Separation agent for RNA with 5' cap structure

Abstract

To provide a separation agent for RNA with a 5' cap structure or the like, enabling the efficient and economical separation of RNA with a 5' cap structure.SOLUTION: The present invention provides a separation agent for RNA with a 5' cap structure or the like in which the terminal unit is a 7-methylguanylic acid residue, where the agent comprises a compound represented by formula (I): A-L-R1 (I) (where A represents a separation carrier residue with a reactive functional group, L represents a linker, and R1 represents a group that reacts with the 7-methylguanine moiety of 7-methylguanosine to form a protecting group).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an agent for separating RNA having a 5' cap structure, and the like. [Background technology]

[0002] The mRNA has a 5'-cap structure derived from 7-methylguanosine at the 5'-end. For example, when producing an mRNA vaccine, it is desirable to separate the mRNA having this 5'-cap structure from the mRNA not having the 5'-cap structure.

[0003] Known methods for separating mRNA having a 5' cap structure from a mixture containing mRNA having a 5' cap structure and mRNA not having a 5' cap structure include, for example, a method in which only uncapped RNA is digested with RNA polyphosphatase to isolate capped RNA (Patent Document 1, etc.), and a method which includes a step of binding a dinucleotide having a reporter moiety (e.g., biotin) to capped RNA, further binding the reporter moiety to a substrate, and then separating the resulting product from uncapped RNA (Patent Document 2). However, these methods have problems such as the need to use expensive dinucleotides and unsatisfactory purification efficiency. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2011-521626 A [Patent Document 2] International Publication No. 2009 / 058911 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention provides a separating agent for RNA having a 5' cap structure, which can efficiently separate RNA having a 5' cap structure at low cost, and the like. [Means for solving the problem]

[0006] As a result of intensive research into the above-mentioned problems, the inventors found that RNA having a 5' cap structure can be easily separated by attaching a compound having an oxalyl group to the 5' cap structure of RNA via a linker to a separation support having a reactive functional group, and by conducting further research based on this finding, they have completed the present invention. That is, the present invention is as follows.

[0007] [1] Formula (I) ALR 1 (I) (In the formula, A represents a separation carrier residue having a reactive functional group, L represents a linker, R 1 represents a group that reacts with the 7-methylguanosine moiety to form a protecting group). [2] R 1 The separating agent according to the above-mentioned [1], wherein is -C(=O)-C(=O)H. [3] The group at the bonding portion of A to L is -C(=O)-, and L is represented by the formula (II).

[0008] [ka]

[0009] (In the formula, R 2 represents hydrogen or methyl, R 3 and R 4 are the same or different and represent hydrogen, lower alkyl, lower alkoxy, or halogen; n represents an integer of 1 to 10; when n is 2 or more, each R 2 The separating agent according to the above [1] or [2], wherein R is a group represented by the formula (I) or (II), which may be the same or different. [4] R 2 , R 3 and R 4 The separating agent according to the above [3], wherein is hydrogen and n is 4. [5] The separating agent according to any one of the above [1] to [4], wherein the separation carrier is a magnetic bead-shaped separation carrier, a glass bead-shaped separation carrier, or a latex bead-shaped separation carrier. [6] The separating agent according to any one of the above [1] to [4], wherein the separation carrier is a magnetic bead-shaped separation carrier. [7] The separating agent according to any one of the above [1] to [6], wherein the RNA is mRNA. [8] Formula (Ia)

[0010] [ka]

[0011] (In the formula, A' represents a separation carrier residue in which the group at the bonding portion with -NH- to which A' is bonded is -C(=O)-, and R 2 represents hydrogen or methyl, R 3 and R 4 are the same or different and represent hydrogen, lower alkyl, lower alkoxy or halogen, and n represents an integer of 1 to 10. [9] R 2 , R 3 and R 4 is hydrogen and n is 4.

[10] (1) Formula (I) ALR 1 (I) (In the formula, A represents a separation carrier residue having a reactive functional group, L represents a linker, R 1 represents a group that reacts with the 7-methylguanosine moiety to form a protecting group) (hereinafter, the compound represented by formula (I) will be referred to as compound (I). The same applies to the compounds having other compound numbers) Formula (III)

[0012] [ka]

[0013] (In the formula, R 5 represents hydrogen or methyl, R 6 is hydrogen or a group represented by the formula (IV)

[0014] [ka]

[0015] (In the formula, R 7 is R 5 When is hydrogen, it represents hydrogen, and R 5 represents hydrogen or methyl when is methyl, to obtain a compound represented by formula (V)

[0016] [ka]

[0017] (In the formula, A, L, R 5 and R 6 each of which has the same meaning as defined above, (2) preparing a solution containing RNA having a 5' cap structure based on compound (V) using compound (V) obtained in step (1) as a transcription initiation site; (3) isolating RNA having a 5' cap structure based on compound (V) from the solution obtained in step (2); and (4) removing compound (I) from the RNA having a 5' cap structure based on compound (V) separated in step (3); A method for producing RNA having a 5' cap structure at the end of which is a 7-methylguanylic acid residue.

[11] The method for producing the above-mentioned

[10] , wherein the RNA is mRNA.

[12] Formula (V)

[0018] [ka]

[0019] (In the formula, A represents a separation carrier residue having a reactive functional group, L represents a linker, R 5 represents hydrogen or methyl, R 6 is hydrogen or a group represented by the formula (IV)

[0020] [ka]

[0021] (In the formula, R 7 is R 5 When is hydrogen, it represents hydrogen, and R 5 When is methyl, it represents hydrogen or methyl.

[13] AL- is the formula

[0022] [ka]

[0023] (In the formula, A' represents a separation carrier residue in which the group at the bonding portion with -NH- to which A' is bonded is -C(=O)-, and R 2 represents hydrogen or methyl, R 3 and R 4 and n is an integer of 1 to 10.

[14] (1) Formula (I) ALR 1 (I) (In the formula, A represents a separation carrier residue having a reactive functional group, L represents a linker, R 1represents a group which reacts with the 7-methylguanosine moiety to form a protecting group) (hereinafter, the compound represented by formula (I) will be referred to as compound (I)) with a mixture containing RNA having a 5' cap structure whose terminal is a 7-methylguanylic acid residue and RNA not having the 5' cap structure, thereby binding compound (I) only to the RNA having the 5' cap structure; (2) isolating the RNA having a 5'-cap structure bound to compound (I) obtained in step (1); and (3) removing compound (I) from the RNA having a 5'-cap structure to which compound (I) is bound, which has been separated in step (2); A method for separating RNA having a 5' cap structure, the terminus of which is a 7-methylguanylic acid residue, from a mixture containing RNA having the 5' cap structure and RNA not having the 5' cap structure.

[15] The method according to

[14] above, wherein the RNA is mRNA. Effect of the Invention

[0024] INDUSTRIAL APPLICABILITY The present invention can provide a separating agent for RNA having a 5' cap structure, which can efficiently separate RNA having a 5' cap structure at low cost, and the like. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] 1. Isolation agent for RNA with 5' cap structure The present invention relates to Formula (I) ALR 1 (I) (In the formula, A represents a separation carrier residue having a reactive functional group, L represents a linker, R 1The present invention provides a separation agent for RNA having a 5' cap structure whose terminal is a 7-methylguanylic acid residue (hereinafter, also referred to as "the separation agent of the present invention"), which contains a compound represented by formula (I) (hereinafter, the compound represented by formula (I) will be referred to as compound (I); the same applies to compounds having other formula numbers),

[0026] In the definition of each group in formula (I), The separation carrier is not particularly limited as long as it can physically separate the separation carrier-bound RNA from a mixture containing a separation carrier-bound RNA bound via a linker to an RNA having a 5' cap structure at the end of which is a 7-methylguanylic acid residue, and an RNA not having the 5' cap structure, and the like. For example, plastics such as containers, plates, and beads, glass, latex, polysaccharides, metals, ceramics, etc. are used, and a plurality of these materials may be used. Examples of plastics include acrylic polymers, silicone rubber, polystyrene, polyethylene terephthalate, polycarbonate, etc., and examples of metals include magnetic metals (metals having magnetism). Among them, the separation carrier is preferably a bead-shaped one, and more preferably a magnetic bead-shaped separation carrier, a glass bead-shaped separation carrier, or a latex bead-shaped separation carrier, and even more preferably a magnetic bead-shaped separation carrier.

[0027] The reactive functional group is not particularly limited as long as it is a group that can be bonded to a linker moiety, and examples thereof include a carboxyl group, a formyl group, a hydroxyl group, an amino group, and an azide group. For example, when the reactive functional group is a carboxyl group, the group at the bonding portion in A to L is -C(=O)-, and when the reactive functional group is an amino group, the group at the bonding portion in A to L is -NH-.

[0028] The separation carrier having a reactive functional group used in the present invention may be a commercially available product, and specific examples include Dynabeads (registered trademark) MyOne (trademark) carboxylic acid (800-1600 nmol COOH / mg) (Veritas Corporation), FG COOH beads (Tamagawa Seiki Co., Ltd.), IMMUTEX (trademark) Carboxyl (Medical and Biological Laboratories Co., Ltd.), etc., but are not limited to these.

[0029] The linker is not particularly limited as long as it can achieve the object of the present invention. For example, a structure having a residue derived from polyethylene glycol or polypropylene glycol is preferable. More specifically, for example, when the group at the bonding portion of A to L is -C(=O)-, the linker may be represented by the formula (II):

[0030] [ka]

[0031] (In the formula, R 2 represents hydrogen or methyl, R 3 and R 4 are the same or different and represent hydrogen, lower alkyl, lower alkoxy, or halogen; n represents an integer of 1 to 10; when n is 2 or more, each R 2 may be the same or different).

[0032] In the definition of each group in formula (II), Examples of the lower alkyl and the lower alkyl moiety of the lower alkoxy include straight-chain or branched alkyl having 1 to 6 carbon atoms, specifically, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, etc.

[0033] Halogen means fluorine, chlorine, bromine and iodine atoms.

[0034] In formula (II), R 2 , R3 and R 4 is preferably hydrogen, and n is preferably 4. 2 , R 3 and R 4 It is more preferred that n is hydrogen and n is 4.

[0035] Examples of groups that react with the 7-methylguanosine moiety to form a protecting group include -C(=O)-C(=O)H.

[0036] The 5' cap structure refers to a structure or a related structure that contains a triphosphate group linked in order to the 5'-carbon of the 5'-terminal nucleotide and 7-methylguanosine linked to the triphosphate group, which is found at the 5' end of most mRNAs of eukaryotic cells and most viral mRNAs that infect eukaryotes (e.g., JP 2011-521626 A), and this structure inhibits the degradation of mRNAs having a 5' cap structure by enzymes (e.g., exonucleases) and promotes the binding of translation initiation factors to the mRNA.

[0037] RNA is a molecule containing at least one ribonucleotide residue, and includes double-stranded RNA or single-stranded RNA, synthetic RNA or natural RNA, or recombinant RNA that has been modified differently from natural RNA by the addition, deletion, substitution, etc. of one or more nucleotides, as well as mRNA, rRNA, tRNA, etc., but is preferably mRNA.

[0038] Compound (I) may exist in the form of a salt and / or an adduct with water or various solvents, and these salts and / or adducts are also included in the concept of compound (I). Examples of salts include metal salts, base addition salts such as organic amines, and acid addition salts such as organic or inorganic acids.

[0039] Next, a method for producing compound (I) will be described.

[0040] Compound (I) is a compound obtained by reacting a separation carrier having a reactive functional group derived from A in formula (I) with LR in formula (I) 1 (ii) a method of reacting a separation carrier having a reactive functional group from which A in formula (I) is derived with a compound from which LR in formula (I) is derived by a conventional method; 1 is reacted with a part of the compound derived from the formula (I) by a conventional method, 1 The compound (Ia) can be produced by further reacting the remaining portion of the compound from which it is derived by a conventional method (for example, the method for producing compound (Ia) described in the section "2. Compound bound to a separation carrier" below).

[0041] The separating agent of the present invention may consist of compound (I), but may also contain other components such as excipients, diluents, water, physiological saline, etc., within the scope of the achievement of the object of the present invention. When the separating agent of the present invention contains such other components, it can be prepared from compound (I) and such other components by appropriate conventional methods.

[0042] The content of the above-mentioned other components in the separating agent of the present invention is not particularly limited, but the total amount in the separating agent of the present invention is preferably 0.000001% by weight to 90% by weight, more preferably 0.00001% by weight to 70% by weight, even more preferably 0.00001% by weight to 50% by weight, even more preferably 0.0001% by weight to 30% by weight, and particularly preferably 0.0001% by weight to 10% by weight.

[0043] Next, a method for using the separating agent of the present invention will be described.

[0044] The separation agent of the present invention can be used to separate RNA having a 5' cap structure whose terminal is a 7-methylguanylic acid residue from a mixture containing RNA having the 5' cap structure, and is particularly preferably used to separate RNA having a 5' cap structure from a mixture containing RNA having a 5' cap structure whose terminal is a 7-methylguanylic acid residue and RNA not having the 5' cap structure.

[0045] Examples of mixtures containing RNA having a 5' cap structure whose terminal is a 7-methylguanylic acid residue and RNA not having the 5' cap structure include (1) a mixture containing RNA having the 5' cap structure produced when RNA is produced using a nucleotide, dinucleotide, etc. having a 5' cap structure whose terminal is a 7-methylguanylic acid residue as the transcription initiation site, and by-product RNA not having the 5' cap structure, (2) a mixture containing RNA having a 5' cap structure produced when RNA having a 5' cap structure whose terminal is a 7-methylguanylic acid residue is produced from RNA, and unreacted RNA not having the 5' cap structure.

[0046] Methods for producing RNA using a nucleotide, dinucleotide, or the like as a transcription initiation point and having a 5' cap structure with a 7-methylguanylic acid residue at the end include the methods described in Jemielity, J. et al., Novel "anti-reverse" cap analogs with superior translational properties, RNA 9, 1108-1122(2003) and Roland, C., Hilde, C., Wim, D. & Walter, F. Simple, efficient in vitro synthesis of capped RNA useful for direct expression of cloned eukaryotic genes, Nucleic Acids Res., 10, 6353-6362(1982), or methods modified therefrom.

[0047] Methods for producing RNA having a 5' cap structure with a 7-methylguanylic acid residue at the end from RNA include the methods described in Ramanathan, A., Robb, GB & Chan, SH, mRNA capping: Biological functions and applications, Nucleic Acids Res. 44, 7511-7526(2016) and JP 2011-521626 A, or methods similar thereto.

[0048] 2. Compounds bound to separation carriers The present invention also relates to a compound of formula (Ia)

[0049] [ka]

[0050] (In the formula, A' represents a separation carrier residue in which the group at the bonding portion with -NH- to which A' is bonded is -C(=O)-, and R 2 represents hydrogen or methyl, R 3 and R 4 are the same or different and represent hydrogen, lower alkyl, lower alkoxy or halogen, and n represents an integer of 1 to 10) (hereinafter also referred to as "the compound of the present invention").

[0051] The terms "separation carrier", "lower alkyl", "lower alkoxy" and "halogen" are as described above for the separation agent of the present invention. 2 , R 3 and R 4 is preferably hydrogen, and n is preferably 4. 2 , R 3 and R 4 It is more preferred that n is hydrogen and n is 4.

[0052] Next, the method for producing compound (Ia) will be described.

[0053] Compound (Ia) can be produced, for example, according to the following scheme.

[0054] [ka]

[0055] (In the formula, A', R 2 , R 3 , R 4 and n are as defined above.

[0056] Process 1 Compound (VIII) can be obtained by an amide formation reaction between compound (VI) and compound (VII).

[0057] Compound (VI) is commercially available, for example, as Dynabeads (registered trademark) MyOne (trademark) carboxylic acid (800-1600 nmol COOH / mg) (Veritas Corporation). Compound (VII) is also commercially available.

[0058] Many methods are known for the amide-forming reaction. For example, compound (VI) is converted into its reactive equivalent, such as an acid chloride, an active ester, or a mixed acid anhydride, in a solvent inert to the reaction, and this is reacted with 1 to 5 equivalents of compound (VII) in a solvent inert to the reaction in the presence or absence of 1 to 10 equivalents of a base to obtain compound (VIII).

[0059] When compound (VI) is converted into an acid chloride, for example, thionyl chloride, oxalyl chloride, phosphorus pentachloride, etc. are used to convert it into an acid chloride, when compound (VI) is converted into an active ester, for example, N-hydroxysuccinimide (NHS), 1-hydroxybenzotriazole, 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine, etc. are used to convert it into an active ester, when compound (VI) is converted into a mixed acid anhydride, for example, ethyl chloroformate, etc. Also, when compound (VI) is converted into an active ester, for example, it can be converted into an active ester in the presence of an activator such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI).

[0060] The solvent inert to the reaction may be any solvent inert to the reaction, and is not particularly limited. For example, dimethylformamide (DMF), dimethylacetamide, dimethylsulfoxide, tetrahydrofuran, dioxane, diethyl ether, ethylene glycol, dichloromethane, chloroform, toluene, xylene, ethyl acetate, acetonitrile, pyridine, etc. can be used alone or in combination, and among these, DMF, etc. is preferred.

[0061] Examples of the base that can be used include sodium hydroxide, potassium hydroxide, sodium methoxide, potassium ethoxide, potassium tert-butoxide, butyllithium, lithium diisopropylamide, and organic bases such as triethylamine, diisopropylamine, diisopropylethylamine, tributylamine, dicyclohexylmethylamine, N-methylmorpholine, N-methylpiperidine, diazabicycloundecene, diazabicyclononene, pyridine, and dimethylaminopyridine, and among these, triethylamine or pyridine is preferred.

[0062] The reaction is carried out at a temperature between 0° C. and 100° C., preferably between room temperature and 50° C., more preferably at room temperature, and is usually completed within about 5 minutes to 48 hours.

[0063] Compound (VIII) can also be obtained by reacting compound (VI) with 1 to 5 equivalents of compound (VII) in the presence of 1 to 10 equivalents of a condensing agent in a solvent inert to the reaction.

[0064] The solvent inert to the reaction may be any solvent inert to the reaction, and is not particularly limited. For example, DMF, dimethylacetamide, dimethylsulfoxide, tetrahydrofuran, dioxane, diethyl ether, ethylene glycol, dichloromethane, chloroform, toluene, xylene, ethyl acetate, acetonitrile, pyridine, etc. can be used alone or in combination, and among these, DMF, etc. is preferred.

[0065] As the condensing agent, for example, dicyclohexylcarbodiimide, diisopropylcarbodiimide, N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide or its hydrochloride, benzotriazol-1-yl-tris(dimethylamino)phosphonium hexafluorophosphide salt, diphenylphosphoryl azide, etc. can be used.

[0066] If necessary, this reaction is carried out in the presence of 1 to 5 equivalents of an additive, such as NHS, 1-hydroxybenzotriazole, or 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine.

[0067] The reaction is carried out at a temperature between 0° C. and 150° C., preferably between room temperature and 50° C., more preferably at room temperature, and is usually completed in about 5 minutes to 48 hours.

[0068] Process 2 Compound (Ia) can be obtained, for example, by subjecting compound (VIII) obtained in the above step 1 and compound (IX) to a Huisgen cycloaddition reaction.

[0069] Compound (IX) is commercially available.

[0070] The Huisgen cycloaddition reaction can be carried out, for example, by reacting compound (VIII) with 1 to 5 equivalents of compound (IX) in a reaction-inert solvent in the presence or absence of 0.01 to 1 equivalent of a copper catalyst.

[0071] The solvent inert to the reaction may be any solvent inert to the reaction, and is not particularly limited. For example, water, a buffer solution such as a phosphate buffer, DMF, dimethylacetamide, dimethylsulfoxide, tetrahydrofuran, dioxane, diethyl ether, ethylene glycol, dichloromethane, chloroform, toluene, xylene, ethyl acetate, acetonitrile, pyridine, etc. can be used alone or in combination, and among these, a mixed solvent of water and DMF, etc. is preferred.

[0072] The copper catalyst may, for example, be copper sulfate, copper chloride, copper bromide, copper iodide, etc., and among these, copper sulfate, etc. is preferred.

[0073] A copper ion stabilizer can be used together with the copper catalyst. Examples of the copper ion stabilizer include 2-(4-((bis((1-(tert-butyl)-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)acetic acid (BTTAA), tris(benzimidazol-1-ylmethyl)amine ((BimH)3), tris(1-benzyltriazol-4-ylmethyl)amine (TBTA), tris(1-(3-hydroxypropyl)triazol-4-ylmethyl)amine (THPTA), L-histidine, and the like. Of these, BTTAA and the like are preferred.

[0074] Furthermore, when a copper catalyst is used, it is preferable to add sodium ascorbate or the like to reductively generate copper(I) in the system.

[0075] The reaction is carried out at a temperature between 0° C. and 100° C., preferably between room temperature and 50° C., more preferably at room temperature, and is usually completed in about 30 seconds to 24 hours.

[0076] 3. Method for producing RNA having a 5' cap structure The present invention also provides (1) Formula (I) ALR 1 (I) (In the formula, A represents a separation carrier residue having a reactive functional group, L represents a linker, R 1 represents a group that reacts with the 7-methylguanosine moiety to form a protecting group), Formula (III)

[0077] [ka]

[0078] (In the formula, R 5 represents hydrogen or methyl, R 6 is hydrogen or a group represented by the formula (IV)

[0079] [ka]

[0080] (In the formula, R 7 is R 5 When is hydrogen, it represents hydrogen, and R 5 represents hydrogen or methyl when is methyl, to obtain a compound represented by formula (V)

[0081] [ka]

[0082] (In the formula, A, L, R 5 and R 6 each of which has the same meaning as defined above, (2) preparing a solution containing RNA having a 5' cap structure based on compound (V) using compound (V) obtained in step (1) as a transcription initiation site; (3) isolating RNA having a 5' cap structure based on compound (V) from the solution obtained in step (2); and (4) removing compound (I) from the RNA having a 5' cap structure based on compound (V) separated in step (3); The present invention provides a method for producing RNA having a 5' cap structure at the end of which is a 7-methylguanylic acid residue (hereinafter also referred to as the "production method of the present invention").

[0083] The "separation carrier," "reactive functional group," "linker," "group that reacts with the 7-methylguanosine moiety to form a protecting group," "5' cap structure," and "RNA" are as described above for the separation agent of the present invention.

[0084] Process (1) Compound (V) can be obtained by reacting compound (I) with 1 to 5 equivalents of compound (III) in a solvent inert to the reaction.

[0085] The solvent inert to the reaction may be any solvent inert to the reaction, and is not particularly limited. For example, DMF, dimethylacetamide, dimethylsulfoxide, methanol, ethanol, isopropyl alcohol, butanol, tetrahydrofuran, dioxane, diethyl ether, ethylene glycol, dichloromethane, chloroform, toluene, xylene, ethyl acetate, acetonitrile, pyridine, etc. can be used alone or in combination, and among these, DMF, etc. is preferred.

[0086] The reaction is usually carried out at a temperature between 0°C and 50°C, preferably between room temperature and 37°C, more preferably at room temperature, and is completed usually in about 1 minute to 6 hours, preferably about 5 minutes to 2 hours, more preferably about 5 minutes to 30 minutes.

[0087] Process (2) Preparation of a solution containing RNA having a 5' cap structure based on compound (V) using compound (V) obtained in step (1) as the transcription initiation point can be carried out by the methods described in Huang, F., Efficient incorporation of CoA, NAD and FAD into RNA by in vitro transcription, Nucleic Acids Res. 31, e8(2003), Roland, C., Hilde, C., Wim, D. & Walter, F Simple, efficient in vitro synthesis of capped RNA useful for direct expression of cloned eukaryotic genes, Nucleic Acids Res., 10, 6353-6362(1982), etc., or methods similar thereto.

[0088] As the solvent for the solution containing RNA having a 5'-cap structure based on compound (V), water, a buffer solution such as phosphate buffered saline, or a Tris buffer solution, etc. can be used.

[0089] Process (3) The separation of RNA having a 5'-cap structure based on compound (V) from the solution obtained in step (2) varies depending on the separation carrier. For example, when the separation carrier is a magnetic separation carrier, it can be performed by collecting the RNA by magnetic force. When the separation carrier is latex, the separation can be performed by precipitation over time, sedimentation by centrifugation, or the like. When the separation carrier is in the form of a container or plate, or made of glass, the separation can be performed simply by removing the separation carrier from the above solution.

[0090] After the above separation, it is preferable to wash the separated RNA having a 5' cap structure based on compound (V). The washing may be carried out with any suitable washing solution that does not adversely affect the RNA having a 5' cap structure, including, for example, the buffer solutions described in step (2) above.

[0091] Process (4) The elimination of compound (I) from the RNA having a 5'-cap structure based on compound (V) separated in step (3) can be carried out, for example, by heat-treating the RNA having a 5'-cap structure based on compound (V) in a solvent usually at a temperature between 50° C. and 100° C., preferably between 80° C. and 100° C., more preferably at about 95° C., usually for 1 minute to 2 hours, preferably 5 minutes to 1 hour, more preferably about 10 minutes. As the solvent, water, a buffer solution such as phosphate buffered saline, or a Tris buffer solution, etc. can be used.

[0092] In one aspect, the present invention also provides compound (V) above.

[0093] In the compound (V), AL- in the formula (V) is

[0094] [ka]

[0095] (In the formula, A' represents a separation carrier residue in which the group at the bonding portion with -NH- to which A' is bonded is -C(=O)-, and R 2 represents hydrogen or methyl, R 3 and R 4 are the same or different and represent hydrogen, lower alkyl, lower alkoxy or halogen, and n represents an integer of 1 to 10).

[0096] Here, the terms "separation carrier", "lower alkyl", "lower alkoxy" and "halogen" are as described above for the separation agent of the present invention. 2 , R 3 and R 4 is preferably hydrogen, and n is preferably 4. 2 , R 3 and R 4 It is more preferred that n is hydrogen and n is 4.

[0097] Compound (V) may exist in the form of a salt and / or an adduct with water or various solvents, and these salts and / or adducts are also included in the concept of compound (V). Examples of salts include metal salts, base addition salts such as organic amines, and acid addition salts such as organic or inorganic acids.

[0098] 4. Method for isolating RNA with a 5' cap structure The present invention also provides (1) Formula (I) ALR 1 (I) (In the formula, A represents a separation carrier residue having a reactive functional group, L represents a linker, R 1 represents a group which reacts with the 7-methylguanosine moiety to form a protecting group) with a mixture containing RNA having a 5' cap structure whose terminal is a 7-methylguanylic acid residue and RNA not having the 5' cap structure, thereby binding compound (I) only to the RNA having the 5' cap structure; (2) isolating the RNA having a 5'-cap structure bound to compound (I) obtained in step (1); and (3) removing compound (I) from the RNA having a 5'-cap structure to which compound (I) is bound, which has been separated in step (2); A method for separating RNA having a 5' cap structure, the terminus of which is a 7-methylguanylic acid residue, from a mixture containing RNA having the 5' cap structure and RNA not having the 5' cap structure is provided.

[0099] The "separation carrier," "reactive functional group," "linker," "group that reacts with the 7-methylguanosine moiety to form a protecting group," "5' cap structure," and "RNA" are as described above for the separation agent of the present invention.

[0100] Process (1) Compound (I) can be bound only to RNA having a 5' cap structure by reacting compound (I) with a mixture containing RNA having a 5' cap structure whose terminal is a 7-methylguanylic acid residue and RNA not having the 5' cap structure in a solvent inert to the reaction.

[0101] The above reaction can be carried out in the same manner as in step (1) described above in the production method of the present invention.

[0102] In addition, a method for obtaining a compound in which compound (I) is bound only to RNA having the 5' cap structure includes the steps of: 1 and a mixture containing RNA having a 5' cap structure whose terminal is a 7-methylguanylic acid residue and RNA not having the 5' cap structure, are simultaneously reacted in accordance with standard methods and the above-described step (1), etc.

[0103] Process (2) The RNA having a 5'-cap structure bound to compound (I) obtained in step (1) can be separated in the same manner as in step (3) described above for the production method of the present invention.

[0104] Process (3) The detachment of compound (I) from the RNA having a 5'-cap structure to which compound (I) is bound, which has been separated in step (2), can be carried out in the same manner as in step (4) described above for the production method of the present invention.

[0105] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples in any way. EXAMPLES

[0106] Example 1 2 mg of Dynabeads® MyOne® carboxylic acid (800-1600 nmol COOH / mg; hereinafter also referred to as "magnetic beads") (Veritas Corporation) was washed with DMF, and a solution of 18 mg of NHS and 30 mg of EDCI dissolved in 0.4 mL of DMF was added thereto, and the mixture was stirred at room temperature for 2 hours, after which the magnetic beads were washed with DMF to obtain magnetic beads having NHS esters. Next, 0.08 mL of a 10 mg / mL acetylene-PEG4-amine (Sigma-Aldrich) DMF solution and 0.003 mL of diisopropylethylamine (DIPEA) were added thereto, and the mixture was stirred at room temperature for 2 hours, after which the magnetic beads were collected by magnetic separation. Next, 0.05 mL of a 1 M 4-amino-1-butanol DMF solution was added thereto, and the mixture was stirred at room temperature to neutralize the unreacted NHS esters, to obtain magnetic beads having acetylene groups. The acetylene-PEG4-amine remaining in the supernatant was subjected to the ninhydrin reaction to develop color, and the yield was estimated to be about 82%.

[0107] The acetylene-containing magnetic beads obtained above were suspended in 1 mL of DMF, and 0.125 mL was transferred to a new tube. After removing the DMF, 0.126 mL of 100 mM phosphate buffer pH 7.0, 0.04 mL of 50 mM BTTAA (JENA Bioscience) aqueous solution, 0.004 mL of 100 mM copper sulfate aqueous solution, 0.02 mL of 1 M sodium ascorbate aqueous solution, and 0.01 mL of 10 mM p-azidophenylglycosal (APG) (Nacalai Tesque, Inc.) DMF solution were added and shaken at room temperature for 1 hour. The decrease in the amount of APG in the reaction solution after 0 and 1 hour was monitored by HPLC (Alliance 2695, Waters) using ODS-SR-5 (4.6 x 250 mm; Nomura Chemical Co., Ltd.) (mobile phase A: 100% water, mobile phase B: 100% ethanol; 0-8 min 100% mobile phase A retention, 8-48 min linear gradient to mobile phase B; flow rate: 0.5 ml / min). As a result, it was estimated that about 90% of the APG was immobilized.

[0108] Synthesis Example 1 0.125 mL of the acetylene group-containing magnetic bead suspension was transferred to a new tube in the same manner as in Example 1. After removing the DMF, 0.116 mL of 100 mM phosphate buffer pH 7.0, 0.04 mL of 50 mM BTTAA solution, 0.004 mL of 100 mM copper sulfate aqueous solution, 0.02 mL of 1 M sodium ascorbate aqueous solution, 0.01 mL of 10 mM m7G(5')ppp(5')A RNA Cap Structure Analog (NEB) aqueous solution, and 0.01 mL of 10 mM APG DMF solution were added and shaken at room temperature for 1 hour. The decrease in the amount of m7G(5')ppp(5')A RNA Cap Structure Analog in the reaction solution after 0 and 1 hour was monitored by HPLC (Alliance2695, Waters) using ODS-SR-5 (4.6 x 250 mm; Nomura Chemical Co., Ltd.) (mobile phase A: 100% water, mobile phase B: 100% ethanol; 0-8 min 100% mobile phase A retention, 8-48 min linear gradient to mobile phase B; flow rate: 0.5 ml / min). As a result, it was estimated that about 60% of m7G(5')ppp(5')A RNA Cap Structure Analog was immobilized.

[0109] As shown in Synthesis Example 1, the 5' cap structure with a 7-methylguanylic acid residue at the end could be efficiently bound to the separation carrier. This suggests that RNA having a 5' cap structure with a 7-methylguanylic acid residue at the end can be efficiently separated at low cost. [Industrial Applicability]

[0110] INDUSTRIAL APPLICABILITY The present invention can provide a separating agent for RNA having a 5' cap structure, which can easily separate RNA having a 5' cap structure, and the like.

Claims

1. Formula (I) A-L-R １ (I) (In the formula, A represents a separation carrier residue having a reactive functional group, L represents a linker, R １ represents a group that reacts with the 7-methylguanine moiety of 7-methylguanosine to form a protecting group),

2. R １ The separating agent according to claim 1, wherein is -C(=O)-C(=O)H.

3. The group at the bonding portion of A to L is -C(=O)-, and L is represented by the formula (II). 【Chemistry 1】 (In the formula, R ２ represents hydrogen or methyl; R ３ and R ４ are the same or different and represent hydrogen, lower alkyl, lower alkoxy, or halogen; n represents an integer of 1 to 10; when n is 2 or more, each R ２ The separating agent according to claim 2 , wherein R 1 is a group represented by the formula (I) and R 2 is a group represented by the formula (II) and R 3 is a group represented by the formula (II) and R 4 is a group represented by the formula (II) and

4. R ２ , R ３ and R ４ The separating agent according to claim 3 , wherein: is hydrogen and n is 4.

5. The separating agent according to any one of claims 1 to 4, wherein the separation carrier is a magnetic bead-shaped separation carrier, a glass bead-shaped separation carrier, or a latex bead-shaped separation carrier.

6. The separating agent according to any one of claims 1 to 4, wherein the separation carrier is a bead-shaped separation carrier having magnetism.

7. The separating agent according to any one of claims 1 to 4, wherein the RNA is mRNA.

8. Formula (Ia) 【Chemistry 2】 (In the formula, A' represents a separation carrier residue in which the group at the bonding portion with -NH- to which A' is bonded is -C(=O)-, R ２ represents hydrogen or methyl; R ３ and R ４ are the same or different and represent hydrogen, lower alkyl, lower alkoxy or halogen, and n represents an integer of 1 to 10.

9. R ２ , R ３ and R ４ The compound of claim 8 , wherein is hydrogen and n is 4.

10. Formula (V) 【Chemistry 3】 (In the formula, A represents a separation carrier residue having a reactive functional group, L represents a linker, R ５ represents hydrogen or methyl; R 6 is hydrogen or a group represented by formula (IV) 【Chemistry 4】 (In the formula, R ７ is R ５ represents hydrogen when R ５ When is methyl, it represents hydrogen or methyl.

11. A-L- is a formula 【Chemistry 5】 (In the formula, A' represents a separation carrier residue in which the group at the bonding portion with -NH- to which A' is bonded is -C(=O)-, R ２ represents hydrogen or methyl; R ３ and R ４ and n is an integer of 1 to 10.

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