Specific tyrosinase targeting ASO (Accelerated Solvent Oxidase) as well as preparation method and application thereof

Abstract

The invention provides a specific tyrosinase targeting ASO as well as a preparation method and application thereof, the specific tyrosinase targeting ASO can specifically target tyrosinase mRNA and degrade an hTYR mRNA sequence, the expression of tyrosinase is inhibited, so that the production of melanin is inhibited for a long time, the whitening effect is achieved, the specificity is high, the safety is high, special chemical modification is realized, the stability is high, and the ASO is not easy to degrade; a liposome delivery carrier is not needed, and the liposome can automatically penetrate through a cell membrane and enter the cell to play a role. The effective use concentration is low, the cost is controllable and the use is convenient; the cosmetic composition can be independently used or used in combination with other conventional cosmetics, and is suitable for various product forms, such as paste, spray or freeze-dried powder.

Description

Technical Field

[0001] The invention relates to the fields of cosmetics, medicines and food chemistry, and in particular to a specific targeted tyrosinase, a preparation method and application thereof. Background Art

[0002] Tyrosinase (EC 1.14.18.1, Tyrosinase, TYR for short), also known as polyphenol oxidase, is a copper-binding protein that is a key enzyme protein that causes human skin pigmentation and related diseases.

[0003] Research on inhibitors of tyrosinase activity has received strong attention from many fields at home and abroad for many years. However, among the many existing compounds used to inhibit tyrosinase activity, practice has shown that most compounds have low efficacy and obvious toxic side effects.

[0004] Kojic acid, chemical name is 5-hydroxy-2-hydroxymethyl-1,4-pyrone. Kojic acid has the function of inhibiting the activity of melanin-producing enzyme tyrosinase, has a significant whitening effect, and does not inhibit other enzymes. It has a wide range of application value in the field of daily chemical industry. Traditional whitening cosmetic raw materials, such as kojic acid, have certain whitening effects, but have disadvantages such as safety issues and unstable effects. Summary of the invention

[0005] In order to overcome the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a specific targeted tyrosinase, a preparation method and an application thereof.

[0006] The technical solution adopted by the present invention to solve the technical problem is: a method for preparing a specifically targeted tyrosinase ASO, comprising the following synthetic route:

[0007]

[0008] The synthesis steps include deprotection, coupling, oxidation and capping reaction steps; For a solid phase synthesis link.

[0009] As a further improvement of the present invention: wherein the deprotection step reaction formula is as follows:

[0010]

[0011] The first base at the 3' end of the oligonucleotide is selected, and CPG linked to one of the bases A, G, C, and T is selected as a solid phase carrier. The 5'OH of this base is protected by a DMT protecting group. Before starting the synthesis, the DMT of the first base is first removed with trichloroacetic acid to expose the active 5'OH; For a solid phase synthesis link.

[0012] As a further improvement of the present invention: the coupling reaction formula is as follows:

[0013]

[0014] The phosphite bond formed in the coupling step is unstable under acidic conditions, and I2 is needed to oxidize trivalent phosphorus into pentavalent phosphorus to form a stable phosphodiester bond. There are two ways of oxidation: oxidative and thioactive. The residual trichloroacetic acid on the solid support is cleaned with anhydrous acetonitrile, and tetrazole and the corresponding phosphoramidite monomer are added. The phosphoramidite monomer activated by tetrazole reacts with the 5'OH on the solid support to form a phosphite bond. In order to ensure that only one base is coupled in each cycle, the 5'OH of the phosphoramidite monomer is protected by DMT. For a solid phase synthesis link.

[0015] As a further improvement of the present invention: the oxidation reaction formula is as follows:

[0016]

[0017] I2 oxidizes trivalent phosphorus to pentavalent phosphorus to form a phosphodiester bond. After the removal of DMT, the residual trichloroacetic acid on the solid support is cleaned with anhydrous acetonitrile, and tetrazole and the corresponding phosphoramidite monomer are added. The phosphoramidite monomer activated by tetrazole reacts with 5'OH on the solid support to form a phosphite bond.

[0018] As a further improvement of the present invention: the capping reaction formula is as follows:

[0019]

[0020] The 5'OH that does not participate in the reaction is blocked with acetyl group, and the phosphoramidite method is used to synthesize oligonucleotides. The average coupling efficiency of each cycle can reach more than 99.5%. However, no matter how perfect the synthesis process is, the coupling efficiency cannot reach 100%. In each round of coupling reaction, there will be a trace amount of 5'OH that does not participate in the coupling reaction. It is necessary to block the 5'OH that does not participate in the reaction with acetyl group to prevent it from participating in the next round of reaction. For a solid phase synthesis link.

[0021] As a further improvement of the present invention: the deprotection, coupling, oxidation and capping reaction steps are cyclically reacted, with one base added in each cyclic reaction until the entire sequence is completed.

[0022] As a further improvement of the present invention, it also includes a step of removing the solid phase carrier, and the reaction formula is as follows:

[0023]

[0024] As a further improvement of the present invention: the chemical reaction equation for removing the amino protecting group is as follows:

[0025]

[0026] The chemical reaction equation for removing the phosphate backbone protecting group is as follows:

[0027]

[0028] As a further improvement of the present invention: chemically synthesized oligonucleotides usually use butadiene amino groups to fix the 3' end of the oligonucleotide on a solid phase carrier. This group is stable to all reagents in the synthesis process. After the synthesis is completed, ammonia water can be used to remove the group, so that the oligonucleotide is separated from the solid phase carrier to form an oligonucleotide with 3'OH. ​​At the same time, ammonia water can remove the protecting groups of the primary amino groups of pyrimidine and purine and the cyanoethyl protecting group on the phosphate backbone of the oligonucleotide at room temperature. Only oligonucleotides with completely removed amino protecting groups and phosphate backbone protecting groups have biological activity.

[0029] The present invention also includes an ASO specifically targeting tyrosinase, which is prepared by using the above-mentioned preparation method of a specifically targeting tyrosinase.

[0030] The specific sequences of the above-mentioned ASOs specifically targeting tyrosinase are shown in SEQ ID NOs: 1-3;

[0031] The specific sequence of hTYRASO-1 is:

[0032] / +G / * / +A / * / +G / * / +C / *T*G*A*T*G*G*T*A* / +T / * / +G / * / +C / * / +T / -3'Chol(SEQ IDNO:1);

[0033] The specific sequence of hTYRASO-2 is:

[0034] / +A / * / +G / * / +C / * / +T / *G*T*G*G*T*A*A*T* / +C / * / +C / * / +T / * / +C / -3'Chol(SEQ IDNO:2);

[0035] The specific sequence of hTYRASO-3 is

[0036] / +C / * / +T / * / +G / * / +A / * / *C*C*T*C*C*C*A*T / +G / * / +T / * / +A / * / +C / -3'Chol(SEQID NO:3);

[0037] The specific sequence of ASO-NC is:

[0038] / +G / * / +G / * / +C / * / +T / *A*C*T*A*C*G*C*C* / +G / * / +T / * / +C / * / +A / -3'Chol;

[0039] Among them, + represents LNA modification, * represents phosphorothioate modification, and 3'Chol represents 3' terminal cholesterol modification.

[0040] The preferred ASO specifically targeting tyrosinase is any one or a combination thereof;

[0041] A more preferred ASO specifically targeting tyrosinase is shown in SEQ ID NO:3.

[0042] The present invention also includes the use of the ASO specifically targeting tyrosinase prepared by the above-mentioned preparation method of the ASO specifically targeting tyrosinase in the preparation of cosmetics.

[0043] Preferably, the cosmetics are whitening cosmetics.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] 1. The ASO specifically targeting tyrosinase of the present invention can specifically target tyrosinase mRNA, inhibit the expression of tyrosinase, thereby long-term inhibiting the production of melanin, achieving the effect of whitening, and specifically degrading hTYRmRNA sequence, with high specificity, high safety, special chemical modification, high stability, and not easy to degrade;

[0046] 2. No liposome delivery carrier is required, it can pass through the cell membrane and enter the cell to exert its effect;

[0047] 3. Antisense nucleic acid combines the latest RNA advanced structure prediction algorithm to design multiple antisense nucleic acid sequences in the single-stranded region of tyrosinase mRNA. After screening, sequences with high inhibition efficiency and strong specificity are obtained;

[0048] 4. The 4 bases at the 3' end and the 4 bases at the 5' end of the antisense nucleic acid are modified with locked nucleic acid (LNA) or 2'-O-(2-Methoxyethyl) to improve the complementary binding ability of the antisense nucleic acid and the tyrosinase mRNA sequence, effectively enhancing the stability and affinity of the antisense nucleic acid; the middle 8-10 DNA bases are modified with thiophosphate throughout the chain to improve biological stability.

[0049] Phosphorothioate modification greatly improves the stability of antisense nucleic acids in vivo;

[0050] 5. Since phosphorothioate bonds are more resistant to nuclease degradation than unmodified phosphate bonds (P=O), the modified antisense nucleic acid can exist in the body for a longer time, thereby improving its inhibitory effect. Phosphorothioate modification can also enhance the binding affinity of antisense nucleic acid to target RNA. This is because the introduction of sulfur atoms changes the three-dimensional structure of the antisense nucleic acid molecule, which helps it to bind more stably to the target RNA.

[0051] Antisense nucleic acids modified with thiophosphate are more easily absorbed by cells. This modification changes the negative charge distribution of antisense nucleic acids, which helps them pass through the cell membrane, thereby increasing the concentration inside the cell. The 3' end or 5' end of the antisense nucleic acid is chemically modified with cholesterol or C16 to improve the cell membrane penetration of the antisense nucleic acid, so that it can enter the cell and exert its effect without the assistance of other delivery materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the technical solution, the drawings required for use in the implementation will be briefly introduced below. Obviously, the drawings described below are only some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0053] Figure 1 This is a schematic diagram of western blotting detection in a melanin inhibition test of a TRY protein inhibitor that specifically targets tyrosinase in the present invention.

[0054] Figure 2 This is a grayscale analysis diagram of estern blotting detection in a melanin inhibition test of a TRY protein inhibitor specifically targeting tyrosinase of the present invention, wherein NC ASO is a random ASO and TRYASO is ASO-3.

[0055] Figure 3 This is a melanin detection analysis diagram in a melanin inhibition test of a TRY protein inhibitor that specifically targets tyrosinase according to the present invention.

[0056] Figure 4 This is the QRT-PCR test result of a long-term inhibition test result specifically targeting tyrosinase in the present invention.

[0057] Figure 5 This is a schematic diagram of the expression level of tyrosinase in a long-term inhibition test result of specifically targeting tyrosinase of the present invention.

[0058] Figure 6The figure is a schematic diagram of the results of measuring the L* value of the model after the apparent chromaticity detection in a whitening efficacy test specifically targeting tyrosinase of the present invention - a melanin skin model whitening test, wherein BC is a blank control, NC is a negative control, PC is a positive control, and 200nM ASO is 200nM hTYRASO-3.

[0059] Figure 7 This is a schematic diagram summarizing the apparent chromaticity results in a whitening efficacy test specifically targeting tyrosinase in the present invention - a melanin skin model whitening test. DETAILED DESCRIPTION

[0060] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention and the corresponding drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0061] The present invention is further described in conjunction with the accompanying drawings and examples: The present invention provides a specific targeted tyrosinase, a preparation method and an application thereof, which is obtained by a specific chemical synthesis method and subjected to special chemical modification to improve its stability and biological activity.

[0062] Example 1 A method for preparing an ASO specifically targeting tyrosinase,

[0063] Specifically include the following synthetic routes:

[0064]

[0065] The phosphoramidite method synthesizes from the 3' to the 5' end of the oligonucleotide, adding one base in each cycle; each cycle reacts with one base until the entire sequence is completed.

[0066] The synthesis steps include deprotection, coupling, oxidation and capping reaction steps.

[0067] The deprotection reaction includes the following steps: according to the first base at the 3' end of the synthesized oligonucleotide, CPG linked to one of the bases A, G, C, and T is selected as a solid phase carrier, and the 5'OH of this base is protected by a DMT protecting group. Before starting the synthesis, the DMT of the first base is first removed with trichloroacetic acid to expose the active 5'OH. The reaction equation is shown below:

[0068]

[0069] The coupling reaction includes the following steps: after the removal of DMT, the residual trichloroacetic acid on the solid phase carrier is cleaned with anhydrous acetonitrile, tetrazole and the corresponding phosphoramidite monomer are added, and the phosphoramidite monomer activated by tetrazole reacts with the 5'OH on the solid phase carrier to form a phosphite bond. To ensure that only one base is coupled in each cycle, the 5'OH of the phosphoramidite monomer is protected by DMT. It is a solid phase synthesis link; the reaction equation is shown below:

[0070]

[0071] The oxidation reaction includes the following steps. The oxidation reaction includes oxygenation and thiogenation. The phosphite bond formed after the coupling reaction is unstable under acidic conditions. It is necessary to use I2 to oxidize trivalent phosphorus into pentavalent phosphorus to form a stable phosphodiester bond; For a solid phase synthesis link.

[0072] The oxygenation reaction equation is shown below:

[0073]

[0074] The thiolation reaction equation is shown below:

[0075]

[0076] In the above synthesis method, Tyrosinase_001-003ASO was successfully obtained, and its specific sequence is shown in SEQ ID NO: 1-3.

[0077] As an embodiment of the present invention, it also includes a purification step, using ion-pair reverse-phase high performance liquid chromatography to purify oligonucleotides, utilizing the difference in hydrophobicity of oligos of different lengths, and as the elution intensity increases, eluting salts, short fragments that failed to be synthesized, long fragments, target fragments and fragments that failed to be removed from protecting groups in sequence, thereby achieving the purpose of purifying oligonucleotides, and the purity after purification can reach more than 95%.

[0078] Wherein, vacuum drying is performed after the purification step.

[0079] As an embodiment of the present invention, a specific targeted tyrosinase of the present invention can be added to products of different forms such as ointments, sprays or freeze-dried powders as needed in cosmetic formulas. Specific use methods include but are not limited to: as an active ingredient of whitening cosmetics, used to make whitening creams, whitening essences, etc.; combined with sunscreen ingredients to make sunscreen sprays, providing dual whitening and sunscreen effects.

[0080] The present invention specifically targets tyrosinase and designs multiple antisense nucleic acid sequences in the single-stranded region of tyrosinase mRNA, and obtains sequences with high inhibition efficiency and strong specificity after screening. The 3'-end 4 bases and 5'-end 4 bases of the antisense nucleic acid are modified with locked nucleic acid (LNA) or 2'-O-(2-Methoxyethyl) to improve the complementary binding ability of the antisense nucleic acid and the tyrosinase mRNA sequence, and effectively enhance the stability and affinity of the antisense nucleic acid; the middle is 8-10 DNA bases, and the whole chain is modified with thiophosphate to improve biological stability. The thiophosphate modification greatly improves the stability of the antisense nucleic acid in the organism. Since the thiophosphate bond is more resistant to nuclease degradation than the unmodified phosphate bond (P=O), the modified antisense nucleic acid can exist in the body for a longer time, thereby improving its inhibitory effect. The thiophosphate modification can also enhance the binding affinity of the antisense nucleic acid to the target RNA. This is because the introduction of sulfur atoms changes the three-dimensional structure of the antisense nucleic acid molecule, which helps it to bind more stably to the target RNA. Antisense nucleic acids modified with thiophosphate are more easily absorbed by cells. This modification changes the negative charge distribution of antisense nucleic acids, which helps them pass through the cell membrane, thereby increasing the concentration inside the cell. The 3' end or 5' end of the antisense nucleic acid is chemically modified with cholesterol or C16 to improve the cell membrane penetration of the antisense nucleic acid, so that it can enter the cell and exert its effect without the assistance of other delivery materials.

[0081] Example 2 The effective sequences of the species-specific tyrosinase-targeting ASO of the present invention were tested.

[0082] The specific test steps are as follows:

[0083] Experimental materials: Human melanoma cells SK-MEL-28 (provided by Guangzhou Yeshan Biotechnology Co., Ltd.);

[0084] Tyrosinase ASO is provided by epigenetic biosynthesis.

[0085] Experimental reagents:

[0086] Cell culture reagents: fetal bovine serum (Hyclone, Cat. No. SH30087.01), DMEM-high glucose medium (Hyclone, Cat. No. SH30022.01B), penicillin-streptomycin (Hyclone, Cat. No. SH30010), PBS potassium phosphate buffer (Hyclone, Cat. No. SH30256.01B).

[0087] Experimental consumables:

[0088] 6-well cell culture plate (CORNING, Cat. No. 040810004), Pasteur pipette (JET BIOFIL 3 ml).

[0089] Test method:

[0090] (I) Cell recovery

[0091] 1). Preheat the water bath to 37°C;

[0092] 2). Use 75% alcohol to wipe the clean bench surface that has been exposed to ultraviolet light for 30 minutes;

[0093] 3). Place sterilized centrifuge tubes, pipettes, culture bottles, etc. in order on the clean bench;

[0094] 4). Take out the cryotube;

[0095] 5) Thaw quickly. Put the cryotube into a preheated water bath and shake it constantly to melt the liquid in the tube. Take it out when there is still a little bit of liquid left in the cryotube.

[0096] 6). Wipe the outer wall of the cryopreservation tube with an alcohol cotton ball and then put it into the clean bench;

[0097] 7). Prepare cell suspension, transfer cells to a 15ml centrifuge tube, add preheated culture medium drop by drop, while shaking the centrifuge tube; the amount of culture medium added should reach more than 10ml;

[0098] 8). Centrifuge at 800 rpm for 5 minutes in a low-speed centrifuge; remove the supernatant and resuspend the cells in 1 ml of culture medium;

[0099] 9). Divide the cell suspension into culture dishes and place the culture dishes in a 37℃ incubator containing CO2 for culture. The time for changing the medium depends on the cell sedimentation rate.

[0100] (II) Cell culture

[0101] 2.1). Preheat the water bath to 37°C;

[0102] 2.2). Use 75% alcohol to wipe the clean bench surface that has been exposed to ultraviolet light for 30 minutes;

[0103] 2.3) Place sterilized centrifuge tubes, pipettes, culture bottles, etc. in order on the clean bench;

[0104] 2.4). Take out the cell culture flask and operate aseptically;

[0105] 2.5). Open the bottle cap and suck out the old culture medium;

[0106] 2.6). Wash the cells once or twice with PBS;

[0107] 2.7) For cell trypsinization: add trypsin-EDTA solution (1ml / 25cm2, 2ml / 75cm2) to the rinsed cells and gently wash the bottom of the cell dish. Aspirate the trypsin-EDTA solution, put it in a 37℃ incubator for 2-3 minutes, tap the wall of the culture bottle to make most of the cells fall off, observe under an inverted microscope, and when the cells are about to separate and appear granular, add an appropriate amount of fresh culture medium containing serum to terminate the trypsin effect;

[0108] 2.8) Use a pipette to pipette up and down several times to break up the cell clumps. After mixing evenly, add 3n (n is the number of subculture bottles) ml of culture medium (MEM) and transfer to a new culture bottle according to the dilution ratio;

[0109] 2.9). Place in a CO2 incubator (culture conditions: 5% CO2, saturated humidity, 37°C) and replace the culture medium every 3 days.

[0110] (III) Cell transfection and ASO screening

[0111] 3.1). ASO screening transfection scheme:

[0112] (1) Plate: Seed the cells at a density of 5 × 10^5 cells / well and shake thoroughly;

[0113] Sample setup:

[0114] Plate 1:

[0115]

[0116] Plate 2:

[0117] Tyrosinase_002ASO25nM Tyrosinase_002ASO50nM Tyrosinase_002ASO100nM Tyrosinase_003ASO25nM Tyrosinase_003ASO50nM Tyrosinase_003ASO100nM

[0118] The nucleotide sequence of Tyrosinase_001ASO is shown in SEQ ID NO: 1, the nucleotide sequence of Tyrosinase_002ASO is shown in SEQ ID NO: 2; and the nucleotide sequence of Tyrosinase_003ASO is shown in SEQ ID NO: 3.

[0119] (2) On the third day, the cell confluence was 40%, and transfection was performed;

[0120] Transfection process:

[0121] a. Cell culture medium replacement: aspirate the complete culture medium, wash twice with PBS, and add 1 ml of DMEM high-glucose culture medium containing 20% ​​fetal bovine serum to each well;

[0122] b. Dissolve ASO in RNAase-free deionized water to a final concentration of 20uM, dissolve ASO in 500ul opti-MEM, mix well, and let stand ----- Tube A;

[0123] Final concentration 25nM 50nM 100nM Take 20uMASO 2.5ul 5.0ul 10.0ul

[0124] c. Add 5ul Lipofectamine TM Add RNAiMAX to 500ul of opti-MEM, mix gently, and let stand for no more than 5 minutes-----Tube B;

[0125] d. Mix tube A and tube B, mix well and let stand for 20 minutes;

[0126] e. Add to each well respectively;

[0127] f. Mix well and place in cell culture incubator;

[0128] g. After 4-6 hours, remove the transfection medium, wash twice with PBS, and add 2 ml of complete medium to each well;

[0129] (3) 24 hours after transfection, the culture medium was discarded and 1 ml of Trizol was added to each well to prepare for the quantitative PCR method to detect the interference efficiency of ASO;

[0130] Note: (1) During the ASO dissolution and ASO transfection process, RNAase-free consumables must be used.

[0131] (IV) PCR testing

[0132] 1. Total RNA Extraction

[0133] Collect the cells, add 1 ml of Trizol solution, mix by pipetting to fully lyse the cells, and let stand for 5 min;

[0134] Add 200 μl of chloroform, shake vigorously for 30 seconds to allow the aqueous phase and the organic phase to fully contact, and let stand at room temperature for 2 minutes;

[0135] Centrifuge at 14,000 g for 15 min at 4°C. The tube can be separated into three layers. RNA is in the upper aqueous phase and transferred to another new RNasefree EP tube.

[0136] Precipitate RNA: Add an equal volume of isopropanol, mix gently and thoroughly, and let stand at room temperature for 10 minutes;

[0137] Centrifuge at 14,000 g for 10 min at 4°C to collect RNA precipitate and discard supernatant;

[0138] Wash twice with 75% ethanol and air dry in a clean bench;

[0139] Add 20 μl of DEPC water to dissolve the precipitate.

[0140] 2. Total RNA purity and integrity testing

[0141] 1) Purity test: Take 1 μl RNA sample and dilute it 50 times. Measure the OD value on the BioPhotometerplus nucleic acid protein analyzer. If the OD260 / OD280 ratio is greater than 1.8, it means that the prepared RNA is relatively pure and has no protein contamination.

[0142] 2) Total RNA integrity test: Take 1 μl of RNA sample, run 1% agarose gel electrophoresis at 80V×20min, and use a gel imaging system to observe the 5s rRNA, 18s rRNA and 28s rRNA bands of the total RNA. If the three bands are intact, it can be proved that the total RNA extraction is relatively complete.

[0143] 3. Reverse transcription

[0144] Prepare the following solutions in an RNase free PCR tube:

[0145] (EasyScriptFirst-Strand cDNA Synthesis SuperMix)

[0146]

[0147] (1) Incubate the above 20 μl reaction solution at 25°C for 10 min;

[0148] (2) Keep warm at 42℃ for 30min;

[0149] (3).Keep warm at 85℃ for 5 seconds.

[0150] 4. Quantitative PCR

[0151] 1. Detect sequence fragment size

[0152] Internal reference fragment: 18s-112bp;

[0153] Target fragment: tyrosinase-140bp;

[0154]

[0155]

[0156] 2. Reaction system:

[0157]

[0158] 3. Reaction conditions:

[0159] 95℃5min; 95℃15s, plate reading at 60℃32s, 40 cycles;

[0160] Melting curve analysis: temperature 60℃-95℃;

[0161] Each sample was repeated 3 times;

[0162] Among them, quantitative PCR instrument: ABI 7500 Sequence Detection System; Suzhou Antai clean bench (SW-CJ-IFD), low-speed centrifuge (Zhongjia, SC3614), inverted optical microscope (OLYMPUS CKX41, U-CTR30-2), cell constant temperature incubator (Thermo scientific, HERACELL150i) inverted fluorescence microscope manufacturer: Leica model: DMI6000B.

[0163] Example 3 Melanin inhibition test of a TRY protein inhibitor specifically targeting tyrosinase of the present invention,

[0164] The specific test steps are as follows:

[0165] Experimental materials: Human melanoma cells SK-MEL-28 (provided by Guangzhou Yeshan Biotechnology Co., Ltd.);

[0166] Tyrosinase ASO is provided by epigenetic biosynthesis.

[0167] Experimental reagents:

[0168] Cell culture reagents: fetal bovine serum (Hyclone, Cat. No. SH30087.01), DMEM-high glucose medium (Hyclone, Cat. No. SH30022.01B), penicillin-streptomycin (Hyclone, Cat. No. SH30010), PBS potassium phosphate buffer (Hyclone, Cat. No. SH30256.01B).

[0169] Test method:

[0170] 1. Cell Recovery

[0171] 1.1. Preheat the water bath to 37°C;

[0172] 1.2. Use 75% alcohol to wipe the clean bench surface that has been exposed to ultraviolet light for 30 minutes;

[0173] 1.3. Place sterilized centrifuge tubes, pipettes, culture bottles, etc. in order on the clean bench;

[0174] 1.4. Take out the cryotube;

[0175] 1.5. Thaw quickly. Put the cryotube into a preheated water bath and shake it constantly to melt the liquid in the tube. Take it out when there is still a little bit of liquid left in the cryotube.

[0176] 1.6. Wipe the outer wall of the cryotube with an alcohol cotton ball and then put it into the clean bench;

[0177] 1.7. Prepare cell suspension, transfer cells to a 15ml centrifuge tube, add preheated culture medium drop by drop, and shake the centrifuge tube at the same time; the amount of culture medium added should reach more than 10ml;

[0178] 1.8. Centrifuge at 800 rpm for 5 minutes in a low-speed centrifuge; remove the supernatant and resuspend the cells in 1 ml of culture medium;

[0179] 1.9. Divide the cell suspension into culture dishes and place the culture dishes in a 37℃ incubator containing CO2 for culture. The time for changing the medium depends on the cell sedimentation rate.

[0180] 2. Cell Culture

[0181] 2.1. Preheat the water bath to 37°C;

[0182] 2.2. Use 75% alcohol to wipe the clean bench surface that has been exposed to ultraviolet light for 30 minutes;

[0183] 2.3. Place sterilized centrifuge tubes, pipettes, culture bottles, etc. in order on the clean bench;

[0184] 2.4. Take out the cell culture flask and operate aseptically;

[0185] 2.5. Open the bottle cap and suck out the old culture medium;

[0186] 2.6. Wash the cells once or twice with PBS;

[0187] 2.7. For cell trypsinization: add trypsin-EDTA solution (1ml / 25cm2, 2ml / 75cm2) to the rinsed cells and gently wash the bottom of the cell dish. Aspirate the trypsin-EDTA solution, put it in a 37℃ incubator for 2-3 minutes, tap the wall of the culture bottle to make most of the cells fall off, observe under an inverted microscope, and when the cells are about to separate and appear granular, add an appropriate amount of fresh culture medium containing serum to terminate the trypsin effect;

[0188] 2.8. Use a pipette to pipette up and down several times to break up the cell clumps. After mixing evenly, add 3n (n is the number of subculture bottles) ml of culture medium (MEM) and transfer to a new culture bottle according to the dilution ratio;

[0189] 2.9. Place the cells in a CO2 incubator (culture conditions: 5% CO2, saturated humidity, 37°C) and replace the culture medium every 3 days.

[0190] 3. Experimental Grouping

[0191]

[0192] The sequence of the ASO is shown in SEQ ID NO: 3;

[0193] 4. Western blotting

[0194] 4.1 Experimental Materials

[0195] Antibody: Anti-Tyrosinase antibody [EPR10141] (ab170905).

[0196] 4.2 Experimental instruments

[0197]

[0198]

[0199] 4.3 Experimental methods

[0200] 4.3.1. Protein sample preparation

[0201] Use appropriate lysis buffer such as RIPA lysis buffer to lyse adherent cells, suspension cells or tissue samples;

[0202] After collecting the protein samples, in order to ensure that the loading amount of each protein sample is consistent, the protein concentration of each protein sample needs to be measured. Depending on the lysate used, an appropriate protein concentration determination method needs to be used.

[0203] 4.3.2. Electrophoresis

[0204] (1) SDS-PAGE gel preparation

[0205] (2) Sample processing

[0206] Add an appropriate amount of concentrated SDS-PAGE protein loading buffer to the collected protein samples.

[0207] Heat at 100°C or in a boiling water bath for 3-5 minutes to fully denature the protein.

[0208] (3) Sample loading and electrophoresis

[0209] After cooling to room temperature, the protein sample can be directly loaded into the SDS-PAGE gel loading well.

[0210] In order to facilitate the observation of electrophoresis and membrane transfer effects, as well as to determine the molecular weight of the protein, it is best to use a prestained protein molecular weight standard.

[0211] During electrophoresis, it is usually recommended to use low voltage constant voltage electrophoresis when the upper gel is in the upper gel, and high voltage constant voltage electrophoresis when bromophenol blue enters the lower gel. For Bio-Rad's standard electrophoresis device or similar electrophoresis devices, the low voltage can be set at 80-100V, and the high voltage can be set at around 120V. For the convenience of electrophoresis, the constant voltage method can also be used throughout the SDS-PAGE process, usually setting the voltage at 100V, and then setting the timing time to 90-120 minutes. Setting the timing can avoid electrophoresis overshoot, which often occurs.

[0212] Usually, electrophoresis can be stopped when bromophenol blue reaches the bottom of the gel, or it can be stopped when it is estimated that the target protein has been properly separated based on the electrophoresis of the pre-stained protein molecular weight standard.

[0213] 4.3.3. Transfer

[0214] Usually, if you use Bio-Rad's standard wet transfer device, you can set the transfer current to 300-400mA and the transfer time to 30-60 minutes. You can also transfer overnight at 15-20mA. The specific transfer time depends on the size of the target protein. The larger the molecular weight of the target protein, the longer the transfer time required, and the smaller the molecular weight of the target protein, the shorter the transfer time required.

[0215] During the transfer process, especially when transferring at high current and quickly, there is usually very serious heating. It is best to place the transfer tank in an ice bath for transfer.

[0216] The effect of membrane transfer can be observed by observing the pre-stained protein molecular weight standards used. Usually, the 1-2 bands with the largest molecular weight are difficult to transfer completely to the membrane.

[0217] 4.3.4. Blocking

[0218] After the transfer is completed, immediately place the protein membrane in the pre-prepared Western washing solution and rinse for 1-2 minutes to wash off the transfer solution on the membrane. In all steps after the transfer, be sure to keep the membrane moisturized and avoid drying the membrane, otherwise it will easily produce a high background.

[0219] Slowly shake on a shaker and block at room temperature for 60 minutes. For some antibodies with high background, block overnight at 4°C.

[0220] 4.3.5. Primary antibody incubation

[0221] Refer to the instructions of the primary antibody and use the Western primary antibody diluent according to the appropriate ratio.

[0222] Use a micro desktop vacuum pump or dropper to suck out the blocking solution, immediately add the diluted primary antibody, and incubate slowly on a side-swinging shaker at room temperature or 4°C for one hour. If the primary antibody incubation is not effective after one hour, you can incubate slowly at 4°C overnight. Or choose the appropriate incubation temperature and time according to the antibody instructions.

[0223] Recover the primary antibody. Add Western washing solution and wash slowly on a side-swing shaker for 5-10 minutes. After the washing solution is completely absorbed, add washing solution again and wash for 5-10 minutes. Wash 3 times in total. If the background is high, the washing time can be appropriately extended and the number of washes can be increased.

[0224] 4.3.6. Secondary antibody incubation

[0225] Refer to the instructions of the secondary antibody and dilute the horseradish peroxidase (HRP) labeled secondary antibody with Western secondary antibody diluent according to the appropriate ratio. The secondary antibody should be selected according to the primary antibody. Use a micro desktop vacuum pump or dropper to suck out the washing solution, immediately add the diluted secondary antibody, and incubate slowly on a side-swinging shaker at room temperature or 4°C for one hour.

[0226] Recover the secondary antibody. Add Western washing solution and wash slowly on a side-swing shaker for 5-10 minutes. After the washing solution is completely absorbed, add washing solution again and wash for 5-10 minutes. Wash 3 times in total. If the background is high, the washing time can be appropriately extended and the number of washes can be increased.

[0227] 4.3.7. Detection of proteins

[0228] Use ECL reagents such as BeyoECL Plus to detect proteins. Tablets can be pressed using a dedicated tableting cassette.

[0229] You can use an automatic X-ray film developer to develop the film. If you don't have an automatic film developer, you can prepare the developer and fixer yourself using the developer and fixer kit for manual film development. For X-ray films, it is recommended to use Kodak's original Kodak X-OMAT BT film for biological experiments.

[0230] 4.3.8. Gel image analysis

[0231] Scan or photograph the film and analyze the molecular weight and net optical density of the target band using a gel imaging system, such as Quantity One from Bio-Rad.

[0232] 5. Grayscale analysis of WB bands using ImagJ

[0233] 6. Determination of melanin content

[0234] 6.1 According to the method proposed by Hideya Ando et al., the melanin content was determined after some improvements. Take the reserved cell suspension and add PBS buffer to adjust the cell density to 5×10^5 / ml;

[0235] 6.2 Take 1 mL of cell suspension from each group and place it in 3 parallel stoppered colorimetric tubes. After centrifugation, discard the supernatant and add 200uL of distilled water to resuspend the cells.

[0236] 6.3 Then add 1 mL of 1:1 ethanol-ether solution to dissolve the non-melanin opaque particles, leave at room temperature for 15 minutes, centrifuge and discard the supernatant;

[0237] 6.4 Add 1 mL of 2 mol / L NaOH solution and place in an 80°C water bath for 30 min to dissolve the cells;

[0238] 6.5 Measure the absorbance at 420 nm and divide the absorbance by the number of cells as an indicator for evaluating the melanin content.

[0239] Melanin synthesis inhibition rate = [1-(drug treatment group value / cell number) / (blank control group value / cell number)]*100%.

[0240] 7. Experimental Results

[0241] Western blotting test results Figure 1 As shown, the grayscale analysis of western blotting is shown in the following table:

[0242]

[0243] Melanin detection and data analysis are shown in the following table:

[0244]

[0245] This indicates that ASO-3 targeting TYR has a significant inhibitory effect on the expression of TYR mRNA and its protein products.

[0246] Among them, the instruments used are: Suzhou Antai clean workbench (SW-CJ-IFD), low-speed centrifuge (Zhongjia, SC3614), inverted optical microscope (OLYMPUS CKX41, U-CTR30-2), cell constant temperature incubator (Thermo scientific, HERACELL150i) inverted fluorescence microscope manufacturer: Leica model: DMI6000B.

[0247] Reagents used:

[0248] 1.SDS-PAGE reagents:

[0249] 1.1 5x sample buffer (10ml): 0.6ml 1mol / L Tris-HCl (pH6.8), 5ml 50% glycerol, 2ml 10% SDS, 0.5ml mercaptoethanol, 1ml 1% bromophenol blue, 0.9ml distilled water. It can be stored at 4℃ for several weeks or at -20℃ for several months;

[0250] 1.2 Gel storage solution: In a fume hood, weigh 30g acrylamide and 0.8g methylene bisacrylamide, add heavy distilled water to dissolve, and then adjust the volume to 100ml. Filter and place in a brown bottle, store at 4℃, generally can be stored for 1 month;

[0251] 1.3 pH 8.9 separation gel buffer: 36.3 g Tris, add 48 ml 1 mol / L HCl, add 80 ml of heavy distilled water to dissolve, adjust pH to 8.9, make up to 100 ml, and store at 4°C;

[0252] 1.4 pH 6.7 stacking gel buffer: Tris 5.98 g, add 1 mol / L HCl 48 ml, add heavy distilled water 80 ml to dissolve, adjust pH 6.7, make up to 100 ml, and store at 4°C;

[0253] 1.5 TEMED (tetraethylethylenediamine) stock solution;

[0254] 1.610% ammonium persulfate (freshly prepared with double distilled water);

[0255] 1.7 pH 8.3 Tris-Glycine Electrode Buffer: Weigh 6.0 g Tris, 28.8 g glycine, add about 900 ml of distilled water, adjust pH to 8.3, and make up to 1000 ml with distilled water. Store at 4°C and dilute 10 times before use.

[0256] 2. Homogenization buffer: 1.0 M Tris-HCl (pH 6.8) 1.0 ml; 10% SDS 6.0 ml; β-mercaptoethanol 0.2 ml; ddH2O 2.8 ml.

[0257] 3. Transfer buffer: 2.9 g glycine; 5.8 g Tris; 0.37 g SDS; 200 ml methanol; add ddH2O to make up to 1000 ml.

[0258] 4.0.01M PBS (pH 7.4): NaCl 8.0g; KCl 0.2g; Na2HPO4 1.44g; KH2PO4 0.24g; add ddH2O to 1000ml.

[0259] 5. Membrane staining solution: 0.2 g Coomassie brilliant blue; 80 ml methanol; 2 ml acetic acid; 118 ml ddH2O. Coating solution (5% skim milk powder, freshly prepared): 1.0 g skim milk powder dissolved in 20 ml 0.01 M PBS.

[0260] 6. Color developing solution: DAB 6.0 mg; 0.01 M PBS 10.0 ml; nickel ammonium sulfate 0.1 ml; H202 1.0 μl.

[0261] Example 4 Melanin inhibition test of a TRY protein inhibitor specifically targeting tyrosinase of the present invention

[0262] The specific test steps are as follows:

[0263] Experimental materials: Human melanoma cells SK-MEL-28 (provided by Guangzhou Yeshan Biotechnology Co., Ltd.);

[0264] Tyrosinase ASO is provided by epigenetic biosynthesis.

[0265] Experimental reagents:

[0266] Cell culture reagents: fetal bovine serum (Hyclone, Cat. No. SH30087.01), DMEM-high glucose medium (Hyclone, Cat. No. SH30022.01B), penicillin-streptomycin (Hyclone, Cat. No. SH30010), PBS potassium phosphate buffer (Hyclone, Cat. No. SH30256.01B).

[0267] Test method:

[0268] 1. Cell Recovery

[0269] 1.1. Preheat the water bath to 37°C;

[0270] 1.2. Use 75% alcohol to wipe the clean bench surface that has been exposed to ultraviolet light for 30 minutes;

[0271] 1.3. Place sterilized centrifuge tubes, pipettes, culture bottles, etc. in order on the clean bench;

[0272] 1.4. Take out the cryotube;

[0273] 1.5. Thaw quickly. Put the cryotube into a preheated water bath and shake it constantly to melt the liquid in the tube. Take it out when there is still a little bit of liquid left in the cryotube.

[0274] 1.6. Wipe the outer wall of the cryotube with an alcohol cotton ball and then put it into the clean bench;

[0275] 1.7. Prepare cell suspension, transfer cells to a 15ml centrifuge tube, add preheated culture medium drop by drop, and shake the centrifuge tube at the same time; the amount of culture medium added should reach more than 10ml;

[0276] 1.8. Centrifuge at 800 rpm for 5 minutes in a low-speed centrifuge; remove the supernatant and resuspend the cells in 1 ml of culture medium;

[0277] 1.9. Divide the cell suspension into culture dishes and place the culture dishes in a 37℃ incubator containing CO2 for culture. The time for changing the medium depends on the cell sedimentation rate.

[0278] 2 Cell culture

[0279] 2.1. Preheat the water bath to 37°C;

[0280] 2.2. Use 75% alcohol to wipe the clean bench surface that has been exposed to ultraviolet light for 30 minutes;

[0281] 2.3. Place sterilized centrifuge tubes, pipettes, culture bottles, etc. in order on the clean bench;

[0282] 2.4. Take out the cell culture flask and operate aseptically;

[0283] 2.5. Open the bottle cap and suck out the old culture medium;

[0284] 2.6. Wash the cells once or twice with PBS;

[0285] 2.7. For cell trypsinization: add trypsin-EDTA solution (1ml / 25cm2, 2ml / 75cm2) to the rinsed cells and gently wash the bottom of the cell dish. Aspirate the trypsin-EDTA solution, put it in a 37℃ incubator for 2-3 minutes, tap the wall of the culture bottle to make most of the cells fall off, observe under an inverted microscope, and when the cells are about to separate and appear granular, add an appropriate amount of fresh culture medium containing serum to terminate the trypsin effect;

[0286] 2.8. Use a pipette to pipette up and down several times to break up the cell clumps. After mixing evenly, add 3n (n is the number of subculture bottles) ml of culture medium (MEM) and transfer to a new culture bottle according to the dilution ratio;

[0287] 2.9. Place the cells in a CO2 incubator (culture conditions: 5% CO2, saturated humidity, 37°C) and replace the culture medium every 3 days.

[0288] 3 Experimental Grouping

[0289]

[0290] 4PCR detection

[0291] 1. Total RNA Extraction

[0292] (1) Collect cells, add 1 ml of Trizol solution, mix by pipetting to fully lyse the cells, and let stand for 5 min;

[0293] (2) Add 200 μl of chloroform, shake vigorously for 30 seconds to allow the aqueous phase and organic phase to fully contact, and let stand at room temperature for 2 minutes;

[0294] (3) Centrifuge at 14,000 g for 15 min at 4°C. The tube can be separated into three layers. RNA is in the upper aqueous phase. Transfer the mixture to another new RNase-free EP tube.

[0295] (4) Precipitate RNA: Add an equal volume of isopropanol, mix gently and thoroughly, and let stand at room temperature for 10 min;

[0296] (5) Centrifuge at 14,000 g for 10 min at 4°C to collect the RNA precipitate and discard the supernatant;

[0297] (6) Wash twice with 75% ethanol and air dry in a clean bench;

[0298] (7) Add 20 μl of DEPC water to dissolve the precipitate.

[0299] 2. Total RNA purity and integrity testing

[0300] 1) Purity test: Take 1 μl RNA sample and dilute it 50 times. Measure the OD value on the BioPhotometerplus nucleic acid protein analyzer. If the OD260 / OD280 ratio is greater than 1.8, it means that the prepared RNA is relatively pure and has no protein contamination.

[0301] 2) Total RNA integrity test: Take 1 μl of RNA sample, run 1% agarose gel electrophoresis at 80V×20min, and use a gel imaging system to observe the 5s rRNA, 18s rRNA and 28s rRNA bands of the total RNA. If the three bands are intact, it can be proved that the total RNA extraction is relatively complete.

[0302] 3. Reverse transcription

[0303] Prepare the following solutions in an RNase free PCR tube:

[0304] (EasyScriptFirst-Strand cDNA Synthesis SuperMix)

[0305]

[0306] The above 20 μl reaction solution was incubated at 25°C for 10 min;

[0307] Keep warm at 42℃ for 30min;

[0308] Keep warm at 85℃ for 5s.

[0309] 4. Quantitative PCR

[0310] 1. Detect sequence fragment size

[0311] Internal reference fragment: 18s-112bp.

[0312] Target fragment: tyrosinase-140bp;

[0313]

[0314]

[0315] 2. Reaction system:

[0316]

[0317] 3. Reaction conditions:

[0318] 95℃5min; 95℃15s, plate reading at 60℃32s, 40 cycles;

[0319] Melting curve analysis: temperature 60℃-95℃.

[0320] Each sample was repeated 3 times.

[0321] Quantitative PCR instrument: ABI 7500Sequence Detection System.

[0322] Test results:

[0323] QRT-PCR test results are shown in Figure 4 In the figure, Lane 1 is day 0, Lane 2 is day 4, Lane 3 is day 8, Lane 4 is day 12, and Lane 5 is day 16. The expression level of tyrosinase is shown in Figure 5 .

[0324] The cosmetics prepared by the ASO specifically targeting tyrosinase prepared by the preparation method of the ASO specifically targeting tyrosinase of the present invention were tested for whitening efficacy - melanin skin model whitening test, and the testing organization was Shanghai Microspectra Testing Technology Group Co., Ltd.;

[0325] The test steps are as follows:

[0326]

[0327] Prepare a 24-well plate, add 0.5 mL of maintenance culture medium to each well, and transfer the model to the marked 24-well plate; each experimental group requires 3 models, and transfer all 24-well plates with models to an incubator for culture (37°C, 5% CO2).

[0328] Since the day the model was received (Day 0), the negative control (NC), positive control (PC) and sample groups were irradiated with UVB (50 mJ / cm2) every day, and the blank control group (BC) was not irradiated with UVB. The culture medium of the model was changed every day. The positive control group (kojic acid, 500 μg / mL) and the sample group were administered twice on Day 3 and Day 5, respectively, by surface administration, and the administration volume was 100 μL.

[0329] After the model was cultured continuously for 7 days (Day 7), samples were collected for testing.

[0330] Apparent color test:

[0331] After the model is cultured, use a camera to take photos of the appearance. The specific standard operation of taking photos is as follows: (1) Camera mode: manual; Photo parameters; Settings: focal length = 5.8 mm, aperture = f / 8, aperture F22, shutter speed = 1 / 80s, ISO = 1600. (2) Place the melanin skin model at the center of the colorimetric card and take photos.

[0332] Apparent brightness (L* value) test:

[0333] After the apparent colorimetric test, the L* value of the model is tested. The specific test operation is as follows: Place the model on a flat and hard white surface with the stratum corneum facing upwards, align the colorimeter detection hole vertically with the model surface for testing, repeat the reading three times for each model, and take the average value as the L* value reading of the single model. The tested model is placed in a clean EP tube for melanin content determination.

[0334] Melanin relative content test:

[0335] After the L* value test is completed, the melanin content of the model is tested. The test operation is as follows:

[0336] (1) Place the model in a 1.5 mL EP tube, label it, add 1 mL PBS buffer to each tube, vortex for 3 min, centrifuge at 2000 r / min at low temperature for 10 min, and discard the supernatant;

[0337] (2) Add 200 μL of distilled water, 500 μL of anhydrous ethanol and 500 μL of ether to the EP tube in step (1) in sequence, mix thoroughly, let stand at room temperature for 20 min, centrifuge at 3000 r / min for 5 min, and discard the supernatant;

[0338] (3) Add 1 mL of 1 mol / L NaOH aqueous solution containing 10% DMSO and heat in a water bath at 80°C for 40 min;

[0339] (4) After the heat incubation, pipette 200 μL of supernatant into the corresponding wells of a clearly labeled 96-well plate and read the OD value at 405 nm. Test two replicates of each model on a 96-well plate.

[0340] According to the apparent colorimetric results, after the culture was completed, the melanin model photos showed that compared with the BC group, the apparent colorimetric of the melanin model in the NC group was darker, indicating that more melanin was deposited on the surface of the model; compared with the NC group, the apparent colorimetric of the melanin model in the PC group and the sample group was lighter, indicating that less melanin was deposited on the surface of the model.

[0341] Apparent brightness L* value test results:

[0342] The L* value of the model after the apparent chromaticity test is measured, and the results are as follows: Figure 6 shown.

[0343] Group average value SD p-value Blank control (BC) 77.73 1.75 / Negative control (NC) 63.63 2.24 0.021# Positive Control (PC) 78.16 0.58 0.012* hTYRASO-3200nM 77.73 1.26 0.020*

[0344] When using the t-test two-tailed test method for statistical analysis, the significance of the NC group compared with the BC group is indicated by #, p-value < 0.05 is indicated by #, and p-value < 0.01 is indicated by ##. The significance of the sample group and PC group compared with the NC group is indicated by *, p-value < 0.05 is indicated by *, and p-value < 0.01 is indicated by **.

[0345] Based on the 3D melanin model apparent chromaticity L* value test, the apparent brightness of the NC group was significantly reduced compared with the BC group (p < 0.05), indicating that the UVB stimulation conditions of this experiment were effective.

[0346] Compared with the NC group, the apparent brightness of the PC group was significantly improved (p < 0.01), indicating that the positive control in this experiment was effective.

[0347] Compared with the NC group, the apparent brightness of the sample group was significantly improved, with a statistically significant difference (p < 0.05), indicating that cosmetics containing tyrosinase-targeted ASO have a good whitening effect.

[0348] The size of the absorbance value (OD) can reflect the relative content of melanin, and the test results are shown in the following table:

[0349] Group Average OD value SD p-value Blank control (BC) 0.1302 0.000 / Negative control (NC) 0.1860 0.002 0.000## Positive Control (PC) 0.1489 0.004 0.000** hTYRASO-3200nM 0.1371 0.007 0.001**

[0350] When using the t-test two-tailed test method for statistical analysis, the significance of the NC group compared with the BC group is indicated by #, p-value < 0.05 is indicated by #, and p-value < 0.01 is indicated by ##. The significance of the sample group and PC group compared with the NC group is indicated by *, p-value < 0.05 is indicated by *, and p-value < 0.01 is indicated by **.

[0351] based on 3D melanin skin model, sample hTYRASO-3 can improve the apparent chromaticity and apparent brightness (L* value) and reduce the melanin content after acting on the melanin skin model, and there is a statistical difference compared with the NC group (p<0.05). This shows that sample hTYRASO-3 has a whitening effect.

[0352] Example 5 The stability test of a specific tyrosinase targeting ASO of the present invention is carried out, and the test steps are as follows:

[0353] Using ESI source ionization spray technology, the sample is converted into moving charged ion fragments in negative ion mode, which are separated and recorded according to the mass-to-nuclear ratio (m / z).

[0354] The steps of mass spectrometry detection are as follows:

[0355] (1) Prepare 100 uM concentration series of hTYRASO for use;

[0356] (2) Use a 2 ml centrifuge tube with a well-sealed screw cap to take 500 μl of the nucleic acid solution and heat it in a 60°C water bath;

[0357] (3) Take 20ul of sample every week and dilute it 6 times with 100ul of ultrapure water;

[0358] (4) Mass spectrometry was used to detect base deficiency, thiolation deficiency, and depurination, with three replicates performed each time.

[0359] The results are as follows:

[0360]

[0361] The mass spectrometry results show that as the heating treatment time increases, part of the thio group is converted to oxo group. After 4 weeks of heating treatment, the proportion of thio groups successfully converted to oxo groups increased from 2% to 7.23%; as the heating treatment time increases, the proportion of missing bases does not increase, and the sequence is still a full-length sequence; as the heating treatment time increases, depurination is stable, and the aqueous solution is slightly acidic. Depurination will only occur under acidic conditions. It is expected that dissolving the probe with alkaline TE buffer can avoid depurination. This shows that the stability of the tyrosinase-targeting ASO of the present invention is good.

[0362] The liquid phase detection steps are as follows:

[0363] (1) Prepare 100 uM concentration series of hTYRASO for use;

[0364] (2) Use a 2 ml centrifuge tube with a well-sealed screw cap to take 500 μl of the nucleic acid solution and heat it in a 60°C water bath;

[0365] (3) Take 20ul samples every week;

[0366] (4) The liquid phase purity of the sequence was detected by Waters 2795+PDA996 detector.

[0367]

[0368] The results of HPLC showed that the samples were basically stable after 3 weeks of heating treatment, and the purity began to decrease rapidly in the fourth week; the deterioration of purity was mainly due to the conversion of part of the thio skeleton into an oxo skeleton; and part of the depurination caused the purity to decrease, which could be avoided by dissolving in alkaline TE buffer.

[0369] In the high performance liquid chromatography detection, the equipment was: waters 2795 + PDA996 detector; chromatographic column: XBridge Oligonucleotide BEH Column, 130A, 3.5 μm, 4.6 mm × 50 mm; column temperature: 60 ° C; flow rate: 0.5 ml / min; detection wavelength: 260 nm; loading concentration: 100 uM; loading volume: 10 ul; mobile phase A: 0.1 M TEAA, pH 7.2; mobile phase B: acetonitrile; elution gradient is as follows:

[0370]

[0371]

[0372] Example 6 A specific targeted tyrosinase of the present application was also subjected to MTC (maximum tolerated concentration test), irritation (zebrafish neutrophil inhibition rate test), sensitization (zebrafish trypsin content test) and developmental toxicity (zebrafish embryo short-term developmental toxicity test) tests. When the MTC (maximum tolerated concentration test) was 0.5 nM, no death or deformity was observed, it was not irritating in irritation (zebrafish neutrophil inhibition rate test), it was not sensitizing at a concentration of 200 nM in sensitization (zebrafish trypsin content test), and it was not developmentally toxic in the developmental toxicity (zebrafish embryo short-term developmental toxicity test) test.

[0373] The ASO specifically targeting tyrosinase of the present invention can specifically target tyrosinase mRNA, inhibit the expression of tyrosinase, thereby inhibiting the production of melanin for a long time, achieving the effect of whitening, and specifically degrading hTYR mRNA sequence, with high specificity, high safety, special chemical modification, high stability, and not easy to degrade; without the need for liposome delivery carriers, it can pass through the cell membrane and enter the cell nucleus to exert its effect.

[0374] The effective use concentration is low, the cost is controllable, and it is easy to use: it can be used alone or in combination with other conventional cosmetics, and is suitable for a variety of product forms, such as ointments, sprays, or freeze-dried powders.

[0375] In summary, after reading the present invention document, ordinary technicians in this field can make various other corresponding transformation schemes based on the technical scheme and technical concept of the present invention without creative mental work, which all fall within the scope of protection of the present invention.

Claims

1. An ASO specifically targeting tyrosinase, wherein the specific sequence thereof is any one of SEQ ID NO: 1-3 or a combination thereof.

2. The ASO according to claim 1, whose specific sequence is shown in SEQ ID NO:

3.

3. The method for preparing ASO according to any one of claims 1 to 2, characterized in that: The following synthetic routes are included: Wherein, the synthesis steps include deprotection, coupling, oxidation and capping reaction steps; in For a solid phase synthesis link.

4. The preparation method according to claim 3, characterized in that: The deprotection step reaction formula is as follows: The first base at the 3' end of the oligonucleotide is selected, and CPG linked to one of the bases A, G, C, and T is selected as a solid phase carrier. The 5'OH of this base is protected by a DMT protecting group. Before starting the synthesis, the DMT of the first base is first removed with trichloroacetic acid to expose the active 5'OH; For a solid phase synthesis link.

5. The preparation method according to claim 3, characterized in that: The coupling reaction formula is as follows: The residual trichloroacetic acid on the solid phase carrier is cleaned with anhydrous acetonitrile, tetrazole and the corresponding phosphoramidite monomer are added, and the phosphoramidite monomer activated by tetrazole reacts with 5'OH on the solid phase carrier to form a phosphite bond; wherein For a solid phase synthesis link.

6. The preparation method according to claim 1, characterized in that: The oxidation reaction formula is as follows: I2 oxidizes trivalent phosphorus to pentavalent phosphorus, forming a phosphodiester bond.

7. The preparation method according to claim 3, characterized in that: The capping reaction is as follows: The 5'OH group that did not participate in the reaction was blocked with an acetyl group.

8. The preparation method according to claim 3, characterized in that: It also includes a step of removing the solid phase carrier, and the reaction formula is as follows:

9. The preparation method according to claim 3, characterized in that: The chemical reaction equation for removing the amino protecting group is as follows:

10. The preparation method according to claim 3, characterized in that: The chemical reaction equation for removing the phosphate backbone protecting group is as follows:

11. A cosmetic, characterized in that: Comprising the ASO according to any one of claims 1-2.

12. Use of the ASO according to any one of claims 1 to 2 or the cosmetic according to claim 11 in skin whitening.

Images