Compositions comprising hot spot derived peptide-nucleic acid hybrid molecules for treatment of infections caused by mutant coronaviruses

Abstract

The present invention relates to compositions comprising peptide-nucleic acid hybrid molecules of hot spot origin for use in the prevention or treatment of coronavirus infections. It has been proved that the in vitro evolution-based hot spot source peptide-nucleic acid hybrid molecule constructed by the construction method of the invention has high binding affinity to SARS-CoV-2VOC (alpha, beta, gamma, delta and o). In particular, hybrid molecules are demonstrated to exhibit the highest binding resistance in cos with the most mutations. In addition, even when competing with RBD-binding aptamers, macrocyclic peptides, and monoclonal antibodies, hybrid molecules have been observed to have high binding affinity for RBD. In addition, the hybrid molecules prove excellent nuclease resistance and serum stability, indicating that the hybrid molecules not only have anti-SARS-CoV-2, but also have excellent potential as an antiviral neutralizer.

Description

Technical Field

[0001] The present invention relates to a composition for preventing or treating coronavirus infection, comprising a peptide-nucleic acid hybrid molecule derived from a hotspot. This application claims the rights and priority of Korean Patent Application No. 10-2022-0052389 filed on April 27, 2022 and Korean Patent Application No. 10-2023-0044897 filed on April 5, 2023, the disclosures of which are incorporated herein by reference in their entirety. Background Art

[0002] To infect host cells, many viruses specifically recognize cellular membrane proteins as they enter host cells. For example, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is covered with thousands of spike proteins that strongly bind to human angiotensin-converting enzyme 2 (hACE2), a membrane receptor, leading to membrane fusion. To achieve higher infectivity, viruses, including SARS-CoV-2, often mutate, which enhances the infectivity of the virus and causes immune escape over time. Since the first report of SARS-CoV-2 in 2019, many variants have emerged, including variants of concern (VOCs) of α (B.1.1.7), β (B.1.351), γ (P.1), δ (B.1.617.2), and ο (B.1.1.529) that have occurred independently on different continents. The receptor binding domain (RBD) of the spike protein, which facilitates specific hACE2 recognition, is known to contain many common mutations. In particular, some mutations located in binding hotspots (e.g., N501Y) have been identified to increase the binding affinity of RBD to its target hACE2, thereby increasing the infectivity of SARS-CoV-2.

[0003] The frequent emergence of variants may be a major obstacle to the development of antiviral prevention and treatment strategies. In order to avoid viral infection, the use of affinity reagents can be an effective method to block the specific interaction between the virus and the host cell. For SARS-CoV-2, many neutralizing affinity reagents have been developed to recognize various epitopes of the spike protein, some of which are known to overlap with the hACE2 contact surface. However, since some escape mutations cause structural changes in the spike protein, affinity reagents will inevitably lose specific recognition of their binding sites, resulting in reduced neutralization efficacy. As an alternative, non-competitive affinity reagents can be co-administered, and due to the rapid emergence of SARS-CoV-2 variants, the U.S. Food and Drug Administration (FDA) has issued emergency use authorizations for antibody mixtures such as REGN-COV2. As viral variants accumulate escape mutations, they tend to become more resistant to antibody mixtures while producing stronger binding interactions with host cell receptors. Therefore, it is necessary to develop effective and efficient neutralizers with high affinity for the target virus, while taking into account the binding tolerance to its variants. Summary of the invention

[0004] [Technical issues]

[0005] Inspired by the improved receptor recognition of viral variants, the inventors invented the generation of receptor-mimicking synthetic reagents that can effectively interact with target viruses and their variants. Specifically, they focused on peptide motifs on host cell receptors that contribute significantly to the binding free energy at the center of the virus-receptor interface. Without a stable but insoluble transmembrane domain, the short hotspot peptide cannot maintain optimal binding ability to the target virus. In this process, it is synergistically integrated with soluble nucleic acids that can act as binding cooperators and structural stabilizers. Random nucleic acids (~10 14 ), hybrid ligands can be easily discovered by selectively isolating and amplifying aptamer-like scaffolds to maximize hotspot interactions, which can lead to strong binding to viral variants.

[0006] In addition, the inventors successfully created a hACE2 receptor mimic hybrid ligand that directly interacts with the hotspot of SARS-CoV-2 by using a novel in vitro evolution technology called "hotspot-oriented ligand display" (HOLD). Compared with reported affinity reagents (such as peptides, aptamers or neutralizing antibodies), the synergistic interaction between the hotspot peptide and the aptamer scaffold achieves effective blocking of SARS-CoV-2 by more effectively binding to RBD. In addition, when identifying various SARS-CoV-2 variants (such as α, β, γ, δ and ο), the inventors confirmed that the hotspot-binding hACE2 mimics maintained or even enhanced their binding ability to SARS-CoV-2 variants, and completed the present invention.

[0007] Therefore, an object of the present invention is to provide a composition for preventing or treating coronavirus infection, comprising a peptide-nucleic acid hybrid molecule, wherein the peptide is a hotspot-derived peptide comprising an amino acid sequence of SEQ ID No.15, and wherein the nucleic acid comprises a sequence selected from the group consisting of SEQ ID No.7, 8, 18, 19 and 20.

[0008] Another object of the present invention is to provide a composition for preventing or treating coronavirus infection, comprising a peptide-nucleic acid hybrid molecule prepared by a method comprising the following steps:

[0009] (a) site-specifically conjugating a hotspot-derived peptide consisting of the amino acid sequence of SEQ ID No. 15 with a random nucleic acid library to prepare a peptide-nucleic acid hybrid;

[0010] (b) co-incubating the target protein-coated magnetic beads with the peptide-nucleic acid hybrid of step (a); and

[0011] (c) Using a magnet to screen peptide-nucleic acid hybrid molecules that bind to the target protein.

[0012] Another object of the present invention is to provide a quasi-drug composition for preventing or inhibiting coronavirus infection, comprising a peptide-nucleic acid hybrid molecule, wherein the peptide is a hotspot-derived peptide comprising an amino acid sequence of SEQ ID No.15, and wherein the nucleic acid comprises a base sequence selected from the group consisting of SEQ ID Nos.7, 8, 18, 19 and 20.

[0013] Another object of the present invention is to provide an antiviral composition against coronavirus, comprising a peptide-nucleic acid hybrid molecule, wherein the peptide is a hotspot-derived peptide comprising an amino acid sequence of SEQ ID No.15, and wherein the nucleic acid comprises a sequence selected from the group consisting of SEQ ID No.7, 8, 18, 19 and 20.

[0014] Another object of the present invention is to provide a composition for neutralizing coronavirus, comprising a peptide-nucleic acid hybrid molecule, wherein the peptide is a hotspot-derived peptide comprising an amino acid sequence of SEQ ID No.15, and wherein the nucleic acid comprises a base sequence selected from the group consisting of SEQ ID No.7, 8, 18, 19 and 20.

[0015] Another object of the present invention is to provide a method for preventing, inhibiting or treating coronavirus infection, comprising the step of administering a peptide-nucleic acid hybrid molecule to an individual in need thereof, wherein the peptide is a hotspot-derived peptide comprising an amino acid sequence of SEQ ID No.15, and wherein the nucleic acid comprises a base sequence selected from the group consisting of SEQ ID No.7, 8, 18, 19 and 20.

[0016] Another object of the present invention is a method for preventing, inhibiting or treating coronavirus infection, comprising administering to an individual in need thereof a peptide-nucleic acid hybrid molecule prepared by a method comprising the following steps:

[0017] (a) site-specifically conjugating a hotspot-derived peptide consisting of the amino acid sequence of SEQ ID No. 15 with a random nucleic acid library to prepare a peptide-nucleic acid hybrid;

[0018] (b) co-incubating the target protein-coated magnetic beads with the peptide-nucleic acid hybrid of step (a); and

[0019] (c) Using a magnet to screen peptide-nucleic acid hybrid molecules that bind to the target protein.

[0020] Another object of the present invention is to provide a method for inhibiting or neutralizing coronavirus, comprising the step of administering a peptide-nucleic acid hybrid molecule to an individual in need thereof, wherein the peptide is a hotspot-derived peptide comprising an amino acid sequence of SEQ ID No. 15, and wherein the nucleic acid comprises a base sequence selected from the group consisting of SEQ ID No. 7, 8, 18, 19 and 20.

[0021] However, technical challenges of the present invention are not limited to those mentioned above, and other challenges not mentioned will be apparent to those of ordinary skill in the art in view of the following description.

[0022] [Technical solution]

[0023] The terms used in this specification are for illustrative purposes only and should not be construed as limiting the present invention. Unless the context clearly indicates otherwise, singular expressions include plural expressions. In this specification, the terms "including" or "having" etc. represent the presence of features, numbers, steps, actions, components, parts or combinations thereof, and do not exclude the presence or addition of one or more other features, numbers, steps, actions, components, parts or combinations thereof.

[0024] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by those of ordinary skill in the art. Unless explicitly defined in this application, terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant art, and should not be interpreted in an idealized or overly formal sense.

[0025] Hereinafter, the present invention will be described in detail.

[0026] The present invention provides a composition for preventing or treating coronavirus infection, comprising a peptide-nucleic acid hybrid molecule, wherein the peptide is a hotspot-derived peptide comprising an amino acid sequence of SEQ ID No.15, and wherein the nucleic acid comprises a base sequence selected from the group consisting of SEQ ID Nos.7, 8, 18, 19 and 20.

[0027] In the present invention, the peptide-nucleic acid hybrid molecule can be prepared by the following method, but is not limited thereto:

[0028] (a) site-specifically conjugating a hotspot-derived peptide consisting of the amino acid sequence of SEQ ID No. 15 with a random nucleic acid library to prepare a peptide-nucleic acid hybrid;

[0029] (b) co-incubating the target protein-coated magnetic beads with the peptide-nucleic acid hybrid of step (a); and

[0030] (c) Using a magnet to screen peptide-nucleic acid hybrid molecules that bind to the target protein.

[0031] The method may further include but is not limited to the following steps:

[0032] (d) selectively amplifying and cloning the nucleic acid portion of the peptide-nucleic acid hybrid molecule selected in step (c);

[0033] (e) preparing the double-stranded DNA produced in step (d) into single-stranded DNA by using an exonuclease, and purifying it; and

[0034] (f) preparing a new random nucleic acid library comprising the single-stranded DNA of step (e), and repeating the selection, amplification and purification processes of steps (a) to (f).

[0035] In the present invention, hotspot-derived peptides refer to peptide residues that are considered to be highly associated with the binding between a target protein and its receptor. Hotspot-derived peptides may be amino acids at the binding site between a target protein and its receptor; all or part of the amino acids involved in the binding site in the target protein sequence; or all or part of the amino acids involved in the binding site in the receptor sequence. In the present invention, hACE2-derived hotspot peptides or hACE2-derived RBD-binding peptides are used as preferred embodiments of hotspot-derived peptides, and more specifically, among the RBD contact residues of hACE2, seven amino acid fragments L351 to R357 (LGKGDFR, SEQ ID No. 15) are used as hotspot-derived peptides, wherein the hotspot-derived peptides may include, but are not limited to, sequences having 80%, 85%, 90%, 95%, 99% or 100% identity with SEQ ID No. 15.

[0036] In the present invention, the peptide-nucleic acid hybrid molecule can bind to the receptor binding domain (RBD) of the spike protein of the coronavirus. In addition, the peptide-nucleic acid hybrid molecule can show excellent binding to the RBD in competition with the RBD binding affinity reagent, and can show excellent binding in the presence of mutations in the RBD of the spike protein. Therefore, the peptide-nucleic acid hybrid molecule can inhibit the interaction between the RBD of the coronavirus and the human angiotensin converting enzyme 2 (hACE2) receptor, and can neutralize the coronavirus, and can also neutralize the variant of concern (VOC) of the coronavirus, but is not limited to this.

[0037] In the present invention, RBD may be a wild type (SEQ ID No. 23) or a mutant, wherein the mutant may include one or more selected from the following: N501Y, E484K, K417N, K417T, T478K, L452R, E484A, G339D, S371L, S373P, S375F, N440K, S477N, G446S, Q493R, G496S, Q498R and Y505H.

[0038] [RBD sequence: amino acid sequence [319-537] of SARS-CoV-2 spike protein]

[0039] 319-rv qptesivrfp nitnlcpf g e vfnatrfasv yawnrkrisn cvadysvlyn s a s f s tfkcy gvsptklndl cftnvyadsf virgdevrqi apgqtg kiad ynyklpddft gcviawnsn n ldskv g gnyn y l yrlfrksn lkpferdist eiyqag st pc ngv e gfncyf pl q sy g f q pt n gvg y qpyrvvvlsfellha patvcgpkks tnlvknk-537(SEQ ID No.23)

[0040] In the present invention, peptide-nucleic acid hybrid molecules can meet the characteristics of nuclease resistance or serum stability, but are not limited thereto. In addition, peptide-nucleic acid hybrid molecules can be administered by various routes of administration according to treatment purposes and formulation methods. For example, it can be administered by the following: oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, perispinal space injection (intrathecal injection), sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, eye administration, ear administration, nasal administration, inhalation, atomization by mouth or nose, skin administration or transdermal administration, preferably by intravenous or respiratory route, but not limited thereto. In addition, peptide-nucleic acid hybrid molecules can be hybrid aptamers, hotspot peptide binding aptamer scaffolds, receptor mimicking hybrid ligands or hACE2 mimicking hybrid ligands, but are not limited thereto.

[0041] In the present invention, nucleic acids can be coupled to hotspot source peptides in a click reaction to prepare peptide-nucleic acid hybrid molecules. In addition, nucleic acids can be isolated from a library of many other nucleic acids that are site-specifically connected to hotspot peptides in vitro by repeated selection and amplification cycles, wherein nucleic acids that can provide higher binding to peptide-nucleic acid hybrid molecules can be selected. In addition, the nucleic acid can enhance the binding affinity of the peptide-nucleic acid hybrid molecule to the binding site (RBD) between the spike protein and its receptor, and can structurally stabilize the RBD binding peptide from hACE2. In addition, the nucleic acid may include a base sequence selected from the group consisting of SEQ ID No. 7, 8, 18, 19 and 20, and the nucleic acid may include a base sequence selected from the group consisting of SEQ ID No. 7, 8, 18, 19 and 20 with 80%, 85%, 90%, 95%, 99% or 100% identity, but is not limited thereto. In addition, the nucleic acid may be, but is not limited to, ssDNA, aptamer scaffolds, or aptamer scaffolds based on hotspot peptides modified with hexynyl.

[0042] In the present invention, the aptamer scaffold is a nucleic acid that binds two or more molecules together to form a functional unit and can strongly and specifically bind to a specific molecule. In one embodiment of the present invention, a hybrid molecule of a peptide and an aptamer scaffold, also known as a peptide-based aptamer scaffold, can be combined with a hotspot-derived peptide to have a virus neutralizing function and bind to the spike protein.

[0043] In the present invention, the coronavirus can be one selected from the group consisting of, but not limited to, human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus NL63 (HCoV-NL63, New Haven coronavirus), human coronavirus HKU1, Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and variants thereof, wherein the SARS-CoV-2 variant can be any one selected from the group consisting of, but not limited to, alpha (α), beta (β), gamma (γ), delta (δ) and omicron (ο) variants.

[0044] In the present invention, "coronavirus" is a species of virus genus included in the order Nidovirales, the family Coronaviridae, the subfamily Coronavirinae or the subfamily Torovirinae. Coronavirus is a virus with a +ssRNA and a helical symmetric nucleopeptide envelope. In addition, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the seventh coronavirus to infect humans so far, and the others are human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus NL63 (HCoV-NL63, New Haven coronavirus), human coronavirus HKU1 and Middle East respiratory syndrome coronavirus (MERS-CoV). The proteins that make up the overall structure of coronavirus are spikes, envelopes and nucleocapsids. In the case of SARS coronavirus, the established ligand receptor domain on the spike (S) mediates the attachment of the virus to its cellular receptor, angiotensin converting enzyme 2 (ACE2). Some coronaviruses (especially the beta coronavirus subgroup) also have a short spike of a protein called antisense esterase. Coronaviruses can cause viral pneumonia or secondary bacterial pneumonia, and they can also cause direct viral bronchitis or secondary bacterial bronchitis. The human coronavirus discovered in 2003 is the severe acute respiratory syndrome coronavirus (SARS-CoV), which causes severe acute respiratory syndrome (SARS), an infection of the upper and lower respiratory tracts.

[0045] In addition, as used herein, the term "SARS-CoV-2" refers to a new coronavirus, which is an RNA virus and a variant of SARS and MERS. SARS-CoV-2 has about 79.7% sequence identity with SARS and about 50% sequence identity with MERS. However, compared with SARS and MERS, the spike glycoprotein of 2019-nCoV forms a structure with an upwardly protruding RBD domain, resulting in a binding strength with its target receptor ACE2 (angiotensin) that is 100 to 1,000 times that of SARS and MERS. This stronger binding allows better penetration into cells, thereby enhancing infectivity.

[0046] In addition, for the purposes of the present invention, variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represent variants of COVID-19. Variants are divided into major variants [variants of concern (VOC)] and other variants [variants of interest (VOI)], and variants are named with Greek letters (α, β, γ, etc.) to prevent the use of localized names and promote communication. Major variants are those variants that have been determined to have increased transmission or negative epidemiological changes, increased pathogenicity or clinically significant changes in disease severity, or reduced effectiveness of diagnosis, vaccines, treatments, etc. At present, VOC is o variant, and in the past, they included α, β, γ and δ variants. Specifically, the alpha (α) variant of SARS-CoV-2 was identified in the UK in September 2020, and has a pedigree classification of B.1.1.7. In addition, the beta (β) variant of SARS-CoV-2 was identified in South Africa in May 2020, and has a pedigree classification of B.1.351. In addition, the gamma (γ) variant of SARS-CoV-2 was identified in Brazil in November 2020 and has a pedigree classification of P.1. In addition, the delta (δ) variant of SARS-CoV-2 was identified in India in October 2020 and has a pedigree classification of B.1.617.2. In addition, the omicron (o) variant of SARS-CoV-2 was identified in multiple countries in November 2021 and has a pedigree classification of B.1.1.529.

[0047] In the present invention, coronavirus infection can be coronavirus respiratory tract infection disease.Viral respiratory tract infection disease may show symptoms such as cough, sneezing, headache, nasal congestion, sore throat, diarrhea, discoloration of fingers or toes, conjunctivitis, high fever, wheezing, bronchitis, bronchiolitis, pneumonia, asthma, loss of smell and taste or respiratory failure.If coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), fever and respiratory symptoms (cough, sore throat, shortness of breath) may be cardinal symptoms, accompanied by headache, muscle aches, hemoptysis and nausea, chills, chest pain or diarrhea.In addition, coronavirus infection can be coronavirus disease 19 (COVID-19).

[0048] The present invention provides a composition for preventing or treating coronavirus infection, comprising a peptide-nucleic acid hybrid molecule prepared by a method comprising the following steps:

[0049] (a) site-specifically conjugating a hotspot-derived peptide consisting of the amino acid sequence of SEQ ID No. 15 with a random nucleic acid library to prepare a peptide-nucleic acid hybrid;

[0050] (b) co-incubating the target protein-coated magnetic beads with the peptide-nucleic acid hybrid of step (a); and

[0051] (c) Using a magnet to screen peptide-nucleic acid hybrid molecules that bind to the target protein.

[0052] In the present invention, the method may further include but is not limited to the following steps:

[0053] (d) selectively amplifying and cloning the nucleic acid portion of the peptide-nucleic acid hybrid molecule selected in step (c);

[0054] (e) preparing the double-stranded DNA produced in step (d) into single-stranded DNA by using an exonuclease, and purifying it; and

[0055] (f) preparing a new random nucleic acid library comprising the single-stranded DNA of step (e), and repeating the selection, amplification and purification processes of steps (a) to (f).

[0056] In the present invention, the method may further include, after step (a), heat denaturation and cooling of the peptide-nucleic acid hybrid to induce 3D folding of the nucleic acid. This step allows the hotspot peptide to be positioned in the correct position and orientation, and a stable 3D aptamer scaffold can be prepared, which can maintain similar or stronger binding properties to the receptor.

[0057] Step (a) is to prepare a peptide-nucleic acid hybrid, wherein the hotspot-derived peptide can be a peptide having any functional group that can be used in a click chemistry reaction, which functional group is bound to the C-terminus or N-terminus of the isolated hotspot-derived peptide, and it can be, for example, a peptide conjugated with at least one functional group selected from the group consisting of azidolysine, azidobutyric acid, azidoacetic acid, azide, hexynyl, 5-octadiynyl and alkyne, preferably a peptide conjugated (labeled) with azide at the C-terminus or N-terminus, more preferably an azide-labeled LGKGDFR (L351 to R357, having SEQ ID NO: 15) peptide, but is not limited thereto.

[0058] The randomized nucleic acid library of step (a) can be a nucleic acid having any functional group at its 5' end that can be used for click chemistry reaction, such as a single-stranded nucleic acid having one or more functional groups selected from the group consisting of but not limited to: hexynyl, 5-octadiynyl, alkyne, azidolysine, azidobutyric acid, azidoacetic acid and azide, preferably a nucleic acid in which the hexynyl is bonded to the 5' end. In addition, the functional groups bound to the hotspot source peptide and the nucleic acid library can be interchangeable. In addition, the random nucleic acid library can be characterized by including random nucleic acids having the structure shown below:

[0059] 5'-Functional Group-Forward Primer-[N x ]-Reverse Primer-3',

[0060] wherein the functional group is selected from the group consisting of hexynyl, 5-octadiynyl, alkyne, azidolysine, azidobutyric acid, azidoacetic acid and azide,

[0061] wherein N is A, T, C or G, and x is an integer from 25 to 100.

[0062] The length of the random nucleic acid library is minimized to accurately bind to a target protein having a diameter of 10 nm or less, and can be any length suitable for maximizing the diversity of the library, but specifically, the number of sequences in the random nucleic acid library can be, but is not limited to, 55 to 130, 55 to 110, 55 to 90, 55 to 80, 60 to 130, 60 to 110, 60 to 90, 60 to 80, 65 to 130, 65 to 110, 65 to 90, 65 to 80, 65 to 75 or 70.

[0063] Furthermore, x may be, but is not limited to, 25 to 100, 25 to 80, 25 to 60, 25 to 50, 30 to 100, 30 to 80, 30 to 60, 30 to 50, 35 to 100, 35 to 80, 35 to 60, 35 to 50, 35 to 45, or 40.

[0064] A preferred embodiment of the present invention utilizes a forward primer as shown in SEQ ID No. 1 and a reverse primer as shown in SEQ ID No. 2, wherein the randomized nucleic acid library comprises a structural formula of GGAAGAGATGGCGAC- 40 -A randomized nucleic acid of AGCTGATCCTGATGG.

[0065] The selective amplification of the nucleic acid portion of step (d) can be amplified using a forward primer conjugated to one or more functional groups attached to the 5' end, the functional group being selected from the group consisting of, but not limited to, hexynyl, 5-octadiynyl, and alkyne. Because the functional group is bound to the forward primer, any PCR product amplified can be generated with the functional group attached to the 5' end, which allows site-specific conjugation by a click reaction, but is not limited thereto.

[0066] In the present invention, the hotspot derived peptide and the single-stranded nucleic acid may be characterized by, but not limited to, site-specific coupling via a click reaction. The click reaction may be, but not limited to, a reaction in which the functional groups of the hotspot derived peptide and the functional groups of the nucleic acid are chemically cross-linked with each other or bonded via a copper-catalyzed cycloaddition in a short period of time.

[0067] The pharmaceutical composition according to the present invention may further include suitable carriers, excipients and diluents conventionally used in the preparation of the pharmaceutical composition. The excipient may be one or more selected from the group consisting of the following, for example: diluents, binders, disintegrants, glossing agents, adsorbents, humectants, film coating materials and controlled release additives.

[0068] The pharmaceutical composition according to the present invention can be formulated into the following forms according to conventional methods known in the art: powder, granules, sustained-release granules, enteric-coated granules, liquid, eye drops, elixir, emulsion, suspension, injection, trochanter, fragrance, lemonade, tablet, sustained-release tablet, enteric-coated tablet, sublingual tablet, hard capsule, soft capsule, sustained-release capsule, enteric-coated capsule, pill, tincture, soft extract, dry extract, liquid extract, injection, capsule, infusion, alert agent, lotion, paste, spray, inhalant, patch, sterile injection solution, or external application such as aerosol, and the external application can have the following preparations such as cream, gel, patch, spray, ointment, alert agent, lotion, liniment, paste or poultice.

[0069] Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, glucose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate and mineral oil.

[0070] When formulated, they are prepared using commonly used diluents or excipients, such as fillers, enhancers, binders, humectants, disintegrants or surfactants.

[0071] The additives of the tablets, powders, granules, capsules and lozenges according to the present invention may include excipients such as corn starch, potato starch, wheat starch, lactose, white sugar, glucose, fructose, dimannitol, precipitated calcium carbonate, synthetic aluminum silicate, monocalcium phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolinite, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC) 1928, HPMC2208, HPMC2906, HP MC2910, propylene glycol, casein, calcium lactate or primogel; binders such as gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethyl cellulose, carboxymethyl cellulose calcium, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethyl cellulose, purified shellac, starch paste, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl alcohol or polyvinyl pyrrolidone; disintegrants such as hydroxypropyl methyl Cellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, silicic anhydride, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginate, amylose, guar gum, sodium bicarbonate, polyvinyl pyrrolidone, calcium phosphate, gelling starch, gum arabic, pullulan, pectin, sodium polyphosphate, ethylcellulose, white sugar, magnesium aluminum silicate, disorbitol solution or hard silicic anhydride; and lubricants (gly dents), such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, petrolatum, kaolinite, petrolatum, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol 4000, polyethylene glycol 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, polyethylene glycol, synthetic aluminum silicate, anhydrous silicic acid, high-quality fatty acids, high-quality alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ethyl ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, light anhydrous silicic acid.

[0072] The additives of the liquid according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitan fatty acid esters (diesters), polyoxyethylene monoalkyl esters, lanolin esters, lanolin esters, acetic acid, hydrochloric acid, ammonia solution, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamin, polyvinyl pyrrolidone, ethyl cellulose and sodium carboxymethyl cellulose.

[0073] The syrup according to the present invention may include a solution of white sugar or other sugars, or a sweetener, and may contain flavors, colorants, preservatives, stabilizers, suspending agents, emulsifiers or thickeners as needed.

[0074] Purified water may be used in the emulsion according to the present invention, and an emulsifier, a preservative, a stabilizer or a flavor may be used as needed.

[0075] The suspending agent according to the present invention may include gum arabic, gum tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropyl methylcellulose, HPMC 1828, HPMC 2906 and HPMC 2910, and a surfactant, a preservative, a stabilizer, a colorant or a flavor may be used as needed.

[0076] The injection according to the present invention includes a solvent such as distilled water for injection, 0.9% sodium chloride injection solution, Ringer's solution, glucose injection solution, glucose + sodium chloride injection solution, PEG, lactated Ringer's injection solution, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate or phenyl benzoate; a dissolving aid such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, Tween, nicotinamide, hexamine or dimethylacetamide; a buffer such as a weak acid and its salt (acetic acid or acetate), a weak base and its salt (ammonia or ammonium acetate), an organic compound, protein, albumin, peptone or gum; an isotonic agent such as sodium chloride; a stabilizer such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium pyrosulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen (N2) or ethylenediaminetetraacetic acid; a sulfurizing agent such as 0.1% sodium hydrosulfide, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid or sodium acetyl nitrate bisulfite; an analgesic such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose or calcium gluconate; and a suspending agent such as sodium CMC, sodium alginate, Tween 80 or aluminum monostearate.

[0077] The suppositories according to the invention comprise bases such as cocoa butter, lanolin, semi-synthetic fatty acid esters (Witepsol), polyethylene glycol, glycerinated gelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, lanet wax, glyceryl monostearate, Tween or Span, Imhausen, hydrocarbon oil (propylene glycol monostearate), glycerol, stearyl triglyceride (Adeps solidus), Buytyrum Tego-G, Cebes Pharma 16, Hexaride Base 95, Cotomar, Hydrokote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrokote 25, Hydrokote 711, Idropostal, Massaestrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramount-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppositories type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), Test triglyceride base (TG-95, MA, 57).

[0078] Solid preparations for oral administration include tablets, pills, powders, granules and capsules, which are prepared by mixing the extract with at least one excipient such as starch, calcium carbonate, sucrose or lactose or gelatin. In addition to simple excipients, lubricants such as magnesium stearate talc are also used.

[0079] Liquid preparations for oral administration include suspensions, solutions, emulsions and syrups, which can also include multiple excipients, such as humectants, sweeteners, flavorings or preservatives, in addition to commonly used simple diluents such as water or liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized materials and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil and injectable esters such as ethanol esters (ethylolate).

[0080] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a condition at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined based on the following: the type and severity of the patient's condition, the activity of the drug, sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, factors including combined medication, and other factors well known in the medical field.

[0081] The pharmaceutical composition according to the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered in single doses or multiple doses. Taking all of the above factors into account, it is important to administer the amount that produces the maximum effect with the minimum amount without side effects, which can be easily determined by those of ordinary skill in the art.

[0082] Pharmaceutical composition of the present invention can be administered to individuals by various routes. Any method of administration is predictable, such as by oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, perispinal space injection (intrathecal injection), sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, ear administration, nasal administration, inhalation, by oral or nasal atomization, skin administration or transdermal administration.

[0083] The pharmaceutical composition of the present invention is determined by the type of drug as the active ingredient together with many other relevant factors such as the disease to be treated, the route of administration, and the patient's age, sex, weight, and severity of the disease.

[0084] As used herein, "subject" means a subject in need of treatment for a disease, and more specifically, a mammal, such as a human or non-human primate, mouse, rat, dog, cat, horse, or cow.

[0085] As used in the present invention, "administration" means providing a predetermined amount of the composition of the present invention to an individual by any suitable method. In the present invention, "prevention" means any behavior that inhibits or delays the onset of the target disease, "treatment" means any behavior that improves or beneficially changes the target disease and its metabolic abnormalities by administering a pharmaceutical composition according to the present invention, and "improvement" means any behavior that reduces parameters associated with the target disease (such as the severity of symptoms) by administering a composition according to the present invention.

[0086] The present invention provides a pharmaceutical composition for preventing or inhibiting coronavirus infection, comprising a peptide-nucleic acid hybrid molecule, wherein the peptide is a hotspot source peptide comprising an amino acid sequence of SEQ ID No.15, and wherein the nucleic acid comprises a base sequence selected from the group consisting of SEQ ID No.7, 8, 18, 19 and 20.

[0087] In addition, quasi-drug products may include topical or personal care products. For example, they may include, but are not limited to, cleansers, shower foams, mouthwashes, wipes, washing soaps, hand soaps or ointments.

[0088] When the quasi-drug composition according to the present invention is used as a quasi-drug additive, the composition can be added alone or in combination with other quasi-drugs or quasi-drug components, and can be appropriately used according to conventional methods. The blending amount of the active ingredient can be appropriately determined according to the purpose of use.

[0089] The quasi-drug composition of the present invention can be formulated, for example, in the form of a conventional emulsified preparation or a solubilized preparation. For example, they may have a preparation such as an emulsion, such as a lotion, cream, ointment, spray, oil gel, gel, oil, aerosol or mist, but can be used without limitation as long as they show the pest control induction effect of the present invention. In addition, each of the above-mentioned quasi-drug compositions can be appropriately formulated with oil, water, a surfactant, a moisturizer, a lower alcohol having 1 to 4 carbon atoms, a thickener, a chelating agent, a colorant, a preservative or a fragrance, which are generally formulated in the quasi-drug composition as needed.

[0090] The present invention is an antiviral composition for coronavirus, comprising a peptide-nucleic acid hybrid molecule, wherein the peptide is a hotspot source peptide including an amino acid sequence of SEQ ID No.15, and wherein the nucleic acid includes a base sequence selected from the group consisting of SEQ ID No.7, 8, 18, 19 and 20.

[0091] In the present invention, "antiviral" means inhibiting the proliferation of viruses in the body, thereby weakening or destroying the effects of viruses that have already invaded the body, and more specifically, inhibiting the proliferation of viruses by inhibiting the nucleic acid synthesis process, gene expression process or viral replication process of the virus, and in the present invention, coronavirus is targeted.

[0092] In the present invention, the antiviral composition may have antiviral activity against: human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus NL63 (HCoV-NL63, New Haven coronavirus), human coronavirus HKU1, Middle East respiratory syndrome coronavirus (MERS-CoV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or 2019 novel coronavirus or 2019-nCoV).

[0093] The present invention provides a composition for neutralizing coronavirus, comprising a peptide-nucleic acid hybrid molecule, wherein the peptide is a hotspot-derived peptide comprising an amino acid sequence of SEQ ID No. 15, and wherein the nucleic acid comprises a base sequence selected from the group consisting of SEQ ID Nos. 7, 8, 18, 19 and 20.

[0094] The description of the composition for preventing or treating coronavirus infection can be applied to the pharmaceutical composition for preventing or inhibiting coronavirus infection, the antiviral composition for coronavirus, and the neutralizing composition for coronavirus, and redundant descriptions are omitted to avoid complexity of the description.

[0095] [Beneficial Effects]

[0096] It is confirmed that the hotspot-derived peptide-nucleic acid hybrid molecules prepared by the method of the present invention based on in vitro evolution have high binding affinity to SARS-CoV-2 VOC (α, β, γ, δ and ο), and in particular, the greatest binding tolerance is presented in ο with the highest degree of mutation. In addition, the hybrid molecule shows high RBD binding affinity when competing with RBD binding nucleic acid aptamers, macrocyclic peptides or monoclonal antibodies. The hybrid molecule also shows excellent nuclease resistance and serum stability, indicating that it has the potential to be a SARS-CoV-2 virus neutralizer. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1a The role of nucleic acid scaffolds in inducing stable binding of hotspot peptides to viral RBD is illustrated.

[0098] Figure 1b Schematic diagram of the in vitro evolution of receptor mimicking hybrid ligands including hotspot peptides and aptamer scaffolds.

[0099] Figure 2a Analysis of contact residues between the hACE2 receptor and SARS-CoV-2 RBD is shown.

[0100] Figure 2b Depicted is the N501Y mutant of the RBD, which results in higher binding affinity to hACE2.

[0101] Figure 2c Results of quantitative assessment of RBD binding in round 7 (R7) pools of hotspot peptide binding are shown: random DNA library bound to hotspot peptide (light grey), R7 ssDNA pool without hotspot peptide (dark grey), and R7 ssDNA pool with hotspot peptide (green).

[0102] Figure 3a Comparison of the RBD binding fractions of R7-01, R7-02, R7-03 and the hybridized random library is shown.

[0103] Figure 3b Shown are RBD binding affinity characterization analyses of R7-02: RBD and serum albumin coated beads (green and grey, respectively) and naked beads (black).

[0104] Figure 3c The RBD binding affinity properties (K) of R7-01 and R7-03 are shown. d value) analysis results.

[0105] Figure 3d The energy-minimized structure of R7-02 binding to RBD by molecular docking simulation is shown: LGKGDFR peptide (red), hACE2 (white), RBD (blue), and aptamer scaffold (green).

[0106] Figure 3e and 3f The results of co-incubation and competitive binding analysis of hybrid ligands with RBD-binding nucleic acid aptamers (CoV2-RBD-1C, CoV2-6C3, aptamer-1), macrocyclic peptides (peptide 4) or monoclonal antibodies (P05Dhu, AM122) are shown.

[0107] Figure 3g The equilibrium dissociation constants K for RBD binding to nucleic acid aptamers (CoV2-RBD-1C, CoV2-6C3, aptamer-1), macrocyclic peptides (peptide 4), or monoclonal antibodies (P05Dhu, AM122) are shown. d The analysis results of the value.

[0108] Figure 4a is a schematic diagram and list of mutants for variants of concern (VOC) of α, β, γ, δ, and ο.

[0109] Figure 4b The K values ​​of α, β, γ, δ and ο VOCs in R7-02 are shown. d The analysis results of the value.

[0110] Figure 4c The results of the analysis of the RBD binding portion of R7-02 and several types of RBD binders (aptamer-1, peptide 4, P05DHu and AM122) for wild-type and oVOC are shown.

[0111] Figure 4d The binding fraction of R7-02 and several types of RBD binders (aptamer-1, peptide 4, P05DHu and AM122) against wild type and oVOC via flow cytometry are shown.

[0112] Figure 5aShown is the inhibitory effect of R7-02 on RBD-hACE2 binding by enzyme-linked immunosorbent assay (ELISA).

[0113] Figure 5b Inhibition of RBD-hACE2 interaction by LGKGDFR peptide, R7-02 DNA domain, R7-02, random DNA library, and hACE2 is shown.

[0114] Figure 5c Schematic diagram of neutralization assay based on pseudotyped SARS-CoV-2.

[0115] Figure 5d and 5f Pseudotyped SARS-CoV-2 infected hACE2-293T ( Figure 5d : LGKGDFR peptide, R7-02 DNA domain, R7-02; Figure 5f : Confocal microscopy observation results of CoV2-RBD-1C, CoV2-6C3, aptamer-1, peptide 4, P05Dhu, AM122).

[0116] Figure 5e and 5g The percentage of pseudotyped SARS-CoV-2 infected cells expressing GFP relative to total hACE2-293T cells is shown ( Figure 5e : LGKGDFR peptide, R7-02 DNA domain, R7-02; Figure 5g : CoV2-RBD-1C, CoV2-6C3, aptamer-1, peptide 4, P05Dhu, AM122).

[0117] Figure 5h The results of the nuclease stability analysis of R7-02 are shown. DETAILED DESCRIPTION

[0118] Below, preferred embodiments of the present invention are provided to facilitate understanding of the present invention. However, the following embodiments are provided only to facilitate understanding of the present invention, and the scope of the present invention is not limited to the following embodiments.

[0119] [Experimental methods]

[0120] 1. Experimental Materials

[0121] DNA oligonucleotides were synthesized and purified by Bioneer (Korea). The sequences of all oligonucleotides used in the present invention are shown in Table 1 below. C-terminal-(4-azidobutyryl (lysine))-labeled LGKGDFR peptides (with or without N-terminal FITC modification) were synthesized and purified by Peptron (Korea). EDC (1-ethyl-3-(3-365 dimethylaminopropyl) carbodiimide hydrochloride), NHS (N-hydroxysuccinimide), 1M magnesium chloride solution, human angiotensin converting enzyme 2 (hACE2), bovine serum albumin (BSA), high glucose DMEM, Tween-20, dimethyl sulfoxide (DMSO), tris (3-hydroxypropyltriazolylmethyl) amine (THPTA) and copper sulfate (II) pentahydrate were purchased from Sigma-Aldrich. L-ascorbic acid sodium salt was purchased from Alfa Aesar. λ exonuclease and 10xλ exonuclease buffer were purchased from Thermo Fisher Scientific. 1M Tris-HCl (pH 7.4) was purchased from Bioneer (South Korea). 10x PBS, 8M urea solution, 10x TBE and nuclease-free purified water were purchased from T&I (South Korea). 1x PBSMT was used as a binding buffer (composed of 1x PBS with a pH of 7.4, containing 2.5mM MgCl2 and 0.02% Tween-20 (v / v)). Wild-type, α (B.1.1.7), β (B.1.351), γ (P.1) and δ (B.1.617.2) SARS-CoV-2 RBD were expressed and purified. ο (B.1.1.529) SARS-CoV-2 RBD was purchased from Abbexa (UK). Alexa-488-labeled monoclonal RBD conjugate antibody (P05DHu) and Alexa Fluor 488 mini protein labeling kit were purchased from Invitrogen. RBD-conjugated monoclonal antibody (AM122) was purchased from ACROBiosystems. A macrocyclic peptide (peptide 4) with N-terminal chloroacetyl-modified D-tyrosine was linearly synthesized by Peptron (Korea). 1 mg of linear peptide 4 was dissolved in 86 μL of DMSO containing 3 μL of N,N-diisopropylethylamine and incubated at 60°C for 24 hours to cyclize.

[0122] [Table 1]

[0123]

[0124] 2. Site-specific conjugation of azide-labeled hotspot peptides and hexyne-modified ssDNA

[0125] For ultra-efficient copper-catalyzed alkyne-azide addition cycloaddition, 1 μL of 50 mM copper sulfate (II) aqueous solution and 1.67 μL of 150 mM THPTA aqueous solution were premixed 10 min before conjugation. 3 μL of DMSO, 1 μL of 10x PBS, 3 μL of 0.1 mM hexynyl-modified ssDNA aqueous solution, 1 μL of 2.5 mM azide-labeled LGKGDFR peptide, 2.67 μL of premix (copper sulfate (II) and THPTA) and 1 μL of 500 mM sodium ascorbate aqueous solution (freshly prepared) were added in sequence and mixed thoroughly. The solution was shaken overnight at room temperature and purified by ethanol precipitation.

[0126] 3. Confirmation of Peptide-ssDNA Conjugation Hybridization Library

[0127] After coupling the FITC-modified azide-labeled LGKGDFR peptide, the formation of the peptide-ssDNA conjugate was confirmed by fluorescence. 1 μL of the reaction product (~50 ng), 7 μL of 8M urea water and 1 μL of 6x loading dye were mixed and heat denatured at 95°C for 10 min. The denatured products were analyzed by electrophoresis at 300V for 40 min in 10% urea polyacrylamide gel in 1x TBE buffer and imaged by Azure C600 (Azure Biosystems). Equal amounts of unconjugated hexyl-modified ssDNA libraries were stained with 10,000x SYBR gold stain (Invitrogen) and imaged simultaneously.

[0128] 4.HOLD (Hotspot-Oriented Ligand Display)

[0129] The peptide-nucleic acid hybridization library was heat denatured at 95°C for 10 minutes and then rapidly cooled in ice to form the most stable 3D structure. The heat-treated hybridization library was then gently rotated and incubated with RBD-coated beads at room temperature for 1 hour. In order to prepare RBD-coated beads, the target RBD was fixed on DynabeadsM-270 carboxylic acid (Invitrogen) via the EDC-NHS coupling process using the manufacturer's protocol, and then quantified by the NanoOrange protein quantification kit (Thermo Fisher Scientific). After incubation, the magnetic beads were thoroughly washed with 1x PBSMT to remove unbound hybrid molecules, and the hybrid ligands bound to the magnetic beads were selectively separated with a DynaMag-2 magnet (Invitrogen). 5'-hexyne-modified forward primers and 5'-phosphorylated reverse primers were used to perform PCR amplification only on the DNA domains of the isolated hybrid ligand candidates (see Table 1 above). The optimal number of PCR cycles was determined by the pilot PCR process. PCR amplified dsDNA was purified using QIAquick PCR purification kit (Qiagen) and digested with lambda exonuclease to generate a pool of hexyl-modified ssDNA for the next round of HOLD. The ssDNA was purified by phenol / chloroform extraction and ethanol precipitation and quantified by UV-vis measurement at 260 nm. In order to provide the same format of hybridization library pool for each round of HOLD, the click reaction between azide-labeled LGKGDFR peptide and purified hexyl-modified ssDNA pool was repeated in each round of HOLD.

[0130] 5. Volume Dilution Challenge

[0131] The volume dilution challenge (VDC) technique is used to remove unbound hybrid molecules very efficiently. After incubation for 1 hour and subsequent washing of the bead-bound hybrid library, it is immediately diluted with increasing volumes (100-500x) of 1xPBSMT and incubated for an additional 30 minutes to remove the unbound hybrids with a high rate dissociation constant (k off After VDC, the beads were collected and washed three times with 200 μL 1xPBSMT.

[0132] 6. Amplification of DNA domains to enrich for high-affinity hybrid ligands

[0133] The 100 μL PCR reaction mixture consisted of the following: 5 units of tag polymerase (T&I, Korea), 0.5 μL of 0.1 mM 5'-hexynyl modified forward primer and 5'-phosphorylated reverse primer, 8 μL of 10 mM dNTP (T&I, Korea), 2 μL of 10x PCR buffer (T&I, Korea), 10 μL of collected hybridization ligands, and additional nuclease-free purified water (up to 100 μL). The PCR reaction mixture was pre-denatured at 95°C for 600 seconds, followed by repeated cycles of denaturation at 95°C for 30 seconds, annealing at 51°C for 30 seconds, and extension at 72°C for 60 seconds. 5 μL of the PCR mixture was collected and dissolved in a 10% polyacrylamide gel to determine the optimal PCR amplification cycle number (pilot PCR) with the least side products. The DNA domains of the hybridization ligands collected in all rounds of HOLD were PCR amplified at the optimized cycle number.

[0134] 7. ssDNA Generation

[0135] After PCR amplification, hexynyl-modified double-stranded DNA (dsDNA) was purified using the QIAquick PCR purification kit (Qiagen), and 2.5 μg of purified dsDNA was digested with 5 units of lambda exonuclease (Thermo Fisher Scientific) in 50 μL. The mixture was incubated at 37°C for 10 min and at 80°C for 10 min, and the hexynyl-modified ssDNA was purified by phenol / chloroform extraction and ethanol precipitation, and quantified by UV-vis measurement at 260 nm.

[0136] 8. Relative Binding Affinity Analysis of R7 Pool

[0137] After 7 rounds of HOLD, the relative binding capacity of the hybrid ligand pool was compared. Equal amounts of the peptide-nucleic acid hybridization library bound to the hotspot peptide, the R7 ssDNA pool without the hotspot peptide, and the R7 ssDNA pool bound to the hotspot peptide were heat denatured at 95 ° C for 10 min, followed by rapid cooling on ice. After heat denaturation, the RBD-coated magnetic beads were attacked for 1 hour at room temperature, followed by washing three times with 1xPBSMT. RBD binding was analyzed by quantitative PCR (LightCycler 480, Roche). Each PCR reaction contained 10 μL of Light cycler 480SYBR green I master (Roche), 0.1 μL of 0.1 mM forward primer and reverse primer, 2 μL of RBD-binding ligand, and 7 μL of nuclease-free purified water. Based on the standard curve obtained previously, the threshold cycle was extended to the amount of RBD-binding ligand.

[0138] 9. High-throughput sequencing

[0139] After seven rounds of selection, the enrichment pool was PCR amplified with forward and reverse primers containing adapter sequences, and the optimal number of PCR cycles was determined by pilot PCR. These PCR products were purified using a gel extraction kit (Qiagen) and sequenced by Illumina MiSeq next-generation sequencing by SYSGENLAB (Korea). The sequencing library was analyzed in 101-bp double-end (PE) sequencing mode on the Illumina Novasek6000 platform. The sequencing read quality was measured with the FastQC tool, and the adapter sequence was removed with the cutadapt trimmer with the -e 0.1-j 20 option. Only fully matched reads were used for further analysis. High-quality sequencing reads were passed through the fastq_quality_filter (≥Q30) of the FASTX toolkit (RRID:SCR_005534).

[0140] 10. Affinity and specificity analysis of hybrid ligands

[0141] To evaluate the binding affinity of hybrid ligands to wild-type RBD and its variants, a DNA sequence of R7-02 was synthesized with a 5'-hexynyl group for hotspot peptide conjugation and a 3'-FAM for flow cytometry-based binding assays. FAM-labeled R7-02 in 1x PBSMT was heat denatured and rapidly cooled. After heat denaturation, different concentrations of R7-02 were challenged with RBD-coated beads with gentle rotation for 1 hour at room temperature. The beads were washed twice with 1x PBSMT to exclude nonspecific binding, and then the bead-bound R7-02 was collected with a DynaMag-2 magnet (Invitrogen). The collected beads were resuspended in 1xPBSMT and analyzed by measuring the mean fluorescence of the beads using CytoFLEX S (Beckman Coulter). 15,000 cases were analyzed for each measurement. Finally, K was calculated using nonlinear regression analysis (Prism software, GraphPad Prism version 9.3.1) d . To confirm their binding specificity, BSA-coated beads and uncoated beads (naked beads) were similarly characterized simultaneously. When preparing uncoated beads, Dynabeads M-270 carboxylic acid beads were Tris-blocked in 50mM Tris-HCl (pH7.4) at room temperature for 15min. A similar characterization process was performed for the high-density sequence families (R7-01 and R7-03) and reported RBD binding affinity reagents; aptamer-1, CoV2-RBD-1C, CoV2-6C3, peptide 4, P05DHu and AM122.

[0142] 11. Molecular docking simulation of R7-02 and RBD complex

[0143] To analyze the high binding affinity of R7-02 to RBD, the molecular structure of R7-02 in complex with RBD was constructed using molecular docking simulations. The crystal structures of individual RBDs of hACE2-derived hotspot peptides, LGKGDFR, and SARS-CoV-2 spike protein were obtained from the RCSB PDB database (PDB ID: 6M0J) with a single-site mutation (N501Y) in RBD. The secondary structure of the aptamer scaffold in R7-02 was predicted using the mfold web server (see Table 1) and entered into RNAcomposer for 3D structure. The peptide-nucleic acid hybrid structure of R7-02 was constructed by connecting a triazole-modified linker residue between the hexyne-modified 5' end of the folded DNA and the butyramide-modified lysine C-terminus of the hotspot peptide. Molecular docking simulations were subsequently performed to find the best fit of the hotspot peptide-binding aptamer scaffold with the RBD while restraining the hotspot peptide and RBD in the crystal position. The resulting structure was energy minimized by using the AMBER14 force field using the molecular dynamics simulation package OpenMM version 7.4.

[0144] 12. Competitive Binding Assays Using Reported RBD Binding Affinity Reagents

[0145] For competitive binding assays, R7-02 and reported RBD-binding aptamers were modified with FAM and TAMRA, respectively. Specifically, after thermal denaturation and rapid cooling, they were co-incubated with 5nM RBD beads (final volume: 100μL) and gently rotated at room temperature for 1 hour. The beads were washed three times and resuspended in 100μL of 1x PBSMT and analyzed with CytoFLEX S (Beckman Coulter). The competitive RBD binding fraction was calculated as the ratio of the competitive mean fluorescence intensity to the non-competitive (free binding) mean fluorescence intensity. In order to competitively bind with the reported RBD-binding cyclic peptides and antibodies, TAMRA-modified R7-02 was co-incubated with FAM-modified peptides or Alexa-488-modified RBD antibodies and analyzed in the same manner.

[0146] 13. Comparison of wild-type and oRBD binding fractions

[0147] In order to analyze the binding tolerance of the selected RBD binders (aptamer-1, peptide 4, P05DHu and AM122) to SARS-CoV-2 variants, the binding fractions to wild-type and oRBD were quantitatively measured at 10nM of R7-02 and antibodies (P05DHu and AM122) and at 100nM of aptamers (aptamer-1) and cyclic peptides (peptide 4). The RBD-coated magnetic beads were gently rotated and incubated with each binder at room temperature for 1 hour, and the beads were then washed three times and resuspended in 100 μL of 1xPBSMT. The binding fractions of each binder to wild-type and oRBD were directly measured using CytoFLEX S (Beckman Coulter).

[0148] 14. ELISA-based RBD-hACE2 binding inhibition test

[0149] To characterize the inhibitory efficiency, a SARS-CoV-2 inhibitor screening kit (ACROBiosystems) was used according to the manufacturer's protocol. Microplates were pre-coated with hACE2, and different concentrations of R7-02 (1 pM to 1 μM) were added to the plates. HRP-conjugated SARS-CoV-2 wild-type RBD was added immediately. After incubation for 1 hour at room temperature, the wells were washed three times, and substrate was added to quantify RBD-hACE2 binding. The reaction was terminated by adding stop solution, and the absorbance was measured at 450 nm with a microplate reader (Tecan). After logarithmic transformation of the hybridization ligand concentration, a sigmoidal dose-response nonlinear regression analysis (Prism software, GraphPad Prism version 9.3.1) was used to determine the maximum half-inhibitory concentration (IC50) of R7-02-induced RBD-hACE2 binding inhibition. 50 In the same way, RBD-hACE2 inhibition was compared after the addition of 3 μM of individual ligands (LGKGDFR peptide, DNA domain of R7-02, and R7-02) to analyze the synergistic effect between the hotspot peptides and the aptamer scaffolds in inhibiting the RBD-hACE2 interaction.

[0150] 15. Cell Culture and Sample Preparation

[0151] The hACE2-293T cell line was obtained from Takara (Japan). hACE2-293T cells were cultured in high glucose DMEM (Sigma Aldrich) supplemented with 10% fetal bovine serum (Gibco) and 1% sodium pyruvate (Sigma Aldrich) at 37°C and 5% CO2 atmosphere. Subsequently, hACE2-293T from passage 5 to 15 was used for pseudotyped SARS-CoV-2 neutralization assays.

[0152] 16. Neutralization Assay of Pseudotyped SARS-CoV-2

[0153] To confirm the neutralization capacity of R7-02, a neutralization assay with pseudotyped SARS-CoV-2 was performed. hACE2-293T cells (~4 × 10 3 ) were pre-seeded into 96-well plates and incubated overnight. After 24 hours, 5 μL of GFP reporter pseudotype SARS-CoV-2 (4×10 5 Transfection units per mL, BPS bioscience) were pre-incubated with 3 μM of R7-02 in 1x PBSM (1x PBS, containing 2.5 mM magnesium) for 30 minutes. Cell culture medium was removed from pre-inoculated hACE2-293T cells, and the cells were washed twice with pre-warmed 1xPBSM. After washing, a mixture of pseudovirus and R7-02 was added to the cells and infected at 37 ° C for 6 hours. After infection, 1X PBSM was replaced with fresh cell culture medium, and the cells were incubated at 37 ° C for another 48 hours. Finally, GFP fluorescence images and bright field images were observed using a confocal microscope (Leica Dmi8, source wavelength: 488 nm). The percentage of transfected cells expressing GFP in total hACE2-293T cells was calculated and analyzed by CellProfiler version 4.2.1. The neutralization efficacy of other RBD-binding ligands (aptamer-1, CoV2-RBD-1C, CoV2-6C3, P05DHu, AM122, and peptide 4) was analyzed by the same method.

[0154] 17. Serum stability test

[0155] To characterize serum stability, nucleases were allowed to degrade FAM-labeled R7-02 for up to 24 hours at 37°C in 10% fetal bovine serum. For comparison, R7-02 introduced a phosphorothioate backbone and other modifications, such as 2'-O-methyl and 2'-F modifications; and other unmodified single-stranded DNAs were tested together. 4 μL of 10 μM sample was mixed with 10x PBS (4 μL), fetal bovine serum (4 μL), and nuclease-free water (28 μL). After incubation at 37°C for 0, 0.5, 2, 4, 8, 18, and 24 hours, 5 μL of each aliquot from the different mixtures was immediately treated at 90°C for 5 min and stored in ice before analysis. Aliquots were analyzed by electrophoresis in 10% urea polyacrylamide gels in 1xTBE buffer, stained with 10,000x SYBR gold stain (Invitrogen), and imaged by Azure C600 (Azure Biosystems).

[0156] [Implementation Method]

[0157] Example 1. Receptor-mimicking hybrid ligands and hotspot-directed in vitro selection

[0158] With proper folding, nucleic acids can provide a highly stable and soluble 3D scaffold that places the hotspot peptide in the correct position and orientation to maintain receptor-like binding properties. Figure 1a As shown in . For specific RBD-hACE2 interactions, several hotspot peptides are reported to be about 30 amino acids or less in length, but the flexible motif cannot strongly bind to the hotspot surface of the receptor binding domain (RBD) except for the insoluble hACE2 transmembrane receptor. In addition to negligible immunogenicity, it is well known that nucleic acids have abnormal hydrophilicity due to the polarity of the phosphate backbone. In addition, iterative cycles of selection and amplification of system design can lead to the selection of unique nucleic acids in vitro that are able to structurally stabilize hACE2-derived RBD-binding peptides. Without a large number of insoluble domains, compact and highly available peptide stabilizers will allow high-dose intravenous administration for virus neutralization. In addition, the role of nucleic acids can be further extended to hotspot binding enhancers, synergistically interacting with the target RBD. That is, there can be additional aptamer-like interactions around the monovalent peptide binding, further enhancing the hotspot binding ability of the hACE2 mimic hybrid ligand.

[0159] From a large number of nucleic acids (~10 14 ), aptamer scaffolds that support hotspot peptides can be isolated in vitro through repeated cycles of selection and amplification. Figure 1b Shown in.

[0160] like Figure 1b As shown, initially, the azide-tagged C-terminus of the peptide can be site-specifically conjugated to the 5′-hexynyl terminus of a single-stranded DNA (ssDNA) library via an efficient copper-catalyzed cycloaddition ( Figure 1b Step 1 in ). After purification and heat denaturation, the peptide-nucleic acid hybrid library is rapidly cooled for the unique 3D folding of individual ssDNAs. After the introduction of RBD-coated magnetic beads, the RBD surface attracts hotspot interactions of the hybridization ligand candidates, and magnetic separation ensures that unbound peptide-DNA conjugates are systematically separated ( Figure 1b Subsequently, the DNA domain of the bead-bound hybridization ligand is selectively amplified by PCR using a 5'-hexynyl-modified forward primer and a 5'-phosphorylated reverse primer ( Figure 1b Using lambda exonuclease, 5'-hexynyl-modified ssDNA was generated, and click reaction with azide-labeled hotspot peptides again provided a pool of hybridization libraries of the same format for the next round of evolutionary HOLD process ( Figure 1b Step 5 in the above example).

[0161] Example 2. Preparation of SARS-CoV-2 RBD binding hotspot peptides and hACE2 mimetics

[0162] Among the RBD contact residues of hACE2, seven amino acid fragments L351 to R357 (LGKGDFR, SEQ ID No. 15) were selected to be fused to the random DNA library due to the strong hot spot interaction with the RBD of SARS-CoV-2. Cryo-electron microscopy (cryo-EM) observation revealed that within a short and continuous chain, four amino acids (K353, G354, D355, and R357) were in direct contact with the RBD surface (see Figure 2a ). Importantly, the above hotspot regions correlated with the binding affinity of SARS-CoV-2 variants of concern (VOC). VOC bound to hACE2 more tightly and strongly due to the N501Y mutant in the RBD, because the central amino acid of the selected hotspot peptide (K353 of hACE2) had an unexpected hydrophobic interaction with the replaced tyrosine (see Figure 2b ). In addition, this LGKGDFR peptide contains two hydrophobic amino acids and two positively charged amino acids, which cannot provide the properties of completely hydrophilic and negatively charged DNA. Therefore, as the hot peptide-nucleic acid hybridization library evolves to have high affinity for the RBD of SARS-CoV-2, it will provide a variety of electrostatic interactions.

[0163] After seven rounds of in vitro selection with increasing stringency, the hybrid ligand pool was successfully enhanced with strong binding affinity to RBD due to the synergistic effect between the hotspot peptides and the aptamer scaffold. Figure 2c Further, the parameters in each round of HOLD are shown in Table 2 below.

[0164] [Table 2]

[0165]

[0166] like Figure 2c As shown in the figure, the RBD binding of the 7th round (R7) pool linked to the hotspot peptide was quantitatively evaluated in a relative binding assay. Despite the presence of the hotspot peptide linker, the random DNA library exhibited significantly lower levels of RBD binding. When the peptide-nucleic acid hybridization library was challenged with RBD-coated magnetic beads, the binding fraction was only 0.38%, indicating that the hotspot peptide cannot strongly bind to the RBD in the absence of a stabilizing scaffold. In the absence of the LGKGDFR peptide, the R7 pool slightly increased RBD binding, resulting in a binding fraction of 5.74%, which may be due to its own aptamer binding effect. On the other hand, the peptide-based R7 pool significantly enhanced its affinity for RBD, with its binding fraction (22.6%) being 59.37-fold and 3.94-fold higher than that of the random DNA-peptide conjugate and peptide-deficient R7 pools, respectively. The significant improvement in RBD binding is attributed to the synergistic interaction between the hotspot peptide and the newly evolved aptamer scaffold.

[0167] Example 3. Identification and characterization of peptide-supported aptamer scaffolds

[0168] Using high-throughput sequencing and flow cytometry-based binding assays, the best peptide-supported aptamer scaffolds were identified in the R7 hybrid ligand pool. When more than 4 million DNAs in the R7 pool were sequenced, the three most abundant sequence families were found to account for a high percentage (>18%). These are shown in Table 3 below (SEQ ID No.16 to 22). Among these three sequence families, the most representative DNA sequences were synthesized with 5'-hexynyl for site-specific peptide conjugation and 3'-FAM dye for flow cytometry-based binding assays. For 5'-linked hotspot peptides, the second most abundant DNA sequence (DNA domain of R7-02) had the strongest RBD binding. In relative binding analysis, the RBD binding fractions of R7-02 were approximately 5 times and 8 times that of R7-01 and R7-03, respectively (see Figure 3a ).

[0169] [Table 3]

[0170]

[0171] As a hACE2 mimetic, the strongest hybrid ligand R7-02 showed high affinity and specificity for RBD at the correct hACE2 binding site. d When RBD K d is 5.702nM (see Figure 3b , green), while the K of R7-01 (27.01 nM) and R7-03 (93.8 nM) d The value is much higher than R7-02 (see Figure 3c ).

[0172] Additionally, molecular docking simulations were performed and the results were Figure 3d Shown in.

[0173] like Figure 3d As shown, the high binding affinity of R7-02 to RBD is predicted to be attributed to the synergistic binding effect. In the energy minimization simulation snapshot, the LGKGDFR peptide (red) is precisely located at the binding hotspots of the original hACE2 (white) and RBD (blue), and the conjugated aptamer scaffold (green) effectively stabilizes the binding. The DNA folding at the 3' end provides an additional binding motif in the loop structure (A475-N487) of RBD (yellow arrow), resulting in strong and specific binding of R7-02 to RBD. The precise positioning of R7-02 at the interface between RBD and hACE2 demonstrates its potential to neutralize SARS-CoV-2 at low nanomolar concentrations in physiological fluids.

[0174] R7-02 exhibited sustained binding retention on the target surface of the RBD even when competed with previously reported RBD binding affinity reagents (see Figure 3e and 3f ). To induce competition for RBD binding, the hybrid ligands of the hotspot peptide and aptamer scaffold were co-incubated with RBD-binding nucleic acid aptamers (CoV2-RBD-1C, CoV2-6C3, aptamer-1), macrocyclic peptides (peptide 4), or monoclonal antibodies (P05DHu and AM122). Most of them are known to block the binding interface between RBD and hACE2 (see Figure 3e ). In addition, for R7-02, the K values ​​of various RBD binders under the same conditions were confirmed. d Value (see Figure 3g ).

[0175] To assess competitive binding, the fraction of R7-02 bound by RBD with or without potential binding competitors was measured, and the reduction in RBD binding was quantitatively compared to that of other affinity reagents (see Figure 3f ). After incubation with RBD-binding aptamers or cyclic peptides for 1 hour, R7-02 was able to maintain RBD binding, and the ratio of competitive binding to free binding was reduced to a low level (approximately 6.8%). At the same time, the RBD binding of all other aptamers to the cyclic peptide was significantly inhibited, and the ratio between competitive binding and free binding decreased by up to 77.2%, indicating that the binding of monomeric aptamers to the cyclic peptide was relatively weak and could not occupy the restricted RBD binding site for competition with the cooperative hybrid ligand. In addition, due to the different binding sites, the competition between R7-02 and P05DHu and the AM122 antibody was not significant.

[0176] Example 4. Binding tolerance of hACE2 mimetics to SARS-CoV-2 variants of concern (VOC)

[0177] The hotspot peptide-nucleic acid hybrid (R7-02) was found to strongly bind to all reported VOCs of SARS-CoV-2 due to its high variant tolerance to binding. Although initially evolved to bind to the wild-type RBD via the HOLD process, the binding ability of the hybrid ligand is also able to recognize highly mutated RBDs of VOCs. This enables them to interact strongly with the hotspot interface of the RBD. VOCs were found to have several key mutants in the RBD, including N501Y, which highly interacts with the lysine of the LGKGDFR peptide in the hybrid ligand, enhancing its binding to the hACE2 receptor and infectivity to enter host cells (see Figure 4a ).

[0178] When the binding of R7-02 to five VOCs was quantitatively evaluated, the K dThe values ​​(α, β, γ, δ and ο; 5.486 nM, 3.376 nM, 1.614 nM, 2.757 nM and 1.209 nM) were lower than those of wild-type RBD (5.702 nM), confirming the binding tolerance of R7-02 to all variants (see Figure 4b Interestingly, o, which had the most RBD mutations, was most strongly recognized by R7-02, whereas several selected types of RBD binders (aptamer-1, peptide 4, P05DHu, and AM122) lost their ability to bind to RBD due to severe mutations in the variants (see Figure 4c and 4d ).

[0179] Therefore, the binding tolerance of R7-02 to SARS-CoV-2 VOC was confirmed. In addition, this suggests that the binding of hot peptide-derived RBD may be affected by the enhanced hACE2 recognition mechanism of SARS-CoV-2 VOC.

[0180] Example 5. Inhibition of RBD-hACE2 interaction and neutralization of pseudotyped SARS-CoV-2

[0181] Due to the strong binding to RBD, the hot peptide-nucleic acid hybrid molecule can effectively inhibit the interaction between the RBD of SARS-CoV-2 and the hACE2 receptor even at nanomolar concentrations. To characterize the inhibition efficiency, an enzyme-linked immunosorbent assay (ELISA) was performed while changing the concentration of R7-02 from 1pM to 1μM, and R7-02 presented a half-maximal inhibitory concentration (IC 50 ) is 138.9nM (see Figure 5a ).

[0182] At the same time, R7-02's RBD inhibition is guided by the synergistic effect of the hotspot peptides and in vitro evolved aptamer scaffolds of the system combination; the individual components of R7-02 (LGKGDFR peptide and aptamer scaffold) showed 11.69% and 26.82% inhibition of RBD-hACE2 interaction at 3 μM concentration, while the entire structure of R7-02 showed 89.60% inhibition. As evidence of aptamer behavior, the DNA scaffold alone showed better inhibition efficiency (inhibition rate of 3.77%) compared with the random DNA library (see Figure 5b ).

[0183] Previously, several RBD-binding affinity reagents failed to block the precise hACE2 contact residues on the RBD surface and therefore failed to effectively inhibit the RBD-hACE2 interaction. In contrast, the hotspot-derived RBD binding of R7-02 demonstrated effective inhibition of the RBD in hACE2 binding, indicating great potential for neutralizing SARS-CoV-2.

[0184] Therefore, the SARS-CoV-2 neutralization ability of the hot peptide-nucleic acid hybrid molecule was further confirmed. For the neutralization assay, a pseudotype SARS-CoV-2 was prepared that expresses the SARS-CoV-2 spike protein and green fluorescent protein (GFP) but lacks the ability to replicate itself. The neutralization efficiency of R7-02 was analyzed fluorescently by measuring the fluorescence intensity of GFP, which is only expressed in cells infected by the pseudotype SARS-CoV-2 (see Figure 5c Briefly, the hACE2-overexpressing HEK-293T cell line (hACE2-293T) was pre-seeded into microtiter plates overnight and then challenged with pseudotyped SARS-CoV-2 (0.5 multiplicity of infection, MOI) for 6 hours for viral infection. Figure 5d and 5e Shown in.

[0185] like Figure 5d and 5e As shown, in the absence of RBD inhibitors, hACE2-293T was infected with pseudotyped SARS-CoV-2, and 58.4% of all cells quantified emitted strong green fluorescence as evidence of successful viral infection. In contrast, in the presence of aptamer scaffolds composed of R7-02 or hotspot peptides, the level of RBD binding was limited, thereby reducing the infection rate of pseudotyped SARS-CoV-2 to 22.0% and 12.6%, respectively. In addition, R7-02 (an aptamer scaffold based on hotspot peptides) exhibited negligible green fluorescence and a significantly lower percentage of infected cells (1.9%).

[0186] When virus neutralization was compared with that of a number of different RBD binders, R7-02 was found to have significantly higher or comparable neutralization efficiencies (see Figure 5f and 5g On the other hand, it should be taken into account that, unlike the most commonly used neutralizing antibodies, the hybrid ligand of the present invention is completely synthetic. That is, the hybrid ligand of the present invention has a low molecular weight (20 kDa), which is one-tenth of that of a monoclonal antibody, but it ensures effective infection inhibition. In addition, without additional modification, the chimeric structure of the peptide-aptamer hybrid ligand shows good nuclease resistance and serum stability compared to well-known oligonucleotide modifications (such as phosphorothioate backbone, 2'-O-methyl and 2'-F modifications) (see Figure 5h ), indicating its great potential as a SARS-CoV-2 neutralizing agent.

[0187] The present invention relates to a method for preventing, inhibiting or treating coronavirus infection, comprising the step of administering a peptide-nucleic acid hybrid molecule to an individual in need thereof, wherein the peptide is a hotspot-derived peptide comprising an amino acid sequence of SEQ ID No.15, and wherein the nucleic acid comprises a base sequence selected from the group consisting of SEQ ID Nos.7, 8, 18, 19 and 20.

[0188] The present invention provides a method for preventing, inhibiting or treating coronavirus infection, comprising the step of administering to an individual in need thereof a peptide-nucleic acid hybrid molecule prepared by a method comprising the following steps:

[0189] (a) site-specifically binding a hotspot-derived peptide consisting of the amino acid sequence of SEQ ID No. 15 to a random nucleic acid library to prepare a peptide-nucleic acid hybrid;

[0190] (b) co-incubating the magnetic beads coated with the target protein with the peptide-nucleic acid hybrid of step (a); and

[0191] (c) Using a magnet to screen peptide-nucleic acid hybrid molecules that bind to the target protein.

[0192] The present invention provides a method for inhibiting or neutralizing coronavirus, comprising the step of administering a peptide-nucleic acid hybrid molecule to an individual in need thereof, wherein the peptide is a hotspot-derived peptide comprising an amino acid sequence of SEQ ID No.15, and wherein the nucleic acid comprises a base sequence selected from the group consisting of SEQ ID Nos.7, 8, 18, 19 and 20.

[0193] The above description of the present invention is for illustrative purposes only, and it will be appreciated by those skilled in the art that other specific forms may be easily adopted without changing the technical ideas or essential features of the present invention. Therefore, it should be understood that the above embodiments are exemplary and non-restrictive in all aspects.

Claims

1. A composition for preventing or treating coronavirus infection, comprising a peptide-nucleic acid hybrid molecule, wherein: The peptide is a hotspot-derived peptide comprising an amino acid sequence of SEQ ID No. 15, wherein the nucleic acid comprises a base sequence selected from the group consisting of SEQ ID Nos. 7, 8, 18, 19 and 20.

2. The composition according to claim 1, wherein The peptide-nucleic acid hybrid molecule is prepared by the following method: (a) site-specifically conjugating a hotspot-derived peptide consisting of the amino acid sequence of SEQ ID No. 15 with a random nucleic acid library to prepare a peptide-nucleic acid hybrid; (b) co-incubating the target protein-coated magnetic beads with the peptide-nucleic acid hybrid of step (a); and (c) Using a magnet to screen peptide-nucleic acid hybrid molecules that bind to the target protein.

3. The composition according to claim 2, wherein The method further comprises the following steps: (d) selectively amplifying and cloning the nucleic acid portion of the peptide-nucleic acid hybrid molecule selected in step (c); (e) preparing the double-stranded DNA produced in the above step (d) into single-stranded DNA by using an exonuclease, and purifying it; and (f) preparing a new random nucleic acid library consisting of the single-stranded DNA of step (e) above, and repeating the selection, amplification and purification processes of steps (a) to (f) above.

4. The composition according to claim 1, wherein The peptide-nucleic acid hybrid molecule binds to the receptor binding domain (RBD) of the spike protein of the coronavirus.

5. The composition according to claim 4, wherein The RBD is wild type or mutant.

6. The composition according to claim 5, wherein The mutants include at least one selected from the group consisting of N501Y, E484K, K417N, K417T, T478K, L452R, E484A, G339D, S371L, S373P, S375F, N440K, S477N, G446S, Q493R, G496S, Q498R and Y505H.

7. The composition according to claim 1, wherein The peptide-nucleic acid hybrid molecule neutralizes coronavirus.

8. The composition according to claim 1, wherein The peptide-nucleic acid hybrid molecule inhibits the interaction between the RBD of the coronavirus and the human angiotensin converting enzyme 2 (hACE2) receptor.

9. The composition according to claim 1, wherein The nucleic acid enhances binding affinity and solubility to the RBD of the spike protein.

10. The composition according to claim 1, wherein The peptide-nucleic acid hybrid molecule satisfies any one of the following characteristics: (a) nuclease resistance; and (b) Serum stability.

11. The composition according to claim 1, wherein The peptide-nucleic acid hybrid molecule is used for intravenous or respiratory administration.

12. The composition according to claim 1, wherein The coronavirus is at least one selected from the group consisting of human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus NL63 (HCoV-NL63, New Haven coronavirus), human coronavirus HKU1, Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and variants thereof.

13. A composition for preventing or treating coronavirus infection, comprising a peptide-nucleic acid hybrid molecule prepared by a method comprising the following steps: (a) site-specifically conjugating a hotspot-derived peptide consisting of the amino acid sequence of SEQ ID No. 15 with a random nucleic acid library to prepare a peptide-nucleic acid hybrid; (b) co-incubating the target protein-coated magnetic beads with the peptide-nucleic acid hybrid of step (a); and (c) Using a magnet to screen peptide-nucleic acid hybrid molecules that bind to the target protein.

14. The composition according to claim 13, wherein The method further comprises the following steps: (d) selectively amplifying and cloning the nucleic acid portion of the peptide-nucleic acid hybrid molecule selected in step (c); (e) preparing the double-stranded DNA produced in the above step (d) into single-stranded DNA by using an exonuclease, and purifying it; and (f) preparing a new random nucleic acid library consisting of the single-stranded DNA of step (e) above, and repeating the selection, amplification and purification processes of steps (a) to (f) above.

15. The composition according to claim 13, wherein The method further comprises the steps of heat denaturing and cooling the peptide-nucleic acid hybrid to induce 3D folding of the nucleic acid after the above step (a).

16. The composition according to claim 13, wherein The hotspot-derived peptide of the above step (a) is a peptide having at least one functional group selected from the group consisting of azidolysine, azidobutyric acid, azidoacetic acid and azide at the C-terminus or N-terminus, The random nucleic acid library of step (a) above comprises single-stranded nucleic acids having at least one functional group selected from the group consisting of hexynyl, 5-octadiynyl and alkyne at the 5' end, The hotspot-derived peptide and the single-stranded nucleic acid are site-specifically conjugated via a click reaction.

17. The composition according to claim 13, wherein The hotspot-derived peptide of the above step (a) is a peptide having at least one functional group selected from the group consisting of hexynyl, 5-octadiynyl and alkyne at the C-terminus or N-terminus, The random nucleic acid library of step (a) above comprises single-stranded nucleic acids having at least one functional group selected from the group consisting of azidolysine, azidobutyric acid, azidoacetic acid and azide at the 5' end, The hotspot-derived peptide and the single-stranded nucleic acid are site-specifically conjugated via a click reaction.

18. The composition according to claim 13, wherein The random nucleic acid library has the following structure: 5'-Functional Group-Forward Primer-[N x ]-Reverse Primer-3', wherein the functional group is selected from the group consisting of hexynyl, 5-octadiynyl, alkyne, azidolysine, azidobutyric acid, azidoacetic acid and azide, wherein N is A, T, C or G, and x is an integer from 25 to 100.

19. A quasi-drug composition for preventing or inhibiting coronavirus infection, comprising a peptide-nucleic acid hybrid molecule, wherein: The peptide is a hotspot-derived peptide comprising an amino acid sequence of SEQ ID No. 15, wherein the nucleic acid comprises a base sequence selected from the group consisting of SEQ ID Nos. 7, 8, 18, 19 and 20.

20. An antiviral composition for coronavirus, comprising a peptide-nucleic acid hybrid molecule, wherein: The peptide is a hotspot-derived peptide comprising an amino acid sequence of SEQ ID No. 15, wherein the nucleic acid comprises a base sequence selected from the group consisting of SEQ ID Nos. 7, 8, 18, 19 and 20.

21. A composition for neutralizing coronavirus, comprising a peptide-nucleic acid hybrid molecule, wherein: The peptide is a hotspot-derived peptide comprising an amino acid sequence of SEQ ID No. 15, wherein the nucleic acid comprises a base sequence selected from the group consisting of SEQ ID Nos. 7, 8, 18, 19 and 20.

22. A method for preventing, inhibiting or treating coronavirus infection, comprising the step of administering a peptide-nucleic acid hybrid molecule to an individual in need thereof, wherein: The peptide is a hotspot-derived peptide comprising an amino acid sequence of SEQ ID No. 15, wherein the nucleic acid comprises a sequence selected from the group consisting of SEQ ID Nos. 7, 8, 18, 19 and 20.

23. A method for preventing, inhibiting or treating coronavirus infection, comprising administering to an individual in need thereof a peptide-nucleic acid hybrid molecule prepared by a method comprising the following steps: (a) site-specifically conjugating a hotspot-derived peptide consisting of the amino acid sequence of SEQ ID No. 15 with a random nucleic acid library to prepare a peptide-nucleic acid hybrid; (b) co-incubating the target protein-coated magnetic beads with the peptide-nucleic acid hybrid of step (a); and (c) Using a magnet to screen peptide-nucleic acid hybrid molecules that bind to the target protein.

24. A method for inhibiting or neutralizing coronavirus, comprising the step of administering a peptide-nucleic acid hybrid molecule to an individual in need thereof, wherein: The peptide is a hotspot-derived peptide comprising an amino acid sequence of SEQ ID No. 15, wherein the nucleic acid comprises a base sequence selected from the group consisting of SEQ ID Nos. 7, 8, 18, 19 and 20.

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