DNA aptamer specifically binding to envelope protein of sarscov2 and use thereof

Abstract

The present invention relates to a DNA aptamer specifically binding to an envelope protein of SARS-CoV-2 and use thereof. Specifically, the present invention relates to a DNA aptamer specifically binding to an envelope protein of SARS-CoV-2 virus, a composition for detecting an envelope protein of SARS-CoV-2 virus, comprising the DNA aptamer as an active ingredient, a kit for detecting SARS-CoV-2 virus, a chip or microarray for detecting SARS-CoV-2 virus, and a method for detecting an envelope protein of SARS-CoV-2 virus, using the DNA aptamer. Also, the present invention relates to: a composition for diagnosing coronavirus infection-19 and a pharmaceutical composition for preventing or treating coronavirus infection-19, each composition comprising the DNA aptamer of the present invention as an active ingredient; a method for producing a DNA aptamer specifically binding to an envelope protein of SARS-CoV-2 virus; and a method for providing information necessary for diagnosing coronavirus infection-19.

Description

DNA aptamer specifically binding to the coat protein of SRSC2 and uses thereof

[0001] The present invention relates to a DNA aptamer that specifically binds to the SARS-CoV-2 envelope protein and uses thereof.

[0002] COVID-19, which emerged in December 2019, is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. It has caused high morbidity and mortality rates worldwide and continues to spread through numerous mutations during reproduction and transmission. SARS-CoV-2 has an incubation period of approximately two days to two weeks, after which symptoms appear. The most common symptoms include lethargy, dry cough, fever, muscle pain, and nasal congestion. In severe cases, it has caused complications such as myocardial damage, arrhythmia, stroke, and septic shock, and death has also been reported.

[0003] The coronavirus family, coronaviridae, to which SARS-CoV-2 belongs, has four genera, and coronaviruses classified into the alpha and beta genera cause infections in humans and animals. Alphacoronaviruses, including types 229D, OC43, NL63, and HKU1, are seasonal respiratory viruses that mostly cause upper respiratory tract infections, while betacoronaviruses include SARS-CoV, MERS-CoV, and SARS-CoV-2. The genome of SARS-CoV-2 shares approximately 79% with SARS-CoV and 50% with MERS-CoV.

[0004] Currently, the antiviral drugs Paxrobid and Lagebrio are being used in Korea to treat SARS-CoV-2. Paxrobid is an oral coronavirus treatment developed by Pfizer. However, its combination with 28 contraindications makes prescribing it difficult, and in June 2022, several studies reported the emergence of viral mutations resistant to Paxrobid. Lagebrio is a treatment developed by the US pharmaceutical company Merck (MSD) to inhibit viral replication and is one of the most widely used treatments in Korea. However, according to a joint research team from the Francis Crick Institute and the University of Cambridge in the UK, the frequency of mutations has increased significantly since the use of this treatment has increased, and the related mutation patterns were confirmed to be lower in countries that have not approved the use of Lagebrio's active ingredient, molnupiravir. Furthermore, there are concerns that prescribing Paxrobid and molnupiravir together could lead to the generation of other dangerous mutant viruses.

[0005] SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA virus. The virion is composed of the structural proteins nucleocapsid (N), membrane (M), envelope (E), and spike (S). The S protein is the most important protein for cell entry, as it contains an immune recognition site and binds to surface receptors during host cell entry. The N protein packages the viral RNA into a helical ribonucleocapsid and is involved in the CoV replication cycle and the host cell response to viral infection. The M protein promotes viral assembly through interactions with other structural proteins, and in particular, induces the formation of stable virus-like particles even when only the E and M proteins interact. The E protein forms an ion channel that selectively transports cations. Through self-interactions and interactions with other structural, nonstructural, accessory, and host proteins, it modulates the host immune response by activating the NLRP3 inflammasome and the PDZ-binding motif in the C-terminus. Additionally, as a protein that is well conserved across various virus subtypes, deletion of the E protein of SARS-CoV and SARS-CoV-2 weakens or eliminates the virulence of the virus.

[0006] The E protein of SARS-CoV-2 is 98.8% conserved across numerous SARS-CoV-2 variants that have emerged since the outbreak. This high conservation not only makes it a potential vaccine target, but also suggests potential applications in diagnosis and treatment. These E proteins interact with other E proteins, the M protein, and the PALS1 protein, forming dimers, trimers, and pentamers through disulfide bonds. The formed E proteins form quintuple ion channels, which trigger a cytokine storm that leads to acute respiratory distress syndrome (ARS). The interaction between the E and M proteins is involved in viral assembly and infection, while the interaction with the PALS1 protein facilitates viral transmission. Furthermore, the interactions between the E proteins allow SARS-CoV-2 to multiply more easily, stably, and rapidly.

[0007] Meanwhile, respiratory infections caused by SARS-CoV-2 infection have continued to occur since 2019, necessitating the development of technologies that can more effectively detect and treat SARS-CoV-2.

[0008] Accordingly, the present inventors have completed the present invention by developing a novel DNA aptamer that can specifically bind to the E protein of SARS-CoV-2 using an aptamer, which is a short single-stranded oligonucleotide that can bind various molecules with high affinity, specificity, and cost-effectiveness, and can shorten the treatment time for severe patients by stopping the spread of the virus and improving excessive immune responses such as cytokine storm in patients.

[0009] Therefore, the purpose of the present invention is to provide a DNA aptamer that specifically binds to the envelope protein of the SARS-CoV-2 virus.

[0010] Another object of the present invention is to provide a composition for detecting the envelope protein of SARS-CoV-2 virus, which comprises the DNA aptamer of the present invention as an active ingredient.

[0011] Another object of the present invention is to provide a kit for detecting SARS-CoV-2 virus comprising the above detection composition as an active ingredient.

[0012] Another object of the present invention is to provide a chip or microarray for detecting SARS-CoV-2 virus, characterized in that the DNA aptamer of the present invention is immobilized on a substrate and specifically reacts with a biological sample isolated from an individual infected with the SARS-CoV-2 virus.

[0013] Another object of the present invention is to provide a method for detecting the envelope protein of a SARS-CoV-2 virus, comprising: (1) a step of contacting a biological sample isolated from a living body with the DNA aptamer of the present invention; and (2) a step of measuring the presence or content of the envelope protein of the SARS-CoV-2 virus in the biological sample by confirming a specific binding reaction between the biological sample and the DNA aptamer and the envelope protein of the SARS-CoV-2 virus.

[0014] Another object of the present invention is to provide a composition for diagnosing coronavirus infection-19 (COVID-19), comprising the DNA aptamer of the present invention as an active ingredient.

[0015] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating coronavirus infection-19 (COVID-19), comprising the DNA aptamer of the present invention as an active ingredient.

[0016] Another object of the present invention is to provide a method for producing a DNA aptamer that specifically binds to the envelope protein of a SARS-CoV-2 virus, comprising the steps of: (1) amplifying a DNA aptamer using PCR and asymmetric PCR techniques and selecting a single-stranded DNA aptamer; (2) inducing binding of the single-stranded DNA aptamer to the envelope protein of a SARS-CoV-2 virus; (3) binding the envelope protein of the SARS-CoV-2 virus bound to the single-stranded DNA aptamer to Ni-NTA agarose resin; (4) removing DNA aptamers that fail to bind to the envelope protein of the SARS-CoV-2 virus; and (5) recovering and selecting a DNA aptamer that specifically binds to the envelope protein of the SARS-CoV-2 virus using a SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technique.

[0017] Another object of the present invention is to provide a method for providing information necessary for diagnosing coronavirus disease-19 (COVID-19), comprising: (1) a step of contacting a biological sample isolated from a living body with the DNA aptamer of the present invention; and (2) a step of measuring the presence or content of the envelope protein of the SARS-CoV-2 virus in the biological sample by confirming a specific binding reaction between the biological sample and the DNA aptamer and the envelope protein of the SARS-CoV-2 virus.

[0018] To achieve the above purpose, the present invention provides a DNA aptamer that specifically binds to the envelope protein of the SARS-CoV-2 virus.

[0019] In one embodiment of the present invention, the DNA aptamer may be composed of any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 16.

[0020] In one embodiment of the present invention, the DNA aptamer may further include a labeling substance.

[0021] In one embodiment of the present invention, the labeling material may be any one labeling material selected from the group consisting of a fluorescent material, an amine group, biotin, a thiol group, and digoxigenin, labeled at the 5' end or the 3' end of the DNA aptamer.

[0022] In one embodiment of the present invention, the DNA aptamer may be single-stranded DNA.

[0023] In addition, the present invention provides a composition for detecting the envelope protein of SARS-CoV-2 virus, which comprises the DNA aptamer of the present invention as an active ingredient.

[0024] In addition, the present invention provides a kit for detecting SARS-CoV-2 virus comprising the detection composition of the present invention as an active ingredient.

[0025] In addition, the present invention provides a chip or microarray for detecting SARS-CoV-2 virus, characterized in that the DNA aptamer of the present invention is immobilized on a substrate and specifically reacts with a biological sample isolated from an individual infected with the SARS-CoV-2 virus.

[0026] In addition, the present invention provides a method for detecting the envelope protein of a SARS-CoV-2 virus, comprising: (1) a step of contacting a biological sample isolated from a living body with the DNA aptamer of the present invention; and (2) a step of measuring the presence or content of the envelope protein of the SARS-CoV-2 virus in the biological sample by confirming a specific binding reaction between the biological sample and the DNA aptamer and the envelope protein of the SARS-CoV-2 virus.

[0027] The present invention also provides a composition for diagnosing coronavirus infection-19 (COVID-19), comprising the DNA aptamer of the present invention as an active ingredient.

[0028] The present invention also provides a pharmaceutical composition for preventing or treating coronavirus infection-19 (COVID-19), comprising the DNA aptamer of the present invention as an active ingredient.

[0029] In addition, the present invention provides a method for producing a DNA aptamer that specifically binds to the envelope protein of a SARS-CoV-2 virus, comprising: (1) a step of amplifying a DNA aptamer using PCR and asymmetric PCR techniques and selecting a single-stranded DNA aptamer; (2) a step of inducing binding of the single-stranded DNA aptamer to the envelope protein of a SARS-CoV-2 virus; (3) a step of binding the envelope protein of the SARS-CoV-2 virus bound to the single-stranded DNA aptamer to a Ni-NTA agarose resin; (4) a step of removing DNA aptamers that fail to bind to the envelope protein of the SARS-CoV-2 virus; and (5) a step of recovering and selecting a DNA aptamer that specifically binds to the envelope protein of the SARS-CoV-2 virus using a SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technique.

[0030] In one embodiment of the present invention, the step (5) may include a real-time PCR step for selecting a SELEX round having optimal affinity; a PCR and cloning step for selecting a group of DNA aptamer candidates that bind to the envelope protein of the SARS-CoV-2 virus in the optimal SELEX round; a step for measuring the affinity of the selected group of DNA aptamer candidates for the envelope protein of the SARS-CoV-2 virus; and a step for determining the DNA sequence of the selected group of DNA aptamer candidates that bind to the envelope protein of the SARS-CoV-2 virus.

[0031] Furthermore, the present invention provides a method for providing information necessary for diagnosing coronavirus disease-19 (COVID-19), comprising: (1) a step of contacting a biological sample isolated from a living body with the DNA aptamer of the present invention; and (2) a step of measuring the presence or content of the envelope protein of the SARS-CoV-2 virus in the biological sample by confirming a specific binding reaction between the biological sample and the DNA aptamer and the envelope protein of the SARS-CoV-2 virus.

[0032] The DNA aptamer of the present invention has the characteristic of being able to specifically bind to the envelope protein of SARS-CoV-2, and is not only non-immunogenic and non-toxic and highly stable, but also can bind to the envelope protein of the SARS-CoV-2 virus to inhibit the spread of the virus and improve excessive immune responses such as cytokine storms in infected individuals, so that it can be used not only for the detection and diagnosis of the SARS-CoV-2 virus, but also for the prevention, improvement, or treatment of coronavirus infection-19.

[0033] Figure 1 shows the results of selectively recovering only ssDNA using streptavidin beads after amplifying random DNA aptamers using PCR and asymmetric PCR techniques. Lane M: 100 bp DNA marker, Lane 1: The result of amplifying the DNA aptamer pool using PCR technique and purifying it using a purification kit, Lane 2: The result of amplifying the DNA aptamer pool by diluting it by 1 / 2 using PCR technique and purifying it using a purification kit, Lane 3: The result of amplifying the DNA aptamer pool by diluting it by 1 / 5 using PCR technique and purifying it using a purification kit.

[0034] Figure 2 shows the amount of DNA aptamer that binds to the SARS-CoV-2 envelope protein recovered in each round during the SELEX process for producing a DNA aptamer of the present invention that specifically binds to the envelope protein of SARS-CoV-2, quantitatively measured using a nanodrop.

[0035] Figure 3 shows the secondary structure of DNA aptamers according to the present invention selected to exhibit specific binding affinity to the envelope protein of SARS-CoV-2.

[0036] Figure 4 shows the binding structures of five types selected as optimal aptamers in one embodiment of the present invention.

[0037] Figure 5 shows an image of the site of interaction between the Apta-E-36 aptamer, selected as the optimal aptamer in one embodiment of the present invention, and four proteins, namely, Wild type, Beta, Omicron BA2.75, and Omicron BA1.

[0038] Figure 6A is an image showing optimized aptamers including the Apta-E-36 sequence that specifically binds to the envelope protein of SARS-CoV-2, and Figure 6B is an image showing the binding structure and sequence of the optimized aptamer Apta-E-36-3 that can bind to all mutants.

[0039] The present invention relates to a DNA aptamer that specifically binds to the envelope protein of SARS-CoV-2 and uses thereof.

[0040] The present inventors have discovered a novel aptamer that can rapidly and accurately detect the SARS-CoV-2 virus while solving the problems of existing antibody-based target substance detection techniques.

[0041] The DNA aptamer of the present invention capable of detecting the SARS-CoV-2 virus is a DNA aptamer that specifically binds to the envelope protein of the SARS-CoV-2 virus, and it was confirmed that the DNA aptamer designed in the present invention specifically binds to the envelope protein of the SARS-CoV-2 virus, so that the OP protein of the SARS-CoV-2 virus can be easily detected from a biological sample, and it was confirmed that it can also be used for the diagnosis of COVID-19 infection, a disease caused by SARS-CoV-2 virus infection.

[0042] The DNA aptamer that specifically binds to the envelope protein of the SARS-CoV-2 virus of the present invention refers to a single-stranded DNA that can bind to a target substance with high specificity and affinity, and can be produced through the SELEX (Systematic Evolution of Ligand of Exponential Enrichment) technique.

[0043] As used herein, the term "DNA aptamer" refers to a DNA nucleic acid molecule capable of binding to a specific molecule with high affinity and specificity. The term "DNA aptamer" is used interchangeably with "DNA oligonucleotide." Aptamers are short oligomers characterized by forming a stable tertiary structure and exhibiting specific binding affinity to a target substance. Furthermore, aptamers are composed of nucleic acids (DNA or RNA), making them more stable than antibodies, which are composed of proteins. They also possess the advantage of being able to bind specifically to various target substances (proteins, peptides, metals, chemicals, etc.). Furthermore, because they can be manufactured using chemical synthesis techniques, they can be mass-produced in a short period of time and at low cost. Furthermore, aptamers have the advantage of continuously producing aptamers with the same properties after a single production run. Furthermore, they are highly stable across a wide range of pH and temperature conditions, leading to their potential applications in various fields, including environmental and medical fields, such as the development of target substance detection and disease diagnostic sensors.

[0044] In one embodiment of the present invention, the DNA aptamer that specifically binds to the envelope protein of the SARS-CoV-2 virus may be an oligonucleotide having any one base sequence selected from the group consisting of base sequences of SEQ ID NO: 1 to SEQ ID NO: 16, or having a base sequence having 90% or more identity with such base sequence. The DNA aptamer may be one in which the hydroxyl group at the 2' position of the ribose of one or more nucleotides constituting the DNA aptamer is substituted with any one selected from the group consisting of a hydrogen atom, a fluorine atom, -OR, -COOR, and an amino group.

[0045] In addition, the DNA aptamer may further include a labeling substance, and the labeling substance may be any one labeling substance selected from the group consisting of a fluorescent substance, an amine group, biotin, a thiol group, and digoxigenin, which may be labeled at the 5' end or the 3' end of the DNA aptamer.

[0046] As used herein, the term "oligonucleotide" generally refers to a nucleotide polymer having a length of less than about 200 nucleotides, which may include DNA and RNA, and is preferably a DNA nucleic acid molecule. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substrate that can be incorporated into the polymer by a DNA or RNA polymerase or by a synthetic reaction. If modifications to the nucleotide structure are present, such modifications may be added before or after the synthesis of the oligonucleotide polymer. The nucleotide sequence may be interrupted by non-nucleotide components. The oligonucleotide may be further modified after synthesis, for example, by conjugation to a label.

[0047] The DNA aptamer of the present invention can typically be obtained by an in vitro selection method for binding to a target molecule. Methods for selecting aptamers that specifically bind to a target molecule are well known in the art. For example, organic molecules, nucleotides, amino acids, polypeptides, cell surface marker molecules, ions, metals, salts, and polysaccharides can be suitable target molecules for isolating aptamers that can specifically bind to each ligand. Aptamer selection can utilize a known in vivo or in vitro selection technique, such as the SELEX method (Ellington et al., Nature 346, 818-22, 1990; and Tuerk et al., Science 249, 505-10, 1990). Specific methods for screening and preparing aptamers are described in U.S. Pat. No. 5,582,981, WO 00 / 20040, U.S. Pat. No. 5,270,163, Lorsch and Szostak, Biochemistry, 33:973 (1994), Mannironi et al., Biochemistry 36:9726 (1997), Blind, Proc. Natl. Acad. Sci. USA 96:3606-3610 (1999), Huizengan and Szostak, Biochemistry, 34:656-665 (1995), WO 99 / 54506, WO 99 / 27133, WO 97 / 42317, and U.S. Pat. No. 5,756,291, which are incorporated herein by reference.

[0048] Additionally, the DNA aptamer that specifically binds to the envelope protein of the SARS-CoV-2 virus of the present invention forms a structure as shown in FIG. 3 of the present specification.

[0049] The DNA aptamer of the present invention that specifically binds to the envelope protein of the SARS-CoV-2 virus is interpreted to also include an oligonucleotide having a base sequence that exhibits substantial identity with any one base sequence selected from the group consisting of base sequences of SEQ ID NOs: 1 to 16, while maintaining the property of binding to the envelope protein of the SARS-CoV-2 virus.

[0050] The substantial identity is determined by aligning the nucleotide sequence of the present invention with any other sequence to the greatest extent possible, and by using an algorithm commonly used in the art (Smith and Waterman, Adv. Appl. Math. 2:482 (1981) Needleman and Wunsch, J.Mol. Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc. Acids Res. 16:10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8:155-65 (1992) and Pearson et al., Meth. Mol. When the aligned sequence is analyzed using the method of Biol. 24:307-31 (1994)), it refers to a nucleotide sequence that exhibits at least 90% identity, more preferably at least 95% identity, and most preferably at least 98% identity.

[0051] In addition, the envelope protein of the SARS-CoV-2 virus to which the DNA aptamer of the present invention can bind may preferably be composed of the amino acid sequence of SEQ ID NO: 17.

[0052] Furthermore, the present invention can provide a method for producing a DNA aptamer that specifically binds to the envelope protein of the SARS-CoV-2 virus, the method preferably comprising: (1) a step of amplifying a DNA aptamer using PCR and an asymmetric PCR technique and selecting a single-stranded DNA aptamer; (2) a step of inducing binding of the single-stranded DNA aptamer to the envelope protein of the SARS-CoV-2 virus; (3) a step of binding the envelope protein of the SARS-CoV-2 virus bound to the single-stranded DNA aptamer to a Ni-NTA agarose resin; (4) a step of removing DNA aptamers that do not bind to the envelope protein of the SARS-CoV-2 virus; and (5) a step of recovering and selecting a DNA aptamer that specifically binds to the envelope protein of the SARS-CoV-2 virus using a SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technique.

[0053] Here, the step (5) above specifically comprises a real-time PCR step for selecting a SELEX round having optimal affinity; a PCR and cloning step for selecting a group of DNA aptamer candidates binding to the envelope protein of the SARS-CoV-2 virus in the optimal SELEX round; a step for measuring the affinity of the group of DNA aptamer candidates binding to the envelope protein of the SARS-CoV-2 virus; and a step for determining the DNA sequence of the selected DNA aptamer candidates.

[0054] In one embodiment of the present invention, in order to select the DNA aptamer of the present invention, an asymmetric PCR was performed to amplify only ssDNA (single strand DNA) among dsDNA (double strand DNA) amplified using PCR, and at this time, the asymmetric PCR was performed using 10 μl of the forward primer and 2 μl of the reverse primer at a 10:2 ratio of the forward primer and the reverse primer at the same concentration (25 uM), thereby obtaining ssDNA. In addition, the method for selecting the ssDNA aptamer was performed by attaching biotin to the reverse primer during PCR to amplify dsDNA, and treating the 3' end of the amplification product with streptavidin to form a biotin-streptavidin complex, and selectively removing the complex, thereby obtaining only the ssDNA aptamer on the opposite end to which biotin is not bound.

[0055] Afterwards, a DNA aptamer having binding ability to the envelope protein of the SARS-CoV-2 virus was selected using the SELEX technique using the envelope protein of the SARS-CoV-2 virus among the selected ssDNA aptamers. According to one embodiment of the present invention, after amplifying and securing the aptamer sequence, a step of immobilizing the envelope protein of the SARS-CoV-2 virus on a resin; a step of binding the immobilized envelope protein and the DNA aptamer; and a step of recovering only the binding DNA aptamers reactive with the envelope protein were performed as one round to select an optimal SELEX round having high affinity and specificity.

[0056] In addition, the selection process for the optimal SELEX round was performed through the following steps: an optimal round selection step using a NanoDrop spectrophotometer (NanoDrop, USA) and quantitative analysis using real-time PCR; a PCR and cloning step to secure a group of SARS-CoV-2 viral envelope protein-specific binding DNA aptamer candidates in the optimal SELEX round; and a DNA sequencing step of the secured DNA aptamer candidates.

[0057] In the present invention, the “SELEX method” refers to a method of identifying the DNA binding sequence of a specific molecule by selecting and amplifying DNA having a high binding affinity for the specific molecule from a collection of randomly synthesized DNAs (Louis et al. 1992. Nature 355, 564-566).

[0058] The present invention can select the SARS-CoV-2 virus envelope protein-specific binding DNA aptamer recovered from each round for the purpose of continuing SELEX and selecting the optimal round after completing the SELEX process up to 12 times for the optimal SELEX round selection through Nanodrop. After that, in order to secure the sequence of the SARS-CoV-2 virus envelope protein-specific binding DNA aptamer candidate in the selected optimal SELEX round, PCR and cloning were performed, and then the DNA sequence of the selected SARS-CoV-2 virus envelope protein-specific binding DNA aptamer candidate was determined.

[0059] The DNA aptamer of the present invention, which specifically binds to the envelope protein of the SARS-CoV-2 virus discovered through the above method, can be used to directly detect the envelope protein of the SARS-CoV-2 virus or to detect the envelope protein of the SARS-CoV-2 virus in a biological sample of a patient, and can be used to diagnose coronavirus disease-19 (COVID-19).

[0060] The detection of the envelope protein of the SARS-CoV-2 virus of the present invention is based on a method for detecting a complex of a DNA aptamer that specifically binds to the envelope protein of the SARS-CoV-2 virus and the envelope protein of the SARS-CoV-2 virus. In one embodiment of the present invention, the DNA aptamer that specifically binds to the envelope protein of the SARS-CoV-2 virus of the present invention may include a nucleotide that is labeled with a chemical substance such as a fluorescent substance (e.g., fluorescein, Cy3, Cy5, or HRP), a radioactive substance, or biotin, or modified with a primary amine, to facilitate detection of the complex.

[0061] In addition, the DNA aptamer of the present invention can be manufactured in the form of a biosensor, kit, and chip including an immobilized substrate and used to detect the envelope protein of the SARS-CoV-2 virus.

[0062] As used herein, the term “sensor and chip” refers to a sensor and chip in which a specific material is densely attached to a specific area of ​​a substrate. As used herein, the term “substrate” of the biosensor and sensor chip refers to a support having suitable rigidity or semi-rigidity, and includes, but is not limited to, glass, a membrane, a slide, a filter, a chip, a wafer, a fiber, a magnetic bead or a non-magnetic bead, a gel, a tubing, a plate, a polymer, a microparticle, and a capillary. The DNA aptamer of the present invention can be arranged and immobilized on the substrate. Such immobilization can be achieved by a chemical bonding method or a covalent bonding method such as UV. For example, the DNA oligonucleotide can be bound to a glass surface modified to include an epoxy compound or an aldehyde group, and can also be bound to a polylysine-coated surface by UV. In addition, the DNA oligonucleotide can be bound to the substrate via a linker (e.g., an ethylene glycol oligomer and a diamine).

[0063] The DNA aptamer that specifically binds to the envelope protein of the SARS-CoV-2 virus of the present invention can be, for example, biotinylated and successfully immobilized on a streptavidin-coated substrate. The DNA aptamer that specifically binds to the envelope protein of the SARS-CoV-2 virus of the present invention immobilized on the substrate can bind to and capture the envelope protein of the SARS-CoV-2 virus, and the captured envelope protein of the SARS-CoV-2 virus can be visualized again using the DNA aptamer that specifically binds to the envelope protein of the SARS-CoV-2 virus to determine whether it has been captured.

[0064] In addition, the composition for detecting the envelope protein of the SARS-CoV-2 virus in the present invention may be provided in the form of a kit. In the present invention, the kit comprises, as an active ingredient, a DNA oligonucleotide having a base sequence of SEQ ID NO: 1 to SEQ ID NO: 16 or a base sequence having at least 90% identity thereto. The kit in the present invention may additionally include an instruction manual or label for using the kit to detect the envelope protein of the SARS-CoV-2 virus in a sample.

[0065] According to another aspect of the present invention, the present invention can provide useful information for diagnosing coronavirus disease-19 (COVID-19) or determining its prognosis through specific detection of the envelope protein of the SARS-CoV-2 virus.

[0066] In addition, the present invention can provide a method for detecting the envelope protein of a SARS-CoV-2 virus, comprising: (1) a step of contacting a biological sample isolated from a living body with the DNA aptamer of the present invention; and (2) a step of measuring the presence or content of the envelope protein of the SARS-CoV-2 virus in the biological sample by confirming a specific binding reaction between the biological sample and the DNA aptamer and the envelope protein of the SARS-CoV-2 virus.

[0067] Furthermore, the present invention can provide a composition for diagnosing coronavirus infection-19 (COVID-19) comprising the DNA aptamer of the present invention as an active ingredient and a method for providing information necessary for diagnosing coronavirus infection-19 (COVID-19) using the DNA aptamer of the present invention.

[0068] The method for providing information necessary for diagnosing coronavirus infection-19 using the DNA aptamer of the present invention comprises: (1) a step of contacting a biological sample isolated from a living body with the DNA aptamer of the present invention; and (2) a step of measuring the presence or content of the envelope protein of the SARS-CoV-2 virus in the biological sample by confirming a specific binding reaction between the biological sample and the DNA aptamer and the envelope protein of the SARS-CoV-2 virus.

[0069] Here, if the level or content of the envelope protein of the SARS-CoV-2 virus contained in the sample is higher than that of the normal control group (sample from a normal person), it can be predicted or diagnosed that the patient has been infected with the SARS-CoV-2 virus and has developed coronavirus infection-19.

[0070] In the present invention, the “biological sample” may include blood, saliva, tears, urine, synovial fluid, mucus, cells, tissues, and other tissues and body fluids, and also includes, but is not limited to, cell culture supernatants, ruptured eukaryotic cells, and bacterial expression systems.

[0071] Furthermore, a composition comprising the DNA aptamer of the present invention as an active ingredient can also be used as a pharmaceutical composition for preventing or treating coronavirus infection-19 (COVID-19).

[0072] The DNA aptamer of the present invention is not only non-immunogenic, non-toxic, and highly stable, but can also bind to the envelope protein of the SARS-CoV-2 virus to inhibit the spread of the virus and improve excessive immune responses such as cytokine storms in infected individuals, and thus can be used for the prevention, improvement, or treatment of coronavirus disease-19.

[0073]

[0074] Hereinafter, the present invention will be described in more detail through examples. These examples are intended to more specifically illustrate the present invention, and the scope of the present invention is not limited to these examples.

[0075]

[0076] <Example 1>

[0077] DNA aptamer production

[0078] <1-1> Amplification of DNA random library using PCR technique

[0079] To generate a single-stranded DNA aptamer that specifically binds to the envelope protein (E) of SARS-CoV-2, two primers capable of amplifying a 76-bp template DNA (5'-ATACCAGCTTATTCAATT -N40-AGATAGTAAGTGCAATCT-3') containing 40 random sequences in the ratio of dA: dG: dC: dT = 1.5:1.15:1.25:1 were custom-made by Bioneer (Korea) (forward primer: 5'-ATACCAGCTTATTCAATT-3', biotinylated reverse primer: 5'-biotin-AGATAGTAAGTGCAATCT-3'). The random DNA library was amplified using the PCR technique. The PCR reaction composition for amplification of the 76 bp DNA library was a mixture containing 1 μl of template DNA, 5 μl of 10X PCR buffer, 4 μl of dNTP mixture, 2 μl of 25 μM forward primer, 2 μl of 25 μM biotinylated reverse primer, 0.25 μl (1 unit / μl) of Ex Taq polymerase (TaKaRa, Japan), and 35.75 μl of distilled water. The PCR reaction conditions were as follows: denaturation at 95°C for 5 min, followed by 4 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, followed by an additional extension reaction at 72°C for 5 min. After the PCR reaction, 4 μl was taken and the amplification product was confirmed through 2% agarose gel electrophoresis. The DNA library obtained through PCR was purified using a PCR purification kit (Qiagen, USA) and then recovered using 50 μl of distilled water (see Figure 1).

[0080]

[0081] <1-2> ssDNA amplification using asymmetric PCR technique

[0082] Among the dsDNA amplified using the PCR technique, an asymmetric PCR reaction was performed to amplify only ssDNA. The asymmetric PCR reaction composition was a mixture containing 7 μl of the template DNA obtained in <1-1> above, 10 μl of 10X PCR buffer, 8 μl of 2.5 mM dNTP mixture, 10 μl of 25 μM forward primer, 2 μl of 25 μM biotinylated reverse primer, 0.5 μl (1 unit / μl) of Ex Taq polymerase (Takara, Japan), and 62.5 μl of distilled water. The asymmetric PCR reaction conditions were as follows: first, denaturation at 95°C for 5 minutes, followed by 17 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, followed by an additional extension reaction at 72°C for 7 minutes. After the PCR reaction, 4 μl was taken and checked using a 2% agarose gel to see if a band of the correct size appeared, and the remaining DNA was subjected to the PCI extraction and ethanol precipitation methods commonly used to recover a pure DNA aptamer pool.

[0083] Specifically, the reaction solution was treated with an equal volume of PCI (Phenol: Chloroform: Isoamylalcohol = 25:24:1) solution, stirred vigorously, and centrifuged at 13,000 rpm at 4°C for 15 minutes to recover only the supernatant. 1 / 100 volume of tRNA (sigma aldrich, USA) and 3 times the volume of 100% ethanol were added to the supernatant, and the reaction was performed at -70°C for more than 1 hour. After the reaction, centrifugation was performed at 13,000 rpm at 4°C for 20 minutes to recover only the DNA. The recovered DNA was dried at 65°C and then dissolved in 50 μl of distilled water. 4 μl of the recovered DNA was taken and subjected to 2% agarose gel electrophoresis to confirm whether a band of the correct size appeared.

[0084]

[0085] <1-3> Production and recovery of ssDNA using heating-cooling technique

[0086] In order to remove biotin-tagged dsDNA and ssDNA from the PCR products amplified using asymmetric PCR and to secure only pure forward ssDNA, 50 ㎕ of distilled water was added to 50 ㎕ of the DNA obtained in <1-2>, and then dsDNA was denatured into ssDNA using the heating-cooling technique. dsDNA was denatured into ssDNA by reacting at 85℃ for 5 minutes, and then the reaction solution was immediately cooled to 4℃ after the reaction was completed to obtain ssDNA.

[0087] After this, 50 ㎕ of streptavidin (Pierce, USA) was added and reacted at room temperature for 1 hour. After the reaction was completed, the reaction solution was centrifuged for 10 minutes using a centrifuge at 4℃ and 13,000 rpm, and only the supernatant was recovered to secure ssDNA. To secure pure ssDNA from the reaction solution, PCI extraction and ethanol precipitation were utilized. The reaction solution was treated with an equal volume of PCI (Phenol: Chloroform: Isoamylalcohol = 25:24:1) solution, stirred vigorously, and centrifuged at 4℃ and 13,000 rpm for 15 minutes to recover only the supernatant. 1 / 100 volume of tRNA (Sigma Aldrich, USA) and 3 times the volume of 100% ethanol were added to the supernatant, and the reaction was performed at -70℃ for more than 1 hour. After the reaction, the mixture was centrifuged at 13,000 rpm at 4°C for 20 minutes to recover only ssDNA. The recovered ssDNA was dried at 65°C and then dissolved in 50 μl of distilled water. Ten μl of the recovered ssDNA was subjected to 10% acrylamide gel electrophoresis to confirm that a band of the correct size appeared.

[0088]

[0089] <Example 2>

[0090] Screening of ssDNA aptamers that specifically bind to the envelope protein of SARS-CoV-2 using the SELEX technique.

[0091] <2-1> Composition of each solution used in SELEX

[0092] The composition of each solution used in the SELEX technique is as follows.

[0093] 1X SELEX Buffer: 50mM Tris-HCl (pH 7.5), 150mM NaCl, 5mM KCl, 1mM CaCl 2, 1 mM MgCl2

[0094] 1X Binding Buffer: 5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl (pH 7.5)

[0095] 1X Wash Buffer: 60 nM imidazole, 0.5 M NaCl, 20 mM Tris-HCl (pH 7.5)

[0096] 1X Elution Buffer: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM KCl, 1 mM CaCl 2, 1 mM MgCl2

[0097]

[0098] <2-2> Production of ssDNA aptamer structure for use in SELEX and induction of binding of ssDNA aptamer to E protein of SARS-CoV-2

[0099] In order to select a DNA aptamer that specifically binds to the SARS-CoV-2 E protein, 60 μl of distilled water was added to 40 μl of the ssDNA obtained in the above example, 100 μl of 1X SELEX buffer was added, and then boiled at 85°C for 5 minutes to denature, and then slowly cooled to room temperature to form a stable three-dimensional structure of the ssDNA aptamer.

[0100] 10 μl of SARS-CoV-2 E protein was added to the ssDNA aptamer with a stably completed three-dimensional structure, and binding between the ssDNA aptamer and SARS-CoV-2 E protein was induced at 4°C for 12 hours.

[0101]

[0102] <2-3> Selection of ssDNA aptamers binding to the envelope protein (E) of SARS-CoV-2 using Ni-NTA agarose resin activation and SELEX method for the selection of DNA aptamers binding to the envelope protein (E) of SARS-CoV-2

[0103] To select a DNA aptamer that specifically binds to the E protein of SARS-CoV-2, a Ni-NTA agarose resin (QIAGEN, German) that specifically binds to the his-tag of the recombinant protein was used. First, 500 μl of 1X SELEX buffer was added to 200 μl of Ni-NTA agarose resin, and then stirred. After that, the supernatant was removed through centrifugation (4°C, 13,000 rpm, 10 min), and the E protein of SARS-CoV-2 bound to the ssDNA aptamer prepared in <2-2> above was added to the activated Ni-NTA agarose resin, and binding of the Ni-NTA agarose resin was induced at 4°C for 1 hour. To remove the E protein of SARS-CoV-2 that did not bind to the Ni-NTA agarose resin, the supernatant was removed through centrifugation (4°C, 13,000 rpm, 10 min). Afterwards, all ssDNA aptamers that non-specifically bound to the E protein of SARS-CoV-2 were removed by washing three times using a washing buffer. The ssDNA aptamer that specifically bound to the SARS-CoV-2 E protein was denatured by adding 200 μl of 1X elution buffer and reacting at 85°C for 5 minutes, and then recovered through centrifugation (4°C, 13,000 rpm, 10 min). The recovery process was performed twice in total, and to recover a pure DNA aptamer pool from the elution solution of the DNA aptamer that specifically binds to the E protein of SARS-CoV-2, PCI extraction and ethanol precipitation were performed, and then recovered in 50 μl of distilled water.

[0104]

[0105] <2-4> Removal of non-specific ssDNA aptamers through negative SELEX

[0106] In order to remove ssDNA aptamers that nonspecifically adsorb to the Ni-NTA agarose resin rather than the envelope protein of SARS-CoV-2, negative SELEX was performed using Ni-NTA resin on which the E protein of SARS-CoV-2 was not immobilized between SELEX rounds 6 and 7. The ssDNA aptamer cooled to room temperature was reacted with the activated Ni-NTA resin using 1X PBS buffer for 1 hour, and the ssDNA aptamer solution that came out without binding to the resin was used in the 7th SELEX. Thereafter, SELEX was performed up to 12 rounds, and the binding conditions of SELEX were such that the amount of ssDNA aptamer and the reaction time were reduced as the number of rounds increased, and the reaction conditions were roughened as the number of rounds increased in order to obtain an aptamer that specifically binds to the E protein of SARS-CoV-2, which is the target substance.

[0107]

[0108] <Example 3>

[0109] Affinity testing to select optimal SELEX rounds that specifically bind to the envelope protein of SARS-CoV-2.

[0110] <3-1> Checking the concentration of each round using nano drop

[0111] After completing 12 rounds of SELEX, the concentration of ssDNA specifically bound to the E protein of SARS-CoV-2 recovered in each round was measured using Nano-drop to quantitatively confirm whether SELEX was in progress and the affinity of the ssDNA aptamer eluted in each round.

[0112] As a result of measuring the concentration of ssDNA aptamer eluted in each round using Nano-drop, the concentration in the 7th round after the Negative round was 589.2 ng / ㎕, the concentration in the 8th round was 793.1 ng / ㎕, the concentration in the 9th round was 688.5 ng / ㎕, the concentration in the 10th round was 701 ng / ㎕, the concentration in the 11th round was 762.3 ng / ㎕, and the concentration in the 12th round was 1067.9 ng / ㎕ (see Fig. 2).

[0113]

[0114] <3-2> Confirming the optimal round using real-time PCR technique

[0115] After confirming the concentration of each round using nanodrop, experiments were conducted to identify the optimal round using real-time PCR. To this end, after completing 12 rounds of SELEX, real-time PCR was performed to quantitatively determine whether SELEX was in progress and the affinity of the DNA aptamer eluted after the negative round.

[0116] First, each ssDNA aptamer eluted in rounds 7, 8, 9, 10, 11, and 12 after the negative round was amplified using the PCR technique, and ssDNA was secured using the heating-cooling technique. The ssDNA aptamer pools secured in each of rounds 7, 8, 9, 10, 11, and 12 were prepared at the same concentration. Each ssDNA aptamer obtained through this was 10 0 , 10 -1 , 10 -2It was diluted and used as a template DNA for real-time PCR. Real-time PCR was performed using Biorad's iQ SYBR Green Supermix (Bio-rad, USA), and the real-time PCR reaction conditions were as follows: first, denaturation at 94°C for 5 minutes, then 30 cycles of 94°C for 20 seconds, 52°C for 20 seconds, and 72°C for 20 seconds, followed by an extension at 72°C for 5 minutes. The amplification curves of rounds 7, 8, 9, 10, 11, and 12 were checked, and the Ct value was checked using the Tershold value as an indicator. It was confirmed that the result of round 10 showed the smallest number. This means that the concentration of the aptamer pool recovered in that round is the highest, and it means that a large number of aptamer molecules with high affinity for the E protein of SARS-CoV-2 are included.

[0117] Therefore, round 10 was selected as the optimal round for SELEX (see Table 1).

[0118] RoundRound 7Round 8Round 9Round 10Round 11Round 12Ct value12.2711.9811.917.329.317.43

[0119]

[0120] <Example 4>

[0121] Screening and cloning of DNA aptamer candidates that specifically bind to the envelope protein of SARS-CoV-2.

[0122] dsDNA was obtained by PCR using the forward primer (5'-ATACCAGCTTATTCAATT-3') and the reverse primer (5'-AGATAGTAAGTGCAATCT-3') for the 10-round ssDNA aptamer that was judged to have the highest binding efficiency to the envelope protein of SARS-CoV-2. The dsDNA obtained in this way was cloned using Solgent's T-blunt cloning kit. Cloning was performed using the conditions of mixing 1 ㎕ of T-vector (10 ng / ㎕), 4 ㎕ of PCR product (20 ng / ㎕), and 1 ㎕ of 6× T-blunt buffer and incubating at 25°C for 5 minutes. 6 ㎕ of the T-blunt cloning reaction solution was mixed with 100 ㎕ of DH5α, heat-shocked at 42°C for 30 seconds, and then incubated on ice for 2 minutes. Afterwards, 900 ㎕ of SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose) was added and incubated at 37℃ for 40 minutes. After incubation, 200 ㎕ of the solution was spread on an LB culture plate containing ampicillin (50 ㎍ / ㎖), kanamycin (50 ㎍ / ㎖), X-gal (50 ㎍ / ㎖), and IPTG (5 ㎍ / ㎖), and incubated at 37℃ for 15 hours. Only white colonies were selected and the base sequence of the aptamer was determined by Solgent (Korea). Through sequence analysis, 13 DNA aptamer sequences that bind to the E protein of SARS-CoV-2 without duplication were obtained, and the 13 obtained DNA aptamer sequences are shown in Table 2 below.

[0123]

[0124]

[0125] <Example 5>

[0126] Structure determination of a DNA aptamer that binds to the envelope protein of SARS-CoV-2

[0127] Next, the inventors of the present invention analyzed the structure of the DNA aptamer candidates that bind to the envelope protein (E) of SARS-CoV-2 obtained in Example 4 above. The structural analysis was visualized using the DNA mfold program provided by Rensselear Polytechnic Institute, and the results are shown in Fig. 3.

[0128]

[0129] Through the above results, the inventors of the present invention were able to find out that the DNA aptamers designed in the present invention have excellent binding ability to the envelope protein (E) of SARS-CoV-2, and thus the aptamers of the present invention can be usefully used for the detection of SARS-CoV-2 by binding to the envelope protein of SARS-CoV-2, and can also be useful for the treatment of infections caused by SARS-CoV-2 virus infection, as they can stop the spread of the SARS-CoV-2 virus and improve excessive immune responses such as cytokine storms in infected patients.

[0130]

[0131] <Example 6>

[0132] 3D binding score and structural conformation analysis of DNA aptamers binding to the envelope protein of SARS-CoV-2

[0133] <6-1> Securing a model for analyzing the three-dimensional structure of the SARS-CoV-2 envelope protein and the DNA aptamer binding to it.

[0134] Based on the two-dimensional structure of the DNA aptamer binding to the SARS-CoV-2 envelope protein through the above experiment, a three-dimensional structure was modeled using the RNA composer website (http: / rnacomposer.cs.put.poznan.pl / ). The RNA structure was converted to a DNA structure using the Pymol program, and the three-dimensional structure was predicted. In addition, the structure of the SARS-CoV-2 envelope protein was analyzed using the model predicted through alphafold.

[0135]

[0136] <6-2> Analysis of binding scores between the SARS-CoV-2 envelope protein and DNA aptamer

[0137] The binding score was analyzed through 3D structure prediction using a 3D model of the obtained SARS-CoV-2 envelope protein and DNA aptamer. The MOE2020.09 program was used for structural prediction. In the case of SARS-CoV-2, mutations continuously occur, resulting in changes in the envelope protein sequence and structure. To ensure that the aptamer can bind to all mutants and bind to all SARS-CoV-2, the amino acid sequences of the E protein of SARS-CoV-2 variants that occurred up to September 2023 were analyzed. By analyzing the amino acid sequences and confirming the mutant part excluding the conserved sequence, four types of mutants, including the wild type, Beta, Omicron BA2.75, and Omicron BA1, were selected for the E protein to analyze all SARS-CoV-2 envelope proteins.

[0138] Therefore, in order to confirm whether the selected aptamers also bind to the mutant structure of the E protein, binding to four types of proteins, Wild type, Beta, Omicron BA2.75, and Omicron BA1, was analyzed, and the binding scores of the 13 aptamers of the present invention binding to four types of SARS-CoV-2 envelope proteins were analyzed, and five types of optimal aptamers were secured, and the secured binding scores are shown in Table 3 below.

[0139] Additionally, the binding scores of aptamers binding to the wild type and mutant envelope proteins of SARS-CoV-2 were compared, and Apta-E-36 was selected as the optimal aptamer.

[0140]

[0141]

[0142] <6-3> Predicting the binding form of the SARS-CoV-2 envelope protein and DNA aptamer in a three-dimensional structure

[0143] The binding form of the obtained SARS-CoV-2 envelope protein and DNA aptamer was analyzed through 3D structure prediction using a 3D model. The MOE2020.09 program was used for structural prediction, and the Pymol structure program and Chimera X program were used for structural analysis to diagram it. The aptamer-bound site is marked in green. Before structural prediction, the 3D structure of the SARS-CoV-2 envelope protein and DNA aptamer was prepared by minimizing all energies, and the binding relationship between the SARS-CoV-2 envelope protein and DNA aptamer was predicted using the Triangle Matcher method. All structural predictions were repeated 10 times, and the result with the most structure predictions was diagrammed. The binding structures of the five optimal aptamers selected are shown in Figure 4.

[0144]

[0145] <6-4> Analysis of the binding site of the optimal DNA aptamer that specifically binds to the envelope protein of SARS-CoV-2

[0146] In order for the selected aptamer to bind to the mutant structure of the E protein, the aptamer must interact with the conserved region that has not been mutated. To analyze this, the MOE2020.09 program and the Ligplot program were used to analyze which amino acid site of each mutant the Apta-E-36 aptamer binds to. The amino acid sequence alignment results of 20 mutants and the interaction site of Apta-E-36 with four proteins, Wild type, Beta, Omicron BA2.75, and Omicron BA1, were imaged and are shown in Figure 5.

[0147]

[0148] <Example 7>

[0149] Optimization of an optimal DNA aptamer that specifically binds to the envelope protein of SARS-CoV-2, analysis of its binding affinity and three-dimensional binding structure conformation.

[0150] <7-1> Sequence optimization of the optimal aptamer that specifically binds to the envelope protein of SARS-CoV-2

[0151] Apta-E-36, which specifically binds to the envelope protein of the secured SARS-CoV-2, was analyzed for amino acids interacting with the aptamer sequence through a two-dimensional structure and binding prediction, including the primer sequence, and the aptamer sequence that was not involved was removed and optimized. The secured aptamer sequence is shown in Table 4, and this is imaged and shown in Fig. 6 (A).

[0152]

[0153]

[0154] <7-2> SPR-based binding affinity analysis of optimal aptamers that specifically bind to the envelope protein of SARS-CoV-2

[0155] In order to evaluate the affinity of the aptamer obtained in the above example with the envelope protein of SARS-CoV-2, surface plasmon resonance (SPR) analysis was performed using an SPR detection system instrument, BIAcore X-100 (Cytiva). To quantify the affinity between the envelope protein of SARS-CoV-2 and the DNA aptamer, a sensor needle CM5 (Cytiva, UK) whose surface was coated with a carboxyl group was used. A mixture of 0.1 M NHS and 0.4 M EDC was flowed through the sensor chip CM5 at a rate of 10 μl / min for 10 minutes to activate the carboxyl groups on the sensor chip surface into more reactive NHS esters. To immobilize the SARS-CoV-2 envelope protein onto the NHS-ester-activated sensor chip CM5 surface, the chip surface was coated with a solution of the SARS-CoV-2 envelope protein in a 10 mM sodium acetate (pH 4.0) buffer for 120 s. Subsequently, 1 M ethanolamine was flowed over the sensor chip with the SARS-CoV-2 envelope protein immobilized thereon to inactivate the remaining carboxyl groups on the sensor chip surface. This prevented direct binding of other reagents and DNA aptamers to the chip surface. After each experiment, the sensor chip was regenerated with 1 M NaCl and 50 mM NaOH. The rate parameters were obtained and quantified using the BIA evaluation program (Cytiva).

[0156] To quantify the affinity of the aptamer with the envelope protein of SARS-CoV-2, the obtained aptamers were dissolved in HBS-EP buffer (Cytiva) at concentrations of 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, and 50 nM, respectively. The affinity between the DNA aptamer that specifically binds to the envelope protein of SARS-CoV-2 was quantified by flowing various concentrations of aptamers on a sensor chip (channel 1) that was not bound to anything and a sensor chip (channel 2) on which the envelope protein of SARS-CoV-2 was immobilized. The binding affinity of the Apta-E-36-3 aptamer was 1.970 x 10 -9 The results of obtaining the dissociation constant (KD) of the aptamer are shown in Table 5.

[0157]

[0158]

[0159] <7-3> Predicting the binding form of the optimal DNA aptamer to the envelope protein of SARS-CoV-2 in a three-dimensional structure.

[0160] In order to analyze the binding structure of the secured optimized aptamer Apta-E-36-3, the analysis technique of Example <6-3> was utilized, and through interaction analysis with the envelope protein of SARS-CoV-2, it was analyzed that it binds to a conserved sequence so that it can bind to all mutants, and this was imaged and diagrammed in Figure 6(B).

[0161]

[0162] Through the above results, the inventors of the present invention were able to find out that the DNA aptamers designed in the present invention have excellent binding ability to the envelope protein (E) of SARS-CoV-2, and thus the aptamers of the present invention can be usefully used for the detection of SARS-CoV-2 by binding to the envelope protein of SARS-CoV-2, and can also be useful for the treatment of infections caused by SARS-CoV-2 virus infection, as they can stop the spread of the SARS-CoV-2 virus and improve excessive immune responses such as cytokine storms in infected patients.

[0163]

[0164] The present invention has been described above, focusing on preferred embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than limiting. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

[0165] In addition, the present invention is a result of the following national research project.

[0166] Assignment ID: 2400424307

[0167] Subproject Number: RS-2024-00424307

[0168] Ministry of Science and ICT

[0169] Research Management Agency: Korea Institute for Science and Technology Promotion

[0170] Research Project Name: University Technology Management Promotion (IP Star Scientist Support Type)

[0171] Research Project Name: IP Enhancement and Commercialization for Virus Diagnosis and Treatment Commercialization of Aptamer Technology

[0172] Host institution: Chungbuk National University

[0173] Research period: April 1, 2024 - December 31, 2025

Claims

1. DNA aptamer that specifically binds to the envelope protein of the SARS-CoV-2 virus.

2. In paragraph 1, A DNA aptamer, characterized in that the DNA aptamer comprises any one base sequence selected from the group consisting of sequence numbers 1 to 16.

3. In paragraph 1, A DNA aptamer, characterized in that the DNA aptamer further comprises a labeling substance.

4. In paragraph 3, A DNA aptamer, characterized in that the labeling material is selected from the group consisting of a fluorescent substance, an amine group, biotin, a thiol group, and digoxigenin, and is labeled at the 5' end or the 3' end of the DNA aptamer.

5. In paragraph 1, A DNA aptamer, characterized in that the above DNA aptamer is a single-stranded DNA.

6. A composition for detecting the envelope protein of SARS-CoV-2 virus, comprising the DNA aptamer of clause 1 as an active ingredient.

7. A kit for detecting SARS-CoV-2 virus containing the composition of Article 6 as an active ingredient.

8. A chip or microarray for detecting SARS-CoV-2 virus, characterized in that the DNA aptamer of clause 1 is fixed on a substrate and specifically reacts with a biological sample isolated from an individual infected with the SARS-CoV-2 virus. 9.(1) A step of contacting a biological sample separated from a living organism with the DNA aptamer of the first paragraph; and (2) a step of measuring the presence or content of the envelope protein of the SARS-CoV-2 virus in the biological sample by confirming a specific binding reaction between the biological sample and the DNA aptamer and the envelope protein of the SARS-CoV-2 virus; including; Method for detecting the envelope protein of SARS-CoV-2 virus.

10. A composition for diagnosing coronavirus infection-19 (COVID-19), comprising the DNA aptamer of clause 1 as an active ingredient.

11. A pharmaceutical composition for preventing or treating coronavirus infection-19 (COVID-19), comprising the DNA aptamer of clause 1 as an active ingredient. 12.(1) A step of amplifying DNA aptamers using PCR and asymmetric PCR techniques and selecting single-stranded DNA aptamers; (2) a step of inducing binding of the single-stranded DNA aptamer to the envelope protein of the SARS-CoV-2 virus; (3) A step of binding the envelope protein of the SARS-CoV-2 virus combined with a single-stranded DNA aptamer to Ni-NTA agarose resin; (4) a step of removing DNA aptamers that fail to bind to the envelope protein of the SARS-CoV-2 virus; and (5) A step of recovering and selecting a DNA aptamer that specifically binds to the envelope protein of the SARS-CoV-2 virus using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technique; A method for producing a DNA aptamer that specifically binds to the envelope protein of the SARS-CoV-2 virus.

13. In paragraph 12, The above step (5) is a real-time PCR step for selecting a SELEX round with optimal affinity; PCR and cloning steps for screening DNA aptamer candidates that bind to the envelope protein of SARS-CoV-2 virus in the optimal SELEX round; A step of measuring the affinity of the selected DNA aptamer candidates to the SARS-CoV-2 viral envelope protein; and A method for producing a DNA aptamer that specifically binds to the envelope protein of a SARS-CoV-2 virus, characterized by comprising the step of determining the DNA sequence of a group of DNA aptamer candidates that bind to the selected SARS-CoV-2 virus envelope protein. 14.(1) A step of contacting a biological sample separated from a living organism with the DNA aptamer of the first paragraph; and (2) a step of measuring the presence or content of the envelope protein of the SARS-CoV-2 virus in the biological sample by confirming a specific binding reaction between the biological sample and the DNA aptamer and the envelope protein of the SARS-CoV-2 virus; including; A method of providing information necessary for the diagnosis of coronavirus disease-19 (COVID-19).

Images