APTAMER TEMPLATE AND METHOD FOR PREPARING APTAMER BY USING SAME

Abstract

Provided are a novel nucleic acid structure, its use as an aptamer template, and a method for preparing an aptamer using thereof.

Claims

1. A nucleic acid structure represented by the following general formula 2:
5-[(N).sub.p][(X).sub.1][G(N).sub.n][(X).sub.m][(N).sub.q]-3(general formula 2) wherein, N is each independently selected from A, T or U, G, and C randomly, and X is each independently selected from A, T or U, G, and C randomly, and the ratio of C or G is 55% or higher based on the total nucleotides of [(X).sub.l] or [(X).sub.m], and [G(N).sub.n] is a site forming a loop structure, and n is the number of nucleotide N and an integer of 3 to 29, and [(X).sub.l] and [(X).sub.m] are sites forming a stem structure by forming a base pair with antiparallel complementary sequences each other, and 1 and m are numbers of nucleotide X and each is an integer of 3 to 10, and [(N).sub.p] and [(N).sub.q] are 5 end region and 3 end region, respectively, and p and q are numbers of nucleotide N, and each is independently an integer of 1 to 50, and p<q.

2. The nucleic acid structure according to claim 1, wherein the [(X).sub.l] is 5-GGAC-3, and [(X).sub.m] is 5-GTCC-3 or 5-GUCC-3 which is anti-parallel complementary to the [(X).sub.l], and the condition p<q is satisfied, and the p is selected from integers of 1 to 20, and the q is selected from integers of 15 to 50.

3. The nucleic acid structure according to claim 2, wherein the p is an integer of 1 to 10, and the q is an integer of 15 to 50.

4. The nucleic acid structure according to claim 1, wherein the nucleic acid structure is represented by the following general formula 3 or general formula 4: TABLE-US-00015 (generalformula3;SEQIDNO:44) NNNGGACGNNNNNNNGTCCNNNNNNNNNNNNNNNNNNNNN, (generalformula4;SEQIDNO:45) NNNGGACGNNNNNNNGUCCNNNNNNNNNNNNNNNNNNNNN, wherein the N is each independently selected from A, T or U, G, and C.

5. An aptamer template comprising the nucleic acid structure of claim 1.

6. A composition for preparing an aptamer comprising the nucleic acid structure of claim 1.

7. A nucleic acid aptamer, prepared from the aptamer template of claim 5.

8. A method for preparing a target material-specific binding nucleic acid molecule, comprising (1) contacting the nucleic acid structure of claim 1 with a target material; and (2) measuring whether to bind between the nucleic acid structure and target material.

9. The method for preparing a target material-specific binding nucleic acid molecule according to claim 8, further comprising (3) determining the nucleic acid structure as a nucleic acid molecule specifically binding to the target material, when binding between the nucleic acid structure and target material is confirmed.

10. The method for preparing a target material-specific binding nucleic acid molecule according to claim 8, wherein the nucleic acid structure is two or more kinds having different sequences each other.

11. The method for preparing a target material-specific binding nucleic acid molecule according to claim 10, wherein the target material-specific binding nucleic acid molecule is a target material-specific nucleic acid aptamer.

12. A method for preparing a nucleic acid aptamer specifically binding to a target material, comprising (i) contacting a library comprising one or more kinds of the nucleic acid structures of claim 1 with beads in which a target material is immobilized; (ii) washing the beads of the step (i); and (iii) confirming the nucleic acid structure bound to the beads of the step (ii).

13. The method for preparing a nucleic acid aptamer according to claim 12, further comprising (iv) determining the nucleic acid structure confirmed in the step (iii) as a nucleic acid aptamer specifically binding to the target material.

14. The method for preparing a nucleic acid aptamer according to claim 12, wherein the nucleic acid structure is two or more kinds having different sequences each other.

15. The method for preparing a nucleic acid aptamer according to claim 13, wherein the nucleic acid structure is two or more kinds having different sequences each other.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0112] FIG. 1 schematically shows the structure of the aptamer template according to one example.

[0113] FIG. 2 is a graph showing the Tm value of the aptamer obtained by using the aptamer template (HFL) according to one example, by comparing with the Tm value of the aptamer obtained by using a random library.

[0114] FIG. 3 is a graph showing the free energy (AG) of the aptamer obtained by using the aptamer template (HFL) according to one example, by comparing with the free energy (AG) of the aptamer obtained by using a random library.

[0115] FIG. 4 is a graph showing binding of the VEGF-specific aptamer obtained by using the aptamer template (HFL) according to one example to VEGF (measured by the degree of luminescence) as a fold of the background value.

[0116] FIG. 5 is a graph showing binding of the CBF1-specific aptamer obtained by using the aptamer template (HFL) according to one example to CBF1 (measured by the degree of luminescence) as a fold of the background value.

[0117] FIG. 6 is a graph showing binding of the TGFBRII ectodomain-specific aptamer obtained by using the aptamer template (HFL) according to one example to TGFBRII ectodomain (measured by the degree of luminescence) as a fold of the background value.

MODE FOR CARRYING OUT THE INVENTION

[0118] Hereinafter, the present invention will be described in more detail by examples, but they are illustrative and are not intended to limit the scope of the present invention. It is apparent to those skilled in the art that the examples described below can be modified without departing from the essential gist of the invention.

Example 1: Aptamer library production

[0119] Based on a aptamer template of stem-loop structure having the following general formula, any base was introduced to N position to prepare a DNA aptamer library with a total length of 86mer (hereinafter, HFL (High Functional Library)):

TABLE-US-00003 (generalformula5;SEQIDNO:46) 5-TAGGGAAGAGAAGGACATATGAT [NNNGGACGNNNNNNNGTCCNNNNNNNNNNNNNNNNNNNNN] TTGACTAGTACATGACCACTTGA-3

[0120] (in the general formula, N is each independently selected from A, T, G, and C, and the bolded part has anti-parallel complementary sequences and forms a stem structure, and the underlined part forms a loop structure; the part indicated in italics is an added sequence that can bind to forward and reverse primers).

[0121] For comparison, an oligonucleotide having the following general formula (86mer in total; hereinafter, Random library) was prepared:

TABLE-US-00004 (SEQIDNO:47 5-TAGGGAAGAGAAGGACATATGAT[N].sub.40 TTGACTAGTACATGACCACTTGA-3;
N is each independently selected from A, T, G, and C, and is different from the sequence in [ ] of the general formula 5)

[0122] All oligomers were produced in a form of Biotinylated-ssDNA(86mer) by commissioning to Bioneer corporation.

Example 2: DNA Aptamer Analysis

[0123] For the ssDNA (aptamer) library prepared using HFL and Random library obtained in Example 1, PCR was performed using the following primers.

TABLE-US-00005 Forward primer: 5-biotin-TAGGGAAGAGAAGGACATATGAT3(23mer) Reverseprimer: 5-TCAAGTGGTCATGTACTAGTCAA-3(23mer)

[0124] The PCR process is summarized as follows:

[0125] (1) PCR mixture: Reverse primer(10 uM), forward primer(10 uM), dNTP(each 2 mM), pfu(+) DNA buffer (CANCERROP Co., Ltd.), pfu(+) DNA polymerase(1 U; CANCERROP Co., Ltd.), and Library prepared in Example 1 (100 ng).

[0126] (2) First cycles: 95 C. for 3 min, 53 C. for 30 sec, 72 C. for 30 min, 4 C. hold.

[0127] (3) add the forward primer and mix.

[0128] (4) Second cycles: 95 C. for 2 min, start cycle (4 cycles)95 C. for 30 sec, 53 C. for 30 sec, 72 C. for 10 sec-end cycle, 72 C. for 5 min, 4 C. hold.

[0129] For the obtained PCR products, cloning was performed using ToPcloner PCR Cloning kit (enzynomics Co., Ltd.). For cloning, pTOP Blunt V2(10 ng) 1 ul, TOPcloner buffer (1.2M NaCl, 0.06M MgCl.sub.2) 1 ul, and PCR product(4 ul) were mixed, and then it was left for 5 minutes at room temperature to prepare cloning product 6 ul.

[0130] Using the prepared cloning product, DH5a chemically competent E. coli was transformed. More specifically, cloning product 6 ul and DH5a chemically competent E. coli (CANCERROP Co., Ltd.) 510.sup.7 were mixed, and it was left in ice for 30 minutes and left at 42 C. for 2 minutes, and then it was immediately put in ice. After that, the cells were put in SOC media (2.0% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM Glucose) and mixed, and then they were left at 37 C. for 1 hour, and then were under spin down at 5000g for 2 minutes, and the supernatant was removed, and then SOC media 200 ul was added again and mixed. The 510.sup.7 prepared cells were spread on 90 LB plate (+Ampicillin 50 ug/ml) and then were cultured at 37 C. overnight. The colony was picked and it was cultured at 37 C. in a shaking incubator overnight.

[0131] To extract plasmid DNA in the cultured cells, Mini-prep was performed using mini-prep kit (CANCERROP Co., Ltd.). More specifically, the cell culture was centrifuged at 13000g for 2 minutes to collect cells. The supernatant was removed and 51 buffer 250 ul of mini-prep kit (CANCERROP Co., Ltd.) was added and mixed well, and then S2 buffer 250 ul was added, and 4-6 times inverts were performed and S3 buffer 350 ul was added and 6 times inverts were performed, and then it was centrifuged at 13000g for 10 minutes and only the supernatant was collected and was loaded in a column, and then it was centrifuged at 13000 rpm for 1 minute. PW buffer (CANCERROP Co., Ltd.) 700 ul was loaded in a column, and then it was centrifuged at 13000 rpm for 1 minutes twice, and then the column was put in a new tube, and D.W 50 ul was loaded in the center of the column membrane, and it was left for 2 minutes, and then it was centrifuged at 13000 rpm for 1 minute.

[0132] The sequence of DNA (20 aptamer DNAs selected in HFL and 20 aptamer DNAs obtained using Random library) obtained by the above method was analyzed (commissioned to Macrogen Co., Ltd.; reading the sequence with Universal M13 forward primer). In addition, free energy value and the Tm value of the aptamer section were obtained through Mfold program (http://unafold.rna.albany.edu/?q=mfold) and the result of comparing the average value was shown in the following Tables 1 and 2, and FIG. 2 and FIG. 3.

TABLE-US-00006 TABLE1 Freeenergy(G),Tm,andsequence ofaptamersobtainedusingHFL. SEQ ID Tm G sequence NO. (C.) (kcal/mol) ATAGGACGGATGAGCGTCCG 1 77.3 10.29 GCCGGATGGGTTTTCCATCA CTGGGACGCGGATGAGTCCG 2 66.2 6.35 CTCCACCAGCCATCCATGTA TTAGGACGCGAAGATGTCCT 3 60.1 5.34 TGCAAGGGCTCATCGCATCA ATGGGACGACCCTGTGTCCC 4 57.1 5.48 TATATGCGCGGAGAGCCATC CTTGGACGCCCTCGTGTCCC 5 57.1 4.30 TTGCTCCTTCACATACGCAA GCTGGACGTTCGTGGGTCCC 6 56.8 2.10 ATTCTGAAGCCTAAGACTGG GAGGGACGAACAAGCGTCCA 7 64.2 6.07 GCGGTTATGCACCTTCGGTA TACGGACGCACCTCCGTCCG 8 75.8 10.33 GTTCTCAGCGTGGGGCGCAC GCAGGACGTACTGATGTCCC 9 57.0 3.74 GTTGCATGTCGCTAAGCTCC CGCGGACGGGGTTTTGTCCC 10 70.8 6.93 CCGTAGCTGACTAAATACAT ACGGGACGCGTTCTGGTCCC 11 66.5 3.88 TACTTCTTGTGTGTCTATTC AATGGACGCATCCCAGTCCC 12 56.5 2.20 CCAAGCTTCCGTATCGTAAC TGCGGACGCACGCCTGTCCT 13 50.1 3.05 CAACCCATGATTACACGTTT TGGGGACGGGTATACGTCCA 14 63.9 5.59 TCTCGTATCCAACTAGGCGA GTGGGACGCGTGTAGGTCCA 15 60.7 5.20 AACGGTGCGGTTTGGCGCTC GGAGGACGAGTGGTCGTCCC 16 70.8 7.77 GCTGGGCAATAGCTCGGGGC ACAGGACGCCAGAGCGTCCA 17 73.8 9.86 AGGTCGCCGCTAGTAGACCC TCCGGACGTAGCCCGGTCCC 18 71.9 7.08 AGCGCACGGTACCACCGTCG CCAGGACGACTCTAGGTCCT 19 68.1 3.02 CATGAGATTAGATATCGGTT GTCGGACGGAATAACGTCCT 20 69.7 4.61 AAATATTTATGCAACGGGGA average 64.72 5.66

TABLE-US-00007 TABLE2 Freeenergy(G),Tm,andsequenceofaptamers obtainedusingRandomlibrary. SEQ ID Tm G sequence NO. (C.) (kcal/mol) CCTCTTGCGCGCTCCGTATC 21 47.1 1.55 ACATTTATATGATGGCCAGT TCTCTTACAACGGGTCTTGG 22 50.9 1.11 CCATAACACAATAATCCTGC CCTAACTCGACCAGTGGGCT 23 48.2 1.19 ACGGACGGATGTTAACCCTA CCCCTACATCCCTTTTTGCC 24 41.3 0.67 GCAAATCACCCAGCTCCTGA CTTCCTTGATCGCTGCCCGA 25 61.6 4.78 TTTTTGGAATAGGTTCCTAG CTCAGGTTGTCGATCTAACC ATTGGCGGGTACGTTGTACC 26 56.4 4.24 CCCATGTCAGCCGGTTAATC 27 43.0 0.63 TTAATGTTGATTCGAGTCAC CTCTTGTCGCAGTACCTCAA 28 55.1 1.74 TTGAGCGTTGTACACATTGT TGTATTTCAGTAATCCAACG 29 58.2 1.39 GCACTTGTGACACTGCTACT TACTCAGACGAATCAAATAT 30 62.9 2.38 CGTGTCCAAAGTCCTCCGAG CCCCCTCAGGATTTTCCGTA 31 52.9 2.79 GCTTGTCTGGCAGCAGCTGT TGGCATTTAATAAACACCCT 32 50.5 1.96 CGTTAACGCGTGTGTGGATT GCAGCTCCATTGAGGCTAGA 33 59.8 3.12 ACGTGAGTCGTACCCCACCA CGCTATTGGGTAAAGGATCT 34 67.1 3.31 CTCGTAGCTCTGCATGAGCC GCTGGTGTGGCCGCCGGAAG 35 61.6 2.43 ATGACTTATACCCTGCACTC CTTTTTTCCAAACCGGGTGT 36 53.3 2.28 GCCACGGCCTGCGAATCGGT TGGGAAGAAATCAATGAATG 37 64.9 2.67 TCTCACTGATAGCTGGTGGT TCCCGATTTGATGGGGTGCT 38 54.7 1.76 GCTTATGATAGTCACGAAAC AGGCCCGTTTTTACCTACTC 39 48.4 2.46 TCATATTTGGACAACCGGGT TTATTTGTCCATACATTATA 40 50.9 2.69 GCGCGACGTGGAACAGGTAA average 54.44 2.26

[0133] As shown in Tables 1 and 2, and FIG. 2 and FIG. 3, the aptamer obtained from the aptamer template having a stem-loop structure of the present invention (HFL), was confirmed to have higher Tm value and lower free energy value, compared to the aptamer obtained from the conventional random library, and this result shows that an aptamer with enhanced stability can be prepared without an additional stabilization process, using the aptamer template provided herein.

Example 3: Protein SELEX Using LibraryTGFBRII Ectodomain, VEGF, & CBF1

[0134] pI value means the pH at which the net charge of protein becomes 0, and the value is determined by kinds of amino acids composing protein. Since an acidic amino acid has one more carboxyl group (COOH) and a basic amino acid has one more amino group (NH2), the more the acidic amino acid is in protein, the smaller the pI value is, and the more the basic amino acid is, the bigger the pI value is. When specific protein is present in a higher pH environment (for example, buffer) than its pI value, the protein has a negative charge (), and in the lower pH environment (for example, buffer), it has a positive charge (+).

[0135] In the following example, three proteins belonging to different pI ranges (acidic/basic/hydrophobic), respectively, in the buffer environment of physiological pH 7.5 of human were selected to progress an experiment, and thereby it was confirmed that the HFL library of the present invention had high binding affinity to target protein than Random library in all different charge environments.

[0136] More specifically, using the DNA aptamer library prepared in Example 1 (Biotinylated-ssDNA (86mer) library), SELEX (Systematic evolution of ligands by exponential enrichment) was performed for the following three proteins:

[0137] TGFBRII ectodomain: N terminal 6His-tagged, C terminal Thioredoxin-tagged, 35 KDa, pi value: 5.74

TABLE-US-00008 (MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIA DEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGAL SKGQLKEFLDANLALAGSGSGHMHHHHHHSSGLVPRGSGMKQTAAAKF GRQHMDSPDLNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNC SITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASP KCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLEHHHH HH;SEQIDNO:41);

[0138] VEGF: AbcamCo., Ltd., N terminal 6His-tagged VEGFA protein, 22 KDa, pi value:

7.6

TABLE-US-00009 (HHHHHHAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVD IFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEES NITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQE NPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELN ERTCRCDKPRR;SEQIDNO:42);

[0139] CBF1: C terminal 6His-tagged, 54 KDa, pi value: 8.3

TABLE-US-00010 (MAWIKRKFGERPPPKRLTREAMRNYLKERGDQTVLILHAK VAQKSYGNEKRFFCPPPCVYLMGSGWKKKKEQMERDGCSE QESQPCAFIGIGNSDQEMQQLNLEGKNYCTAKTLYISDSD KRKHFMLSVKMFYGNSDDIGVFLSKRIKVISKPSKKKQSL KNADLCIASGTKVALFNRLRSQTVSTRYLHVEGGNFHASS QQWGAFFIHLLDDDESEGEEFTVRDGYIHYGQTVKLVCSV TGMALPRLIIRKVDKQTALLDADDPVSQLHKCAFYLKDTE RMYLCLSQERIIQFQATPCPKEPNKEMINDGASWTIISTD KAEYTFYEGMGPVLAPVTPVPVVESLQLNGGGDVAMLELT GQNFTPNLRVWFGDVEAETMYRCGESMLCVVPDISAFREG WRWVRQPVQVPVTLVRNDGIIYSTSLTFTYTPEPGPRPHC SAAGAILRANSSQVPPNESNTNSEGSYTNASTLEHHHHHH; SEQIDNO:43).

[0140] Protein SELEX was progressed in 3 rounds for each protein according to the scheme of the following Tables 3 to 5:

TABLE-US-00011 TABLE 3 ssDNA (DNA aptamer) TGFBRII Molar Ratio Round pM g pM g ssDNA : protein round 1 500 13.95 50 1.75 10 : 1 round 2 100 2.8 10 0.35 10 : 1 round 3 100 2.8 5 0.18 20 : 1

TABLE-US-00012 TABLE 4 ssDNA (DNA aptamer) VEGF Molar Ratio Round pM g pM g ssDNA : protein round 1 500 13.95 50 1.1 10 : 1 round 2 100 2.8 10 0.22 10 : 1 round 3 100 2.8 5 0.11 20 : 1

TABLE-US-00013 TABLE 5 ssDNA (DNA aptamer) CBF1 Molar Ratio Round pM g pM g ssDNA : protein round 1 500 13.95 50 2.7 10 : 1 round 2 100 2.8 10 0.54 10 : 1 round 3 100 2.8 5 0.27 20 : 1

[0141] Ni-NTA magnetic bead 20 ul of Qiagen Co., Ltd. was transferred to an E-tube, and the supernatant was removed using a magnetic rack. Using SELEX buffer (P B/W buffer) 100 ul, beads were washed three times. The composition of SELEX buffer used for each protein herein was as follows: TGFBRII SELEX buffer (P B/W buffer): 12.5 mM Tris-HCl (pH 7.5), 125 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1 mM PMSF (phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluoride);

[0142] VEGF SELEX buffer (P B/W buffer): 12.5 mM Tris-HCl(pH 7.5), 125 mM NaCl, 0.25 mM DTT(dithiothreitol), 50 mM KCl,

[0143] CBF1 SELEX buffer (P B/W buffer): 12.5 mM Tris-HCl(pH 7.5), 125 mM NaCl, 50 mM KCl, 1 mM DTT (dithiothreitol).

[0144] Then, the protein prepared in advance (TGFBRII ectodomain, VEGF, or CBF1) was added in an amount of 50 pM to the SELEX buffer 100 ul, and then it was under binding to beads at 4 C. for 1 hour. After completing protein binding, the supernatant was removed, and beads were washed three times using SELEX buffer 100 ul. Then, after adding SELEX buffer 100 ul and biotinylated-ssDNA library 500 pM prepared in Example 1 to the protein-bound Ni-NTA beads, it was reacted at 37 C. for 1 hour, to bind DNA library (Example 1; HFL library and Random library) to protein-beads (DNA:protein=10:1; molar ratio). After that, the beads were washed with SELEX buffer 100 ul three times, and then ssDNA elution buffer (50 mM NaOH) 20 ul was added, and it was heated at 95 C. for 10 minutes, and ssDNA (single-stranded DNA) bound to the protein bound to beads was eluted. For the eluted ssDNA, symmetric PCR was performed under the following conditions, and thereby selected ssDNA was amplified:

[0145] (1) PCR mixture: Reverse primer(20 pM), biotinylated-forward primer(20 pM), dNTP(each 1 nM), pfu(+) buffer(CANCERROP Co., Ltd.), pfu(+) DNA polymerase (1U; CANCERROP Co., Ltd.), template-ssDNA(20 ng) prepared in Example 1;

[0146] (2) cycles: 95 C. for 5 min, start cycle (15 cycles)95 C. for 30 sec, 53 C. for 30 sec, 72 C. for 10 sec, end cycle72 C. for 5 min, 4 C. hold.

TABLE-US-00014 Forwardprimer: 5-biotin-TAGGGAAGAGAAGGACATATGAT3(23mer) Reverseprimer: 5-TCAAGTGGTCATGTACTAGTCAA-3(23mer).

[0147] The dsDNA produced after performing PCR as above was used to obtain biotinylated-ssDNA (biotinylated-ssDNA for progressing SELEX 1 round: 86mer biotinylated-ssDNA prepared by commissioning to Bioneer Co., Ltd. described in Example 1; then, biotinylated-ssDNA for progressing SELEX 2 round and 3 round: biotinylated-ssDNA obtained by the process described below (method using streptavidin beads).

[0148] For the obtained PCR products, using streptavidin magnetic beads (NEB Co., Ltd.), biotinylated ssDNA was isolated.

[0149] More specifically, PCR product Mug obtained by the method and D.W, 200 mM NaOH, and 4 Wash/Binding Buffer (W/B buffer; 0.5 M NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA (Ethylenediaminetetraacetic acid)) were mixed at a volume ratio of 1:1:1:1, and it was left at 37 C. for 10 minutes, to prepare PCR product solution (Final concentration: 50 mM NaOH, lx wash/binding buffer). On the other hand, streptavidin magnetic bead (NEB Co., Ltd.) solution 250 ul (pure bead amount: 1 mg) was taken to remove the supernatant, and it was washed with 1W/B buffer 200 ul three times. The streptavidin magnetic bead in which the supernatant was removed was mixed well with the prepared PCR product (dsDNA state) solution 10 ug, and it was reacted at 37 C. for 1 hour to allow biotinylated-ssDNA to bind. After downing the streptavidin magnetic beads using a magnetic rack and then removing the supernatant, the beads were washed with 1W/B buffer 200 ul three times. After putting D.W 200 ul to the biotinylated-ssDNA-bead obtained like this, it was left at 95 C. for 20 minutes to elute biotinylated-ssDNA. The ssDNA obtained like this was used for the next SELEX round.

Example 4. Ka Value Measurement Through Indirect ELISA

[0150] The degree of binding (Ka value) between protein and the aptamer obtained for each protein by performing SELEX 3 round (See Example 3) by using the HFL or Random library prepared in Example 1 was measured by performing Indirect ELISA as follows.

[0151] Coating buffer (100 mM sodium carbonate/bicarbonate (pH 9.6), 3.03 g Na2CO3, 6.0 g NaHCO.sub.3, 1000 ml distilled water) 100 ul and each protein 25 pM/well were loaded to a plate (Pierce 96-Well Polystyrene Plates, White Opaque), and it was incubated at a room temperature for 2 hours. Then, after removing the buffer in the plate by vigorously spraying it on the sink, 200 ul of wash buffer (PBS) was added to each well and it was washed 3 times repeatedly. Blocking buffer (5%(v/v) BSA in PBS) of 200 ul was added to each well and it was incubated at a room temperature for 3 hours. Then, after removing the blocking buffer in the plate by vigorously spraying it on the sink, 200 ul of wash buffer (PBS) was added to each well and it was washed three times.

[0152] The biotinylated-ssDNA 0.2 pM selected per SELEX round in Example 3 was loaded with 100 ul of the SELEX buffer of each target protein (See Example 3) into each well of the prepared plate in which each protein was coated, and it was incubated at 37 C. for 1 hour (in the present example, 3 rounds were performed in total). After removing the solution in the plate by vigorously spraying it on the sink, 200 ul of wash buffer (PBS) was added to each well, and it was washed three times repeatedly. The streptavidin-HRP protein (abcam) was diluted in blocking buffer (5%(v/v) BSA in PBS) to 10000:1 (v:v), and 200 ul was loaded to each well, and then it was incubated at a room temperature under the darkroom condition for 2 hours. After removing the solution in the plate by vigorously spraying it on the sink, 200 ul of wash buffer (PBS) was added to each well, and it was washed three times. After that, 100 ul of ECL Substrate solution (SuperSignal ELISA Femto Substrate) was added to each well and the generated luminescence value was measured (measured by SPARK (TECAN)). For comparison, the luminescence value in case of no-treatment of the target protein was measured by the method the same as above and it was used as a background value. All tests were performed three times, and the average value was calculated and used for analysis of results. The result of dividing the obtained luminescence value by the background value was shown in FIG. 4 to FIG. 6.

[0153] As shown in FIG. 4 to FIG. 6, it could be confirmed that the aptamer obtained from the aptamer template having a stem-loop structure (HFL) had excellent binding ability to a target protein, even when performing a relatively less number of SELEX round (for example, 3 round), compared to the aptamer obtained from the conventional random library, and it could be confirmed that it showed high binding ability, for all the tested proteins having various pI values including protein having a low pI value with weak binding ability to the aptamer by electrostatic repulsive power (for example, TGFBRII ectodomain), compared to the aptamer obtained from the conventional random library.