DNA APTAMER SPECIFICALLY BINDING TO GLUTATHIONE AND USE THEREOF

Abstract

The present invention relates to a single-stranded DNA aptamer that binds to glutathione to inhibit oxidation of the glutathione or stabilize the glutathione, characterized in that the single-stranded DNA aptamer has one or more stem-loop structures; a method for oxidation prevention and stabilization of glutathione using the aptamer; and application to various fields such as pharmaceuticals, cosmetics, and food, using the aptamer. The aptamer of the present invention can be applied to various fields, such as pharmaceuticals, cosmetics, and food, which require oxidation prevention and stabilization of glutathione.

Claims

1. A DNA aptamer selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 1 to 26 that binds to glutathione to inhibit oxidation of glutathione or stabilize glutathione.

2. A method of stabilizing glutathione by treating the aptamer of claim 1 to glutathione.

3. A cosmetic composition comprising the aptamer of claim 1 as an active ingredient.

4-6. (Canceled)

Description

DESCRIPTION OF DRAWINGS

[0060] FIG. 1 is a diagram of maintaining a stable state of glutathione through hydrogen bonding between the base constituting the aptamer and the thiol group of glutathione.

[0061] FIG. 2 shows the SELEX process for producing an aptamer that specifically binds to glutathione.

[0062] FIG. 3 is a graph confirming the Tyrosinase activity rate of Aptamin G1 (SEQ ID NO: 24), Aptamin G4 (SEQ ID NO: 25), Aptamin G12 (SEQ ID NO: 26) that specifically binds to GSH;

[0063] FIG. 4 is a graph confirming the tyrosinase activity rate of GSH-specific binding Aptamin G1 (SEQ ID NO: 17), Aptamin G4 (SEQ ID NO: 18), Aptamin G12 (SEQ ID NO: 19) by a cell test,

[0064] FIG. 5 is a graph of the antioxidant efficacy test (DTNB assay) of Aptamin G1 (SEQ

[0065] ID NO: 24), Aptamin G4 (SEQ ID NO: 25), and Aptamin G12 (SEQ ID NO: 26) that specifically binds to GSH.

MODE FOR INVENTION

[0066] Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are described with the intention of illustrating the present invention, and the scope of the present invention is not to be construed as being limited by the following examples.

[0067] In the present invention, in order not to rapidly oxidize the target (glutathione), all buffers and solutions were stirred in Chelex®100 resin (BioRad) for 1 hour, filtered through a 0.2 μm filter, and sprayed with N.sub.2 gas (Praxair) for 10 minutes. Thus, it was prepared using molecular biology grade water (Phenix Research) from which incidental metals were removed.

Example 1: DNA Aptamer Selection and Sequencing

Glutathione SELEX:

[0068] Nine rounds of SELEX against glutathione were performed using a DNA library composed of ˜10.sup.15 unique oligonucleotides. The buffer composition used was as follows: 50 mM Sodium Acetate pH 5.5 (Sigma), Phosphate-buffered saline (PBS) with 1 mM MgCl.sub.2, 0.05% Tween 20 (Sigma), 1% BSA (Sigma) and 1 mM glutathione (Sigma). The stringency of SELEX was changed by reducing the binding time of the aptamer to the target, changing the buffer composition, and reducing the concentration of the target in free molecule elution (FIG. 2).

DNA Aptamer Selection

[0069] Bioinformation analysis of the abundant library produced by the SELEX method obtained candidate aptamers, screened for the ability to protect glutathione from oxidation from these top 26, and the results are shown in Table 1 below.

TABLE-US-00001 TABLE 1 SEQ sequence ID size NO: selected sequence (bp) 1 GACCAACGGAAGCGCGGCACCACA 30 ACGGTG 2 CGAACAGCATGGAGGCGCGCCCGT 43 TGTGCCGTGCGCGCGGGAT 3 GGCACGCAGTGTGACGCGCCTCGT 43 CGTTCACTCGGCGCGGGAT 4 GCACGGCACAACGGGCGCGCCTCC 43 ATGCTGTTCGGCGCGGGAT 5 CGAGTGAACGACGAGGCGCGTCAC 43 ACTGCGTGCCGCGCGGGAT 6 CGAGTCAGTGCGAGGCGCTCCCCT 41 GTCGGTGCGCGCGGGAT 7 GCACCGACAGGGGAGCGCCTCGCA 41 CTGACTCGGCGCGGGAT 8 ACGCATGCCGGGCGCGCTCCCTGTC 38 GTCCGCGCGGGAT 9 CGACTACGAGGAGGCGCGCACCAC 29 ACGTT 10 AACGTACGTGGAGCGGCTCCCTGC 38 ACTGCGCGCGGGAT 11 GCAGTGCAGGGAGCCGCTCCACGT 38 ACGTTGCGCGGGAT 12 GGTCTGCCGGGGCCGCACCTCCTGT 39 CGTCGGCGCGGGAT 13 GCACAATCGGGGCGCGCTCGTCCTC 40 TGGCCGGCGCGGGAT 14 CGGCCAGAGGACGAGCGCGCCCCG 40 ATTGTGCGCGCGGGAT 15 GGACGACAGGGAGCGCGCCC 20 16 CGACGACAGGAGGTGCGGCCCCGG 39 CAGACCGCGCGGGAT 17 CGAGTGAGGGCGAGGCGCGACGTC 43 CCTTCGGTCCGCGCGGGAT 18 CGAGTCAGTGCGAGGCGCGCTCCT 41 GCCGTTGCGCGCGGGAT 19 GGACCGAAGGGACGTCGCGCCTCG 43 CCCTCACTCGGCGCGGGAT 20 CGACGGAGGGAGGCGCGCACCACA 28 CGTT 21 GACCAACGGAGCGCGGCCCACAAC 27 GGT 22 GACATCAGGAGCGCGCCCCGTCAC 25 G 23 GACCAACGGAGCGCGGCCCACAAC 28 GGTG 24 CGAACAGCATGGAGGCGCGCCCGT 34 TGTGCCGTGC 25 CGAGTGAACGACGAGGCGCGTCAC 30 ACTGCG 26 GGACGACAGGGAGCGCGCCCGGCA 30 TGCGTG

Example 2: Whitening Efficacy Test In vitro Tyrosinase Activity (N=5)

[0070] The final concentrations of each component in this experiment were 20 μM GSH, 0.2 μM Apt G, and 0.01% arbutin.

[0071] The experimental method is as follows.

[0072] After dissolving in 3.sup.th distilled water that reacts specifically with GSH (glutathione) obtained through the SELEX, it was boiled at 95° C. for 5 minutes, and then the temperature was gradually lowered to room temperature to form a tertiary structure.

[0073] 300 μM GSH and 3 μM of the prepared aptamer (molar concentration of 100:1) were dissolved in tertiary distilled water and reacted at room temperature for 30 minutes.

[0074] In a 96 well plate, 200 μL of 0.1 M potassium phosphate buffer (pH6.5), 20 μL of sample, 20 μL of 2 KU/mL tyrosinase, and 60 μL of 1.0 mM tyrosine were sequentially added and reacted at 37° C. for 10 minutes.

[0075] After the reaction, absorbance was measured at 490 nm using a plate reader.

[0076] The results are shown in FIG. 3. FIG. 3 is a graph confirming the Tyrosinase activity rate of Aptamin G1 (SEQ ID NO: 24), Aptamin G4 (SEQ ID NO: 25), Aptamin G12 (SEQ ID NO: 26) that specifically binds to GSH,

[0077] As a result of FIG. 3, it was confirmed that GSH inhibited the activity of tyrosinase by about 75%, and when complex with the Aptamin G sequence, it was confirmed that the inhibition of tyrosinase activity of GSH was increased by more than 10%.

[0078] For reference, the G1 sequence used in the present invention, SEQ ID NO: 24: CGAACAGCATGGAGGCGCGCCCGTTGTGCCGTGC, is a part of SEQ ID NO: 2 in the patent,

[0079] The G4 sequence, SEQ ID NO:25:CGAGTGAACGACGAGGCGCGTCACACTGCG, is part of SEQ ID NO:5 in the patent.

Example 3: Whitening Efficacy Test In vitro Cell-Based Tyrosinase Activity (N=5, Arb only N=3)

[0080] The final concentration of each component in this experiment was 100 μM GSH, 1 μM Apt G, 0.01% arbutin,

[0081] The experimental method is as follows.

[0082] After dissolving in 3.sup.th distilled water that reacts specifically with GSH (glutathione) obtained through the SELEX, it was boiled at 95° C. for 5 minutes, and then the temperature was gradually lowered to room temperature to form a tertiary structure. 100 μM GSH and 1 μM of the prepared aptamer (molar concentration of 100:1) were added to the media and reacted at room temperature for 30 minutes.

[0083] MNT-1 cells were seeded on a 6-well plate and incubated for 24 hours at 37° C., 5% CO.sub.2 incubator.

[0084] After treating each of the prepared samples, the samples were again incubated at 37° C. and 5% CO.sub.2 incubator for 72 hours.

[0085] After incubation, ice-cold lysis buffer (RIPA buf.+protease inhibitor) was used to remove the cells from the plate, and the removed cells were transferred to an EP-tube and centrifuged to remove only the supernatant.

[0086] Lysate, L-DOPA, potassium phosphate buffer (pH6.5) was added to a 96 well plate and mixed (each test group used the same amount of protein through protein quantification).

[0087] After incubation for 1 hour at 37° C., 5% CO.sub.2 incubator, absorbance was measured at 475 nm using a plate reader.

[0088] The results are shown in FIG. 4.

[0089] FIG. 4 is a graph confirming the tyrosinase activity rate of Aptamin G1 (SEQ ID NO: 24), Aptamin G4 (SEQ ID NO: 25), and Aptamin G12 (SEQ ID NO: 26) that specifically binds to GSH by cell test;

[0090] As a result of FIG. 4, GSH showed a tendency not to inhibit the activity of tyrosinase, and when complexed with the Aptamin G sequence, it inhibited about 25% of the tyrosinase activity of GSH, which had similar inhibitory efficacy as 0.01% Arbutin, a positive control. visible was confirmed.

Example 4: Antioxidant Efficacy Test DTNB Assay (Sulfhydryl Group (—SH) Determination, n=5)

[0091] In this experiment, the final concentration of each component was 125 μM GSH and 1.25 μM aptamin G

[0092] The experiment is as follows.

[0093] After dissolving in 3th distilled water that reacts specifically with GSH (glutathione) obtained through the SELEX, and then boiled at 95° C. for 5 minutes, and then slowly lowered the temperature to the room temperature to form a tertiary structure.

[0094] The prepared aptamer (100: 1) of the prepared aptamer (100: 1) of 250 μM GSH and 2.5 μM was dissolved in the 3.sup.th distilled water for 30 minutes at room temperature.

[0095] The above GSH-Aptamin G complex was stored for a certain period of time under severe conditions (37° C.), and samples were obtained every 4 days and measured for 16 days.

[0096] Just before the sample measurement, 10 mm DTNB (4 mg/ml, buffer: 0.1 M potassium Phosphate buffer, pH 8.0) was prepared to mix the sample (50 μL) of DTNB solution and shaking incubation.

[0097] After the reaction, the UV Absorbance value was measured at 412 nm and REF wavelength 605 nm using the Plate Reader.

[0098] The result was shown in FIG. 5.

[0099] FIG. 5 is an antioxidant efficacy test of Aptamin G1 (SEQ ID NO: 24), Aptamin G4 (SEQ ID NO: 25), and Aptamin G12 (SEQ ID NO: 26).

[0100] As a result of FIG. 5, the GSH was steadily oxidized for 16 days, and the amount of GSH remaining was about 60%, but on day 16, it was confirmed that about 85% or more of GSH remained unoxidized by Aptamins G1, G4, and G12 (aptamers of SEQ ID NOs: 24, 25 and 26, respectively).