Nucleic acid for transfection

Abstract

Provided are: a nucleic acid for transfection having excellent cell membrane permeability; a method for producing the nucleic acid for transfection; and a transfection method. The present invention is a nucleic acid for transfection in which a target nucleic acid introduced into a cell and a cell membrane-permeable group are linked, the cell membrane-permeable group containing a perfluoroalkyl group having 2-10 carbon atoms, the perfluoroalkyl group optionally having 1-5 ether-linkable oxygen atoms between carbon atoms, and a method for transfecting a nucleic acid, the method for transfecting the nucleic acid involves bringing the nucleic acid for transfection into contact with a cell and introducing the nucleic acid into the cell.

Description

Technical Field

[0001] The present invention relates to a nucleic acid for transfection having excellent cell membrane permeability and a method for producing the same.

[0002] This application claims priority based on Japanese Patent Application No. 2022-130213 filed in Japan on August 17, 2022, the contents of which are incorporated herein by reference. Background Art

[0003] In recent years, the nucleic acid medicine using oligonucleotides has been studied. Nucleic acid medicine has the advantages of high specificity to target molecules and few side effects. However, the cell membrane permeability of nucleic acid medicine is low, and it is difficult to reach the target molecule present in the cell. In particular, since siRNA is double-stranded, the molecular weight and negative charge are both large compared with antisense RNA, and the cell membrane permeability is lower than antisense RNA, and it is necessary to utilize the drug delivery of a carrier. As a drug delivery agent, it is known to have a delivery agent (patent document 1) using lipid nanoparticles and a delivery agent (patent document 2) using cationic polymer nanoparticles. However, from the efficiency of cell membrane permeability and the aspect of worrying about toxicity, most of them should be improved.

[0004] On the other hand, it is known that compounds having a polyfluorinated structure are stable in vivo and have low toxicity, and are excellent in uptake into cells and release from endosomes (Non-patent Document 1). Studies have also been conducted on utilizing this property to introduce a polyfluorinated structure into oligonucleotides or peptide nucleic acids as a portion having cell membrane permeability (Patent Documents 3 and 4, Non-patent Documents 2 to 5).

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: International Publication No. 2011 / 036557

[0008] Patent Document 2: International Publication No. 2017 / 212006

[0009] Patent Document 3: International Publication No. 2012 / 130941

[0010] Patent Document 4: International Publication No. 2021 / 060506

[0011] Patent Document 5: Japanese Patent Application Publication No. 2006-321797

[0012] Patent Document 6: International Publication No. 2000 / 056694

[0013] Non-patent literature

[0014] Non-patent literature 1: Zhang et al., MRS Communications, 2018, vol. 8, p. 303-313.

[0015] Non-patent document 2: Godeau et al., Medicinal Chemistry Communications, 2010, vol. 1. p. 76-78.

[0016] Non-patent document 3: Ellipilli et al., Chemical Communications, 2016, vol. 52, p. 521-524.

[0017] Non-patent document 4: Rochambeaua et al., Polymer Chemistry, 2016, vol. 7, p. 4998-5003.

[0018] Non-patent document 5: Metelev et al., Theranostics, 2017, vol. 7, p. 3354-3368. Summary of the invention

[0019] The difficulty of the nucleic acid to which the cell membrane permeable group is bonded described in Non-Patent Document 2 is that it cannot be easily synthesized by the phosphoramidite method.

[0020] Non-patent document 3 describes a peptide nucleic acid that is endowed with excellent cell membrane permeability by utilizing a polyfluorinated structure, but does not describe nucleic acids.

[0021] Non-patent document 4 describes nucleic acids to which a polyfluorinated structure is added, but does not involve cell membrane permeability, and a transfection reagent is used separately for intracellular introduction.

[0022] Non-patent document 5 describes nucleic acids to which a perfluorinated structure is added, but further improvement in cell membrane permeability is desired.

[0023] An object of the present invention is to provide a nucleic acid for transfection having excellent cell membrane permeability and a method for producing the same.

[0024] The present inventors have found that by introducing a perfluoroalkyl group into a target nucleic acid to be introduced into cells, the cell membrane permeability of the nucleic acid is improved, and the nucleic acid can be introduced into cells without using a transfection reagent, thereby completing the present invention.

[0025] That is, the present invention is as follows.

[0026] [1] A nucleic acid for transfection, comprising a nucleic acid to be introduced into a cell and a cell membrane permeable group linked thereto,

[0027] The cell membrane permeable group contains a perfluoroalkyl group having 1 to 10 carbon atoms.

[0028] The perfluoroalkyl group may have 1 to 5 ether-bonded oxygen atoms between carbon atoms.

[0029] [2] The nucleic acid for transfection according to [1] above, wherein the cell membrane permeable group has a structure represented by the following general formula (A1):

[0030]

[0031] [Where R FE is a perfluoroalkyl group having 1 to 10 carbon atoms without an ether-bonded oxygen atom between carbon atoms, or a perfluoroalkyl group having 2 to 10 carbon atoms with 1 to 5 ether-bonded oxygen atoms between carbon atoms, na is an integer of 1 to 10, and a black dot represents a bonding site].

[0032] [3] The nucleic acid for transfection according to [1] above, wherein the cell membrane permeable group has a structure represented by the following general formula (A2):

[0033]

[0034] [Where R FE is a perfluoroalkyl group having 1 to 10 carbon atoms without an ether-bonded oxygen atom between carbon atoms, or a perfluoroalkyl group having 2 to 10 carbon atoms with 1 to 5 ether-bonded oxygen atoms between carbon atoms, na is an integer of 1 to 10, and a black dot represents a bonding site].

[0035] [4] The nucleic acid for transfection according to [3] above, which has a structure in which two or more structures represented by the general formula (A2) are connected in series.

[0036] [5] A method for producing a nucleic acid for transfection, comprising: bonding a compound represented by the following general formula (A2-0) to a target nucleic acid to be introduced into a cell by a phosphoramidite method, thereby synthesizing a nucleic acid for transfection,

[0037]

[0038] [Where R FE is a perfluoroalkyl group having 1 to 10 carbon atoms without an ether-bonded oxygen atom between carbon atoms, or a perfluoroalkyl group having 2 to 10 carbon atoms with 1 to 5 ether-bonded oxygen atoms between carbon atoms, na is an integer of 1 to 10, DMTr is 4,4'-dimethoxytriphenylmethyl, and i-Pr is isopropyl].

[0039] [6] The method for producing a nucleic acid for transfection according to [5] above, wherein two or more compounds represented by the general formula (A2-0) are bonded in series to the nucleic acid.

[0040] [7] A method for transfection of a nucleic acid, comprising bringing the nucleic acid for transfection according to any one of [1] to [4] above into contact with a cell for introduction.

[0041] The nucleic acid for transfection according to the present invention has excellent cell membrane permeability due to the introduction of a perfluoroalkyl group, so the nucleic acid for transfection can be introduced into cells without using other transfection reagents in combination.

[0042] According to the production method of the present invention, the nucleic acid for transfection can be produced. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a graph showing the results of measuring the mean fluorescence intensity at each culture time of cells that have taken up the fluorine-added nucleic acid (PFC-ssDNA-FAM) modified with N[C8-PFC] at the 5' end and the control nucleic acid (ssDNA-FAM) in Example 1.

[0044] Figure 2 The images are fluorescent staining images of cells into which the fluorine-added nucleic acid (PFC-ssDNA-FAM) was introduced in Example 1 (left), and fluorescent staining images of cells into which the control nucleic acid (ssDNA-FAM) was introduced using Lipofectamine (right). DETAILED DESCRIPTION

[0045] In the present invention and the specification of this application, "nucleic acid" refers to a molecule formed by phosphodiester bonding of nucleotides. The nucleotides include not only natural nucleotides (naturally occurring nucleotides) such as DNA and RNA, but also artificial nucleotides that are modified from natural nucleotides and can form phosphodiester bonds with natural nucleotides. Examples of artificial nucleotides include artificial nucleotides in which the side chains of natural nucleotides are modified with functional groups such as amino groups, artificial nucleotides in which the hydroxyl group at the 2'-position of the ribose backbone is substituted with a methoxy group, a fluoro group, a methoxyethyl group, or the like, phosphorothioate nucleotides (nucleotides in which the oxygen atom of the phosphate group is substituted with a sulfur atom), morpholino nucleotides (nucleotides in which ribose or deoxyribose is substituted with a morpholine ring), BNA (Bridged Nucleic Acid), HNA (Hexitol Nucleic Acid), LNA (Locked Nucleic Acid), PNA (Peptide Nucleic Acid), TNA (Threose Nucleic Acid), GNA (Glycerol Nucleic Acid), CeNA (Cyclohexenyl Nucleic Acid), and the like. In addition, "nucleic acid" also includes any of the following molecules: molecules such as DNA and RNA that are composed of only one or more natural nucleotides bonded by phosphodiester bonds, molecules that are composed of one or more natural nucleotides and one or more artificial nucleotides bonded by phosphodiester bonds, and molecules that are composed of only one or more artificial nucleotides bonded by phosphodiester bonds.

[0046] In the present invention and the specification of this application, “C p1-p2 "(p1 and p2 are positive integers satisfying p1<p2) refers to a group having a carbon atom number of p1~p2.

[0047] In the present invention and the specification of this application, “C 1-10 "Alkyl" refers to an alkyl group having 1 to 10 carbon atoms, which may be a straight chain or a branched chain. 2-10 "Alkyl" refers to an alkyl group having 2 to 10 carbon atoms, which may be a straight chain or a branched chain. 1-10 Examples of the alkyl group include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl and decyl.

[0048] In the present invention and the specification of this application, “C 1-6 "Alkyl" refers to an alkyl group having 1 to 6 carbon atoms, which may be a straight chain or a branched chain. 1-6Examples of the alkyl group include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl and hexyl.

[0049] In the present invention and the specification of this application, an "alkylene group" is a divalent group obtained by removing two hydrogen atoms from a saturated hydrocarbon, and may be a linear or branched group. Examples of the alkylene group include methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, nonamethylene, methylmethylene, ethylmethylene, methylethylene, methylpropylene, ethylethylene, dimethylmethylene, 1,2-dimethylethylene, 1,1-dimethylethylene, 1-ethylpropylene, 2-ethylpropylene, 1,2-dimethylpropylene, 2,2-dimethylpropylene, 1-propylpropylene, 2-propylpropylene, 1-methyl-1-ethylpropylene, 1-methyl-2-ethyl-propylene, 1-ethyl-2-methyl-propylene, 2-methyl-2-ethyl-propylene, 1-methylbutylene, 2-methylbutylene, 3-methylbutylene, 2-ethylbutylene, 1-methylpentylene, 2-ethylpentylene, and 1-methylhexylene.

[0050] In the present invention and the specification of this application, “C 1-10 A "perfluoroalkyl group" is a group in which all hydrogen atoms of an alkyl group having 1 to 10 carbon atoms are substituted with fluorine atoms. 1-10 Examples of the perfluoroalkyl group include perfluoromethyl, perfluoroethyl, perfluoropropyl, perfluoroisopropyl, perfluorobutyl, perfluoroisobutyl, perfluorosec-butyl, perfluorotert-butyl, perfluoropentyl, perfluoroisopentyl, perfluoroneopentyl, perfluorotert-pentyl, perfluorohexyl, perfluoroheptyl, perfluorooctyl, perfluorononyl, and perfluorodecyl.

[0051] In the present invention and the specification of the present application, "perfluoroalkylene" is a group in which all hydrogen atoms of an alkylene group are substituted with fluorine atoms. Examples of perfluoroalkylene include groups in which all hydrogen atoms of the alkylene group listed above are substituted with fluorine atoms.

[0052] In the present invention and the specification of this application, "ether-bonded oxygen atoms" refer to oxygen atoms connecting carbon atoms, and do not include oxygen atoms connected in series. An alkyl group having Nc carbon atoms (Nc is an integer greater than 2) may have a maximum of Nc-1 ether-bonded oxygen atoms. 2-10 "Alkyl" refers to 2-10 A group having at least one ether-bonded oxygen atom between carbon atoms of the alkyl group. Hereinafter, this may be referred to as an "alkyl group containing an ether bond".

[0053] In the present invention and the specification of this application, "C 2-10"Perfluoroalkyl" refers to 2-10 The alkyl group has at least one ether-bonded oxygen atom between carbon atoms. 2-10 A group in which all hydrogen atoms of an alkyl group are substituted with fluorine atoms. Hereinafter, a "perfluoroalkyl group having an ether-bonded oxygen atom between carbon atoms" may be referred to as an "ether-bonded perfluoroalkyl group".

[0054] In addition, hereinafter, "compound n" means a compound represented by formula (n).

[0055] The nucleic acid for transfection of the present invention is a nucleic acid having a target nucleic acid (target nucleic acid for introduction) to be introduced into a cell and a cell membrane permeable group connected thereto. The target nucleic acid for introduction generally has low cell membrane permeability, but by connecting it to a cell membrane permeable group, the cell membrane permeability can be significantly improved. Therefore, the nucleic acid for transfection of the present invention can be introduced into a cell by contacting the surface of a cell without using other transfection reagents.

[0056] In the present invention and the specification of this application, a "cell membrane permeable group" refers to a group that can improve the cell membrane permeability of a nucleic acid by adding to the nucleic acid. Specifically, a perfluoroalkyl group (C 1-10 Since the perfluoroalkyl group itself has excellent cell membrane permeability, the cell membrane permeability of the nucleic acid can be improved by adding a group containing a perfluoroalkyl group to the nucleic acid.

[0057] The perfluoroalkyl group possessed by the cell membrane permeable group may have 1 to 5 ether-bonded oxygen atoms between carbon atoms. 1-10 A perfluoroalkyl group, or a perfluoroalkyl group having 2 to 10 carbon atoms and having 1 to 5 ether-bonded oxygen atoms between carbon atoms (C 2-10 perfluoroalkyl).

[0058] As a C having no ether bond oxygen atom between carbon atoms 1-10 Perfluoroalkyl groups can be straight-chain or branched. The larger the number of carbon atoms in a perfluoroalkyl group, the higher the cell membrane permeability. However, if the number of carbon atoms is too large, it may cause cytotoxicity. 1-10 The perfluoroalkyl group can exert sufficient cell membrane permeability and can suppress toxicity to cells. As the cell membrane permeable group possessed by the nucleic acid for transfection according to the present invention, a linear C 2-10 Perfluoroalkyl, preferably linear C 3-10 Perfluoroalkyl, preferably linear C 4-10 Perfluoroalkyl.

[0059] As C containing ether-bonded oxygen 2-10The perfluoroalkyl group is preferably a group represented by any one of the following general formulae (F1) to (F3), for example.

[0060] -R F1 -OR F2 (F1)

[0061] -R F3 -(O-CF2-CF2-)n1-OR F4 (F2)

[0062] -R F5 -(O-CF2-CF2-CF2-)n2-OR F6 (F3)

[0063] In the general formula (F1), R F1 is a perfluoroalkylene group having q1 carbon atoms, R F2 The perfluoroalkyl group has q2 carbon atoms. The sum of q1 and q2 is a natural number of 2 to 10.

[0064] In the general formula (F2), R F3 is a perfluoroalkylene group having q3 carbon atoms, R F4 It is a perfluoroalkyl group having q4 carbon atoms. n1, q3 and q4 are natural numbers (2×n1+q3+q4) of 4 to 10.

[0065] In the general formula (F3), R F5 is a perfluoroalkylene group having q5 carbon atoms, R F6 It is a perfluoroalkyl group having q6 carbon atoms. n2, q5 and q6 are natural numbers of 5 to 10 (3×n2+q5+q6).

[0066] As R in the general formula (F1) F1 , R in the general formula (F2) F3 and R in the general formula (F3) F5 , may be a linear perfluoroalkylene group or a branched perfluoroalkylene group. F1 , R F3 and R F5 , preferably C 1-3 The perfluoroalkylene group is more preferably a group in which all the hydrogen atoms of a methylene group, an ethylene group, a methylmethylene group, an ethylmethylene group, a dimethylmethylene group or a methylethylene group are substituted with fluorine atoms; further preferably a group in which all the hydrogen atoms of a methylene group, a methylmethylene group, an ethylmethylene group or a dimethylmethylene group are substituted with fluorine atoms; further preferably a group in which all the hydrogen atoms of a methylene group or a methylmethylene group are substituted with fluorine atoms.

[0067] As R in the general formula (F1) F2 , R in the general formula (F2) F4and R in the general formula (F3) F6 , may be a linear perfluoroalkyl group or a branched perfluoroalkyl group. F2 , R F4 and R F6 , preferably a linear or branched C 1-5 Perfluoroalkyl, more preferably linear C 1-5 Perfluoroalkyl, preferably linear C 2-4 Perfluoroalkyl.

[0068] As a C having ether-bonded oxygen in the cell membrane permeable group possessed by the nucleic acid for transfection according to the present invention, 2-10 Perfluoroalkyl, preferably R in the general formula (F1) F1 is a methylene group or a methylmethylene group in which all hydrogen atoms are replaced by fluorine atoms, R F2 A straight chain C 1-5 A perfluoroalkyl group; R in the general formula (F2) F3 is a methylene group or a group in which all hydrogen atoms of a methylmethylene group are replaced by fluorine atoms, n1 is 1, 2 or 3, R F4 A straight chain C 1-7 A perfluoroalkyl group; R in the general formula (F3) F5 is a methylene group or a group in which all hydrogen atoms of a methylmethylene group are replaced by fluorine atoms, n2 is 1 or 2, R F6 A straight chain C 1-6 A perfluoroalkyl group; more preferably R in the general formula (F1) F1 is a methylene group or a group in which all hydrogen atoms of a methylmethylene group are replaced by fluorine atoms, R F2 A straight chain C 1-5 A perfluoroalkyl group; R in the general formula (F2) F3 is a methylene group or a methylmethylene group in which all hydrogen atoms are replaced by fluorine atoms, n1 is 1, R F4 A straight chain C 1-6 A perfluoroalkyl group; R in the general formula (F2) F3 is a methylene group or a methylmethylene group in which all hydrogen atoms are replaced by fluorine atoms, n1 is 2, R F4 A straight chain C 1-4 A perfluoroalkyl group; R in the general formula (F2) F3 is a methylene group or a methylmethylene group in which all hydrogen atoms are replaced by fluorine atoms, n1 is 3, R F4 A straight chain C 1-3 A perfluoroalkyl group; R in the general formula (F3) F5 is a methylene group or a methylmethylene group in which all hydrogen atoms are replaced by fluorine atoms, n2 is 1, R F6 A straight chain C1-6 Perfluoroalkyl group.

[0069] In the present invention, as the ether bond-containing perfluoroalkyl group introduced into the nucleic acid, for example, the ether bond-containing perfluoroalkyl group possessed by the fluorine compound described together with the synthesis method thereof in Patent Document 5 or 6 can be used.

[0070] The cell membrane permeable group possessed by the nucleic acid for transfection according to the present invention preferably has a linking group for connecting to the nucleic acid to be introduced, in addition to the perfluoroalkyl group that bears the cell membrane permeability. The linking group is not particularly limited as long as it is a group that can connect the nucleic acid and the perfluoroalkyl group, and any divalent or trivalent organic group can be used. Examples of the linking group include alkylene groups, alkenylene groups, carbonyl groups, amino groups, ether bonds, thioether bonds, ester bonds, amide bonds, polyethylene glycol groups (PEG: -(C2H4O)n-), siloxane bonds, silyl ether bonds, sugars, and peptides. In addition, pyrrole rings, pyrazole rings, imidazole rings, triazole rings, pyridine rings, pyrimidine rings, pyrazine rings, A group obtained by removing two or three hydrogen atoms from a ring such as an azole ring, a thiazole ring, a furan ring, a thiophene ring, or a benzene ring can also be used as a linking group. These groups can also be used in combination as appropriate.

[0071] As the cell membrane permeable group possessed by the nucleic acid for transfection according to the present invention, for example, a group having a structure represented by the following general formula (A1) is preferred. FE is the above-mentioned ether bond-containing perfluoroalkyl group. In addition, na is an integer of 1 to 10. The black dots represent the bonding sites.

[0072]

[0073] As the cell membrane permeable group possessed by the nucleic acid for transfection involved in the present invention, it is preferably a group that is connected to the target nucleic acid connected to the group by various coupling reactions. For example, by using a phosphoramidite containing a perfluoroalkyl group containing an ether bond as a raw material and performing the phosphoramidite method, it is possible to easily introduce a perfluoroalkyl group containing an ether bond into a nucleic acid. A general nucleic acid automatic synthesis device utilizes the phosphoramidite method. Therefore, by using a phosphoramidite containing a perfluoroalkyl group containing an ether bond as a raw material, it is possible to easily synthesize a nucleic acid containing a perfluoroalkyl group containing an ether bond at a desired position in a nucleic acid of various base sequences using an automatic synthesis device.

[0074] As the phosphoramidite containing an ether bond perfluoroalkyl group, for example, a compound obtained by replacing the nucleoside part in the phosphoramidite generally used for nucleic acid synthesis with an organic group containing an ether bond perfluoroalkyl group can be cited. As such a compound, for example, a compound represented by the following general formula (A2-0) can be cited.

[0075]

[0076] In the general formula (A2-0), R FE is the above-mentioned ether bond-containing perfluoroalkyl group. In addition, na is an integer of 1 to 10. i-Pr represents an isopropyl group, and DMTr represents a 4,4'-dimethoxytriphenylmethyl group.

[0077] The nucleic acid synthesized by the phosphoramidite method using compound (A2-0) has the structure of the following general formula (A2). FE is the above-mentioned ether bond-containing perfluoroalkyl group. In addition, na is an integer of 1 to 10. The black dots represent the bonding sites.

[0078]

[0079] As the nucleic acid for transfection involved in the present invention, it is also preferred that the nucleic acid obtained by connecting two or more structures represented by the general formula (A2) in series to the nucleic acid to be introduced. By having multiple structures represented by the general formula (A2) connected in series, the cell membrane permeability of the nucleic acid to be introduced can be significantly improved. The nucleic acid for transfection can be synthesized by connecting multiple compounds (A2-0) to the nucleic acid to be introduced by the phosphoramidite method.

[0080] Furthermore, according to the methods described in Non-Patent Documents 2 to 5, a cell membrane permeable group can be introduced into a nucleic acid to be introduced.

[0081] The synthesized target nucleic acid can be separated and purified by various methods such as ion chromatography, gel filtration chromatography, reverse phase chromatography, normal phase chromatography, etc.

[0082] As the nucleic acid for transfection of the present invention, there is no particular limitation as long as it is a nucleic acid for introduction into a cell. As the nucleic acid for introduction, it is preferably a functional nucleic acid that exhibits certain physiological activities by being taken into a target cell in a living body. Specifically, for example, nucleic acids for expressing proteins in cells, nucleic acids for inhibiting gene expression in cells, nucleic acids for genetic modification, nucleic acid aptamers that specifically bind to biological molecules as targets, functional nucleic acids that affect the physiological functions of cells, etc. can be cited. As nucleic acids for protein expression, for example, cDNA and mRNA encoding proteins can be cited, and expression plasmid vectors with regions encoding target proteins can be introduced. As nucleic acids for gene expression inhibition, for example, functional nucleic acids for RNA interference such as siRNA, miRNA, shRNA, and antisense oligonucleotides can be cited, and RNAi vectors can be cited. As nucleic acids for genetic modification, fragments of genomic DNA can be cited. As functional nucleic acids that affect the physiological functions of cells, Decoy nucleic acids, CpG (cytosine-phosphate-guanine) oligonucleotides, etc. can be cited. In addition, a fluorescent substance or a functional molecule such as a functional peptide may be linked to a nucleic acid. For example, a nucleic acid molecule having a fluorescent substance linked to the end of a single-stranded nucleic acid having a guanine quadruplex (G-quadruplex) structure may be used as the nucleic acid to be introduced.

[0083] The nucleic acid to be introduced in the nucleic acid for transfection of the present invention is not particularly limited, and the nucleotides contained may be nucleic acids composed entirely of natural nucleotides, or nucleic acids composed partially or entirely of artificial nucleotides. In addition, it may be single-stranded nucleic acids, or double-stranded nucleic acids. It may be DNA, RNA, or a chimeric nucleic acid of DNA and RNA.

[0084] In the nucleic acid for transfection of the present invention, the position of introducing the cell membrane permeable group is not particularly limited, and the cell membrane permeable group can be introduced at any position within the range that does not impair the function of the nucleic acid. For example, the cell membrane permeable group can be bonded to the 5' end or 3' end of the nucleic acid to be introduced, or can be introduced between two nucleotides in the nucleic acid to be introduced.

[0085] The nucleic acid for transfection according to the present invention may be modified in various ways as long as the cell membrane permeability brought about by the perfluoroalkyl group is not impaired and the function of the nucleic acid to be introduced is not impaired. Examples of such modifications include sugar chain modification, lipid modification, and peptide modification.

[0086] The nucleic acid for transfection of the present invention has excellent cell membrane permeability compared with nucleic acids not containing perfluoroalkyl groups due to cell membrane permeability groups. Therefore, the nucleic acid for transfection of the present invention can be introduced into cells simply by contacting with cells without using common transfection reagents such as Lipofectamine. In the case of cultured cells, the target cells to be introduced with the nucleic acid for transfection of the present invention can be introduced into the cells by simply adding the nucleic acid for transfection to the culture medium for culturing. In addition, for animal tissues, for example, the nucleic acid for transfection of the present invention can be introduced into the cells constituting the tissue by spraying or applying a solution in which the nucleic acid for transfection of the present invention is dissolved to the tissue surface.

[0087] Example

[0088] Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to these examples.

[0089] <Cell culture>

[0090] In the following experiments, cell culture was performed as follows.

[0091] HeLa cells (Riken Cell Bank) were cultured in low-glucose Dulbecco's modified Eagle's medium (DMEM, manufactured by FUJIFILM Wako) supplemented with 10% FBS and a 0.5% penicillin-streptomycin mixed solution at 37°C in a humidified atmosphere (5% CO2 by volume).

[0092] 24 hours before the introduction of nucleic acid into cells, the cells were plated at a concentration of 1.0×10 4 The cells were seeded in a 96-well culture plate (manufactured by VIOLAMO) at a cell / well format and allowed to adhere to the plate and grow (pre-culture).

[0093] The cells used for image analysis were cultured in a 35 mm glass-bottom culture dish (manufactured by IWAKI).

[0094] [Synthesis example 1]

[0095] Synthesis of perfluoroalkyl groups containing 8 carbon atoms (C8F 17 -: hereinafter referred to as C8-PFC group) is used to produce a nucleic acid containing a C8-PFC group by the phosphoramidite method.

[0096] <Synthesis of phosphoramidites containing C8-PFC groups>

[0097] The phosphoramidite containing a C8-PFC group (N[C8-PFC]phosphoramidite) represented by the following formula was synthesized by the method described in Non-Patent Document 4.

[0098]

[0099] [Synthesis example 2]

[0100] Synthesis of perfluoroalkyl groups containing 6 carbon atoms (C6F 17 -: hereinafter referred to as C6-PFC group) is used to produce a nucleic acid containing a C6-PFC group by the phosphoramidite method.

[0101] <Synthesis of phosphoramidites containing C6-PFC groups>

[0102] The phosphoramidite containing a C6-PFC group represented by the following formula (N[C6-PFC]phosphoramidite) was synthesized in the same manner as in Synthesis Example 1.

[0103]

[0104] 1 H-NMR (400 MHz, deuterated chloroform) δ7.45-7.39 (m, 2H), 7.35-7.26 (m, 6H), 7.23-7.17 (m, 1H), 6.85-6.79 (m, 4H), 3.83-3.52 (m, 6H), 3.79 (s, 6H), 3.79 (t, J = 16.6 Hz, 2H), 3.20 (t, J = 5.7 Hz, 2H), 2.94 (dt, J = 15.6, 5.9 Hz, 4H), 2.57 (t, J = 6.5 Hz, 2H), 1.16 (dd, J = 15.3, 6.8 Hz, 12H).

[0105] 19 F-NMR (376 MHz, deuterated chloroform) δ-80.64 (t, J = 10.1 Hz, 3F), -117.31--117.72 (m, 2F), -121.86--121.91 (m, 2F), -122.55--122.94 (m, 2F), -123.32--123.40 (m, 2F), -125.93--126.06 (m, 2F).

[0106] 31 P-NMR (162MHz, deuterated chloroform) δ148.35.

[0107] [Synthesis example 3]

[0108] Phosphoramidite containing a perfluoropolyether group having 5 carbon atoms (CF3-(OCF2)4-: hereinafter referred to as C5-PFPE group) was synthesized, and a nucleic acid containing a C5-PFPE group was produced by the phosphoramidite method using the synthesized phosphoramidite.

[0109] <Synthesis of phosphoramidites containing C5-PFPE groups>

[0110] The phosphoramidite containing a C5-PFPE group represented by the following formula (N[C5-PFPE]phosphoramidite) was synthesized in the same manner as in Synthesis Example 1.

[0111]

[0112] 1 H-NMR (400 MHz, deuterated chloroform) δ7.44-7.39 (m, 2H), 7.35-7.27 (m, 6H), 7.23-7.17 (m, 1H), 6.89-6.75 (m, 4H), 3.82-3.45 (m, 6H), 3.79 (s, 6H), 3.27 (t, J = 11.1 Hz, 2H), 3.18 (t, J = 5.8 Hz, 2H), 2.93 (dt, J = 14.2, 6.0 Hz, 4H), 2.57 (tt, J = 6.5, 0.9 Hz, 2H), 1.15 (dd, J = 15.4, 6.8 Hz, 12H).

[0113] 19 F-NMR (376 MHz, deuterated chloroform) δ -52.93 (p, J = 10.8 Hz, 2F), -54.85 (p, J = 10.1 Hz, 2F), -55.36 (qt, J = 9.4, 9.3 Hz, 2F), -56.76 (t, J = 8.8 Hz, 3F), -77.18 (td, J = 11.40, 11.39 Hz, 2F).

[0114] 31 P-NMR (162 MHz, deuterated chloroform) δ148.40.

[0115] <DNA synthesis>

[0116] DNA synthesis was performed using NTS H-8 DNA / RNA synthesizer (manufactured by NIHON TECHNO SERVICE) using commercially available reagents, various phosphoramidites (acetonitrile solution, 0.1 M), and 5-ethylthio-1H-tetrazole (acetonitrile solution, 0.25 M) as an activator.

[0117] [Example 1]

[0118] The cellular uptake efficiency of the fluorescein-modified nucleic acids containing a C8-PFC group and the fluorescein-modified nucleic acids not containing a C8-PFC group was investigated by quantitative analysis using flow cytometry and observation using a confocal microscope.

[0119] <Synthesis of Fluorescein-modified Nucleic Acid>

[0120] A fluorescein-modified nucleic acid (ssDNA-FAM) having fluorescein introduced into the 3' end of a single-stranded DNA (G-quadruplex DNA) consisting of sequence number 1 (5'-AGGGTTAGGGTTAGGGTTA GGG-3') was synthesized using a DNA synthesizer. Next, one, two or five molecules of N[C8-PFC] phosphoramidite synthesized in Synthesis Example 1 were linked to the 5' end of the obtained ssDNA-FAM by manual synthesis in a glove box to obtain ssDNA-FAM (PFC-ssDNA-FAM) having two cell membrane permeable groups containing a C8-PFC group linked in series at the 5' end.

[0121]

[0122] <Preparation of sample solution>

[0123] ssDNA-FAM (control nucleic acid) and PFC-ssDNA-FAM (fluorine-added nucleic acid) were prepared in a DMEM medium (low glucose, 10% FBS, 1% penicillin-streptomycin mixed solution) at a concentration of 1.0 μM as sample solutions. A 1.0 μM solution of ssDNA-FAM was used as a negative control, and a mixture of ssDNA-FAM and a commercially available transfection reagent ("Lipofectamine LTX", manufactured by Thermo Fisher Scientific) was used as a positive control. The mixing ratio and amount of each reagent of Lipofectamine LTX followed the manufacturer's standard protocol. The blank group was measured using a DMEM medium (low glucose, 10% FBS, 1% penicillin-streptomycin mixed solution) without the introduction of nucleic acid.

[0124] <Evaluation of cell membrane permeability using flow cytometry>

[0125] The culture medium of HeLa cells previously cultured at 37°C for 24 hours was replaced with the sample solution, and the cell membrane permeability after culture at 37°C for 1 hour or 4 hours was evaluated. After each predetermined time of culture, the cell surface was washed three times with PBS (phosphate saline), and the cells were peeled and recovered with trypsin-EDTA 0.05% (Gibco). Next, the recovered cells were applied to flow cytometry (guava easyCyte (trademark) 8) to measure the green 2 fluorescence (448nm) of the fluorescent pigment fluorescein (FAM) introduced into the synthetic nucleic acid.

[0126] The cells without fluorescein-modified nucleic acid added to the culture medium were used as the blank group, the cells with only control nucleic acid (ssDNA-FAM) added to the culture medium were used as the negative control, and the cells with both control nucleic acid and Lipofectamine LTX added to the culture medium were used as the positive control.

[0127] The average fluorescence intensity of each cell at each culture time is shown in Figure 1 In the figure, "Blank" indicates the results of cells in which no fluorescein-modified nucleic acid was added to the culture medium, "Naked" indicates the results of cells in which only control nucleic acid was added to the culture medium, "PFCs" indicates the results of cells in which only fluorine-added nucleic acid (PFC-ssDNA-FAM formed by linking two molecules of N[C8-PFC] phosphoramidite to ssDNA-FAM) was added to the culture medium, and "Lipofectamine" indicates the results of cells in which both control nucleic acid and Lipofectamine LTX were added to the culture medium. Figure 1 As shown, the average fluorescence intensity of the fluorine-added nucleic acid (PFC-ssDNA-FAM) was 6.7 times that of the control nucleic acid (ssDNA-FAM) without fluorine addition in cells cultured for 1 hour and 24.4 times that in cells cultured for 4 hours, and the average fluorescence intensity of each cell was higher than that in the case of using Lipofectamine. These results indicate that nucleic acids introduced with cell membrane permeable groups containing perfluoroalkyl groups have better cell membrane permeability than general transfection reagents.

[0128] Lipofectamine is a widely used transfection reagent, but it has the following problems: the culture solution needs to be made serum-free, and the added culture medium needs to be replaced within a few hours. In addition, the solution mixture needs to be adjusted according to the number of experiments, and the permeability results fluctuate greatly with the experiment. On the other hand, the transfection nucleic acid of the present invention containing perfluoroalkyl does not need to be serum-free, and does not require culture medium replacement, and it is known that it has excellent properties as a reagent for cell introduction of nucleic acid.

[0129] <Confocal microscopy observation>

[0130] In order to confirm that the fluorine-added nucleic acid was not attached to the cell surface but was actually introduced into the cell, confocal microscopy observation was performed.

[0131] Use the sample solution prepared in the same way as above. Replace the culture medium of HeLa cells cultured at 37°C for 24 hours with the sample solution and culture at 37°C for 2 hours. Next, wash the cell surface three times with PBS, and culture at 37°C for 30 minutes in Hoechst 33258 (manufactured by Dojin Scientific Research Institute) diluted 5000 times with OPTI-MEM culture medium for nuclear staining. Thereafter, wash the cell surface three times with PBS, replace the culture medium with Leibovitz's L-15 culture medium, and observe with a confocal microscope (SPARKSP8, manufactured by Leica).

[0132] The fluorescence staining image of cells into which the fluorine-added nucleic acid (PFC-ssDNA-FAM) was introduced is shown in Figure 2 (Left) Fluorescence staining images of cells into which control nucleic acid (ssDNA-FAM) was introduced using Lipofectamine are shown in Figure 2 (Right). In the figure, the large circles in the Hoechst staining image are nuclei, and the white dots in the FAM image are fluorescein that labels nucleic acids. It was possible to confirm that the fluorine-added nucleic acids were indeed introduced into the cells rather than attached to the cell surface 2 hours after the introduction (replacing the culture medium with the sample solution). In addition, when transfection was performed using a mixture of Lipofectamine and control nucleic acid, the nucleic acids were surrounded by the lipid membrane in the cells and became circular. This circle was co-localized with the nuclear stain Hoechst, so it is believed that a large number of nucleic acids formed aggregates and were surrounded by the lipid membrane ( Figure 2 (right figure)). On the other hand, the fluorinated nucleic acid exists as a nucleic acid monomer in the form of dots or chains, and is not co-localized with the nuclear stain Hoechst33258, so it is estimated that it functions rapidly in the cytoplasm within 2 hours of introduction. In addition, it can be confirmed that part of the nucleic acid has also entered the nucleus ( Figure 2 (Left figure)) In summary, it is shown that the transfection nucleic acid according to the present invention containing a perfluoroalkyl group is a nucleic acid that functions as a siRNA or a functional nucleic acid for controlling transcription at an earlier time than nucleic acid introduction using a liposome transfection reagent.

[0133] [Example 2]

[0134] The efficiency of fluorescein-modified nucleic acids containing a C8-PFC group into cells was studied by quantitative analysis using flow cytometry.

[0135] <Synthesis of Fluorescein-modified Nucleic Acid>

[0136] A fluorescein-modified nucleic acid (ssDNA-FAM) in which fluorescein was introduced into the 3' end of a single-stranded DNA consisting of sequence number 2 (5'-TTTTTCAGTTGACCATAT A-3') was synthesized in the same manner as in Example 1. Next, two molecules of N[C8-PFC] phosphoramidite synthesized in Synthesis Example 1 were linked to the 5' end of the obtained ssDNA-FAM by manual synthesis in a glove box to obtain ssDNA-FAM (PFC-ssDNA-FAM) in which two cell membrane permeable groups containing a C8-PFC group were linked in series at the 5' end.

[0137] <MTT assay and LDH assay>

[0138] The cell viability of the fluorine-modified nucleic acid (PFC-ssDNA-FAM) synthesized using the single-stranded DNA consisting of sequence number 2 was measured by the MTT assay, and the cytotoxicity was measured by the LDH assay. The cells to which the fluorescein-modified nucleic acid was not added to the culture medium were used as the blank group, the cells to which only the control nucleic acid (ssDNA-FAM) was added to the culture medium were used as the negative control, and the cells to which both the control nucleic acid and Lipofectamine LTX were added to the culture medium were used as the positive control.

[0139] HeLa cells were cultured at a rate of 1.0 × 10 4 The cells / wells were inoculated in 96-well culture plates to adhere to the wall and propagated. After culturing for 24 hours, the fluorescein-modified nucleic acid (fluorine-added nucleic acid or control nucleic acid) was added to the culture medium in a final concentration of 1 μM and cultured for 24, 48 or 72 hours. For the cultured cells, the cell viability was determined by the MTT assay, and the cytotoxicity was determined by the LDH assay. The MTT assay uses a commercially available MTT cell count assay kit (manufactured by Nacalai Tesque) and is measured according to the protocol. The LDH assay uses a commercially available cytotoxicity assay kit (manufactured by Dojindo) and is measured according to the protocol.

[0140] The relative cell viability of the measurement results of the MTT assay (the cells of the blank group were set as 100%) is shown in Table 1, and the cell injury rate (%) of the measurement results of the LDH assay (([number of viable cells in the blank group] - [number of viable cells of the measurement target cells]) / [number of viable cells in the blank group] × 100%) is shown in Table 2. In the table, "Blank" indicates the results of cells to which no fluorescein-modified nucleic acid was added to the culture medium, "Naked" indicates the results of cells to which only the control nucleic acid was added to the culture medium, "PFCs" indicates the results of cells to which only the fluorine-added nucleic acid was added to the culture medium, and "Lipofectamine" indicates the results of cells to which both the control nucleic acid and Lipofectamine LTX were added to the culture medium.

[0141] [Table 1]

[0142]

[0143] [Table 2]

[0144]

[0145] As shown in Tables 1 and 2, when the control nucleic acid and Lipofectamine LTX were added to the culture medium at the same time, the cell survival rate dropped to less than 50% in 72 hours of culture. In contrast, the cell survival rate of cells to which only fluorescein-modified nucleic acid was added to the culture medium was more than 90%. Based on these results, it was confirmed that the transfection nucleic acid involved in the present invention is different from the case of using Lipofectamine, and the cytotoxicity is very low.

[0146] [Example 3]

[0147] Since it was confirmed that the fluorine-added nucleic acid (PFC-ssDNA-FAM) synthesized in Example 2 has high cell permeability to HeLa cells, it was confirmed whether this high permeability is also effective in other cell lines. As cell types, HeLa cells, HepG2 cells (Riken Cell Bank), HEK293T cells (Riken Cell Bank), HT-1080 cells (JCRB Cell Bank), MCF7 cells (Riken Cell Bank), A549 cells (Riken Cell Bank), A431 cells (Riken Cell Bank), Huvec cells (Takara bio), MDA-MB-231 cells (ANGIO-PROTEOMIE), or MDA-MB-453 cells (Riken Cell Bank) were used.

[0148] The cell culture of HeLa cells was carried out in the same manner as in Example 1. HepG2 cells, HEK293T cells, and HT-1080 cells were cultured in the same medium and culture conditions as HeLa cells. MCF7 cells were cultured in the same manner as HeLa cells using EMEM (Eagle's minimum essential medium) medium (manufactured by FUJIFILM Wako) supplemented with 1% non-essential amino acids (manufactured by Nacalai Tesque), 1mM sodium pyruvate, 0.01mg / mL insulin (manufactured by FUJIFILM Wako), 10% FBS, and 1% penicillin-streptomycin mixed solution. A549 cells were cultured in the same manner as HeLa cells using EMEM medium supplemented with 1% non-essential amino acids, 10% FBS, and 1% penicillin-streptomycin mixed solution. A431 cells were cultured in the same manner as HeLa cells using DMEM high glucose (manufactured by FUJIFILM Wako) supplemented with 10% FBS and 1% penicillin-streptomycin as a culture medium. Huvec cells were cultured in endothelial cell growth medium 2 (SupplementPack Enndothelial Cell GM2, manufactured by Takara Bio) supplemented with 1% penicillin-streptomycin mixed solution, 0.04% trypsin / 0.03% EDTA was used for cell detachment, trypsin inhibitor / 0.1% BSA (manufactured by Takara Bio) was used for inactivation of trypsin, and other culture conditions were the same as those for HeLa cells. MDA-MB-231 cells and MDA-MB-453 cells were cultured in Leibovitz's L-15 medium (manufactured by FUJIFILM Wako) supplemented with 10% FBS and 1% penicillin-streptomycin mixed solution at 37°C in a carbon dioxide-free environment.

[0149] Specifically, the cell membrane permeability was studied in the same manner as in Example 1, except that the fluorine-added nucleic acid (PFC-ssDNA-FAM) or the control nucleic acid (ssDNA-FAM) synthesized in Example 1 was added to the culture medium at a concentration of 1 μM and the cells were subjected to flow cytometry after 24 hours of culture.

[0150] Based on the measured mean fluorescence intensity of each cell, the relative mean fluorescence intensity was determined with the mean fluorescence intensity of the cells in the blank group set to 1. The relative mean fluorescence intensity of each cell is shown in Table 3. In addition, based on the results of flow cytometry, the ratio (%) of cells into which fluorescein-modified nucleic acid was introduced (cells in which green fluorescence was confirmed) relative to the whole cells is shown in Table 4. In the table, "Blank" indicates the results of cells to which fluorescein-modified nucleic acid was not added to the culture medium, "Naked" indicates the results of cells to which only control nucleic acid was added to the culture medium, "PFCs" indicates the results of cells to which only fluorine-added nucleic acid was added to the culture medium, and "Lipofectamine" indicates the results of cells to which both control nucleic acid and Lipofectamine LTX were added to the culture medium.

[0151] [Table 3]

[0152]

[0153] [Table 4]

[0154]

[0155] As shown in Table 3, regardless of the cell strain, the average fluorescence intensity of cells to which fluorine-added nucleic acid (PFC-ssDNA-FAM) was added to the culture medium was greater than that of cells in the blank group or cells to which only control nucleic acid was added, and it was confirmed that fluorine-added nucleic acid was introduced into these cells. In particular, MDA-MB-453 cells, Huvec cells, HepG2 cells, and A431 cells, like HeLa cells, had a higher average fluorescence intensity than cells treated with Lipofectamine. In addition, as shown in Table 4, the proportion of cells to which nucleic acid was introduced in cells to which fluorine-added nucleic acid (PFC-ssDNA-FAM) was added to the culture medium was the same as or higher than that in the case of using Lipofectamine in most cells. Based on these results, it was confirmed that the transfection nucleic acid of the present invention has excellent cell membrane permeability compared with the existing Lipofectamine reagent.

[0156] [Example 4]

[0157] The cellular uptake efficiency of the fluorescein-modified nucleic acids containing a C8-PFC group and the fluorescein-modified nucleic acids not containing a C8-PFC group was investigated by quantitative analysis using flow cytometry.

[0158] <Synthesis of Fluorescein-modified Nucleic Acid>

[0159] A fluorescein-modified nucleic acid (ssDNA-FAM) in which fluorescein is introduced into the 3' end of a single-stranded DNA consisting of sequence number 2 is synthesized in the same manner as in Example 1. In the same manner as in Example 1, one, two, three, four or five molecules of N[C8-PFC] phosphoramidite synthesized in Synthesis Example 1 are linked to the 5' end of the obtained ssDNA-FAM to obtain ssDNA-FAM (PFC-ssDNA-FAM) in which one, two, three, four or five cell membrane permeable groups containing a C8-PFC group are linked in series to the 5' end.

[0160]

[0161] <Preparation of sample solution>

[0162] ssDNA-FAM (control nucleic acid) and PFC-ssDNA-FAM (fluorine-added nucleic acid) were prepared in a DMEM medium (low glucose, 10% FBS, 1% penicillin-streptomycin mixed solution) at a concentration of 1.0 μM as sample solutions. DMEM medium (low glucose, 10% FBS, 1% penicillin-streptomycin mixed solution) was also used for blank group measurement.

[0163] <Evaluation of cell membrane permeability using flow cytometry>

[0164] The cell membrane permeability of HeLa cells was evaluated after culturing with each sample solution for 1 hour, 6 hours, or 24 hours in the same manner as in Example 1. Cells to which no fluorescein-modified nucleic acid was added to the culture medium were used as a blank control.

[0165] The mean fluorescence intensity (MFI) of each cell at each culture time is shown in Table 5. In the table, "Blank" indicates the result of cells in which no fluorescein-modified nucleic acid was added to the culture medium, and "N[PFC]n-DNA" indicates the result of cells in which n molecules of fluorine-added nucleic acid ssDNA-FAM were added to the culture medium. As shown in Table 5, the mean value of the fluorescence intensity of fluorine-added nucleic acid (PFC-ssDNA-FAM) reached a maximum value when n=2 in the results after 1 hour. These results indicate that in the case of the C8-PFC group, excellent cell membrane permeability can be achieved by performing a 2-molecule modification.

[0166] [Table 5]

[0167]

[0168] [Example 5]

[0169] The efficiency of fluorescein-modified nucleic acids containing a C5-PFPE group incorporating into cells was investigated by quantitative analysis using flow cytometry and observation using a confocal microscope.

[0170] <Synthesis of Fluorescein-modified Nucleic Acid>

[0171] A fluorescein-modified nucleic acid (ssDNA-FAM) in which fluorescein was introduced into the 3' end of the single-stranded DNA consisting of sequence number 2 was synthesized in the same manner as in Example 1. In the same manner as in Example 1, two molecules of N[C5-PFPE] phosphoramidite synthesized in Synthesis Example 3 were linked to the 5' end of the obtained ssDNA-FAM to obtain ssDNA-FAM (PFPE-ssDNA-FAM) in which two cell membrane permeable groups containing a C5-PFPE group were linked in series at the 5' end.

[0172]

[0173] <Preparation of sample solution>

[0174] PFPE-ssDNA-FAM (fluorine-added nucleic acid) was prepared in a DMEM medium (low glucose, 10% FBS, 1% penicillin-streptomycin mixed solution) at a concentration of 1.0 μM as a sample solution. DMEM medium (low glucose, 10% FBS, 1% penicillin-streptomycin mixed solution) was also used for blank group measurement.

[0175] <Evaluation of cell membrane permeability using flow cytometry>

[0176] The cell membrane permeability of HeLa cells was evaluated after culturing with each sample solution at 37° C. for 28 hours in the same manner as in Example 1. Cells to which no fluorescein-modified nucleic acid was added to the culture medium were used as a blank control.

[0177] The mean fluorescence intensity (MFI) of each cell at each culture time is shown in Table 6. In the table, "Blank" indicates the results of cells in which no fluorescein-modified nucleic acid was added to the culture medium, and "C5-PFPE" indicates the results of cells in which ssDNA-FAM to which two molecules of fluorine-added nucleic acid were added was added to the culture medium. As shown in Table 6, fluorine-added nucleic acid (PFPE- ss The average fluorescence intensity of DNA-FAM) was about twice that of the blank group, indicating that modification with the C5-PFPE group can exert excellent cell membrane permeability.

[0178] [Table 6]

[0179] Blank <![CDATA[C5-PFPE]]> Mean fluorescence intensity (MFI) 44 94

[0180] Industrial Applicability

[0181] The present invention provides a transfection nucleic acid having cell membrane permeability due to a perfluoroalkyl group and a method for producing the same. The transfection nucleic acid of the present invention has excellent cell membrane permeability and can be introduced into cells without using a transfection reagent, so it is expected to be used in the medical field or the bioengineering field.

Claims

1. A nucleic acid for transfection, comprising a nucleic acid to be introduced into a cell and a cell membrane permeable group linked thereto, The cell membrane permeable group contains a perfluoroalkyl group having 2 to 10 carbon atoms, The perfluoroalkyl group may have 1 to 5 ether-bonded oxygen atoms between carbon atoms.

2. The nucleic acid for transfection according to claim 1, wherein The cell membrane permeable group has a structure represented by the following general formula (A1): In formula (A1), R FE It is a perfluoroalkyl group having 2 to 10 carbon atoms without an ether-bonded oxygen atom between carbon atoms, or a perfluoroalkyl group having 2 to 10 carbon atoms with 1 to 5 ether-bonded oxygen atoms between carbon atoms, na is an integer of 1 to 10, and a black dot represents a bonding site.

3. The nucleic acid for transfection according to claim 1, wherein The cell membrane permeable group has a structure represented by the following general formula (A2): In formula (A2), R FE It is a perfluoroalkyl group having 2 to 10 carbon atoms without an ether-bonded oxygen atom between carbon atoms, or a perfluoroalkyl group having 2 to 10 carbon atoms with 1 to 5 ether-bonded oxygen atoms between carbon atoms, na is an integer of 1 to 10, and a black dot represents a bonding site.

4. The nucleic acid for transfection according to claim 3, wherein It has a structure in which two or more structures represented by the general formula (A2) are connected in series.

5. A method for producing a nucleic acid for transfection, comprising bonding a compound represented by the following general formula (A2-0) to a target nucleic acid to be introduced into a cell by a phosphoramidite method, thereby synthesizing a nucleic acid for transfection, In formula (A2-0), R FE It is a perfluoroalkyl group having 2 to 10 carbon atoms without an ether-bonded oxygen atom between carbon atoms, or a perfluoroalkyl group having 2 to 10 carbon atoms with 1 to 5 ether-bonded oxygen atoms between carbon atoms, na is an integer of 1 to 10, DMTr is 4,4'-dimethoxytriphenylmethyl, and i-Pr is isopropyl.

6. The method for producing a nucleic acid for transfection according to claim 5, wherein: Two or more compounds represented by the general formula (A2-0) are bonded in series to the nucleic acid.

7. A method for transfection of a nucleic acid, comprising bringing the nucleic acid for transfection according to any one of claims 1 to 4 into contact with a cell for introduction.

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