Method for producing polynucleotide linked product

Abstract

The present disclosure provides a technique that enables the simple preparation and / or purification of a desired polynucleotide linked product. Specifically, a desired polynucleotide linked product can be simply prepared and / or isolated by using a polynucleotide fragment containing a hydrophobic tag for the linking reaction.

Description

Methods for producing polynucleotide ligation products

[0001] The present disclosure relates to methods for producing polynucleotide ligation products, methods for purifying polynucleotides, and polynucleotides comprising hydrophobic tags, among others.

[0002] In recent years, nucleic acid medicines have been attracting attention as the next generation of medicines following antibody drugs and small molecule drugs. In particular, mRNA medicines are being used as vaccines against the new coronavirus, and have attracted much public attention amid the COVID-19 pandemic.

[0003] Further improvements to mRNA drugs are being investigated from the viewpoints of in vivo stability, translation efficiency, purity, etc. For example, Patent Document 1 investigates a technique for purifying nucleotides having a desired 5'-end structure by introducing a hydrophobic protecting group into the 5'-end of the nucleotides.

[0004] International Publication No. 2023 / 282245

[0005] An mRNA drug is composed of, in order from the 5' end of RNA, a cap structure (5' cap), a 5' untranslated region (5' UTR), a translated region, a 3' untranslated region (3' UTR), and a poly(A) tail. Patent Document 1 primarily aims to obtain nucleotides with a desired 5' end structure, and does not specifically consider the control of the 3' end structure.

[0006] Generally, mRNA is synthesized by transcribing from the 5'-end cap structure to the 3'UTR using DNA as a template (in vitro transcription: IVT), followed by the addition of a poly(A) tail. However, with mRNA synthesis using IVT, it is difficult to control the 3'-end sequence and the length of the poly(A) tail added. Therefore, with conventional IVT mRNA synthesis, it is difficult to obtain homogeneous mRNA, especially mRNA with a controlled 3'-end structure.

[0007] Furthermore, in mRNA medicines, it is important to prepare long-chain RNA with high purity. One method for preparing long-chain polynucleotides is to ligate multiple polynucleotide fragments. When attempting to obtain long-chain polynucleotides by ligating polynucleotide fragments, it is extremely difficult to proceed with the ligation reaction quantitatively, and therefore, the reaction system after the ligation reaction contains a mixture of the desired polynucleotide ligation product and unreacted polynucleotide fragments. Therefore, in this method, it is necessary to purify the desired polynucleotide ligation product after the ligation reaction of the polynucleotide fragments.

[0008] In view of the above circumstances, the present inventors aimed to provide a technique that allows for the simple preparation and / or purification of a desired polynucleotide ligation product.

[0009] The present inventors have discovered that the use of polynucleotide fragments containing hydrophobic tags in ligation reactions may enable the convenient preparation and / or isolation of desired polynucleotide ligation products. They have further improved the method and have completed the present disclosure.

[0010] The present disclosure includes, for example, the subject matter described in the following items: Item 1. Step (α): A step of ligating a polynucleotide fragment (A) containing a hydrophobic tag and a polynucleotide fragment (B) containing a hydrophobic tag, wherein the polynucleotide fragment (A) is represented by the general formula (1): [wherein k represents an integer of 1 or more. YA R represents a k-valent group obtained by removing k atoms or groups from a polynucleotide. A are the same or different and are represented by the general formula (2): (In the formula: R 1A represents an alkyl group. 2A represents a hydrogen atom or an alkyl group. 3A and R 5A One of the R groups represents a nitro group, and the other represents a hydrogen atom, an alkyl group, or an alkoxy group. 4A , R 6A , and R 7A are the same or different and represent a hydrogen atom, an alkyl group, or an alkoxy group (provided that R 3A is a nitro group, R4A and R 7A indicates a hydrogen atom). XA represents a linker. represents a group represented by the general formula (3): [wherein m represents an integer of 1 or more. YB R represents an m-valent group obtained by removing m atoms or groups from a polynucleotide. B are the same or different and are represented by the general formula (4): (In the formula: R 1B represents an alkyl group. 2B represents a hydrogen atom or an alkyl group. 3B and R 5B One of the R groups represents a nitro group, and the other represents a hydrogen atom, an alkyl group, or an alkoxy group. 4B , R 6B , and R 7B are the same or different and represent a hydrogen atom, an alkyl group, or an alkoxy group (provided that R 3B is a nitro group, R 4B and R 7B indicates a hydrogen atom). XB Item 2. A method for producing a polynucleotide ligation product, which is a polynucleotide fragment represented by the formula: Item 1. Item 3. A method for producing a polynucleotide ligation product according to Item 1, further comprising: step (β): purifying the polynucleotide ligation product based on the degree of hydrophobicity of the ligation product. Item 4. A method for producing a polynucleotide ligation product according to Item 1, further comprising: step (γ): removing the hydrophobic tag. Item 5. R A R YA Item 4. The method according to any one of Items 1 to 3, wherein the R B R YBItem 6. The method of any of Items 2 to 5, wherein step (β) comprises a separation step by liquid chromatography. Item 7. The method of any of Items 3 to 6, wherein step (γ) comprises a step of removing the hydrophobic tag by light irradiation and / or a step of removing the hydrophobic tag by reduction treatment. Item 8. The method of any of Items 1 to 4, wherein the linker R of the polynucleotide fragment (A) comprises a linker R of the polynucleotide fragment (A) XA and a linker R contained in the polynucleotide fragment (B). XB are the same or different and are a single bond, —O—CH2—, —O—C(═O)—, —C(═O)—, —O—(CH2) n -R 8 - or -O-C(=O)-(CH2) n -R 8 - [wherein n represents an integer of 1 or more. R 8 is represented by the general formula (5): (X 1 and X 2 and R are the same or different and represent O or S. Item 9. The method for producing a polynucleotide fragment (A) according to any one of Items 1 to 7, wherein R is the same or different and represents a divalent group represented by the general formula (1): [wherein k represents an integer of 1 or more. YA R represents a k-valent group obtained by removing k atoms or groups from a polynucleotide. A are the same or different and are represented by the general formula (2): (In the formula: R 1A R represents an alkyl group having 1 to 30 carbon atoms. 2A represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. 3A and R 5A One of the groups represents a nitro group, and the other represents a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, or an alkoxy group having 1 to 10 carbon atoms. 4A , R 6A , and R 7A are the same or different and represent a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, or an alkoxy group having 1 to 10 carbon atoms (wherein R 3A is a nitro group, R 4A and R 7Aindicates a hydrogen atom). XA represents a linker. represents a group represented by the formula (3): [wherein m represents an integer of 1 or more. YB R may be the same or different and represent an m-valent group obtained by removing m atoms or groups from a polynucleotide. B is represented by the general formula (4): (In the formula: R 1B R represents an alkyl group having 1 to 30 carbon atoms. 2B represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. 3B and R 5B One of the groups represents a nitro group, and the other represents a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, or an alkoxy group having 1 to 10 carbon atoms. 4B , R 6B , and R 7B are the same or different and represent a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, or an alkoxy group having 1 to 10 carbon atoms (wherein R 3B is a nitro group, R 4B and R 7B indicates a hydrogen atom). XB Item 10. The method according to any one of Items 1 to 9, wherein the polynucleotide ligation product is a polynucleotide fragment represented by the formula: Item 11. The method according to any one of Items 1 to 10, wherein in step (α), the reaction system comprises a polynucleotide having a sequence complementary to at least a portion of the polynucleotide fragment (A) and a sequence complementary to at least a portion of the polynucleotide fragment (B). Item 12. The method according to any one of Items 1 to 11, wherein the polynucleotide ligation product is an mRNA drug. Item 13. A polynucleotide ligation product represented by the formula (1): [wherein k represents an integer of 1 or more. YA R represents a k-valent group obtained by removing k atoms or groups from a polynucleotide. A are the same or different and are represented by the general formula (2): (In the formula: R 1Arepresents an alkyl group. 2A represents a hydrogen atom or an alkyl group. 3A and R 5A One of the R groups represents a nitro group, and the other represents a hydrogen atom, an alkyl group, or an alkoxy group. 4A , R 6A , and R 7A are the same or different and represent a hydrogen atom, an alkyl group, or an alkoxy group (provided that R 3A is a nitro group, R 4A and R 7A indicates a hydrogen atom). XA represents a linker. ) represents a group represented by the formula: A R YA Item 14. A polynucleotide in which R is bound to at least one nucleotide located 1 to 30 bases from the 3' end of R A R YA Item 15. The polynucleotide according to Item 13, wherein R 1A represents an alkyl group having 1 to 30 carbon atoms, and R 2A represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, and R 3A and R 5A one of the groups represents a nitro group, and the other represents a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, or an alkoxy group having 1 to 10 carbon atoms; R 4A , R 6A , and R 7A are the same or different and represent a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, or an alkoxy group having 1 to 10 carbon atoms (wherein R 3A is a nitro group, R 4A and R 7A Item 16. The polynucleotide according to Item 13 or 14, wherein the linker R XA are the same or different and are a single bond, —O—CH2—, —O—C(═O)—, —C(═O)—, —O—(CH2) n -R 8 - or -O-C(=O)-(CH2) n -R 8- [wherein n represents an integer of 1 or more. R 8 is represented by the general formula (5): (X 1 and X 2 Item 16. The polynucleotide of any one of Items 13 to 15, wherein R, R, and R are the same or different and represent O or S. Item 17. The polynucleotide of any one of Items 13 to 16, which is 20 to 2000 bases in length. Item 18. The polynucleotide of any one of Items 13 to 17, which is an mRNA drug. Item 19. A method for producing an RNA ligation product, comprising: step (α): ligating an RNA fragment (A) containing a hydrophobic tag and an RNA fragment (B) containing a hydrophobic tag; step (β): purifying the RNA ligation product based on the degree of hydrophobicity of the ligation product; and step (γ): removing the hydrophobic tag, wherein in step (α), the reaction system comprises a polynucleotide having a sequence complementary to at least a portion of the RNA fragment (A) and a sequence complementary to at least a portion of the RNA fragment (B), and wherein the RNA fragment (A) is a polynucleotide represented by the general formula (1): [wherein k represents an integer of 1 or more. YA R represents a k-valent group obtained by removing k atoms or groups from RNA. A are the same or different and are represented by the general formula (2): (In the formula: R 1A represents an alkyl group. 2A represents a hydrogen atom or an alkyl group. 3A and R 5A One of the R groups represents a nitro group, and the other represents a hydrogen atom, an alkyl group, or an alkoxy group. 4A , R 6A , and R 7A are the same or different and represent a hydrogen atom, an alkyl group, or an alkoxy group (provided that R 3A is a nitro group, R 4A and R 7A indicates a hydrogen atom). XA represents a linker. represents a group represented by the general formula (3): [wherein m represents an integer of 1 or more. YBR represents an m-valent group obtained by removing m atoms or groups from RNA. B are the same or different and are represented by the general formula (4): (In the formula: R 1B represents an alkyl group. 2B represents a hydrogen atom or an alkyl group. 3B and R 5B One of the R groups represents a nitro group, and the other represents a hydrogen atom, an alkyl group, or an alkoxy group. 4B , R 6B , and R 7B are the same or different and represent a hydrogen atom, an alkyl group, or an alkoxy group (provided that R 3B is a nitro group, R 4B and R 7B indicates a hydrogen atom). XB Item 20. A method for producing an RNA ligation product, wherein the RNA fragment (A) is an RNA fragment represented by the formula: [R(R)-R(R)) (where R(R)-R(R)) represents a linker. ) represents a group represented by the formula: ], wherein the step (β) includes a separation step by liquid chromatography, and the step (γ) includes a step of removing the hydrophobic tag by light irradiation and / or a step of removing the hydrophobic tag by reduction treatment. XA and a linker R contained in the RNA fragment (B). XB are the same or different and are a single bond, —O—CH2—, —O—C(═O)—, —C(═O)—, —O—(CH2) n -R 8 - or -O-C(=O)-(CH2) n -R 8 - [wherein n represents an integer of 1 or more. R 8 is represented by the general formula (5): (X 1 and X 2 Item 21. The method according to Item 19, wherein the RNA fragment (A) is a divalent group represented by general formula (1): [wherein k represents an integer of 1 or more. YA R represents a k-valent group obtained by removing k atoms or groups from RNA. A are the same or different and are represented by the general formula (2): (In the formula: R 1AR represents an alkyl group having 1 to 30 carbon atoms. 2A represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. 3A and R 5A One of the groups represents a nitro group, and the other represents a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, or an alkoxy group having 1 to 10 carbon atoms. 4A , R 6A , and R 7A are the same or different and represent a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, or an alkoxy group having 1 to 10 carbon atoms (wherein R 3A is a nitro group, R 4A and R 7A indicates a hydrogen atom). XA represents a linker. represents a group represented by the formula (3): [wherein m represents an integer of 1 or more. YB R may be the same or different and represent an m-valent group obtained by removing m atoms or groups from RNA. B is represented by the general formula (4): (In the formula: R 1B R represents an alkyl group having 1 to 30 carbon atoms. 2B represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. 3B and R 5B One of the groups represents a nitro group, and the other represents a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, or an alkoxy group having 1 to 10 carbon atoms. 4B , R 6B , and R 7B are the same or different and represent a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, or an alkoxy group having 1 to 10 carbon atoms (wherein R 3B is a nitro group, R 4B and R 7B indicates a hydrogen atom). XB Item 21. The method for producing an RNA fragment according to Item 19 or 20, wherein the RNA fragment is represented by the general formula (1): [wherein k represents an integer of 1 or more. YA R represents a k-valent group obtained by removing k atoms or groups from RNA. Aare the same or different and are represented by the general formula (2): (In the formula: R 1A represents an alkyl group having 1 to 30 carbon atoms, and R 2A represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, and R 3A and R 5A one of the groups represents a nitro group, and the other represents a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, or an alkoxy group having 1 to 10 carbon atoms; R 4A , R 6A , and R 7A are the same or different and represent a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, or an alkoxy group having 1 to 10 carbon atoms (wherein R 3A is a nitro group, R 4A and R 7A indicates a hydrogen atom). XA represents a linker. ) represents a group represented by the formula: A R YA is bound to at least one nucleotide located 1 to 30 bases from the 3' end of R A R YA Item 23. The linker R is bound to at least one nucleotide located 1 to 30 bases from the 5' end of the XA are the same or different and are a single bond, —O—CH2—, —O—C(═O)—, —C(═O)—, —O—(CH2) n -R 8 - or -O-C(=O)-(CH2) n -R 8 - [wherein n represents an integer of 1 or more. R 8 is represented by the general formula (5): (X 1 and X 2 and R are the same or different and represent O or S. The RNA according to Item 22,

[0011] As described above, mRNA drugs are composed of, in order from the 5' end of the RNA, a cap structure (5' cap), a 5' untranslated region (5' UTR), a translated region, a 3' untranslated region (3' UTR), and a poly(A) tail. It is known that structures other than the translated region affect mRNA stability and translation efficiency. However, conventional mRNA synthesis methods have made it difficult to control the 3' end sequence and the length of the poly(A) tail added. In other words, mRNA obtained using conventional mRNA synthesis methods is not homogeneous, particularly in terms of the 3' end sequence and poly(A) tail length. In contrast, the technology disclosed herein allows for the ligation of RNA fragments containing hydrophobic tags and subsequent purification based on the degree of hydrophobicity, thereby producing highly purified, homogeneous mRNA. Furthermore, because the hydrophobic tag can be removed under mild conditions, it is possible to ligate additional RNA fragments after removing the hydrophobic tag from the purified ligated product. In other words, the process of ligating RNA fragments, purifying the ligated product, and removing the hydrophobic tag can be repeated until an RNA ligation product of the desired length is obtained (Figure 1).

[0012] This figure shows an overview of the method for preparing and purifying polynucleotide ligation products using the technology of the present disclosure. The right side of the figure shows the expected peak variations when reversed-phase HPLC is performed at each stage. The results of reversed-phase HPLC before and after the reduction treatment performed in Test Example 2 are shown. This figure shows the 25-mer RNA synthesized using an automated nucleic acid synthesizer in Test Example 3, with CPR-Nb at the 5' end and compound 26 at the 3' end. This figure shows the results of reversed-phase HPLC performed on the 25-mer RNA obtained in Test Example 3, with CPR-Nb at the 5' end and compound 26 at the 3' end. * indicates the peak of the target product (RNA with hydrophobic tags at the 5' and 3' ends). Other peaks are believed to represent the target product from which at least one hydrophobic tag has been removed. The results of reversed-phase HPLC performed again on the target product shown in Figure 4 are shown. This figure shows the results of reversed-phase HPLC performed on the target RNA fractionated in Test Example 3 after UV irradiation. Schematic diagrams of the DNA template (i), 25-mer RNA fragment (ii), and 25-mer RNA fragment (iii) with a hydrophobic tag at the 3'-end used in the ligation reaction in Test Example 4, as well as the ligation product produced by the ligation reaction, are shown. The results of reverse-phase HPLC performed in Test Example 4 are shown. An outline of Test Example 5 is shown. A conventional co-transcriptional transfer method using ARCA (anti-reverse cap analog) (left) and an outline of the transcription reaction using "Pure Cap analog" as a substrate (right) performed in Test Example 5-1 are shown. The sequence encoding NanoLuc™ luciferase (Promega) transcribed in Test Example 5-1 is shown. The results of reverse-phase HPLC of the transcription product obtained in Test Example 5-1 (before fractionation and UV irradiation) are shown. The peak of the target product is indicated by *. The results of reverse-phase HPLC of the target product after fractionation (*) and the product after fractionation and UV irradiation (*') performed in Test Example 5-1 are shown. The results of denaturing PAGE performed in Test Example 5-1 are shown. The sequence of the RNA containing a polyA chain synthesized in Test Example 5-2-1 is shown. PS represents a phosphorothioate group. The results of separating the RNA containing a polyA chain synthesized in Test Example 5-2-1 by reverse-phase HPLC (before UV irradiation) are shown. * represents the peak of the target product. The results of denaturing PAGE of the target product (*) after separation, performed in Test Example 5-2-1, are shown.18 shows the results of reverse-phase HPLC for the target product after fractionation (*) and the product after fractionation and UV irradiation (*') performed in Test Example 5-2-1. 19 shows the results of reverse-phase HPLC before and after post-modification performed in Test Example 5-2-3. Note that the results before post-modification are the same as those shown in Figure 18. The left figure shows the results before post-modification, and the right figure shows the results after post-modification. 19 shows the results of denaturing PAGE performed in Test Example 5-2-3. 3-OME: RNA with three 2'-O-methyl adenosines at the 3' end. 5-OME: RNA with five 2'-O-methyl adenosines at the 3' end. 19 shows an overview of Test Example 5-2. 19 shows the results of reverse-phase HPLC (before fractionation and UV irradiation) performed in Test Example 5-3. 19 shows the results of denaturing PAGE performed in Test Example 5-3. 19 shows the results of denaturing PAGE performed in Test Example 5-3. 19 shows the results of reverse-phase HPLC (before fractionation and UV irradiation) performed in Test Example 5-3. 19 shows the results of denaturing PAGE performed in Test Example 5-3. 19 shows the results of reverse-phase HPLC (after fractionation and UV irradiation) ... The results of Test Example 5-4 are shown. The structures at the bottom of the graph indicate the structures of the 3'-end side of each RNA used in Test Example 5-4.

[0013] Each embodiment of the present disclosure will be described in more detail below. The present disclosure preferably includes, but is not limited to, a method for producing a polynucleotide ligation product, a method for purifying a polynucleotide ligation product, and a polynucleotide comprising a hydrophobic tag. The present disclosure includes all of the disclosures herein that can be recognized by a person skilled in the art.

[0014] The method for producing a polynucleotide ligation product encompassed by the present disclosure includes a step of ligating polynucleotide fragments containing hydrophobic tags (sometimes referred to as step (α) in the present disclosure).

[0015] In this disclosure, the terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to a polymer of two or more nucleotides of any length. The term "polynucleotide" also includes "polynucleotide derivatives." That is, the term "polynucleotide" in this disclosure includes polynucleotides containing nucleotide derivatives; polynucleotides in which the internucleotide bond is different from the usual one; and nucleotide derivatives, and further includes polynucleotides in which the internucleotide bond is different from the usual one.

[0016] More specific examples of polynucleotide derivatives include those that have been subjected to known chemical modifications. For example, to prevent degradation by hydrolases such as nucleases, the phosphate residues of each nucleotide can be substituted with chemically modified phosphate residues such as phosphorothioate (PS), methylphosphonate, and phosphorodithioate. The hydroxyl group at the 2-position of the sugar (ribose) can also be substituted with -OR (where R represents, for example, CH3(2'-O-Me), CH2CHOCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, or CH2CH2CN). Furthermore, the base moiety (pyrimidine or purine) can also be chemically modified, for example, by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a thiocarbonyl group for the carbonyl group at the 2-position. Specific examples of polynucleotide derivatives include, but are not limited to, those in which the phosphate moiety or hydroxyl moiety is modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, etc. In addition, BNA (LNA), in which the conformation of the sugar moiety of a nucleotide is fixed to N-type by bridging the 2' oxygen and 4' carbon of the sugar moiety, can also be used as a polynucleotide derivative.

[0017] More specific examples of nucleic acid bases constituting nucleotides include not only typical bases in DNA and RNA (adenine (A), uracil (U), guanine (G), cytosine (C), etc.), but also other bases such as hypoxanthine (I) and modified bases. Modified bases include, for example, pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (e.g., 5-methylcytosine), 5-alkyluracil (e.g., 5-ethyluracil), 5-halouracil (5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidine (6-methyluracil), 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, 1-methyladenine, 1-methylhypoxanthine (I), and the like. Examples include xanthine, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyladenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, 2-thiocytosine, purine, 2-aminopurine, isoguanine, indole, imidazole, and xanthine.

[0018] As the polynucleotide, RNA is particularly preferred from the viewpoint that there is a high need for the technology of the present disclosure and that the technology of the present disclosure can provide a polynucleotide with excellent functions.

[0019] In this disclosure, "polynucleotide fragment" refers to a polynucleotide that serves as a substrate for the ligation reaction described below. Furthermore, "polynucleotide ligation product" refers to the product of the ligation reaction. Therefore, when ligation reactions are performed sequentially, the "polynucleotide ligation product" produced by the first ligation reaction can be referred to as the "polynucleotide fragment" in the subsequent ligation reaction. For example, when polynucleotide (a), polynucleotide (b), and polynucleotide (c) are ligated in this order, in the first ligation reaction, polynucleotide (a) and polynucleotide (b) correspond to the "polynucleotide fragment" of this disclosure, and polynucleotide (a+b) formed by ligating polynucleotide (a) and polynucleotide (b) corresponds to the "polynucleotide ligation product" of this disclosure. Meanwhile, in the second ligation reaction, polynucleotide (a+b) and polynucleotide (c) correspond to the "polynucleotide fragment," and polynucleotide (a+b+c) formed by ligating polynucleotide (a+b) and polynucleotide (c) corresponds to the "polynucleotide ligation product."

[0020] The "ligation reaction" in the present disclosure is not particularly limited as long as it is a reaction in which two or more polynucleotides are ligated (i.e., an intermolecular reaction between polynucleotides), and may be a chemical synthetic ligation reaction or an enzymatic ligation reaction. Examples of chemical synthetic ligation reactions include ligation reactions performed using a commercially available automated nucleic acid synthesizer. Examples of enzymatic ligation reactions include ligation reactions using DNA ligases such as T4 DNA ligase, E. coli DNA ligase, and Tfi DNA ligase, and ligation reactions using RNA ligases such as T4 RNA ligase 1 and T4 RNA ligase 2. The "ligation reaction" in the present disclosure is preferably an enzymatic ligation reaction, more preferably a ligation reaction using an RNA ligase, and particularly preferably a ligation reaction using T4 RNA ligase 2. Furthermore, the "ligation reaction" in the present disclosure is preferably a ligation reaction in which a new phosphodiester bond is generated.

[0021] When the ligation reaction is an enzymatic ligation reaction, it is typically carried out in a solution (preferably further containing a template) containing multiple types (e.g., 2 to 5 types, 2 to 3 types, or 2 types) of polynucleotide fragments to be ligated and an enzyme (e.g., ligase). In this case, the reaction temperature is not particularly limited as long as it allows the enzyme activity to be exerted, and is, for example, 10 to 80°C, 15 to 50°C, 15 to 40°C, or 15 to 35°C. The reaction time is also not particularly limited as long as a detectable level of ligation product is produced, and is, for example, 1 to 48 hours or 4 to 24 hours.

[0022] Although not particularly limited, in the ligation reaction of the present disclosure, the reaction system preferably includes a polynucleotide having a sequence complementary to at least a portion of polynucleotide fragment (A) described below and a sequence complementary to at least a portion of polynucleotide fragment (B) described below. The polynucleotide functions as a "template" in the ligation reaction and can improve the efficiency of the ligation reaction. More specifically, when polynucleotide fragment (A) and polynucleotide fragment (B) anneal to complementary sites on the template, the ends of the two polynucleotide fragments are brought into close proximity. Therefore, for example, when the above-mentioned ligation reaction is an enzymatic ligation reaction, the efficiency of the reaction in which the enzyme ligates the ends of the two polynucleotide fragments can be improved.

[0023] Furthermore, the 3'-terminal sequences of the RNA fragments obtained by IVT are not uniform. However, by performing the ligation reaction in the presence of a template, it is expected that the other RNA fragment will be ligated only to the 3'-end of the RNA fragment with the desired sequence. In other words, especially when RNA fragments obtained by IVT are used in ligation reactions, the presence of a template in the reaction system can suppress the production of ligation products with unintended sequences.

[0024] The method for preparing polynucleotide fragments used in the present disclosure is not particularly limited, and any conventionally known method or a method easily derived from a conventionally known method can be employed. For example, chemical synthesis can be performed using a commercially available automated nucleic acid synthesizer, or enzymatic synthesis can be performed using a polynucleotide serving as a template encoding a desired sequence and a polynucleotide synthesizing enzyme, or multiple polynucleotides can be ligated together. Examples of polynucleotide synthesizing enzymes include DNA polymerase and RNA polymerase. Furthermore, (mono- or poly)nucleotide derivatives can be used as raw materials or substrates for the synthesis, and polynucleotides can be modified after synthesis.

[0025] In the present disclosure, the length of the polynucleotide fragments subjected to the ligation reaction is not particularly limited as long as the effects of the present disclosure are not impaired. For example, the length may be 2 to 100,000 bases, or 5 to 50,000 bases, preferably 10 to 10,000 bases, more preferably 15 to 5,000 bases, and particularly preferably 20 to 1,000 bases. The upper or lower limit of the range may be 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, or 90000 bases in length.

[0026] In the present disclosure, the length of the polynucleotide ligation product obtained by the ligation reaction is not particularly limited as long as the effects of the present disclosure are not impaired. For example, the length may be 5 to 200,000 bases, or 10 to 100,000 bases, preferably 20 to 50,000 bases, more preferably 50 to 10,000 bases, and particularly preferably 100 to 5,000 bases. The upper or lower limit of the range may be 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, or 900000 bases in length.

[0027] More specifically, the method for producing a polynucleotide ligation product encompassed by the present disclosure comprises the step of ligating the following polynucleotide fragment (A) and the following polynucleotide fragment (B):

[0028] Polynucleotide fragment (A): a polynucleotide fragment of the following general formula (1): [wherein k represents an integer of 1 or more. YA R represents a k-valent group obtained by removing k atoms or groups from a polynucleotide. A are the same or different and are represented by the following general formula (2): (In the formula, R 1A represents an alkyl group. 2A represents a hydrogen atom or an alkyl group. 3A and R 5A One of the R groups represents a nitro group, and the other represents a hydrogen atom, an alkyl group, or an alkoxy group. 4A , R 6A , and R 7A are the same or different and represent a hydrogen atom, an alkyl group, or an alkoxy group (provided that R 3A is a nitro group, R 4A and R 7A indicates a hydrogen atom). XArepresents a linker. represents a group represented by the formula:

[0029] Polynucleotide fragment (B): a polynucleotide fragment of the following general formula (3): [In the formula, m represents an integer of 1 or more. YB R represents an m-valent group obtained by removing m atoms or groups from a polynucleotide. B are the same or different and are represented by the following general formula (4): (In the formula, R 1B represents an alkyl group. 2B represents a hydrogen atom or an alkyl group. 3B and R 5B One of the R groups represents a nitro group, and the other represents a hydrogen atom, an alkyl group, or an alkoxy group. 4B , R 6B , and R 7B are the same or different and represent a hydrogen atom, an alkyl group, or an alkoxy group (provided that R 3B is a nitro group, R 4B and R 7B indicates a hydrogen atom). XB represents a linker. represents a group represented by the formula:

[0030] In the present disclosure, the structure of the above general formula (2) or (4) excluding the linker may be particularly referred to as a "hydrophobic tag."

[0031] In the present disclosure, k is an integer of 1 or greater and is not particularly limited as long as the effects of the present disclosure are achieved. k may be 1 to 100, or 1 to 50, preferably 1 to 20, more preferably 1 to 10, and particularly preferably 1 to 5. The upper or lower limit of the range may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, or 45. The above description of k applies to m. Note that k and m may be the same or different.

[0032] R 1Ais not particularly limited as long as it is an alkyl group. It may be either a linear alkyl group or a branched alkyl group. Furthermore, the number of carbon atoms is not particularly limited, and may be, for example, 1 to 30 carbon atoms, preferably 1 to 20, more preferably 1 to 10, and particularly preferably 1 to 5. The upper or lower limit of the range may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. 1A The description of R 1B It is incorporated by reference in R 1A and R 1B may be the same or different.

[0033] R 2A is not particularly limited as long as it is a hydrogen atom or an alkyl group, but is preferably a hydrogen atom. 2A When R is an alkyl group, it may be a straight-chain alkyl group or a branched-chain alkyl group. Furthermore, the number of carbon atoms is not particularly limited, and may be, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 4. The upper or lower limit of the range may be 2, 3, 4, 5, 6, 7, 8, or 9. 2A The description of R 2B It is incorporated by reference in R 2A and R 2B may be the same or different.

[0034] R 3A and R 5A One of the R groups represents a nitro group, and the other represents a hydrogen atom, an alkyl group, or an alkoxy group. 3A When R is a nitro group, the hydrophobic tag has an o-nitrobenzyl backbone. 5AWhen is a nitro group, the hydrophobic tag has a p-nitrobenzyl skeleton. In this disclosure, the structure represented by general formula (6) will be referred to as an o-nitrobenzyl skeleton, and the structure represented by general formula (7) will be referred to as a p-nitrobenzyl skeleton. In this disclosure, the moiety having an o-nitrobenzyl skeleton or a p-nitrobenzyl skeleton may be referred to as a "nitrobenzyl portion." General formula (6): General formula (7): [In the formula, R represents any substituent other than a nitro group or a hydrogen atom.]

[0035] The polynucleotide fragment of the present disclosure may be, but is not limited to, R 3A is preferably a nitro group, i.e., the hydrophobic tag has an o-nitrobenzyl backbone. 3A and R 5A The non-nitro group among the above is not particularly limited as long as it is a hydrogen atom, an alkyl group, or an alkoxy group, but is preferably a hydrogen atom. When it is an alkyl group, it may be a straight-chain alkyl group or a branched-chain alkyl group. When it is an alkoxy group, it may be a straight-chain alkoxy group or a branched-chain alkoxy group. When it is an alkyl group or an alkoxy group, the number of carbon atoms is not particularly limited, and may be, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 4. The upper or lower limit of the range may be 2, 3, 4, 5, 6, 7, 8, or 9. 3A and R 5A The description of R 3B and R 5B It is incorporated by reference in R 3A and R 3B , and R 5A and R 5B may be the same or different.

[0036] R 4A , R 6A , and R 7A are the same or different and represent a hydrogen atom, an alkyl group, or an alkoxy group, and R 3A is a nitro group, R 4A and R 7A represents a hydrogen atom.4A , R 6A , and R 7A is preferably a hydrogen atom. When it is an alkyl group, it may be a straight-chain alkyl group or a branched-chain alkyl group. When it is an alkoxy group, it may be a straight-chain alkoxy group or a branched-chain alkoxy group. When it is an alkyl group or an alkoxy group, the number of carbon atoms is not particularly limited, and may be, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 4. The upper or lower limit of the range may be 2, 3, 4, 5, 6, 7, 8, or 9. 4A , R 6A , and R 7A The description of R 4B , R 6B , and R 7B It is incorporated by reference in R 4A and R 4B , R 6A and R 6B , and R 7A and R 7B may be the same or different.

[0037] In this specification, examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, n-hexyl, 3-methylpentyl, etc. Examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, etc.

[0038] R XA R represents a linker, and its structure is not particularly limited as long as the hydrophobic tag can be removed in the hydrophobic tag removal step described below. XA Examples of the structure include a single bond, -O-CH2-, -O-C(=O)-, -C(=O)-, and -O-(CH2) n -R 8 -, -O-C (=O) - (CH2) n -R 8 - [wherein n represents an integer of 1 or more. R 8is represented by the general formula (5): (X 1 and X 2 are the same or different and represent O or S). XA The description of R XB It is incorporated by reference in R XA and R XB may be the same or different.

[0039] R YA represents a k-valent group obtained by removing k atoms or groups from a polynucleotide. As mentioned above, the term "polynucleotide" in the present disclosure includes "polynucleotide derivatives." Therefore, R YA may be a k-valent group obtained by removing k atoms or groups from a polynucleotide derivative. Examples of the polynucleotide derivative include those modified by alkylation such as methylation.

[0040] R YB R represents an m-valent group obtained by removing m atoms or groups from a polynucleotide. YB may be an m-valent group obtained by removing m atoms or groups from a polynucleotide derivative, and examples of the polynucleotide derivative include those modified by alkylation such as methylation.

[0041] Examples of atoms or groups to be removed from a polynucleotide include a phosphate group, a hydroxyl group or hydrogen atom constituting a phosphate group, a hydroxyl group or hydrogen atom of a sugar moiety, and an amino group or hydrogen atom of a nucleic acid base.

[0042] Although not particularly limited, R A is R YAPreferably, the hydrophobic tag is bound to at least one of the nucleotides located 1 to 30 bases from the 3'-end of the polynucleotide fragment (A). In other words, the polynucleotide fragment (A) of the present disclosure preferably has at least one hydrophobic tag located 1 to 30 bases from the 3'-end. In step (β): purifying the polynucleotide ligation product based on the degree of hydrophobicity, from the viewpoint of improving the purification efficiency, the polynucleotide fragment (A) preferably has the hydrophobic tag located 1 to 20 bases from the 3'-end, more preferably located 1 to 10 bases, and particularly preferably located 1 to 5 bases. From the same viewpoint, and also from the viewpoint of production efficiency of the polynucleotide fragment (A), the polynucleotide fragment (A) preferably has the hydrophobic tag at the 3'-end. The upper or lower limit of the range may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29.

[0043] Although not particularly limited, R B is R YB Preferably, the hydrophobic tag is bound to at least one of the nucleotides located 1 to 30 bases from the 5' end of the polynucleotide fragment (B). In other words, the polynucleotide fragment (B) of the present disclosure preferably has at least one hydrophobic tag located 1 to 30 bases from the 5' end. In step (β): purifying the polynucleotide ligation product based on the degree of hydrophobicity, from the viewpoint of improving the purification efficiency, the polynucleotide fragment (B) preferably has the hydrophobic tag located 1 to 20 bases from the 5' end, more preferably located 1 to 10 bases, and particularly preferably located 1 to 5 bases. From the same viewpoint, and also from the viewpoint of production efficiency of the polynucleotide fragment (B), the polynucleotide fragment (B) preferably has the hydrophobic tag at the 3' end. The upper or lower limit of the range may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29.

[0044] The polynucleotide fragment (A) preferably contains a coding sequence for a polypeptide, and also preferably contains a 5' untranslated region (5'UTR) in addition to the coding sequence. The polynucleotide fragment (B) preferably contains a polyA region (a region formed by linking multiple nucleotides (e.g., 3 to 500 bases, preferably 10 to 300 bases, more preferably 20 to 250 bases) in which the nucleic acid base is adenine). By performing a ligation reaction using an RNA fragment as the above fragment, homogeneous mRNA, particularly mRNA with a controlled structure on the 3' end, can be obtained.

[0045] Preferably, the method for producing a polynucleotide ligation product encompassed by the present disclosure further comprises a step of purifying the polynucleotide ligation product based on the degree of hydrophobicity of the ligation product (sometimes referred to as step (β) in the present disclosure). The method used in step (β) is not particularly limited as long as it is capable of separating compounds according to the degree of hydrophobicity, and known methods or methods that can be easily derived from known methods can be used. Examples of such methods include liquid chromatography and membrane separation. Step (β) preferably comprises a separation step by liquid chromatography, more preferably a separation step by reversed-phase liquid chromatography, and particularly preferably a separation step by reversed-phase high-performance liquid chromatography (reverse-phase HPLC).

[0046] When step (β) includes a separation step using reversed-phase liquid chromatography, the packing material packed in the column preferably contains an alkyl group having 1 to 40 carbon atoms as a bonded phase. The number of carbon atoms in the alkyl group may be 2 to 30, preferably 3 to 20, more preferably 4 to 18, and particularly preferably 4 to 8. The upper or lower limit of the range may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39. An example of a column packed with a packing material containing an alkyl group having 4 carbon atoms as a bonded phase is YMC-Triart Bio C4 (YMC Corporation).

[0047] The mobile phase used in reversed-phase liquid chromatography is not particularly limited as long as it can separate compounds according to their degree of hydrophobicity. For example, water and organic solvents such as methanol and acetonitrile (ACN) can be used. Alternatively, gradient elution can be performed by continuously changing the water / organic solvent ratio. Furthermore, the temperature at which the chromatography is performed is also not particularly limited. For example, the temperature can be about 10 to 50°C, about 15 to 40°C, preferably about 20 to 30°C, and more preferably about 25°C.

[0048] Preferably, the method for producing a polynucleotide ligation product encompassed by the present disclosure further comprises a step of removing the hydrophobic tag by light irradiation and / or a step of removing the hydrophobic tag by reduction treatment (sometimes referred to as step (γ) in the present disclosure). The site to be removed in step (γ) is not particularly limited as long as it contains a hydrophobic tag. Therefore, the site to be removed may or may not contain the linker.

[0049] As described above, the hydrophobic tag of the present disclosure has an o-nitrobenzyl skeleton or a p-nitrobenzyl skeleton. When the hydrophobic tag has an o-nitrobenzyl skeleton, the hydrophobic tag can be released by light irradiation or reduction treatment. On the other hand, when the hydrophobic tag has a p-nitrobenzyl skeleton, the hydrophobic tag is not released by light irradiation but can be released by reduction treatment. Therefore, when a polynucleotide ligation product contains both a hydrophobic tag having an o-nitrobenzyl skeleton and a hydrophobic tag having a p-nitrobenzyl skeleton, a "separate release" method is possible: first, the hydrophobic tag having the o-nitrobenzyl skeleton is selectively released by light irradiation, and then the hydrophobic tag having the p-nitrobenzyl skeleton is released by reduction treatment.

[0050] The conditions for the light irradiation are not particularly limited as long as the hydrophobic tag can be removed, and can be appropriately set depending on the properties of the hydrophobic tag, etc. For example, light having a wavelength of 300 nm to 400 nm is irradiated at 0.5 to 10 mW / cm 2 It is also possible to irradiate the specimen with a light intensity of 1 to 60 minutes.

[0051] The conditions for the reduction treatment are not particularly limited as long as the hydrophobic tag can be removed, and can be appropriately set depending on the properties of the hydrophobic tag, etc. For example, the reduction treatment may be performed by incubation in a 1 to 1000 mM sodium dithionite solution at 20 to 45°C for 10 to 60 minutes.

[0052] The polynucleotide from which the hydrophobic tag has been removed exhibits reduced hydrophobicity (i.e., increased hydrophilicity), and this can be used to confirm the removal of the hydrophobic tag. For example, if the retention time of the sample after step (γ) in reverse-phase HPLC is shorter than that of the sample before step (γ), it can be determined that the hydrophobic tag has been removed.

[0053] The polynucleotide ligation product obtained by the production method of the present disclosure may be an mRNA drug. As described above, an mRNA drug is composed of, in order from the 5' end of the RNA, a cap structure (5' cap), a 5' untranslated region (5' UTR), a translated region, a 3' untranslated region (3' UTR), and a poly(A) tail. The production method of the present disclosure can obtain a polynucleotide ligation product with a homogeneous structure from the 5' cap to the poly(A) tail. In particular, due to the characteristics of mRNA drugs, the expression efficiency (translation efficiency) of the target protein in the body must be highly controlled. The structure of the terminal end of the mRNA is known to have a significant impact on the stability and translation efficiency of the mRNA. Therefore, it can be said that the polynucleotide ligation product obtained by the production method of the present disclosure, with a highly controlled structure from the 5' cap to the poly(A) tail, is suitable as an mRNA drug.

[0054] In this specification, the term "comprising" includes "essentially consisting of" and "consisting of" in addition to "containing." Furthermore, the present disclosure includes any and all combinations of the constituent elements described in this specification.

[0055] Furthermore, the various characteristics (properties, structures, values, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to identify the subject matter encompassed by the present disclosure. In other words, the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described in this specification.

[0056] Hereinafter, the embodiments of the present disclosure will be described in more detail with reference to examples, but the embodiments of the present disclosure are not limited to the following examples. Note that, unless otherwise specified, "%" hereinafter refers to "% by mass."

[0057] Test Example 1 Synthesis of Hydrophobic Tag An amidite compound (compound 26) for the hydrophobic tag to be introduced at the 3' end was synthesized. The synthesis scheme is shown below.

[0058] 1-1. Synthesis of Compound 22: 2-methyl-2-(2-nitrophenyl)propanoic acid 2-Nitrophenylacetic acid (0.500 g, 2.76 mmol, 1.0 eq.) was dissolved in methanol (MeOH) (15.0 mL) and cooled in an ice bath. Thionyl chloride (300 μL, 4.14 mmol, 1.5 eq.) was added dropwise to the solution at 0 °C. The mixture was stirred at room temperature for 2 hours. The mixture was diluted with ethyl acetate and washed with water and saturated brine. The organic layer was dried over Na2SO4 and concentrated. The residue was dissolved in N,N-dimethylformamide (DMF) (15.0 mL), and sodium hydride (0.166 g, 6.90 mmol, 2.5 eq.) was added portionwise to the solution. The mixture was stirred at room temperature for 30 minutes, and then iodomethane (395 μL, 6.35 mmol, 2.3 eq.) was added and stirred at room temperature overnight. The mixture was diluted with ethyl acetate and washed with water and saturated brine. The organic layer was dried over Na2SO4 and concentrated. The residue was dissolved in MeOH (5.00 mL) and aqueous NaOH (8 M, 5.00 mL) was added. The mixture was stirred at 85 °C for 3 hours, and then aqueous HCl (2 M) was added to adjust the pH to 3.0-4.0. The mixture was diluted with ethyl acetate and washed with water and saturated brine. The organic layer was dried over Na2SO4 and concentrated to give the target compound (521 mg, 90.2% yield) as a yellow powder. 1 [HRMS] (ESI) m / z Cal. for C 10 H 11 NNaO4 + [M+Na] + ; 232.0580 found 232.0655

[0059] 1-2. Compound 23: 3',5'-O-bis(t-butylsilyl)-2'-O-(t-butyldimethylsilyl)-N 6Synthesis of -[2-nitrobenzyl(dimethyl)carbonyl]-adenosine 2-Methyl-2-(2-nitrophenyl)propanoic acid (compound 22) (1.20 g, 5.75 mmol, 3.0 eq.) was dissolved in dichloromethane (DCM) (30.0 mL) and cooled in an ice bath. To this solution, thionyl chloride (1.25 mL, 17.3 mmol, 9.0 eq.) was added dropwise at 0 °C. The mixture was stirred at 40 °C for 4 h. The solution was concentrated using an evaporator, and the residue was dissolved in DCM / pyridine (5 / 1, 10.0 mL) (solution A). 4-Dimethylaminopyridine (70.2 mg, 0.575 mmol, 0.3 eq.) was added to solution A and stirred for 20 min. 3',5'-O-bis(t-butylsilyl)-2'-O-(t-butyldimethylsilyl)adenosine (compound 13) (1.00 g, 1.92 mmol, 1.0 eq.) was dissolved in pyridine (5.00 mL) (solution B), and triethylamine (801 μL, 5.75 mmol, 3.0 eq.) was added at room temperature. Solution A was added dropwise to solution B, and the mixture was stirred at 80 °C overnight. The reaction mixture was diluted with ethyl acetate, water, NaHCO 3 . 3飽和 The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography eluting with 12.5% ​​acetonitrile / dichloromethane to give the target compound (0.507 g, 36.9% yield) as a brown foam. 1 H NMR] (594 MHz, DMSO, ppm, mixture of rotamer) δ 9.92-9.78 (m, 1H, NH), 8.66-8.56 (m, 2H, C 2 H.C. 8 H), 7.85-7.51(m, 4H, aromatic), 6.05-6.00(m, 1H, C 1' H), 4.73-4.64 (m, 2H, C 2' H.C. 5' H), 4.37-4.34 (m, 1H, C 3'H), 4.03-3.97 (m, 2H, C 4' H.C. 5' H), 1.95-1.72 (m, 6H, (CH3)2), 1.044, 1.040 (2s, 9H, tBu), 0.984, 0.980 (2s, 9H, tBu), 0.85 (s, 9H, tBu), 0.087, 0.083 (2s, 3H, Si-CH3), 0.060, 0.056(2s, 3H, Si-CH3) [ 13 C NMR] (149 MHz, DMSO, ppm, mixture of rotamer) δ 173.63, 152.12, 151.80, 150.58, 149.51, 143.63, 138.65, 133.81, 129.87, 128.79, 125.97, 125.61, 91.74, 75.72, 75.27, 74.76, 67.51, 48.42, 27.86, 27.56, 27.35, 26.26, 22.77, 20.50, 18.55, 14.62, -4.02, -4.62 [HRMS] (ESI) m / z Cal. for C 34 H 52 N6NaO7Si2 + [M+Na] + ; 735.3328 found 735.3303

[0060] 1-3. Compound 24: 2'-O-(t-butyldimethylsilyl)-N 6 Synthesis of -[2-nitrobenzyl(dimethyl)carbonyl]-adenosine Hydrogen fluoride-pyridine (43.8 μL, 1.05 mmol, 2.5 eq.) was carefully diluted with pyridine (250 μL) under cooling. The resulting solution was dissolved in DCM (10 mL) and 3',5'-O-bis(t-butylsilyl)-2'-O-(t-butyldimethylsilyl)-N 6The resulting mixture was slowly added to a stirred suspension of 2-[2-nitrobenzyl(dimethyl)carbonyl]-adenosine (compound 23) (0.300 g, 0.421 mmol, 1.0 eq) and allowed to react at 0°C for 9 hours. The reaction mixture was diluted with ethyl acetate and washed with water, saturated aqueous NaHCO3, and saturated brine. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography eluting with 4-100% ethyl acetate / hexane to give the target compound (0.270 g, 44.1% yield) as a brown foam. 1 H NMR] (594 MHz, CH3OD, ppm, mixture of rotamer) δ8.64-8.59 (m, 2H, C 2 H.C. 8 H), 7.90-7.85 (m, 2H, aromatic), 7.76-7.73 (m, 1H, aromatic), 7.55-7.52 (m, 1H, aromatic), 6.12-6.05 (m, 1H, C 1' H), 4.86-4.81 (m, 1H, C 2' H), 4.27-4.26 (m, 1H, C 3' H), 4.17-4.12 (m, 1H, C 4' H), 3.90-3.84 (m, 1H, C 5' H), 3.77-3.72 (m, 1H, C 5' H), 1.82 (s, 6H, (CH3)2), 0.78-0.77 (m, 9H, tBu), -0.05--0.06 (m, 3H, Si-CH3), -0.17--0.19 (m, 3H, Si-CH3) [ 13C NMR] (149 MHz, CDCl3, ppm, mixture of rotamer) δ 174.29, 151.61, 151.36, 149.67, 149.21, 143.29, 137.73, 133.33, 129.07, 128.40, 125.43, 89.60, 86.58, 75.99, 71.06, 61.63, 26.20, 25.05, 24.80, 17.61, -6.14, -6.48 [HRMS] (ESI) m / z Cal. for C 26 H 36 N6NaO7Si + [M+Na] + ; 595.2307 found 595.2278

[0061] 1-4. Compound 25: 5'-O-dimethoxytrityl-2'-O-(t-butyldimethylsilyl)-N 6 Synthesis of -[2-nitrobenzyl(dimethyl)carbonyl]-adenosine 2'-O-(t-butyldimethylsilyl)-N6-[2-nitrobenzyl(dimethyl)carbonyl]-adenosine (compound 24) (0.500 g, 0.932 mmol, 1.0 eq.) was coevaporated with benzene three times. This compound was dissolved in pyridine (10 mL). 4,4'-dimethoxytrityl chloride (0.349 g, 0.1.03 mmol, 1.1 eq.) and 4-dimethylaminopyridine (0.002 g, 0.0187 mmol, 0.02 eq.) were added to this solution at room temperature and stirred overnight. The reaction mixture was diluted with ethyl acetate and washed with water, saturated aqueous NaHCO3, and saturated brine. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography eluting with 4-50% ethyl acetate / hexane to give the target compound (0.604 g, 74.0% yield) as a yellow foam. 1H NMR](594 MHz, DMSO, ppm, mixture of rotamer)δ 9.90(s, 1H, NH), 8.60-8.53(m, 2H, C 2 H・C 8 H), 7.851, 7.848(2s, 1H, aromatic), 7.78(dd, J=0.8, 5.6, 1H, aromatic), 7.73-7.70(m, 1H, aromatic), 7.53-7.51(m, 1H, aromatic), 7.35-7.34(m, 2H, aromatic), 7.23-7.15(m, 7H, aromatic), 6.82-6.79(m, 4H, aromatic), 6.01(d, J=4.75, 1H, C 1' H), 5.15(d, J=6.53, 1H, OH), 4.82(t, J=3.20, 1H, C 2' H), 4.25-4.22(m, 1H, C 3' H), 4.09-4.08(m, 1H, C 4' H), 3.69-3.68(m, 6H, OCH3), 3.27-3.22(m, 2H, C 5' H), 1.95, 1.72(2s, 6H, CH3), 0.73, 0.72(2s, 9H, tBu), -0.067, -0.072(2s, 3H, Si-CH3), -0.15, -0.17(2s, 3H, Si-CH3) [ 1313C NMR (149 MHz, CDCl3, ppm, mixture of rotamer) δ 172.69, 158.63, 152.76, 151.59, 149.54, 149.27, 144.62, 141.63, 137.70, 135.73, 133.58, 130.17, 130.14, 128.95, 128.84, 128.22, 127.99, 127.01, 126.16, 123.32, 113.29, 88.65, 86.70, 84.12, 75.56, 71.37, 63.24, 55.33, 48.55, 27.10, 25.67, 17.98, 14.28, -4.78, -5.10 [HRMS] (ESI) m / z Cal. for C 47 H 54 N6NaO9Si + [M+Na] + ; 897.3614 found 897.3652

[0062] 1 - 5. Compound 26: 5'-O-dimethoxytrityl-N 6 -[2-nitrobenzyl(dimethyl)carbonyl]-adenosine, 2'-O-(t-butyldimethylsilyl)-3'-O-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite synthesis 5'-O-dimethoxytrityl-2'-O-(t-butyldimethylsilyl)-N 62-[2-nitrobenzyl(dimethyl)carbonyl]-adenosine (compound 25) (0.604 g, 0.691 mmol, 1.0 eq.) was coevaporated with benzene three times. This compound was dissolved in DCM (7.0 mL). N,N-diisopropylethylamine (0.722 mL, 4.15 mmol, 6.0 eq.) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.409 mL, 1.73 mmol, 2.5 eq.) were added to this solution at 0 °C, and the mixture was stirred overnight. The reaction mixture was diluted with ethyl acetate and washed with water, saturated aqueous NaHCO3, and saturated brine. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography eluting with 12.5% ​​acetonitrile / dichloromethane to give the desired compound (0.612 g, 82.3% yield) as a brown foam. 1 H NMR] (594 MHz, DMSO, ppm, mixture of rotamers and stereoisomers) δ 9.91, 9.76 (2s, 1H, NH), 8.65-8.48 (m, 2H, C 2 H.C. 8 H), 7.84 (dd, J=0.80, 5.20, 1H, aromatic), 7.78 (d, J=7.72, 1H, aromatic), 7.71 (t, J=5.2, 1H, aromatic), 7.53-7.51 (m, 1H, aromatic), 7.38-7.36 (m, 2H, aromatic), 7.26-7.16(m, 7H, aromatic), 6.83-6.79(m, 4H, aromatic), 6.02-5.97(m, 1H, C 1' H), 5.15-5.13 (m, 1H, C 2' H), 4.42-4.21 (m, 2H, C 3' H.C. 4'H), 3.83 - 3.76 (m, 2H, CH2), 3.68 - 3.65 (m, 6H, OCH3), 3.57 - 3.54 (m, 2H, CH-iPr), 3.40 - 3.36 (m, 1H, C 5' H), 3.24 - 3.21 (m, 1H, C 5' H), 2.86 - 2.74 (m, 2H, CH2), 1.90, 1.72 (2s, 6H, CH3), 1.19 - 0.98 (m, 12H, iPr), 0.674 (s, 9H, tBu), -0.11, -0.14 (2s, 3H, Si-CH3), -0.30, -0.31 (2s, 3H, Si-CH3) 13 C NMR] (149 MHz, DMSO, ppm, mixture of rotamer and stereoisomers) δ 173.575, 158.65, 151.89, 149, 52, 145.30, 130.25, 129.92, 128.32, 128.12, 125.61, 119.36, 113.68, 88.56, 86.31, 82.07, 73.69, 73.69, 63.66, 59.37, 59.25, 55.55, 52.42, 48.45, 27.51, 25.98, 25.01, 24.95, 24.80, 24.76, 18.09, -4.37, -4.82 31 P NMR] (241 MHz, DMSO, ppm, mixture of rotamer and stereoisomers) δ 150.23, 148.78 [HRMS] (ESI) m / z Cal. for C 56 H 71 N8NaO 10 PSi + [M + Na] + ; 1097.4692 found 1097.4745

[0063] Test Example 2: Desorption reaction of nitrobenzyl part by reduction treatment

[0064] The present inventors investigated the elimination reaction of the nitrobenzyl moiety under reducing conditions using dithionite for compound 24 obtained in Test Example 1-3. As mentioned above, in this disclosure, the term "nitrobenzyl moiety" refers to a moiety having an o-nitrobenzyl skeleton or a p-nitrobenzyl skeleton.

[0065] Specifically, compound 24 was dissolved in 0.1 M NaSO solution (water / acetonitrile = 1 / 1, 1 mL) and then incubated at 37°C for 30 minutes for reduction. The results of reverse-phase HPLC before and after the reduction are shown in Figure 2. As can be seen from Figure 2, the retention time of the compound before reduction was around 20 minutes, while after reduction, it eluted around 16 minutes. These results indicated that the removal of the nitrobenzyl moiety proceeded quantitatively. [HRMS] (ESI) m / z Cal. for C 16 H 27 N5NaO4Si + [M+Na] + ; 404.1724 found 404.1679

[0066] Test Example 3: Evaluation of the stability and hydrophobicity of Compound 26 The present inventors synthesized RNA with Compound 26 introduced at the 3' end using an automated nucleic acid synthesizer, and evaluated its stability and hydrophobicity. Specifically, RNA with CPR-Nb introduced at the 5' end of a 25-mer RNA and Compound 26 introduced at the 3' end was synthesized using an automated nucleic acid synthesizer. The structure of the synthesized RNA is shown in Figure 3 (SEQ ID NO: 1). The structure of CPR-Nb is also shown below. In Figure 3, p Nb 2-cyanoethyl (2,2-dimethyl-1-(2-nitrophenyl)propyl) diisopropylphosphoramidite was used to Nb 5'-O-dimethoxytrityl-N 6It was introduced into RNA using 2-[2-nitrobenzyl(dimethyl)carbonyl]-adenosine and 2'-O-(t-butyldimethylsilyl)-3'-O-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (compound 26).

[0067] After synthesizing a 25-mer RNA with CPR-Nb at the 5' end and compound 26 at the 3' end, analysis by reverse-phase HPLC confirmed that the target RNA was synthesized in high yield (Figures 4 and 5). Furthermore, when the isolated target RNA was irradiated with UV light, the retention time on reverse-phase HPLC was shortened by approximately 4 minutes (Figure 6). Specifically, UV irradiation was performed using 365 nm UV light at 4,000 μW / cm. 2 The UV irradiation was carried out under the above conditions for 15 minutes, unless otherwise specified. LC-MS analysis confirmed that the UV irradiation selectively eliminated the 5'-terminal nitrobenzyl moiety ((positive mode) [M+H] + Cal. 8375.0725 found 8375.42).

[0068] As described in Test Example 2, the nitrobenzyl moiety of Compound 26 can be removed by reduction treatment. Therefore, the results of Test Examples 2 and 3 demonstrated that the nitrobenzyl moiety can be "separately removed" by light irradiation and reduction treatment.

[0069] Experimental Example 4: Purification of RNA Ligation Products Using Hydrophobic Tags Next, we ligated a polynucleotide fragment bearing a hydrophobic tag at its 5' end to a polynucleotide fragment bearing a hydrophobic tag at its 3' end, and evaluated the hydrophobicity of the ligation product. A summary is shown in Figure 7. Specifically, a 25-mer RNA fragment bearing a hydrophobic tag at its 5' end ((ii) in Figure 7), a 25-mer RNA fragment bearing a hydrophobic tag at its 3' end ((iii) in Figure 7), a DNA template having a sequence complementary to each of the polynucleotide fragments ((i) in Figure 7), and T4 RNA ligase 2 were mixed and incubated at 25°C for 16 hours. After ligation, the reaction mixture was extracted with TE-saturated phenol and chloroform, and the resulting proteinaceous insoluble matter was removed by vigorously mixing. The aqueous layer was extracted with chloroform, desalted by alcohol precipitation, and then subjected to reverse-phase HPLC and denaturing PAGE. The reverse-phase HPLC results are shown in Figure 8. As is clear from Figure 8, the ligation product having two hydrophobic tags ((iv) in Figures 7 and 8) had a longer retention time on the column in reversed-phase HPLC than the RNA fragments used as substrates ((ii) and (iii) in Figures 7 and 8).

[0070] From the above results, it was found that by using polynucleotide fragments having hydrophobic tags as substrates for ligation reactions, the difference in hydrophobicity can be exploited to separate the polynucleotide ligation product from unreacted polynucleotide fragments, and the polynucleotide ligation product can be easily purified.

[0071] Test Example 5. Purification of mRNA using hydrophobic tags Next, the present inventors investigated the possibility of preparing mRNA by ligating RNA fragments having hydrophobic tags, and purifying the mRNA using the hydrophobic tags.

[0072] Specifically, mRNA was prepared and purified in the following three steps. Step 1: Using a plasmid DNA containing the target sequence as a template, the 5'-end cap structure to the 3'UTR was prepared by the IVT method. Step 2: A sequence containing a poly(A) tail was chemically synthesized using an automated nucleic acid synthesizer, and a hydrophobic tag was then introduced to its end. Step 3: The RNAs obtained in Steps 1 and 2 were ligated to prepare full-length mRNA, which was then purified by reverse-phase HPLC. An overview of the process is shown in Figure 9.

[0073] Test Example 5-1. Transcriptional Synthesis of Target RNA (Step 1) Common methods for introducing a cap structure into mRNA include co-transcriptional incorporation using ARCA (anti-reverse cap analog) and post-transcriptional incorporation using a capping enzyme. In the co-transcriptional incorporation method, reverse transcription is performed with RNA polymerase using ARCA and nucleotide triphosphates (NTPs) as substrates, resulting in a mixture of capped and uncapped RNA (Figure 10). Due to the constraints of constructing the reaction system, the co-transcriptional incorporation method achieves only about 80% RNA capping efficiency. In contrast, in the post-transcriptional incorporation method, the transcribed 5' triphosphate RNA serves as a substrate for the capping enzyme, enzymatically introducing the cap structure. However, because the post-transcriptional incorporation method is an enzymatic reaction, the reaction efficiency is unstable.

[0074] Therefore, the inventors performed a transcription reaction using a "Pure Cap analog" as a substrate, which is a conventional ARCA with a hydrophobic tag. When a transcription reaction is performed using a Pure Cap analog as a substrate, the transcription product is obtained as a mixture of capped and uncapped RNA. Because capped RNA contains a hydrophobic tag, but uncapped RNA does not, capped RNA can be isolated by reverse-phase HPLC. An overview of the above is shown in Figure 10. The structures of ARCA and the Pure Cap analog are shown below.

[0075] Specifically, RNA (671mer) was transcribed using the "Pure Cap analog." The transcribed sequence encoded NanoLuc™ luciferase (Promega) (Figure 11, SEQ ID NO: 2). First, a transcription reaction mixture containing the Pure Cap analog, ATP, UTP, GTP, CTP, template DNA, and T7 RNA polymerase was prepared and incubated at 37°C for 2 hours. After the transcription reaction, recombinant DNase was added and incubated at 37°C for 30 minutes. The reaction mixture was extracted with TE-saturated phenol and chloroform, and then vigorously mixed to remove the resulting proteinaceous insoluble material. The aqueous layer was extracted with chloroform and desalted by alcohol precipitation. The target RNA was then isolated by reverse-phase HPLC (Figures 12 and 13) (YMC-Triart Bio C4 (TB30S05-2546PTH, serial #101DA90001) 4.6 x 250 mm, S-5 μm, 30 nm, A: 50 mM TEAA, 5% ACN, B: ACN, gradient: [B%] 0 to 20% (0 to 20 min), flow rate: 1.0 mL / min, detection wavelength: 260 nm, column temperature: 50°C). The retention time difference between capped and uncapped RNA in reverse-phase HPLC was approximately 2 min, which was sufficient for isolation of capped RNA. Furthermore, when the sample was irradiated with UV light after fractionation, the RNA retention time was shortened by approximately 2 minutes, confirming the removal of the nitrobenzyl moiety (Figure 13). The RNA length of the fractionated sample was confirmed by denaturing PAGE (Figure 14).

[0076] Test Example 5-2. Chemical Synthesis of Poly(A) Tail and Post-Modification of the 3' End (Step 2) 5-2-1. Chemical Synthesis of Poly(A) Tail RNA containing a poly(A) tail was chemically synthesized using an automated nucleic acid synthesizer. During chemical synthesis, three 2'-O-methyl adenosine groups and a phosphorothioate group were introduced at the 3' end of the RNA. The RNA sequence is shown in Figure 15 (SEQ ID NO: 3). The 5' end of the synthesized RNA was phosphorylated using the CPR-Nb described above. The CPR-Nb-bound RNA was then isolated by reverse-phase HPLC (Figure 16). Denaturing PAGE was also performed to confirm that the isolated sample had the desired RNA length (Figure 17). The RNA was irradiated with UV light to remove the nitrobenzyl moiety from the 5' end. The removal of the nitrobenzyl moiety was confirmed by a 3.5-minute decrease in the retention time of the sample after UV irradiation in reverse-phase HPLC (Figure 18).

[0077] 5-2-2. Synthesis of bromoacetic acid derivative (2,2-dimethyl-1-(2-nitrophenyl)propyl 2-bromoacetate) As shown in the scheme below, bromoacetyl bromide was reacted with nitrobenzyl alcohol to synthesize the bromoacetic acid derivative (2,2-dimethyl-1-(2-nitrophenyl)propyl 2-bromoacetate).

[0078] Nitrobenzyl alcohol (100 mg, 0.478 mmol, 1.0 eq.) was dissolved in acetonitrile (4.00 mL). To this solution, N,N-diisopropylethylamine (DIPEA) (166 μL, 0.956 mmol, 2.0 eq.) and 2-bromoacetyl bromide (248 μL, 2.87 mmol, 6.0 eq.) were added and stirred at 50°C for 6 hours. The reaction mixture was diluted with ethyl acetate and washed with water and saturated brine. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography eluting with 16.7% acetonitrile / dichloromethane to give the target compound (0.125 g, 79.1% yield) as a yellow oil. 1H NMR] (594 MHz, CDCl3, ppm) δ 7.88 (d, J=11.88, 1H, aromatic), 7.58 (d, J=5.94, 2H, aromatic), 7.45-7.41 (m, 1H, aromatic), 6.51 (s, 1H, CH-tBu), 3.85 (dd, J=0.8, 3.6, 2H, CH2-Br), 0.95(s, 9H, tBu) [ 13 C NMR] (149 MHz, CDCl3, ppm) δ 166.04, 149.29, 132.61, 132.50, 128.97, 128.82, 124.72, 77.82, 77.47, 77.15, 76.82, 36.39, 25.80, 25.68, 25.59 [HRMS] (ESI) m / z Cal. for C 13 H 16 BrNNaO4 + [M+Na] + ; 352.0155 found 352.0240

[0079] 5-2-3. Post-modification of the 3' end The RNA obtained in 5-2-1 and the bromoacetic acid derivative obtained in 5-2-2 were incubated in DMF at room temperature for 2 hours. The results of reverse-phase HPLC before and after the reaction are shown in Figure 19. As shown in Figure 19, the retention time of the sample after the reaction was approximately 10 minutes longer than that of the sample before the reaction. These results confirmed that a hydrophobic tag had been introduced at the 3' end of the synthesized RNA (post-modification of the 3' end).

[0080] RNA with five 2'-O-methyl adenosines at the 3' end was also synthesized in the same manner and used in Test Examples 5-3 and 5-4 described below. Furthermore, RNA with unmethylated poly(A) was also synthesized in the same manner and used in Test Example 5-4 described below.

[0081] The RNA length of the post-modification sample was confirmed by denaturing PAGE (FIG. 20). An overview of the above Test Example 5-2 is shown in FIG.

[0082] Test Example 5-3. Ligation of RNA fragments and isolation and purification by reverse-phase HPLC (third stage) Ligation was performed using T4 RNA ligase 2 using the RNA fragments transcribed and synthesized in Test Example 5-1 and the RNA fragment containing poly(A) synthesized chemically in Test Example 5-2. Note that the 3'-terminal sequences of the RNA fragments obtained by the IVT method are not uniform. However, by performing ligation in the presence of a template strand, it is expected that the RNA fragment containing poly(A) will be ligated only to the 3'-end of the RNA fragment having the desired sequence.

[0083] The RNA fragments transcribed and synthesized in Test Example 5-1 ((ii) in Figure 22), the poly(A)-containing RNA fragments chemically synthesized in Test Example 5-2 ((iv) in Figure 22), DNA templates having sequences complementary to each of the RNA fragments ((i) in Figure 22), and T4 RNA ligase 2 were mixed and incubated at 25°C for 2 hours. After ligation, the reaction mixture was extracted with TE-saturated phenol and chloroform and vigorously mixed to remove the resulting proteinaceous insoluble matter. The aqueous layer was extracted with chloroform and desalted by alcohol precipitation, followed by reverse-phase HPLC and denaturing PAGE. The results are shown in Figures 22 and 23. As mentioned above, Test Example 5-2 yielded RNA fragments with three 2'-O-methyl adenosines at the 3' end, RNA fragments with five 2'-O-methyl adenosines at the 3' end, and RNA fragments with unmethylated poly(A). In this test example, the results obtained using an RNA fragment with five 2'-O-methyl adenosines at the 3' end are described as a representative example, but similar procedures were also performed on an RNA fragment with three 2'-O-methyl adenosines at the 3' end and an RNA fragment with unmethylated poly(A).

[0084] The results of denaturing PAGE confirmed that the target ligation product (756 mer (671 + 85 mer)) was (iii) in Figure 22. As mentioned above, the ligation product was a full-length mRNA containing the 5' cap structure through the poly(A) tail.

[0085] In Test Example 5-3, unlike Test Example 4, the retention time of ligated product (iii) in reverse-phase HPLC was shorter than that of substrate (iv). In this test example, ligated product (iii) has one more hydrophobic tag than substrate (iv), but its base length is significantly longer than that of (iv). Therefore, it is thought that the hydrophilicity-enhancing effect of the longer base length (nucleic acids generally have many hydrophilic groups) exceeded the hydrophobicity-enhancing effect of the increased hydrophobic tag.

[0086] Next, the inventors irradiated the full-length mRNA (RNA ligation product) obtained by the above procedure with UV light and then analyzed it by reverse-phase HPLC (Figure 24). As is clear from a comparison of Figures 22 and 24, the retention time of the full-length mRNA after UV irradiation was approximately 3 minutes shorter than the retention time before UV irradiation. Furthermore, the full-length mRNA after UV irradiation showed a single peak (Figure 24). These results demonstrate that the nitrobenzyl moiety containing the hydrophobic tag is efficiently cleaved from the full-length mRNA by UV irradiation.

[0087] 5-4. Evaluation of Translation Efficiency The present inventors evaluated the intracellular translation efficiency of mRNA obtained by a conventional transcription synthesis method using ARCA ((i) in Figure 25); mRNA obtained in Test Example 5-3 by ligating, with RNA ligase, the 5'-end fragment prepared by the IVT method and the 3'-end fragment prepared by an automated nucleic acid synthesizer (without purification by reverse-phase HPLC after ligation; (ii) and (iii) in Figure 25); mRNA obtained by ligating, with RNA ligase, the 5'-end fragment prepared by the IVT method and a 3'-end fragment containing poly(A) prepared by an automated nucleic acid synthesizer, followed by post-modification of the 3'-end with a hydrophobic tag, purification by reverse-phase HPLC, and subsequent removal of the hydrophobic tag by UV irradiation ((iv) in Figure 25); and the 5'-end fragment prepared by the IVT method ((v) in Figure 25). As mentioned above, each mRNA encodes NanoLuc™ luciferase (Promega). In this test example, the Nano-Glo™ Luciferase Assay System (Promega) was used to evaluate translation efficiency using luminescence intensity as an indicator.

[0088] More specifically, the translation efficiency was evaluated by the following procedure: Day 1: 2.0 × 10 4 HeLa cells were seeded at 100 μL / well. Day 2: 20 ng / well mRNA was transfected using Lipofectamine™ Messenger MAX (Thermo Fisher Scientific). After 5 hours of incubation at 37°C, luciferase assays were performed using the Nano-Glo™ Luciferase Assay System (Promega). The results are shown in Figure 25.

[0089] As is clear from Figure 25, the translation efficiency of mRNA that was obtained by ligating the 5'-end fragment and the 3'-end fragment and then purifying by reverse-phase HPLC using the hydrophobic tag added to the 3'-end was approximately 14.9-fold higher than the translation efficiency of mRNA that was not purified by reverse-phase HPLC after ligation, and approximately 3.7-fold higher than the translation efficiency of mRNA obtained by conventional transcription synthesis methods.

[0090] 6. Discussion The above experimental results demonstrated that: 1) selectively using hydrophobic tags with an o-nitrobenzyl backbone and hydrophobic tags with a p-nitrobenzyl backbone enables the selective removal of hydrophobic tags by light irradiation and reduction treatment; 2) using polynucleotide fragments containing hydrophobic tags as substrates for ligation reactions allows for easy purification of the ligated product from a mixture of unreacted polynucleotide fragments and ligated products based on changes in the degree of hydrophobicity; 3) after the purification, the hydrophobic tag can be removed from the ligated product with high efficiency; 4) introducing a moiety such as a phosphorothioate group to which a hydrophobic tag can be added during the preparation of polynucleotide fragments allows for the subsequent introduction of a hydrophobic tag into the polynucleotide fragment (post-modification); 5) full-length mRNA can be prepared by ligating RNA fragments with hydrophobic tags; and 6) the translation efficiency of mRNA purified using hydrophobic tags after ligation is significantly higher than that of mRNA obtained by conventional transcription synthesis and that of mRNA not purified using hydrophobic tags after ligation.

[0091] As described above, mRNA drugs are composed of, in order from the 5' end of the RNA, a cap structure (5' cap), a 5' untranslated region (5' UTR), a translated region, a 3' untranslated region (3' UTR), and a poly(A) tail. It is known that structures other than the translated region affect mRNA stability and translation efficiency. However, conventional mRNA synthesis methods have made it difficult to control the 3' end sequence and the length of the poly(A) tail added. In other words, mRNA obtained using conventional mRNA synthesis methods is not homogeneous, particularly in terms of the 3' end sequence and poly(A) tail length. In contrast, the technology disclosed herein allows for the ligation of RNA fragments containing hydrophobic tags and purification based on the degree of hydrophobicity, thereby producing highly purified, homogeneous mRNA. Furthermore, because the hydrophobic tag can be removed under mild conditions, it is possible to ligate additional RNA fragments after removing the hydrophobic tag from the purified ligated product. In other words, the steps of ligating RNA fragments, purifying the ligated product, and removing the hydrophobic tag can be repeated until an RNA ligation product of the desired length is obtained. Therefore, the technology of the present disclosure can be particularly suitably used in the production of mRNA pharmaceuticals, which require the preparation of long-chain RNA with high purity.

[0092] Hydrophobic tags having an o-nitrobenzyl backbone can be removed by either photoirradiation or reduction treatment. On the other hand, hydrophobic tags having a p-nitrobenzyl backbone cannot be removed by photoirradiation but can be removed by reduction treatment. In other words, by selectively using hydrophobic tags having an o-nitrobenzyl backbone and hydrophobic tags having a p-nitrobenzyl backbone, it is possible to selectively remove the hydrophobic tags by photoirradiation and reduction treatment. Therefore, for example, when ligating polynucleotide fragments sequentially from the 3'-end, a hydrophobic tag having a p-nitrobenzyl backbone can be added to the 3'-end polynucleotide fragment and a hydrophobic tag having an o-nitrobenzyl backbone can be added to the 5'-end polynucleotide fragment before ligation. After purifying the ligation product based on the degree of hydrophobicity, only the hydrophobic tag having an o-nitrobenzyl backbone can be removed by photoirradiation. Subsequently, the steps of ligating polynucleotide fragments to which hydrophobic tags having an o-nitrobenzyl backbone have been added, purifying the ligation product, and removing the hydrophobic tag having an o-nitrobenzyl backbone by photoirradiation can be repeated. The above steps are repeated until the desired ligation product is obtained, and then reduction treatment is performed to remove the hydrophobic tag having an o-nitrobenzyl skeleton and the hydrophobic tag having a p-nitrobenzyl skeleton (Figure 1).

[0093] As mentioned above, in Test Example 5-3, unlike Test Example 4, the retention time of ligation product (iii) in reverse-phase HPLC was shorter than that of substrate (iv). This is presumably because the improved hydrophilicity due to the longer base length outweighed the improved hydrophobicity due to the increased hydrophobic tags. It is believed that by increasing or decreasing the number of hydrophobic tags added to each polynucleotide fragment depending on the length of the polynucleotide fragment and the desired polynucleotide ligation product, it is possible to control the separation of the ligation products in the step of purifying the ligation products based on the degree of hydrophobicity.

Claims

1. Step (α): A step of ligating a polynucleotide fragment (A) containing a hydrophobic tag and a polynucleotide fragment (B) containing a hydrophobic tag, The polynucleotide fragment (A) is represented by the general formula (1): 【Chemistry 1】 [wherein k represents an integer of 1 or more. ＹＡ represents a k-valent group obtained by removing k atoms or groups from a polynucleotide. Ａ are the same or different and are represented by the general formula (2): 【Chemistry 2】 (In the formula: R １Ａ represents an alkyl group. ２Ａ represents a hydrogen atom or an alkyl group. ３Ａ and R ５Ａ One of R represents a nitro group, and the other represents a hydrogen atom, an alkyl group, or an alkoxy group. ４Ａ , R ６Ａ , and R ７Ａ are the same or different and represent a hydrogen atom, an alkyl group, or an alkoxy group (provided that R ３Ａ is a nitro group, R ４Ａ and R ７Ａ represents a hydrogen atom). ＸＡ represents a linker.) represents a group represented by the formula: is expressed as R Ａ is R ＹＡ a polynucleotide fragment bound to at least one nucleotide located 1 to 10 bases from the 3' end of The polynucleotide fragment (B) is represented by the general formula (3): 【Transformation 3】 [wherein m represents an integer of 1 or more. ＹＢ represents an m-valent group obtained by removing m atoms or groups from a polynucleotide. Ｂ are the same or different and are represented by the general formula (4): 【Chemistry 4】 (In the formula: R １Ｂ represents an alkyl group. ２Ｂ represents a hydrogen atom or an alkyl group. ３Ｂ and R ５Ｂ One of R represents a nitro group, and the other represents a hydrogen atom, an alkyl group, or an alkoxy group. ４Ｂ , R ６Ｂ , and R ７Ｂ are the same or different and represent a hydrogen atom, an alkyl group, or an alkoxy group (provided that R ３Ｂ is a nitro group, R ４Ｂ and R ７Ｂ represents a hydrogen atom). ＸＢ represents a linker.) represents a group represented by the formula: is a polynucleotide fragment represented by Methods for producing polynucleotide ligation products.

2. Step (β): Purifying the polynucleotide ligation product based on the degree of hydrophobicity of the ligation product. The method of claim 1 further comprising:

3. Step (γ): A step of removing the hydrophobic tag The method of claim 1 or 2, further comprising:

4. (delete)

5. R Ｂ is R ＹＢ 3. The method according to claim 1, wherein the nucleotide is bound to at least one of the nucleotides located 1 to 30 bases from the 5'-end of

6. The method according to claim 2 , wherein the step (β) comprises a separation step by liquid chromatography.

7. The method according to claim 3 , wherein the step (γ) comprises a step of removing the hydrophobic tag by light irradiation and / or a step of removing the hydrophobic tag by reduction treatment.

8. The linker R contained in the polynucleotide fragment (A) ＸＡ and the linker R contained in the polynucleotide fragment (B). ＸＢ are the same or different and are a single bond, —O—CH ２ -, -OC(=O)-, -C(=O)-, -O-(CH ２ ) ｎ -R ８ -, or -O-C(=O)-(CH ２ ) ｎ -R ８ - [wherein n represents an integer of 1 or more. ８ is represented by the general formula (5): 【Transformation 5】 (X １ and X ２ and R 1 and R 2 are the same or different and each represents O or S. The method according to claim 1 or 2,

9. The polynucleotide fragment (A) is represented by the general formula (1): 【Transformation 6】 [wherein k represents an integer of 1 or more. ＹＡ represents a k-valent group obtained by removing k atoms or groups from a polynucleotide. Ａ are the same or different and are represented by the general formula (2): 【Transformation 7】 (In the formula: R １Ａ represents an alkyl group having 1 to 30 carbon atoms. ２Ａ represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. ３Ａ and R ５Ａ One of the groups represents a nitro group, and the other represents a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, or an alkoxy group having 1 to 10 carbon atoms. ４Ａ , R ６Ａ , and R ７Ａ are the same or different and represent a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, or an alkoxy group having 1 to 10 carbon atoms (wherein R ３Ａ is a nitro group, R ４Ａ and R ７Ａ represents a hydrogen atom). ＸＡ represents a linker.) represents a group represented by the formula: and The polynucleotide fragment (B) is represented by the general formula (3): 【Transformation 8】 [wherein m represents an integer of 1 or more. ＹＢ R may be the same or different and represent an m-valent group obtained by removing m atoms or groups from a polynucleotide. Ｂ is represented by the general formula (4): 【Chemistry 9】 (In the formula: R １Ｂ represents an alkyl group having 1 to 30 carbon atoms. ２Ｂ represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. ３Ｂ and R ５Ｂ One of the groups represents a nitro group, and the other represents a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, or an alkoxy group having 1 to 10 carbon atoms. ４Ｂ , R ６Ｂ , and R ７Ｂ are the same or different and represent a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, or an alkoxy group having 1 to 10 carbon atoms (wherein R ３Ｂ is a nitro group, R ４Ｂ and R ７Ｂ represents a hydrogen atom). ＸＢ represents a linker.) represents a group represented by the formula: The method according to claim 1 or 2, wherein the polynucleotide fragment is represented by the formula:

10. The method according to claim 1 or 2, wherein the polynucleotide ligation product has a base length of 20 to 2000 bases.

11. 3. The production method according to claim 1 or 2, wherein in step (α), the reaction system comprises a polynucleotide having a sequence complementary to at least a portion of the polynucleotide fragment (A) and a sequence complementary to at least a portion of the polynucleotide fragment (B).

12. The method of claim 1 or 2, wherein the polynucleotide ligation product is an mRNA drug.

13. A polynucleotide having a structure in which a polynucleotide fragment (A) and a polynucleotide fragment (B) are linked together, It contains both a hydrophobic tag having an o-nitrobenzyl skeleton and a hydrophobic tag having a p-nitrobenzyl skeleton, 20 to 50,000 bases in length, The polynucleotide fragment (A) is represented by the general formula (1): 【Chemistry 10】 [In the formula, k represents an integer of 1 or more. ＹＡ represents a k-valent group obtained by removing k atoms or groups from a polynucleotide. Ａ are the same or different and are represented by the general formula (2): 【Chemistry 11】 (In the formula: R １Ａ represents an alkyl group. ２Ａ represents a hydrogen atom or an alkyl group. ３Ａ and R ５Ａ One of R represents a nitro group, and the other represents a hydrogen atom, an alkyl group, or an alkoxy group. ４Ａ , R ６Ａ , and R ７Ａ are the same or different and represent a hydrogen atom, an alkyl group, or an alkoxy group (provided that R ３Ａ is a nitro group, R ４Ａ and R ７Ａ represents a hydrogen atom). ＸＡ represents a linker.) represents a group represented by the formula: and R Ａ is R ＹＡ and the nucleic acid is bound to at least one nucleotide located 1 to 10 bases from the 3' end of the nucleic acid; The polynucleotide fragment (B) is represented by the general formula (3): 【Chemistry 12】 [wherein m represents an integer of 1 or more. ＹＢ represents an m-valent group obtained by removing m atoms or groups from a polynucleotide. Ｂ are the same or different and are represented by the general formula (4): 【Chemistry 13】 (In the formula: R １Ｂ represents an alkyl group. ２Ｂ represents a hydrogen atom or an alkyl group. ３Ｂ and R ５Ｂ One of R represents a nitro group, and the other represents a hydrogen atom, an alkyl group, or an alkoxy group. ４Ｂ , R ６Ｂ , and R ７Ｂ are the same or different and represent a hydrogen atom, an alkyl group, or an alkoxy group (provided that R ３Ｂ is a nitro group, R ４Ｂ and R ７Ｂ represents a hydrogen atom). ＸＢ represents a linker.) represents a group represented by the formula: and R Ｂ is R ＹＢ and the nucleic acid is bound to at least one nucleotide located 1 to 20 bases from the 5' end of the nucleic acid; Here, either the polynucleotide fragment (A) or the polynucleotide fragment (B) contains a hydrophobic tag having an o-nitrobenzyl backbone, and the other contains a hydrophobic tag having a p-nitrobenzyl backbone.

14. (delete)

15. In the general formula (1), R １Ａ represents an alkyl group having 1 to 30 carbon atoms, R ２Ａ represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, R ３Ａ and R ５Ａ one of the groups represents a nitro group, and the other represents a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, or an alkoxy group having 1 to 10 carbon atoms; R ４Ａ , R ６Ａ , and R ７Ａ are the same or different and represent a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, or an alkoxy group having 1 to 10 carbon atoms (wherein R ３Ａ is a nitro group, R ４Ａ and R ７Ａ The polynucleotide according to claim 13, wherein represents a hydrogen atom.

16. The linker R ＸＡ are the same or different and are a single bond, —O—CH ２ -, -OC(=O)-, -C(=O)-, -O-(CH ２ ) ｎ -R ８ -, or -O-C(=O)-(CH ２ ) ｎ -R ８ - [wherein n represents an integer of 1 or more. ８ is represented by the general formula (5): 【Chemistry 14】 (X １ and X ２ and R are the same or different and represent O or S. The polynucleotide according to claim 13 or 15,

17. The polynucleotide according to claim 13 or 15, which has a length of 20 to 2000 bases.

18. The polynucleotide of claim 13 or 15, which is an mRNA drug.

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