Single-stranded nucleic acid adsorption inhibitor, nucleic acid solution, and nucleic acid amplification method

Abstract

The present invention provides a single-stranded nucleic acid adsorption inhibitor containing one or more polymers selected from the group A: [group A] Polymer A1: a copolymer containing a structural unit (a) derived from 2-(meth) acryloyloxyethyl phosphorylcholine and a structural unit (b) derived from a C2-C6 alkyl (meth) acrylate having two or more hydroxyl groups as substituents; polymer A2: a copolymer containing the constituent unit (a) and a constituent unit (c) derived from a polyethylene glycol (meth) acrylate; and polymer A3: a copolymer containing the constituent unit (a) and a constituent unit (d) derived from a C1-C6 alkyl (meth) acrylate having a phenyl group or a phenoxy group as a substituent.

Description

TECHNICAL FIELD

[0001] The present application relates to a single-stranded nucleic acid adsorption inhibitor, a nucleic acid solution, and a nucleic acid amplification method. BACKGROUND

[0002] RNA (Ribonucleic acid) is a biological polymer in which ribonucleotides as monomers are connected by a phosphodiester bond. RNA is composed of ribonucleotides and derivatives thereof of four nucleic acid bases of adenine, guanine, cytosine, and uracil, which are naturally contained, and has functions of transmitting genetic information, transducing signals, transporting amino acids in protein synthesis, regulating metabolism, constituting ribozymes, constituting cell organelles, and the like in a living body, or exists as an RNA of unknown function, or constitutes a genome in a part of viruses.

[0003] As examples of using RNA in society, there are a therapeutic target in the medical field, a component of a nucleic acid drug, a marker for virus / microorganism detection in environmental investigation, a component material of an mRNA vaccine, and the like. In addition, as other examples of use, there are a raw material for a genome editing tool in genetic engineering, and a guide RNA as a site-specific nuclease such as CRISPR / Cas9 (Clustered regularly interspaced short palindromic repeats / crispr associated protein 9). Further, as other examples, there are uses as a raw material for a health food or a food additive.

[0004] Among the above, in any of the applications, non-specific adsorption of RNA often becomes a problem. That is, in the handling, storage, and transfer of RNA, RNA is adsorbed on the surface of a container, a microchip, or the like, and in particular, on the surface of a plastic member, resulting in a decrease in the concentration of free RNA, and such a situation often becomes a problem.

[0005] In particular, in performing detection, quantification, and base sequence analysis of RNA, in which a target RNA at an extremely low concentration of micromole or less than attomole is used as an analysis target, cases where the remaining RNA is difficult to obtain are also frequent, and the loss of RNA adsorbed on the inner wall of a container or the like becomes a significant problem. In addition, the same problems as described above also occur with respect to single-stranded DNA.

[0006] In such a background, various modification techniques of plastics are studied with the aim of suppressing adsorption of nucleic acids onto plastic containers. For example, a plastic tube product to which a specific modification is applied is introduced in Non-Patent Literature 1, and it is described that when a nucleic acid concentration standard is prepared and stored using this product, adsorption of nucleic acids onto the container is suppressed, the yield of good nucleic acids is improved, and the precision and sensitivity of an application using the standard can be improved.

[0007] Further, in Patent Literatures 1 and 2, techniques of suppressing adsorption of nucleic acids by coating the surface of a container or a biochip with a specific coating material, and techniques of suppressing adsorption of nucleic acids by forming a layer containing a specific high molecular substance are disclosed. Prior Art Documents Patent Literatures

[0008] Patent Literature 1: Japanese Patent Application Publication No. 2012-78365 Patent Literature 2: Japanese Patent Application Publication No. 2006-258630 Non-Patent Literatures

[0009] Non-Patent Literature 1: Eppendorf PCR Consumables-Compatibility Guide for PCR and qPCR Cyclers SUMMARY Problems to be Solved by the Invention

[0010] However, by the approach of modifying plastics as in Non-Patent Literature 1, it is necessary to replace all components with which the nucleic acid is expected to come into contact with components to which the technique is applied in advance, and this is inconvenient. Similarly, the techniques described in Patent Literatures 1 and 2 also require that all components with which the nucleic acid is expected to come into contact be applied with the technique in advance, and this is inconvenient.

[0011] The present application was made in view of the above problems, and aims to provide a technique of suppressing adsorption of single-stranded nucleic acids by modifying a solution of nucleic acids. That is, the present application aims to provide a single-stranded nucleic acid adsorption suppressor which can suppress adsorption of a nucleic acid to a container or a component by adding to a solution of the nucleic acid in advance, a solution of the nucleic acid containing the suppressor, and a nucleic acid amplification method using the solution of the nucleic acid. By this approach, it is possible to adjust the concentration of addition according to the characteristics of a sample, and it has the advantage that the degree of freedom is high. Means for Solving the Problems

[0012] The present application which can achieve the above object is as follows. [1] A single-stranded nucleic acid adsorption suppressor containing one or more polymers selected from Group A below, [Group A] Polymer A1: a copolymer containing the structural unit (a) derived from 2- (methyl) acryloyloxyethylphosphocholine and the structural unit (b) derived from a (methyl) acrylate C2-C6 alkyl ester having 2 or more hydroxyl groups as substituents; Polymer A2: a copolymer containing the structural unit (a) and the structural unit (c) derived from a polyethylene glycol (methyl) acrylate; and Polymer A3: a copolymer containing the structural unit (a) and the structural unit (d) derived from a (methyl) acrylate C1-C6 alkyl ester having a phenyl group or a phenoxy group as a substituent.

[0013] [2] The single-stranded nucleic acid adsorption inhibitor according to the above [1], wherein the 2-(methyl) acryloyloxyethylphosphocholine is 2-methyl acryloyloxyethylphosphocholine.

[0014] [3] The single-stranded nucleic acid adsorption inhibitor according to the above [1] or [2], wherein the (methyl) acrylate C2-C6 alkyl ester having 2 or more hydroxyl groups as substituents is a glycerol mono(methyl) acrylate, a threitol mono(methyl) acrylate, an erythritol mono(methyl) acrylate, a xylitol mono(methyl) acrylate, an arabitol mono(methyl) acrylate, a mannitol mono(methyl) acrylate, a galactitol mono(methyl) acrylate, or a sorbitol mono(methyl) acrylate, preferably a glycerol mono(methyl) acrylate, more preferably a glycerol monomethyl acrylate.

[0015] [4] The single-stranded nucleic acid adsorption inhibitor according to any one of the above [1] to [3], wherein the polyethylene glycol (methyl) acrylate is a polyethylene glycol mono(methyl) acrylate, a methoxypolyethylene glycol (methyl) acrylate, or an ethoxypolyethylene glycol (methyl) acrylate, preferably a methoxypolyethylene glycol (methyl) acrylate or an ethoxypolyethylene glycol (methyl) acrylate, more preferably a methoxypolyethylene glycol methyl acrylate or an ethoxypolyethylene glycol methyl acrylate, further preferably a methoxypolyethylene glycol methyl acrylate.

[0016] [5] The single-stranded nucleic acid adsorption inhibitor according to any one of the above [1] to [4], wherein the number average molecular weight of the polyethylene glycol chain contained in the polyethylene glycol (methyl) acrylate is 50 to 10,000, preferably 50 to 5,000, more preferably 50 to 1,000, further preferably 100 to 1,000.

[0017] [6] The single-stranded nucleic acid adsorption inhibitor according to any one of the above [1] to [5], wherein the (meth)acrylic acid C1-C6 alkyl ester having a phenyl group or a phenoxy group as a substituent is benzyl (meth)acrylate, 1-phenylethyl (meth)acrylate, 2-phenylethyl (meth)acrylate, or phenoxyethyl glycol (meth)acrylate, preferably benzyl (meth)acrylate or 2-phenylethyl (meth)acrylate, more preferably benzyl (meth)acrylate, and further more preferably benzyl methacrylate.

[0018] [7] The single-stranded nucleic acid adsorption inhibitor according to any one of the above [1] to [6], wherein the weight average molecular weight of the polymer A1 is 10,000 to 500,000, preferably 10,000 to 100,000, and more preferably 10,000 to 50,000. [8] The single-stranded nucleic acid adsorption inhibitor according to any one of the above [1] to [7], wherein the weight average molecular weight of the polymer A2 is 50,000 to 1,000,000, preferably 100,000 to 500,000, and more preferably 100,000 to 300,000. [9] The single-stranded nucleic acid adsorption inhibitor according to any one of the above [1] to [8], wherein the weight average molecular weight of the polymer A3 is 5,000 to 2,000,000, preferably 50,000 to 1,000,000, and more preferably 100,000 to 500,000.

[0019]

[10] The single-stranded nucleic acid adsorption inhibitor according to any one of the above [1] to [9], wherein the proportion of the structural unit (a) with respect to the total structural units of the polymer A1 is 10 to 70 mol%, preferably 20 to 60 mol%, and more preferably 30 to 50 mol%.

[11] The single-stranded nucleic acid adsorption inhibitor according to any one of the above [1] to

[10] , wherein the proportion of the structural unit (b) with respect to the total structural units of the polymer A1 is 2 to 50 mol%, preferably 5 to 40 mol%, and more preferably 10 to 30 mol%.

[0020]

[12] The single-stranded nucleic acid adsorption inhibitor according to any one of the above [1] to

[11] , wherein the polymer A1 is a copolymer composed of the structural unit (a), the structural unit (b), and a structural unit (e) derived from a monomer other than 2-(meth)acryloyloxyethylphosphocholine, a (meth)acrylic acid C2-C6 alkyl ester having 2 or more hydroxyl groups as substituents, a polyethylene glycol (meth)acrylate, and a (meth)acrylic acid C1-C6 alkyl ester having a phenyl group or a phenoxy group as a substituent.

[13] The single-stranded nucleic acid adsorption inhibitor according to the above

[12] , the other monomer being a C1 to C6 alkyl (meth)acrylate, (meth)acrylic acid, or isobornyl (meth)acrylate, preferably a C1 to C6 alkyl (meth)acrylate, more preferably butyl (meth)acrylate, further more preferably butyl methacrylate.

[14] The single-stranded nucleic acid adsorption inhibitor according to the above

[12] or

[13] , the proportion of the structural unit (e) with respect to the total structural units of the polymer A1 being 10 to 60 mol%, preferably 20 to 50 mol%, more preferably 30 to 45 mol%.

[0021]

[15] The single-stranded nucleic acid adsorption inhibitor according to any one of the above [1] to

[14] , the proportion of the structural unit (a) with respect to the total structural units of the polymer A2 being 60 to 95 mol%, preferably 80 to 95 mol%, more preferably 85 to 95 mol%.

[16] The single-stranded nucleic acid adsorption inhibitor according to any one of the above [1] to

[15] , the proportion of the structural unit (c) with respect to the total structural units of the polymer A2 being 5 to 40 mol%, preferably 5 to 20 mol%, more preferably 5 to 15 mol%.

[0022]

[17] The single-stranded nucleic acid adsorption inhibitor according to any one of the above [1] to

[16] , the polymer A2 being a copolymer composed of the structural unit (a) and the structural unit (c).

[0023]

[18] The single-stranded nucleic acid adsorption inhibitor according to any one of the above [1] to

[17] , the proportion of the structural unit (a) with respect to the total structural units of the polymer A3 being 60 to 95 mol%, preferably 70 to 90 mol%, more preferably 75 to 85 mol%.

[19] The single-stranded nucleic acid adsorption inhibitor according to any one of the above [1] to

[18] , the proportion of the structural unit (d) with respect to the total structural units of the polymer A3 being 5 to 40 mol%, preferably 10 to 30 mol%, more preferably 15 to 25 mol%.

[0024]

[20] The single-stranded nucleic acid adsorption inhibitor according to any one of the above [1] to

[19] , the polymer A3 being a copolymer composed of the structural unit (a) and the structural unit (d).

[0025]

[21] The single-stranded nucleic acid adsorption inhibitor according to any one of the above [1] to

[20] , the single-stranded nucleic acid being RNA.

[0026]

[22] A nucleic acid solution containing the single-stranded nucleic acid adsorption inhibitor and the single-stranded nucleic acid according to any one of [1] to

[20] .

[23] The nucleic acid solution according to the above-mentioned

[22] , which is an aqueous nucleic acid solution.

[24] The nucleic acid solution according to the above-mentioned

[22] or

[23] , wherein the single-stranded nucleic acid is RNA.

[25] A nucleic acid amplification method using the nucleic acid solution according to any one of

[22] to

[24] .

[0027]

[26] A method for inhibiting adsorption of a single-stranded nucleic acid in a nucleic acid solution to a member contacted with the nucleic acid solution, comprising the step of mixing the single-stranded nucleic acid adsorption inhibitor according to any one of [1] to

[20] , the single-stranded nucleic acid, and a solvent.

[27] The method according to the above-mentioned

[26] , wherein the nucleic acid solution is an aqueous nucleic acid solution, and the solvent is water.

[28] The method according to the above-mentioned

[26] or

[27] , wherein the single-stranded nucleic acid is RNA. Effects of the Invention

[0028] When the single-stranded nucleic acid adsorption inhibitor of the present invention is used, adsorption of free single-stranded nucleic acid present in a solution to which the single-stranded nucleic acid adsorption inhibitor has been added to a surface of a container, a microchip, or the like can be inhibited. Therefore, in various examinations in which, for example, a sample containing a single-stranded nucleic acid is used as a detection sample, improvement in stability during transport of the detection sample, improvement in stability during storage of the detection sample, improvement in detection sensitivity, and the like can be expected. In addition, from another viewpoint, when the single-stranded nucleic acid adsorption inhibitor of the present invention is used, improvement in stability during transport and storage of a pharmaceutical product containing a single-stranded nucleic acid can be expected. DETAILED DESCRIPTION

[0029] The present invention will be described in detail below. In addition, each description in the present specification can be combined with each other except for cases where it is explicitly not combinable.

[0030] In the present specification, in cases where a range of values at stages is described, the lower limit value and the upper limit value of each range of values can be combined. For example, in cases where "10 to 100 is preferable, and 20 to 90 is more preferable" is described, the "lower limit value of the preferable range: 10" and the "upper limit value of the more preferable range: 90" can be combined (i.e., the range of values of "10 to 90" is also within the scope of the present specification).

[0031] In the present specification, "single-stranded nucleic acid" means "RNA or single-stranded DNA". In the present specification, "single-stranded nucleic acid adsorption inhibitor" means an additive used in a solution of a single-stranded nucleic acid in order to inhibit adsorption of the nucleic acid onto the surface of a member in contact with the solution in a container, a microchip, or the like, which handles the solution.

[0032] In the present specification, "structural unit" means a repeating unit in a polymer derived from a monomer. Therefore, a structure not repeated in a polymer (for example, a structure derived from a polymerization initiator or the like) is not included in the "structural unit".

[0033] In the present specification, "(meth)acrylate" basically means "acrylate or methacrylate". In the case where a plurality of (meth)acrylates can also be present, "(meth)acrylate" means "acrylate and / or methacrylate". Other terms similar to "(meth)acrylate" also have the same meaning as "(meth)acrylate".

[0034] In the present specification, "(meth)acrylic acid C2-C6 alkyl ester" means "alkyl ester of (meth)acrylic acid in which the number of carbon atoms of the alkyl group is 2 to 6". Other terms similar to "(meth)acrylic acid C2-C6 alkyl ester" also have the same meaning as "(meth)acrylic acid C2-C6 alkyl ester".

[0035] In the present specification, "(meth)acrylic acid C2-C6 alkyl ester having 2 or more hydroxyl groups as a substituent" means "(meth)acrylic acid C2-C6 alkyl ester in which the alkyl group has 2 or more hydroxyl groups as a substituent".

[0036] In the present specification, "polyethylene glycol (meth)acrylate" means "ester of 1 polyethylene glycol and 1 (meth)acrylic acid". The terminal on the opposite side to the (meth)acrylate terminal of "polyethylene glycol (meth)acrylate" can be a hydroxyl group (for example, polyethylene glycol mono(meth)acrylate) or a methoxyl group or the like (for example, methoxyl polyethylene glycol (meth)acrylate).

[0037] In the present specification, "polyethylene glycol mono(meth)acrylate" means "monoester of polyethylene glycol and (meth)acrylic acid, in which the terminal on the opposite side to the (meth)acrylate terminal is a hydroxyl group".

[0038] In the present specification, "methoxyl polyethylene glycol (meth)acrylate" means "compound in which the hydroxyl group of the terminal of polyethylene glycol mono(meth)acrylate is replaced with a methoxyl group". Other terms similar to "methoxyl polyethylene glycol (meth)acrylate" also have the same meaning as "methoxyl polyethylene glycol (meth)acrylate".

[0039] In the present specification, "(methyl) acrylate C1-C6 alkyl ester having phenyl or phenoxy as a substituent" means "(methyl) acrylate C1-C6 alkyl ester having phenyl or phenoxy as a substituent of the alkyl group". The number of carbon atoms refers to the number of carbon atoms of the alkyl group, excluding the number of carbon atoms of the substituent (phenyl or phenoxy).

[0040] [Single-stranded nucleic acid adsorption inhibitor] The single-stranded nucleic acid adsorption inhibitor of the present application contains one or more polymers selected from the following Group A. [Group A] Polymer A1: a copolymer containing structural unit (a) derived from 2- (methyl) acryloyloxyethylphosphocholine (hereinafter referred to as "monomer a" in cases where such a description is present) and structural unit (b) derived from (methyl) acrylate C2-C6 alkyl ester having two or more hydroxyl groups as substituents (hereinafter referred to as "monomer b" in cases where such a description is present); Polymer A2: a copolymer containing the structural unit (a) and structural unit (c) derived from polyethylene glycol (methyl) acrylate (hereinafter referred to as "monomer c" in cases where such a description is present); and Polymer A3: a copolymer containing the structural unit (a) and structural unit (d) derived from (methyl) acrylate C1-C6 alkyl ester having phenyl or phenoxy as a substituent (hereinafter referred to as "monomer d" in cases where such a description is present).

[0041] The monomer a forming the structural unit (a) contained in the polymers A1 to A3 (hereinafter referred to as "polymers of the present application" in cases where such a description is present) is 2-methacryloyloxyethylphosphocholine or 2-acryloyloxyethylphosphocholine, and from the viewpoint of storage stability of the polymers of the present application, 2-methacryloyloxyethylphosphocholine is preferred.

[0042] The monomer a can be used alone or in combination of two kinds. In addition, the monomer a can be used as a commercially available product or can be produced according to a publicly known method.

[0043] As examples of the monomer b forming the structural unit (b) contained in the polymer A1, there can be mentioned glycerol 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, sorbitol mono(meth)acrylate and the like. Of these, from the viewpoints of raw material availability and single-stranded nucleic acid adsorption inhibitor effect, glycerol mono(meth)acrylate is preferred, and glycerol monomethacrylate is more preferred. In the present specification, "glycerol mono(meth)acrylate" means "a monoester of glycerol and (meth)acrylic acid". Other terms similar to "glycerol mono(meth)acrylate" also have the same meaning as "glycerol mono(meth)acrylate".

[0044] The monomer b can be used singly or in combination of two kinds. In addition, the monomer b can be used as a commercially available product or can be produced according to a publicly known method.

[0045] From the viewpoint of single-stranded nucleic acid adsorption inhibition effect, the proportion of the structural unit (a) with respect to the total structural units of the polymer A1 is preferably 10 to 70 mol%, more preferably 20 to 60 mol%, and further preferably 30 to 50 mol%.

[0046] From the viewpoint of single-stranded nucleic acid adsorption inhibition effect, the proportion of the structural unit (b) with respect to the total structural units of the polymer A1 is preferably 2 to 50 mol%, more preferably 5 to 40 mol%, and further preferably 10 to 30 mol%.

[0047] The weight average molecular weight of the polymer A1 is not particularly limited, but from the viewpoint of single-stranded nucleic acid adsorption inhibition effect, it is preferably 10,000 to 500,000, more preferably 10,000 to 100,000, and further preferably 10,000 to 50,000. In addition, the weight average molecular weight can be determined by, for example, gel filtration chromatography using an EcoSEC system (manufactured by Tosoh Corporation) or the like, converted to polyethylene glycol.

[0048] As examples of the monomer c forming the structural unit (c) contained in the polymer A2, there can be mentioned polyethylene glycol mono(meth)acrylate, methoxypolyethylene glycol (meth)acrylate, ethoxypolyethylene glycol (meth)acrylate and the like. Of these, from the viewpoint of storage stability of the polymer of the present application, methoxypolyethylene glycol (meth)acrylate, ethoxypolyethylene glycol (meth)acrylate are preferred, methoxypolyethylene glycol methacrylate, ethoxypolyethylene glycol methacrylate are more preferred, and methoxypolyethylene glycol methacrylate is further preferred.

[0049] From the viewpoints of raw material availability and single-stranded nucleic acid adsorption inhibitory effect, the number average molecular weight of the polyethylene glycol chain contained in monomer c is preferably from 50 to 10,000, more preferably from 50 to 5,000, further preferably from 50 to 1,000, and particularly preferably from 100 to 1,000. In addition, the number average molecular weight of the polyethylene glycol chain can be determined, for example, based on the hydroxyl value calculated by the method described in JIS K 1557, or can be determined, for example, by gel filtration chromatography by polyethylene glycol conversion.

[0050] Monomer c can be used singly or in combination of two or more. In addition, monomer c can be used as a commercially available product, or can be produced according to a publicly known method.

[0051] From the viewpoint of single-stranded nucleic acid adsorption inhibitory effect, the proportion of structural unit (a) with respect to the total structural units of polymer A2 is preferably from 60 to 95 mol%, more preferably from 80 to 95 mol%, and further preferably from 85 to 95 mol%.

[0052] From the viewpoint of single-stranded nucleic acid adsorption inhibitory effect, the proportion of structural unit (c) with respect to the total structural units of polymer A2 is preferably from 5 to 40 mol%, more preferably from 5 to 20 mol%, and further preferably from 5 to 15 mol%.

[0053] The weight average molecular weight of polymer A2 is not particularly limited, and is preferably from 50,000 to 1,000,000, more preferably from 100,000 to 500,000, and further preferably from 100,000 to 300,000.

[0054] As examples of monomer d forming the structural unit (d) contained in polymer A3, for example, benzyl (meth)acrylate, 1-phenylethyl (meth)acrylate, 2-phenylethyl (meth)acrylate, phenoxy ethylene glycol (meth)acrylate can be given. Among these, from the viewpoints of raw material availability and single-stranded nucleic acid adsorption inhibitory effect, benzyl (meth)acrylate, 2-phenylethyl (meth)acrylate are preferred, and benzyl (meth)acrylate is more preferred. In addition, from the viewpoint of storage stability of polymer A3, benzyl methacrylate is further preferred.

[0055] Monomer d can be used singly or in combination of two or more. In addition, monomer d can be used as a commercially available product, or can be produced according to a publicly known method.

[0056] From the viewpoint of single-stranded nucleic acid adsorption inhibitory effect, the proportion of structural unit (a) with respect to the total structural units of polymer A3 is preferably from 60 to 95 mol%, more preferably from 70 to 90 mol%, and further preferably from 75 to 85 mol%.

[0057] From the viewpoint of single-stranded nucleic acid adsorption inhibition effect, the proportion of the structural unit (d) with respect to the total structural units of the polymer A3 is preferably 5 to 40 mol%, more preferably 10 to 30 mol%, and further preferably 15 to 25 mol%.

[0058] The weight average molecular weight of the polymer A3 is not particularly limited, and is preferably 5,000 to 2,000,000, more preferably 50,000 to 1,000,000, and further preferably 100,000 to 500,000.

[0059] Among the polymers of the present application, the copolymer containing the structural unit (b) (for example, the copolymer containing the structural unit (a) to the structural unit (c), the copolymer containing the structural unit (a), the structural unit (b), and the structural unit (d), and the copolymer containing the structural unit (a) to the structural unit (d)) is not the polymer A2 or A3, but is classified as the polymer Al.

[0060] The proportion of the structural unit (c) with respect to the total structural units of the polymer Al is preferably 30 mol% or less, more preferably 20 mol% or less, and further preferably 10 mol% or less. The polymer Al is particularly preferably free of the structural unit (c).

[0061] The proportion of the structural unit (d) with respect to the total structural units of the polymer Al is preferably 30 mol% or less, more preferably 20 mol% or less, and further preferably 10 mol% or less. The polymer Al is particularly preferably free of the structural unit (d).

[0062] Among the polymers of the present application, the copolymer containing the structural unit (c) (wherein the copolymer containing the structural unit (b) is excluded) (for example, the copolymer containing the structural unit (a), the structural unit (c), and the structural unit (d)) is not the polymer A3, but is classified as the polymer A2.

[0063] The proportion of the structural unit (d) with respect to the total structural units of the polymer A2 is preferably 30 mol% or less, more preferably 20 mol% or less, and further preferably 10 mol% or less. The polymer A2 is particularly preferably free of the structural unit (d).

[0064] The polymers Al to A3 can also contain a structural unit (e) derived from another monomer different from the monomers a to d, within a range not impairing the effects of the present application.

[0065] As the other monomer, for example, (meth)acrylic acid C1-C6 alkyl ester, (meth)acrylic acid, isobornyl (meth)acrylate, and the like can be mentioned. Among these, (meth)acrylic acid C1-C6 alkyl ester is preferable, (meth)acrylic acid butyl ester is more preferable, and methyl methacrylate is further preferable.

[0066] The other monomer can be used singly or in combination of two or more. In addition, the other monomer can be used as a commercially available product or can be produced according to a publicly known method.

[0067] The proportion of the structural unit (e) with respect to the total structural units of the polymer A1 is preferably 10 to 60 mol%, more preferably 20 to 50 mol%, and further preferably 30 to 45 mol%.

[0068] The proportion of the structural unit (e) with respect to the total structural units of the polymer A2 or the polymer A3 is preferably 30 mol% or less, more preferably 20 mol% or less, and further preferably 10 mol% or less. Both the polymer A2 and the polymer A3 are particularly preferably free of the structural unit (e).

[0069] The polymer A1 is preferably a copolymer composed of the structural unit (a), the structural unit (b), and the structural unit (e). Among these, the "copolymer composed of the structural unit (a), the structural unit (b), and the structural unit (e)" means a copolymer in which all the structural units (repeating units) in the copolymer are composed of the structural unit (a), the structural unit (b), and the structural unit (e). Other terms similar to the "copolymer composed of the structural unit (a), the structural unit (b), and the structural unit (e)" also have the same meaning as the "copolymer composed of the structural unit (a), the structural unit (b), and the structural unit (e)".

[0070] The polymer A1 is more preferably a copolymer composed of the following structural units: one or two structural units (a) derived from 2-(meth)acryloyloxyethylphosphocholine; one or two or more structural units (b) derived from propane-1, 2, 3-triol 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, or sorbitol mono(meth)acrylate; and one or two or more structural units (e) derived from (meth)acrylic acid C1-C6 alkyl ester.

[0071] The polymer A1 is further preferably a copolymer composed of the following structural units: one or two structural units (a) derived from 2-(meth)acryloyloxyethylphosphocholine; one or two structural units (b) derived from glycerol mono(meth)acrylate; and one or two structural units (e) derived from butyl (meth)acrylate.

[0072] Polymer A1 is particularly preferably a copolymer composed of the following structural units: structural unit (a) derived from 2-methacryloyloxyethylphosphocholine; structural unit (b) derived from glycerol monomethacrylate; and structural unit (e) derived from butyl methacrylate.

[0073] Polymer A2 is preferably a copolymer composed of structural unit (a) and structural unit (c).

[0074] Polymer A2 is more preferably a copolymer composed of the following structural units: one or two structural units (a) derived from 2-(meth)acryloyloxyethylphosphocholine; and one or more than two structural units (c) derived from polyethylene glycol mono(meth)acrylate, methoxypolyethylene glycol (meth)acrylate or ethoxypolyethylene glycol (meth)acrylate.

[0075] Polymer A2 is further preferably a copolymer composed of one or two structural units (a) derived from 2-(meth)acryloyloxyethylphosphocholine and one or more than two structural units (c) derived from methoxypolyethylene glycol (meth)acrylate or ethoxypolyethylene glycol (meth)acrylate.

[0076] Polymer A2 is particularly preferably a copolymer composed of the following structural units: structural unit (a) derived from 2-methacryloyloxyethylphosphocholine; and one or two structural units (c) derived from methoxypolyethylene glycol methacrylate or ethoxypolyethylene glycol methacrylate.

[0077] Polymer A2 is most preferably a copolymer composed of the following structural units: structural unit (a) derived from 2-methacryloyloxyethylphosphocholine; and structural unit (c) derived from methoxypolyethylene glycol methacrylate.

[0078] Polymer A3 is preferably a copolymer composed of structural unit (a) and structural unit (d).

[0079] Polymer A3 is more preferably a copolymer composed of the following structural units: one or two structural units (a) derived from 2-(meth)acryloyloxyethylphosphocholine; and one or two or more structural units (d) derived from benzyl (meth)acrylate, 1-phenylethyl (meth)acrylate, 2-phenylethyl (meth)acrylate, or phenoxyethanediol (meth)acrylate.

[0080] Polymer A3 is more preferably a copolymer composed of the following structural units: one or two structural units (a) derived from 2-(meth)acryloyloxyethylphosphocholine; and one or two or more structural units (d) derived from benzyl (meth)acrylate, 1-phenylethyl (meth)acrylate, or 2-phenylethyl (meth)acrylate.

[0081] Polymer A3 is more preferably a copolymer composed of the following structural units: one or two structural units (a) derived from 2-(meth)acryloyloxyethylphosphocholine; and one or two structural units (d) derived from benzyl (meth)acrylate.

[0082] Polymer A3 is more preferably a copolymer composed of the following structural units: one or two structural units (a) derived from 2-(meth)acryloyloxyethylphosphocholine; and one or two structural units (d) derived from benzyl (meth)acrylate.

[0083] The polymer of the present application can be any of a random copolymer, an alternating copolymer, a block copolymer, a graft polymer, or a copolymer having a structure of two or more of these, and from the viewpoint of the manufacturability of the polymer of the present application, a random copolymer is preferred.

[0084] The polymers A1 to A3 can be produced by a known method (for example, the method described in International Publication No. 2018 / 216628, etc.).

[0085] The nucleic acid adsorption inhibitor of the present application can contain components other than the polymers A1 to A3, within a range that does not impair the nucleic acid adsorption inhibitory effect based on the polymers A1 to A3. The other components are not particularly limited, and can be appropriately selected from the components exemplified as "other components" in the nucleic acid solution of the present application described later.

[0086] By previously containing the polymers A1 to A3 in the single-stranded nucleic acid solution, it can be easily used as a single-stranded nucleic acid adsorption inhibitor.

[0087] As a method of making a nucleic acid solution containing the polymer of the present application, a method of adding the polymer Al to A3 to a prepared single-stranded nucleic acid solution and dissolving it, a method of previously dissolving the polymer of the present application in a solvent such as a buffer solution for dissolving a single-stranded nucleic acid, a method of previously putting the polymer of the present application into a container for preparing a single-stranded nucleic acid solution, and then putting a single-stranded nucleic acid solution into it and dissolving it, and the like can be considered.

[0088] The concentration (final concentration) of the nucleic acid adsorption inhibitor of the present application added to a single-stranded nucleic acid solution is preferably 0.01 to 5 w / v%, more preferably 0.1 to 1 w / v%, and further preferably 0.1 to 0.5 w / v%. When the amount of addition is too small, there is a possibility that the single-stranded nucleic acid adsorption inhibitory effect cannot be obtained; and when the amount of addition is too large, there is a possibility that problems such as reaction inhibition occur when the single-stranded nucleic acid solution is used for enzyme reactions and the like.

[0089] The single-stranded nucleic acid to which the single-stranded nucleic acid adsorption inhibitor of the present application can be applied can be any kind of RNA or single-stranded DNA, and is preferably RNA.

[0090] The single-stranded nucleic acid can be a nucleic acid artificially synthesized by chemical synthesis, in vitro synthesis (for example, reverse transcription reaction), PCR, and the like, or can be a nucleic acid prepared from cells, microorganisms, viruses, and the like by a publicly known method. The cells, microorganisms, viruses, and the like can be materials extracted from nature or the environment, from humans or animals and plants, and in addition, can be substances isolated / cultured.

[0091] [Nucleic acid solution] The present application further provides a nucleic acid solution containing the single-stranded nucleic acid adsorption inhibitor of the present application and a single-stranded nucleic acid.

[0092] The concentration (final concentration) of the single-stranded nucleic acid adsorption inhibitor in the nucleic acid solution of the present application is as described above, and is preferably 0.01 to 5 w / v%, more preferably 0.1 to 1 w / v%, and further preferably 0.1 to 0.5 w / v%.

[0093] The single-stranded nucleic acid contained in the nucleic acid solution of the present application can be any kind of RNA or single-stranded DNA, and is preferably RNA.

[0094] The single-stranded nucleic acid can be, for example, a nucleic acid (e.g., RNA, complementary DNA, etc.) artificially synthesized by chemical synthesis, in vitro synthesis (e.g., reverse transcription reaction, solid-phase synthesis, etc.), PCR, etc., and can also be a substance provided as a virus, a bacterium, a cell, a body fluid, a tissue, etc., or a suspension of these or a nucleic acid extract prepared from these. In addition, the virus, the bacterium, the cell, the body fluid, the tissue, etc. can be a substance extracted from nature or the environment, from a human or an animal or a plant, and can also be a substance isolated / cultured. In addition, from other viewpoints, the single-stranded nucleic acid can be a single-stranded nucleic acid existing in a living organism, such as messenger RNA, transfer RNA, ribosomal RNA, non-coding RNA, micro RNA, ribozyme, single-stranded genomic RNA, single-stranded genomic DNA, etc., and can also be a single-stranded nucleic acid having a primary structure equivalent to or complementary to all or a part of the above-described single-stranded nucleic acid, and can also be a single-stranded nucleic acid having a primary structure entirely artificially designed. The concentration of the single-stranded nucleic acid can be appropriately determined depending on the application of the nucleic acid.

[0095] The nucleic acid solution of the present application can contain other components within a range not impairing the effects of the present application.

[0096] As the other components, for example, polyhydric alcohols, polyethers, proteins, salts, buffers, surfactants, solvents, biochemical reagents, pigments, preservatives, oils, solid-phase carriers, etc. can be mentioned.

[0097] As the polyhydric alcohols, for example, glycerol, sucrose, glucose, etc. can be mentioned. As the polyethers, for example, polyethylene glycol, etc. can be mentioned. As the proteins, for example, albumin, gelatin, casein, enzymes, etc. can be mentioned.

[0098] As the salts, for example, alkali metal salts, alkaline earth metal salts, salts of amino acids, salts of peptides, salts of organic acids such as ethylenediaminetetraacetic acid, etc. can be mentioned. As the buffers, for example, Tris-hydrochloric acid buffer, Good's buffer, glycine buffer, boric acid buffer, TE buffer, TAE buffer, TBE buffer, SSC buffer, etc. can be mentioned. As the surfactants, for example, polyoxyethylene alkyl ether, polyoxyethylene sorbitan monoalkyl ether, alkyl betaine, etc. can be mentioned.

[0099] As the solvents, water and organic solvents can be mentioned. As the organic solvents, for example, ethanol, propanol, isopropyl alcohol, glycerol, dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, chloroform, phenol, etc. can be mentioned. The solvent is preferably water, and the nucleic acid solution of the present application is preferably a nucleic acid aqueous solution. The nucleic acid aqueous solution can further contain an organic solvent.

[0100] As a biochemical reagent, for example, flavins and the like can be given. As a colorant, for example, ethidium bromide, SYBR TM Green I and the like nucleic acid staining reagents, ROX TM Dye and the like fluorescent dyes, Orange G, bromophenol blue, xylene cyanol FF and the like coloring agents and the like.

[0101] As a preservative, for example, sodium azide, p-hydroxybenzoic acid preparations, dehydroacetic acid preparations, ProClin preparations and the like can be given. As an oil, for example, mineral oil and the like can be given. As a solid phase carrier, for example, silica spheres, magnetic beads and the like can be given.

[0102] [Nucleic acid amplification method] The nucleic acid solution of the present application can be maintained to contain the nucleic acid adsorption inhibitor of the present application directly for an enzyme reaction.

[0103] As an enzyme reaction, for example, cleavage using a nuclease, reverse transcription using a reverse transcriptase, replication using a DNA polymerase, and reactions such as nucleic acid amplification using these can be given. The enzyme reaction referred to in the present application is preferably replication of single-stranded DNA or RNA, and more preferably reverse transcription PCR.

[0104] [Method for inhibiting adsorption of single-stranded nucleic acid] The present application provides a method for inhibiting adsorption of single-stranded nucleic acid in a nucleic acid solution to a member contacted with the nucleic acid solution, which comprises a step of mixing the single-stranded nucleic acid adsorption inhibitor of the present application, single-stranded nucleic acid and a solvent. The mixing is not particularly limited, and for example, (1) the nucleic acid adsorption inhibitor of the present application, single-stranded nucleic acid and a solvent can be mixed; (2) a solution containing single-stranded nucleic acid and a solvent and the single-stranded nucleic acid adsorption inhibitor of the present application can be mixed; or (3) a solution containing the single-stranded nucleic acid adsorption inhibitor of the present application and a solvent and single-stranded nucleic acid can be mixed. The description of the nucleic acid solution obtained by mixing the single-stranded nucleic acid adsorption inhibitor of the present application, single-stranded nucleic acid and a solvent (for example, the kind of single-stranded nucleic acid to be used, the concentration of the single-stranded nucleic acid adsorption inhibitor of the present application and the description of other components) is the same as the description of the nucleic acid solution of the present application described above. Examples

[0105] The present application is specifically described below by way of examples and the like, but the present application is not limited to these.

[0106] [Synthesis of polymer] The polymers A1-1 to A3-1 within the scope of the present application and the polymer Z-1 outside the scope of the present application were prepared according to the following points. The resulting polymer was dissolved in water (Nuclease free, manufactured by NIPPON GENE Co., Ltd., hereinafter referred to as "PW") up to 10 times the final concentration described in each condition of the Examples and Comparative Examples described later, and the resulting aqueous polymer solution was used. The five polymers prepared were summarized in Table 1.

[0107] [Synthesis Example 1] The polymer A1-1 belonging to the polymer A1 was prepared according to the following points. In a polymerization glass flask, 57 g of 2-methacryloyloxyethylphosphocholine (hereinafter referred to as "MPC"), 15 g of glycerol monomethacrylate (hereinafter referred to as "GLM"), and 28 g of butyl methacrylate (hereinafter referred to as "BMA") (MPC / GLM / BMA = 40 / 20 / 40 (molar ratio)) were weighed, and 410 g of ethanol and 200 g of purified water were added to dissolve. The solution was heated to 60°C, 1.9 g of 2,2'-azobis(isobutyronitrile) (hereinafter referred to as "AIBN") was added under a nitrogen atmosphere, and polymerization was performed by stirring for 5 hours. The resulting polymerization solution was purified by dialysis using a semi-permeable membrane with a molecular weight cut-off of 3,000, and then freeze-dried to obtain the polymer A1-1. The weight average molecular weight of the polymer A1-1 was determined by GPC under the conditions described later, and was 21,000 in terms of polyethylene glycol.

[0108] [Synthesis Example 2] The polymer A2-1 belonging to the polymer A2 was prepared according to the following points. In a polymerization glass flask, 84 g of MPC and 16 g of methoxypolyethylene glycol methacrylate (number average molecular weight of polyethylene glycol chain: about 500, hereinafter referred to as "PEG-MA") (MPC / PEG-MA = 90 / 10 (molar ratio)) were weighed, and 233 g of purified water was added to dissolve. To the resulting solution, 2.6 g of 4,4'-azobis(4-cyanopentanoic acid) was added. The solution was heated to 70°C, and polymerization was performed by stirring for 6 hours under a nitrogen atmosphere. The resulting polymerization solution was purified by dialysis using a semi-permeable membrane with a molecular weight cut-off of 20,000, and then freeze-dried to obtain the polymer A2-1. The weight average molecular weight of the polymer A2-1 was determined by GPC under the conditions described later, and was 133,000 in terms of polyethylene glycol.

[0109] [Synthesis Example 3] The polymer A3-1 belonging to the polymer A3 was prepared according to the following points. In a polymerization glass flask, 87 g of MPC and 13 g of benzyl methacrylate (hereinafter referred to as "BzMA") (MPC / BzMA = 80 / 20 (molar ratio)) were weighed, and 233 g of ethanol (hereinafter referred to as "EtOH") was added to dissolve. The solution was heated to 60°C, and polymerization was performed by adding 0.33 g of AIBN under a nitrogen atmosphere while stirring for 6 hours. The resulting polymerization solution was purified by dialysis using a semi-permeable membrane having a molecular weight cut-off of 3,000, and then freeze-dried to produce Polymer A3-1. The weight average molecular weight of Polymer A3-1 was determined by GPC under the following conditions, and was 240,000 in terms of polyethylene glycol.

[0110] [Synthesis Example 4] Polymer Z-1 outside the scope of the present application was prepared according to the following points. In a polymerization glass flask, 47 g of MPC and 53 g of BMA (MPC / BMA = 30 / 70 (molar ratio)) were weighed, and 900 g of EtOH was added to dissolve. The solution was heated to 60°C, and polymerization was performed by adding 2.4 g of AIBN under a nitrogen atmosphere while stirring for 7 hours. The resulting polymerization solution was purified by dialysis using a semi-permeable membrane having a molecular weight cut-off of 3,000, and then freeze-dried to produce Polymer Z-1. The weight average molecular weight of Polymer Z-1 was determined by GPC under the following conditions, and was 94,000 in terms of polyethylene glycol. [GPC measurement]

[0111] The GPC measurement of each of the polymers produced in Synthesis Examples 1 to 4 was performed under the following conditions. GPC system: EcoSEC system (manufactured by Tosoh Corporation) Chromatography column: Shodex OHpak SB-802.5HQ (manufactured by Showa Denko K.K.) and SB-806MHQ (manufactured by Showa Denko K.K.) were connected in series Elution 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 resulting polymer was diluted with the elution solvent to a final concentration of 0.1% by weight. Injection amount: 100 μL

[0112] [Table 1]

[0113] [Experimental Example 1] To a solution of RNA, the polymer A1-1 to polymer A3-1 or polymer Z-1 prepared in Synthesis Examples 1 to 4 was added for preparation. This was put in a glass tube, and the entire amount was transferred to a new glass tube, and such operation was repeated, and then the solution was developed by electrophoresis. By comparing the quantitative value of RNA with a control, the residual rate of RNA in the solution was compared. That is, the amount of RNA remaining unadsorbed to the inner surface of the glass tube, the microchip, and the like, to which the solution was subjected, was compared. In detail, the following experiment was performed. 1) As the RNA, RNA Ladder (manufactured by NIPPON GENE Co., Ltd.) was used, and was added to the RNA solution described later to a final concentration of 2.5 ng / μL. 2) As the 10X buffer solution, 10X TE (manufactured by Sanko Junya Co., Ltd.) was used. 3) In a DNA LoBind tube (manufactured by Eppendorf Co., Ltd.), each of the RNA solutions shown in Table 2 was prepared. 4) The entire amount of each of the solutions of 3) was transferred to a LABORAN screw tube No. 2 (manufactured by AS ONE Co., Ltd., hereinafter referred to as "glass tube"). 5) The entire amount of each of the solutions of 4) was transferred to a new glass tube. 6) The entire amount of each of the solutions of 5) was transferred to a new glass tube. 7) The entire amount of each of the solutions of 6) was transferred to a new glass tube. 8) The entire amount of each of the solutions of 7) was transferred to a new glass tube. 9) As the electrophoresis buffer solution, a buffer solution (1X TAE) diluted from 50X TAE (manufactured by NIPPON GENE Co., Ltd.) was prepared according to the instruction manual; and as the electrophoresis gel, 1X TAE 100 mL was added in a ratio of 1 g of Agarose S (manufactured by NIPPON GENE Co., Ltd.) to 1X TAE 100 mL, and was boiled and dissolved to prepare a solidified gel. 10) From each of the solutions of 8), 7.5 μL was taken out and transferred to a DNA LoBind tube. 11) To each of the solutions of 10), 7.5 μL of 2X RNA loading buffer solution (without ethidium bromide) (manufactured by FUJIFILM and Otsuka Pharmaceutical Co., Ltd.) was added. 12) Each of the solutions of 11) was heated in a water bath at 70°C for 10 minutes, and was cooled on ice for 1 minute or more directly, and was used as an electrophoresis sample. 13) The sample of 12) was developed with agarose gel electrophoresis. In addition, the sample was applied at 5 μL using the electrophoresis buffer and the gel of 9), and electrophoresis was performed at 100 V for about 30 minutes. 14) Staining of the gel was performed by immersion in Midori Green Advance (manufactured by Genetics, Japan) diluted 10,000 times with IX TAE for about 15 minutes, followed by decolorization by immersion in pure water for about 15 minutes. 15) Imaging was performed with FASdigi Compact (manufactured by Genetics, Japan) under Blue Green LED irradiation, and quantitative values were calculated using ImageJ (National Institutes of Health, USA) for the bands of 6 kbase. The quantitative values were expressed as relative values with the control set to 100. The results were summarized in Table 2.

[0114] [Table 2] *1 Each of the copolymers was added to a final concentration of 0.1% (w / v). *2 The quantitative values were expressed as relative values with the control set to 100.

[0115] Examples 1-1 to 1-3 and Comparative Example 1-1 used each of Polymer Al-1 to Polymer A3-1 or Polymer Z-1 as the single-stranded nucleic acid adsorption inhibitor. From these results, it was found that, by using the polymer of the present application as the single-stranded nucleic acid adsorption inhibitor, the adsorption of RNA on the glass tube, the microchip was inhibited, and the residual amount of RNA was increased.

[0116] [Experimental Example 2] The same experiment as Experimental Example 1 was performed using a polypropylene screw cap tube 2 mL (self-standing) (manufactured by AS ONE Corporation, hereinafter referred to as "PP tube") instead of the glass tube. 1) Each of the RNA aqueous solutions shown in Table 3 was prepared, and was injected into the PP tube. 2) The other conditions and steps were the same as in Experimental Example 1. The results are shown in Table 3.

[0117] [Table 3] *1 Each of the copolymers was added to a final concentration of 0.1% (w / v). *2 The quantitative values were expressed as relative values with the control set to 100.

[0118] Examples 2-1 to 2-3 and Comparative Example 2-1 were each conducted using a PP tube instead of a glass tube to perform the same experiment as Examples 1-1 to 1-3 and Comparative Example 1-1. From the results, it was found that if the polymer of the present application is used as a single-stranded nucleic acid adsorption inhibitor, even when the material of the container is polypropylene, adsorption of single-stranded nucleic acid can be inhibited. Industrial applicability

[0119] By simply adding the single-stranded nucleic acid adsorption inhibitor of the present application to a single-stranded nucleic acid solution in advance, adsorption of the nucleic acid to the components of a container, a microchip, or the like can be inhibited. Therefore, in genetic testing in the fields of medicine, veterinary medicine, and forensic medicine, for example, it is expected that the loss due to adsorption to a container or the like when nucleic acid extraction is performed will be reduced, that is, it is expected that the recovery rate will be improved, and that it is expected that the detection sensitivity will be improved. Furthermore, when a detection sample for genetic testing containing a single-stranded nucleic acid is stored or transported, by inhibiting the decrease in effective concentration due to adsorption to a container or the like, it is expected that the storage stability and the transport stability will be improved. Furthermore, it is expected that the stability during transport and storage of a pharmaceutical or the like containing a single-stranded nucleic acid will be improved.

[0120] This application is based on Japanese Patent Application No. 2023-074988, the contents of which are incorporated herein in their entirety.

Claims

1. A single-chain nucleic acid adsorption inhibitor comprising one or more polymers selected from group A below: Group A Polymer A1: A copolymer containing a structural unit (a) derived from 2-(meth)acryloyloxyethyl phosphocholine and a structural unit (b) derived from a (meth)acrylic acid C2-C6 alkyl ester having two or more hydroxyl groups as substituents; Polymer A2: a copolymer containing the structural unit (a) and a structural unit (c) derived from polyethylene glycol (meth)acrylate; and Polymer A3: A copolymer containing the structural unit (a) and structural units (d) derived from C1-C6 alkyl esters of (meth)acrylic acid having phenyl or phenoxy as substituents.

2. A nucleic acid solution comprising the single-stranded nucleic acid adsorption inhibitor and single-stranded nucleic acid as described in claim 1.

3. A nucleic acid amplification method using the nucleic acid solution described in claim 2.

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