Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F230/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal
  • C08F230/02—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal containing phosphorus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions

Definitions

• the present invention relates to a single-stranded nucleic acid adsorption inhibitor, a nucleic acid solution, and a nucleic acid amplification method.
• RNA ribonucleic acid
• RNA is a biopolymer in which monomeric ribonucleotides are linked together via phosphodiester bonds.
• RNA is naturally composed of ribonucleotides containing the four nucleic acid bases adenine, guanine, cytosine, and uracil, and their derivatives.In the body, RNA has functions such as transmitting genetic information, signal transduction, transporting amino acids in protein synthesis, metabolic control, forming ribozymes, and forming organelles.It also exists as RNA with unknown functions, and forms the genomes of some viruses.
• RNA in society examples include therapeutic targets in the medical field, components of nucleic acid medicines, markers for detecting viruses and microorganisms in infectious disease treatment and environmental surveys, and components of mRNA vaccines.
• Another example of use is the components of genome editing tools in genetic engineering, such as guide RNA for site-specific nucleases such as CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/crispr associated protein 9).
• CRISPR/Cas9 Clustered regularly interspaced short palindromic repeats/crispr associated protein 9
• Yet another example is its use as a raw material for health foods and food additives.
• RNA is often adsorbed to the surfaces of containers, microchips, etc., particularly the surfaces of plastic parts, resulting in a decrease in the concentration of free RNA.
• target RNA is analyzed at extremely low concentrations, ranging from micromolar to attomole or less, and it is often difficult to obtain surplus RNA, so loss of RNA due to adsorption to the inner walls of containers, etc., is a serious problem.
• the same problem occurs with single-stranded DNA.
• Non-Patent Document 1 introduces a plastic tube product that has been specifically modified, and describes how using this product to prepare and store a nucleic acid concentration standard suppresses the adsorption of nucleic acids to the container, improves the nucleic acid yield, and improves the accuracy and sensitivity of applications that use the standard.
• Patent documents 1 and 2 disclose a technique for suppressing nucleic acid adsorption by coating the surface of a container or biochip with a specific coating material, and a technique for suppressing nucleic acid adsorption by forming a layer containing a specific polymeric substance.
• Non-Patent Document 1 the approach of improving plastics as described in Non-Patent Document 1 is inconvenient because it requires all components that will come into contact with nucleic acids to be replaced with components to which the technology has been applied in advance.
• the technologies described in Patent Documents 1 and 2 are inconvenient because it requires the technology to be applied in advance to all components that will come into contact with the nucleic acid.
• the present invention aims to provide a technology for suppressing single-stranded nucleic acid adsorption by an approach that improves the nucleic acid solution. That is, the present invention aims to provide a single-stranded nucleic acid adsorption inhibitor that can be added to a solution of single-stranded nucleic acid to suppress the adsorption of the nucleic acid to a container or component, a nucleic acid solution containing the same, and a nucleic acid amplification method using the nucleic acid solution.
• This approach also has the advantage of allowing the concentration of the additive to be adjusted according to the characteristics of the sample, providing a high degree of flexibility.
• An agent for inhibiting adsorption of single-stranded nucleic acid comprising one or more polymers selected from the following 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 C 2 -C 6 alkyl (meth)acrylate having two or more hydroxy 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 a structural unit (d) derived from a C 1 -C 6 alkyl (meth)acrylate having a phenyl group or a phenoxy group as a substituent.
• the C 2 -C 6 alkyl (meth)acrylate having two or more hydroxy groups as substituents is 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, or sorbitol mono(meth)acrylate; Preferred is glycerin mono(meth)acrylate.
• the agent for inhibiting adsorption of single-stranded nucleic acid according to the above [1] or [2] is more preferably glycerin monomethacrylate.
• the polyethylene glycol (meth)acrylate is polyethylene glycol mono(meth)acrylate, methoxypolyethylene glycol (meth)acrylate, or ethoxypolyethylene glycol (meth)acrylate; Preferred is methoxypolyethylene glycol (meth)acrylate or ethoxypolyethylene glycol (meth)acrylate, More preferably, it is methoxypolyethylene glycol methacrylate or ethoxypolyethylene glycol methacrylate. More preferably, the agent for inhibiting adsorption of single-stranded nucleic acid according to any one of the above [1] to [3] is methoxypolyethylene glycol methacrylate.
• [7] The agent for inhibiting adsorption of single-stranded nucleic acid according to any one of the above [1] to [6], wherein the weight-average molecular weight of polymer A1 is 10,000 to 500,000, preferably 10,000 to 100,000, and more preferably 10,000 to 50,000.
• the weight-average molecular weight of polymer A2 is 50,000 to 1,000,000, preferably 100,000 to 500,000, and more preferably 100,000 to 300,000.
• [10] The agent for inhibiting adsorption of single-stranded nucleic acid according to any one of [1] to [9] above, wherein the ratio of the structural unit (a) to all structural units of polymer A1 is 10 to 70 mol %, preferably 20 to 60 mol %, and more preferably 30 to 50 mol %.
• the proportion of the structural unit (b) relative to all structural units of polymer A1 is 2 to 50 mol %, preferably 5 to 40 mol %, and more preferably 10 to 30 mol %.
• polymer A1 is a copolymer consisting of the structural unit (a), the structural unit (b), and a structural unit (e) derived from another monomer different from 2-(meth)acryloyloxyethyl phosphorylcholine, a C2 - C6 alkyl (meth)acrylate having two or more hydroxyl groups as substituents, polyethylene glycol (meth)acrylate, and a C1- C6 alkyl (meth)acrylate having a phenyl group or a phenoxy group as a substituent.
• the other monomer is C 1 -C 6 alkyl (meth)acrylate, (meth)acrylic acid, or isobornyl (meth)acrylate; Preferably it is a C 1 -C 6 alkyl (meth)acrylate; More preferably, it is butyl (meth)acrylate. More preferably, the agent for inhibiting adsorption of single-stranded nucleic acid according to the above item [12] is butyl methacrylate.
• [15] The agent for inhibiting adsorption of single-stranded nucleic acid according to any one of [1] to [14] above, wherein the ratio of the structural unit (a) to all structural units of polymer A2 is 60 to 95 mol %, preferably 80 to 95 mol %, and more preferably 85 to 95 mol %.
• the proportion of the structural unit (c) relative to all structural units of polymer A2 is 5 to 40 mol %, preferably 5 to 20 mol %, and more preferably 5 to 15 mol %.
• polymer A3 is a copolymer consisting of structural unit (a) and structural unit (d).
• nucleic acid solution comprising the single-stranded nucleic acid adsorption inhibitor according to any one of [1] to [20] above and a single-stranded nucleic acid.
• the nucleic acid solution according to [22] above which is an aqueous solution of nucleic acid.
• nucleic acid solution according to [22] or [23] above, wherein the single-stranded nucleic acid is RNA.
• a method for suppressing adsorption of single-stranded nucleic acid in a nucleic acid solution to a member in contact with the nucleic acid solution comprising mixing a single-stranded nucleic acid adsorption inhibitor according to any one of [1] to [20] above, single-stranded nucleic acid, and a solvent.
• the nucleic acid solution is an aqueous nucleic acid solution
• the solvent is water.
• the method according to [26] or [27] above, wherein the single-stranded nucleic acid is RNA.
• the use of the single-stranded nucleic acid adsorption inhibitor of the present invention can inhibit free single-stranded nucleic acid present in a solution to which it is added from being adsorbed to the surfaces of containers, microchips, etc. Therefore, for example, in various tests in which a sample containing single-stranded nucleic acid is used as a specimen, it is expected to improve the stability during specimen transportation, improve the stability during specimen storage, and improve detection sensitivity. From another perspective, the use of the single-stranded nucleic acid adsorption inhibitor of the present invention can be expected to improve the stability during transportation and storage of pharmaceuticals and other products containing single-stranded nucleic acid.
• single-stranded nucleic acid refers to "RNA or single-stranded DNA.”
• single-stranded nucleic acid adsorption inhibitor refers to an additive used in a solution in order to inhibit the adsorption of nucleic acid to the surface of a component that comes into contact with the solution in a container, microchip, or the like that handles the solution of single-stranded nucleic acid.
• structural unit means a repeating unit in a polymer that is derived from a monomer. Therefore, “structural unit” does not include non-repeating structures in a polymer (for example, structures derived from a polymerization initiator, etc.).
• (meth)acrylate basically means “acrylate or methacrylate.” In cases where multiple (meth)acrylates may be present, "(meth)acrylate” means “acrylate and/or methacrylate.” Other terms similar to “(meth)acrylate” also have the same meaning as “(meth)acrylate.”
• C2 - C6 alkyl (meth)acrylate means “an alkyl (meth)acrylate having an alkyl group with 2 to 6 carbon atoms.”
• Other terms similar to “ C2 - C6 alkyl (meth)acrylate” also have the same meaning as “ C2 - C6 alkyl (meth)acrylate.”
• C 2 -C 6 alkyl (meth)acrylate having two or more hydroxy groups as a substituent means “C 2 -C 6 alkyl (meth)acrylate in which the alkyl group has two or more hydroxy groups as a substituent”.
• polyethylene glycol (meth)acrylate means "an ester of one polyethylene glycol and one (meth)acrylic acid.”
• the end of the “polyethylene glycol (meth)acrylate” opposite the (meth)acrylic acid ester end may be a hydroxy group (e.g., polyethylene glycol mono(meth)acrylate) or a methoxy group (e.g., methoxypolyethylene glycol (meth)acrylate).
• polyethylene glycol mono(meth)acrylate means "a monoester of polyethylene glycol and (meth)acrylic acid, in which the end opposite the (meth)acrylic acid ester end is a hydroxyl group.”
• methoxypolyethylene glycol (meth)acrylate means "a compound in which the terminal hydroxyl group of polyethylene glycol mono(meth)acrylate is replaced with a methoxy group.”
• methoxypolyethylene glycol (meth)acrylate also have the same meaning as “methoxypolyethylene glycol (meth)acrylate.”
• C 1 -C 6 alkyl (meth)acrylate having a phenyl group or a phenoxy group as a substituent means “C 1 -C 6 alkyl (meth)acrylate in which the alkyl group has a phenyl group or a phenoxy group as a substituent.” Note that the number of carbon atoms is the number of carbon atoms in the alkyl group, and does not include the number of carbon atoms in the substituent (phenyl group or phenoxy group).
• the agent for inhibiting adsorption of single-stranded nucleic acid of the present invention contains one or more polymers selected from the following Group A.
• Group A Polymer A1: A copolymer including a structural unit (a) derived from 2-(meth)acryloyloxyethyl phosphorylcholine (hereinafter may be referred to as "monomer a") and a structural unit (b) derived from a C 2 -C 6 alkyl (meth)acrylate having two or more hydroxy groups as substituents (hereinafter may be referred to as "monomer b"); Polymer A2: a copolymer containing the structural unit (a) and a structural unit (c) derived from polyethylene glycol (meth)acrylate (hereinafter may be referred to as "monomer c"); and Polymer A3: a copolymer containing the structural unit (a) and a structural unit
• the monomer a forming the structural unit (a) contained in the polymers A1 to A3 (hereinafter sometimes referred to as the "polymers of the present invention") is 2-methacryloyloxyethyl phosphorylcholine or 2-acryloyloxyethyl phosphorylcholine, and from the viewpoint of the storage stability of the polymers of the present invention, 2-methacryloyloxyethyl phosphorylcholine is preferred.
• Monomer a may be used alone or in combination of two types.
• Monomer a may be a commercially available product or may be produced by a known method.
• Examples of monomer b forming the structural unit (b) contained in polymer A1 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. From the viewpoint of raw material availability and the effect of the single-stranded nucleic acid adsorption inhibitor, glycerin mono(meth)acrylate is preferred, and glycerin monomethacrylate is more preferred.
• glycolin mono(meth)acrylate means “monoester of glycerin and (meth)acrylic acid.”
• Other terms similar to “glycerin mono(meth)acrylate” also have the same meaning as “glycerin mono(meth)acrylate.”
• Monomer b may be used alone or in combination of two or more types.
• Monomer b may be a commercially available product or may be produced by a known method.
• the ratio of structural unit (a) to all structural units of polymer A1 is preferably 10 to 70 mol%, more preferably 20 to 60 mol%, and even more preferably 30 to 50 mol%, from the viewpoint of the effect of inhibiting the adsorption of single-stranded nucleic acid.
• the ratio of structural unit (b) to all structural units of polymer A1 is preferably 2 to 50 mol%, more preferably 5 to 40 mol%, and even more preferably 10 to 30 mol%, from the viewpoint of the effect of inhibiting adsorption of single-stranded nucleic acid.
• the weight-average molecular weight of polymer A1 is not particularly limited, but from the viewpoint of the effect of inhibiting the adsorption of single-stranded nucleic acid, it 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 filtration chromatography using, for example, an EcoSEC system (manufactured by Tosoh Corporation).
• Examples of monomer c forming the structural unit (c) contained in polymer A2 include polyethylene glycol mono(meth)acrylate, methoxypolyethylene glycol (meth)acrylate, ethoxypolyethylene glycol (meth)acrylate, etc.
• methoxypolyethylene glycol (meth)acrylate and ethoxypolyethylene glycol (meth)acrylate are preferred, methoxypolyethylene glycol methacrylate and ethoxypolyethylene glycol methacrylate are more preferred, and methoxypolyethylene glycol methacrylate is even more preferred.
• the number average molecular weight of the polyethylene glycol chain contained in monomer c is preferably 50 to 10,000, more preferably 50 to 5,000, even more preferably 50 to 1,000, and particularly preferably 100 to 1,000, from the viewpoints of raw material availability and the effect of inhibiting adsorption of single-stranded nucleic acid.
• the number average molecular weight of the polyethylene glycol chain can be determined based on the hydroxyl value calculated, for example, by the method described in JIS K 1557, or can be determined in terms of polyethylene glycol by, for example, gel filtration chromatography.
• Monomer c may be used alone or in combination of two or more types.
• Monomer c may be a commercially available product or may be produced by a known method.
• the ratio of structural unit (a) to all structural units of polymer A2 is preferably 60 to 95 mol%, more preferably 80 to 95 mol%, and even more preferably 85 to 95 mol%, from the viewpoint of the effect of inhibiting adsorption of single-stranded nucleic acid.
• the ratio of structural unit (c) to all structural units of polymer A2 is preferably 5 to 40 mol%, more preferably 5 to 20 mol%, and even more preferably 5 to 15 mol%, from the viewpoint of the effect of inhibiting adsorption of single-stranded nucleic acid.
• the weight average molecular weight of polymer A2 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.
• Examples of monomer d forming the structural unit (d) contained in polymer A3 include benzyl (meth)acrylate, 1-phenylethyl (meth)acrylate, 2-phenylethyl (meth)acrylate, and phenoxyethylene glycol (meth)acrylate.
• benzyl (meth)acrylate and 2-phenylethyl (meth)acrylate are preferred, and benzyl (meth)acrylate is more preferred.
• benzyl methacrylate is even more preferred.
• Monomer d may be used alone or in combination of two or more types.
• Monomer d may be a commercially available product or may be produced by a known method.
• the ratio of structural unit (a) to all structural units of polymer A3 is preferably 60 to 95 mol%, more preferably 70 to 90 mol%, and even more preferably 75 to 85 mol%, from the viewpoint of the effect of inhibiting adsorption of single-stranded nucleic acid.
• the ratio of structural unit (d) to all structural units of polymer A3 is preferably 5 to 40 mol%, more preferably 10 to 30 mol%, and even more preferably 15 to 25 mol%, from the viewpoint of the effect of inhibiting adsorption of single-stranded nucleic acid.
• the weight average molecular weight of polymer A3 is not particularly limited, but is preferably 5,000 to 2,000,000, more preferably 50,000 to 1,000,000, and even more preferably 100,000 to 500,000.
• copolymers containing structural unit (b) are classified as polymer A1, not polymer A2 or A3.
• the ratio of the structural unit (c) to all structural units of polymer A1 is preferably 30 mol% or less, more preferably 20 mol% or less, and even more preferably 10 mol% or less. It is particularly preferable that polymer A1 does not contain structural unit (c).
• the ratio of the structural unit (d) to all structural units of polymer A1 is preferably 30 mol% or less, more preferably 20 mol% or less, and even more preferably 10 mol% or less. It is particularly preferable that polymer A1 does not contain structural unit (d).
• copolymers containing structural unit (c) (excluding copolymers containing structural unit (b)) (for example, copolymers containing structural units (a), (c) and (d)) are classified as polymer A2, not polymer A3.
• the ratio of the structural unit (d) to all structural units of polymer A2 is preferably 30 mol% or less, more preferably 20 mol% or less, and even more preferably 10 mol% or less. It is particularly preferable that polymer A2 does not contain structural unit (d).
• Polymers A1 to A3 may contain a structural unit (e) derived from a monomer other than monomers a to d, as long as the effect of the present invention is not impaired.
• Examples of the other monomers include C 1 to C 6 alkyl (meth)acrylates, (meth)acrylic acid, isobornyl (meth)acrylate, etc. Among these, C 1 to C 6 alkyl (meth)acrylates are preferred, butyl (meth)acrylate is more preferred, and butyl methacrylate is even more preferred.
• the other monomers may be used alone or in combination of two or more.
• the other monomers may be commercially available products or may be produced by known methods.
• the ratio of structural unit (e) to all structural units of polymer A1 is preferably 10 to 60 mol%, more preferably 20 to 50 mol%, and even more preferably 30 to 45 mol%.
• the ratio of the structural unit (e) to all structural units of polymer A2 or polymer A3 is preferably 30 mol % or less, more preferably 20 mol % or less, and even more preferably 10 mol % or less. It is particularly preferable that neither polymer A2 nor polymer A3 contains the structural unit (e).
• Polymer A1 is preferably a copolymer consisting of structural units (a), (b), and (e).
• a copolymer consisting of structural units (a), (b), and (e) means a copolymer in which all of the structural units (repeating units) in the copolymer consist of structural units (a), (b), and (e).
• Other terms similar to "a copolymer consisting of structural units (a), (b), and (e)” also have the same meaning as “a copolymer consisting of structural units (a), (b), and (e)”.
• the polymer A1 more preferably comprises one or two structural units (a) derived from 2-(meth)acryloyloxyethyl phosphorylcholine, (b) one or more structural units derived from 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, or sorbitol mono(meth)acrylate; and (e) one or more structural units derived from a C 1 -C 6 alkyl(meth)acrylate. It is a copolymer consisting of:
• the polymer A1 further preferably comprises one or two structural units (a) derived from 2-(meth)acryloyloxyethyl phosphorylcholine, one or two structural units (b) derived from glycerin mono(meth)acrylate, and one or two structural units (e) derived from butyl (meth)acrylate. It is a copolymer consisting of:
• the polymer A1 is particularly preferably A structural unit (a) derived from 2-methacryloyloxyethyl phosphorylcholine, A structural unit (b) derived from glycerin monomethacrylate, and a structural unit (e) derived from butyl methacrylate. It is a copolymer consisting of:
• Polymer A2 is preferably a copolymer consisting of structural units (a) and (c).
• Polymer A2 more preferably comprises one or two types of structural units (a) derived from 2-(meth)acryloyloxyethyl phosphorylcholine, and one or more types of structural units (c) derived from polyethylene glycol mono(meth)acrylate, methoxypolyethylene glycol (meth)acrylate, or ethoxypolyethylene glycol (meth)acrylate. It is a copolymer consisting of:
• the polymer A2 further preferably comprises one or two structural units (a) derived from 2-(meth)acryloyloxyethyl phosphorylcholine, and one or more structural units (c) derived from methoxypolyethylene glycol (meth)acrylate or ethoxypolyethylene glycol (meth)acrylate. It is a copolymer consisting of:
• the polymer A2 particularly preferably comprises a structural unit (a) derived from 2-methacryloyloxyethyl phosphorylcholine, and one or two structural units (c) derived from methoxypolyethylene glycol methacrylate or ethoxypolyethylene glycol methacrylate. It is a copolymer consisting of:
• the polymer A2 most preferably comprises a structural unit (a) derived from 2-methacryloyloxyethyl phosphorylcholine, and a structural unit (c) derived from methoxypolyethylene glycol methacrylate. It is a copolymer consisting of:
• Polymer A3 is preferably a copolymer consisting of structural units (a) and (d).
• Polymer A3 more preferably contains one or two structural units (a) derived from 2-(meth)acryloyloxyethyl phosphorylcholine, and one or more structural units (d) derived from benzyl (meth)acrylate, 1-phenylethyl (meth)acrylate, 2-phenylethyl (meth)acrylate, or phenoxyethylene glycol (meth)acrylate. It is a copolymer consisting of:
• Polymer A3 more preferably contains one or two structural units (a) derived from 2-(meth)acryloyloxyethyl phosphorylcholine, and one or more structural units (d) derived from benzyl (meth)acrylate, 1-phenylethyl (meth)acrylate, or 2-phenylethyl (meth)acrylate. It is a copolymer consisting of:
• Polymer A3 particularly preferably comprises one or two structural units (a) derived from 2-(meth)acryloyloxyethyl phosphorylcholine, and one or two structural units (d) derived from benzyl (meth)acrylate. It is a copolymer consisting of:
• the polymer A3 most preferably comprises a structural unit (a) derived from 2-methacryloyloxyethyl phosphorylcholine, and a structural unit (d) derived from benzyl methacrylate. It is a copolymer consisting of:
• the polymer of the present invention may be a random copolymer, an alternating copolymer, a block copolymer, a graft polymer, or a copolymer having two or more of these structures, but from the viewpoint of manufacturability of the polymer of the present invention, a random copolymer is preferred.
• Polymers A1 to A3 can be produced by known methods (e.g., the method described in WO 2018/216628).
• the nucleic acid adsorption inhibitor of the present invention can contain components other than the polymers A1 to A3 to the extent that the nucleic acid adsorption inhibitory effect of the polymers A1 to A3 is not impaired.
• the other components are not particularly limited, but may be appropriately selected from those exemplified as "other components" in the nucleic acid solution of the present invention described below.
• Polymers A1 to A3 can be easily used as single-stranded nucleic acid adsorption inhibitors by being contained in a single-stranded nucleic acid solution.
• Possible methods for incorporating the polymer of the present invention into a nucleic acid solution include adding polymers A1 to A3 to a prepared single-stranded nucleic acid solution and dissolving them, dissolving the polymer of the present invention in advance in a solvent such as a buffer that dissolves single-stranded nucleic acid, and placing the polymer of the present invention in advance in a container for preparing the single-stranded nucleic acid solution, and then adding the single-stranded nucleic acid solution thereto and dissolving it.
• a solvent such as a buffer that dissolves single-stranded nucleic acid
• the concentration (final concentration) of the nucleic acid adsorption inhibitor of the present invention added to the single-stranded nucleic acid solution is preferably 0.01 to 5 w/v%, more preferably 0.1 to 1 w/v%, and even more preferably 0.1 to 0.5 w/v%. If the amount added is too small, the effect of inhibiting single-stranded nucleic acid adsorption may not be obtained, and if the amount added is too large, problems such as reaction inhibition may occur when the single-stranded nucleic acid solution is used in applications such as enzyme reactions.
• the single-stranded nucleic acid to which the single-stranded nucleic acid adsorption inhibitor of the present invention can be applied may be either RNA or single-stranded DNA, and is preferably RNA.
• the single-stranded nucleic acid may be artificially synthesized by chemical synthesis, in vitro synthesis (e.g., reverse transcription reaction), PCR, etc., or may be prepared by a known method from cells, microorganisms, viruses, etc.
• 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.
• the present invention further provides a nucleic acid solution containing the agent for inhibiting adsorption of single-stranded nucleic acid of the present invention and a single-stranded nucleic acid.
• the concentration (final concentration) of the single-stranded nucleic acid adsorption inhibitor in the nucleic acid solution of the present invention is preferably 0.01 to 5 w/v%, more preferably 0.1 to 1 w/v%, and even more preferably 0.1 to 0.5 w/v%.
• the single-stranded nucleic acid contained in the nucleic acid solution of the present invention may be either RNA or single-stranded DNA, with RNA being preferred.
• the single-stranded nucleic acid may be, for example, chemically synthesized, in vitro synthesized (e.g., reverse transcription reaction, solid-phase synthesis, etc.), artificially synthesized by PCR or the like (e.g., RNA, complementary DNA, etc.), or may be provided as a virus, bacterial body, cell, body fluid, tissue, etc., or a suspension thereof, or a nucleic acid extract prepared from these.
• the virus, bacterial body, cell, body fluid, tissue, etc. may be collected from the natural world or environment, humans, animals, or plants, or may be isolated and cultured.
• the single-stranded nucleic acid may be a single-stranded nucleic acid present in a living body, such as messenger RNA, transfer RNA, ribosomal RNA, non-coding RNA, microRNA, ribozyme, single-stranded genomic RNA, or single-stranded genomic DNA, or may be a single-stranded nucleic acid having a primary structure equivalent to or complementary to the entire or a part of the single-stranded nucleic acid, or may be a single-stranded nucleic acid having a completely artificially designed primary structure.
• the concentration of the single-stranded nucleic acid is appropriately determined depending on the application in which the nucleic acid is used.
• the nucleic acid solution of the present invention may contain other components to the extent that they do not impair the effects of the present invention.
• Other components include, for example, polyols, polyethers, proteins, salts, buffer solutions, surfactants, solvents, biochemical reagents, dyes, preservatives, oils, solid phase carriers, etc.
• polyols examples include glycerol, sucrose, glucose, and the like.
• polyether is polyoxyethylene glycol.
• proteins include albumin, gelatin, casein, enzymes, and the like.
• salts include alkali metal salts, alkaline earth metal salts, salts of amino acids, salts of peptides, and salts of organic acids such as ethylenediaminetetraacetic acid.
• buffer solution include Tris-HCl buffer, Good's buffer, glycine buffer, borate buffer, TE buffer, TAE buffer, TBE buffer, SSC buffer and the like.
• surfactant include polyoxyethylene alkyl ether, polyoxyethylene sorbitan monoalkyl ether, and alkyl betaine.
• the solvent may be water or an organic solvent.
• the organic solvent include ethanol, propanol, isoamyl alcohol, glycerin, dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, chloroform, and phenol.
• the solvent is preferably water, and the nucleic acid solution of the present invention is preferably an aqueous nucleic acid solution.
• the aqueous nucleic acid solution may further contain an organic solvent.
• the biochemical reagents include, for example, flavins.
• dyes include nucleic acid staining reagents such as ethidium bromide and SYBR TM Green I, fluorescent dyes such as ROX TM Dye, and colorants such as Orange G, bromophenol blue, and xylene cyanol FF.
• preservatives include sodium azide, paraoxybenzoic acid preparations, dehydroacetic acid preparations, and Proclin preparations.
• the oil may, for example, be mineral oil.
• the solid phase carrier include silica beads and magnetic beads.
• nucleic acid amplification method The nucleic acid solution of the present invention can be subjected to an enzyme reaction while still containing the agent for inhibiting adsorption of single-stranded nucleic acid of the present invention.
• enzymatic reactions include cleavage by nuclease, reverse transcription by reverse transcriptase, replication by DNA polymerase, and reactions that utilize these reactions for nucleic acid amplification.
• the enzymatic reaction referred to in the present invention is preferably replication of single-stranded DNA or RNA, and more preferably reverse transcription PCR.
• the present invention provides a method for suppressing the adsorption of single-stranded nucleic acid in a nucleic acid solution to a member in contact with the nucleic acid solution, comprising mixing the single-stranded nucleic acid adsorption inhibitor of the present invention, single-stranded nucleic acid, and a solvent.
• the mixing there is no particular limitation on the mixing, and for example, (1) the single-stranded nucleic acid adsorption inhibitor of the present invention, single-stranded nucleic acid, and a solvent may be mixed, (2) a solution containing single-stranded nucleic acid and a solvent may be mixed with the single-stranded nucleic acid adsorption inhibitor of the present invention, or (3) a solution containing single-stranded nucleic acid adsorption inhibitor of the present invention and a solvent may be mixed with single-stranded nucleic acid.
• the description of the nucleic acid solution obtained by mixing the single-stranded nucleic acid adsorption inhibitor of the present invention, single-stranded nucleic acid, and a solvent is the same as the description of the nucleic acid solution of the present invention described above.
• MPC 2-methacryloyloxyethyl phosphorylcholine
• GLM glycerin monomethacrylate
• BMA butyl methacrylate
• This solution was heated to 60°C, and 1.9 g of 2,2'-azobis(isobutyronitrile) (hereinafter referred to as "AIBN”) was added under a nitrogen atmosphere, and polymerization was carried out by stirring for 5 hours.
• AIBN 2,2'-azobis(isobutyronitrile)
• the obtained polymerization solution was purified by dialysis using a semipermeable membrane with a molecular weight cutoff of 3,000, and then freeze-dried to obtain polymer A1-1.
• the weight average molecular weight of polymer A1-1 was 21,000 in terms of polyethylene glycol, as determined by GPC measurement under the conditions described below.
• Polymer A2-1 which corresponds to Polymer A2, was prepared in the following manner.
• the obtained polymerization solution was purified by dialysis using a semipermeable membrane with a molecular weight cutoff of 20,000, and then freeze-dried to obtain polymer A2-1.
• the weight average molecular weight of polymer A2-1 was 133,000 in terms of polyethylene glycol, as determined by GPC measurement under the conditions described below.
• BzMA benzyl methacrylate
• EtOH ethanol
• the obtained polymerization solution was purified by dialysis using a semipermeable membrane with a molecular weight cutoff of 3,000, and then freeze-dried to obtain polymer A3-1.
• the weight average molecular weight of polymer A3-1 was 240,000 in terms of polyethylene glycol, as determined by GPC measurement under the conditions described below.
• RNA Ladder manufactured by Nippon Gene Co., Ltd.
• 10X TE manufactured by Dojindo Laboratories, Ltd.
• Each RNA solution shown in Table 2 was prepared in a DNA LoBind Tube (Eppendorf).
• the entire amount of each solution in 3) was transferred to a Labolan screw cap vial No. 2 (manufactured by AS ONE Corporation, hereinafter referred to as "glass tube”).
• glass tube 5) The entire amount of each solution in 4) was transferred to a new glass tube.
• the electrophoresis buffer and gel from 9) were the same as those used in 12), the amount of sample applied was 5 ⁇ L, and the electrophoresis was performed at 100 V for about 30 minutes. 14)
• the gel was stained by immersing it in Midori Green Advance (manufactured by Nippon Genetics Co., Ltd.) diluted 10,000-fold with 1 ⁇ TAE for about 15 minutes, and then destained by immersing it in pure water for about 15 minutes.
• Images were taken using FASdigi Compact (manufactured by Nippon Genetics Co., Ltd.) under BlueGreen LED irradiation, and the quantitative values of the 6 kbase band were calculated using ImageJ (National Institutes of Health, USA). The quantitative values were expressed as relative values with the control set at 100. The results are summarized in Table 2.
• Example 1-1 to 1-3 and Comparative Example 1-1 Polymer A1-1 to Polymer A3-1 or Polymer Z-1 were used as single-stranded nucleic acid adsorption inhibitors, respectively. These results show that by using the polymer of the present invention as a single-stranded nucleic acid adsorption inhibitor, RNA adsorption to glass tubes and microchips is suppressed and the amount of remaining RNA is increased.
• Example 2 An experiment similar to that in Experimental Example 1 was carried out using a 2 mL polypropylene screw cap tube (self-supporting type) (manufactured by AS ONE Corporation, hereinafter referred to as "PP tube") instead of a glass tube. 1) Each aqueous RNA solution shown in Table 3 was prepared and dispensed into PP tubes. 2) Other conditions and procedures were the same as in Experimental Example 1. The results are shown in Table 3.
• Examples 2-1 to 2-3 and Comparative Example 2-1 the same experiments as in Examples 1-1 to 1-3 and Comparative Example 1-1 were carried out, respectively, using PP tubes instead of glass tubes. These results show that by using the polymer of the present invention as an agent for inhibiting single-stranded nucleic acid adsorption, inhibition of single-stranded nucleic acid adsorption can be achieved even when the container material is polypropylene.
• single-stranded nucleic acid adsorption inhibitor of the present invention can inhibit the nucleic acid from adsorbing to components such as containers and microchips. Therefore, for example, in genetic testing in the fields of medical care, veterinary medicine, and forensic medicine, it is expected that loss due to adsorption to containers, etc. during nucleic acid extraction can be reduced, i.e., recovery rates can be improved, and detection sensitivity can be improved. Furthermore, by inhibiting the decrease in effective concentration due to adsorption to containers, etc. during storage and transportation of specimens for genetic testing containing single-stranded nucleic acid, it is expected that storage stability and transportation stability can be improved. It is also expected that the stability of pharmaceuticals containing single-stranded nucleic acid during transportation and storage can be improved.

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