNA The effect of reaction temperature on short-chain RNA ligation reactions was investigated. Oligonucleotides listed in Table 2, item 1 were used as substrates and reacted with T4 RNA ligase 2. The reaction mixture consisted of 50 mM Tris-HCl, 2 mM MgCl2, 1 mM dithiothreitol, 400 μM ATP, pH 7.5, enzyme concentration of 0.2 U / μL, and 10 μM RNA fragment, with a reaction volume of 10 μL. The reaction temperature was set to 16°C, 25°C, 30°C, and 37°C, and after reaction for 1, 2, and 4 hours, respectively, the reaction was stopped by heating at 80°C for 5 minutes. The concentration of the ligation product in the reaction mixture was analyzed by HPLC under the conditions described in Example 1.

[0067] The results are shown in Figure 3. At reaction temperatures above 25°C, approximately 10 μM of ligation product accumulated in both the sense and antisense chains after 1 hour of reaction. On the other hand, at 16°C, the rate of ligation product formation, particularly on the sense chain, was lower compared to above 25°C.

[0068] [Example 3] Confirmation of reaction progress using modified RNA 1) Synthesis of substrates and product preparations The enzymatic ligation reaction of siRNA from four fragments of modified oligonucleotides was evaluated. The target siRNA was a double-stranded molecule consisting of a sense strand (MOD1-S) and an antisense strand (MOD1-A), as shown in Table 3. This siRNA has the same base sequence as the native RNA used in Examples 1 and 2, but all residues are modified with 2'-F or 2'-O-methyl groups, and some phosphate groups are substituted with thiophosphate groups. Four fragments, as shown in Table 3, were synthesized. The base sequences of these four fragments are identical to the combination numbered 1 in Table 2.

[0069] [Table 3]

[0070] 2) Ligation reaction by T4 RNA ligase 2 Four modified RNA fragments were reacted with T4 RNA ligase 2. The reaction mixture consisted of 50 mM Tris-HCl, 2 mM MgCl2, 1 mM dithiothreitol, 400 μM ATP, pH 7.5, and 10 μM modified RNA fragments, in a volume of 50 μL. The enzyme concentration was set to 0.2 or 1.0 U / μL, and the reaction was also performed under conditions without enzyme addition as a negative control. The reaction was stopped by heating at 25°C for 1 hour using a thermal cycler, followed by heating at 80°C for 5 minutes.

[0071] 3) Analysis by HPLC and LC-TOF / MS The reaction solution was analyzed by HPLC using an ACQUITY UPLC Oligonucleotide BEH C18 Column (Waters, 2.1 × 100 mm, 1.7 μm). The analytical conditions were: column temperature 80°C, detection wavelength 260 nm, injection volume 10 μL, and flow rate 0.4 mL / min. The mobile phase was analyzed using a linear gradient with solution A (hexafluoroisopropanol-triethylamine) and solution B (methanol). The formation of ligation products was confirmed by similar analysis of the sense and antisense strand standards. In addition, the ligation products were subjected to mass spectrometry using an Agilent 6230 TOF LC / MS system (Agilent Technologies).

[0072] 4) Results The results of the HPLC analysis are shown in Figure 4. HPLC analysis showed that the addition of the enzyme resulted in the acquisition of a ligation product peak at a retention time consistent with the standard, and the peak area of ​​the modified RNA of the substrate decreased compared to the negative control. Furthermore, the peak area of ​​the ligation product increased with increasing enzyme addition. Additionally, LC-TOF / MS analysis of the reaction mixture revealed the formation of sense and antisense strands under enzyme addition conditions. Sense strand LC / MS m / z: calcd 2266.13, found 2265.9703[M-3H] 3- Antisense chain LC / MS m / z: calcd 2531.04, found 2531.0199[M-3H] 3-

[0073] The above demonstrates that siRNA can be generated from four fragments of modified RNA using T4 RNA ligase 2.

[0074] [Example 4] Confirmation of reaction progress using DNA ligase The enzymatic ligation reaction of siRNA from four fragments of modified oligonucleotides was evaluated using DNA ligase. T4 DNA ligase (New England Biolabs) was used as the DNA ligase.

[0075] The reaction solution was composed of 50 mM Tris-HCl DNA ligase, 10 mM MgCl2, 10 mM dithiothreitol, 1 mM ATP, and pH 7.5. The enzyme concentration was 470 nM, and each oligonucleotide fragment was 10 μM. The reaction volume was 30 μL. The oligonucleotide fragments used were the combinations shown in Table 3. The reaction was carried out at 25°C using a thermal cycler, and after 4 hours, 10 μL was collected and the reaction was stopped by heating at 80°C for 5 minutes. The concentration of the ligation product in the reaction solution was analyzed by HPLC under the conditions described in Example 3. The concentration of the ligation product was quantified by similar analysis of the standard sample.

[0076] HPLC analysis confirmed the accumulation of ligation products, with the sense strand showing a concentration of 0.58 μM and the antisense strand 5.1 μM after 4 hours of reaction. As described above, the siRNA generation reaction from four modified RNA fragments proceeded even in DNA ligase.

[0077] [Example 5] Preparation of Deinococcus radiodurans RNA ligase and ligation reaction (1) Construction of recombinant expression strains using E. coli We constructed a strain expressing DraRnl, an RNA ligase belonging to the Rnl5 family derived from Deinococcus radiodurans, in E. coli, and prepared purified enzyme. First, we synthesized a plasmid containing a nucleotide sequence optimized for E. coli codons from the amino acid sequence of DraRnl (SEQ ID NO: 17) by total gene synthesis. Next, we subcloned this sequence into the NdeI / BamHI site of the pET16b vector. We transformed E. coli BL21(DE3) with this expression plasmid to obtain a DraRnl expression strain. In this expression strain, DraRnl with a His-tag attached to the N-terminus is expressed.

[0078] (2) Preparation of recombinant enzymes Each expression strain was grown overnight at 37°C on LB agar medium containing 100 mg / L ampicillin. The resulting colonies were inoculated into 100 mL of LB medium containing 100 mg / L ampicillin and cultured with shaking using a Sakaguchi flask. After 2 hours of incubation at 37°C, IPTG and ethanol were added to achieve final concentrations of 0.1 mM and 2%, respectively. The cells were then incubated at 17°C for a further 16 hours.

[0079] After culturing was complete, the bacterial cells were collected from the culture medium by centrifugation and suspended in a buffer consisting of 50 mM Tris-HCl (pH 7.6), 250 mM NaCl, 10% sucrose, 15 mM imidazole, 1% lysozyme, and 0.1% Triton-X100. The cells were then sonicated. The bacterial residue was removed from the lysate by centrifugation, and the resulting supernatant was used as the soluble fraction.

[0080] The obtained soluble fraction was subjected to a His-tag protein purification column, HisTALON Superflow Cartridge (Takara Bio), equilibrated with the buffer solution, and adsorbed onto a support. Proteins that did not adsorb to the support (unadsorbed proteins) were washed off with a buffer solution consisting of 50 mM Tris-HCl (pH 7.6), 250 mM NaCl, 10% sucrose, and 15 mM imidazole. Then, the adsorbed proteins were eluted with a buffer solution consisting of 50 mM Tris-HCl (pH 8.0), 250 mM NaCl, 10% glycerol, and 200 mM imidazole.

[0081] The elution fraction containing the enzyme was collected and buffered using Amicon Ultra-15 10kDa (Merck Millipore) with a buffer consisting of 50mM Tris-HCl (pH 8.0), 200mM NaCl, 2mM DTT, 2mM EDTA, 10% glycerol, and 0.1% Triton X-10 to obtain a purified enzyme solution.

[0082] (3) Ligation reaction by DraRnl Four modified RNA fragments were reacted with DraRnl. The reaction mixture consisted of 50 mM Tris-HCl (pH 7.5), 10 mM MnSO4, 1 mM dithiothreitol, 400 μM ATP, and 10 μM modified RNA fragments, in a volume of 25 μL. The modified RNA fragments used were the combinations shown in Table 3. The enzyme concentration was set to 72 μg / mL, and the reaction was also performed under conditions without enzyme addition as a negative control. After reacting at 25°C for 3 hours using a thermal cycler, the reaction was stopped by adding EDTA to a final concentration of 1 mM.

[0083] (4) Analysis by HPLC The reaction solution was analyzed by HPLC using an ACQUITY HPLC Oligonucleotide BEH C18 Column (Waters, 2.1 × 100 mm, 1.7 μm). The analytical conditions were: column temperature 60°C, detection wavelength 260 nm, injection volume 10 μL, and flow rate 0.4 mL / min. The mobile phase was analyzed using a linear gradient with solution A (hexafluoroisopropanol-triethylamine) and solution B (methanol). The formation of ligation products was confirmed by similar analysis of the sense and antisense strand standards.

[0084] The results of the HPLC analysis are shown in Figure 5. HPLC analysis showed that the addition of the enzyme resulted in a ligation product peak at a retention time consistent with the standard, and the peak area of ​​the modified RNA of the substrate decreased compared to the negative control. These results demonstrate that DraRnl can generate the desired modified oligonucleotide from four fragments of modified RNA.

[0085] [Example 6] Generation of modified oligonucleotides with loop structure The reaction to generate modified oligonucleotides with loop structures from four oligonucleotide fragments via enzymatic ligation was evaluated. The sequences of the target product and the synthesized substrate fragments are shown in Table 4.

[0086] [Table 4]

[0087] The four substrate oligonucleotide fragments listed in Table 4 were reacted with T4 RNA ligase 2 (New England Biolabs). The reaction mixture consisted of 50 mM Tris-HCl, 2 mM MgCl2, 1 mM dithiothreitol, 400 μM ATP, pH 7.5, and 10 μM oligonucleotide fragments, with a reaction volume of 100 μL. The product concentration was compared using an enzyme addition concentration of 17.8 μg / μL. A reaction was also performed under conditions without enzyme addition as a negative control. The reaction was stopped by heating at 80 °C for 5 minutes after reacting at 25 °C using a thermal cycler. The reaction mixture was analyzed by HPLC and LC-TOF / MS under the conditions of Example 5.

[0088] As shown in Figure 6, HPLC analysis revealed that, compared to the negative control, the addition of the enzyme reduced the peak area of ​​the modified RNA of the substrate, and a peak with a longer retention time (around 5.6 minutes) could be detected. Furthermore, LC-TOF / MS analysis of the reaction mixture showed that the target product was observed under the enzyme-added conditions. LC / MS m / z: calcd 2727.33, found 2727.21[M-6H] 6-

[0089] From the above, it was shown that T4 RNA ligase 2 can generate the desired modified oligonucleotide with a loop structure from four fragments.

[0090] [Example 7] Generation of heteroduplexes consisting of DNA and RNA strands A heteroduplex consisting of a modified DNA strand and a modified RNA strand was generated using a double-stranded RNA ligase. T4 RNA ligase 2 (New England Biolabs) was used as the double-stranded RNA ligase.

[0091] The reaction solution was composed of 50 mM Tris-HCl, 2 mM MgCl2, 1 mM dithiothreitol, 400 μM ATP, and pH 7.5. The enzyme concentration was 3.56 μg / mL, and the substrates were 10 μM each of the four oligonucleotide fragments shown in Table 5. The reaction volume was 40 μL. The reaction was carried out at 37°C using a thermal cycler, and the reaction was stopped after 18 hours by adding EDTA to a final concentration of 10 mM. The reaction was also carried out under conditions without enzyme addition as a negative control. The concentration of the ligation product in the reaction solution was analyzed by HPLC and LC-TOF / MS under the conditions described in Example 5.

[0092] HPLC analysis revealed that, compared to the negative control, the peak area of ​​each substrate decreased upon enzyme addition, and two new peaks could be detected. Furthermore, LC-TOF / MS analysis of the reaction mixture showed the generation of modified DNA and modified RNA strands under enzyme addition conditions. Modified DNA strand LC / MS m / z: calcd 2119.50, found 2119.34[M-2H] 2- Modified RNA strand LC / MS m / z: calcd 2126.89, found 2126.36[M-2H] 2-

[0093] From the above, it was shown that T4 RNA ligase 2 can generate heteroduplexes consisting of modified DNA strands and modified RNA strands.

[0094] [Table 5]

[0095] [Example 8] Reaction using oligonucleotide fragments containing mismatched base pairs The reaction to generate double-stranded modified oligonucleotides with base pair mismatches from four oligonucleotide fragments via enzymatic ligation was evaluated. The respective chains of the target product were designated as chain A and chain B. Table 6 shows the sequences of the target product and the synthesized substrate fragments, and Figure 7 shows the combinations of the four fragments.

[0096] The reaction mixture was composed of 1.78 μg / mL T4 RNA ligase 2 (New England Biolabs), 50 mM Tris-HCl, 2 mM MgCl2, 1 mM dithiothreitol, 400 μM ATP, and pH 7.5. Four oligonucleotide fragments were added as substrates to a final concentration of 10 μM each, and the reaction was carried out in 40 μL of solution. The reaction was carried out at 25°C using a thermal cycler, and the reaction was stopped after 4 hours by adding EDTA to a final concentration of 10 mM. The reaction was also carried out under conditions without enzyme addition as a negative control. The concentration of the ligation product in the reaction mixture was analyzed by HPLC and LC-TOF / MS under the conditions described in Example 5.

[0097] HPLC analysis revealed that the addition of the enzyme reduced the peak area of ​​each substrate compared to the negative control, allowing for the detection of two new peaks. Furthermore, LC-TOF / MS analysis of the reaction solution confirmed the formation of A and B chains under the enzyme-added conditions. A chain LC / MS m / z: calcd 1361.77, found 1361.75[M-5H] 5- B chain LC / MS m / z: calcd 1517.41, found 1517.35[M-5H] 5-

[0098] From the above, it was shown that T4 RNA ligase 2 can generate double-stranded modified oligonucleotides containing mismatch sequences.

[0099] [Table 6]

[0100] [Example 9] Reaction using oligonucleotide fragments 5 and 6 The reaction process for generating double-stranded modified oligonucleotides from 5- and 6-fragment oligonucleotides via enzymatic ligation was evaluated. The respective chains of the target product were designated as chain A and chain B. Table 7 shows the sequences of the target product and the synthesized substrate fragments, and Figure 8 shows the combinations of the four fragments.

[0101] The reaction mixture was composed of 1.78 μg / mL T4 RNA ligase 2 (New England Biolabs), 50 mM Tris-HCl, 2 mM MgCl2, 1 mM dithiothreitol, 400 μM ATP, and pH 7.5. Oligonucleotide fragments were added as substrates to a final concentration of 10 μM, and the reaction was carried out in 30 μL of solution. The reaction was carried out at 37°C using a thermal cycler, and the reaction was stopped after 16 hours by adding EDTA to a final concentration of 10 mM. The reaction was also carried out under conditions without enzyme addition as a negative control. The concentration of the ligation product in the reaction mixture was analyzed by HPLC under the conditions described in Example 5.

[0102] HPLC analysis, as shown in Figure 9, revealed that the addition of the enzyme reduced the peak area of ​​each substrate compared to the negative control, allowing for the detection of two new peaks. The retention times of these peaks were consistent with those of the product standard.

[0103] From the above, it was shown that T4 RNA ligase 2 can generate double-stranded modified oligonucleotides from 5-fragment and 6-fragment substrate oligonucleotides.

[0104] [Table 7]

[0105] [Example 10] Reaction using an oligonucleotide with a DMTr group attached to its 5' end The reaction to synthesize double-stranded oligonucleotides from four oligonucleotide fragments, including two fragments with dimethoxytrityl (DMTr) groups added to the 5' end, via enzymatic ligation was evaluated. The respective chains of the target product were designated as chain A and chain B. Table 8 shows the sequences of the target product and the synthesized substrate fragments, and Figure 10 shows the combinations of the four fragments.

[0106] The reaction mixture was composed of 1.78 μg / mL T4 RNA ligase 2 (New England Biolabs), 50 mM Tris-HCl, 2 mM MgCl2, 1 mM dithiothreitol, 400 μM ATP, and pH 7.5. Four oligonucleotide fragments were added as substrates to a final concentration of 10 μM each, and the reaction was carried out in 40 μL of solution. The reaction was carried out at 25°C using a thermal cycler, and the reaction was stopped after 4 hours by adding EDTA to a final concentration of 10 mM. The reaction was also carried out under conditions without enzyme addition as a negative control. The concentration of the ligation product in the reaction mixture was analyzed by HPLC and LC-TOF / MS under the conditions described in Example 5.

[0107] HPLC analysis revealed that the addition of the enzyme reduced the peak area of ​​each substrate compared to the negative control, allowing for the detection of two new peaks. Furthermore, LC-TOF / MS analysis of the reaction solution confirmed the formation of A and B chains under the enzyme-added conditions. A chain LC / MS m / z: calcd 1764.80, found 1764.79[M-4H] 4- B chain LC / MS m / z: calcd 1738.76, found 1738.75[M-4H] 4-

[0108] From the above, it was shown that double-stranded modified oligonucleotides can be generated from substrate fragments containing DMTr groups using T4 RNA ligase 2.

[0109] [Table 8]

[0110] [Example 11] Reaction using carrier-added oligonucleotide fragments The reaction to produce double-stranded modified oligonucleotides by enzymatic ligation from four oligonucleotide fragments, one of which had N-acetylgalactosamine (GalNAc) added to its 5' end, was evaluated. The respective chains of the target product were designated as A and B. Table 9 shows the sequences of the target product and the synthesized substrate fragments, and Figure 11 shows the combinations of the four fragments. The GalNAc-modified fragment was synthesized by linking Trivalent β-D-GalNAc with carboxyl-functionalized PEG5 Linker (Sussex) to the 5' end of the oligonucleotide via an amino C6 linker.

[0111] The reaction mixture was composed of 1.78 μg / mL T4 RNA ligase 2 (New England Biolabs), 50 mM Tris-HCl, 2 mM MgCl2, 1 mM dithiothreitol, 400 μM ATP, and pH 7.5. Four oligonucleotide fragments were added as substrates to a final concentration of 10 μM each, and the reaction was carried out in 40 μL of solution. The reaction was carried out at 25°C using a thermal cycler, and the reaction was stopped after 4 hours by adding EDTA to a final concentration of 10 mM. The reaction was also carried out under conditions without enzyme addition as a negative control. The ligation product contained in the reaction mixture was analyzed by HPLC and LC-TOF / MS under the conditions described in Example 5.

[0112] HPLC analysis revealed that, compared to the negative control, the peak area of ​​each substrate decreased upon enzyme addition, and two new peaks could be detected. Furthermore, LC-TOF / MS analysis of the reaction solution detected both A and B chains under enzyme addition conditions, confirming their formation. A chain LC / MS m / z: calcd 1730.40, found 1730.37[M-5H] 5- B chain LC / MS m / z: calcd 1330.38 found 1330.36[M-5H]5-

[0113] From the above, it was shown that double-stranded oligonucleotides modified with N-acetylgalactosamine at their ends can be generated by reacting four fragments using T4 RNA ligase 2.

[0114] [Table 9]

[0115] [Example 12] Reaction for the production of double-strand modified oligonucleotides having a thiophosphate diester bond in the linking portion The reaction process for generating double-stranded oligonucleotides from four oligonucleotide fragments in which phosphate groups were replaced with thiophosphate groups via enzymatic ligation was evaluated. The respective chains of the target product were designated as chain A and chain B. Table 10 shows the sequences of the target product and the synthesized substrate fragments, and Figure 12 shows the combinations of the four fragments.

[0116] The reaction mixture was composed of 1.78 μg / mL T4 RNA ligase 2 (New England Biolabs), 50 mM Tris-HCl, 2 mM MgCl2, 1 mM dithiothreitol, 400 μM ATP, and pH 7.5. Four oligonucleotide fragments were added as substrates to a final concentration of 10 μM each, and the reaction was carried out in 40 μL of solution. The reaction was carried out at 25°C using a thermal cycler, and the reaction was stopped after 4 hours by adding EDTA to a final concentration of 10 mM. The reaction was also carried out under conditions without enzyme addition as a negative control. The ligation product contained in the reaction mixture was analyzed by HPLC and LC-TOF / MS under the conditions described in Example 5.

[0117] HPLC analysis revealed that, compared to the negative control, the peak area of ​​each substrate decreased upon enzyme addition, and two new peaks could be detected. Furthermore, LC-TOF / MS analysis of the reaction solution detected both A and B chains under enzyme addition conditions, confirming their formation. A chain LC / MS m / z: calcd 1769.15, found 1769.13[M-4H] 4- B chain LC / MS m / z: calcd 1743.11 found 1743.10[M-4H] 4-

[0118] From the above, it was shown that double-stranded modified oligonucleotides with thiophosphate bonds at the ligation site can be generated by reacting four fragments using T4 RNA ligase 2.

[0119] [Table 10]

[0120] [Example 13] Reaction for the production of hairpin-shaped oligonucleotides The reaction to generate hairpin-shaped oligonucleotides from four oligonucleotide fragments via enzymatic ligation was evaluated. The sequences of the target product and synthesized substrate fragments are shown in Table 11, and the combinations of the four fragments are shown in Figure 13. A proline derivative described in the literature (Hamasaki T, Suzuki H, Shirohzu H, et al. Efficacy of a novel class of RNA interference therapeutic agents. PLoS ONE. 2012;7(8):e42655.) was used as the linker.

[0121] The reaction mixture was composed of 1.78 μg / mL T4 RNA ligase 2 (New England Biolabs), 50 mM Tris-HCl, 2 mM MgCl2, 1 mM dithiothreitol, 400 μM ATP, and pH 7.5. Four oligonucleotide fragments were added as substrates to a final concentration of 10 μM each, and the reaction was carried out in 40 μL of solution. The reaction was carried out at 25°C using a thermal cycler, and the reaction was stopped after 4 hours by adding EDTA to a final concentration of 10 mM. The reaction was also carried out under conditions without enzyme addition as a negative control. The ligation product contained in the reaction mixture was analyzed by HPLC and LC-TOF / MS under the conditions described in Example 5.

[0122] HPLC analysis showed that the addition of the enzyme reduced the peak area of ​​each substrate compared to the negative control, and a new peak could be detected. Furthermore, LC-TOF / MS analysis of the reaction solution detected the target product under the enzyme-added conditions, confirming its formation. LC / MS m / z: calcd 1891.61 found 1891.60[M-9H] 9-

[0123] From the above, it was shown that hairpin-shaped oligonucleotides can be generated by the reaction of four fragments using T4 RNA ligase 2.

[0124] [Table 11]

[0125] [Example 14] Effect of the base length of the protruding end on reactivity The products were identical, and the substrate oligonucleotides were designed to form overhangs of 1 to 6 nucleotides in length. The differences in reactivity due to differences in the length of these overhangs were then compared. The target products were designated as A and B chains. In each combination, the substrate constituting the B chain had a common sequence, while the cleavage positions of the A chain differed. The sequences of the synthesized target product standards and substrate fragments are shown in Table 12, and the combinations of the four fragments are shown in Figure 14.

[0126] The reaction mixture was composed of 1.78 μg / mL T4 RNA ligase 2 (New England Biolabs), 50 mM Tris-HCl, 2 mM MgCl2, 1 mM dithiothreitol, 400 μM ATP, and pH 7.5. Four oligonucleotide fragments were added as substrates to a final concentration of 10 μM each, and the reaction was carried out in 40 μL of solution. The reaction was carried out at 25°C using a thermal cycler, and 5 μL was collected at 15 minutes, 30 minutes, 1 hour, 2 hours, and 4 hours, and the reaction was stopped by adding EDTA to a final concentration of 10 mM. The reaction was also carried out under conditions without enzyme addition as a negative control. The concentrations of the ligation product and product standard in the reaction mixture were analyzed by HPLC under the conditions described in Example 5, and the concentration of the ligation product was calculated.

[0127] HPLC analysis confirmed that the formation of both A and B chains was observed at overhanging ends of all base lengths. The reaction rate tended to be lower at overhanging ends of 1 base length.

[0128] [Table 12]

[0129] [Example 15] Effect of base length on the reactivity of the product The differences in reactivity when short target products consisting of complementary regions of 11-14 nucleotide lengths were prepared were compared. The respective chains of the target product were designated as chain A and chain B. Table 13 shows the sequences of the target product and the synthesized substrate fragments, and Figure 15 shows the combinations of the four fragments.

[0130] The reaction mixture was composed of 1.78 μg / mL T4 RNA ligase 2 (New England Biolabs), 50 mM Tris-HCl, 2 mM MgCl2, 1 mM dithiothreitol, 400 μM ATP, and pH 7.5. Four oligonucleotide fragments were added as substrates to a final concentration of 10 μM each, and the reaction was carried out in 40 μL of solution. The reaction was carried out at 25°C using a thermal cycler, and the reaction was stopped after 4 hours by adding EDTA to a final concentration of 10 mM. The reaction was also carried out under conditions without enzyme addition as a negative control. The ligation product contained in the reaction mixture was analyzed by HPLC and LC-TOF / MS under the conditions described in Example 5.

[0131] HPLC analysis revealed that, compared to the negative control, the addition of the enzyme reduced the peak area of ​​each substrate, and new peaks were detected at later retention times. Furthermore, LC-TOF / MS analysis of the reaction solution showed that, under enzyme-added conditions, divalent or trivalent ions of the A and B chains were detected in products of all base lengths, as shown in Table 14, confirming the progress of the reaction.

[0132] The peak area values ​​of the substrate under each reaction condition were calculated using HPLC, and the substrate retention rate was calculated using the following formula. Remaining percentage (%) = (Total peak area of ​​substrate under enzyme addition conditions) (Total peak area of ​​substrates in negative control) × 100

[0133] Products consisting of complementary regions of 11 nucleotides tended to have a higher substrate retention rate compared to products consisting of complementary regions of 12 nucleotides or longer.

[0134] [Table 13]

[0135] [Table 14]

[0136] [Example 16] Reaction with high-concentration substrate The reaction rates for the formation of modified oligonucleotides in the presence of higher substrate concentrations were compared at various pH levels. The chains of the target product were designated as chain A and chain B. Table 15 shows the sequences of the target product and the synthesized substrate fragments, and Figure 16 shows the combinations of the four fragments.

[0137] The reaction mixture consisted of 1.78 μg / mL T4 RNA ligase 2 (New England Biolabs), 2 mM MgCl2, 1 mM dithiothreitol, and 400 μM ATP, with 50 mM Tris-HCl (pH 7.0-9.0) used as buffer. Oligonucleotide fragments were added as substrates to final concentrations of 10 μM, 300 μM, 500 μM, or 1000 μM, and the reaction was carried out in 40 μL volumes. The reaction was carried out at 25°C using a thermal cycler, and after 15 minutes or 1 hour, samples were taken and the reaction was stopped by adding EDTA to a final concentration of 10 mM. The ligation product contained in the reaction mixture was analyzed by HPLC under the conditions described in Example 5, and the production rate was calculated from the total concentration of the A and B chains. Reaction progress was confirmed even at substrate concentrations of 300 μM or higher, and high reaction rates were observed at pH 8.0 and 8.5 at these concentrations.

[0138] [Table 15]

[0139] [Example 17] Effect of adding surfactant The reaction rate of modified oligonucleotide formation by adding a surfactant was evaluated. The sequences of the target product and synthesized substrate fragments are shown in Table 15, and the combinations of the four fragments are shown in Figure 16. The reaction mixture consisted of 1.78 μg / mL T4 RNA ligase 2 (New England Biolabs), 2 mM MgCl2, 1 mM dithiothreitol, 400 μM ATP, and 50 mM Tris-HCl (pH 7.5). Triton X-100 was used as the surfactant at a final concentration of 0.1%. Oligonucleotide fragments were added as substrates to a final concentration of 20 μM each, and the reaction was carried out in 40 μL of solution. The evaluation compared three conditions: a control condition in which the enzyme solution was diluted to 17.8 μg / mL in a storage buffer (10 mM Tris-HCl, 50 mM KCl, 35 mM ammonium sulfate, 0.1 mM dithiothreitol, 0.1 mM EDTA, 50% glycerol, pH 7.5) and then added to the reaction mixture at a 1 / 10 volume; a test condition 1 in which the enzyme solution was diluted to 17.8 μg / mL in a storage buffer and then added to a reaction mixture containing 0.1% TritonX-100 at a 1 / 10 volume; and a test condition 2 in which the enzyme solution was diluted to 17.8 μg / mL in a storage buffer containing 0.1% TritonX-100 and then added to a reaction mixture containing 0.09% TritonX-100 at a 1 / 10 volume (TritonX-100 final concentration 0.1%). The reaction was carried out at 25°C using a thermal cycler, and after 4 hours, a sample was taken and the reaction was stopped by adding EDTA to a final concentration of 10 mM. The ligation product contained in the reaction solution was analyzed by HPLC under the conditions described in Example 5, and the concentrations of chain A and chain B were calculated. Under test conditions 1 and 2, higher concentrations of A-chain and B-chain products were observed compared to the control condition.

[0140] [Example 18] Comparison of impurity selection properties based on differences in base length of the product Reactions with different base lengths were carried out using substrates and products, and impurities in the substrate oligonucleotide fragments and the solution after the enzymatic reaction were analyzed. The respective chains of the target product were designated as chain A and chain B. The sequences of the target product and the synthesized substrate fragments are shown in Table 16, and the combinations of the four fragments are shown in Figure 17.

[0141] The reaction mixture composition for the reaction using modified oligonucleotides was 8.9 μg / mL T4 RNA ligase 2 (New England Biolabs), 50 mM Tris-HCl, 2 mM MgCl2, 1 mM dithiothreitol, 400 μM ATP, and pH 7.5. For the reaction using DNA, the composition was 22.0 μg / mL T7 DNA ligase (New England Biolabs), 50 mM Tris-HCl, 2 mM MgCl2, 1 mM dithiothreitol, 400 μM ATP, and pH 7.5. Four oligonucleotide fragments were added as substrates to a final concentration of 50 μM each, and the reaction was carried out in 30 μL of solution. The reaction was carried out at 25°C using a thermal cycler, and the reaction was stopped after 8 hours by adding EDTA to a final concentration of 12.5 mM. The reaction mixture was analyzed by HPLC and LC-TOF / MS under the conditions described in Example 5 to confirm the progress of the desired reaction.

[0142] Based on the obtained mass spectrometry results, the content of impurities (N±1mer) relative to the product of the target structure was calculated according to the method described in the previous literature (Roussis et al., Journal of Chromatogr A. 2019;1584:106-114.). Similarly, the substrate oligonucleotide solution used in the reaction was also analyzed by LC-TOF / MS, and the content of impurities (N±1mer) relative to the substrate was calculated.

[0143] Based on the values ​​calculated above, the percentage of remaining impurities was calculated for each of the A and B chains of each reaction using the following formula. Percentage of impurities remaining (%) = (Ratio of N±1 mers to the target product in the reaction solution) / (Ratio of N±1 mers to the substrate in the substrate solution) × 100

[0144] The results are shown in Table 17. In the reaction that produced oligonucleotides with a length of 28 bases, the residual rate of impurities (N±1mer) was 86% or more, while in the reaction that produced oligonucleotides with a length of 25 bases or less, it ranged from 23% to 59%.

[0145] [Table 16]

[0146] [Table 17] [Industrial applicability]

[0147] The present invention is useful for producing modified oligonucleotides (e.g., siRNA and heterodouble-stranded oligonucleotides) that can be used in products such as nucleic acid drugs.

Claims

[Claim 1] A method for producing a modified oligonucleotide containing a complementary portion of 11 to 27 base lengths, The method includes treating a total of four or more oligonucleotide raw material fragments in the presence of an oligonucleotide ligase to produce the modified oligonucleotide, A total of four or more oligonucleotide raw material fragments correspond to oligonucleotide raw material fragments obtained when the modified oligonucleotide is divided at fragment linkages that satisfy the following conditions (i) to (v): (i) There is one or more fragment linkers in each chain of the complementary portion, and there are a total of two or more fragment linkers in the modified oligonucleotide; (ii) When the modified oligonucleotide is separated at the fragment linkage, the protruding end is formed in the complementary portion, and the protruding end is 1 to 10 bases long; (iii) At least one oligonucleotide raw material fragment contains a modified nucleotide, (iv) Of the total of four or more oligonucleotide raw material fragments, four oligonucleotide raw material fragments include a complementary portion of 5 to 25 bases in length, (v) The sum of the base lengths corresponding to each strand of the complementary portion of the oligonucleotide raw material fragment is 11 to 27 base lengths.

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