Application of nucleic acid aptamers in specific recognition of coronaviruses

Abstract

The present invention discloses the application of nucleic acid aptamers in the specific identification of coronaviruses. The present invention provides the application of nucleic acid aptamer 6.8 and its truncated sequence 6.8s in the detection of novel coronaviruses and the preparation of products for the detection of novel coronaviruses. The present invention also provides a method for detecting novel coronaviruses. The nucleic acid aptamer provided by the present invention can specifically bind to the novel coronavirus, has high sensitivity, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to the application of nucleic acid aptamers in the specific identification of coronaviruses. Background Art

[0002] The new coronavirus is a virus discovered after SARS and Middle East Respiratory Syndrome that can cause severe acute respiratory diseases in humans. It has typical characteristics such as strong infectivity and rapid transmission. It has spread in many countries around the world and has become a major disease that seriously endangers human health. It has attracted widespread attention from scholars around the world and has led to investigations and research.

[0003] At present, the detection methods of coronavirus widely used in clinical practice are RT-PCR and serological identification. However, the use of RT-PCR is limited, requiring professionals and equipped with professional instruments. The operation is cumbersome and time-consuming, and it cannot meet the practical requirements of instant, portable, and home use. Serological testing is limited to health care institutions and is not suitable for the determination of suspected cases and rapid screening of close contacts. Therefore, a highly specific, easy-to-operate, instant, and economical coronavirus detection method is urgently needed to cope with the current situation. Summary of the invention

[0004] To make up for the deficiencies of the prior art, the present invention provides the application of nucleic acid aptamer 6.8 in identifying the new coronavirus.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides a nucleic acid aptamer for detecting coronavirus, wherein the nucleic acid aptamer comprises a nucleotide sequence as shown in any one of SEQ ID NO. 1 or 2 or a variant or truncated form thereof.

[0007] Further, the variant comprises a nucleotide sequence that is at least 50% homologous to the nucleotide sequence as shown in any one of SEQ ID NO. 1 or 2.

[0008] Furthermore, the nucleic acid aptamer comprises the nucleotide sequence shown in SEQ ID NO.2.

[0009] Furthermore, the sequence of the nucleic acid aptamer is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0010] The second aspect of the present invention provides a nucleic acid aptamer for detecting coronavirus, wherein the nucleic acid aptamer comprises any one of the following sequences:

[0011] (1) a sequence that hybridizes with the nucleotide sequence of the nucleic acid aptamer disclosed in the first aspect of the present invention;

[0012] (2) An RNA sequence transcribed from the nucleotide sequence of the nucleic acid aptamer shown in the first aspect of the present invention.

[0013] Furthermore, the nucleic acid aptamer comprises one or more modified nucleotides, and / or one or more substitutions.

[0014] Further, the modified nucleotide comprises a chemical substitution or modification at one or more positions independently selected from a ribose position, a deoxyribose position, a phosphate position and a base position.

[0015] The third aspect of the present invention provides a coronavirus nucleic acid aptamer conjugate, which comprises a substance for labeling, detection, diagnosis or treatment connected to the nucleotide sequence of the nucleic acid aptamer described in the first aspect or the second aspect of the present invention.

[0016] Furthermore, the substance used for labeling includes enzyme labeling, fluorescent labeling, isotope labeling, and biotin labeling.

[0017] Furthermore, the substance used for labeling is selected from biotin labeling.

[0018] The fourth aspect of the present invention provides a coronavirus nucleic acid aptamer derivative, which comprises a thiophosphate backbone derived from the backbone of the nucleotide sequence of the nucleic acid aptamer described in the first aspect or the second aspect of the present invention or the nucleic acid aptamer conjugate described in the third aspect of the present invention, and a corresponding locked nucleic acid or peptide nucleic acid modified from the nucleic acid aptamer described in the first aspect or the second aspect of the present invention or the nucleic acid aptamer conjugate described in the third aspect of the present invention.

[0019] The fifth aspect of the present invention provides the use of the nucleic acid aptamer described in the first aspect or the second aspect of the present invention, the nucleic acid aptamer conjugate described in the third aspect of the present invention, or the nucleic acid aptamer derivative described in the fourth aspect of the present invention in enriching coronaviruses.

[0020] Furthermore, the coronavirus includes SARS coronavirus and MERS coronavirus.

[0021] Furthermore, the coronavirus is selected from SARS coronavirus.

[0022] Furthermore, the SARS coronavirus includes SARS CoV 2 coronavirus and SARS CoV 1 coronavirus.

[0023] Furthermore, the SARS coronavirus is selected from SARS CoV 2 coronavirus.

[0024] The sixth aspect of the present invention provides the use of the nucleic acid aptamer described in the first aspect or the second aspect of the present invention, the nucleic acid aptamer conjugate described in the third aspect of the present invention, or the nucleic acid aptamer derivative described in the fourth aspect of the present invention in the preparation of a product for detecting coronavirus.

[0025] Furthermore, the product includes test strips and test kits.

[0026] Furthermore, the coronavirus includes SARS coronavirus and MERS coronavirus.

[0027] Furthermore, the coronavirus is selected from SARS coronavirus.

[0028] Furthermore, the SARS coronavirus includes SARS CoV 2 coronavirus and SARS CoV 1 coronavirus.

[0029] Furthermore, the SARS coronavirus is selected from SARS CoV 2 coronavirus.

[0030] The seventh aspect of the present invention provides the use of the nucleic acid aptamer described in the first aspect or the second aspect of the present invention, the nucleic acid aptamer conjugate described in the third aspect of the present invention, or the nucleic acid aptamer derivative described in the fourth aspect of the present invention in inhibiting coronavirus infection or preparing a pharmaceutical composition for treating coronavirus pneumonia.

[0031] Furthermore, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier.

[0032] Furthermore, the coronavirus includes SARS coronavirus and MERS coronavirus.

[0033] Furthermore, the coronavirus is selected from SARS coronavirus.

[0034] Furthermore, the SARS coronavirus includes SARS CoV 2 coronavirus and SARS CoV 1 coronavirus.

[0035] Furthermore, the SARS coronavirus is selected from SARS CoV 2 coronavirus.

[0036] The eighth aspect of the present invention provides a method for detecting coronavirus, which comprises contacting the nucleic acid aptamer described in the first aspect or the second aspect of the present invention, the nucleic acid aptamer conjugate described in the third aspect of the present invention, or the nucleic acid aptamer derivative described in the fourth aspect of the present invention with a sample, thereby detecting the coronavirus in the sample.

[0037] Furthermore, the coronavirus includes SARS coronavirus and MERS coronavirus.

[0038] Furthermore, the coronavirus is selected from SARS coronavirus.

[0039] Furthermore, the SARS coronavirus includes SARS CoV 2 coronavirus and SARS CoV 1 coronavirus.

[0040] Furthermore, the SARS coronavirus is selected from SARS CoV 2 coronavirus.

[0041] Furthermore, the method is a method for non-diagnostic purposes.

[0042] Advantages and beneficial effects of the present invention:

[0043] The present invention proves through ELISA experiments and Bli technology that nucleic acid aptamer 6.8 and its truncated sequence 6.8s can specifically identify the new coronavirus, and the operation is simple and rapid, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a graph showing the specific binding of aptamer 6.8 to the new coronavirus in an ELISA experiment;

[0045] Figure 2 This is the image of aptamer 6.8s specifically binding to the new coronavirus in the ELISA experiment;

[0046] Figure 3 This is a diagram of aptamer 6.8 specifically binding to the new coronavirus in the Bli experiment;

[0047] Figure 4 This is the equilibrium dissociation constant diagram of aptamer 6.8 binding to the new coronavirus. DETAILED DESCRIPTION

[0048] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0049] The present invention provides a nucleic acid aptamer for detecting coronavirus, wherein the nucleic acid aptamer comprises a nucleotide sequence as shown in any one of SEQ ID NO. 1 or 2 or a variant or truncated form thereof.

[0050] The variant comprises a nucleotide sequence that is at least 50% homologous to the nucleotide sequence as shown in any one of SEQ ID NO. 1 or 2.

[0051] The nucleic acid aptamer comprises the nucleotide sequence shown in SEQ ID NO.2.

[0052] The sequence of the nucleic acid aptamer is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0053] In the present invention, coronaviruses are large enveloped positive-strand RNA viruses divided into four genera: α-, β-, γ- and δ coronaviruses. Coronaviruses have the largest genome (27-32 kb) of all RNA viruses, which is packaged in a helical capsid surrounded by an envelope. At least three structural proteins are associated with the viral envelope: membrane protein (M), envelope protein (E) and spike (glyco) protein (S). Some coronaviruses encode hemagglutinin esterase protein (HE).

[0054] Among them, coronaviruses include SARS coronavirus and MERS coronavirus.

[0055] In some embodiments of the invention, the coronavirus is selected from the SARS coronavirus.

[0056] SARS coronaviruses include SARS-CoV-2 coronavirus and SARS-CoV-1 coronavirus.

[0057] In a specific embodiment of the present invention, the SARS coronavirus is selected from the SARS-CoV-2 coronavirus.

[0058] In the present invention, SARS-CoV-2 coronavirus and novel coronavirus are used interchangeably, and SARS-CoV-2 coronavirus includes variants B.1.1.7, B.1525, B.1.351, and a variant called P1. These variants can be characterized by mutations (e.g., amino acid additions, substitutions, and / or deletions) within the spike protein. For example, the SARS-CoV-2 coronavirus may include any variant having one or more of the following mutations within the spike protein:

[0059] 1) HV69-70 deletion; and / or 2) N501Y; and / or 3) E484K.

[0060] In the present invention, nucleic acid aptamers are used interchangeably with aptamers and aptamers, and refer to nucleic acid molecules that can specifically bind to target molecules. In an embodiment of the present invention, as long as the nucleic acid aptamer has the function of specifically binding to the coronavirus, there is no upper limit to its length, and as long as it does not hinder the function of the nucleic acid aptamer, the same sequence can also be connected to different base sequences and nucleic acid aptamers. However, considering the ease of synthesis, antigenicity, etc., for the length of the nucleic acid aptamer in the present embodiment, as an upper limit, for example, less than 200 bases, preferably less than 150 bases, and more preferably less than 100 bases.

[0061] Specificity refers to the ability of the nucleic acid aptamer of the present invention to bind to coronavirus but not to other viruses. When the aptamer binds to coronavirus at least 2 times, at least 3 times, at least 5 times, at least 7 times, and preferably at least 10 times more than other viruses, the aptamer is considered specific.

[0062] In the present invention, nucleotide refers to a glycoside comprising a sugar portion, a base portion and a covalently linked group (linking group), such as a phosphate or phosphorothioate internucleotide linking group, and includes naturally occurring nucleotides, such as DNA or RNA, and non-naturally occurring nucleotides, comprising modified sugar and / or base portions.

[0063] In the field of biochemistry, the term "nucleoside" is generally used to refer to a glycoside comprising a sugar portion and a base portion, and can therefore be used when referring to a nucleotide unit, covalently linked by nucleotide linkages between nucleotides of an oligonucleotide; the term "nucleotide" refers to a nucleic acid monomer or unit, and itself can refer to a base in the case of an oligonucleotide, such as a "nucleotide sequence", which generally refers to a nucleobase sequence (i.e., the presence of a sugar backbone and an internucleotide linkage is implied), and a nucleotide sequence and a sequence can be used interchangeably in the present invention. Similarly, particularly in the case of an oligonucleotide in which more than one internucleoside linking group is modified, the term "nucleotide" can refer to a "nucleoside", for example even when specifying the presence and nature of the linkage between nucleosides, the term "nucleotide" can be used.

[0064] In the present invention, homology or identity refers to the sequence similarity between two nucleic acid aptamers. Homology and identity can be determined by comparing the positions in each sequence that can be compared for comparison purposes. When the equivalent position in the compared sequence is occupied by the same base, the molecules are identical at that position; when the equivalent site is occupied by the same or similar nucleic acid residues (e.g., similar in space and / or electronic properties), the molecules can be said to be homologous at that position.

[0065] In the present invention, a truncated form generally refers to a nucleic acid aptamer that is truncated by the deletion of nucleotides but still has the desired or even improved binding characteristics. The length of the truncated form can vary depending on the length of the starting aptamer and is defined as the term nucleic acid aptamer in the present invention. The truncation in the truncated form can occur in the fixed region or the variable region of the starting aptamer, or in both the fixed region and the variable region.

[0066] The present invention provides a nucleic acid aptamer for detecting coronavirus, wherein the nucleic acid aptamer comprises any one of the following sequences:

[0067] (1) a sequence that hybridizes with the nucleotide sequence of the nucleic acid aptamer shown above;

[0068] (2) RNA sequence transcribed from the nucleotide sequence of the nucleic acid aptamer shown above.

[0069] In the present invention, hybridization refers to the formation of a duplex structure by complementary base pairing between two single-stranded nucleic acids. Hybridization can occur between exactly complementary nucleic acid chains or between complementary nucleic acid chains containing a low number of mismatches.

[0070] In the present invention, transcription refers to the process by which the genetic code in a DNA sequence is transcribed into RNA.

[0071] Furthermore, the nucleic acid aptamer comprises one or more modified nucleotides, and / or one or more substitutions.

[0072] The modified nucleotide comprises a chemical substitution or modification at one or more positions independently selected from a ribose position, a deoxyribose position, a phosphate position and a base position.

[0073] In the present invention, the one or more (e.g., 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 or 1-2) modified nucleotides can be substituted with a polyethylene glycol moiety or a joint comprising multiple (e.g., 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 or 1-2) polyethylene glycol moieties. In some cases, the joint can be a hexaethylene glycol (H or PEG6) joint. Such substitutions can occur in any nucleotide of the nucleic acid aptamer, as long as it does not substantially affect the activity (e.g., binding activity or inhibitory activity) and / or function of the nucleic acid aptamer.

[0074] The modified nucleotides comprise one or more modifications independently selected from the following: 2'-position sugar modification, 2'-amino (2'-NH2) modification, 2'-fluoro (2'-F) modification, 2'-O-methyl (2'-OMe) modification, 2'-O-(2-methoxyethyl) (2'-O-MOE) modification, 5-position modified pyrimidine, modification at the amine outside the cytosine ring, substitution with 5'-bromouracil, substitution with 5'-bromodeoxyuridine, substitution with 5'-bromodeoxycytidine, backbone modification, locked nucleic acid (LNA), methylation, 3' cap and 5' cap.

[0075] The 5-position modified pyrimidine is selected from 5-carboxy-2'-deoxyuridine, 5-aminoallyl-2'-deoxyuridine, 5-[(3-indolyl)propionamide-N-allyl]-2'-deoxyuridine, 5-carboxy-2'-deoxycytidine, 5-aminoallyl-2'-deoxycytidine, biotin-16-aminoallyl-2'-deoxycytidine, 5-(N-benzylcarboxamide)-2'-deoxyuridine, 5-(N-isobutylcarboxamide)-2'-deoxyuridine, 5-(N-naphthylmethylcarboxamide)-2'-deoxyuridine and 5-(N-tryptaminecarboxamide)-2'-deoxyuridine.

[0076] In the present invention, the Cell-SELEX method or the SELEX method can be used to obtain aptamers on complex targets, living cells or tissues.

[0077] Among them, the SELEX method is a method for selecting oligonucleotides that specifically bind to a target molecule from an oligonucleotide pool with 10-14 different nucleotide sequences. The oligonucleotides used have a random sequence of about 40 residues, which are flanked by primer sequences. This oligonucleotide pool is allowed to mix with the target molecule, and only RNA bound to the target molecule is collected by using a filter, etc. The collected oligonucleotides are amplified by RT-PCR and used as a template for the next cycle. By repeating this operation for about 10 times, an aptamer that specifically binds to the target molecule is obtained. By increasing the number of cycles or using competitive substances, aptamers that show stronger binding potential to the target molecule are concentrated and selected. Therefore, by adjusting the number of cycles of SELEX and / or changing competitive conditions, aptamers with different binding forces or binding modes and aptamers with the same binding forces or binding modes but with different base sequences can be obtained in some cases. The SELEX method includes a process of amplification by PCR; by using manganese ions, etc. to cause mutations in the process, SELEX may have higher diversity.

[0078] In a specific embodiment of the present invention, the SELEX method is used to screen nucleic acid aptamers.

[0079] The present invention provides a coronavirus nucleic acid aptamer derivative, wherein the nucleic acid aptamer derivative comprises a phosphorothioate backbone derived from the backbone of the nucleotide sequence of the nucleic acid aptamer or a corresponding locked nucleic acid or peptide nucleic acid modified from the nucleic acid aptamer.

[0080] In the present invention, the phosphorothioate backbone refers to that the non-bridging oxygen atoms of the phosphodiester backbone of RNA and DNA nucleic acid aptamers can be replaced by one or two sulfur atoms, respectively, to produce a phosphorothioate backbone with a phosphorothioate or phosphorodithioate bond. Such phosphorothioate backbones are known to have increased binding affinity to their targets, as well as enhanced resistance to nuclease degradation.

[0081] Peptide nucleic acid or PNA refers to a synthetic DNA analog in which the normal phosphodiester backbone is replaced by an N-(2-aminoethyl)glycine chain or other optional amino acid chain. Its nucleoside bases complement DNA or RNA in the same AT(U) and GC manner (Nielsen, et al., Science 254:1497-1500 (1991); Hanvey, et al., Science 258:1481-1485 (1992); and Egholm, et al., Nature 365:566-568 (1993)). The artificial backbone makes PNA resistant to nucleases.

[0082] In the present invention, it refers to an oligonucleotide whose backbone is composed of N-(2-aminoethyl)-glycine repeating units linked by peptide bonds, wherein different nitrogenous bases are linked to the main chain by methylene bonds (-CH2-) and carbonyl groups (-(C=O)-). It refers to a nucleic acid analog containing a methylene bridge connecting the 2'-O and 4'-C atoms of the ribose monosaccharide.

[0083] Locked nucleic acid or LNA in the present invention refers to a chemically modified RNA nucleotide with a bridge connecting the 2' and 4' carbons. Such nucleotides maintain base pairing specificity and increase pairing overlap, thereby forming a more stable double helix with a significantly higher melting point.

[0084] The present invention provides the use of the above-mentioned nucleic acid aptamer, the above-mentioned nucleic acid aptamer conjugate or the above-mentioned nucleic acid aptamer derivative in the preparation of a product for detecting coronavirus.

[0085] The products include test strips and test kits.

[0086] In the present invention, the kit may include, in addition to the nucleic acid aptamer of the present invention, a buffer, a labeling reagent and / or instructions, etc.

[0087] The present invention provides the use of the above-mentioned nucleic acid aptamer, the above-mentioned nucleic acid aptamer conjugate or the above-mentioned nucleic acid aptamer derivative in inhibiting coronavirus infection.

[0088] In the present invention, inhibition refers to the ability to inhibit coronavirus by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or a greater overall reduction in the ability to inhibit coronavirus.

[0089] The present invention provides the use of the above-mentioned nucleic acid aptamer, the above-mentioned nucleic acid aptamer conjugate or the above-mentioned nucleic acid aptamer derivative in preparing a pharmaceutical composition for treating coronavirus pneumonia.

[0090] In the present invention, the inhibition of coronavirus infection or treatment of coronavirus pneumonia preferably includes a conjugate of a nucleic acid aptamer, and more preferably refers to a substance used for treatment connected to the nucleotide sequence of the nucleic acid aptamer. The therapeutic substance refers to a substance with medicinal, pharmacological, psychosomatic and therapeutic effects, including but not limited to nanoparticles, RNA drugs, siRNA, and synthetic or biologically produced drugs.

[0091] Treatment generally involves treating and physiotherapy humans or animals (e.g., in veterinary applications), wherein some desired therapeutic effects are achieved, such as inhibiting disease progression, and including reducing progression rate, stopping progression rate, alleviating disease symptoms, improving disease, and curing disease. Treatment as a preventive measure (i.e., prevention) is also included. For example, the term "treatment" also includes the application of patients who have not yet developed the disease but are at risk of developing the disease. Treatment in the present invention refers to the administration of a therapeutically effective amount of a nucleic acid aptamer, a nucleic acid aptamer conjugate, or a nucleic acid aptamer derivative as disclosed in the present invention, which can inhibit coronavirus infection or reduce or inhibit the symptoms of coronavirus pneumonia.

[0092] The therapeutically effective amount relates to the amount of a compound or material, a composition or dosage form containing a compound that is effective in producing some desired therapeutic effect and matches a reasonable benefit / risk ratio when administered according to the desired treatment regimen. The therapeutically effective amount of the present invention refers to the amount of a nucleic acid aptamer, a nucleic acid aptamer conjugate or a nucleic acid aptamer derivative sufficient to reduce or inhibit the infection of coronavirus and / or the symptoms of coronavirus pneumonia.

[0093] The pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier.

[0094] Pharmaceutically acceptable refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0095] Pharmaceutically acceptable carriers include, but are not limited to, diluents, binders, surfactants, wetting agents, adsorbent carriers, lubricants, fillers, disintegrants, excipients, and solubilizers.

[0096] Among them, diluents include but are not limited to lactose, sodium chloride, glucose, urea, starch, water, etc.; adhesives such as starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginate, xanthan gum, hydroxypropyl cellulose and hydroxypropyl methylcellulose.

[0097] Surfactants include, but are not limited to, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, and cetyl alcohol.

[0098] Adsorption carriers include, but are not limited to, starch, lactose, bentonite, silica gel, kaolin, and bentonite.

[0099] Lubricants include, but are not limited to, zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, and magnesium dodecyl sulfate.

[0100] Fillers include but are not limited to mannitol (granular or powdered), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugars, coupling sugars, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate and sodium bicarbonate, etc.; disintegrants such as cross-linked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, and soybean polysaccharides.

[0101] Excipients include, but are not limited to, sugars such as monosaccharides, disaccharides, cyclodextrins and polysaccharides (specifically, without limitation, including glucose, sucrose, lactose, raffinose, mannitol, sorbitol, inositol, dextrin, maltodextrin, starch and cellulose), metal salts (e.g., sodium phosphate or calcium phosphate, calcium sulfate, magnesium sulfate), citric acid, tartaric acid, glycine, low / medium / high molecular weight polyethylene glycol (PEG), Pluronic or a combination thereof.

[0102] The cosolvent includes, but is not limited to, a pharmaceutically acceptable inorganic acid or organic acid, an inorganic base or an organic base, and may also be a Lewis acid or base in a broad sense, and may contain one or more of them, and may be one or more of hydrochloric acid, phosphoric acid, propionic acid, acetic acid, lactic acid, citric acid, tartaric acid, boric acid, polyhydroxycarboxylic acids such as glucuronic acid, gluconic acid, lactobionic acid, malic acid, threonic acid, glucoheptonic acid, 2,5-dihydroxybenzoic acid, acidic amino acids such as aspartic acid, glutamic acid, etc.

[0103] The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.

[0104] Among them, parenteral administration includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intracorporeal, intralesional, and intracranial injection or infusion techniques. As long as the target tissue can be reached, the pharmaceutical composition of the present invention can be administered to the recipient by any route.

[0105] The dosage forms for oral administration include, but are not limited to, solid preparations (including tablets, pills, sublingual tablets, capsules, drops, lozenges), granules, powders, granules, and liquids. In addition, the solid preparations can be in the form of coatings known in the art, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double-layer tablets, and multi-layer tablets, as required.

[0106] The dosage form of the pharmaceutical composition of the present invention is not particularly limited as long as it does not inactivate the active ingredient and can exert its pharmacological action in vivo after administration. It generally varies depending on the administration method and / or formulation conditions.

[0107] It should be noted that the specific shape and size of each of the above-mentioned dosage forms are not particularly limited as long as they are within the range of dosage forms known in the art.

[0108] The pharmaceutical composition of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.Liposomes can be composed of a variety of phospholipids such as cholesterol, stearylamine or phosphatidylcholine.

[0109] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0110] Example 1: Nucleic acid aptamers specifically recognize and inactivate novel coronavirus particles

[0111] 1.1 Experimental Materials

[0112] Inactivated coronavirus particles: provided by Sinovac Vaccine Company.

[0113] 1.2 Experimental methods

[0114] 1.2.1 Synthesis of nucleic acid aptamers

[0115] 6.8, 6.8s and an irrelevant control sequence - A20S were synthesized, and the 5' ends of 6.8, 6.8s and A20s were labeled with Bio by the company. The specific sequences are shown in Table 1.

[0116] Table 1 Aptamer sequences

[0117]

[0118] 1.2.2 ELISA experiment

[0119] 1) Dissolve a certain amount of inactivated SARS-CoV-2 particles (or inactivated HINI virus particles) in a carbonate buffer at pH 9.7, add 100 μl / well to the enzyme-linked strip, and coat overnight at 4°C;

[0120] 2) Discard the coating solution, add 100 μl of 2% BSA blocking solution to each well, and block for 60 min at room temperature;

[0121] 3) Dissolve a certain concentration of Bio-6.8 and an irrelevant control sequence Bio-A20S; or Bio-6.8 and its truncated sequence 6.8s in an appropriate volume of buffer (50 mM HEPES, 100 mM NaCl, 2 mM MgCl2, 5 mM KCl, 1 mM CaCl2), denature at 100°C for 5 min, and immediately place on ice to cool thoroughly;

[0122] 4) Add the denatured Bio-6.8 and an irrelevant control sequence (Bio-A20S library); or Bio-6.8 and its truncated sequence 6.8s to the enzyme-linked strip, and incubate the aptamer and the coated inactivated virus particles at 37°C for 30 minutes;

[0123] 5) Discard the liquid in the wells, wash each well with 200 μl of washing solution, repeat the washing three times, and completely dry the liquid in the wells after the last wash;

[0124] 6) Add 100 μl of HRP enzyme diluted 1:100 to each well, incubate at room temperature for 40 min, discard the liquid in the well, and wash the plate 5 times using the same method as above;

[0125] 7) Add 100 μl TMB colorimetric substrate to each well and develop the color at 37°C in the dark. When there is a clear color change, add 10 μl stop solution and read the result with the enzyme-linked immunosorbent assay.

[0126] 1.2.3Bli Technology

[0127] 100nM Bio-labeled aptamer 6.8 was dissolved in a buffer solution (50mM HEPES, 100mM NaCl, 2mMMgCl2, 5mM KCl, 1mM CaCl2), combined with the probe with streptavidin protein, and then combined with 10ug / ml inactivated SARS-CoV-2 particle solution (dissolved in the loading solution), and finally dissociated with the loading solution. The entire loading, binding, and dissociation process was performed in a molecular interaction instrument.

[0128] 1.2.4 Determination of equilibrium dissociation constant (KD value)

[0129] Inactivated SARS-CoV-2 particles were coated in a microplate (1ug / well), and the biotin-labeled aptamer 6.8 was prepared at concentrations of 0.78nM, 1.56nM, 3.125nM, 6.25nM, 12.5nM, 25nM and 100nM, and then added to the wells coated with SARS-CoV2 virus particles for binding, and the curve was drawn according to the OD450nm absorbance value.

[0130] 1.3 Experimental Results

[0131] The results are as follows Figure 1 As shown, ELISA experiments have shown that 6.8 specifically recognizes inactivated SARS-CoV-2 particles, but does not bind to other inactivated virus particles (HINI virus); 6.8s specifically recognizes inactivated SARS-CoV-2 particles ( Figure 2 ).

[0132] Bli technology proves that 6.8 specifically recognizes and inactivates new coronavirus particles ( Figure 3 ).

[0133] The equilibrium dissociation constant KD value of 6.8 binding to inactivated novel coronavirus particles is about 20.9nM ( Figure 4 ), and the relevant values ​​are shown in Table 2.

[0134] Table 2 Calculated values ​​of equilibrium dissociation constants

[0135] Model Michaelis Menten Equation <![CDATA[y=V max *x / (K m +x)]]> Reduced Chi-Sqr 0.00142 Adj.R-Square 0.9213 Value Standard Erro Mean <![CDATA[V max ]]> 0.52238 0.06587 Mean <![CDATA[K m ]]> 20.92859 6.68597

[0136] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A nucleic acid aptamer for detecting coronavirus, characterized in that: The sequence of the nucleic acid aptamer is shown as SEQ ID NO.1 or SEQ ID NO.

2.

2. A nucleic acid aptamer for detecting coronavirus, characterized in that: The nucleic acid aptamer is selected from any one of the following sequences: (1) a sequence that hybridizes with the nucleotide sequence of the nucleic acid aptamer of claim 1; (2) An RNA sequence transcribed from the nucleotide sequence of the nucleic acid aptamer shown in claim 1.

3. A coronavirus nucleic acid aptamer conjugate, characterized in that: The nucleic acid aptamer conjugate comprises a substance for labeling, detection, diagnosis or treatment connected to the nucleotide sequence of the nucleic acid aptamer according to any one of claims 1 to 2.

4. The conjugate according to claim 3, characterized in that The labeling substances include enzyme labeling, fluorescent labeling, isotope labeling, and biotin labeling.

5. The conjugate according to claim 4, characterized in that The substance used for labeling is selected from biotin labeling.

6. A coronavirus nucleic acid aptamer derivative, characterized in that: The nucleic acid aptamer derivative comprises a phosphorothioate backbone derived from the backbone of the nucleotide sequence of the nucleic acid aptamer described in any one of claims 1-2 or the nucleic acid aptamer conjugate described in any one of claims 3-5, and a corresponding locked nucleic acid or peptide nucleic acid modified from the nucleic acid aptamer described in any one of claims 1-2 or the nucleic acid aptamer conjugate described in any one of claims 3-5.

7. Use of the nucleic acid aptamer described in any one of claims 1-2, the nucleic acid aptamer conjugate described in any one of claims 3-5, or the nucleic acid aptamer derivative described in claim 6 in enriching coronavirus for non-diagnostic purposes.

8. The use according to claim 7, characterized in that: The coronaviruses include SARS coronavirus and MERS coronavirus.

9. The use according to claim 8, characterized in that: The coronavirus is selected from SARS coronavirus.

10. The use according to claim 9, characterized in that: The SARS coronavirus includes SARS-CoV-2 coronavirus and SARS-CoV-1 coronavirus.

11. The use according to claim 10, characterized in that: The SARS coronavirus is selected from the SARS-CoV-2 coronavirus.

12. Use of the nucleic acid aptamer described in any one of claims 1-2, the nucleic acid aptamer conjugate described in any one of claims 3-5, or the nucleic acid aptamer derivative described in claim 6 in the preparation of a product for detecting coronavirus.

13. The use according to claim 12, characterized in that: The products include test strips and test kits.

14. The use according to claim 12, characterized in that: The coronaviruses include SARS coronavirus and MERS coronavirus.

15. The use according to claim 14, characterized in that The coronavirus is selected from SARS coronavirus.

16. The use according to claim 15, characterized in that The SARS coronavirus includes SARS-CoV-2 coronavirus and SARS-CoV-1 coronavirus.

17. The use according to claim 16, characterized in that The SARS coronavirus is selected from the SARS-CoV-2 coronavirus.

18. Use of the nucleic acid aptamer described in any one of claims 1-2, the nucleic acid aptamer conjugate described in any one of claims 3-5, or the nucleic acid aptamer derivative described in claim 6 in the preparation of a pharmaceutical composition for treating coronavirus pneumonia.

19. The use according to claim 18, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

20. The use according to claim 18, characterized in that The coronaviruses include SARS coronavirus and MERS coronavirus.

21. The use according to claim 20, characterized in that The coronavirus is selected from SARS coronavirus.

22. The use according to claim 21, characterized in that The SARS coronavirus includes SARS-CoV-2 coronavirus and SARS-CoV-1 coronavirus.

23. The use according to claim 22, characterized in that The SARS coronavirus is selected from the SARS-CoV-2 coronavirus.

24. A method for detecting coronavirus for non-diagnostic purposes, characterized in that: The method comprises contacting the nucleic acid aptamer described in any one of claims 1 to 2, the nucleic acid aptamer conjugate described in any one of claims 3 to 5, or the nucleic acid aptamer derivative described in claim 6 with a sample, thereby detecting the coronavirus in the sample.

25. The method according to claim 24, characterized in that The coronaviruses include SARS coronavirus and MERS coronavirus.

26. The method according to claim 25, characterized in that The coronavirus is selected from SARS coronavirus.

27. The method according to claim 26, characterized in that The SARS coronavirus includes SARS-CoV-2 coronavirus and SARS-CoV-1 coronavirus.

28. The method according to claim 27, characterized in that The SARS coronavirus is selected from the SARS-CoV-2 coronavirus.

Images