Stabilizer for nucleic acid solution

Abstract

The present invention provides a stabilizer for a nucleic acid solution, which is a polymer containing a structural unit derived from a monomer represented by formula (1), wherein the definition of symbols in the formula is as described in the description.

Description

Stabilizers for nucleic acid solutions

[0001] The present invention relates to a stabilizer for a nucleic acid solution and a method for improving the storage stability of nucleic acids in a nucleic acid solution using the same.

[0002] RNA plays a role in protein biosynthesis based on the genetic information stored in DNA. In recent years, various industrial products, such as useful proteins and pharmaceuticals, have been developed using RNA.

[0003] This RNA is unstable and easily decomposed in solution, making its storage difficult. Therefore, the extracted RNA must be immediately stored at low temperatures. For example, methods of storing RNA by freeze-drying (e.g., Patent Document 1) and storing the RNA at −20° C. without freezing have been used to improve RNA stability.

[0004] Japanese Patent Application Laid-Open No. 2021-119161

[0005] The storage stability of RNA can be improved by freeze-drying a sample containing RNA or by adding a buffer solution to the sample and storing it at a low temperature without freezing. However, the freezing of RNA during freeze-drying or the thawing of the frozen sample before use can cause RNA degradation. Furthermore, adding a buffer solution to a sample containing RNA does not provide sufficient storage stability of RNA.

[0006] The present invention has been made in consideration of the above-mentioned problems, and aims to provide a stabilizer that can improve the storage stability of nucleic acids by simply adding it to a nucleic acid solution (particularly an RNA solution).

[0007] As a result of intensive research, the present inventors have found that the compound represented by the following formula (1):

[0008]

[0009] It has been found that the storage stability of nucleic acids can be improved by adding to a nucleic acid solution a polymer containing a structural unit derived from a monomer represented by the formula (1):

[0010]

[0011] (In the formula, X １ represents a (meth)acryloyloxy group or a (meth)acryloylamino group, L １ represents an alkylene group having 2 to 4 carbon atoms which may have one hydroxy group, or an alkyleneoxyalkylene group having 2 to 4 carbon atoms, and R １ ~R ３ and each independently represent an alkyl group having 1 to 3 carbon atoms.

[0012] [2] The polymer is represented by formula (2):

[0013]

[0014] (In the formula, R ４ represents a hydrogen atom or a methyl group, and R ５ represents a hydrogen atom, an alkyl group having 1 to 20 carbon atoms, or *-(OCH ２ CH ２ ) ｎ OR ６ group (where * represents a bonding position, n represents a number from 1 to 10, and R ６ represents a hydrogen atom, a methyl group, or an ethyl group. ) represents a copolymer further comprising a structural unit derived from a monomer represented by the formula (I).

[0015] [3] The polymer is represented by formula (3):

[0016]

[0017] (In the formula, R ７ represents a hydrogen atom or a methyl group, and R ８ represents an alkyl group having 3 to 6 carbon atoms and having two or more hydroxy groups.

[0018] [4] The stabilizer according to any one of [1] to [3], wherein the nucleic acid solution is an RNA solution.

[0019] [5] A method for improving the storage stability of a nucleic acid by adding the stabilizer according to any one of [1] to [4] above to a nucleic acid solution at a concentration of 0.0001 to 10 w / v %.

[0020] The stabilizer of the present invention can improve the storage stability of a nucleic acid by being contained in the nucleic acid solution (particularly an RNA solution).

[0021] The present invention will be described in detail below. The descriptions in this specification can be combined with each other unless it is clear that they cannot be combined. Furthermore, when stepwise numerical ranges are described in this specification, the lower and upper limits of each numerical range can be combined. For example, when "preferably 10 to 100, more preferably 20 to 90" is described, "preferably lower limit: 10" and "more preferably upper limit: 90" can be combined (i.e., the numerical range "10 to 90" is also within the scope of this specification).

[0022] [Stabilizer for nucleic acid solution] The present invention provides a stabilizer for a nucleic acid solution (hereinafter sometimes abbreviated as "stabilizer of the present invention"), which is a polymer (hereinafter sometimes abbreviated as "polymer of the present invention") containing a constitutional unit derived from a monomer represented by formula (1).

[0023] As used herein, "nucleic acid" refers to "RNA or DNA." As used herein, "stabilizer for nucleic acid solution" refers to an additive used in a nucleic acid solution to improve the storage stability of nucleic acid. The nucleic acid solution is preferably an RNA solution.

[0024] The stabilizer of the present invention has the formula (1):

[0025]

[0026] (In the formula, X １ represents a (meth)acryloyloxy group or a (meth)acryloylamino group, L １ represents an alkylene group having 2 to 4 carbon atoms which may have one hydroxy group, or an alkyleneoxyalkylene group having 2 to 4 carbon atoms, and R １ ~R ３and each independently represent an alkyl group having 1 to 3 carbon atoms. In other words, the present invention provides use of a polymer containing a constitutional unit derived from a monomer represented by formula (1) as a stabilizer for a nucleic acid solution.

[0027] The polymer of the present invention may be used alone or in combination of two or more types. Hereinafter, "monomer represented by formula (1)" may be abbreviated as "monomer (1)". "Monomers represented by other formulas" may also be abbreviated as "monomer represented by formula (1)". Furthermore, "structural unit derived from monomer (1)" may also be abbreviated as "structural unit (1)". "Structural unit derived from other monomer" may also be abbreviated as "structural unit derived from monomer (1)". A "structural unit derived from monomer (1)" refers to a structural unit having a structure formed by reaction of the carbon-carbon double bond of the (meth)acryloyl group contained in monomer (1). A structural unit derived from another monomer has the same meaning as a structural unit derived from monomer (1).

[0028] The monomer (1) may be used alone or in combination of two or more types. That is, the polymer of the present invention may be a homopolymer consisting of one type of structural unit (1), or a copolymer containing two or more types of structural unit (1). The copolymer may be a random copolymer, a block copolymer, or a copolymer containing both random and block portions.

[0029] The groups in formula (1) will be explained below in order. X in formula (1) １ represents a (meth)acryloyloxy group (i.e., CH ２ =CR-CO-O-, where R is a hydrogen atom or a methyl group) or a (meth)acryloylamino group (i.e., CH ２ =CR-CO-NH-, R: hydrogen atom or methyl group). １ is preferably a (meth)acryloyloxy group, more preferably a methacryloyloxy group.

[0030] L in formula (1) １represents an alkylene group having 2 to 4 carbon atoms which may have one hydroxy group, or an alkyleneoxyalkylene group having 2 to 4 carbon atoms. The alkylene group may be linear or branched. Examples of the alkylene group having 2 to 4 carbon atoms which may have one hydroxy group include, for example, -C ２ H ４ Examples of the alkyleneoxyalkylene group having 2 to 4 carbon atoms include -C ２ H ４ -O-C ２ H ４ From the viewpoint of raw material availability, L １ is preferably -C ２ H ４ -or-C ２ H ４ -O-C ２ H ４ -, more preferably -C ２ H ４ - (i.e., an ethylene group).

[0031] R in formula (1) １ ~R ３ each independently represents an alkyl group having 1 to 3 carbon atoms. The alkyl group may be linear or branched. Examples of the alkyl group having 1 to 3 carbon atoms include a methyl group, an ethyl group, and a propyl group. From the viewpoint of raw material availability, R １ ~R ３ and are preferably both methyl groups.

[0032] Preferred monomers (1) are those represented by the formula: X １ is a (meth)acryloyloxy group, and L １ But, -C ２ H ４ -or-C ２ H ４ -O-C ２ H ４ - and R １ ~R ３ is a methyl group. １ is a (meth)acryloyloxy group, and L １ is an ethylene group, and R １ ~R ３is a methyl group (i.e., 2-(meth)acryloyloxyethyl phosphorylcholine). A more preferred monomer (1) is 2-methacryloyloxyethyl phosphorylcholine. Commercially available products can be used as the monomer (1).

[0033] In this specification, "2-(meth)acryloyloxyethyl phosphorylcholine" basically means "2-acryloyloxyethyl phosphorylcholine or 2-methacryloyloxyethyl phosphorylcholine." When multiple 2-(meth)acryloyloxyethyl phosphorylcholines may be present, "2-(meth)acryloyloxyethyl phosphorylcholine" means "2-acryloyloxyethyl phosphorylcholine and / or 2-methacryloyloxyethyl phosphorylcholine." Other terms similar to "2-(meth)acryloyloxyethyl phosphorylcholine" also have the same meaning as "2-(meth)acryloyloxyethyl phosphorylcholine."

[0034] One embodiment of the polymer of the present invention is a polymer composed of structural unit (1) (hereinafter sometimes abbreviated as "polymer (1)"). In this specification, "a polymer composed of structural unit (1)" means a polymer in which all structural units (repeating units) in the polymer chain are composed of structural unit (1). Expressions similar to "a polymer composed of structural unit (1)" also have the same meaning as "a polymer composed of structural unit (1)". Note that the "structural unit" refers to a structural unit derived from a monomer, and does not include structures derived from a monomer (for example, structures derived from a polymerization initiator).

[0035] The polymer (1) may be a homopolymer consisting of one type of structural unit (1), or a copolymer consisting of two or more types of structural units (1), but is preferably a copolymer consisting of two or more types of structural units (1).

[0036] The weight-average molecular weight of polymer (1) is not particularly limited, but is preferably 20,000 to 2,000,000, more preferably 100,000 to 1,500,000, and even more preferably 500,000 to 1,500,000. The weight-average molecular weight can be determined in terms of polyethylene glycol by gel permeation chromatography using, for example, an EcoSEC system (manufactured by Tosoh Corporation).

[0037] The polymer of the present invention contains, in addition to the structural unit (1), a structural unit of the formula (2):

[0038]

[0039] (In the formula, R ４ represents a hydrogen atom or a methyl group, and R ５ represents a hydrogen atom, an alkyl group having 1 to 20 carbon atoms, or *-(OCH ２ CH ２ ) ｎ OR ６ group (where * represents a bonding position, n represents a number from 1 to 10, and R ６ represents a hydrogen atom, a methyl group, or an ethyl group. ) represents a methyl group or an ethyl group. ) may further contain a structural unit derived from a monomer represented by

[0040] The groups in formula (2) will be explained in order below. ４ represents a hydrogen atom or a methyl group. From the viewpoint of storage stability of the polymer, R ４ is preferably a methyl group.

[0041] R in formula (2) ５ represents a hydrogen atom, an alkyl group having 1 to 20 carbon atoms, or *-(OCH ２ CH ２ ) ｎ OR ６ Represents a group.

[0042] R in formula (2) ５ is a hydrogen atom or an alkyl group having 1 to 20 carbon atoms (hereinafter, sometimes abbreviated as "monomer (2a)"). The alkyl group may be linear or branched. In the monomer (2a), R ５is preferably a hydrogen atom or an alkyl group having 10 to 20 carbon atoms, more preferably a hydrogen atom or an alkyl group having 15 to 19 carbon atoms, and even more preferably a hydrogen atom or a linear alkyl group having 17 to 19 carbon atoms. In addition, when the polymer of the present invention further contains a structural unit derived from a monomer represented by formula (3) described below, R ５ is preferably an alkyl group having 3 to 6 carbon atoms.

[0043] Specific examples of the monomer (2a) include (meth)acrylic acid, methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, butyl (meth)acrylate, pentyl (meth)acrylate, hexyl (meth)acrylate, heptyl (meth)acrylate, octyl (meth)acrylate, nonyl (meth)acrylate, decyl (meth)acrylate, undecyl (meth)acrylate, dodecyl (meth)acrylate, tridecyl (meth)acrylate, tetradecyl (meth)acrylate, pentadecyl (meth)acrylate, cetyl (meth)acrylate, heptadecyl (meth)acrylate, and stearyl (meth)acrylate. Only one type of the monomer (2a) may be used, or two or more types may be used in combination. Commercially available products can be used as the monomer (2a).

[0044] Among the specific examples of the monomer (2a), (i) (meth)acrylic acid, ethyl (meth)acrylate, propyl (meth)acrylate, butyl (meth)acrylate, pentyl (meth)acrylate, hexyl (meth)acrylate, decyl (meth)acrylate, undecyl (meth)acrylate, dodecyl (meth)acrylate, tridecyl (meth)acrylate, tetradecyl (meth)acrylate, pentadecyl (meth)acrylate, cetyl (meth)acrylate, heptadecyl (meth)acrylate, and stearyl (meth)acrylate are preferred, and (ii) (meth)acrylic acid, propyl (meth)acrylate, butyl (meth)acrylate, pentyl (meth)acrylate, pentadecyl (meth)acrylate, cetyl (meth)acrylate, heptadecyl (meth)acrylate, and stearyl (meth)acrylate are more preferred. (iii) (meth)acrylic acid, butyl (meth)acrylate, and stearyl (meth)acrylate are more preferred; and (iv) methacrylic acid, butyl methacrylate, and stearyl methacrylate are particularly preferred.

[0045] One embodiment of the polymer of the present invention is a copolymer composed of the structural unit (1) and the structural unit (2a) (hereinafter, sometimes abbreviated as "copolymer (1-2a)"). The monomer (1) and the monomer (2a) may each be used alone, or two or more types may be used in combination. That is, the copolymer (1-2a) may be a copolymer containing one or more types of the structural unit (1) and one or more types of the structural unit (2a). The copolymer may be a random copolymer, a block copolymer, or a copolymer containing both a random portion and a block portion.

[0046] In copolymer (1-2a), relative to a total of 100 moles of structural unit (1) and structural unit (2a) (i.e., a total of 100 moles of monomer (1) and monomer (2a) used in the polymerization), the ratio of structural unit (1) (i.e., the ratio of monomer (1) used in the polymerization) is preferably 5 to 95 moles, more preferably 10 to 95 moles, even more preferably 15 to 95 moles, and particularly preferably 20 to 90 moles, and the ratio of structural unit (2a) (i.e., the ratio of monomer (2a) used in the polymerization) is preferably 5 to 95 moles, more preferably 5 to 90 moles, even more preferably 5 to 85 moles, and particularly preferably 10 to 80 moles.

[0047] The weight-average molecular weight of the copolymer (1-2a) is not particularly limited, but is preferably 10,000 to 2,000,000, more preferably 20,000 to 1,500,000, and even more preferably 30,000 to 1,000,000. The weight-average molecular weight can be determined in terms of polyethylene glycol by gel permeation chromatography using, for example, an EcoSEC system (manufactured by Tosoh Corporation).

[0048] R in formula (2) ５ *-(OCH ２ CH ２ ) ｎ OR ６ group (wherein * represents a bonding position, n represents a number from 1 to 10, and R ６ represents a hydrogen atom, a methyl group, or an ethyl group.) (hereinafter, may be abbreviated as "monomer (2b)"). ６ is preferably a methyl group or an ethyl group, more preferably a methyl group.

[0049] *-(OCH ２ CH ２ ) ｎ OR ６ The group n represents a number from 1 to 10, preferably from 5 to 10, and more preferably from 8 to 10.

[0050] Specific examples of the monomer (2b) include polyethylene glycol mono(meth)acrylate, methoxypolyethylene glycol (meth)acrylate, and ethoxypolyethylene glycol (meth)acrylate. The monomer (2b) may be used alone or in combination of two or more. From the viewpoint of storage stability of the polymer, methoxypolyethylene glycol (meth)acrylate and ethoxypolyethylene glycol (meth)acrylate are preferred, and methoxypolyethylene glycol methacrylate is more preferred. Commercially available products can be used as the monomer (2b).

[0051] One embodiment of the polymer of the present invention is a copolymer composed of the structural unit (1) and the structural unit (2b) (hereinafter, sometimes abbreviated as "copolymer (1-2b)"). The monomer (1) and the monomer (2b) may each be used alone, or two or more types may be used in combination. That is, the copolymer (1-2b) may be a copolymer containing one or more types of the structural unit (1) and one or more types of the structural unit (2b). The copolymer may be a random copolymer, a block copolymer, or a copolymer containing both a random portion and a block portion.

[0052] In copolymer (1-2b), relative to a total of 100 moles of structural unit (1) and structural unit (2b) (i.e., 100 moles of the combined total of monomer (1) and monomer (2b) used in the polymerization), the ratio of structural unit (1) (i.e., the ratio of monomer (1) used in the polymerization) is preferably 40 to 95 moles, more preferably 60 to 95 moles, and even more preferably 80 to 95 moles, and the ratio of structural unit (2b) (i.e., the ratio of monomer (2b) used in the polymerization) is preferably 5 to 60 moles, more preferably 5 to 40 moles, and even more preferably 5 to 20 moles.

[0053] The weight average molecular weight of the copolymer (1-2b) is not particularly limited, but is preferably 50,000 to 1,000,000, more preferably 100,000 to 500,000, and even more preferably 100,000 to 300,000.

[0054] The polymer of the present invention contains, in addition to the structural unit (1), a structural unit of the formula (3):

[0055]

[0056] (In the formula, R ７ represents a hydrogen atom or a methyl group, and R ８ represents an alkyl group having 3 to 6 carbon atoms and having two or more hydroxy groups.)

[0057] The groups in formula (3) will be explained below in order. ７ represents a hydrogen atom or a methyl group. From the viewpoint of storage stability of the polymer, R ７ is preferably a methyl group.

[0058] R in formula (3) ８ represents an alkyl group having 3 to 6 carbon atoms and two or more hydroxy groups. The alkyl group may be linear or branched. Examples of the alkyl group having 3 to 6 carbon atoms include a propyl group, a butyl group, a pentyl group, and a hexyl group.

[0059] Specific examples of the monomer (3) include glycerin mono(meth)acrylate, threitol mono(meth)acrylate, erythritol mono(meth)acrylate, xylitol mono(meth)acrylate, arabitol mono(meth)acrylate, mannitol mono(meth)acrylate, galactitol mono(meth)acrylate, and sorbitol mono(meth)acrylate. Among these, glycerin mono(meth)acrylate and xylitol mono(meth)acrylate are preferred, glycerin mono(meth)acrylate is more preferred, and glycerin monomethacrylate is even more preferred. The monomer (3) may be used alone or in combination of two or more.

[0060] Monomer (3) may be a commercially available product or may be produced by a known method. For example, monomer (3) can be produced by an esterification reaction between (meth)acrylic acid or a derivative thereof (e.g., an acid chloride) and a polyhydric alcohol having three or more hydroxy groups. Esterification reactions are well known, and a person skilled in the art can carry out the reaction by appropriately setting the conditions.

[0061] One embodiment of the polymer of the present invention is a copolymer composed of structural units (1), (2), and (3) (hereinafter sometimes abbreviated as "copolymer (1-2-3)"). Monomer (1), monomer (2), and monomer (3) may each be used alone, or two or more types may be used in combination. That is, copolymer (1-2-3) may be a copolymer containing one or more types of structural units (1), one or more types of structural units (2), and one or more types of structural units (3). The copolymer may be a random copolymer, a block copolymer, or a copolymer containing both random and block portions.

[0062] Among the copolymers (1-2-3), copolymers in which the structural unit (2) is a structural unit derived from the monomer (2a) (hereinafter, sometimes abbreviated as "copolymer (1-2a-3)") are preferred. ５ is preferably an alkyl group having 3 to 6 carbon atoms.

[0063] In copolymer (1-2-3), relative to a total of 100 moles of structural unit (1), structural unit (2), and structural unit (3) (i.e., a total of 100 moles of monomer (1), monomer (2), and monomer (3) used in the polymerization), the ratio of structural unit (1) (i.e., the ratio of monomer (1) used in the polymerization) is preferably 30 to 80 moles, more preferably 30 to 70 moles, and even more preferably 30 to 60 moles; the ratio of structural unit (2) (i.e., the ratio of monomer (2) used in the polymerization) is preferably 10 to 60 moles, more preferably 20 to 60 moles, and even more preferably 30 to 60 moles; and the ratio of structural unit (3) (i.e., the ratio of monomer (3) used in the polymerization) is preferably 10 to 60 moles, more preferably 10 to 50 moles, and even more preferably 10 to 40 moles.

[0064] The weight-average molecular weight of the copolymer (1-2-3) is not particularly limited, but is preferably 10,000 to 500,000, more preferably 10,000 to 100,000, and even more preferably 10,000 to 50,000. The weight-average molecular weight can be determined in terms of polyethylene glycol by gel permeation chromatography using, for example, an EcoSEC system (manufactured by Tosoh Corporation).

[0065] The polymer of the present invention may contain other structural units derived from other monomers different from the monomers (1) to (3), as long as the effects of the present invention are not impaired. Only one type of other monomer may be used, or two or more types may be used in combination. The other monomer is not particularly limited, but examples include isobornyl (meth)acrylate. The amount of other structural units in the polymer of the present invention is preferably 20 mol % or less based on the total number of structural units. It is more preferable that the polymer of the present invention does not contain other structural units.

[0066] The polymer of the present invention is preferably at least one selected from the group consisting of copolymer (1-2a), copolymer (1-2b), and copolymer (1-2-3), more preferably at least one selected from the group consisting of copolymer (1-2b) and copolymer (1-2-3), and even more preferably at least one selected from the group consisting of copolymer (1-2b) and copolymer (1-2a-3). The copolymers (1-2a), (1-2b), and (1-2-3) (including copolymer (1-2a-3)) and their monomers have been described above.

[0067] The polymer of the present invention can be produced by a known method (for example, the method described in WO 2018 / 216628).

[0068] The polymer of the present invention can be easily used as a stabilizer by being contained in a nucleic acid solution.

[0069] Examples of methods for incorporating the polymer of the present invention into a nucleic acid solution include: (i) a method in which the polymer of the present invention is added to a prepared nucleic acid solution to dissolve the polymer; (ii) a method in which the polymer of the present invention is dissolved in advance in a solvent such as a buffer solution for dissolving nucleic acids, and the nucleic acid is then dissolved in the solvent; and (iii) a method in which the polymer of the present invention is placed in advance in a container for preparing a nucleic acid solution, and the nucleic acid solution is then added thereto to dissolve the polymer.

[0070] The amount of the stabilizer of the present invention (i.e., the polymer of the present invention) used is determined by its concentration in a nucleic acid solution, which will be described later. From the viewpoint of stabilizing effect, the concentration of the stabilizer of the present invention in the solution is preferably 0.0001 to 10 w / v%, more preferably 0.001 to 1 w / v%, and even more preferably 0.01 to 0.09 w / v%. When two or more stabilizers of the present invention are used, the above concentration refers to the total concentration of the two or more stabilizers. When two or more components are used, the concentrations described below for other components also refer to the total concentration of the two or more components.

[0071] The nucleic acid stabilized by the stabilizer of the present invention may be either RNA or DNA, preferably RNA. Examples of RNA include mRNA, tRNA, rRNA, siRNA, ddRNA, dsRNA, ssRNA, ssRNA-RT, etc. Here, the nucleic acid may be artificially synthesized by chemical synthesis, in vitro synthesis (e.g., reverse transcription reaction), PCR, etc., or may be prepared by known methods from cells, microorganisms, viruses, etc. Here, the cells, microorganisms, viruses, etc. may be collected from nature or the environment, or from humans, animals, plants, etc., or may be isolated and cultured.

[0072] [Nucleic Acid Solution] The present invention further provides a nucleic acid solution containing the stabilizer of the present invention and nucleic acid.

[0073] As mentioned above, the concentration of the stabilizer of the present invention in the nucleic acid solution is 0.0001 to 10 w / v %, more preferably 0.001 to 1 w / v %, and even more preferably 0.01 to 0.09 w / v %.

[0074] The concentration of nucleic acid in the nucleic acid solution is preferably 1 to 10 ８ copy / μL, more preferably 1 to 10 ５ copy / μL, more preferably 1 to 10 ３ copy / μL.

[0075] From the viewpoint of storage stability of nucleic acids, the storage temperature of the nucleic acid solution is preferably -80°C to 40°C, more preferably -20°C to 40°C, and even more preferably 0°C to 40°C.

[0076] The nucleic acid in the nucleic acid solution may be either RNA or DNA, with RNA being preferred. Examples of RNA include mRNA, tRNA, rRNA, siRNA, ddRNA, dsRNA, ssRNA, and ssRNA-RT. The nucleic acid may be artificially synthesized, for example, by chemical synthesis, in vitro synthesis (e.g., reverse transcription), or PCR, or may be provided as a virus, bacterial cells, cells, body fluids, tissues, or the like, or as a suspension thereof, or as a nucleic acid extract prepared therefrom. The virus, bacterial cells, cells, body fluids, tissues, or the like may be collected from nature, the environment, humans, animals, or plants, or may be isolated and cultured.

[0077] The nucleic acid solution may contain other components, such as polyols, polyethers, proteins, salts, buffer solutions, surfactants, solvents, biochemical reagents, dyes, preservatives, oils, and solid phase carriers, as long as the effects of the present invention are not impaired.

[0078] Examples of polyols include glycerol, sucrose, glucose, etc. Examples of polyethers include polyoxyethylene glycol, etc. Examples of proteins include albumin, gelatin, casein, enzymes, etc.

[0079] Examples of salts include amino acids, peptides, alkali metal salts, alkaline earth metal salts, and organic acids such as ethylenediaminetetraacetic acid. Examples of buffer solutions include Tris-HCl buffer, Good's buffer, glycine buffer, borate buffer, TE buffer, TAE buffer, TBE buffer, and SSC buffer. Examples of surfactants include polyoxyethylene alkyl ether, polyoxyethylene sorbitan monoalkyl ether, and alkyl betaine.

[0080] Examples of the solvent include water and organic solvents. Examples of the organic solvent include ethanol, propanol, isoamyl alcohol, glycerin, dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, chloroform, phenol, etc. The solvent is preferably water. The nucleic acid solution is preferably an aqueous nucleic acid solution, more preferably an aqueous RNA solution. The aqueous nucleic acid solution (particularly the aqueous RNA solution) may further contain an organic solvent.

[0081] Examples of biochemical reagents include flavins, etc. Examples of dyes include ethidium bromide and SYBR ＴＭ Nucleic acid staining reagents such as Green I, ROX ＴＭ Examples of suitable coloring agents include fluorescent dyes such as dye, orange G, bromophenol blue, and xylene cyanol FF.

[0082] Examples of preservatives include sodium azide, parahydroxybenzoic acid preparations, dehydroacetic acid preparations, and Proclin preparations. Examples of oils include mineral oil. Examples of solid phase carriers include silica beads and magnetic beads.

[0083] [Method for improving the storage stability of nucleic acid in a nucleic acid solution] The present invention provides a method for improving the storage stability of nucleic acid by adding the stabilizer of the present invention to a nucleic acid solution at a concentration of 0.0001 to 10 w / v % (hereinafter sometimes abbreviated as "the improving method of the present invention").

[0084] In the nucleic acid improvement method of the present invention, the concentration of the stabilizer of the present invention in the nucleic acid solution is 0.0001 to 10 w / v %, preferably 0.001 to 1 w / v %, and more preferably 0.01 to 0.09 w / v %, from the viewpoint of stabilizing effect. In the nucleic acid improvement method of the present invention, the description of the nucleic acid solution other than the concentration of the stabilizer of the present invention is the same as that described above.

[0085] [Nucleic Acid Amplification Method] The present invention provides a nucleic acid amplification method comprising carrying out a nucleic acid amplification reaction using a solution containing the stabilizer of the present invention and nucleic acid, wherein the nucleic acid is preferably RNA.

[0086] The nucleic acid amplification method is preferably a polymerase chain reaction (PCR), more preferably a reverse transcription polymerase chain reaction (RT-PCR) in which the target to be amplified is RNA. The RT-PCR may be a reverse transcription-quantitative PCR (RT-qPCR).

[0087] PCR (particularly RT-PCR) is well known to those skilled in the art. There are no particular limitations on the conditions and reagents (e.g., buffer, substrate, primer, reverse transcriptase, DNA polymerase, fluorescent DNA staining reagent, fluorescent probe, passive reference, etc.) used in PCR, and those skilled in the art can perform PCR using a nucleic acid solution and known reagents for PCR.

[0088] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these.

[0089] Synthesis Example 1 8.4 g of 2-methacryloyloxyethyl phosphorylcholine (hereinafter referred to as "MPC") as monomer (1), 2.1 g of butyl methacrylate (hereinafter referred to as "BMA") as monomer (2), and 4.5 g of glycerin monomethacrylate (hereinafter referred to as "GLM") as monomer (3) (monomer (1) / monomer (2) / monomer (3) = 40 / 40 / 20 (molar ratio)) were weighed into a glass flask for polymerization, and 42.5 g of purified water and 42.5 g of ethanol were added to dissolve the monomers (1) to (3). To the resulting solution, 0.15 g of Perloyl SA (manufactured by NOF Corporation, hereinafter referred to as "PRSA") was added. The atmosphere in the reaction vessel was thoroughly replaced with nitrogen, and the mixture was heated at 70°C for 6 hours with stirring to carry out polymerization. The resulting reaction solution was ice-cooled and added dropwise to acetone to precipitate a polymer. The precipitate was filtered off, washed with acetone, and then vacuum dried to obtain a white powdery homopolymer (hereinafter referred to as "Polymer 1"). The weight-average molecular weight of Polymer 1 was 21,000 in terms of polyethylene glycol, as measured by gel permeation chromatography (hereinafter referred to as "GPC") under the conditions described below.

[0090] Synthesis Example 2 6.0 g of MPC as monomer (1) and 4.0 g of methacrylic acid (hereinafter referred to as "MAc") as monomer (2) (monomer (1) / monomer (2) = 30 / 70 (molar ratio)) were weighed into a glass flask for polymerization, 90.0 g of purified water was added to dissolve the monomers (1) and (2), and 0.78 g of PRSA was added to the resulting solution. Thereafter, a copolymer (hereinafter referred to as "polymer 2") was obtained in the same manner as in Synthesis Example 1. The weight average molecular weight of polymer 2 was 680,000 in terms of polyethylene glycol, as determined by GPC measurement under the conditions described below.

[0091] Synthesis Example 3 25.3 g of MPC as monomer (1) and 4.7 g of methoxypolyethylene glycol methacrylate (Blemmer PME, manufactured by NOF Corporation) as monomer (2) (number average molecular weight: approximately 500, n in formula (2): approximately 9, hereinafter referred to as "PEGMA") (monomer (1) / monomer (2) = 90 / 10 (molar ratio)) were weighed into a glass flask for polymerization, and 70.0 g of purified water was added to dissolve the monomers (1) and (2). 0.23 g of PRSA was added to the resulting solution. The procedure thereafter was the same as in Synthesis Example 1, and a copolymer (hereinafter referred to as "Polymer 3") was obtained. The weight average molecular weight of Polymer 3 was 135,000 in terms of polyethylene glycol, as determined by GPC measurement under the conditions described below.

[0092] Synthesis Example 4 6.0 g of MPC as monomer (1) and 3.3 g of stearyl methacrylate (hereinafter referred to as "SMA") as monomer (2) (monomer (1) / monomer (2) = 80 / 20 (molar ratio)) were weighed into a glass flask for polymerization, 85.0 g of ethanol was added to dissolve the monomers (1) and (2), and 0.06 g of azobisisobutyronitrile was added as a polymerization initiator to the resulting solution. The subsequent procedures were the same as in Synthesis Example 1, and a copolymer (hereinafter referred to as "Polymer 4") was obtained. The weight-average molecular weight of Polymer 4 was 43,000 in terms of polyethylene glycol, as determined by GPC measurement under the conditions described below.

[0093] <GPC Measurement> GPC measurement of polymers 1 to 4 obtained in synthesis examples 1 to 4 was carried out under the following conditions: GPC system: EcoSEC system (manufactured by Tosoh Corporation) Column: Shodex OHpak SB-802.5HQ (manufactured by Showa Denko K.K.) and SB-806HQ (manufactured by Showa Denko K.K.) connected in series Developing solvent: 20 mM sodium phosphate buffer (pH 7.4) Detector: differential refractive index detector Molecular weight standard: EasiVial PEG / PEO (manufactured by Agilent Technologies) Flow rate: 0.5 mL / min Column temperature: 40°C Sample: The obtained polymer was diluted with the developing solvent to a final concentration of 0.1 wt% Injection volume: 100 μL

[0094] Table 1 shows the monomers used in Synthesis Examples 1 to 4, their molar ratios, and the weight average molecular weights of the resulting polymers.

[0095]

[0096] [Examples 1 to 4] Examples are shown in which the nucleic acid is RNA and the nucleic acid amplification method is RT-qPCR.

[0097] <Preparation of Nucleic Acid Solution> The nucleic acid solutions shown in Table 2 were prepared. The nucleic acid solutions were prepared while cooling on ice. The nucleic acid solutions were prepared using the RNA and polymer solutions. Table 2 lists the amounts of the RNA solution and polymer solution added, as well as the final concentrations of the RNA and polymer.

[0098]

[0099] <Storage of Nucleic Acid Solution> The prepared nucleic acid solution was stored at 4°C for 4 days or at 37°C for 11 days.

[0100] <Preparation of composition for amplifying nucleic acid> A composition for amplifying nucleic acid was prepared by mixing the components shown in Table 3. This composition for amplifying nucleic acid was prepared while being cooled on ice.

[0101]

[0102] <Preparation of RT-qPCR reaction solution> 5 μL per well of the nucleic acid solution before storage (i.e., immediately after production), the nucleic acid solution stored at 4°C for 4 days, or the nucleic acid solution stored at 37°C for 11 days was added to the prepared nucleic acid amplification composition to prepare an RT-qPCR reaction solution.

[0103] <RT-qPCR> The RT-qPCR reaction solution obtained as described above was used in a qPCR device (Applied Biosystems "StepOnePlus" ＴＭ RT-qPCR was performed using a Real-Time PCR System ("Real-Time PCR System") to measure the Ct value of the RT-qPCR reaction solution. Here, "Ct value" refers to the number of cycles required for the detected fluorescent signal to exceed the threshold (background). A lower Ct value indicates a higher amount of RNA. The measurement was performed three times for each RT-qPCR reaction solution. The results are shown in Tables 5 and 6 below. The temperature program is as shown in Table 4 below.

[0104]

[0105] Comparative Example 1 In Comparative Example 1, the same operations as in Examples 1 to 4 were carried out except that 1.0 μL of distilled water, nuclease-free, was used instead of 1.0 μL of the polymer solution. The results are shown in Tables 5 and 6 below.

[0106]

[0107]

[0108] As shown in Tables 5 and 6, in Comparative Example 1, in which the stabilizers of the present invention (Polymers 1 to 4) were not used, no Ct value was obtained after storage of the nucleic acid solution, whereas in Examples 1 to 4, in which the stabilizers of the present invention (Polymers 1 to 4) were used, Ct values ​​were obtained even after storage of the nucleic acid solution. In particular, in Examples 1 and 3, Ct values ​​were obtained for all three RT-qPCR reaction solutions, regardless of whether they were stored at 4°C or 37°C. Furthermore, as shown in Table 5, in Examples 1 to 4, there was almost no change in Ct value between before and after storage at 4°C. These results demonstrate that the stabilizers of the present invention stabilize nucleic acids simply by being added to a nucleic acid solution.

[0109] The stabilizer of the present invention is useful for improving the storage stability of nucleic acids in solution.

[0110] This application is based on Japanese Patent Application No. 2023-166492, the contents of which are incorporated in their entirety herein.

Claims

1. Formula (1): (In the formula, X １ represents a (meth)acryloyloxy group or a (meth)acryloylamino group; L １ represents an alkylene group having 2 to 4 carbon atoms which may have one hydroxy group, or an alkyleneoxyalkylene group having 2 to 4 carbon atoms, and R １ ~R ３ each independently represents an alkyl group having 1 to 3 carbon atoms.

2. The polymer has the formula (2): (In the formula, R ４ represents a hydrogen atom or a methyl group, and R ５ is a hydrogen atom, an alkyl group having 1 to 20 carbon atoms, or *-(OCH ２ CH ２ ) ｎ OR ６ group (wherein * represents a bonding position, n represents a number from 1 to 10, and R ６ represents a hydrogen atom, a methyl group, or an ethyl group. The stabilizer according to claim 1, which is a copolymer further comprising a structural unit derived from a monomer represented by the following formula:

3. The polymer has the formula (3): (In the formula, R ７ represents a hydrogen atom or a methyl group, and R ８ 3. The stabilizer according to claim 2, which is a copolymer further comprising a structural unit derived from a monomer represented by the following formula:

4. The stabilizing agent according to claim 1, wherein the nucleic acid solution is an RNA solution.

5. A method for improving the storage stability of a nucleic acid by adding the stabilizer according to any one of claims 1 to 4 to a nucleic acid solution at a concentration of 0.0001 to 10 w / v %.

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