WO2022009873A1 - Stabilisateur d'arn - Google Patents

Stabilisateur d'arn Download PDF

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Publication number
WO2022009873A1
WO2022009873A1 PCT/JP2021/025441 JP2021025441W WO2022009873A1 WO 2022009873 A1 WO2022009873 A1 WO 2022009873A1 JP 2021025441 W JP2021025441 W JP 2021025441W WO 2022009873 A1 WO2022009873 A1 WO 2022009873A1
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rna
rnase
fatty acid
target rna
squalene
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PCT/JP2021/025441
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English (en)
Japanese (ja)
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哲矢 桑野
高良 井上
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花王株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/99—Enzyme inactivation by chemical treatment

Definitions

  • the present invention relates to an RNA stabilizer.
  • RNA is contained in skin surface lipids (SSL), and the SSL-derived RNA can be used for analysis of a living body.
  • SSL skin surface lipids
  • RNA is more unstable and more easily degraded than DNA.
  • RNase ribonuclease
  • ribonuclease is widely present not only in cells and tissues, but also in body fluids such as sweat and saliva, and in the environment. In the operation of RNA extraction and analysis, it is important to protect RNA from RNase activity.
  • RNase inhibitors are commonly used to suppress RNA degradation by RNases.
  • a chaotropic salt such as a guanidine salt, a protein having an RNase inhibitory activity derived from a living body, or a recombinant thereof is known.
  • Patent Document 1 International Publication No. 2018/0083319
  • the present invention provides an RNA stabilizer containing at least one selected from the group consisting of free fatty acids, squalene, cholesterol esters, and triacylglycerols as an active ingredient.
  • the invention provides an RNA stabilizing method comprising applying to a subject RNA at least one selected from the group consisting of free fatty acids, squalene, cholesterol esters, and triacylglycerols. ..
  • the invention provides the use of at least one selected from the group consisting of free fatty acids, squalene, cholesterol esters, and triacylglycerols for RNA stabilization.
  • the invention provides the use of at least one selected from the group consisting of free fatty acids, squalene, cholesterol esters, and triacylglycerols in the production of RNA stabilizers.
  • RNA stabilization by sebum A: RNA stabilization with sebum extract in the presence of RNase A.
  • CE cholesterol ester
  • WE wax ester
  • TAG triacylglycerol
  • FA free fatty acid
  • Chol cholesterol.
  • Ctrl Control (without sebum extract fraction and RNase), AD: Sebum extract fractions AD added,-: No sebum extract fraction. RNA stabilization by sebum constituent lipid components.
  • Ctrl Control (RNA only),-: No sebum-constituting lipid component (RNA + RNAase only).
  • RNA refers to single-stranded RNA and double-stranded RNA, such as mRNA, tRNA, rRNA, small RNA (for example, microRNA (miRNA), small interfering RNA (siRNA), PIWI-interacting RNA (piRNA). ) Etc.), long intergenic non-coding (link) RNA, and the like.
  • miRNA microRNA
  • siRNA small interfering RNA
  • piRNA PIWI-interacting RNA
  • Etc. long intergenic non-coding (link) RNA, and the like.
  • Ribonuclease is an RNA-degrading enzyme present in living organisms. RNase is contained not only in cells and tissues but also in body fluids such as sweat and saliva, and as a result, it is widely present in the environment including water, dust in the air, and experimental equipment. Various types of RNase have been found. Among them, RNase A (EC 3.1.2.5) is an endoribonuclease that has been often used in research for a long time and is a very stable enzyme. RNase A was first identified in bovine pancreas, and then sequence-homologous RNases have been found in a variety of organisms, which are classified as RNase A superfamily.
  • Human RNase A superfamily includes eight types consisting of RNase 1 to 8, and each type of enzyme has a different expression tissue in the living body.
  • RNA7 is known to be expressed in epithelial tissues including skin (Int J Mol Sci. 2016 Aug; 17 (8): 1278), and was in contact with the skin of the subject or operator, or in contact with it. Through experimental equipment, it can cause RNase contamination of biological RNA.
  • tissue is a general term for regions including tissues such as the stratum corneum, epidermis, dermis, hair follicles, and sweat glands, sebaceous glands, and other glands, unless otherwise specified.
  • SSL skin surface lipids
  • the present invention provides an RNA stabilizer effective for stabilizing RNA in the presence of RNase.
  • the RNA stabilizer of the present invention inhibits RNase, suppresses RNA degradation by RNase, and stabilizes RNA in the presence of RNase.
  • the RNA stabilizer of the present invention also stabilizes RNA in a biological sample (eg, SSL).
  • SSL contains various lipid components such as triacylglycerol (TAG), squalene, cholesterol, cholesterol ester (CE), free fatty acid (FA), and fatty acid ester.
  • TAG triacylglycerol
  • CE cholesterol ester
  • FA free fatty acid
  • SSL contains a component effective for stabilizing RNA. As shown in Examples described later, among the lipid components in SSL, squalene, CE, TAG, and FA suppressed RNA degradation by RNase.
  • the present invention relates to using at least one selected from the group consisting of FA, squalene, CE, and TAG for RNA stabilization.
  • the term "stabilization" of RNA means maintaining RNA in a state in which degradation by RNase or the like is suppressed.
  • FA, squalene, CE, and TAG inhibit RNA activity against RNA, thereby suppressing RNA degradation and stabilizing RNA in the presence of RNase, such as biological samples (eg, SSL). .. Therefore, one aspect of RNA stabilization according to the present invention may be RNase inhibition or RNA degradation inhibition.
  • the FA used for RNA stabilization in the present invention is not particularly limited, but is preferably a linear fatty acid, preferably an unsaturated fatty acid, and more preferably a linear unsaturated fatty acid.
  • the chain length (number of carbon atoms) of the FA is not particularly limited, but is preferably 12 to 22, and more preferably 14 to 18.
  • Preferred examples of FA include myristoleic acid, palmitoleic acid, oleic acid and the like.
  • the FA used in the present invention may be any one of the above-mentioned free fatty acids having different chain lengths and degrees of unsaturation, or a combination of any two or more of them.
  • the FA may be extracted from sebum or chemically synthesized. Alternatively, a commercially available free fatty acid (for example, palmitoleic acid (Sigma Aldrich)) can also be used.
  • the FA may be in the form of a salt. Examples of the salt include sodium salts and the like.
  • the amount of FA used in the present invention is preferably 0.005 mg or more, more preferably 0.5 mg or more, and preferably 100 mg or less, more preferably 100 mg or less, per 1 ⁇ g of the target RNA. Is 50 mg or less. For example, it is preferably 0.005 to 100 mg, more preferably 0.05 mg to 50 mg or 5 mg to 100 mg, and further preferably 0.5 to 50 mg per 1 ⁇ g of the target RNA.
  • the squalene used for RNA stabilization in the present invention may be extracted from sebum or chemically synthesized. Alternatively, commercially available squalene (for example, squalene (Sigma Aldrich)) can also be used.
  • the amount of squalene used in the present invention is preferably 0.05 mg or more, more preferably 5 mg or more, and preferably 100 mg or less, more preferably 50 mg per 1 ⁇ g of the target RNA. It is as follows. For example, it is preferably 0.005 to 100 mg, more preferably 0.05 mg to 50 mg or 5 mg to 100 mg, and further preferably 0.5 to 50 mg per 1 ⁇ g of the target RNA.
  • the CE used for RNA stabilization in the present invention is an ester of cholesterol and fatty acid.
  • the fatty acid moiety constituting the CE is not particularly limited, but is preferably a linear fatty acid, preferably an unsaturated fatty acid, and more preferably a linear unsaturated fatty acid.
  • the chain length (number of carbon atoms) of the fatty acid is not particularly limited, but is preferably 12 to 22, and more preferably 14 to 18.
  • Preferred examples of fatty acids include myristoleic acid, palmitoleic acid, oleic acid and the like.
  • the CE may be extracted from sebum or chemically synthesized. Alternatively, a commercially available CE (for example, Collestryl palmilate; D-161 (Olbracht Battery Research Laboratories)) can also be used.
  • the amount of CE used in the present invention is preferably 0.05 mg or more, more preferably 5 mg or more, and preferably 100 mg or less, more preferably 50 mg per 1 ⁇ g of the target RNA. It is as follows. For example, it is preferably 0.005 to 100 mg, more preferably 0.05 mg to 50 mg or 5 mg to 100 mg, and further preferably 0.5 to 50 mg per 1 ⁇ g of the target RNA.
  • the TAG used for RNA stabilization in the present invention may be one extracted from sebum or one chemically synthesized. Alternatively, a commercially available TAG (for example, triolein (Fujifilm Wako Pure Chemical Industries, Ltd.)) can be used.
  • the TAG may be in the form of a salt. Examples of the salt include sodium salts and the like.
  • the acyl group contained in the TAG is not particularly limited, but is preferably a linear fatty acid group, preferably an unsaturated fatty acid group, and more preferably a linear unsaturated fatty acid group.
  • the chain length (number of carbon atoms) of the acyl group is not particularly limited, but is preferably 12 to 22, and more preferably 14 to 18.
  • acyl group examples include a 9-tetradecenoyl group, a 9-hexadecenoyl group, an oleoyl group and the like.
  • the individual acyl groups contained in the TAG may be the same or different from each other. Further, the TAG used in the present invention may be a combination of TAGs having different acyl group compositions.
  • the amount of TAG used in the present invention is preferably 0.05 mg or more, more preferably 5 mg or more, and preferably 100 mg or less, more preferably 50 mg per 1 ⁇ g of the target RNA. It is as follows. For example, it is preferably 0.005 to 100 mg, more preferably 0.05 mg to 50 mg or 5 mg to 100 mg, and further preferably 0.5 to 50 mg per 1 ⁇ g of the target RNA.
  • At least one selected from the group consisting of FA, squalene, CE, and TAG preferably at least one selected from the group consisting of FA, squalene, and CE, more preferably FA and /.
  • squalene more preferably FA
  • the amount of each component used in the above may be appropriately adjusted within the range of the amount of each component used described above.
  • the present invention provides an RNA stabilizer containing at least one selected from the group consisting of FA, squalene, CE, and TAG as an active ingredient.
  • the invention provides the use of at least one selected from the group consisting of FA, squalene, CE, and TAG in the production of RNA stabilizers.
  • the invention provides an RNA stabilizing method comprising applying the RNA stabilizer of the invention to a subject RNA.
  • the RNA stabilizer of the present invention may consist of at least one selected from the group consisting of FA, squalene, CE, and TAG.
  • the RNA stabilizer of the present invention can be a composition comprising at least one selected from the group consisting of FA, squalene, CE, and TAG.
  • Specific examples of the composition include oil and fat compositions containing at least one of FA, squalene, CE, or TAG, for example, oil and fat compositions derived from plants, animals, microorganisms, and the like, preferably plants. Examples include edible oils and fats of origin.
  • the content of FA, squalene, CE, and TAG in the composition may be any amount as long as the amount used for the above-mentioned target RNA can be achieved.
  • the means for applying the RNA stabilizer of the present invention to the target RNA is not particularly limited, and any means may be used, for example, any means capable of adding or mixing the RNA stabilizer to the sample containing the target RNA.
  • the target RNA to be stabilized is RNA in a biological sample
  • the RNA stabilizer of the present invention is a stabilizer for RNA in a biological sample.
  • the biological sample may be a sample containing RNA separated, secreted or released from the living body, for example, cells, tissues, body fluids (blood, etc.), feces and urine, secretions (saliva, etc.), horns collected from the living body. Layers, skin and secretions on the skin of living organisms (sweat, SSL, etc.), and the like.
  • the RNA stabilizer of the present invention may be applied to the biological sample.
  • the biological sample when the biological sample is a liquid (for example, body fluid or secretion), it is preferable to mix the sample with the RNA stabilizer of the present invention.
  • the biological sample when the biological sample is a solid (for example, cell or tissue), the disrupted sample may be mixed with the RNA stabilizer of the present invention, or the sample may be disrupted together with the stabilizer of the present invention. preferable.
  • the biological sample is SSL.
  • SSL can be taken from a subject having SSL on the skin, such as a human or non-human mammal.
  • the skin part from which SSL is collected include skin of any part of the body such as the head, face, neck, trunk, and limbs.
  • any means used to recover or remove SSL from the skin can be employed.
  • an SSL absorbent material, an SSL adhesive material, or an instrument that scrapes SSL from the skin can be used.
  • the SSL absorbent material or the SSL adhesive material is not particularly limited as long as it is a material having an affinity for SSL, and examples thereof include polypropylene and pulp.
  • More detailed examples of the procedure for collecting SSL from the skin include a method of absorbing SSL into a sheet-like material such as oil blotting paper and oil blotting film, a method of adhering SSL to a glass plate, tape, etc., a spatula, a scraper, etc. There is a method of scraping off the SSL and collecting the SSL.
  • an SSL-absorbing material pre-impregnated with a highly lipophilic solvent may be used.
  • the SSL-absorbent material contains a highly water-soluble solvent or water, the adsorption of SSL is hindered, which is not preferable.
  • the SSL absorbent material is preferably used in a dry state.
  • the RNA stabilizer of the present invention is added to SSL separated from the subject. In one example, the RNA stabilizer of the present invention is added to an SSL-absorbing material or an SSL-adhesive material from which SSL has been recovered. In another embodiment, the RNA stabilizer of the present invention is added to the supraventricular lipid on the skin of a subject. In one example, the RNA stabilizer of the invention is applied externally (eg, applied or sprayed) onto the skin of a subject and mixed with SSL, after which the SSL is recovered with the RNA stabilizer. In another example, an SSL-absorbing material or an SSL-adhesive material containing the RNA stabilizer of the present invention is brought into contact with the skin of a subject to recover the SSL and add the RNA stabilizer to the SSL.
  • an SSL-absorbing material or an SSL-adhesive material containing the RNA stabilizer of the present invention is brought into contact with the skin of a subject to recover the SSL and add the RNA stabilizer to the SSL.
  • RNA from a biological sample to which the RNA stabilizer of the present invention is applied may be carried out according to a normal procedure for recovering RNA from a biological sample.
  • a commercially available RNA purification reagent suitable for a biological sample can be used.
  • RNA stabilizer containing at least one selected from the group consisting of free fatty acids, squalene, cholesterol esters, and triacylglycerols as an active ingredient.
  • the fatty acid moiety constituting the cholesterol ester is preferably a linear fatty acid or an unsaturated fatty acid, more preferably a linear unsaturated fatty acid.
  • the chain length of the fatty acid portion constituting the cholesterol ester is preferably 12 to 22, more preferably 14 to 18.
  • acyl group contained in the triacylglycerol is preferably a linear fatty acid group or an unsaturated fatty acid group, and more preferably a linear unsaturated fatty acid group.
  • RNA stabilizer according to any one of [1] to [6], wherein the free fatty acid has a chain length of preferably 12 to 22, more preferably 14 to 18.
  • the RNase is It is preferably an RNase belonging to RNase A superfamily, and is preferably an RNase.
  • the RNA stabilizer according to any one of [8] to [10].
  • the RNA is It is preferably RNA in a biological sample, and is preferable. More preferably, it is RNA in lipids on the surface of the skin.
  • the RNA stabilizer according to [12] which is preferably added to the skin surface lipid separated from the subject or added to the skin surface lipid on the subject's skin.
  • RNA stabilizer according to any one of [1] to [13], wherein the squalene is applied to the target RNA in the following amounts: Per 1 ⁇ g of the target RNA, preferably 0.05 mg or more, more preferably 5 mg or more, and preferably 100 mg or less, more preferably 50 mg or less, or Per 1 ⁇ g of the target RNA, preferably 0.05 to 100 mg, more preferably 0.05 mg to 50 mg or 5 mg to 100 mg, still more preferably 5 to 50 mg.
  • the cholesterol ester is applied to the target RNA in the following amount.
  • Per 1 ⁇ g of the target RNA preferably 0.05 mg or more, more preferably 5 mg or more, and preferably 100 mg or less, more preferably 50 mg or less, or Per 1 ⁇ g of the target RNA, preferably 0.05 to 100 mg, more preferably 0.05 mg to 50 mg or 5 mg to 100 mg, still more preferably 5 to 50 mg.
  • RNA stabilizer according to any one of [1] to [15], wherein the triacylglycerol is applied to the target RNA in the following amount: Per 1 ⁇ g of the target RNA, preferably 0.05 mg or more, more preferably 5 mg or more, and preferably 100 mg or less, more preferably 50 mg or less, or Per 1 ⁇ g of the target RNA, preferably 0.05 to 100 mg, more preferably 0.05 mg to 50 mg or 5 mg to 100 mg, still more preferably 5 to 50 mg.
  • the free fatty acid is applied to the target RNA in the following amount.
  • Per ⁇ g of the target RNA preferably 0.005 mg or more, more preferably 0.5 mg or more, and preferably 100 mg or less, more preferably 50 mg or less, or Per ⁇ g of the target RNA, preferably 0.005 to 100 mg, more preferably 0.05 mg to 50 mg or 5 mg to 100 mg, still more preferably 0.5 to 50 mg.
  • RNA stabilizer preferably contains an oil / fat composition containing at least one selected from the group consisting of the free fatty acid, squalene, cholesterol ester and triacylglycerol.
  • RNA stabilizing method comprising applying the RNA stabilizer according to any one of [1] to [18] to the target RNA.
  • the RNA stabilization preferably comprises inhibition of RNase or inhibition of RNA degradation in the presence of RNase.
  • RNA stabilizers Use of at least one selected from the group consisting of free fatty acids, squalene, cholesterol esters, and triacylglycerols in the production of RNA stabilizers.
  • the use according to [21] or [22], wherein the fatty acid moiety constituting the cholesterol ester is preferably a linear fatty acid or an unsaturated fatty acid, more preferably a linear unsaturated fatty acid.
  • the free fatty acid is preferably a linear fatty acid or an unsaturated fatty acid, more preferably a linear unsaturated fatty acid.
  • the free fatty acid has a chain length of preferably 12 to 22, more preferably 14 to 18.
  • the RNA stabilization is preferably RNA stabilization by suppressing RNA degradation in an environment in which RNase is present.
  • the RNA stabilization is preferably RNA stabilization by RNase inhibition.
  • RNA stabilization preferably comprises inhibition of RNase or inhibition of RNA degradation in the presence of RNase.
  • the RNase is It is preferably an RNase belonging to RNase A superfamily, and is preferably an RNase. More preferably, it is RNase A or RNase 7. Use according to any one of [29] to [31].
  • the RNA is It is preferably RNA in a biological sample, and is preferable. More preferably, it is RNA in lipids on the surface of the skin. Use according to any one of [21] to [32].
  • At least one selected from the group consisting of the free fatty acid, squalene, cholesterol ester, and triacylglycerol is added to or subject to the superficial lipid on the skin separated from the subject.
  • [36] The use according to any one of [21] to [35], wherein the cholesterol ester is applied to the target RNA in the following amounts: Per 1 ⁇ g of the target RNA, preferably 0.05 mg or more, more preferably 5 mg or more, and preferably 100 mg or less, more preferably 50 mg or less, or Per 1 ⁇ g of the target RNA, preferably 0.05 to 100 mg, more preferably 0.05 mg to 50 mg or 5 mg to 100 mg, still more preferably 5 to 50 mg.
  • RNA stabilizing activity of sebum 1 Preparation of sebum extract Sebum was collected from the faces of four human subjects with an oil-removing film (5.0 cm ⁇ 8.0 cm, oil clear film, Hakugen Earth). The oil-removing film from which sebum was collected was shredded with scissors, mixed in a DDW / t-Butyl Methyl Ether (1: 4 v / v) solution (Wako) with a test tube mixer, and centrifuged at 3,000 rpm for 10 minutes. .. The upper layer (t-Butyl Methyl Ether layer) was recovered and the solvent was removed under a nitrogen gas stream at 50 ° C. to obtain a sebum extract.
  • oil-removing film 5.0 cm ⁇ 8.0 cm, oil clear film, Hakugen Earth
  • the oil-removing film from which sebum was collected was shredded with scissors, mixed in a DDW / t-Butyl Methyl Ether (1: 4 v / v) solution (W
  • RNA stabilization activity of sebum extract RNA extracted from Human Epidermal Keratinocytes, neonal (HEKn) (Cascade Biologicals) using RNeasy mini kit (QIAGEN) was used as a test RNA.
  • RNase bovine RNase A (Fujifilm Wako Pure Chemical Industries, Ltd.) or human RNase 7 (Recombinant human RNASE 7 protein; NOVUS) was used.
  • the test RNA solution final concentration 20 ⁇ g / mL
  • 10 mM Tris-HCl (pH 8.0) as a solvent was mixed with 1 ⁇ L of the sebum extract prepared in 1) (sebum extract final concentration 2% v / v).
  • RNA was recovered by the same method as other samples.
  • the recovered RNA is subjected to the Agilent 4200 TapeStation system (Agilent technologies), using the High Sensitivity RNA Screen Tape (Agilent Technologies) and the High Sensitivity Fibers (Agilent Technologies).
  • RNA degradation by RNase A or RNase 7 The electrophoretic image of RNA is shown in FIG.
  • the addition of sebum extract suppressed RNA degradation by RNase A or RNase 7, indicating that sebum has the effect of inhibiting RNase activity and stabilizing RNA.
  • Example 2 Evaluation of RNA Stabilization Activity of Sebum Component 1 Fractionation of Sebum Extract by Thin Layer Chromatography (TLC) 5 mg of sebum extract prepared in 1) of Example 1 was added to Chloroform / methanol (2: 1 v). / V) Dissolved in 50 ⁇ L. The obtained solution was applied to an HPTLC plate (Glass HPTLC silica gel 60 plates; 10 ⁇ 20 cm, Merck). Appropriate amounts of sebum extract and lipid preparations were applied to the same HPTLC plate for reference. Separation was performed in a horizontal developing tank using a developing solvent Hexane / diethyl ether / acetic acid (70: 30: 1 v / v / v).
  • TLC Thin Layer Chromatography
  • the plates were cut into reference and extraction using a glass cutter. A 10% copper sulfate / 8% phosphoric acid solution was sprayed on a reference plate and then heated at 180 ° C. for 3 minutes to detect spots.
  • cholesterol ester (sigma, C6072) as cholesterol ester [CE]
  • Lauryl palmitoleate (santa cruz biotechnology, sc-280908) as wax ester [WE]
  • Palmetric acid sigma, P9417
  • squalene [SQ] sigma, S3626
  • cholesterol [Chol] sigma, C8667
  • TLC TLC
  • Spots such as squalene (SQ), cholesterol ester (CE), triacylglycerol (TAG), free fatty acid (FA), and cholesterol (Chol) were detected. It was also confirmed that the wax ester (WE) was separated, although no clear spot was detected.
  • SQL squalene
  • CE cholesterol ester
  • TAG triacylglycerol
  • FA free fatty acid
  • Chol cholesterol
  • the extraction plate contains fraction (A) containing SQ, CE and WE, fraction (B) containing TAG, and FA.
  • fraction (C) and fraction (D) containing Chol were scraped, dissolved in 8 mL of chloroform / methanol (2: 1 v / v), and sonicated. The obtained solution was centrifuged at 3,000 rpm for 5 minutes, the supernatant was collected, and dried with nitrogen at 50 ° C. to obtain a sebum extract fraction.
  • RNA stabilizing activity of the sebum extract fraction was evaluated by the same procedure as in 2) of Example 1.
  • the sebum extract fraction to be mixed with the test RNA solution final concentration 20 ⁇ g / mL
  • the redissolved solution and a diluted solution obtained by diluting it 3 times in 2 steps were used, and each solution had a final concentration of 4%. It was added at (v / v).
  • RNase human RNase 7 (Recombinant human RNASE 7 protein; NOVUS) was used.
  • the final concentration of RNase was 1 ⁇ g / mL and the treatment time was 30 minutes.
  • FIG. 3 shows an electrophoretic image of RNA treated with RNase in the presence of a sebum extract fraction. RNA degradation was suppressed in the fraction (A) containing SQ, CE and WE, the fraction (B) containing TAG, and the fraction (C) containing FA.
  • RNA stabilizing activity of sebum-constituting lipid components was examined by the same procedure as in Example 1.
  • the sebum-constituting lipid component include squalene [SQ] (Squarene; sigma, S3626), cholesterol ester [CE] (Cholesteryl platinum; Olbracht Sardy Research Labelories, D-161), and wax ester [WE] Bleh.
  • each lipid was 1 or 100 mg / mL (CE, WE and Chol were only 1 mg / mL).
  • the test RNA the RNA extracted in Example 1 (final concentration 20 ⁇ g / mL) was used.
  • RNase human RNase 7 (Recombinant human RNASE 7 protein; NOVUS) was used.
  • the final concentration of RNase was 1 ⁇ g / mL and the treatment time was 30 minutes.
  • the ratio of RNA of 200 bases or more to the total RNA which is known as an index of the degree of RNA degradation, was calculated from an electrophoretic image using an Agilent 4200 TapeStation system.
  • TAG and FA include triacylglycerol [TAG 16: 1] (Glyceryl tripalmitolate; sigma, T5888), triacylglycerol [TAG 18: 1] (Glyceryl trioleate; sima, T7140), C14 free fatty acid [FA 14: 1]. ] (Myristoleic acid; sigma, M3525), C16 free fatty acid [FA 16: 1] (Palmitoleic acid; sima, P9417), and C18 free fatty acid [FA 18: 1] (Oleic acid; sigma, O1008).
  • each lipid was 100 mg / mL.
  • human RNase 7 Recombinant human RNASE 7 protein; NOVUS
  • the final concentration of RNase was 1 ⁇ g / mL and the treatment time was 30 minutes.
  • RNA degradation was suppressed by SQ, CE, TAG, and FA.
  • all of the monounsaturated free fatty acids C14 (myristoleic acid), C16 (palmitoleic acid), and C18 (oleic acid), which are abundant in sebum, have RNA degradation inhibitory activity.
  • any of the TAGs having a monounsaturated acyl group of C16 (palmitrail group) or C18 (oleyl group) had RNA degradation inhibitory activity.

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Abstract

Stabilisateur d'ARN comprenant, comme principe actif, au moins un élément choisi dans le groupe constitué par les acides gras libres, le squalène, les esters de cholestérol et les triacylglycérols.
PCT/JP2021/025441 2020-07-06 2021-07-06 Stabilisateur d'arn WO2022009873A1 (fr)

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