KR20220082125A - Dna aptamer specifically binding to coronavirus and using the same - Google Patents

Abstract

The present invention relates to a DNA aptamer that specifically binds to a coronavirus and a use thereof. Specifically, the DNA aptamer according to the present invention specifically binds to the antigen protein of the novel coronavirus that causes coronavirus, in particular, COVID-19, so that it can be usefully used not only for detection and diagnosis of coronavirus, but also for treatment.

Description

DNA aptamer that specifically binds to coronavirus and its use {DNA APTAMER SPECIFICALLY BINDING TO CORONAVIRUS AND USING THE SAME}

The present invention relates to a DNA aptamer that specifically binds to a coronavirus and a use thereof.

Coronaviruses have single-stranded positive RNA as their genome and are enveloped viruses that have been isolated from a variety of animals, including humans, since they were first discovered in 1937. Coronaviruses are classified into α, β, γ, or δ groups according to the nucleotide sequence of RNA replication and transcription enzyme (RNA dependent RNA polymerase, RdRp), of which α-coronavirus and β-coronavirus mainly infect mammals and , γ-coronavirus and δ-coronavirus infect birds. However, there are cases in which infection of δ-coronavirus has been confirmed in pigs recently.

As such, the coronavirus is a virus that can transmit heterogeneously, the SARS coronavirus that causes severe acute respiratory syndrome that was prevalent worldwide in 2003 and the MERS coronavirus that spread and spread in Korea in 2015 there is All of these coronaviruses are known to come from bats. In addition, the novel coronavirus (2019-nCoV), which recently occurred in Wuhan, China and is spreading around the world, was also confirmed to be caused by a heterogeneous infection.

However, among mammals, bats have the ability to fly and can live in a wide area, and due to the nature of a large number of individuals living in a group in a narrow space such as a cave, when one individual becomes infected with the virus, the infection is transmitted to the group. . In addition, they can travel over a wide area by flying, allowing the infection to spread to other animals. In addition, unlike other mammals, bats have a high body temperature, which makes them resistant to viruses, so they are not affected by the coronavirus and can continuously spread infections.

The novel coronavirus is a virus having an RNA gene of about 27 to 32 kb. Infectious disease caused by the novel coronavirus (COVID-19) has an incubation period of about 2 weeks, followed by respiratory symptoms such as fever, cough, difficulty breathing, shortness of breath, and sputum. is mainly accompanied by In addition, it may show symptoms such as headache, chills, muscle pain, loss of appetite, vomiting, and abdominal pain.

In other words, it is impossible to rule out the possibility that the coronavirus, which is known to not infect humans, can infect humans, as was the case with the great chaos caused by the SARS and MERS virus pandemics. Therefore, the development of therapeutic agents for the treatment of these coronaviruses is actively progressing. In this regard, Republic of Korea Patent Registration No. 10-2145197 discloses a composition for treating coronavirus comprising L-nucleoside having a specific chemical formula as an active ingredient.

Korean Patent Registration No. 10-2145197

It is an object of the present invention to provide a DNA aptamer that specifically binds to a coronavirus and a use thereof.

In order to achieve the above object, the present invention provides a DNA aptamer that specifically binds to the coronavirus.

In addition, the present invention provides a composition comprising the DNA aptamer.

In addition, the present invention provides a composition for detecting a coronavirus comprising the DNA aptamer.

In addition, the present invention provides a method of detecting a coronavirus using the DNA aptamer.

Furthermore, the present invention provides a method of providing information necessary for diagnosis of a disease caused by coronavirus infection using the DNA aptamer.

The DNA aptamer according to the present invention can be usefully used for treatment as well as detection and diagnosis of coronavirus by specifically binding to the antigen protein of the novel coronavirus that causes coronavirus, particularly COVID-19.

1 is a result of confirming the optimization of a biological sample for SARS-CoV-2 detection through agarose gel electrophoresis in an embodiment of the present invention.
2 is a result of confirming the optimization of a biological sample for SARS-CoV-2 detection by a capillary electrophoresis method in an embodiment of the present invention.
3 is a result of confirming the optimization of a biological sample for SARS-CoV-2 detection through agarose gel electrophoresis according to dissolution time and enzyme treatment in an embodiment of the present invention.
4 is a result confirming the optimization of a biological sample for SARS-CoV-2 detection by a capillary electrophoresis method according to dissolution time and enzyme treatment in an embodiment of the present invention.
5 is a graph showing the result of confirming the SARS-CoV-2 detection effect of the DNA aptamer selected in an embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a DNA aptamer that specifically binds to a coronavirus.

In addition, the present invention provides a composition comprising the DNA aptamer.

In addition, the present invention provides a composition for detecting a coronavirus comprising the DNA aptamer.

In addition, the present invention provides a method of detecting a coronavirus using the DNA aptamer.

Furthermore, the present invention provides a method of providing information necessary for diagnosis of a disease caused by coronavirus infection using the DNA aptamer.

Hereinafter, the present invention will be described in detail by the following examples, provided that the following examples are only for illustrating the present invention, and the present invention is not limited thereto. Anything that has substantially the same configuration as the technical idea described in the claims of the present invention and achieves the same operation and effect is included in the technical scope of the present invention.

Example 1. Amplification of random DNA library

In order to select a DNA aptamer that specifically binds to the antigen protein of the novel coronavirus, a DNA library was first prepared as described below.

Specifically, 100 bp template DNA (SEQ ID NO: 31: 5'-GGTAATACGACTCACTATAGGGAGATACCAGCTTATTCAATT) including 40 random nucleotide sequences (N ₄₀ ) in which dA: dG: dC: dT is 1.5:1.15:1.25:1 in a ratio -N ₄₀ -AGATAGTAAGTGCAATCT-3 ') and a forward primer (SEQ ID NO: 32: 5'-GGTAATACGACTCACTATAGGGAGATACAGCTTATTCAATT-3'), a reverse primer (SEQ ID NO: 33: 5'-AGATTGCACTTACTATCT-3') to Bioneer Corporation (Korea) Made to order. 1 μl template DNA, 5 μl 10x PCR buffer, 4 μl dNTP mixture (2.5 mM), 2 μl 10 μM forward primer, 2 μl 10 μM reverse primer, 0.3 μl ExTaq polymerase (Takara, Japan) ) (1 unit/μl) and 35.7 μl of distilled water were mixed to prepare a PCR reaction. PCR was performed on the prepared reactants under the conditions described in Table 1 below. The obtained PCR product was confirmed through 2% agarose gel electrophoresis.

early metamorphic stage 94°C, 5 minutes 1 time metamorphic stage 94℃, 30 seconds 5 times Annealing step 52℃, 30 seconds elongation step 72℃, 30 seconds Late Stretching Stage 72°C, 5 minutes 1 time

Example 2. Preparation of a DNA aptamer of a three-dimensional structure

In order to select a DNA aptamer that specifically binds to the antigen protein of the novel coronavirus, a three-dimensional DNA aptamer was prepared.

Specifically, 10 μl of 2x PBS was added to 10 μl of DNA aptamer. After denaturing the mixture by boiling at 85° C. for 5 minutes, it was left at room temperature for 1 hour or more to cool slowly, thereby preparing a three-dimensional RNA aptamer.

Example 3. Selection of DNA aptamers that specifically bind to novel coronavirus protein

3-1. Screening of DNA aptamers that specifically bind to novel coronavirus proteins

DNA aptamers that specifically bind to the antigenic protein of the novel coronavirus were selected using the SELEX technique.

Specifically, the antigen protein of novel coronavirus (84 pmole) and the DNA aptamer pool (840 pmole) that formed the three-dimensional structure in Example 2 were mixed, and the total volume was adjusted to 200 μl with 1× PBS. This was reacted with stirring at 4° C. for 1 hour.

Meanwhile, in order to activate the Ni-NTA agarose purification kit, 200 μl of 1X aptamer selection solution (500mM NaCl, 20mM Tris-HCl, 5mM imidazole) was added to 200 μl of Ni-NTA agarose and reacted with stirring at 4°C for 30 minutes. After activation, the supernatant was removed by centrifugation at 13,000 rpm at 4°C for 10 minutes. After repeating the above process twice, the reaction solution in which the histidine fusion antigen protein and the DNA aptamer pool were reacted was reacted with activated Ni-NTA agarose at 4°C for 1 hour with stirring. Thereafter, the supernatant was removed by centrifugation at 13,000 rpm at 4°C for 10 minutes, and 1 ml of a washing solution (500mM NaCl, 60mM imidazole, 20mM Tris-HCl) was added and the washing process was repeated 5 times to repeat the histidine fusion antigen protein DNA aptamers that did not bind to were removed. To elute the histidine fusion antigen protein and DNA aptamer that specifically binds to it from Ni-NTA agarose, 200 μl of a DNA aptamer elution solution (300 mM NaCl, 20 mM Tris-HCl, 250 mM imidazole) was added at 4°C for 1 hour. The reaction was stirred and then centrifuged at 13,000 rpm at 4° C. for 10 minutes to obtain an elution solution of the upper layer. The above process was repeated twice to obtain an elution solution of the upper layer. In order to remove the histidine fusion antigen protein from the binding product between the histidine fusion antigen protein and the DNA aptamer specifically binding thereto, the elution solution was treated with an equal volume of PCI solution and then centrifuged at 4 ° C. at 13,000 rpm for 15 minutes. Only the supernatant was recovered. Then, to recover only the DNA aptamer that specifically binds to the histidine fusion antigen protein, 1/10 volume of 3M NaOAc (pH4.5) and 2 times the volume of 100% ethanol were added to the supernatant recovered through the PCI method. After reacting at -70°C for 2 hours, the reaction solution was centrifuged at 13,000 rpm at 4°C for 20 minutes to recover only the DNA aptamer. The recovered DNA aptamer was dried in air and then dissolved in 50 μl of nuclease free water.

3-2. Removal of non-specifically binding DNA aptamers

In order to remove the DNA aptamer that non-specifically binds to the composition in the column and buffer, not the antigen protein of the coronavirus, and at the same time improve the specificity of the DNA aptamer that binds to the antigen protein, the following experiment was performed.

First, the DNA aptamer obtained in Example 3-1 was amplified by a conventional method, and the process of Example 3-1 was repeated 6 times using the amplified product to select a DNA aptamer with improved specificity. Negative SELEX (negative SELEX) was performed to select a DNA aptamer to which an antigen protein is not bound using the selected DNA aptamer. The experiment was performed under the same conditions and methods as in Example 3-1, except that a column to which only the histidine protein was bound was used. At this time, only DNA aptamers that do not specifically bind under the above conditions were obtained. Using the obtained aptamer, the process of Example 3-1 was repeated 4 more times to perform SELEX a total of 9 times to finally obtain an aptamer that specifically binds to an antigen protein.

Example 4. Selection of high affinity DNA aptamer pool

The affinity for the RdRp protein of the DNA aptamer obtained in each round through SELEX was confirmed by a conventional nanodrop method.

Experimental Example 1. Quantitative confirmation of the affinity of novel coronavirus protein and DNA aptamer

1-1. Preparation of SARS-CoV-2 antigen protein coating sensor chip

Through a surface plasma resonance (SPR) experiment, the binding ability of the selected DNA aptamer to the SARS-CoV-2 antigen protein was quantitatively confirmed.

First, a mixture of 0.1 M NHS (Nhydroxysuccinimide) and 0.4 M EDC (N-ethyl-N' (dimethylaminopropyl) carbodiimide) was added to the sensor chip CM5 (GE healthcare, UK) coated with a carboxyl group at 5 μl/min. The carboxyl group on the surface was activated by flowing it at a speed for 10 minutes. On the surface of the carboxyl group-activated sensor chip CM5, a 10 mM sodium acetate (pH 4.5) solution containing 44 ng/μl of SARS-CoV-2 antigen protein was diluted 1/10 at a rate of 5 μl/min. The treatment was repeated 3 times for 10 minutes. 1 M ethanolamine hydrochloride (pH 8.5) was flowed to the sensor chip CM5 coated with the SARS-CoV-2 antigen protein at a rate of 5 μl/min for 10 minutes to inactivate the activated carboxyl groups on the surface. As a result, a sensor chip CM5 having a surface coated with SARS-CoV-2 antigen protein was fabricated.

1-2. Affinity confirmation of DNA aptamers

The affinity of the selected DNA aptamer to the SARS-CoV-2 antigen protein was quantitatively confirmed using the SPR method.

First, the RNA aptamer whose sequence was confirmed above was prepared by diluting it in HBS-EP buffer (GE Healthcare, BR-1001-88) to a concentration of 300, 500, 700 or 1,000 nM. The experiment was performed using BIAcore 3000 (BIACORE) according to the manufacturer's protocol, and the binding force of the DNA aptamer to the sensor chip CM5 coated with the SARS-CoV-2 antigen protein prepared in Experimental Example 1-1 and nothing was coated. The difference in the binding force of the DNA aptamer to the non-existent sensor chip CM5 was confirmed. At this time, the rate variables were obtained and quantified using the BIA evaluation program (BIACORE), and after each experiment, the sensor chip was regenerated using 1 M NaCl and 50 mM NaOH buffer. As a result, the affinity of the DNA aptamer to the SARS-CoV-2 antigen protein is shown in Table 2 below as a dissociation constant (K _D ) value.

name order Kd SEQ ID NO: CoV-S1-1 TCATCACGCAACTTTCATCAGTTCAGTACTAACATCGCGC 6.87×10 ^-9 SEQ ID NO: 1 Cov-S1-2 CGTCTGCCAGATCCACCACACCAGTGCCCCTGACTAGCTA 8.70×10 ^-8 SEQ ID NO: 2 Cov-S1-3 CCCTAGGCAAGTATACGCTGCAATGGCACTGTGCTTCAGG 1.49×10 ^-8 SEQ ID NO: 3 Cov-S1-4 TGGAACGCTGCAGTTGCGATAATGGGAGGAAGGTTTAGA 3.51×10 ^-8 SEQ ID NO: 4 Cov-S1-5 GGCAGCGTGTTTTGCGGTTGTAGTTGCCCCCAGTTCTGCA 2.06×10 ^-10 SEQ ID NO: 5 Cov-S2-1 CGGTACGGTGACAATCTCTGGAATGTTGGGAGAGTTTTTG 1.45×10 ^-8 SEQ ID NO: 6 Cov-S2-2 CACGTATAGGTGAATGTGCCATGTCATGCCCTCATGCCAG 2.06× 10 ^-10 SEQ ID NO: 7 Cov-S2-3 ACCGGGATGCGGTGCGGCGATAGCTCTTGGGAGTGTTCCG 1.81×10 ^-8 SEQ ID NO: 8 Cov-S2-4 GGCGCGGGGGGCAGCTCTGCCGTGTGTTGTTTGGCCAGAG 1.08×10 ^-9 SEQ ID NO: 9 Cov-S2-5 AGTGTCTGCGATTTCATTATACGGCTTGCCCCGAAGCACG 1.77×10 ^-7 SEQ ID NO: 10 Cov-S1+2-1 AAAGGAGTGGGTAGCATGTTCAGGACTATTCTGAGTTACC 1.45×10 ^-8 SEQ ID NO: 11 Cov-S1+2-2 CGCCTCTTCAGTGTGATCGAGGTTGACCTTACCAAATCTA 1.32×10 ^-8 SEQ ID NO: 12 Cov-S1+2-3 CACTCAGATGCGCCGCCCGAAGGTATGTCACGCGTGAGGA 3.97×10 ^-9 SEQ ID NO: 13 Cov-S1+2-4 ACCCACAACGGCGAGTTTATCCCATGAAGTTCTTGCTTGG 5.84×10 ^-8 SEQ ID NO: 14 Cov-S1+2-5 AAAAAAACTATATCCCAGGCATCGTGGGTGTATTGCGAAG 6.87×10 ^-9 SEQ ID NO: 15 Cov-RBD-1 CTGCAGTATAGGAAAACACCAAGATATCATGATACTCGAG 8.70×10 ^-8 SEQ ID NO: 16 Cov-RBD-2 CTGAGAGCCACCAGACGTTAGAAGTGGTGTTGAAATGGGG 1.31×10 ^-10 SEQ ID NO: 17 Cov-RBD-3 TGTATAAACAAACCAATTGACATGCTGTCCACCTGCCGAC 6.18×10 ^-9 SEQ ID NO: 18 Cov-RBD-4 CACTTGGCCTCTGTTGTAGTCTGGTAGTGGTAAACATAGG 1.76×10 ^-9 SEQ ID NO: 19 Cov-RBD-5 CGGCCGACTACCCTTTTGACTTTTTGTAGCTAGCTGGGCA 1.16×10 ^-8 SEQ ID NO: 20 Cov-NP-1 ACGTAGCCATGCTGTGTTATCAGAATCGTTGGATATTTTC 4.86×10 ^-9 SEQ ID NO: 21 Cov-NP-2 TCAGTTCAGTACTAACATCGCGCTCATCACGCAACTTTCA 5.13×10 ^-8 SEQ ID NO: 22 Cov-NP-3 TGCCAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCC 1.50×10 ^-8 SEQ ID NO: 23 Cov-NP-4 CCTATTCCTTATTATATTTTCTTTTTTTGTAATTTGGTCG 1.11×10 ^-8 SEQ ID NO: 24 Cov-NP-5 GGTGCGAACAATGCCTGGTTGAGAATGCTCAGAATGGGCG 3.12×10 ^-9 SEQ ID NO: 25 CoV-RdRp-1 GCTTTATGCACACGATTGTTATCATATTCGCTCCTACGTC 1.10×10 ^-9 SEQ ID NO: 26 CoV-RdRp-2 GCACAGACTTTTCAATCCGGTTGTTAAGGGGTAAAGGGCG 1.12×10 ^-9 SEQ ID NO: 27 CoV-RdRp-3 GGACGACGACGCACTGATAGGGTGTTGTGGCAATTGCTCG 5.42×10 ^-8 SEQ ID NO: 28 CoV-RdRp-4 CCAGCACTCGATCGTATTCCATTTCGGCTAATGCGTTCTA 1.33×10 ^-10 SEQ ID NO: 29 CoV-RdRp-5 CGCGTGGTGAATTTTAAGCATCCGAAATGGTATTCAGCTA 4.25×10 ^-10 SEQ ID NO: 30

Experimental Example 2. Preparation of biological sample lysis reagent for SARS-CoV-2 detection

2-1. Optimization of biological sample lysis reagent for SARS-CoV-2 detection through STR analysis

For the optimization of the SARS-CoV-2 infection biological sample lysis reagent, chelex® 100 powder (sigma Aldrich, USA) was diluted in 10 ml of distilled water to a concentration of 3, 5, 8, or 10% to prepare a lysis reagent. For the biological sample, the oral epithelial cells of the experimenter were used, and a sterilized cotton swab was prepared by rubbing it into the mouth of the experimenter for at least 1 minute. The surface of the cotton swab imbued with oral epithelial cells was cut with a flame-sterilized scalpel, only the outermost part was recovered in the e-tube, and 500 ul of the prepared lysis reagent was added to the recovered sample. The e-tube containing the lysis reagent was reacted on a heat block at 95°C for 10 minutes and 15 minutes, respectively, and then cooled at room temperature, and only the supernatant was recovered through centrifugation. The recovered sample supernatant was used as a template DNA in the PCR amplification process preceding for short tandem repeat (STR) analysis. PCR process is performed by mixing 5ul of 2X HS prime Taq premix (Genet Bio, Korea), 2ul of distilled water, 1ul of primer mixture, and 2ul of template DNA to prepare a total of 10ul of PCR mixture. Pre-denaturation 96℃, 1 minute; Denaturation 94° C., 1 min; Annealing 60°C, 1 min; Extension 72°C, 1 min; Final extension 60℃, 1 hour process was performed, and the Denaturation ~ Extension process was repeated 28 times. The PCR product was loaded on a 2% agarose gel to confirm the DNA band, and the results are shown in FIG. 1 .

As shown in FIG. 1, PCR products were observed in all samples subjected to 3-10% chelex concentration conditions and dissolution conditions of 85°C and 95°C, and it was confirmed that there was no problem in DNA extraction from biological samples under the conditions.

2-2. Confirmation of amplification using capillary electrophoresis method

It was verified through STR profiling analysis that the PCR product was detected correctly in the STR-based detection method. The size of the PCR product obtained as a result of PCR was confirmed by using a capillary electrophoresis method. The DNA concentration of the PCR amplification product was diluted with distilled water to 600 ng/ul. Specifically, 9.3 μl of Hi-Di foramide, 0.05 μl of GeneScan-500LIZ (ABI, USA) and 0.3 μl of the PCR product were mixed, and heated at 95° C. for 3 minutes to transform the PCR product into a single base strand. The obtained single nucleotide strand was applied to a capillary tube of 3130XL Genetic Analyzer or 3730 Genetic Analyzer, and the amplification product developed by electrophoresis was analyzed using GeneMapper ID software, and the results are shown in FIG. 2 .

As shown in FIG. 2 , as the concentration of chelex increased, there was no tendency to increase the STR peak by locus, but the peaks of some loci (D3S1358, TH01, D21S11, Amelogenin) at low concentrations of chelex were very low. A distinguishable STR peak was observed from a minimum of 5% chelex concentration. The difference in STR peak according to the dissolution temperature did not show much, so the dissolution condition was fixed at 95°C.

2-3. Optimization of dissolution conditions according to dissolution time and enzyme treatment

In order to optimize the dissolution time and the dissolution conditions of biological samples by enzyme treatment such as proteinase K, the dissolution time was increased from 5 minutes to 10 minutes and 20 minutes, and the dissolution results were confirmed with or without proteinase K treatment. The dissolution result was confirmed in the same manner as in the previous method, but proteinase K treatment was added after the chelex dissolution process. After dissolution, proteinase K (Biofact, Korea) was added and reacted at 63° C. for 10 minutes or 20 minutes. After the dissolution process was completed, the sample was loaded on a 2% agarose gel to confirm the DNA band of the PCR product, and the results are shown in FIG. 3 .

Meanwhile, the size of the PCR product obtained as a result of PCR was confirmed by using a capillary electrophoresis method. The DNA concentration of the PCR amplification product was diluted with distilled water to 600 ng/ul. Specifically, 9.3 μl of Hi-Di foramide, 0.05 μl of GeneScan-500LIZ (ABI, USA) and 0.3 μl of the PCR product were mixed, and heated at 95° C. for 3 minutes to transform the PCR product into a single base strand. The obtained single nucleotide strand was applied to a capillary of 3130XL Genetic Analyzer or 3730 Genetic Analyzer, and the amplification product developed by electrophoresis was analyzed using GeneMapper ID software, and the results are shown in FIG. 4 .

3 and 4, when the dissolution time was increased from 5 minutes to 10 minutes on the STR analysis result, the STR peaks of some loci (TH01, D21S11) were increased. In the sample in which the dissolution time was increased to more than 10 minutes, no additional STR peak amplification tendency was observed. In terms of dissolution rate of biological samples according to the presence or absence of proteinase K addition, when protease K was added, the overall peak level increased compared to the STR peak of the sample without addition of proteinase K. In particular, the peak of the locus (D3S1358, TH01, D21S11, Amelogenin) that showed a low peak under the previous conditions was higher than before. The dissolution reagent conditions for dissolution of the final biological sample were 5% chelex and proteinase K, and the dissolution conditions were selected as the dissolution conditions at 95°C for 10 minutes and then at 63°C for 10 minutes.

Experimental Example 3. Confirmation of SARS-CoV-2 detection power of DNA aptamer

Using an aptamer pair having high affinity with each antigen protein selected above (spike protein (S1, S2, S1+S2), Receptor Binding Domain (RBD), nucleocapsid protein, RNA-dependent RNA polymerase (RdRp)) Thus, the detection ability for the SARS-CoV-2 antigen protein was confirmed through an experiment. CoV-1-5, CoV-S2-2, CoV-S1+2-5, CoV-RBD-2, CoV labeled with an amine group (NH2) in each well coated with NOS group (N-oxysuccinimide esters group) -NP-1, Cov-RdRp-5 aptamer was calculated at a concentration of 1uM, mixed with the aptamer screening solution, adjusted to 100ul of reaction volume, stirred at room temperature for 30 minutes to fix the aptamer on the bottom of the plate, and then the solution was removed. Wash wells in which CoV-1-5, CoV-S2-2, CoV-S1+2-5, CoV-RBD-2, CoV-NP-1, and Cov-RdRp-5 are fixed with 100ul of 1X PBS buffer 3 times After giving out, the antigen protein binding specifically to each aptamer was calculated at a concentration of 100 nM, respectively, and mixed with 1X PBS buffer to make a total of 100 ul, and then put into each well and reacted at 4 ° C. for 1 hour. As a negative control, 100ul of 1X PBS was added instead of the antigen protein. After that, all solutions in the wells were removed and the wells were washed 3 times with 100ul of 1X PBS buffer. Another selected aptamer, CoV-1-1, CoV-S2-4, CoV-S1+2-3, CoV-RBD-3, CoV-NP-5, Cov-RdRp-4, is Cy5 at the 5' end. The fluorescent material was labeled, calculated as 1 uM, and mixed with 1X aptamer selection solution so that the final volume was 100 ul. After dispensing 100 μl of each target antigen protein to a well in which each specific antigen protein was immobilized, the plate was wrapped in foil to block light, and then reacted at room temperature for 20 minutes. After removing all the solutions in the wells and washing with 100ul of 1X PBS buffer, the fluorescence values were measured for each well at Exitation 646 nm and Emission 662 nm with a microplate reader. As a negative control, a sample treated with only the DNA aptamer labeled with the Cy5 fluorescent substance and the DNA aptamer labeled with an amine group was also measured.

As a result, as shown in FIG. 5 , fluorescence values were hardly observed in the wells to which the antigenic protein was not added, but high fluorescence values were measured in the wells in which the antigenic proteins were fixed. In addition, although the antigen protein and the fluorescent substance were treated together, the fluorescence value was hardly observed even in the wells to which the amine-labeled aptamer was not immobilized.

<110> HPBIO Inc. <120> DNA APTAMER SPECIFICALLY BINDING TO CORONAVIRUS AND USING THE SAME <130> DP-2020-0310-KR <160> 33 <170> KoPatentIn 3.0 <210> 1 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> CoV-S1-1 <400> 1 tcatcacgca actttcatca gttcagtact aacatcgcgc 40 <210> 2 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Cov-S1-2 <400> 2 cgtctgccag atccaccaca ccagtgcccc tgactagcta 40 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Cov-S1-3 <400> 3 ccctaggcaa gtatacgctg caatggcact gtgcttcagg 40 <210> 4 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Cov-S1-4 <400> 4 tggaacgctg cagttgcgat aatgggagga aggtttaga 39 <210> 5 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Cov-S1-5 <400> 5 ggcagcgtgt tttgcggttg tagttgcccc cagttctgca 40 <210> 6 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Cov-S2-1 <400> 6 cggtacggtg acaatctctg gaatgttggg agagtttttg 40 <210> 7 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Cov-S2-2 <400> 7 cacgtatagg tgaatgtgcc atgtcatgcc ctcatgccag 40 <210> 8 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Cov-S2-3 <400> 8 accgggatgc ggtgcggcga tagctcttgg gagtgttccg 40 <210> 9 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Cov-S2-4 <400> 9 ggcgcggggg gcagctctgc cgtgtgttgt ttggccagag 40 <210> 10 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Cov-S2-5 <400> 10 agtgtctgcg atttcattat acggcttgcc ccgaagcacg 40 <210> 11 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Cov-S1+2-1 <400> 11 aaaggagtgg gtagcatgtt caggactatt ctgagttacc 40 <210> 12 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Cov-S1+2-2 <400> 12 cgcctcttca gtgtgatcga ggttgacctt accaaatcta 40 <210> 13 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Cov-S1+2-3 <400> 13 cactcagatg cgccgcccga aggtatgtca cgcgtgagga 40 <210> 14 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Cov-S1+2-4 <400> 14 acccacaacg gcgagtttat cccatgaagt tcttgcttgg 40 <210> 15 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Cov-S1+2-5 <400> 15 aaaaaaacta tatcccaggc atcgtgggtg tattgcgaag 40 <210> 16 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Cov-RBD-1 <400> 16 ctgcagtata ggaaaacacc aagatatcat gatactcgag 40 <210> 17 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Cov-RBD-2 <400> 17 ctgagagcca ccagacgtta gaagtggtgt tgaaatgggg 40 <210> 18 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Cov-RBD-3 <400> 18 tgtataaaca aaccaattga catgctgtcc acctgccgac 40 <210> 19 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Cov-RBD-4 <400> 19 cacttggcct ctgttgtagt ctggtagtgg taaacatagg 40 <210> 20 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Cov-RBD-5 <400> 20 cggccgacta cccttttgac tttttgtagc tagctgggca 40 <210> 21 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Cov-NP-1 <400> 21 acgtagccat gctgtgttat cagaatcgtt ggatattttc 40 <210> 22 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Cov-NP-2 <400> 22 tcagttcagt actaacatcg cgctcatcac gcaactttca 40 <210> 23 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Cov-NP-3 <400> 23 tgccaaaagg ccgcgttgct ggcgtttttc cataggctcc 40 <210> 24 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Cov-NP-4 <400> 24 cctattcctt attatatttt ctttttttgt aatttggtcg 40 <210> 25 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Cov-NP-5 <400> 25 ggtgcgaaca atgcctggtt gagaatgctc agaatgggcg 40 <210> 26 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> CoV-RdRp-1 <400> 26 gctttatgca cacgattgtt atcatattcg ctcctacgtc 40 <210> 27 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> CoV-RdRp-2 <400> 27 gcacagactt ttcaatccgg ttgttaaggg gtaaagggcg 40 <210> 28 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> CoV-RdRp-3 <400> 28 ggacgacgac gcactgatag ggtgttgtgg caattgctcg 40 <210> 29 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> CoV-RdRp-4 <400> 29 ccagcactcg atcgtattcc atttcggcta atgcgttcta 40 <210> 30 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> CoV-RdRp-5 <400> 30 cgcgtggtga attttaagca tccgaaatgg tattcagcta 40 <210> 31 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> template DNA <400> 31 ggtaatacga ctcactatag ggagatacca gcttattcaa ttnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nnnnnnnnnn nnagatagta agtgcaatct 100 <210> 32 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 32 ggtaatacga ctcactatag ggagatacca gcttattcaa tt 42 <210> 33 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 33 agattgcact tactatct 18

Claims (5)

DNA aptamer that specifically binds to coronavirus.
A composition comprising the DNA aptamer of claim 1.
A composition for detecting a coronavirus comprising the DNA aptamer of claim 1.
A method of detecting a coronavirus using the DNA aptamer of claim 1.
A method of providing information necessary for diagnosis of a disease caused by coronavirus infection using the DNA aptamer of claim 1.

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