ASO specifically targeting tyrosinase, preparation method thereof, and application thereof

Abstract

The present invention provides an ASO specifically targeting tyrosinase, a preparation method, and applications thereof. The ASO can specifically target tyrosinase mRNA and degrade the hTYR mRNA sequence, thereby inhibiting tyrosinase expression and thus long-term suppressing melanin production, achieving a whitening effect. The ASO has high specificity and safety, is specially chemically modified, has high stability, and is not easily degraded. It does not require a liposome delivery carrier and can independently penetrate the cell membrane and enter the cell to exert its effect. It has a low effective concentration, manageable costs, and is easy to use. It can be used alone or in combination with other conventional cosmetics and is adaptable to a variety of product forms, such as ointments, sprays, or freeze-dried powders.

Description

Technical Field

[0001] The present invention relates to the fields of cosmetics, medicine 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, abbreviated as TYR), also known as polyphenol oxidase, is a copper-binding protein that is a key enzyme in 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, chemically known as 5-hydroxy-2-hydroxymethyl-1,4-pyrone, inhibits the activity of the melanin-producing enzyme tyrosinase, resulting in a significant whitening effect without inhibiting other enzymes. Kojic acid has broad application in the daily chemical industry. While traditional whitening cosmetic ingredients, such as kojic acid, have some whitening effects, they suffer from safety concerns and unstable results. Summary of the Invention

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

[0006] The technical solution adopted by the present invention to solve the technical problem is: a method for preparing an ASO specifically targeting tyrosinase, 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 synthesized, and CPG linked to one of the bases A, G, C, and T is selected as the 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 needs to be oxidized to pentavalent phosphorus with I2 to form a stable phosphodiester bond. There are two ways of oxidation: oxo and thio; the residual trichloroacetic acid on the solid support is cleaned with anhydrous acetonitrile, and tetrazole and the corresponding phosphoramidite monomer are added. The tetrazole-activated phosphoramidite monomer reacts with the 5'OH on the solid support 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; 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 de-DMT is completed, the residual trichloroacetic acid on the solid support is cleaned with anhydrous acetonitrile, and tetrazole and the corresponding phosphoramidite monomer are added. The tetrazole-activated phosphoramidite monomer reacts with the 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 groups, 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 groups 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 synthesized.

[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 typically use a butadienylamino group to immobilize the 3' end of the oligonucleotide on a solid support. This group stabilizes all reagents during the synthesis process. After synthesis, ammonia is used to remove the group, separating the oligonucleotide from the solid support, forming an oligonucleotide with a 3'OH group. Simultaneously, ammonia can remove the protecting groups of the primary amino groups of pyrimidines and purines, as well as the cyanoethyl protecting group on the oligonucleotide's phosphate backbone, at room temperature. Only oligonucleotides with both the amino protecting group and the phosphate backbone protecting group completely removed exhibit biological activity.

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

[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 ASOs specifically targeting tyrosinase are 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 method for preparing the tyrosinase-specific target 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 tyrosinase-specific ASO of the present invention can specifically target tyrosinase mRNA, inhibiting the expression of tyrosinase, thereby long-term inhibiting melanin production, achieving a whitening effect, and specifically degrading the hTYR mRNA sequence. It has high specificity, high safety, special chemical modification, high stability, and is not easily degraded;

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

[0047] 3. By combining the latest RNA advanced structure prediction algorithms, we designed multiple antisense nucleic acid sequences in the single-stranded region of tyrosinase mRNA. After screening, we obtained sequences with high inhibition efficiency and strong specificity.

[0048] 4. 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 between 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. Because phosphorothioate bonds are more resistant to nuclease degradation than unmodified phosphate bonds (P=O), modified antisense nucleic acids can persist in the body for longer periods of time, thereby improving their inhibitory efficacy. Phosphorothioate modification can also enhance the binding affinity of antisense nucleic acids to target RNA. This is because the introduction of sulfur atoms changes the three-dimensional structure of the antisense nucleic acid molecule, helping it to bind more stably to the target RNA.

[0051] Phosphorothioate-modified antisense nucleic acids are more easily absorbed by cells. This modification alters the negative charge distribution of the antisense nucleic acid, facilitating its passage through cell membranes and thereby increasing its intracellular concentration. Chemical modification with cholesterol or C16 at the 3' or 5' end of the antisense nucleic acid enhances its cell membrane penetration, allowing it to enter cells and exert its effects without the need for additional delivery materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to illustrate the technical solution more clearly, the following is a brief introduction to the drawings required for use in the implementation. 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 any 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 that specifically targets tyrosinase according to 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 specifically targeting tyrosinase of the present invention.

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

[0058] Figure 6This is a schematic diagram of the L* value measurement results of the model after the apparent chromaticity test is completed 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 melanin skin model whitening test, a whitening efficacy test specifically targeting tyrosinase according to the present invention. DETAILED DESCRIPTION

[0060] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments of the present invention and corresponding drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts 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 synthesized.

[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 the 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. The reaction equation is shown below:

[0068]

[0069] The coupling reaction includes the following steps: After the DMT is removed, the residual trichloroacetic acid on the solid support is cleaned with anhydrous acetonitrile, tetrazole and the corresponding phosphoramidite monomer are added, and the tetrazole-activated phosphoramidite monomer reacts with the 5'OH on the solid support 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 oxo and thio; the phosphite bond formed after the coupling reaction is unstable under acidic conditions; I2 is required to oxidize trivalent phosphorus to 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, a purification step is further included, in which ion-pair reversed-phase high-performance liquid chromatography is used to purify oligonucleotides. The hydrophobicity of oligonucleotides of different lengths is different. As the elution intensity increases, salts, short fragments that failed to be synthesized, long fragments, target fragments, and fragments from which the protecting group removal failed are eluted in sequence, thereby achieving the purpose of purifying the oligonucleotides. 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 specifically targeted tyrosinase can be incorporated into cosmetic formulations, such as ointments, sprays, or freeze-dried powders, as needed. Specific uses include, but are not limited to: using it as an active ingredient in whitening cosmetics, such as in whitening creams and serums; and combining it with sunscreen ingredients to create sunscreen sprays, providing both whitening and sun protection.

[0080] The present invention is a method for specifically targeting tyrosinase by designing 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. 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, effectively enhancing 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), this allows the modified antisense nucleic acid to 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 the sulfur atom changes the three-dimensional structure of the antisense nucleic acid molecule, which helps it to bind more stably to the target RNA. Phosphorothioate-modified antisense nucleic acids are more easily absorbed by cells. This modification alters the negative charge distribution of the antisense nucleic acid, facilitating its passage through cell membranes and thereby increasing its intracellular concentration. Chemical modification with cholesterol or C16 at the 3' or 5' end of the antisense nucleic acid enhances its cell membrane penetration, allowing it to enter cells and exert its effects without the need for additional 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 supplies:

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

[0089] Test method:

[0090] (1) Cell recovery

[0091] 1) Preheat a 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). Remove the cryotube;

[0095] 5) Thaw quickly by placing the cryovial into a preheated water bath and shaking it constantly to melt the liquid in the tube. Remove the tube when there is still a little bit of liquid left in the tube.

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

[0097] 7) Prepare a cell suspension by transferring the cells to a 15ml centrifuge tube and adding preheated culture medium drop by drop while shaking the tube. The amount of culture medium added should reach at least 10ml.

[0098] 8) Centrifuge at 800 rpm for 5 minutes in a low-speed centrifuge; aspirate 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°C incubator containing CO2 for culture. The time for changing the medium is determined by the cell sedimentation rate.

[0100] (2) Cell culture

[0101] 2.1) Preheat a 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) Remove the cell culture flask and perform sterile operation;

[0105] 2.5) Open the bottle cap and remove the old culture medium;

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

[0107] 2.7) Trypsinize the cells: Add trypsin-EDTA solution (1 ml / 25 cm², 2 ml / 75 cm²) to the rinsed cells and gently rinse the bottom of the cell dish. Aspirate the trypsin-EDTA solution and place the dish in a 37°C incubator for 2-3 minutes. Gently tap the sides of the culture flask to release most of the cells. Observe under an inverted microscope. When the cells begin to dissociate and appear granular, add an appropriate amount of fresh serum-containing culture medium to terminate the trypsin treatment.

[0108] 2.8) Pipette up and down several times to break up cell clumps. Mix thoroughly, then add 3n (n is the number of flasks to be passaged) ml of MEM medium and transfer to a new culture flask according to the dilution ratio.

[0109] 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.

[0110] (III) Cell transfection and ASO screening

[0111] 3.1). ASO screening and transfection plan:

[0112] (1) Plating: Seed 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 confluency 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 medium containing 20% ​​fetal bovine serum to each well;

[0122] b. Dissolve ASO in RNAase-free deionized water to a final concentration of 20 μM. Dissolve ASO in 500 μL of opti-MEM, mix well, and let stand.

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

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

[0125] d. Combine tubes A and B, mix thoroughly, and let stand for 20 minutes;

[0126] e. Add to each well respectively;

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

[0128] After 4-6 hours, aspirate 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 minutes;

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

[0135] Centrifuge at 14,000 g for 15 min at 4°C until the tube is separated into three layers, with RNA in the upper aqueous phase, which is transferred to another new RNase-free 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 the RNA precipitate and discard the 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 of RNA sample and dilute it 50-fold. Measure the OD value on a BioPhotometerplus nucleic acid and protein analyzer. An OD260 / OD280 ratio greater than 1.8 indicates that the prepared RNA is relatively pure and free of protein contamination.

[0142] 2) Total RNA integrity test: 1 μl of RNA sample was subjected to 1% agarose gel electrophoresis at 80 V for 20 min. The 5s rRNA, 18s rRNA, and 28s rRNA bands of the total RNA were observed using a gel imaging system. If all three bands were intact, the total RNA extraction was considered 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, 60℃32s plate reading, 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 a 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. Remove the cryotube;

[0175] 1.5. Thaw quickly. Place the cryovial in a preheated water bath and shake it constantly to melt the liquid in the tube. Remove the tube when there is still a little bit of liquid left in the tube.

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

[0177] Prepare a cell suspension by transferring the cells to a 15 ml centrifuge tube and adding preheated culture medium dropwise while shaking the tube. Ensure the total volume of culture medium added is at least 10 ml.

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

[0179] 1.9. Divide the cell suspension into culture dishes and place them in a 37°C 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 a 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. Remove the cell culture flask and perform sterile operation;

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

[0186] Wash cells once or twice with PBS.

[0187] 2.7. Trypsinization of cells: Add trypsin-EDTA solution (1 ml / 25 cm², 2 ml / 75 cm²) to the rinsed cells and gently rinse the bottom of the cell dish. Aspirate the trypsin-EDTA solution and place the dish in a 37°C incubator for 2-3 minutes. Gently tap the sides of the culture flask to release most of the cells. Observe under an inverted microscope. When the cells begin to detach and appear spherical, add an appropriate amount of fresh culture medium containing serum to terminate the trypsin treatment.

[0188] 2.8. Pipette up and down several times to break up cell clumps. Mix thoroughly, then add 3n (n is the number of flasks to be passaged) ml of MEM medium and transfer to a new culture flask 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, to ensure consistent loading of each protein sample, the protein concentration of each protein sample needs to be determined. Depending on the lysis buffer 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 prestained protein molecular weight standards.

[0211] During electrophoresis, it is generally recommended to use a low constant voltage for the upper gel and a high constant voltage for the lower gel when bromophenol blue enters the lower gel. For Bio-Rad's standard electrophoresis apparatus or similar, the low voltage can be set to 80-100V, and the high voltage can be set to around 120V. For convenience, it is also possible to use a constant voltage for the entire SDS-PAGE process, typically setting the voltage to 100V and setting a timer of 90-120 minutes. Setting a timer can prevent the common problem of over-electrophoresis.

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

[0213] 4.3.3. Transfer

[0214] Typically, if using a Bio-Rad standard wet transfer apparatus, the transfer current can be set to 300-400 mA for 30-60 minutes. Alternatively, transfer can be performed overnight at 15-20 mA. The specific transfer time depends on the size of the target protein. Larger target proteins require longer transfer times, while smaller target proteins require shorter transfer times.

[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 complete, immediately place the protein membrane in the pre-prepared Western wash solution and rinse for 1-2 minutes to wash away the transfer solution on the membrane. From the transfer to all steps, be sure to keep the membrane moist and avoid drying out, otherwise it will easily produce a high background.

[0219] Block the plate at room temperature for 60 minutes with slow shaking on a shaker. For 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] Aspirate the blocking solution using a micro-tabletop vacuum pump or pipette. Immediately add the diluted primary antibody and incubate for one hour at room temperature or at 4°C on a side-by-side rocker with slow shaking. If the primary antibody is not effective after one hour, incubate overnight at 4°C with slow shaking. Alternatively, select an appropriate incubation temperature and time according to the antibody's instructions.

[0223] Recover the primary antibody. Add Western wash buffer and wash for 5-10 minutes on a rocking platform. Aspirate the wash buffer completely, then add more wash buffer and wash for 5-10 minutes. Wash three times in total. If background is high, extend the wash time and increase the number of washes.

[0224] 4.3.6. Secondary antibody incubation

[0225] Refer to the secondary antibody's instructions and dilute the horseradish peroxidase (HRP)-conjugated secondary antibody in Western secondary antibody diluent at the appropriate ratio. The secondary antibody should be selected based on the primary antibody. Aspirate the wash buffer using a micro-tabletop vacuum pump or pipette. Immediately add the diluted secondary antibody and incubate for one hour at room temperature or 4°C on a side-by-side rocker with gentle agitation.

[0226] Recover the secondary antibody. Add Western wash buffer and wash for 5-10 minutes on a rocking platform with slow rocking. After the wash buffer is completely aspirated, add more wash buffer and wash for 5-10 minutes. Wash three times in total. If the background is high, extend the wash time and increase the number of washes.

[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 cassette.

[0229] Films can be developed using an automatic X-ray film developer. If an automatic film developer is not available, manually develop the films by preparing the developer and fixer using a developer / fixer kit. For X-ray films, we recommend using Kodak's original Kodak X-OMAT BT film, specifically formulated 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 Determine melanin content using the modified method proposed by Hideya Ando et al. Take the reserved cell suspension and add PBS buffer to adjust the cell density to 5×10^5 / ml;

[0235] 6.2 From each group, 1 mL of cell suspension was taken and placed into three parallel stoppered colorimetric tubes. After centrifugation, the supernatant was discarded and 200 μL of distilled water was added to resuspend the cells.

[0236] 6.3 Then add 1 mL of a 1:1 ethanol-ether solution to dissolve the non-melanin opaque particles. Let stand 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 table below:

[0242]

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

[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 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.

[0247] Reagents used:

[0248] 1.SDS-PAGE reagents:

[0249] 1.1 5x sample buffer (10 ml): 0.6 ml 1 mol / L Tris-HCl (pH 6.8), 5 ml 50% glycerol, 2 ml 10% SDS, 0.5 ml mercaptoethanol, 1 ml 1% bromophenol blue, 0.9 ml distilled water. Can be stored at 4°C for several weeks or at -20°C for several months.

[0250] 1.2 Gel stock solution: In a fume hood, weigh 30g acrylamide and 0.8g methylene bisacrylamide, dissolve in heavy distilled water, and adjust the volume to 100ml. Filter and place in a brown bottle and store at 4°C. It can generally 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: Dissolve 5.98 g of Tris in 48 ml of 1 mol / L HCl and add 80 ml of heavy distilled water to adjust the pH to 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 redistilled water);

[0255] 1.7 pH 8.3 Tris-Glycine Electrode Buffer: Weigh 6.0g Tris and 28.8g glycine, add approximately 900ml of distilled water, adjust the pH to 8.3, and then dilute to 1000ml with distilled water. Store at 4°C and dilute 10-fold 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: Coomassie Brilliant Blue 0.2g; Methanol 80ml; Acetic acid 2ml; ddH2O 118ml. Coating solution (5% skim milk powder, freshly prepared): Dissolve 1.0g skim milk powder in 20ml of 0.01M PBS.

[0260] 6. Color development 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 a 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. Remove the cryotube;

[0273] 1.5. Thaw quickly. Place the cryovial in a preheated water bath and shake it constantly to melt the liquid in the tube. Remove the tube when there is still a little bit of liquid left in the tube.

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

[0275] Prepare a cell suspension by transferring the cells to a 15 ml centrifuge tube and adding preheated culture medium dropwise while shaking the tube. Ensure the total volume of culture medium added is at least 10 ml.

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

[0277] 1.9. Divide the cell suspension into culture dishes and place them in a 37°C 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 a 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. Remove the cell culture flask and perform sterile operation;

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

[0284] Wash cells once or twice with PBS.

[0285] 2.7. Trypsinization of cells: Add trypsin-EDTA solution (1 ml / 25 cm², 2 ml / 75 cm²) to the rinsed cells and gently rinse the bottom of the cell dish. Aspirate the trypsin-EDTA solution and place the dish in a 37°C incubator for 2-3 minutes. Gently tap the sides of the culture flask to release most of the cells. Observe under an inverted microscope. When the cells begin to detach and appear spherical, add an appropriate amount of fresh culture medium containing serum to terminate the trypsin treatment.

[0286] 2.8. Pipette up and down several times to break up cell clumps. Mix thoroughly, then add 3n (n is the number of flasks to be passaged) ml of MEM medium and transfer to a new culture flask 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 test

[0291] 1. Total RNA Extraction

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

[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 it stand at room temperature for 2 minutes;

[0294] (3) Centrifuge at 14,000 g for 15 min at 4°C. The solution will be separated into three layers, with RNA 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 minutes;

[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 of RNA sample and dilute it 50-fold. Measure the OD value on a BioPhotometerplus nucleic acid protein analyzer. An OD260 / OD280 ratio greater than 1.8 indicates that the prepared RNA is relatively pure and free of protein contamination.

[0301] 2) Total RNA integrity test: 1 μl of RNA sample was subjected to 1% agarose gel electrophoresis at 80 V for 20 min. The 5s rRNA, 18s rRNA, and 28s rRNA bands of the total RNA were observed using a gel imaging system. If all three bands were intact, the total RNA extraction was considered 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°C for 30 min;

[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, 60℃32s plate reading, 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 according to the preparation method of the present invention were subjected to a whitening efficacy test - a melanin skin model whitening test. The testing institution 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 labeled 24-well plate; each experimental group requires 3 models, and transfer all 24-well plates containing models to an incubator for culture (37°C, 5% CO2).

[0328] Starting from the day the model was received (Day 0), the negative control (NC), positive control (PC), and sample groups were exposed to UVB (50 mJ / cm²) daily. The blank control (BC) group was not exposed to UVB. The culture medium was changed daily. The positive control group (kojic acid, 500 μg / mL) and the sample group were dosed twice, on Day 3 and Day 5, respectively. The administration method was topical administration, and the volume of the drug was 100 μL.

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

[0330] Apparent colorimetry test:

[0331] After the model is incubated, take a photo of the skin using a camera. The standard procedures for taking photos are as follows: (1) Camera mode: Manual; Photo parameters: Focal length = 5.8 mm, Aperture = f / 8, F22, Shutter speed = 1 / 80 s, ISO = 1600. (2) Place the melanin skin model in the center of the color chart and take a photo.

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

[0333] After the apparent colorimetric test, the model is tested for L* value. The specific test procedure is as follows: Place the model on a flat, hard white surface with the cuticle facing upwards. Align the colorimeter aperture vertically with the model surface. Repeat the reading three times for each model, and take the average value as the L* value for each model. After testing, place the model 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 of PBS buffer to each tube, shake on a vortex shaker for 3 minutes, centrifuge at 2000 rpm for 10 minutes, 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 rpm 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 an 80°C water bath for 40 min;

[0339] (4) After the heat incubation, 200 μL of supernatant was transferred to the corresponding wells of a clearly labeled 96-well plate and the OD value was read at 405 nm. Each model was tested in duplicate on the 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 color 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 statistical analysis was performed using the two-tailed t-test, significance was indicated by # when comparing the NC group with the BC group, p-value < 0.05 was indicated by #, and p-value < 0.01 was indicated by ##. When comparing the sample group and the PC group with the NC group, significance was indicated by *, p-value < 0.05 was indicated by *, and p-value < 0.01 was 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 in 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 statistical difference (p < 0.05), indicating that cosmetics containing tyrosinase-targeted ASO have good whitening effects.

[0348] The absorbance value (OD) can reflect the relative content of melanin. 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 statistical analysis was performed using the two-tailed t-test, significance was indicated by # when comparing the NC group with the BC group, p-value < 0.05 was indicated by #, and p-value < 0.01 was indicated by ##. When comparing the sample group and the PC group with the NC group, significance was indicated by *, p-value < 0.05 was indicated by *, and p-value < 0.01 was indicated by **.

[0351] based on In a 3D melanin skin model, hTYRASO-3 increased apparent chromaticity and apparent brightness (L* value) and reduced melanin content, with statistically significant differences compared to the NC group (p<0.05). This suggests that hTYRASO-3 has a whitening effect.

[0352] Example 5 The stability test of a specific tyrosinase-targeting ASO of the present invention was conducted. The test steps were as follows:

[0353] The ESI source ionization spray technology was used in negative ion mode to convert the sample into moving charged ion fragments, which were 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 μM concentration series of hTYRASO for use;

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

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

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

[0359] Here are the results:

[0360]

[0361] Mass spectrometry results indicate that as the heat treatment time increases, some thio groups convert to oxo groups. After four weeks of heat treatment, the percentage of successfully converted thio groups to oxo groups increased from 2% to 7.23%. The percentage of missing bases did not increase with increasing heat treatment time, and the sequence remained full-length. Depurination stabilized with increasing heat treatment time. Depurination only occurs under slightly acidic conditions in the aqueous solution, and dissolving the probe in a slightly alkaline TE buffer is expected to prevent depurination. This demonstrates the excellent stability of the tyrosinase-targeting ASO of the present invention.

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

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

[0364] (2) Place 500 μl of nucleic acid solution in a 2 ml centrifuge tube with a well-sealed screw cap 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 sample was basically stable after three weeks of heat treatment, but 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. There was also a part of depurination that led to the decrease in purity, which could be avoided by dissolving in alkaline TE buffer.

[0369] High-performance liquid chromatography (HPLC) was performed using a Waters 2795 detector and a PDA996 detector. The chromatographic column was an XBridge Oligonucleotide BEH Column, 130A, 3.5 μm, 4.6 mm × 50 mm. The column temperature was 60°C. The flow rate was 0.5 ml / min. The detection wavelength was 260 nm. The sample concentration was 100 μM. The sample volume was 10 μL. The mobile phase A was 0.1 M TEAA, pH 7.2. The mobile phase B was acetonitrile. The elution gradient was as follows:

[0370]

[0371]

[0372] Example 6 A specific targeted tyrosinase of the present application was also tested for MTC (maximum tolerated concentration), irritation (zebrafish neutrophil inhibition rate test), sensitization (zebrafish trypsin content test) and developmental toxicity (zebrafish embryo short-term developmental toxicity test). When the MTC (maximum tolerated concentration test) was 0.5 nM, no death or deformity was observed, it was not irritating in the irritation (zebrafish neutrophil inhibition rate test), it had no sensitizing effect at a concentration of 200 nM in the sensitization (zebrafish trypsin content test), and it had no developmental toxicity 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 long-term inhibiting the production of melanin, achieving the effect of whitening, and specifically degrading the hTYR mRNA sequence. It has high specificity, high safety, special chemical modification, high stability, and is not easy to degrade; it does not require a liposome delivery carrier and 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 solutions and technical concepts of the present invention without creative mental work, and all of them fall within the scope of protection of the present invention.

Claims

1. An ASO specifically targeting tyrosinase, wherein the specific sequence is / +C / * / +T / * / +G / * / +A / * / *C*C*T*C*C*C*A*T / +G / * / +T / * / +A / * / +C / -3' Chol, wherein + represents LNA modification, * represents phosphorothioate modification, and 3'Chol represents 3'-terminal cholesterol modification.

2. A cosmetic, characterized in that: Containing the ASO according to claim 1.

3. Use of the ASO according to claim 1 or the cosmetic according to claim 2 in the preparation of skin whitening products.

Images