Aptamer specifically binding to L-Ascorbic acid and use of the same

Abstract

The present invention relates to a single-stranded DNA aptamer which inhibits oxidation by binding to L-ascorbic acid, characterized in that the single stranded DNA aptamer has at least one of stem-loop structure, has CG bond at the end of the stem-loop structure, and both of the beginning parts of the stem-loop structure are G or C, in which the same bases are faced to each other. The aptamer of the present invention has an anti-oxidative effect of vitamin C, it can be used for functional cosmetics of various formulations, oral nutritional supplements, foods, etc. by maintaining the reduced state of vitamin C using the aptamer which selectively binds to vitamin C to maintain the anti-oxidative function of vitamin C for a long time. In addition, it can be anticipated that the anti-oxidative effect is continued and maximized even with a small amount of vitamin C by using the aptamer selectively binding to vitamin C.

Claims

1. A single-stranded DNA aptamer which inhibits oxidation by binding to L-ascorbic acid, the single stranded DNA aptamer having at least one from 5 to 3 end direction CG-N1-XG step-loop structure, wherein C is prior to an unpaired loop sequence G-N1-X in which N1 is any of 1 to 3 nucleotides and X is G or T, and G is subsequent to the unpaired loop sequence and forms a C-G bond with the C.

2. The single-stranded DNA aptamer of claim 1, the DNA aptamer comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 4, 5, 17, 22, 23 and 26.

3. An anti-oxidative composition comprising the aptamer of claim 1 as an active ingredient.

4. A method for inhibiting oxidation of vitamin C by treating the vitamin C with the aptamer of claim 1.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a photograph of 12% native gel electrophoresis used for isolating a single stranded DNA library. The band indicated by blue squares was cut and purified by the Crush and Soak method.

(2) FIG. 2 is an OPDA measurement graph for identifying ascorbic acid-antioxidant effect of single stranded DNA. The antioxidant effect was observed in the single stranded DNA of SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 5.

(3) FIG. 3 is a graph of Microscale Thermophoresis (MST) measurement results of selected single stranded DNA. As a result of the binding dissociation constant (Kd) measurement, the binding force was confirmed at the level of nano molarity (nM) in the single stranded DNA of SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 5.

(4) FIG. 4 is a secondary folding structure of an aptamer using an M-fold program. The stem-loop structure of SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 5 has common characteristics that it has a CG bond at the end of the stem structure and has G or C in both of the beginning parts of the loop structure, in which the same bases are faced to each other (Red square).

(5) FIG. 5 is a graph of OPDA measurements for 13 optional sequences including a stem-loop structure. The antioxidant effect of ascorbic acid was observed in 13 sequences including a specific stem-loop structure.

(6) FIG. 6 is a comparative graph of OPDA measurement for SEQ ID NOS: 1, 4, 17, and 18 for which the antioxidant effect was confirmed. It was observed that the aptamer sequence of SEQ ID NO: 17 has the greatest antioxidant effect.

(7) FIG. 7 is a graph showing MST measurement results of SEQ ID NOs: 17 and 18 for which the antioxidant effect is confirmed.

MODE FOR INVENTION

(8) Hereinafter, the present invention will be described in detail with reference to Examples. However, the following examples are described for illustrative purposes only and the scope of the present invention is not construed as being limited by the following Examples.

Example 1: Preparation of a Single Stranded DNA Library

(9) A single-stranded DNA library was constructed to screen single-stranded DNA aptamers specifically binding to L-Ascorbic acid using the SELEX method. Single-stranded DNA library sequences were consisted of a total of 60 bases, each containing 15 specific primer sequences at 5 and 3 and having 30 random sequences.

(10) The single stranded DNA library as designed above is as follows.

(11) TABLE-US-00001 5-ATGCGGATCCCGCGC-N30-GCGCGAAGCTTGCGC-3 (singlestrandedDNAlibrarysequence; SEQIDNO:30)

(12) A single stranded DNA library was amplified by an asymmetric PCR procedure using a single oligonucleotide fragment of the synthesized base sequence of SEQ ID NO: 30 as a template. Primers 1 and 2 were constructed to perform the asymmetric PCR.

(13) The base sequences of primers 1 and 2 above are as follows:

(14) TABLE-US-00002 (Primer1;SEQID.NO:31) 5-ATGCGGATCCCGCGC-3 (Primer2;SEQID.NO:32) 5-GCGCAAGCTTCGCGC-3

(15) The asymmetric PCR was performed by mixing Primer 1 and Primer 2 in a ratio of 10:1 in order to amplify a large amount of single stranded DNA. The asymmetric PCR reaction was amplified by reacting it at 94 C. for 5 minutes, followed by repeatedly carrying out 45 cycles under the condition of 94 C. for 40 seconds, 55 C. for 40 seconds, 72 C. for 30 seconds, followed by reacting it at 72 C. for 10 minutes. The amplified PCR product was elctrophoresed on a 2.5% agarose gel, and the band was visually confirmed at first and then separated by the Crush and Soak method. The PCR product was electrophoresed on 12% native gel, the single-stranded DNA bands were cut, and then dissolved in Crush and Soak buffer (500 mM NH.sub.4OAc, 0.1% SDS, 0.1 mM EDTA) and DNA was purified with Ethanol precipitation method (FIG. 1). The purified single-stranded DNA library was heated at 95 C. for 5 minutes, and then formed the folded structure at room temperature for 10 minutes and then used in SELEX.

Example 2: Selex for Selection of Aptamers Specifically Binding to L-Ascorbic Acid

(16) In order to select the single stranded DNA aptamers that specifically bind to L-ascorbic acid to inhibit oxidation, SELEX (rGO-SELEX) technology using graphene was utilized (Lee, A. Y., Ha, N. R., Jung, I. P., Kim, S. H., Kim, A. R., & Yoon, M. Y. (2017). Development of a ssDNA aptamer for detection of residual benzylpenicillin. Analytical biochemistry, 531, 1-7.).

(17) Hereinafter, the SELEX method using graphene is as follows.

(18) At first, in order to remove the single-stranded DNA non-specifically binding to graphene and 1.5-ml tube, 200 pmol of single-stranded DNA library and 4 mg/mL of graphene were mixed in a binding buffer (20 mM Tris, pH 8.0), reacted for 30 minutes, and then the supernatant was removed by two centrifugations. The remaining single-stranded DNA and graphene mixtures was treated with ascorbic acid for 30 min to react them, and the single-stranded DNA bound to ascorbic acid was amplified by the asymmetric PCR procedure. The amplified single-stranded DNA was isolated by the Crush and Soak method at 12% native gel and was obtained as the first SELEX product. The above procedure was repeated 5 times and the symmetric PCR was performed by using the 5.sup.th repeated SELEX product as a template. The amplified double-stranded DNA fragments were sequenced by Next Generation Sequencing (NGS) technology to identify 16 sequences of SEQ ID NOS: 1 to 16, which are analyzed at a high frequency.

Example 3: Assay for the Antioxidant Effect of Ascorbic Acid

(19) phenylenediamine (OPDA) assay was performed to analyze the antioxidant effect of L-ascorbic acid by an aptamer. The antioxidant effect of ascorbic acid by the aptamer was analyzed by using the principle that DHA, which is an oxidized structure of L-ascorbic acid, binds to OPDA to develop fluorescence (Vislisel, J. M., Schafer, F. Q., & Buettner, G. R. (2007). A simple and sensitive assay for ascorbate using a plate reader. Analytical biochemistry, 365(1), 31-39.).

(20) The OPDA analysis procedure is as follows.

(21) Sixteen sequences were synthesized according to the frequency and folding structure of the sequence analyzed by NGS technology in SELEX products. The synthesized single-stranded DNA was heated at 95 C. for 5 minutes and reacted at room temperature for 10 minutes to form a folded structure. Then, 1 M of single stranded DNA and 5 mM ascorbic acid were mixed and reacted for 30 minutes. Then, hydrogen peroxide (H.sub.2O.sub.2) was added to accelerate the oxidation and then the fluorescence intensity was measured by adding OPDA fluorescent dye (Non-Patent Document 3). A control was a sample adding only vitamin C without adding the synthesized single stranded DNA and was oxidized more rapidly than the samples treated with the single stranded DNA of 16 sequences (SEQ ID NOS: 1 to 16). It was observed that oxidation of L-ascorbic acid was prevented in three single stranded DNAs of SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 5 among the 16 sequences (FIG. 2).

Example 4: Measurement of the Binding Force Between L-Ascorbic Acid and Aptamer Sequence

(22) The binding dissociation constant (Kd) was measured using a Microscale Thermophoresis (MST) method to confirm the binding force of the aptamer binding to L-ascorbic acid. Fluorescence of Cy5 was attached to 5 of the single-stranded DNA and the intensity of binding was measured by analyzing the signal difference depending on the binding force under thermal gradient conditions (Entzian, C., & Schubert, T. (2016). Studying small molecule-aptamer interactions using MicroScale Thermophoresis (MST). Methods, 97, 27-34.).

(23) The concentration of single stranded DNA aptamer was fixed at 5 nM and ascorbic acid was added to 16 tubes in a concentration gradient ranging from 50 uM to 1.53 nM and then reacted for 15 minutes. The reacted single-stranded DNA and L-ascorbic acid were inhaled into a scanning capillary tube (4 ul/tube) and then measured using a Monolith NT.11 instrument. The aptamers of SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 5 confirmed by the above OPDA method were measured to have Kd values of 280 nM, 149 nM and 685 nM, respectively, and thus identified as being a binding force of the level of nano molarity (nM) (FIG. 3).

Example 5: Analysis for the Structural Similarity of Single Stranded DNA Aptamer Specifically Binding to Vitamin C

(24) M-fold program was used to identify the secondary structure of the single-stranded DNA aptamers of SEQ ID NO: 1. SEQ ID NO: 4 and SEQ ID NO: 5 the binding power of which was identified. In the case of SEQ ID NO: 1, it is possible to form a secondary structure in two forms, and it was identified that SEQ ID NO: 4 and SEQ ID NO: 5 could form a stem-loop structures in three or two forms, respectively.

(25) These three single-stranded DNA aptamers (SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 5) are characterized in that they have a CG bond at the end of stem structure of the stem-loop structure and have G or C which is faced to each other at both of the beginning parts of the loop structure (FIG. 4, red squares). The structures of the three identified aptamers have stable structures with a Gibbs free energy values of 3.65 and 1.64, 0.86 (kcal/mol), respectively.

Example 6: Measurement of Antioxidant Effect of 13 Variant Sequences

(26) Single stranded DNA was synthesized by designing arbitrary sequences of 32 base sequences based on the specific structural sequences identified through analysis of folding structure of aptamer. An arbitrary single-stranded DNA was prepared so that 10 base sequences were constituted as a unit in a stem-loop structure to be comprised 1-2 of them at both 5 and 3 ends. The antioxidant effects of the single stranded DNAs of SEQ ID NOS: 17 to 29, which were prepared in 13 kinds, were confirmed through the OPDA assay (FIG. 5).

(27) All of the 13 random sequences had antioxidant effects against L-ascorbic acid, and the single-stranded DNA aptamer of SEQ ID NO: 17 with a stem-loop structure at both ends of 5 and 3 was identified as having the greatest anti-oxidative effect among the total of 29 sequences (13 sequences which were arbitrarily synthesized and identified, and 16 sequences which were selected by the SELEX process) (FIG. 6).

Example 7: Kd Measurement and Comparison of Two Modified Sequences

(28) The binding force of the aptamer was confirmed by MST (Microscale Thermophoresis) analysis for two sequences in which the anti-oxidative effect was confirmed as being high among arbitrary sequences (SEQ ID NOS: 17 to 29). Sequences of SEQ ID NOS: 17 and 18 identified by the OPDA method obtained the measurement values as Kd values of 729 nM and 289 nM, respectively (FIG. 7). Unlike the result identified by the OPDA measurement method, the Kd value of SEQ ID NO: 17 was determined as being high, seemingly indicating that the aptamer of SEQ ID NO: 17 had a low binding force but more specifically bound to the OH functional group.

(29) TABLE-US-00003 TABLE1 Number ofbase SEQ.ID.NOS. 5 to3 (mer) Note SEQ.ID.No:1 GCACCGACAGGGGAGCG 30 ABA1 CCTCGCACTGACT SEQ.ID.No:2 GGTGCAAACCAGCGCGC 30 ABA2 CTCTCTGACGTCG SEQ.ID.No:3 ACGCATGCCGGGCGCGC 30 ABA3 TCCCTGTCGTCCG SEQ.ID.No:4 CGAGTCAGTGCGAGGCG 30 ABA4 CTCCCCTGTCGGT SEQ.ID.No:5 GGGTCTGAGGAGTGCGC 30 AA-001 GGTGCCAGTGAGT SEQ.ID.No:6 GAACCAACGGAAGCGCG 30 AA-002 GCACCACAACGGT SEQ.ID.No:7 CGCAACCTGTTCGGCAG 30 AA-003 TGGGCCTCCGGGT SEQ.ID.No:8 GAACTTGCGCACTAGGT 30 AA-005 GATGCGGATCCCG SEQ.ID.No:9 GAAGCTTGCGCACTAGG 30 AA-006 TGGTGCGGATCCC SEQ.ID.No:10 GATCAACGGAAGCGCGG 30 AA-014 CACCACAACGGTA SEQ.ID.No:11 CGAGTCAGGTGGGATGA 30 AA-035 TGTTCGGGGAAGG SEQ.ID.No:12 GGCACAACGGGCGCGCC 30 AAL-1 TCCATGCTGTTCG SEQ.ID.No:13 TGAACGACGAGGCGCGT 30 AAL-2 CACACTGCGTGCC SEQ.ID.No:14 CGCAGTGTGACGCGCCT 30 AAL-3 CGTCGTTCACTCG SEQ.ID.No:15 CACAATCGGGGCGCGCT 30 AAL-4 CGTCCTCTGGCCG SEQ.ID.No:16 GGAACAACGGGCGCGCC 30 AAL-5 TCCATGCTGTTCG SEQ.ID.No:17 GTGGAGGCGGTGGCCAG 32 332-2 TCTCGCGGTGGCGGC SEQ.ID.No:18 GTGGAGGCGGTGGCCGT 32 332-4 GGAGGCGGAGGCCGC SEQ.ID.No:19 GTGGAGGCGGTGGCCAG 32 332-8 TCTGCGGCGCGGCAG SEQ.ID.No:20 GGCGGTGGCCCTGCAAG 32 332-11 TCTCGCGGTGGCGGC SEQ.ID.No:21 GGCGGTGGCCCTGGAAG 32 332-12 TCTCGCGGTGGCGGC SEQ.ID.No:22 GCGGCGGTGGCCAGAAG 32 332-13 TCTCGCGGTGGCGGC SEQ.ID.No:23 CGGGCGGTGGCCAGAAG 32 332-14 TCTCGCGGTGGCGGC SEQ.ID.No:24 GCGGCGGTGGCCTGAAG 32 332-15 TCTGGCGGTGGCCCG SEQ.ID.No:25 GCGGCGGTGGCCTGAAG 32 332-16 TCTGGCGGTGGCCCC SEQ.ID.No:26 GCGGCGGTGGCCTGAAG 32 332-17 TCTGGCGGTGGCCCA SEQ.ID.No:27 GGCGGTGGCCTGGAAGT 32 332-18 CTCATGGCGGTGGCC SEQ.ID.No:28 GTGGCGGTGGCCAGCAT 32 332-19 ACGGGCGGTGGCCAG SEQ.ID.No:29 GTGGCGGTGGCCAGCAT 32 332-20 AGTGGCGGTGGCCAG

(30) Table 1 shows the aptamer sequences of the present invention.