Method for producing modified oligonucleotides comprising complementary moieties

Abstract

Provided is a method for efficiently producing an oligonucleotide including a complementary moiety, such as siRNA or a heteroduplex oligonucleotide. More specifically, the present invention provides a method for producing a modified oligonucleotide comprising a complementary moiety having 11-27 bases in length, the method comprising: treating a total of 4 or more oligonucleotide starting material fragments in the presence of an oligonucleotide ligase to produce the modified oligonucleotide; the total four or more oligonucleotide raw material fragments are equivalent to oligonucleotide raw material fragments obtained when the modified oligonucleotide is cleaved at fragment linkers satisfying specific conditions.

Description

This application is a divisional application of a patent application with the original application date of February 18, 2020, application number 202080014985.7 (international application number PCT / JP2020 / 006366), and invention name “Method for producing modified oligonucleotides containing complementary parts”. Technical Field

[0001] The present invention relates to a method for producing a modified oligonucleotide containing a complementary portion. Background Art

[0002] Oligonucleotides such as siRNA and antisense oligonucleotides have been shown to be useful as nucleic acid drugs, and their development has become increasingly active in recent years. Oligonucleotides are primarily manufactured by synthesis methods, for example, by sequentially extending the nucleotide residues one base at a time in series using solid-phase synthesis methods such as the phosphoramidite method. However, this method has problems such as decreased product purity and yield as the length of the oligonucleotide chain increases, or low production efficiency. Therefore, it is necessary to synthesize oligonucleotides as short chain fragments and condense them to obtain a parallel synthesis method for the target oligonucleotide.

[0003] Patent Document 1 describes a method for producing a single-stranded oligonucleotide by annealing a plurality of oligonucleotide starting fragments corresponding to fragments obtained by cleaving (shearing) a target oligonucleotide with a template oligonucleotide complementary to the target oligonucleotide, condensing the annealed oligonucleotide starting fragments with an enzyme, and separating the resulting target oligonucleotide chain from the template oligonucleotide. Non-Patent Document 1 describes PEGylation of oligoDNA by ligating an oligoDNA fragment to a PEGylated oligoDNA fragment at its cohesive termini using DNA ligase. However, since Non-Patent Document 1 only uses native oligoDNA fragments, it is unclear whether enzyme-based condensation of oligonucleotides containing complementary moieties using short-chain oligonucleotides containing modified bases, which are thought to have reduced annealing ability, is feasible. Non-patent documents 2 and 3 describe the use of ligase to connect nicks formed by annealing one oligonucleotide chain to two complementary oligonucleotide starting fragments. Non-Patent Document 4 describes the use of RNA ligase to ligate 24-mer double-stranded oligoRNAs with sticky ends to form double-stranded RNAs of 48 or greater lengths. However, in Non-Patent Document 4, the substrate is 24 bases long and the product is 48 bases long, both of which are relatively long. Therefore, the method is only performed under conditions where the substrate has a high annealing ability. Therefore, it is unknown whether an enzymatic reaction with more than two condensation points can be performed using short oligonucleotide substrates, which are thought to have significantly reduced annealing ability and are presumed to have reduced enzyme ligation activity due to the introduction of modified bases. Non-patent document 5 describes the production of siRNA by synthesis; Non-Patent Document 6 describes that RNA ligase DraRn1 is included in the Rn15 family.

[0004] Prior art literature Patent Literature Patent Document 1: U.S. Patent Application Publication No. 2018 / 0023122.

[0005] Non-patent literature Non-Patent Literature 1: Sosic A, Pasqualin M, Pasut G, Gatto B. 2014. Enzymatic formation of PEGylated oligonucleotides. Bioconjug Chem 25: 433-441. Non-patent document 2: Bullard, DR, & Bowater, RP (2006). Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4. Biochem. J, 398, 135-144. Non-patent document 3: Nandakumar, J., & Shuman, S. (2004). How an RNA Ligase Discriminates RNA versus DNA Damage. Molecular Cell, 16(2), 211-221. Non-patent literature 4: Nandakumar J, Ho CK, Lima CD, Shuman S. 2004. RNA substrate specificity and structure-guided mutational analysis of bacteriophage T4 RNAligase 2.J Biol Chem 279: 31337-31347. Non-patent document 5: Jayaprakash K. Nair, et al. (2014), Multivalent N-Acetylgalactosamine-Conjugated siRNA Localizes in Hepatocytes and Elicits Robust RNAi-Mediated Gene Silencing. J. Am. Chem. Soc., 136, 16958-16961. Non-patent literature 6: MIHAELA-CARMEN UNCIULEAC and STEWART SHUMAN (2019), RNA 21: 824-832. Summary of the Invention

[0006] Technical problem to be solved by the invention An object of the present invention is to provide an efficient method for producing oligonucleotides containing complementary portions, such as siRNA and heteroduplex oligonucleotides.

[0007] Technical solutions used to solve technical problems After careful investigation, the present inventors discovered that by treating four or more oligonucleotide raw material fragments corresponding to fragments obtained by cleaving two complementary parts of a target oligonucleotide containing complementary parts with an oligonucleotide ligase, double-stranded oligonucleotides containing complementary parts can be directly constructed. Compared with tandem synthesis methods such as solid-phase synthesis, it can be produced with higher manufacturing efficiency and higher purity, thereby completing the present invention.

[0008] In the past, shorter double-stranded oligonucleotides such as siRNA were manufactured by chemical synthesis because chemical synthesis was easy and simple. Specifically, by carrying out chemical synthesis (for example, solid phase synthesis) respectively to 2 oligonucleotide chains of formation, after they were purified, two chains were annealed to manufacture. Therefore, there are few reports using the method for enzymatic condensation, wherein, the method for enzymatic synthesis by more than 4 oligonucleotide raw material fragments is only reported to have a method for manufacturing a double-stranded oligonucleotide with a longer chain of more than 28 bases. For shorter oligonucleotides, the base length of the oligonucleotide raw material fragments used for its manufacture will naturally shorten, but it is believed that the annealing ability of the oligonucleotide raw material fragments with shorter base lengths will also decline. If the nucleotides of formation are modified, it is also believed that the annealing ability will further decline. Therefore, for the enzymatic oligonucleotide synthesis method using ligase, the annealing ability of the oligonucleotide raw material fragments is considered to be very important, so in the past, no one has attempted to use ligase to synthesize the method for oligonucleotides with a length of more than 4 oligonucleotide raw material fragments less than 28 bases, etc., which are shorter chains. However, after intensive research, the present inventors discovered that, contrary to expectations, using a ligase and four or more oligonucleotide starting fragments allows the production of double-stranded oligonucleotides of less than 28 bases in length, without being affected by annealing ability. Furthermore, in the case of short-stranded oligonucleotides, the purity of the produced oligonucleotides was also found to be improved, leading to the completion of the present invention.

[0009] That is, the present invention is as follows; [1] A method for producing a modified oligonucleotide comprising a complementary portion having a length of 11 to 27 bases, wherein: The method comprises treating a total of four or more oligonucleotide raw material fragments in the presence of an oligonucleotide ligase, wherein the total of four or more oligonucleotide raw material fragments correspond to the oligonucleotide raw material fragments obtained by cleaving the modified oligonucleotide at the fragment junction that satisfies the following conditions (i) to (v): (i) one or more fragment junctions are present on each strand side of the complementary portion, and a total of two or more fragment junctions are present in the modified oligonucleotide; (ii) when the modified oligonucleotide is cleaved at the fragment junction, an overhanging end is formed in the complementary portion, and the length of the overhanging end is 1 to 10 bases; (iii) at least one oligonucleotide starting material segment comprises a modified nucleotide; (iv) four of the four or more oligonucleotide starting fragments contain complementary portions with a length of 5 to 25 bases; and (v) the total length of bases corresponding to each strand of the complementary portion of the oligonucleotide starting fragment is 11 to 27 bases in length; [2] The method according to [1], wherein the length of the protruding end in (ii) is 2 to 6 bases; [3] The method according to [1] or [2], wherein the length of the complementary portion of the four oligonucleotide starting fragments specified in (iv) excluding the overhanging ends is 4 to 16 bases; [4] The method according to any one of [1] to [3], wherein the oligonucleotide ligase is an RNA ligase; [5] The method according to [4], wherein the oligonucleotide ligase is a double-stranded RNA ligase; [6] The method according to [5], wherein the double-stranded RNA ligase is an RNA ligase of the Rnl2 family or the Rnl5 family; [7] The method according to any one of [1] to [6], wherein the modified oligonucleotide comprises modified nucleotide residues; [8] The method according to [7], wherein the modified nucleotide residue is a 1', 2', 3' or 4' chemically modified nucleotide residue, a 5'- or 3'-phosphate modified nucleotide residue, a cross-linked modified nucleotide residue, a carrier-added modified nucleotide residue, or a sugar backbone substituted nucleotide residue; [9] The method according to [8], wherein the modified nucleotide residue is the following residue: i) Position 1', 2', 3' or 4' is replaced by C 1～6 Alkyloxy C 1～6 Alkylene, -OC 1～6 Alkyl, -OC 6～14 Aryl, -C-aryl, halogen atom, -OC 1～6 Alkyl N-amide C 1～6 Alkylene, -OC 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) substituted 1', 2', 3', or 4' chemically modified nucleotide residues; ii) a 5'- or 3'-phosphate-modified nucleotide residue in which the hydroxyl group may be substituted with a protecting group, or substituted with -OP(S)(OH)2, -NH-P(O)(OH)2, or -NH-P(S)(OH)2; iii) Positions 2' and 4' are replaced by 2'-OC 1～6 Alkylene-4', 2'-O-ethylene-4', 2'-O-methyl substituted methylene-4', 2'-OC 1～6 Alkylene-OC 1～6 Alkylene-4', 2'-ON(R)-C1～6 Alkylene-4' (where R represents a methyl group, a hydrogen atom, or a benzyl group), 2'-N(R)-C(O)-4', 2'-NH-C 1～6 Alkylene-4', or 2'-C 1～6 Alkylene-4' substitution, or 3' and 5' positions are replaced by 3'-C 1～6 Alkylene-5' substituted cross-linking modified nucleotide residues; or iv) a hexitol nucleic acid (HNA) residue, a cyclohexenyl nucleic acid (CeNA) residue, or a morpholino nucleic acid (PMO) residue;

[10] The method according to any one of [1] to [9], wherein the total molar ratio of any two oligonucleotide starting fragments selected from a total of four or more oligonucleotide starting fragments is 0.5 to 2;

[11] The method according to any one of [1] to

[10] , wherein the oligonucleotide starting material fragment is treated at a monovalent cation salt concentration of 10 mM or less;

[12] The method according to any one of [1] to

[11] , wherein the oligonucleotide raw material fragment mixed solution is not subjected to a high temperature and then cooled before the treatment;

[13] The method according to any one of [1] to

[12] , wherein the generation of impurities in the modified oligonucleotide is suppressed;

[14] The method according to any one of [1] to

[13] , further comprising purifying the modified oligonucleotide.

[0010] Effects of the Invention According to the method of the present invention, modified oligonucleotides such as siRNA and heteroduplex oligonucleotides can be efficiently produced with high purity.

[0011] Brief description of the accompanying drawings Figure 1 ] Figure 1 is a schematic diagram showing an example of the structure of the present invention; [ Figure 2-1 ] Figure 2-1 to Figure 2-6 This is a graph showing the amount of siRNA produced in response to the concentration of T4 RNA ligase 2 when six patterns (combination numbers 1 to 6) of four short natural RNA fragments for producing the same siRNA were reacted using T4 RNA ligase 2 in Example 1; [ Figure 2-2 ] Figure 2-1 to Figure 2-6 As mentioned above; [ Figure 2-3 ] Figure 2-1 to Figure 2-6 As mentioned above; [ Figure 2-4 ] Figure 2-1 to Figure 2-6 As mentioned above; [ Figure 2-5 ] Figure 2-1 to Figure 2-6 As mentioned above; [ Figure 2-6 ] Figure 2-1 to Figure 2-6 As mentioned above; [ Figure 3-1 ] Figure 3-1 to Figure 3-4 This is a graph showing the temporal changes in the amount of siRNA produced at various reaction temperatures when a combination of four short-chain natural RNA fragments was reacted using T4 RNA ligase 2 in Example 2; [ Figure 3-2 ] Figure 3-1 to Figure 3-4 As mentioned above; [ Figure 3-3 ] Figure 3-1 to Figure 3-4 As mentioned above; [ Figure 3-4 ] Figure 3-1 to Figure 3-4 As mentioned above; [ Figure 4 ] Figure 4 This is a graph showing the confirmation of siRNA and oligonucleotide authentic products (standard products, genuine products) generated from modified RNA at various T4 RNA ligase 2 concentrations in Example 3 based on HPLC analysis; [ Figure 5 ] Figure 5 Graphs showing HPLC analysis of reaction products in the absence of enzyme and in the presence of RNA ligase (DraRn1) derived from Deinococcus radiodurans in Example 5, as well as authentic RNA preparations (sense and antisense strands); [ Figure 6 ] Figure 6 A diagram showing a schematic diagram of the reaction product in Example 6, and 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 Graph showing the relationship between four oligonucleotide starting fragments (including oligonucleotide starting fragments containing mismatched base pairs) and double-stranded modified oligonucleotides generated therefrom; [ Figure 8 ] Figure 8 is a diagram showing the relationship between 5 or 6 oligonucleotide starting material fragments and double-stranded modified oligonucleotides generated therefrom; [ Figure 9 ] Figure 9 Graph showing confirmation of the generation of double-stranded modified oligonucleotides in reactions using 5 or 6 oligonucleotide starting material fragments by HPLC analysis; [ Figure 10 ] Figure 10Graph showing the relationship between four oligonucleotide starting material fragments including an oligonucleotide starting material fragment having a DMTr group added to its 5' end and double-stranded modified oligonucleotides generated therefrom; [ Figure 11 ] Figure 11 Graph showing the relationship between four oligonucleotide starting material fragments including an oligonucleotide starting material fragment with a carrier added to the 5' end and double-stranded modified oligonucleotides generated therefrom; [ Figure 12 ] Figure 12 This is a diagram showing the relationship between four oligonucleotide starting material fragments in which the phosphate groups at the linking portions of the nucleotide residues are replaced with phosphorothioate groups and the double-stranded modified oligonucleotides generated therefrom; [ Figure 13 ] Figure 13 Graph showing the relationship between four oligonucleotide starting material fragments and the hairpin-type modified oligonucleotides generated therefrom; [ Figure 14 ] Figure 14 Graph showing the relationship between four oligonucleotide starting fragments and double-stranded modified oligonucleotides generated therefrom for comparison of the effect of the base length of the overhanging end in the oligonucleotide starting fragment on reactivity; [ Figure 15 ] Figure 15 This figure shows the relationship between four oligonucleotide starting material fragments and double-stranded modified oligonucleotides generated therefrom, used to compare the effect of product base length on reactivity; [ Figure 16 ] Figure 16 This is a diagram showing the relationship between four oligonucleotide starting material fragments and double-stranded modified oligonucleotides generated therefrom, used to investigate the initial reaction velocity at high substrate concentrations; [ Figure 17 ] Figure 17 This is a diagram showing the relationship between four oligonucleotide starting material fragments and double-stranded modified oligonucleotides generated therefrom for comparison of product base lengths. DETAILED DESCRIPTION

[0012] (Summary of the Invention) Hereinafter, the present invention will be described. For the convenience of description of the present invention, an example of the structure of the present invention is shown in Figure 1 However, this schematic diagram is merely an example for illustrating 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 bases in length (hereinafter also referred to as a "target modified oligonucleotide"). The method of the present invention comprises 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 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 various treatment conditions for implementing the method of the present invention are described in detail below.

[0014] (Target modified oligonucleotide) The modified oligonucleotide of interest produced by the method of the present invention is a modified oligonucleotide comprising a complementary portion having a length of 11 to 27 bases.

[0015] "Oligonucleotide" refers to an oligomer containing nucleotide residues as monomer units. Examples of "oligonucleotide" include oligo RNA, oligo DNA, and RNA-DNA hybrid oligonucleotides.

[0016] Oligonucleotides can be classified into "natural oligonucleotides" and "modified oligonucleotides". "Natural oligonucleotides" refer to oligonucleotides composed of nucleotide residues (adenosine (A), guanosine (G), cytidine (C), uridine (U), deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC), thymidine (dT, hereinafter referred to as "natural nucleotide residues") that constitute the polynucleotides (RNA and DNA) contained in cells. "Modified oligonucleotides" refer to oligonucleotides other than "natural oligonucleotides", and are oligonucleotides that contain constituent elements (hereinafter referred to as "modified residues") other than natural nucleotide residues. As modified residues, for example, modified nucleotide residues, amino acid residues, and linkers can be listed. As modified nucleotide residues, for example, nucleotide residues containing modifications described later can be listed. Amino acids include derivatives of amino acids. As amino acids, 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 can be listed.Amino acid derivatives refer to amino acids in which any atom or group in an amino acid is replaced by other atoms or groups, and examples thereof include amino acids in which any atom or group in an amino acid, a hydrogen atom in a carboxyl group, an oxygen atom, a hydroxyl group, any atom or group in a side chain, or a hydrogen atom bonded to a backbone carbon atom (e.g., α-, β-, γ-, δ-carbon atom) is replaced by other atoms (e.g., halogen atoms such as fluorine atoms, chlorine atoms, bromine atoms, iodine atoms) or groups (e.g., substituents after substitution in chemical modifications described later).

[0017] The “modification” in “modified nucleotide residue” includes: substitution of a substituent on the sugar portion (ribose or deoxyribose) of the nucleotide residue, substitution of the sugar portion itself (sugar backbone) of the nucleotide residue, and modification of the nucleic acid base portion of the nucleotide residue (for example, substitution of a substituent on the nucleic acid base portion).

[0018] Examples of "substitution of a substituent on the sugar portion of a nucleotide residue" include substitution with 1'-H, 2'-OH (ribose only), 2'-H, 3'-OH, 3'-NH2, 3'-H, 3'-phosphate, 4'-H, 5'-phosphate, or a combination thereof. Here, "phosphate" is not limited to -OP(O)(OH)2, but also includes groups in which an oxygen atom is replaced by a sulfur atom or NH (e.g., -OP(S)(OH)2, -NH-P(O)(OH)2, -NH-P(S)(OH)2). Furthermore, substitution of the hydroxyl group (-OH) in the phosphate group with OR * (Where R * The term "phosphate group" also includes groups (e.g., protected phosphate groups) that represent organic groups such as protective groups for phosphate groups. Examples of such substitutions (replacements) include 1', 2', 3', or 4'-chemical modifications (substitution at the 1', 2', 3', or 4'-position with another substituent), 5'- or 3'-phosphate modifications (substitution of the 5'- or 3'-phosphate group with another substituent), cross-linking modifications (cross-linking of two groups at the 1', 2', 3', or 4'-position), and carrier-added modifications (substitution of the 1', 2', 3', 4', or 5'-position with a carrier).

[0019] For example, chemical modification can be introduced to improve the degradation resistance of oligonucleotides. Examples of the substituents after substitution in chemical modification 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 Alkylene (e.g., -ON-methylacetamide, -O-NMA), -OC 1～ 6-alkyl-(C 1～6 Alkyl-)amino-C 1～6 Alkylene (e.g., -O-dimethylaminoethoxyethyl, -O-DMAEOE), and -O-amino C 1～6Alkyl (e.g., -O-aminopropyl, -O-AP). Chemical modification is preferably 2'-chemical modification (substitution at the 2' position) or 3'-chemical modification (substitution at the 3' position), and more preferably 2'-chemical modification (substitution at the 2' position). As the substituent after substitution in 2'-chemical modification, for example, 2'-C 1～6 Alkyloxy C 1～6 Alkylene (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 atom (e.g., 2'-F), 2'-OC 1～6 Alkyl N-amide C 1～6 Alkylene (e.g., 2'-ON-methylacetamide, 2'-O-NMA), 2'-OC 1～6 Alkyl-(C 1～6 Alkyl-)amino-C 1～6 Alkylene (e.g., 2'-O-dimethylaminoethoxyethyl, 2'-O-DMAEOE), and 2'-O-amino C 1～6 Alkyl (e.g., 2'-O-aminopropyl, 2'-O-AP). As the substituent after substitution in the 3'-chemical modification, for example, 3'-OP(O)(OH)2, 3'-OP(S)(OH)2, 3'-NH-P(O)(OH)2, 3'-NH-P(S)(OH)2, and the hydroxyl group (-OH) in the phosphate group is replaced by OR * (Where R * represents an organic group such as a protective group for a phosphate group described later).

[0020] 5'- or 3'-phosphate modifications can be introduced, for example, to improve the resistance of oligonucleotides to degradation. As 5'- or 3'-phosphate modifications, for example, substitutions in which the oxygen atom in the phosphate group is replaced by a sulfur atom or NH group can be cited. Examples of such groups include: -OP(S)(OH)2 (thiophosphate group: thiophosphate type modification), -NH-P(O)(OH)2, and -NH-P(S)(OH)2. In addition, 5'- or 3'-phosphate modifications also include substitutions in which the hydroxyl group (-OH) in the phosphate group is replaced by OR * (Where R * The phosphate protecting group includes, for example, trityl (Tr), p-methoxyphenyldiphenylmethyl (MMTr), di(p-methoxyphenyl)phenylmethyl (DMTr), and cyanoethyl (CN-C2H4-).

[0021] Cross-linking modification (bridging modification) can be introduced, for example, to improve the three-dimensional stability of nucleotide residues. Examples of cross-linking modification include: 2'4'-cross-linking modification (substitution of cross-linking 2'-OH and 4'-H), 3'5'-cross-linking modification (substitution of cross-linking 3'-H and 5'-H), etc. Examples of 2'4'-cross-linking modification include: substitution of 2'-OH and 4'-H with 2'-OC 1～6 Alkylene-4' (e.g., 2'-O-methylene-4' (locked nucleic acid: LNA), 2'-O-ethylene-4' (ethylene-bridged nucleic acid: ENA), 2'-OH and 4'-H substituted with 2'-O-methyl substituted methylene-4' (constrained ethyl bridged nucleic acid: a type of BNA (cEt-BNA)), 2'-OH and 4'-H substituted with 2'-OC 1～6 Alkylene-OC 1～6 Alkylene-4' (e.g., 2'-O-methylene-O-methylene-4' (a type of cross-linked nucleic acid: BNA) COC ))、2'-OH and 4'-H are replaced by 2'-ON(R)-C 1～6 Alkylene-4' (e.g., 2'-ON(R)-methylene-4' (a type of cross-linked nucleic acid: BNA) NC ), where R represents a methyl group, a hydrogen atom, or a benzyl group), 2'-NH2 and 4'-H are replaced by 2'-N(R)-C(O)-4' (e.g., 2'-N(methyl)-C(O)-4' (amide cross-linked nucleic acid: AmNA)), 2'-NH2 and 4'-H are replaced by 2'-NH-C 1～6 Alkylene-4' (e.g., 2'-NH-methylene-4'), 2'-H and 4'-H are substituted with 2'-C 1～6 Alkylene-4' (for example, 2'-methyl substituted ethylene-4'). In addition, as a 3'5'-crosslinking type modification, for example, 3'-H and 5'-H are replaced by 3'-C 1～6 Alkylene-5' (for example, 3'-ethylene-5' (bicyclic nucleic acid: Bc nucleic acid), a type of Bc nucleic acid: tc nucleic acid, etc.)

[0022] The carrier in the carrier addition modification can be a carrier used to improve or impart stability, targeting, efficacy, and other properties to the modified oligonucleotide of the target. Such a carrier can be appropriately selected from known carriers according to the purpose of use. Examples of carriers include: N-acetylgalactosamine (GalNAc), peptides, phosphate, cholesterol, tocopherol, fatty chains, and folic acid. The addition site in the carrier addition modification is preferably: the 3' or 5' position (site) corresponding to the end of the modified oligonucleotide of the target.

[0023] Examples of modified nucleotide residues comprising "substitution of the sugar moiety of the nucleotide residue itself" (sugar backbone substituted nucleotide residues) include nucleotide residues comprising substitution of a five-membered sugar ring in an oligonucleotide with a six-membered pseudo-sugar ring, such as hexitol nucleic acid (HNA) and cyclohexenyl nucleic acid (CeNA). Furthermore, examples of modified nucleotide residues comprising "substitution of the sugar moiety of the nucleotide residue itself" include morpholino nucleic acid (PMO) residues, which are artificial compounds similar to nucleotides having a morpholine ring structure that is not decomposed by enzymes in the body (e.g., nucleases such as RNase) and does not induce an immune response.

[0024] Examples of "modification of the nucleobase moiety of a nucleotide residue" include modifications in which the nucleobase moiety of a nucleotide residue is substituted with an alkyl group (for example, modification in which the 5-position of cytosine is substituted with a methyl group).

[0025] " Oligonucleotide comprising a complementary portion " refers to an oligonucleotide comprising a structure in which complementary nucleotide sequences are paired with each other. As " oligonucleotide comprising a complementary portion ", for example, double-stranded oligonucleotides, single-stranded oligonucleotides comprising a double-stranded structure (for example, ring (loop) type oligonucleotides such as hairpin oligonucleotides and dumbbell oligonucleotides). Double-stranded oligonucleotides can be a double-stranded oligonucleotide in which each chain is the above-mentioned oligonucleotide, for example, double-stranded oligoRNA, double-stranded oligoDNA, heteroduplex oligonucleotides formed by oligoRNA and oligoDNA, double-stranded oligonucleotides formed by oligoRNA and RNA-DNA hybrid oligonucleotides, double-stranded oligonucleotides formed by oligoDNA and RNA-DNA hybrid oligonucleotides, and double-stranded oligonucleotides formed mutually by RNA-DNA hybrid oligonucleotides. As double-stranded oligonucleotides, for example, siRNA, heteroduplex oligonucleotides can be mentioned. In the oligonucleotide comprising a complementary portion, the part in which complementary nucleotide sequences are paired with each other is referred to as "complementary portion". The term "complementary portion" refers not only to the complementary portion in the oligonucleotide comprising the complementary portion, but also to the portion in the oligonucleotide raw fragment corresponding to the complementary portion in the oligonucleotide comprising the complementary portion when the oligonucleotide comprising the complementary portion is cut into oligonucleotide raw fragments. For convenience, the complementary nucleotide sequence of either side of the complementary portion is sometimes referred to as the "sense strand" and the complementary nucleotide sequence of the other side is referred to as the "antisense strand". In the present invention, the terms "sense" and "antisense" are merely names for the convenience of referring to either side and the other side of the complementary portion and have no biological significance (particularly in RNAi). The oligonucleotide comprising the complementary portion may or may not contain a loop portion. The "loop portion" refers to a linker that connects the sense side and the antisense side of the complementary portion at the same end side (e.g., the 5' end and the 3' end). Oligonucleotides comprising the complementary portion are particularly used for post-transcriptional gene silencing (e.g., RNA interference (RNAi)).

[0026] Target modified oligonucleotide comprises above-mentioned modified residue in complementary portion.As target modified oligonucleotide, can enumerate for example: double-stranded oligonucleotide or the cyclic oligonucleotide (for example, double-stranded oligonucleotide or the cyclic oligonucleotide comprising modified nucleotide residue in complementary portion), the cyclic oligonucleotide (for example, International Publication No. 2012 / 005368) comprising the residue (for example, amino acid residue and joint etc.) other than modified nucleotide residue in cyclic portion.In target modified oligonucleotide, it can be that part of nucleotide residue is modified nucleotide residue, it can be that all nucleotide residues are modified nucleotide residue, in the case that " modified nucleotide residue " is morpholino nucleic acid (PMO) residue, in target modified oligonucleotide, it is better that part of nucleotide residue is morpholino nucleic acid (PMO) residue. In addition, the target modified oligonucleotides include: gapmers, which are oligonucleotides having modified nucleotide residues at both ends of their sequence and a gap region in the center of their sequence that is recognized by RNase; and further include: mixers, which are oligonucleotides having modified nucleotide residues mixed in their sequence; 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 length of the complementary portion in the target modified oligonucleotide is 11 to 27 bases in length, for example, 12 to 27 bases in length, 15 to 27 bases in length, or 18 to 27 bases in length. For example, when the target modified oligonucleotide is a double-stranded modified oligonucleotide formed only by the complementary portion, the length can be 11 to 27 bases in length. Alternatively, the target modified oligonucleotide has a non-complementary portion in addition to the complementary portion. In this case, the length of the non-complementary portion can be 1 to 16 bases in length, for example, 1 to 10 bases in length, preferably 1 to 5 bases in length, and more preferably 1, 2 or 3 bases in length. In the target modified oligonucleotide having a non-complementary portion in addition to the complementary portion having a length of 11 to 27 bases, the complementary portion of 11 to 27 bases in length can be a continuous form or a non-continuous form interrupted by a mismatch base pair as a non-complementary portion.

[0028] The total number of residues in the target modified oligonucleotide can be appropriately selected according to the function of the target modified oligonucleotide and the conditions in the method of the present invention.

[0029] (Oligonucleotide starting fragment) The four or more oligonucleotide starting fragments used as starting materials in the method of the present invention can be designed to correspond to the oligonucleotide starting fragments obtained when the target modified oligonucleotide is cleaved at the fragment junction (also referred to as "cleavage site") that satisfies the following conditions (i) to (v): (i) one or more fragment junctions are present on each strand side of the complementary portion, and a total of two or more fragment junctions are present in the modified oligonucleotide; (ii) when the modified oligonucleotide is cleaved at the fragment junction, an overhanging end (also called a "sticky end") is formed in the complementary portion, and the length of the overhanging end is 1 to 10 bases; (iii) at least one oligonucleotide starting material segment comprises a modified nucleotide; (iv) four of the four or more oligonucleotide starting fragments contain complementary portions with a length of 5 to 25 bases; and (v) The total length of bases corresponding to each strand of the complementary portion of the oligonucleotide raw material fragment is 11 to 27 bases in length.

[0030] The number of oligonucleotide raw material fragments is 4 or more, preferably 4 to 6 (4, 5, 6). The number of oligonucleotide raw material fragments can also be characterized from the perspective of the number of fragments corresponding to the sense strand and antisense strand of the target modified oligonucleotide (mainly a double-stranded nucleic acid). According to the above condition (i), it can be understood that the number of such oligonucleotide raw material fragments corresponding mainly to the sense strand and antisense strand is 2 or more respectively. The number of such oligonucleotide raw material fragments corresponding to the sense strand and antisense strand can be 3 fragments or 4 fragments, preferably 2 fragments or 3 fragments respectively. The protruding end in the above condition (ii) can be any of the 5' protruding end and the 3' protruding end. The "complementary portion" in the above condition (iv) refers to the portion of the oligonucleotide raw material fragment corresponding to the complementary portion in the target modified oligonucleotide. In the present invention, the term "fragment junction" and "cleavage site" have the same meaning. "Fragment junction" ("cleavage site") refers to a site set to facilitate the design of the combination of oligonucleotide raw material fragments, and does not refer to the site actually cut in the method of the present invention. The four oligonucleotide raw material fragments in (iv) above can be designed so that the target modified oligonucleotide includes a complementary portion 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-mentioned condition (iv) can be a base length that can form a pair, and can be more than 1 base in length. In addition, since the purity, yield, and manufacturing efficiency of the product in the oligonucleotide synthesis will decrease with the increase of base length, it is preferred that the length of the 4 oligonucleotide raw material fragments designed to the complementary portion is less than 17 bases. The length of the 2 chains constituting the complementary portion is preferably 5 to 25 bases in length, for example 5 to 22 bases in length, 5 to 20 bases in length, 5 to 17 bases in length, 8 to 25 bases in length, 8 to 22 bases in length, 8 to 20 bases in length, or 8 to 17 bases in length.

[0032] The length of the protruding end is, for example, 1 to 10 bases in length, preferably 1 to 8 bases in length, more preferably 1 to 6 bases in length, further preferably 2 to 6 bases in length, 3 to 6 bases in length, or 4 to 6 bases in length.

[0033] The base length of the "complementary portion" and the base length of the overhang in condition (iv) above are set to values ​​that satisfy the above ranges and are compatible with each other. For example, if the overhang is 5 bases long, the complementary portion may be 6 to 25 bases long to form an overhang. For example, if the overhang is 6 bases long, the complementary portion may be 7 to 25 bases long to form an overhang.

[0034] The length of the portion excluding the protruding ends in the "complementary portion" of the four oligonucleotide raw material fragments specified in the above condition (iv) is preferably 4 to 24 bases in length, 4 to 21 bases in length, 4 to 19 bases in length, or 4 to 16 bases in length.

[0035] In a specific embodiment, the length of each of the four oligonucleotide raw material fragments specified in the above condition (iv) can be 5 bases in length or more, preferably 6 bases in length or more, more preferably 7 bases in length or more, further preferably 8 bases in length or more, and particularly preferably 9 bases in length or more. The length of such four oligonucleotide raw material fragments can also be 19 bases in length or less, preferably 18 bases in length or less, more preferably 17 bases in length or less, further preferably 16 bases in length or less, and particularly preferably 15 bases in length or less. The length of such four oligonucleotide raw material fragments can also be 5 to 19 bases in length, preferably 6 to 18 bases in length, more preferably 7 to 17 bases in length, further preferably 8 to 16 bases in length, and particularly preferably 9 to 15 bases in length.

[0036] The 5' end of the oligonucleotide raw material fragment corresponding to the 5' end of the target modified oligonucleotide can remain as a 5'-phosphate group, can be substituted with a 5'-OH, can also be introduced with a 5'-phosphate group and modified, or can have the same structure as the 5' end of the target modified oligonucleotide. As the 5'-phosphate group modification, for example, the above-mentioned example can be enumerated. From the viewpoint of the ligation reaction adopting oligonucleotide ligase, the 5' end of the oligonucleotide raw material fragment other than this preferably remains as a 5'-phosphate group. The 3' end of the oligonucleotide raw material fragment corresponding to the 3' end of the target modified oligonucleotide can remain as a 3'-OH, can be introduced with a 3'-phosphate group and modified, or can have the same structure as the 3' end of the target modified oligonucleotide. As the 3'-phosphate group modification, for example, the above-mentioned example can be enumerated. From the viewpoint of the ligation reaction adopting oligonucleotide ligase, the 3' end of the oligonucleotide raw material fragment other than this preferably remains as a 3'-OH.

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

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

[0039] When it is desired to add a functional moiety to the target modified oligonucleotide, the functional moiety can be added to the corresponding portion of the oligonucleotide starting fragment.

[0040] (Ligase treatment) Oligonucleotide ligase is an enzyme that connects oligonucleotide raw material fragments to each other. In the method of the present invention, by the catalytic action of oligonucleotide ligase, oligonucleotide raw material fragments are connected to each other at "cleavage site" (also known as "connection site") to generate target modified oligonucleotide. As oligonucleotide ligase, for example, RNA ligase and DNA ligase can be enumerated. RNA ligase can be any of single-stranded RNA ligase or double-stranded RNA ligase, preferably double-stranded RNA ligase. As double-stranded RNA ligase, for example, RNA ligase of Rnl2 family (also known as "RNA ligase 2"), RNA ligase of Rnl5 family can be enumerated. As RNA ligase, as long as the purpose of the present invention is achieved, RNA ligase derived from any biological species or virus species can be used, for example, RNA ligase (T4 RNA ligase 1, T4 RNA ligase 2) derived from T4 bacteriophage can be used. As DNA ligase, as long as the purpose of the present invention is achieved, DNA ligase derived from any biological species or virus species can be used, for example, DNA ligase derived from T4 bacteriophage can be used.

[0041] The treatment in the presence of oligonucleotide ligase (hereinafter referred to as "ligase treatment") is a reaction in which the oligonucleotide raw material fragments are connected by the catalytic action of the oligonucleotide ligase. The operation of the ligase treatment is to mix the oligonucleotide raw material fragments with the oligonucleotide ligase. In the ligase treatment, all the oligonucleotide raw material fragments can be mixed with the oligonucleotide ligase to perform a ligation reaction in one step. In addition, in the ligase treatment, a multi-step ligation reaction can be performed, in which part of the oligonucleotide raw material fragments are mixed with the oligonucleotide ligase to perform a ligation reaction, and then the remaining oligonucleotide raw material fragments are mixed with the reactants to perform a ligation reaction in the next step. Regarding mixing, it can be: adding the oligonucleotide raw material fragments to the oligonucleotide ligase; adding the oligonucleotide raw material fragments to a system containing the oligonucleotide ligase; or adding the oligonucleotide raw material fragments and the oligonucleotide ligase to a system for the reaction.

[0042] An aqueous solution can be used as a system for ligase treatment. A buffer solution is preferred. Examples of the buffer solution include phosphate buffer, Tris buffer, carbonate buffer, acetate buffer, and citrate buffer. The pH can be, for example, approximately 5 to 9. For example, when the concentration of the oligonucleotide starting material fragments during ligase treatment is high, the pH can 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 is as long as the oligonucleotide raw material fragment dissolves and is sufficient to generate a concentration of the target modified oligonucleotide. The concentration of each oligonucleotide raw material fragment can be, for example, more than 1 μM, more than 10 μM, more than 50 μM, more than 100 μM, more than 300 μM, more than 500 μM or more than 1000 μM. The concentration of each oligonucleotide raw material fragment can also be, for example, 1M, 100mM or less than 10mM. In the case of effective mass production of the target modified oligonucleotide, it is preferably to use each oligonucleotide raw material fragment in a concentration of more than 100 μM in the above-mentioned concentration and a pH range of 7.5 to 9.0 in the above-mentioned pH range.

[0044] From the viewpoint of improving manufacturing efficiency by reducing the amount of unreacted oligonucleotide raw material fragments, the molar number of all oligonucleotide raw material fragments in the ligase treatment is preferably substantially equal. In order to make the molar number of all oligonucleotide raw material fragments substantially equal, the total molar ratio of any two oligonucleotide raw material fragments selected from a total of more than 4 oligonucleotide raw material fragments can be, for example, in the range of 0.5 to 2, preferably 1 / 1.8 to 1.8, more preferably 1 / 1.5 to 1.5, further more preferably 1 / 1.2 to 1.2, and particularly preferably 1 / 1.1 to 1.1.

[0045] The concentration of the oligonucleotide raw material fragment in the ligase treatment is as long as it is a concentration sufficient to generate the target modified oligonucleotide. The concentration of the oligonucleotide ligase can be, for example, more than 0.01U / μL, preferably more than 0.02U / μL, more preferably more than 0.03U / μL, and further more preferably more than 0.04U / μL. The concentration of the oligonucleotide ligase can be, for example, less than 1U / μL, preferably less than 0.5U / μL, more preferably less than 0.2U / μL, and further more preferably less than 0.1U / μL. More specifically, the concentration of the oligonucleotide ligase can be, for example, 0.01~1U / μL, preferably 0.02~0.5U / μL, more preferably 0.03~0.2U / μL, and further more preferably 0.04~0.1U / μL.

[0046] The system that carries out ligase treatment can include the cofactor of oligonucleotide ligase. As the cofactor of oligonucleotide ligase, for example, ATP, divalent metal salt (for example, magnesium salts such as magnesium chloride) can be enumerated. The system that carries out treatment can include the stabilizer of oligonucleotide ligase. As the stabilizer of oligonucleotide ligase, for example, antioxidant (for example, reducing agents such as dithiothreitol, mercaptoethanol) can be enumerated. In order to keep enzyme stable and improve reaction speed, the system that carries out ligase treatment can include surfactant. As surfactant, for example, nonionic surfactant (for example, surfactant of Triton series such as Triton X-100) and ionic surfactant can be enumerated. As ionic surfactant, for example, cationic surfactant, anionic surfactant, zwitterionic surfactant can be enumerated. In addition, in order to improve reaction speed, the system that carries out ligase treatment can include polyethylene glycol.

[0047] The system for carrying out the ligase treatment may have a low concentration of monovalent cation salt concentration, or may be substantially free of monovalent cation salts. The monovalent cation salt concentration of the system for treatment may be, for example, 10 mM or less, preferably 1 mM or less, more preferably 0.1 mM or less, and further preferably 0.01 mM or less. It is particularly preferred that the system for treatment be substantially free of monovalent cation salts. 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 the ligase treatment may be any temperature sufficient to activate the oligonucleotide ligase, and may be, for example, 2 to 50°C, preferably 16 to 50°C, and more preferably 25 to 50°C.

[0049] The ligase treatment may be performed for a time sufficient to generate the target modified oligonucleotide, and such a time may be, for example, 1 to 72 hours.

[0050] According to the present invention, for the target modified oligonucleotide, the generation and mixing of impurities having base lengths other than the target base length, such as N-1 polymers or N+1 polymers, 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, heterologous double-stranded oligonucleotide). The sense strand and antisense strand constituting such a single double-stranded nucleic acid are N and M base lengths, respectively. The N and M base lengths are independently 11 to 30 base lengths (e.g., 18 to 30 base lengths). The N and M base lengths can also be independently 11 to 27 base lengths (e.g., 18 to 27 base lengths). In the present invention, impurities in the target modified oligonucleotide refer to nucleic acid inclusions other than the above-mentioned target modified oligonucleotide. The sense and antisense strands constituting such a nucleic acid inclusion are not N and M bases in length, respectively, but may be (N±α) and M bases in length, N and (M±β) bases in length, or (N±α) and (M±β) bases in length. Here, N and M are the same as described above, and α and β are, for example, 1, 2, or 3 bases.

[0051] (Other optional processes) 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). The method of the present invention can suppress the generation of nucleic acid inclusions other than the target modified oligonucleotide, so the purification of the target modified oligonucleotide from the sample of the synthesized oligonucleotide raw material fragment can be omitted. However, for the method of the present invention, even when the target modified oligonucleotide is purified, the generation of double-stranded nucleic acid inclusions (impurities) caused by a small amount of oligonucleotide raw material fragments other than the target that may remain after the purification can be suppressed, and therefore the target modified oligonucleotide can also be purified. Such purification can be performed, for example, by methods such as chromatography (e.g., HPLC, IEX), gel filtration, etc.

[0052] The method of the present invention may include a reaction quenching step after the ligase treatment step. Examples of the reaction quenching step include high temperature treatment (e.g., 80°C), oligonucleotide ligase inactivation treatment by addition of an acid, base, or organic solvent, and removal of metal ions of the cofactor by addition of a chelating agent such as EDTA. Alternatively, the enzyme may be immobilized on a support for reaction, followed by membrane separation to remove the enzyme from the reaction solution.

[0053] The method of the present invention may include a step of purifying the target modified oligonucleotide after the ligase treatment step. For example, this step can be performed by any appropriate method such as chromatography (eg, HPLC) or gel filtration.

[0054] When the oligonucleotide raw material fragments are annealed (renaturation), generally speaking, the following operations are mostly performed: in order to make the oligonucleotide raw material fragments in a denatured state (non-paired state), the oligonucleotide raw material fragment mixed solution is heated to a high temperature, and then, in order to form a pairing of complementary nucleotide sequences, the high-temperature oligonucleotide raw material fragment mixed solution is slowly cooled by air cooling, etc. However, in the method of the present invention, before the ligase treatment step, such an operation of heating to a high temperature for denaturation and a treatment of cooling for forming a pairing (heating-cooling treatment) are not performed, and the target modified oligonucleotide can be manufactured by a simplified operation. As being placed at a high temperature, for example, the oligonucleotide raw material fragment mixed solution can be kept at a temperature of more than 65°C, more than 70°C, more than 75°C, more than 80°C, more than 85°C, more than 90°C, more than 95°C, or more than 100°C (for example, more than 5 minutes, or more than 10 minutes). Examples of cooling include allowing the oligonucleotide raw material fragment mixed solution to stand at room temperature (e.g., 15-25°C, or 20-25°C) or a specified temperature (e.g., 37°C) (e.g., for more than 5 hours), and maintaining the oligonucleotide raw material fragment mixed solution at a specified temperature (e.g., 37°C) (e.g., for more than 15 minutes).

[0055] In order to omit the heating-cooling treatment, the time for which the oligonucleotide raw material fragment mixed solution is exposed to a high temperature before the ligase treatment step can be controlled to, for example, less than 5 minutes, less than 4.5 minutes, less than 4 minutes, less than 3.5 minutes, less than 3 minutes, less than 2.5 minutes, less than 2 minutes, less than 1.5 minutes, less than 1 minute, or less than 0.5 minutes.

[0056] In order to omit the heating-cooling process, in the method of the present invention, the solution containing the oligonucleotide starting fragments can be maintained at 2 to 50°C from the time the oligonucleotide starting 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 starting fragments and the oligonucleotide ligase contained in the above combination, any pauses between mixing, and the ligase reaction can be carried out at 2 to 50°C. In such an embodiment, the method of the present invention includes the following operations: (1) all oligonucleotide raw material fragments and oligonucleotide ligases contained in the above combination in different systems are mixed at 2 to 50° C. and the mixture is mixed under the condition of being maintained at 2 to 50° C. during all mixing intervals to obtain a mixed solution; and (2) The mixed solution is reacted while being maintained at 2 to 50° C. to obtain a solution containing the target modified oligonucleotide.

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

[0058] The method of the present invention can be used, for example, for large-scale industrial production of target modified oligonucleotides. Example

[0059] Hereinafter, the present invention will 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 Combination Patterns Using Natural RNA Fragments 1) Synthesis of credible substrates and products The effect of base length on the enzymatic synthesis of siRNA from four short natural RNA fragments was evaluated. The target siRNAs used were duplexes formed from RNA1-S (21-mer) and RNA1-A (23-mer) listed in Table 1 (hereinafter referred to as the sense strand and antisense strand, respectively). Eighteen RNA fragments were synthesized, as shown in Table 1, and these were used to evaluate combinations of the six fragment patterns shown in Table 2.

[0061] [Table 1] Table 1. Natural RNAs used for evaluation Capital letters in English: represent RNA, Pho: represents the modification of the 5' end based on the phosphate group.

[0062] [Table 2] Table 2. Combinations of natural RNA fragments 1) represents the sequence on the sense strand side of the overhanging terminal portion.

[0063] 2) Ligation reaction using T4 RNA ligase 2 The reaction was carried out using oligonucleotides of four fragments by T4 RNA ligase 2 (New England Biolabs). The composition of the reaction solution was 50mM Tris-HCl, 2mM MgCl2, 1mM dithiothreitol, 400μM ATP, pH7.5, 10μM of each RNA fragment, and the reaction volume was 10μL. The concentration of the product was compared by setting the added concentration of the enzyme to 0.025, 0.05, 0.1, and 0.2U / μL. After reacting at 25°C for 1 hour using a thermal cycler, the reaction was stopped by heating at 80°C for 5 minutes.

[0064] 3) Analysis using 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 analysis conditions were a column temperature of 60°C, a detection wavelength of 254 nm, an injection volume of 10 μL, and a flow rate of 0.4 mL / min. The mobile phase consisted of a linear gradient of eluent A (hexafluoroisopropanol-triethylamine) and eluent B (methanol). Authentic preparations of the sense and antisense strands were also analyzed to quantify the concentration of the ligation product.

[0065] 4) Results The accumulation of the sense and antisense strands for each combination of fragment base lengths is shown in Figure 2. In combination 3, almost no ligation product accumulated, but in the other combinations, the accumulation of the ligation product increased with increasing enzyme concentration.

[0066] [Example 2] Evaluation of the influence of reaction temperature in the ligation reaction of natural RNA The influence of reaction temperature in the ligation reaction of short chains was investigated. Oligonucleotide 1 in Table 2 was used as a substrate, and the reaction was carried out by T4 RNA ligase 2. The composition of the reaction solution was 50mM Tris-HCl, 2mM MgCl2, 1mM dithiothreitol, 400μM ATP, pH7.5, enzyme concentration 0.2U / μL, 10μM of each RNA fragment, and the reaction volume was 10μL. The reaction temperature was set to 16°C, 25°C, 30°C and 37°C, and after reacting 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 contained in the reaction solution was analyzed by HPLC according to 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 products accumulated in both the sense and antisense strands after one hour of reaction. Meanwhile, at 16°C, the rate of ligation product formation, particularly in the sense strand, was lower than at temperatures above 25°C.

[0068] [Example 3] Confirmation of reaction progress using modified RNA (reaction progress) 1) Synthesis of credible substrates and products The progress of the enzymatic ligation reaction of siRNA starting from 4 fragments in the modified oligonucleotide was evaluated. The target siRNA adopts a double strand formed by the sense strand (MOD1-S) and the antisense strand (MOD1-A) shown in Table 3. The base sequence of this siRNA is the same as the natural RNA used in Examples 1 and 2, but all the residues are modified by 2'-F or 2'-O-methyl, and some of the phosphate groups are replaced (substituted by) thiophosphate groups. In addition, as each fragment, the 4 fragments shown in Table 3 were synthesized. The base sequences of these 4 fragments are the same as the combination of No. 1 in Table 2.

[0069] [Table 3] Table 3. Modified RNAs used for evaluation Capital letters indicate RNA, Pho indicates a 5'-terminal modification based on a phosphate group, (F) indicates a 2'-fluoro modification, (Me) indicates a 2'-O-methyl modification, and ^ indicates replacement of a phosphate group with a thiophosphate group.

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

[0071] 3) Analysis using 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 analysis conditions were set as a column temperature of 80°C, a detection wavelength of 260 nm, an injection volume of 10 μL, and a flow rate of 0.4 mL / min. The mobile phase was analyzed using a linear gradient of liquid A (hexafluoroisopropanol-triethylamine) and liquid B (methanol). Authentic preparations of the sense and antisense strands were also analyzed in the same manner to confirm the formation of the ligation product. In addition, the ligation product was analyzed by mass spectrometry using an Agilent 6230TOF LC / MS system (Agilent Technologies).

[0072] 4) Results The results of the HPLC analysis are shown in Figure 4 In the HPLC analysis, the addition of the enzyme resulted in a peak of the ligation product at a retention time consistent with that of the authentic product, 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 also increased with increasing amounts of enzyme added. Furthermore, in the LC-TOF / MS analysis of the reaction solution, the generation of sense and antisense strands was observed under conditions in which the enzyme was added. Sense strand LC / MS m / z: calculated 2266.13, found 2265.9703 [M-3H] 3- Antisense strand LC / MS m / z: calculated 2531.04, found 2531.0199 [M-3H] 3- .

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

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

[0075] The reaction solution is composed of 50mM Tris-HCl, 10mM MgCl2, 10mM dithiothreitol, 1mM ATP, and pH 7.5. The enzyme concentration is 470nM, the oligonucleotide fragments are 10μM, and the reaction solution volume is 30μL. The oligonucleotide fragments are combined as shown in Table 3. The reaction is carried out at 25°C using a thermal cycler. After 4 hours, 10μL is collected and the reaction is stopped by heating at 80°C for 5 minutes. The concentration of the ligation product contained in the reaction solution is analyzed by HPLC according to the conditions described in Example 3. The authentic product is also analyzed in the same way to quantify the concentration of the ligation product.

[0076] HPLC analysis confirmed the accumulation of ligation products, with the accumulation after 4 hours of reaction being 0.58 μM for the sense strand and 5.1 μM for the antisense strand. As described above, the reaction to generate siRNA from the four-fragment modified RNA was also performed using DNA ligase.

[0077] [Example 5] Preparation of Deinococcus radiodurans RNA ligase and ligation reaction (1) Construction of recombinant expression strain using E. coli A strain expressing DraRn1, an RNA ligase from the Rn15 family derived from Deinococcus radiodurans, was constructed to produce a purified enzyme. First, a plasmid containing the amino acid sequence of DraRn1 (SEQ ID NO: 17) optimized for E. coli codons was constructed by total gene synthesis. This sequence was then subcloned into the NdeI / BamHI sites of the pET16b vector. This expression plasmid was transformed into E. coli BL21 (DE3) to obtain a DraRn1 expression strain. This expression strain expressed DraRn1 with a His-tag at the N-terminus.

[0078] (2) Preparation of recombinant enzyme Each expression strain was grown (cultured) overnight at 37°C in LB agar medium containing 100 mg / L ampicillin. The resulting colony was inoculated into 100 mL of LB medium containing 100 mg / L ampicillin and cultured with shaking in a Sakaguchi flask. After culturing at 37°C for 2 hours, IPTG and ethanol were added to a final concentration of 0.1 mM or 2%, respectively. The culture was then continued at 17°C for 16 hours.

[0079] After completion of the culture, the cells were collected from the resulting culture solution by centrifugation, 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, and disrupted by ultrasonication. The cell debris was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as the soluble fraction.

[0080] The resulting soluble fraction was applied to a HisTALON Superflow Cartridge (Takara Bio Inc.), a His-tagged protein purification column equilibrated with the aforementioned buffer, and adsorbed to the carrier. Proteins not adsorbed to the carrier (unadsorbed proteins) were washed with a buffer consisting of 50 mM Tris-HCl (pH 7.6), 250 mM NaCl, 10% sucrose, and 15 mM imidazole. The adsorbed proteins were then eluted with a buffer consisting of 50 mM Tris-HCl (pH 8.0), 250 mM NaCl, 10% glycerol, and 200 mM imidazole.

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

[0082] (3) Ligation reaction using DraRnl The modified RNA of 4 fragments was reacted by DraRnl. The composition of the reaction solution is 50mM Tris-HCl (pH7.5), 10mM MnSO4, 1mM dithiothreitol, 400μM ATP, 10μM of each modified RNA fragment, and the reaction volume is 25μL. The modified RNA fragments are combined in Table 3. The enzyme addition concentration was set to 72μg / mL, and as a negative control, the reaction was also carried out under the condition of no enzyme addition. After reacting at 25°C for 3 hours using a thermal cycler, EDTA was added to stop the reaction at a final concentration of 1mM.

[0083] (4) Analysis using 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 analysis conditions were a column temperature of 60°C, a detection wavelength of 260 nm, an injection volume of 10 μL, and a flow rate of 0.4 mL / min. The mobile phase was a linear gradient consisting of solvent A (hexafluoroisopropanol-triethylamine) and solvent B (methanol). Authentic preparations of the sense and antisense strands were also analyzed to confirm the formation of the ligation product.

[0084] The results of the HPLC analysis are shown in Figure 5 In HPLC analysis, the addition of the enzyme resulted in a peak for the ligation product at a retention time consistent with that of the authentic product, and the peak area for the modified RNA substrate was reduced compared to the negative control. These results demonstrate that using DraRn1, it is possible to generate target modified oligonucleotides from four fragments of modified RNA.

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

[0086] [Table 4] Table 4. Sequences of products and substrates English capital letters: indicate RNA, Pho: indicates 5' end modification based on phosphate group, (F): indicates 2'-fluoro group modification, (Me): indicates 2'-O-methyl group modification, ^: indicates replacement of phosphate group with phosphorothioate group, t: indicates thymidine; The product is a sequence formed by connecting the substrate fragments in the order of ①②③④ from the 5' end to the 3' end.

[0087] The substrate oligonucleotides of the four fragments in Table 4 were reacted by T4 RNA ligase 2 (New England Biolabs). The composition of the reaction solution is 50mM Tris-HCl, 2mM MgCl2, 1mM dithiothreitol, 400μMATP, pH7.5, 10μM of each oligonucleotide fragment, and the reaction volume is 100μL. The concentration of the product was compared by setting the added concentration of the enzyme to 17.8μg / μL. As a negative control, the reaction was also carried out under the condition of no enzyme addition. After reacting at 25°C for 3 hours using a thermal cycler, the reaction was stopped by heating at 80°C for 5 minutes. The reaction solution was analyzed by HPLC and LC-TOF / MS under the conditions of Example 5.

[0088] In the analysis using HPLC, Figure 6 As shown, the addition of the enzyme reduced the peak area of ​​the modified RNA substrate compared to the negative control, and a peak was detected at a position with a longer retention time than the substrate (around 5.6 minutes). Furthermore, in the analysis of the reaction solution using LC-TOF / MS, the target product was observed under the conditions of enzyme addition; LC / MS m / z: Calculated 2727.33, found 2727.21 [M-6H] 6- .

[0089] The above results show that T4 RNA ligase 2 can generate a target modified oligonucleotide with a loop structure from four fragments.

[0090] [Example 7] Generation of heteroduplexes formed by DNA and RNA chains A heteroduplex consisting of a modified DNA strand and a modified RNA strand was generated using 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 50mM Tris-HCl, 2mM MgCl2, 1mM dithiothreitol, 400μM ATP, and pH 7.5. The enzyme concentration was 3.56μg / mL, and the substrates were 10μM each of the four oligonucleotide fragments shown in Table 5. The reaction volume was 40μL. The reaction was carried out using a thermal cycler at 37°C. After 18 hours, EDTA was added to stop the reaction at a final concentration of 10mM. As a negative control, the reaction was also carried out without the addition of enzyme. The concentration of the ligation product contained in the reaction solution was analyzed by HPLC and LC-TOF / MS according to the conditions described in Example 5.

[0092] In HPLC analysis, the addition of the enzyme reduced the peak area of ​​each substrate compared to the negative control, and two new peaks were detected. Furthermore, in LC-TOF / MS analysis of the reaction solution, the formation of modified DNA and modified RNA chains was observed under the conditions of enzyme addition. Modified DNA strand LC / MS m / z: calculated 2119.50, found 2119.34 [M-2H] 2- Modified RNA chain LC / MS m / z: calculated value 2126.89, found value 2126.36 [M-2H] 2- .

[0093] These results indicate that T4 RNA ligase 2 can generate heteroduplexes consisting of modified DNA and modified RNA chains.

[0094] [Table 5] Table 5. Sequences of products and substrates Lowercase letters indicate DNA, uppercase letters indicate RNA, mC indicates 5-methylcytidine, Pho indicates a phosphate-based 5'-terminal modification, (L) indicates locked nucleic acid (LNA), and (Me) indicates a 2'-O-methyl-based modification.

[0095] [Example 8] Reaction using oligonucleotide fragments containing mismatched base pairs The progress of the reaction of generating a double-stranded modified oligonucleotide having a mismatch in a base pair by enzymatic ligation of four oligonucleotide fragments was evaluated. The chains of the target product are referred to as chain A and chain B. The sequences of the target product and the synthesized substrate fragments are shown in Table 6, and the combinations of the four fragments are shown in Figure 7 .

[0096] The composition of the reaction solution is 1.78μg / mL T4 RNA ligase 2 (New England Biolabs), 50mM Tris-HCl, 2mM MgCl2, 1mM dithiothreitol, 400μM ATP, pH7.5. Four oligonucleotide fragments of the substrate were added under the condition that the final concentration became 10μM, and the reaction was carried out with a liquid volume of 40μL. The reaction was carried out at 25°C using a thermal cycler. After 4 hours, EDTA was added under the condition that the final concentration became 10mM to stop the reaction. As a negative control, the reaction was also carried out under the condition that no enzyme was added. The concentration of the ligation product contained in the reaction solution was analyzed by HPLC and LC-TOF / MS according to the conditions described in Example 5.

[0097] In HPLC analysis, the addition of the enzyme reduced the peak area of ​​each substrate compared to the negative control, and two new peaks were detected. Furthermore, in LC-TOF / MS analysis of the reaction solution, the production of both A and B chains was confirmed under the conditions of enzyme addition. A chain LC / MS m / z: calculated 1361.77, found 1361.75 [M-5H] 5- B chain LC / MS m / z: calculated 1517.41, found 1517.35 [M-5H] 5- .

[0098] The above results show that double-stranded modified oligonucleotides with mismatch sequences can be generated by T4 RNA ligase 2.

[0099] [Table 6] Table 6. Sequences of products and substrates Pho: indicates a phosphate-based modification of the 5' end, capital letters: indicate RNA, (F): indicates a 2'-fluoro-based modification, (Me): indicates a 2'-O-methyl-based modification, ^: indicates a phosphorothioate bond.

[0100] [Example 9] Reactions using 5-fragment and 6-fragment oligonucleotides The progress of the reaction to generate double-stranded modified oligonucleotides by enzymatic ligation of oligonucleotides composed of 5 and 6 fragments was evaluated. The chains of the target product are referred to as chain A and chain B. The sequences of the target product and the synthesized substrate fragments are shown in Table 7, and the combinations of the 4 fragments are shown in Table 8. Figure 8 .

[0101] The composition of the reaction solution is 1.78μg / mL T4 RNA ligase 2 (New England Biolabs), 50mM Tris-HCl, 2mM MgCl2, 1mM dithiothreitol, 400μM ATP, pH7.5. Oligonucleotide fragments as substrates were added under the condition that the final concentration became 10μM, and the reaction was carried out with a liquid volume of 30μL. The reaction was carried out at 37°C using a thermal cycler. After 16 hours, EDTA was added under the condition that the final concentration became 10mM to stop the reaction. As a negative control, the reaction was also carried out under the condition that no enzyme was added. The concentration of the ligation product contained in the reaction solution was analyzed by HPLC according to the conditions described in Example 5.

[0102] In the analysis using HPLC, Figure 9 As shown, the addition of enzyme reduced the peak area of ​​each substrate compared to the negative control, and two new peaks were detected. The retention times of these peaks were consistent with those of authentic product preparations.

[0103] The above results show that double-stranded modified oligonucleotides can be generated from substrate oligonucleotides of 5 fragments and 6 fragments using T4 RNA ligase 2.

[0104] [Table 7] Table 7. Sequences of products and substrates Pho: indicates a phosphate-based 5'-terminal modification, capital letters: indicate RNA, (F): indicates a 2'-fluoro-based modification, and (Me): indicates a 2'-O-methyl-based modification.

[0105] [Example 10] Reaction using an oligonucleotide with a DMTr group added to the 5' end The progress of the reaction to generate a double-stranded modified oligonucleotide by enzymatic ligation of four oligonucleotides including two fragments with dimethoxytrityl (DMTr) added to the 5' end was evaluated. The chains of the target product are referred to as chain A and chain B. The sequences of the target product and the synthesized substrate fragments are shown in Table 8, and the combinations of the four fragments are shown in Figure 10 .

[0106] The composition of the reaction solution is 1.78μg / mL T4 RNA ligase 2 (New England Biolabs), 50mM Tris-HCl, 2mM MgCl2, 1mM dithiothreitol, 400μM ATP, pH7.5. Four oligonucleotide fragments of the substrate were added under the condition that the final concentration became 10μM, and the reaction was carried out with a liquid volume of 40μL. The reaction was carried out at 25°C using a thermal cycler. After 4 hours, EDTA was added under the condition that the final concentration became 10mM to stop the reaction. As a negative control, the reaction was also carried out under the condition that no enzyme was added. The concentration of the ligation product contained in the reaction solution was analyzed by HPLC and LC-TOF / MS according to the conditions described in Example 5.

[0107] In HPLC analysis, the addition of the enzyme reduced the peak area of ​​each substrate compared to the negative control, and two new peaks were detected. Furthermore, in LC-TOF / MS analysis of the reaction solution, the production of both A and B chains was confirmed under the conditions of enzyme addition. A chain LC / MS m / z: calculated value 1764.80, found value 1764.79 [M-4H] 4- B chain LC / MS m / z: calculated value 1738.76, found value 1738.75 [M-4H] 4- .

[0108] The above results show that T4 RNA ligase 2 can generate double-stranded modified oligonucleotides from substrate fragments containing DMTr groups.

[0109] [Table 8] Table 8. Sequences of products and substrates Pho: indicates a phosphate-based 5'-terminal modification, DMTr: indicates a DMTr-based 5'-terminal modification, capital letters: indicate RNA, (Me): indicates a 2'-O-methyl-based modification, and t: indicates a thymidine residue.

[0110] [Example 11] Reaction using carrier-added oligonucleotide fragments The progress of the reaction of generating a double-stranded modified oligonucleotide by enzymatic ligation of four oligonucleotides including a fragment with N-acetylgalactosamine (GalNAc) added to the 5' end was evaluated. The chains of the target product are referred to as chain A and chain B. The sequences of the target product and the synthesized substrate fragments are shown in Table 9, and the combinations of the four fragments are shown in Figure 11The GalNAc-modified fragment was synthesized by linking a trivalent β-D-GalNAc with a carboxyl-functionalized PEG5 linker (Sussex Corporation) to the 5' end of the oligonucleotide via an amino C6 linker.

[0111] The composition of the reaction solution is 1.78μg / mL T4 RNA ligase 2 (New England Biolabs), 50mM Tris-HCl, 2mM MgCl2, 1mM dithiothreitol, 400μM ATP, pH7.5. Four oligonucleotide fragments of the substrate were added under the condition that the final concentration became 10μM, and the reaction was carried out with a liquid volume of 40μL. The reaction was carried out at 25°C using a thermal cycler. After 4 hours, EDTA was added under the condition that the final concentration became 10mM to stop the reaction. As a negative control, the reaction was also carried out under the condition that no enzyme was added. The ligation products contained in the reaction solution were analyzed by HPLC and LC-TOF / MS according to the conditions described in Example 5.

[0112] In HPLC analysis, the addition of the enzyme reduced the peak area of ​​each substrate compared to the negative control, and two new peaks were detected. Furthermore, in LC-TOF / MS analysis of the reaction solution, both the A and B chains were detected under the conditions of enzyme addition, confirming their production. A chain LC / MS m / z: calculated value 1730.40, found value 1730.37 [M-5H] 5- B chain LC / MS m / z: calculated 1330.38, found 1330.36 [M-5H] 5- .

[0113] The above results indicate that a double-stranded modified oligonucleotide whose termini are modified with N-acetylgalactosamine can be produced by a four-fragment reaction using T4 RNA ligase 2.

[0114] [Table 9] Table 9. Sequences of products and substrates Pho: indicates a phosphate-based 5'-terminal modification, GalNAc: indicates an N-acetylgalactosamine-based 5'-terminal modification, capital letters: indicate RNA, (Me): indicates a 2'-O-methyl-based modification, and t: indicates a thymidine residue.

[0115] [Example 12] Reaction for producing a double-stranded modified oligonucleotide having a phosphorothioate diester bond at the linker The progress of the reaction of generating double-stranded modified oligonucleotides by enzymatic ligation of four oligonucleotide fragments in which the phosphate groups were replaced by phosphorothioate groups was evaluated. The chains of the target product are respectively referred to as chain A and chain B. The sequences of the target product and the synthesized substrate fragments are shown in Table 10, and the combinations of the four fragments are shown in Figure 12 .

[0116] The composition of the reaction solution is 1.78μg / mL T4 RNA ligase 2 (New England Biolabs), 50mM Tris-HCl, 2mM MgCl2, 1mM dithiothreitol, 400μM ATP, pH7.5. Four oligonucleotide fragments of the substrate were added under the condition that the final concentration became 10μM, and the reaction was carried out with a liquid volume of 40μL. The reaction was carried out at 25°C using a thermal cycler. After 4 hours, EDTA was added under the condition that the final concentration became 10mM to stop the reaction. As a negative control, the reaction was also carried out under the condition that no enzyme was added. The ligation products contained in the reaction solution were analyzed by HPLC and LC-TOF / MS according to the conditions described in Example 5.

[0117] In HPLC analysis, the addition of the enzyme reduced the peak area of ​​each substrate compared to the negative control, and two new peaks were detected. Furthermore, in LC-TOF / MS analysis of the reaction solution, both the A and B chains were detected under the conditions of enzyme addition, confirming their production. A chain LC / MS m / z: calculated value 1769.15, found value 1769.13 [M-4H] 4- B chain LC / MS m / z: calculated value 1743.11, found value 1743.10 [M-4H] 4- .

[0118] The above results indicate that a double-stranded modified oligonucleotide having a phosphorothioate bond at the ligation site can be produced by a reaction using four fragments using T4 RNA ligase 2.

[0119] [Table 10] Table 10. Sequences of products and substrates PS: indicates a 5'-terminal modification based on a phosphorothioate group, capital letters: indicate RNA, ^: indicates a phosphorothioate bond, (Me): indicates a modification based on a 2'-O-methyl group, and t: indicates a thymidine residue.

[0120] [Example 13] Hairpin oligonucleotide production reaction The progress of the reaction of generating a hairpin oligonucleotide by enzymatic ligation of four oligonucleotide fragments was evaluated. The sequences of the target product and the synthesized substrate fragments are shown in Table 11, and the combinations of the four fragments are shown in Figure 13 As a linker, 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.

[0121] The composition of the reaction solution is 1.78μg / mL T4 RNA ligase 2 (New England Biolabs), 50mM Tris-HCl, 2mM MgCl2, 1mM dithiothreitol, 400μM ATP, pH7.5. Four oligonucleotide fragments of the substrate were added under the condition that the final concentration became 10μM, and the reaction was carried out with a liquid volume of 40μL. The reaction was carried out at 25°C using a thermal cycler. After 4 hours, EDTA was added under the condition that the final concentration became 10mM to stop the reaction. As a negative control, the reaction was also carried out under the condition that no enzyme was added. The ligation products contained in the reaction solution were analyzed by HPLC and LC-TOF / MS according to the conditions described in Example 5.

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

[0123] The above results indicate that a hairpin oligonucleotide can be generated by a reaction using four fragments using T4 RNA ligase 2.

[0124] [Table 11] Table 11. Sequences of products and substrates Pho: indicates a phosphate-based modification of the 5' end, capital letters: indicate RNA, and Pro: indicates a proline derivative.

[0125] [Example 14] Effect of the base length of the protruding end on reactivity Oligonucleotides of the substrates were designed under the conditions that the products were the same and that the protruding ends were formed with a length of 1 to 6 bases, and the differences in reactivity caused by the differences in the base lengths of the protruding ends were compared. The chains of the target product were respectively called A chain and B chain. In each combination, the substrate constituting the B chain used a common sequence, and the cleavage position of the A chain used a different position. The sequences of the synthesized target product authentic products and substrate fragments are shown in Table 12, and the combinations of the four fragments are shown in Figure 14 .

[0126] The composition of the reaction solution is 1.78μg / mL T4 RNA ligase 2 (New England Biolabs), 50mM Tris-HCl, 2mM MgCl2, 1mM dithiothreitol, 400μM ATP, pH7.5. Four oligonucleotide fragments of the substrate were added at a final concentration of 10μM, and the reaction was carried out with a liquid volume of 40μL. The reaction was carried out at 25°C using a thermal cycler, and 5μL was recovered after 15 minutes, 30 minutes, 1 hour, 2 hours and 4 hours. EDTA was added to stop the reaction at a final concentration of 10mM. As a negative control, the reaction was also carried out under the condition of no enzyme addition. The concentrations of the ligation product and the authentic product of the product contained in the reaction solution were analyzed by HPLC according to the conditions described in Example 5, and the concentration of the ligation product was calculated.

[0127] HPLC analysis confirmed that the formation of chains A and B was observed in all overhanging ends of all base lengths. Overhanging ends of one base tended to have a low reaction rate.

[0128] [Table 12] Table 12. Sequences of products and substrates Pho: indicates a phosphate-based 5'-terminal modification, capital letters: indicate RNA, (Me): indicates a 2'-O-methyl-based modification.

[0129] [Example 15] Effect of base length of product on reactivity The differences in reactivity when producing shorter target products consisting of complementary segments with a length of 11 to 14 bases were compared. The chains of the target products are referred to as chain A and chain B. The sequences of the target products and the synthesized substrate fragments are shown in Table 13, and the combinations of the four fragments are shown in Figure 15 .

[0130] The composition of the reaction solution is 1.78μg / mL T4 RNA ligase 2 (New England Biolabs), 50mM Tris-HCl, 2mM MgCl2, 1mM dithiothreitol, 400μM ATP, pH7.5. Four oligonucleotide fragments of the substrate were added under the condition that the final concentration became 10μM, and the reaction was carried out with a liquid volume of 40μL. The reaction was carried out at 25°C using a thermal cycler. After 4 hours, EDTA was added under the condition that the final concentration became 10mM to stop the reaction. As a negative control, the reaction was also carried out under the condition that no enzyme was added. The ligation products contained in the reaction solution were analyzed by HPLC and LC-TOF / MS according to the conditions described in Example 5.

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

[0132] The peak area of ​​the substrate under each reaction condition was calculated by HPLC, and the residual rate of the substrate was calculated by the following formula: Residual rate (%)=(total peak area of ​​substrate under the condition of enzyme addition) / (total peak area of ​​substrate in negative control)×100.

[0133] The product formed from the complementary portion having a length of 11 bases tends to have a higher substrate residual rate than the product formed from the complementary portion having a length of 12 bases or longer.

[0134] [Table 13] Table 13. Sequences of products and substrates Pho: indicates a phosphate-based 5'-terminal modification, capital letters: indicate RNA, (Me): indicates a 2'-O-methyl-based modification.

[0135] [Table 14] Table 14. Confirmation of each product based on mass spectrometry analysis

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

[0137] The composition of the reaction solution is 1.78μg / mL T4 RNA ligase 2 (New England Biolabs), 2mM MgCl2, 1mM dithiothreitol, 400μM ATP, and 50mM Tris-HCl (pH7.0-9.0) is used as a buffer. Oligonucleotide fragments as substrates were added under the conditions of final concentrations of 10μM, 300μM, 500μM or 1000μM, and the reaction was carried out with a liquid volume of 40μL. A thermal cycler was used to react at 25°C, samples were taken after 15 minutes or 1 hour, and EDTA was added to stop the reaction under the conditions of a final concentration of 10mM. The ligation products contained in the reaction solution were analyzed by HPLC according to the conditions described in Example 5, and the generation rate was calculated from the total concentration of chain A and chain B. The reaction was confirmed to proceed when the substrate concentration was above 300μM, and a higher reaction rate was shown at pH 8.0 and 8.5 at these concentrations.

[0138] [Table 15] Table 15. Sequences of products and substrates Pho: indicates a phosphate-based modification of the 5' end, capital letters: indicate RNA, (Me): indicates a 2'-O-methyl-based modification, and t: indicates a thymidine residue.

[0139] [Example 17] Effect of adding surfactant The reaction rate of the modified oligonucleotides generated by the addition of surfactant was evaluated. The sequences of the target products and the synthesized substrate fragments are shown in Table 15, and the combinations of the four fragments are shown in Table 16. Figure 16The reaction solution 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 a surfactant at a final concentration of 0.1%. Oligonucleotide fragments were added as substrates to a final concentration of 20 μM, and the reaction was performed in 40 μL of the solution. In the evaluation, the following three conditions were compared: the condition in which the enzyme solution was diluted to 17.8 μg / mL with 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 a 1 / 10 amount was added to the reaction solution (control condition); the condition in which the enzyme solution was diluted to 17.8 μg / mL with storage buffer and a 1 / 10 amount was added to the reaction solution containing a final concentration of 0.1% Triton X-100 (test condition 1); and the condition in which the enzyme solution was diluted to 17.8 μg / mL with storage buffer containing a final concentration of 0.1% Triton X-100 and a 1 / 10 amount was added to the reaction solution containing a final concentration of 0.09% Triton X-100 (test condition 2, final Triton X-100 concentration 0.1%). The reaction was carried out at 25°C using a thermal cycler. Samples were collected after 4 hours and the reaction was terminated by adding EDTA to a final concentration of 10 mM. The ligation products contained in the reaction solution were analyzed by HPLC according to the conditions described in Example 5, and the concentrations of chain A and chain B were calculated. For experimental conditions 1 and 2, higher concentrations of chain A and chain B products were observed compared to the control conditions.

[0140] [Example 18] Comparison of the elimination of impurities based on the base length of the products Reactions with different base lengths were performed on the substrates and products, and the impurities contained in the substrate oligonucleotide fragments and the solution after the enzyme reaction were analyzed. The chains of the target products of the reaction are respectively referred to as chain A and chain B. The sequences of the target products and the synthesized substrate fragments are shown in Table 16, and the combinations of the four fragments are shown in Figure 17 .

[0141] In the reaction using modified oligonucleotides, the composition of the reaction solution was 8.9μg / mL T4 RNA ligase 2 (New England Biolabs), 50mM Tris-HCl, 2mM MgCl2, 1mM dithiothreitol, 400μM ATP, pH7.5. In the reaction using DNA, the composition of the reaction solution was 22.0μg / mL T7 DNA ligase (New England Biolabs), 50mM Tris-HCl, 2mM MgCl2, 1mM dithiothreitol, 400μM ATP, pH7.5. Oligonucleotide fragments of the four fragments used as substrates were added under the condition that the final concentration became 50μM, and the reaction was carried out with a liquid volume of 30μL. The reaction was carried out at 25°C using a thermal cycler. After 8 hours, EDTA was added under the condition that the final concentration became 12.5mM to stop the reaction. The reaction solution was analyzed by HPLC and LC-TOF / MS according to the conditions described in Example 5, and the progress of the target reaction was confirmed.

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

[0143] Based on the values ​​calculated above, the residual rate (%) of impurities for chain A and chain B in each reaction was calculated using the following formula: Residual rate of impurities (%) = (ratio of N±1-mers in the reaction solution to the target product) / (ratio of N±1-mers in the substrate solution to the substrate)×100.

[0144] The results are shown in Table 17. In the reaction producing oligonucleic acids of 28 bases in length, the residual rate of impurities (N±1-mers) was 86% or more, whereas in the reaction producing oligonucleic acids of 25 bases or less in length, the residual rate was 23-59%.

[0145] [Table 16] Table 16. Sequences of products and substrates Pho: indicates a phosphate-based 5'-terminal modification, lowercase letters indicate DNA, uppercase letters indicate RNA, (Me): indicates a 2'-O-methyl-based modification.

[0146] [Table 17] Table 17. Residual rate of impurities (N±1 polymers) (%)

[0147] Industrial applicability The present invention is useful for the production of modified oligonucleotides (eg, siRNA, heteroduplex oligonucleotides, etc.) that can be used in products such as nucleic acid drugs.

Claims

1. A method for producing a modified oligonucleotide comprising a complementary portion having a length of 11 to 27 bases, wherein: The method comprises: treating a total of four or more oligonucleotide raw material fragments in the presence of an oligonucleotide ligase to generate the modified oligonucleotide; The total of 4 or more oligonucleotide raw material fragments corresponds to the oligonucleotide raw material fragments obtained when the modified oligonucleotide is cleaved at the fragment junction that satisfies the following conditions (i) to (v): (i) one or more fragment junctions are present on each strand side of the complementary portion, and a total of two or more fragment junctions are present in the modified oligonucleotide; (ii) when the modified oligonucleotide is cleaved at the fragment junction, an overhanging end is formed in the complementary portion, and the length of the overhanging end is 1 to 10 bases; (iii) at least one oligonucleotide starting material segment comprises a modified nucleotide; (iv) four of the four or more oligonucleotide starting fragments contain complementary portions with a length of 5 to 25 bases; and (v) The total length of bases corresponding to each strand of the complementary portion of the oligonucleotide raw material fragment is 11 to 27 bases in length.

2. The method according to claim 1, wherein The length of the protruding end in (ii) is 2 to 6 bases.

3. The method according to claim 1 or 2, wherein: The length of the complementary portion of the four oligonucleotide raw material fragments specified in (iv) excluding the overhanging ends is 4 to 16 bases.

4. The method according to any one of claims 1 to 3, wherein The oligonucleotide ligase is an RNA ligase.

5. The method according to claim 4, wherein Oligonucleotide ligase is a double-stranded RNA ligase.

6. The method according to claim 5, wherein: The double-stranded RNA ligase is an RNA ligase of the Rnl2 family or the Rnl5 family.

7. The method according to any one of claims 1 to 6, wherein Modified oligonucleotides contain modified nucleotide residues.

8. The method according to claim 7, wherein: The modified nucleotide residues include: 1', 2', 3' or 4' chemically modified nucleotide residues, 5'- or 3'-phosphate modified nucleotide residues, cross-linked modified nucleotide residues, carrier-added modified nucleotide residues, or sugar backbone substituted nucleotide residues.

9. The method according to claim 8, wherein Modified nucleotide residues are the following residues: i) Position 1', 2', 3' or 4' is replaced by C 1～6 Alkyloxy C 1～6 Alkylene, -OC 1～6 Alkyl, -OC 6～14 Aryl, -C-aryl, halogen atom, -OC 1～6 Alkyl N-amide C 1～6 Alkylene, -OC 1～6 Alkyl-(C 1～6 Alkyl-)amino-C 1～6 Alkylene, or -O-amino C 1～6 Alkyl-substituted 1', 2', 3' or 4' chemically modified nucleotide residues, The -O-amino C 1～6 Alkyl groups are exemplified by -O-aminopropyl, -O-AP; ii) a 5'- or 3'-phosphate-modified nucleotide residue in which the hydroxyl group may be substituted with a protecting group, or substituted with -OP(S)(OH)2, -NH-P(O)(OH)2, or -NH-P(S)(OH)2; iii) Positions 2' and 4' are replaced by 2'-OC 1～6 Alkylene-4', 2'-O-ethylene-4', 2'-O-methyl substituted methylene-4', 2'-OC 1～6 Alkylene-OC 1～6 Alkylene-4', 2'-ON(R)-C 1～6 Alkylene-4', 2'-N(R)-C(O)-4', 2'-NH-C 1～6 Alkylene-4', or 2'-C 1～6 Alkylene-4' substitution, or 3' and 5' positions are replaced by 3'-C 1～6 Alkylene-5' substituted cross-linking modified nucleotide residues, Here, R represents a methyl group, a hydrogen atom or a benzyl group; or iv) a hexitol nucleic acid (HNA) residue, a cyclohexenyl nucleic acid (CeNA) residue, or a morpholino nucleic acid (PMO) residue.

10. The method according to any one of claims 1 to 9, wherein The total molar ratio of any two oligonucleotide starting fragments selected from oligonucleotide starting fragments is 0.5-2.

11. The method according to any one of claims 1 to 10, wherein Oligonucleotide starting material fragments were treated at a monovalent cation salt concentration below 10 mM.

12. The method according to any one of claims 1 to 11, wherein Prior to the treatment, the mixed solution of oligonucleotide raw material fragments was not subjected to a high temperature and then cooled.

13. The method according to any one of claims 1 to 12, wherein: The generation of impurities in the modified oligonucleotide is suppressed.

14. The method according to any one of claims 1 to 13, wherein Further comprising purifying the modified oligonucleotide.

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