WO2024131676A1 - 筛选rna核酸适配体的系统及方法 - Google Patents

筛选rna核酸适配体的系统及方法 Download PDF

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WO2024131676A1
WO2024131676A1 PCT/CN2023/139189 CN2023139189W WO2024131676A1 WO 2024131676 A1 WO2024131676 A1 WO 2024131676A1 CN 2023139189 W CN2023139189 W CN 2023139189W WO 2024131676 A1 WO2024131676 A1 WO 2024131676A1
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nucleic acid
sequence
protein
rna
virus
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French (fr)
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王宇
张菊
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深圳大学
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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    • G01—MEASURING; TESTING
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus

Definitions

  • the present invention relates to a system and method for screening RNA aptamers, and in particular to a system and method for screening RNA aptamers based on CRISPR/Cas technology.
  • the present invention also relates to an RNA aptamer, and in particular to an RNA aptamer for detecting or neutralizing SARS-CoV-2 virus and its application.
  • SELEX systematic evolution of ligands by exponential enrichment
  • the process mainly includes the selection of SELEX technical methods, the synthesis and establishment of oligonucleotide random sequence libraries, the acquisition of target molecules, the incubation, binding, elution, recovery, amplification and other repeated selective cycles of target molecules and nucleic acid libraries.
  • the specific affinity sequence i.e., the nucleic acid aptamer, will occupy the majority of the remaining library sequence types and will eventually be identified and obtained, followed by subsequent exploratory work such as functional verification and chemical modification.
  • the final screening product is very sensitive and strict to the screening environment, i.e., the experimental conditions, such as the ratio of the screening library to the target molecule, the buffer composition, the ionic strength, the pH value, the incubation temperature, the incubation time, and the inherent properties of the target molecule.
  • the superposition of these screening variables will produce a specific selection stringency, thereby affecting the true affinity and effectiveness of the nucleic acid aptamer.
  • the SELEX screening method based on purified proteins may not be able to simulate the natural structural state of the target protein; the targets of cell-based SELEX screening are often concentrated on the cell surface, and the targets that can be used for screening only account for a small proportion of the population; animal-based SELEX screening is costly, lacks specificity and throughput, and may become complicated due to the in vivo screening environment.
  • nucleic acid aptamers At present, the number of nucleic acid aptamers with good application prospects is far from meeting actual needs, and there is still room for development in effective screening methods for nucleic acid aptamers.
  • Nucleic acid aptamers can be applied to different scenarios in the biomedical field, mainly including drug therapy, biosensor technology, molecular imaging and in vitro diagnostic technology, biomarker and biological discovery technology, drug delivery technology, and antiviral and vaccine gene therapy technology.
  • the characteristics of nucleic acid aptamers that can be coupled, labeled, and chemically modified make them easier to combine with other new technologies such as endogenous nucleic acid analysis, microfluidic cell separation, flow cytometry, nanoparticles, etc., to improve diagnostic effects and promote clinical research.
  • RNA aptamers for a target protein comprising: (i) Guide RNA, containing a recognition sequence and a random library of nucleic acid aptamers of a predetermined length; (ii) a targeting sequence, which is paired with the recognition sequence of the guide RNA; (iii) a selection marker, located downstream of the targeting sequence, including a basic promoter and a selection marker gene; (iv) a fusion protein, which comprises the target protein and a transcriptional activation element, and the target protein binds to the RNA nucleic acid aptamer in the random library of nucleic acid aptamers, resulting in the binding of the transcriptional activation element to the selection marker; (v) a dCas protein, which specifically recognizes the targeting sequence under the guidance of the guide RNA; and (vi) screening cells.
  • Guide RNA containing a recognition sequence and a random library of nucleic acid aptamers of a predetermined length
  • a targeting sequence which is paired with the recognition
  • the random library of nucleic acid aptamers is composed of a random sequence of single-stranded oligonucleotides with a length of 8 to 60 bases and a magnitude of 10 5 to 10 36. In some embodiments, the random library of nucleic acid aptamers is inserted into the loop region of the guide RNA backbone.
  • the targeting sequence is an exogenous and/or artificial sequence. In some embodiments, the targeting sequence comprises a plurality of copies of a gLuc sequence or a Tet sequence.
  • the selection marker gene is a luciferase gene, a fluorescent protein gene or an antibiotic resistance gene.
  • the luciferase gene is selected from the group consisting of firefly luciferase gene, Renilla luciferase gene and Gaussia Luciferase gene.
  • the antibiotic resistance gene is a puromycin resistance gene or a kanamycin resistance gene.
  • the fluorescent protein gene is a green fluorescent protein gene or a red fluorescent protein gene.
  • the transcriptional activation element comprises a plurality of transcriptional activators. In some embodiments, the transcriptional activation element comprises VP64, P65 and HSF1.
  • the basic promoter is selected from mini-promoter-1 (SEQ ID NO: 28), mini-promoter-2 (SEQ ID NO: 27), mini-TK promoter (SEQ ID NO: 29), mini-CMV promoter (SEQ ID NO: 30) and Crystallin basal promoter (SEQ ID NO: 31).
  • the dCas protein is dCas9 protein or dUn1Cas12f1 protein.
  • the target protein is derived from a virus, bacteria, fungus or animal.
  • the target protein is derived from humans.
  • the target protein is green fluorescent protein or the RBD region of the S1 protein derived from SARS-CoV-2.
  • the screening cell is a human cell HEK293T or a prokaryotic cell Escherichia coli E.coli.
  • RNA aptamers for a target protein comprising: a viral expression vector, the viral expression vector comprising a guide RNA and a nucleic acid sequence encoding a fusion protein, wherein the guide RNA contains a recognition sequence and a random library of nucleic acid aptamers of a predetermined length, the fusion protein comprises the target protein and a transcriptional activation element, and the binding of the target protein to the random library of nucleic acid aptamers results in the binding of the transcriptional activation element to the selection marker; and a screening cell expressing the dCas protein, which comprises a targeting sequence and a selection marker, wherein the targeting sequence is paired with the recognition sequence of the guide RNA, and the selection marker is located downstream of the targeting sequence and includes a basic promoter and a selection marker gene.
  • the viral expression vector is a lentiviral expression vector.
  • the lentiviral expression vector further comprises a first promoter operably linked to the guide RNA.
  • the nucleic acid aptamer random library is inserted into the guide RNA backbone by a first restriction endonuclease.
  • the coding sequence of the target protein is operably connected to the second promoter by a second restriction endonuclease.
  • the lentiviral expression vector comprises a polyT located downstream of the guide RNA.
  • the lentiviral expression vector comprises a nuclear localization sequence (NLS).
  • the NLS includes a first NLS downstream of the second promoter and a second NLS downstream of the coding sequence of the fusion protein.
  • the lentiviral expression vector comprises a P2A, an antibiotic resistance gene, and a post-transcriptional regulatory element located downstream of the second NLS.
  • the P2A is connected to the second NLS upstream and the antibiotic resistance gene downstream by a connector
  • the coding sequence of the target protein is connected to the first NLS upstream and the coding sequence of the transcription activator downstream by a connector.
  • RNA aptamers for a target protein comprising: one or more plasmid vectors, the plasmid vector comprising a nucleic acid sequence selected from the guide RNA; the targeting sequence and the selection marker; a nucleic acid sequence encoding the fusion protein; and one or more of the nucleic acid sequences encoding the dCas protein, wherein the guide RNA contains a recognition sequence and a random library of nucleic acid aptamers of a predetermined length, the targeting sequence is paired with the recognition sequence of the guide RNA, the selection marker is located downstream of the targeting sequence and includes a basic promoter and a selection marker gene, the fusion protein comprises the target protein and a transcriptional activation element, the target protein binds to the RNA nucleic acid aptamer in the random library of nucleic acid aptamers, resulting in the binding of the transcriptional activation element to the selection marker, and the dCas protein specifically recognizes the guide RNA contains a recognition sequence and a random
  • the system comprises a plasmid vector comprising the guide RNA, the targeting sequence, the selection marker, and a nucleic acid sequence encoding the fusion protein and the dCas protein.
  • the screening cell is a prokaryotic cell.
  • Another aspect of the present invention provides a method for screening RNA aptamers for a target protein, which comprises the following steps: (1) providing screening cells, wherein the screening cells have a guide RNA, a targeting sequence, a selection marker, a fusion protein and a dCas protein; wherein the guide RNA contains a recognition sequence and a random library of aptamers of a predetermined length; the targeting sequence is paired with the recognition sequence of the guide RNA; the selection marker is located downstream of the targeting sequence and includes a basic promoter and a selection marker gene; the fusion protein comprises the target protein and a transcriptional activation element, and the target protein binds to the RNA aptamer in the random library of aptamers, resulting in the binding of the transcriptional activation element to the selection marker; the dCas protein specifically recognizes the targeting sequence under the guidance of the guide RNA; (2) screening the screening cells using the selection marker gene; (3) collecting the screening cells expressing the selection marker gene; (4) lysing the screening cells,
  • step (4) further comprises analyzing and verifying the obtained nucleic acid aptamer sequence.
  • the screening cells are obtained by transfecting one or more plasmid vectors into prokaryotic cells, and the plasmid vector comprises one or more of the nucleic acid sequences selected from the guide RNA; the targeting sequence and the selection marker; the nucleic acid sequence encoding the fusion protein; and the nucleic acid sequence encoding the dCas protein.
  • the prokaryotic cell is an Escherichia coli cell.
  • the screening cells are provided by the following specific steps: (1.1) providing a cell expressing the dcas protein, the cell also comprising the targeting sequence and the selection marker; (1.2) providing a viral expression vector encoding the guide RNA and the fusion protein; (1.3) packaging the viral expression vector; and then using the packaged viral expression vector to infect the cell.
  • the viral expression vector is a lentiviral expression vector.
  • RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus, wherein the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, and the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:7.
  • Another aspect of the present invention provides a detection reagent or kit for detecting SARS-CoV-2 virus, which comprises an RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus, wherein the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, and the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:7.
  • Another aspect of the present invention provides a method for detecting SARS-CoV-2 virus, the method comprising contacting a sample to be detected with an RNA nucleic acid aptamer of the present invention that specifically binds to the S1 protein of the SARS-CoV-2 virus, the RNA nucleic acid aptamer sequence comprising a random region in the middle and fixed sequences at both ends, the fixed sequences at both ends being complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:7.
  • RNA nucleic acid aptamer of the present invention that specifically binds to the S1 protein of the SARS-CoV-2 virus in the preparation of a detection reagent or kit for detecting the SARS-CoV-2 virus, wherein the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, and the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:7.
  • RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus
  • the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, and the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 7.
  • the drug further comprises a viral vector or a non-viral vector, wherein the vector comprises or carries the RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus.
  • Another aspect of the present invention provides a method of neutralizing a SARS-CoV-2 virus in a subject, the method comprising An effective amount of the drug of the present invention is administered to a subject in need thereof, wherein the drug comprises an RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus, and the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, and the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 7.
  • the drug further comprises a viral vector or a non-viral vector, wherein the vector comprises or carries the RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus.
  • Another aspect of the present invention provides the use of the drug of the present invention in the preparation of a drug for neutralizing the SARS-CoV-2 virus in a subject, wherein the drug comprises an RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus, and the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, and the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 7.
  • the drug further comprises a viral vector or a non-viral vector, wherein the vector comprises or carries the RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus.
  • Another aspect of the present invention provides a method for treating or preventing SARS-CoV-2 virus infection, the method comprising administering an effective amount of the drug of the present invention to a subject in need thereof, wherein the drug comprises an RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus, the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 7.
  • the drug further comprises a viral vector or a non-viral vector, wherein the vector comprises or carries the RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus.
  • Another aspect of the present invention provides the use of the drug of the present invention in the preparation of a drug for treating or preventing SARS-CoV-2 virus infection in a subject, wherein the drug comprises an RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus, and the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, and the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 7.
  • the drug further comprises a viral vector or a non-viral vector, wherein the vector comprises or carries the RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus.
  • Another aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the drug of the present invention and a pharmaceutically acceptable excipient, wherein the drug comprises an RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus, and the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, and the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 7.
  • the drug further comprises a viral vector or a non-viral vector, wherein the vector comprises or carries the specific RNA aptamers that bind to the S1 protein of the SARS-CoV-2 virus.
  • Another aspect of the present invention provides a method for neutralizing SARS-CoV-2 virus in a subject, the method comprising administering an effective amount of the pharmaceutical composition of the present invention to a subject in need thereof, wherein the pharmaceutical composition comprises the drug of the present invention and a pharmaceutically acceptable excipient, the drug comprises an RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus, the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 7.
  • the drug further comprises a viral vector or a non-viral vector, wherein the vector comprises or carries the RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus.
  • the pharmaceutical composition of the present invention provides the use of the pharmaceutical composition of the present invention in the preparation of a drug for neutralizing the SARS-CoV-2 virus
  • the pharmaceutical composition comprises the drug of the present invention and a pharmaceutically acceptable excipient
  • the drug comprises an RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus
  • the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 7.
  • the drug further comprises a viral vector or a non-viral vector, wherein the vector comprises or carries the RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus.
  • Another aspect of the present invention provides a method for treating or preventing SARS-CoV-2 virus infection in a subject, the method comprising administering an effective amount of the pharmaceutical composition of the present invention to a subject in need thereof, wherein the pharmaceutical composition comprises the drug of the present invention and a pharmaceutically acceptable excipient, the drug comprises an RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus, the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 7.
  • the drug further comprises a viral vector or a non-viral vector, wherein the vector comprises or carries the RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus.
  • the pharmaceutical composition of the present invention provides the use of the pharmaceutical composition of the present invention in the preparation of a drug for treating or preventing SARS-CoV-2 virus infection in a subject
  • the pharmaceutical composition comprises the drug of the present invention and a pharmaceutically acceptable excipient
  • the drug comprises an RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus
  • the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 7.
  • the drug further comprises a viral vector or a non-viral vector, wherein the vector comprises or carries the RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus.
  • Another aspect of the present invention provides a pharmaceutical kit for neutralizing SARS-CoV-2 virus in a subject, wherein the pharmaceutical kit
  • the package contains two or more drugs or pharmaceutical compositions of the present invention that exist independently.
  • Another aspect of the present invention provides a pharmaceutical kit for treating or preventing SARS-CoV-2 virus infection in a subject, wherein the pharmaceutical kit comprises two or more independently existing drugs or pharmaceutical compositions of the present invention.
  • FIG. 1 CRISPR/Cas9-based aptamer screening system.
  • A Schematic diagram of the molecular device of the RNA aptamer screening system.
  • B The process of RNA aptamer screening system for RNA aptamer screening. The aptamer is marked in red; the target protein of interest (X-protein) is marked in orange; the transcriptional activation domain is marked in green; the mini-promoter is marked in blue, and the selection marker gene is marked in pink.
  • Figure 2 Validation of the aptamer screening system.
  • GFP aptamer was inserted into the tetra loop region (sgRNA 1.1) or stem loop 2 region (sgRNA 1.2) of the sgRNA backbone.
  • B Schematic diagram of the luciferase reporter system used to detect the affinity of the GFP target protein and its RNA aptamer.
  • C Based on the luciferase reporter system in B, 1 copy of the gLuc sgRNA target sequence was used to detect the sensitivity of the system (1 ⁇ gLuc).
  • D Based on the luciferase reporter system in B, 9 copies of the gLuc sgRNA target sequence were used to detect the sensitivity of the system (9 ⁇ gLuc).
  • Mock represents the transient transfection plasmid control
  • UR represents the random sequence control
  • NC represents the negative experimental group that delivered the sgRNA 1.2-random aptamer sequence and GFP-VPH lentiviral component
  • PC represents the positive experimental group that delivered the sgRNA 1.2-GFP aptamer sequence and GFP-VPH lentiviral component
  • data are the mean ⁇ standard deviation of three biological replicates. * represents P ⁇ 0.05, ** represents P ⁇ 0.01, *** represents P ⁇ 0.001, two-tailed t test.
  • Figure 3 Schematic diagram of the puromycin reporter system used to detect the affinity between GFP target protein and GFP RNA nucleic acid aptamer and the screening and identification process of dCas9/9 ⁇ gLuc-puro monoclonal cell line.
  • Figure 4 Characterization of the performance of the dCas9/9 ⁇ gLuc-puro monoclonal cell line by quantifying the number of surviving colonies. The three different concentrations of puromycin used are shown in the figure. NC indicates negative control; PC indicates positive control. Data are mean ⁇ SD of three biological replicates.
  • FIG. 5 Schematic diagram of the CRISmers RNA nucleic acid aptamer screening system based on CRISPR/Cas9.
  • Figure 6 Sequence analysis results of deep sequencing after CRISmers screening for the RBD region of the wild-type spike protein of SARS-CoV-2.
  • R number of screening rounds
  • Filtered sequences sequencing sequences after filtering out some erroneous sequences
  • Unique sequences single/unique sequences.
  • Figure 7 Secondary screening results based on the luciferase reporter system. Second, third, fourth and fifth rounds The sequences obtained in the screening were deeply sequenced and analyzed, and the top 0.1% high-frequency sequences and the top 15 high-enrichment index sequences were obtained for secondary screening using the luciferase reporter system. A total of 2 independent CRISmers screenings were performed in the experiment (results A and B, respectively). Data are the mean ⁇ standard deviation of three biological replicates.
  • Figure 8 Active sequences obtained by screening the RBD region of the wild-type spike protein of SARS-CoV-2 using the CRISmers system.
  • A The results of parallel comparison of the highly activated sequences in Figure 7 after secondary screening by the luciferase reporter system.
  • B The two aptamer sequences #2-1-18 and #5-2-15 with the highest activation multiples in the luciferase parallel comparison experiment ( Figure 7A).
  • C The secondary structures and free energies of these two aptamers were analyzed using the Mfold webserver software. Data are the mean ⁇ SD of three biological replicates.
  • Figure 9 Detection ability of nucleic acid aptamers for SARS-CoV-2.
  • A Schematic diagram of the enzyme-linked oligonucleotide assay method.
  • B The dose-dependent activity of nucleic acid aptamers was tested at a fixed concentration of 250ng of SARS-CoV-2 wild-type spike protein RBD. Scrambled RNA was used as a negative control.
  • C, G The detection limit of wild-type SARS-CoV-2 spike protein RBD protein was tested using 100nM nucleic acid aptamers, and recombinant PD-1 protein and SARS-CoV-2 NSP7-8 complex protein were used as negative controls to test the detection specificity of nucleic acid aptamers.
  • D, H The detection limit of SARS-CoV-2 mutant spike protein Delta RBD protein was tested using 100nM nucleic acid aptamers, and recombinant PD-1 protein and SARS-CoV-2 NSP7-8 complex protein were used as negative controls to test the detection specificity of nucleic acid aptamers.
  • E, I The detection limit of the mutant spike protein Omicron BA.1 RBD protein of SARS-CoV-2 was tested using 100nM nucleic acid aptamers.
  • Recombinant PD-1 protein and SARS-CoV-2 NSP7-8 complex protein were used as negative controls to detect the detection specificity of nucleic acid aptamers.
  • TCID50 half tissue culture infectious dose. Data are mean ⁇ SD of three biological replicates. * represents P ⁇ 0.05, ** represents P ⁇ 0.01, *** represents P ⁇ 0.001, **** represents P ⁇ 0.0001, two-tailed t-test, n.s. represents not significant.
  • FIG. 10 Effect of fixed sequences at both ends on the activity of RNA aptamers (ELONA).
  • ELONA was used to detect the binding activity of the intermediate sequences #Core-2-1-18 and #Core-5-2-15 obtained after screening and the complete sequences #2-1-18 and #5-2-15 combined with fixed sequences at both ends against RBD protein.
  • Scrambled RNA was used as a negative control. Data are from the mean ⁇ standard deviation of three biological replicates. ** represents P ⁇ 0.01, **** represents P ⁇ 0.0001, two-tailed t test, ns represents no significant difference.
  • Figure 11 Binding activity assay of nucleic acid aptamers at different temperatures. ELONA was used to detect the binding activity of nucleic acid aptamers to RBD protein at 4°C, 25°C and 37°C. Scrambled RNA was used as a negative control. Data are the mean ⁇ SD of three biological replicates. *** represents P ⁇ 0.001, **** represents P ⁇ 0.0001, two-tailed t-test.
  • Figure 12 Neutralization activity of aptamer (#5-2-15) against SARS-CoV-2 variant live virus beads.
  • A Schematic diagram of the neutralization experiment against SARS-CoV-2 live virus. Quantification results of relative viral RNA copy number of nucleic acid aptamer #5-2-15 neutralizing SARS-CoV-2 Delta (B) and Omicron BA.1 (C) variant virus beads 2 days after virus infection. Quantification results of relative viral RNA copy number of nucleic acid aptamer #5-2-15 neutralizing SARS-CoV-2 Delta (D) and Omicron BA.1 (E) variant virus beads 4 days after virus infection. Negative control readings were used for 100% normalization calculation. Experimental samples were cell supernatants collected 2 days and 4 days after virus infection. IC50 is marked in the figure. Data are the mean ⁇ SD of three biological replicates.
  • Figure 13 Neutralization activity test results of nucleic acid aptamer (#2-1-18) against SARS-CoV-2 variant live virus beads Omicron BA.1. Cell culture supernatants were collected 2 days and 4 days after virus infection, respectively. Quantification results of relative viral RNA copy number 2 days (A) and 4 days (B) after nucleic acid aptamer neutralized SARS-CoV-2 Omicron BA.1 variant virus beads. Negative control readings were used for 100% normalization calculation. The experimental samples were cell supernatants collected 2 days after virus infection. IC50 is marked in the figure. Data are the mean ⁇ standard deviation of three biological replicates.
  • Figure 14 Binding activity and specificity verification of modified aptamers.
  • A, B The binding activity of unmodified and modified aptamers to the recombinant protein of the spike protein RBD of the SARS-CoV-2 variant virus strain Omicron BA.2 was verified by the ELONA method. The concentrations of aptamers were 100nM (A) and 1000nM (B), respectively.
  • Biotin-apt biotinylated nucleic acid aptamer
  • Cold-apt unmodified nucleic acid aptamer.
  • Data are the mean ⁇ standard deviation of three biological replicates. **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001, two-tailed t-test, n.s., no significant difference.
  • Figure 15 Fluorinated methoxy-modified nucleic acid aptamer (#2-1-18-2'-FO) against SARS-CoV-2 variants Neutralization activity test results of virus beads Omicron BA.1. Quantification of relative viral RNA copy number 2 days (A) and 4 days (B) after nucleic acid aptamers neutralized SARS-CoV-2 Omicron BA.1 variant virus beads. Negative control readings were used for 100% normalization calculation. Experimental samples were cell supernatants collected 2 days and 4 days after virus infection. IC50 is marked in the figure. Data are the mean ⁇ SD of three biological replicates.
  • Figure 16 Neutralization activity test results of fluorinated methoxy-modified nucleic acid aptamer (#5-2-15-2’-F-O) against SARS-CoV-2 variant live virus beads Omicron BA.1. Quantification results of relative viral RNA copy number 2 days (A) and 4 days (B) after nucleic acid aptamer neutralized SARS-CoV-2 Omicron BA.1 variant virus beads. Negative control readings were used for 100% normalization calculation. Experimental samples were cell supernatants collected 2 days and 4 days after virus infection. IC50 is marked in the figure. Data are the mean ⁇ SD of three biological replicates.
  • Figure 17 Neutralization activity test results of fluorinated methoxy-modified nucleic acid aptamers (#2-1-18-2’-F-O and #5-2-15-2’-F-O) against SARS-CoV-2 variant live virus beads Omicron BA.2. Quantification results of relative viral RNA copy number after #2-1-18-2’-F-O (A) and #5-2-15-2’-F-O (B) nucleic acid aptamers neutralized SARS-CoV-2 Omicron BA.2 variant virus beads. Negative control readings were used for 100% normalization calculation. The experimental samples were cell supernatants collected 2 days after virus infection. IC50 is marked in the figure. Data are the mean ⁇ SD of three biological replicates.
  • Figure 18 Neutralization activity test results of fluorinated methoxy-modified aptamers (PEG40K-2-1-18-2’-F-O and PEG40K-5-2-15-2’-F-O) with 40kDa PEG attached to the 5’ end against SARS-CoV-2 variant live virus beads Omicron BA.2. Quantification results of relative viral RNA copy number after neutralization of SARS-CoV-2 Omicron BA.2 variant virus beads by PEG40K-2-1-18-2’-F-O (A) and PEG40K-5-2-15-2’-F-O (B) aptamers. Negative control readings were used for 100% normalization calculation. Experimental samples were cell supernatants collected 2 days after virus infection. IC50 is marked in the figure. Data are the mean ⁇ SD of three biological replicates.
  • Figure 19 Neutralization activity test results of fluorinated methoxy-modified nucleic acid aptamer (chol-PEG6-5-2-15-2’-F-O) with cholesterol-PEG6 attached to the 5’ end against SARS-CoV-2 variant live virus beads Omicron BA.2. Quantification results of relative viral RNA copy number after chol-PEG6-5-2-15-2’-F-O nucleic acid aptamer neutralized SARS-CoV-2 Omicron BA.2 variant virus beads. Negative control readings were used for 100% normalization calculation. The experimental samples were cell supernatants collected 2 days after virus infection. IC50 is marked in the figure. Data are the mean ⁇ SD of three biological replicates.
  • Figure 20 Neutralization activity test results of fluorinated methoxy-modified nucleic acid aptamer (chol-PEG24-5-2-15-2'-FO) with cholesterol-PEG24 attached to the 5' end on SARS-CoV-2 variant live virus beads Omicron BA.2. Quantification results of relative viral RNA copy number after chol-PEG24-5-2-15-2'-FO nucleic acid aptamer neutralized SARS-CoV-2 Omicron BA.2 variant virus beads. Negative control readings were used for 100% normalization calculation. The experimental samples were cell supernatants collected 2 days after virus infection. IC50 is marked in the figure. Data are the mean ⁇ SD of three biological replicates.
  • Figure 21 Neutralization activity test results of fluorinated methoxy-modified nucleic acid aptamer (chol-PEG40K-5-2-15-2’-F-O) with cholesterol-40kDa PEG attached to the 5’ end against SARS-CoV-2 variant live virus beads Omicron BA.2. Quantification results of relative viral RNA copy number after chol-PEG40K-5-2-15-2’-F-O nucleic acid aptamer neutralized SARS-CoV-2 Omicron BA.2 variant virus beads. Negative control readings were used for 100% normalization calculation. The experimental samples were cell supernatants collected 2 days after virus infection. IC50 is marked in the figure. Data are the mean ⁇ SD of three biological replicates.
  • Figure 22 Results of the neutralization activity test of cholesterol on SARS-CoV-2 variant live virus beads Omicron BA.2. Quantification of relative viral RNA copy number after cholesterol acted on SARS-CoV-2 Omicron BA.2 variant virus beads. Negative control readings were used for 100% normalization calculation. The experimental samples were cell supernatants collected 2 days after virus infection. IC50 is marked in the figure. Data are the mean ⁇ SD of three biological replicates.
  • Figure 23 Neutralization activity test results of 40kDa PEG on SARS-CoV-2 variant live virus beads Omicron BA.2. Quantification results of relative viral RNA copy number after 40kDa PEG acted on SARS-CoV-2 Omicron BA.2 variant virus beads. Negative control readings were used for 100% normalization calculation. The experimental samples were cell supernatants collected 2 days after virus infection. IC50 is marked in the figure. Data are the mean ⁇ SD of three biological replicates.
  • Figure 24 Neutralization activity test results of cholesterol-40kDa PEG on SARS-CoV-2 variant live virus beads Omicron BA.2. Quantification results of relative viral RNA copy number after cholesterol-40kDa PEG acted on SARS-CoV-2 Omicron BA.2 variant virus beads. Negative control readings were used for 100% normalization calculation. The experimental samples were cell supernatants collected 2 days after virus infection. IC50 is marked in the figure. Data are the mean ⁇ SD of three biological replicates.
  • Figure 25 Animal validation experiments on Chol-PEG40K-5-2-15-2’-F-O.
  • A Schematic diagram of animal experiments to test the preventive and therapeutic effects of Chol-PEG40K-5-2-15-2’-F-O against the live virus variant Omicron BA.2 of SARS-CoV-2 in vivo.
  • B RT-qPCR experiment to detect the relative copy number of Omicron BA.2 viral RNA in lung tissue in the preventive experiment.
  • C FRNT experiment to detect the titer of Omicron BA.2 viral RNA in lung tissue in the preventive experiment.
  • D RT-qPCR experiment to detect the relative copy number of Omicron BA.2 viral RNA in lung tissue in the therapeutic effect experiment.
  • Figure 26 Animal validation experiments on Chol-PEG6-5-2-15-2'-FO.
  • A RT-qPCR experiment to detect the relative copy number of Omicron BA.2 viral RNA in lung tissue in the prevention experiment.
  • B FRNT experiment to detect the titer of Omicron BA.2 viral RNA in lung tissue in the prevention experiment.
  • C RT-qPCR experiment to detect the relative copy number of Omicron BA.2 viral RNA in lung tissue in the treatment effect experiment.
  • D FRNT experiment to detect the titer of Omicron BA.2 viral RNA in lung tissue in the treatment effect experiment.
  • FRNT live virus foci reduction neutralization assay. Negative control readings were used for 100% normalization. Data are mean ⁇ SD of three biological replicates. *P ⁇ 0.05, **P ⁇ 0.01, *** represents P ⁇ 0.001, ns, no significant difference, two-tailed t test.
  • FIG. 27 Validation of the CRISmers system in different CRISPR/Cas systems.
  • the aptamer sequence was inserted into the loop2 position of the sgRNA backbone of the dCasMINI-V4 system.
  • the firefly luciferase reporter experiment was used to detect the activation effect (B).
  • Scrambled RNA nucleic acid aptamers were used as negative controls. Data are the mean ⁇ SD of three biological replicates. *** represents P ⁇ 0.001, **** represents P ⁇ 0.0001, two-tailed t test.
  • Figure 28 Verification of the effect of using different selection markers in the CRISmers system.
  • the affinity binding ability of the aptamer to the target protein will be converted into the expression ability of green fluorescent protein.
  • cell empty cell group
  • NC negative control group
  • GFP% percentage of GFP-positive cells.
  • Figure 29 Validation of the effect of the CRISmers system in different host cells. Schematic diagram of replacing the original puromycin reporter gene in the CRISmers system with the E. coli kanamycin reporter gene and transforming it into E. coli to detect the activation experiment (A). The effect of the application of the CRISmers system in the E. coli host (B).
  • Figure 30 Schematic diagram of the customizable lentiviral vector (phU6-gLuc sgRNA-1.2 BsmBI-BsmBI-polyT-EF1a-NLS-linker-EcoRI-EcoRI-linker-VP64-P65-HSF1-NLS-linker-P2A-linker-Zeo-WPRE) for screening of eukaryotic HEK293T-dCas9 cell lines.
  • phU6-gLuc sgRNA-1.2 BsmBI-BsmBI-polyT-EF1a-NLS-linker-EcoRI-EcoRI-linker-VP64-P65-HSF1-NLS-linker-P2A-linker-Zeo-WPRE for screening of eukaryotic HEK293T-dCas9 cell lines.
  • Figure 31 Schematic diagram of the customizable plasmid vector for prokaryotic Escherichia coli screening (phU6-gLuc sgRNA-1.2BsmBI-BsmBI-polyT-EF1a-NLS-linker-EcoRI-X-protein-EcoRI-linker-VP64-P65-HSF1-NLS-linker-P2A-dCas9-WPRE-SV40promoter-Zeo).
  • Subject or “patient”, “individual” refers to any object for which diagnosis, prognosis or treatment is desired, particularly a mammalian subject. Mammals include humans, domestic animals, farm animals, zoo animals, competitive animals or pets, such as dogs, cats, pigs, rabbits, rats, mice, horses, cattle, cows, etc.
  • the subject referred to herein is preferably a human.
  • treatment refers to therapeutic and preventive measures that prevent or slow down the occurrence of undesirable physiological changes or symptoms in a subject, such as upper respiratory and digestive tract symptoms such as viral infection, fever, dry cough, and fatigue.
  • beneficial or desired clinical effects include, but are not limited to, reduction or elimination of viruses, alleviation of symptoms, reduction of disease severity, stabilization of the disease state (i.e., no deterioration), delay or slowing of disease progression, alleviation or alleviation of the disease state, and partial or complete cure of the disease, regardless of whether the above effects are detectable.
  • Treatment may also refer to prolonged survival compared to no treatment.
  • Subjects in need of treatment include subjects who already have the disease or condition, as well as subjects who are likely to have the disease or condition, or subjects who want to prevent the disease or condition.
  • the term "patient in need of treatment” or “subject in need of treatment” includes subjects, such as mammalian subjects, who would benefit from the administration of a polypeptide or composition thereof of the present invention for, for example, detection, diagnosis and/or therapeutic use.
  • the term "therapeutically effective amount” or “effective amount” refers to an amount of the drug or pharmaceutical composition of the present invention that is effective in preventing or alleviating the disease or condition to be treated when it is administered alone or in combination with another therapeutic agent to a cell, tissue or subject.
  • a therapeutically effective dose further refers to an amount of the compound sufficient to cause a reduction in symptoms, such as treatment, cure, prevention or alleviation of a related medical condition, or to increase the rate of treatment, cure, prevention or alleviation of the symptoms.
  • the therapeutically effective amount refers to that individual ingredient.
  • the therapeutically effective amount refers to the combined amount of active ingredients that produces a therapeutic effect, regardless of whether it is administered in combination, sequentially or simultaneously.
  • a therapeutically effective amount will alleviate symptoms typically by at least 10%; typically by at least 20%; preferably by at least about 30%; more preferably by at least 40% and most preferably by at least 50%.
  • “about” means that the value is within the acceptable error range of the specific value determined by a person of ordinary skill in the art, and the value depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, “about” can mean within 1 or more than 1 standard deviation in each practice in the art. Alternatively, “about” or “substantially including” can mean a range of up to 20%. In addition, for biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. Unless otherwise stated, when a specific value appears in the application and claims, the meaning of "about” or “substantially including” should be assumed to be within the acceptable error range of the specific value.
  • complementary refers to the ability of accurate pairing between two core bases of an oligomeric compound.
  • the core base at a certain position of an oligonucleotide oligomeric compound
  • the target nucleic acid is a DNA, RNA or oligonucleotide molecule
  • the position of hydrogen bond between the oligonucleotide and the target nucleic acid is considered to be a complementary position.
  • oligonucleotide and other DNA, RNA or oligonucleotide molecules complement each other. Therefore, "complementary" is used to represent a sufficient number of core bases of sufficient degree of accurate pairing or complementarity, so that a term of stable and specific combination between an oligonucleotide and a target nucleic acid.
  • the fixed sequences at both ends of the nucleic acid aptamer in the art do not need 100% complementarity to specifically hybridize to form a "stem" structure.
  • oligonucleotides can hybridize on one or more sections so that insertion or adjacent sections do not participate in hybridization events (such as loop structures or hairpin structures).
  • the fixed sequences at both ends of the nucleic acid aptamer of the present invention have at least 70%, or at least 75%, or at least 80%, or at least 85% sequence complementarity, and more preferably they contain at least 90% sequence complementarity, and even more preferably contain at least 95% or at least 99% sequence complementarity.
  • 18 of the 20 core bases of the 5' end fixed sequence are complementary to the 3' end fixed sequence and will represent 90% complementarity.
  • the remaining non-complementary core bases can be clustered or interspersed with complementary core bases, and do not need to be adjacent to each other or adjacent to complementary core bases.
  • an antisense oligomer of 18 nucleobases in length has 4 non-complementary nucleobases flanked by two regions of complete complementarity to the target nucleic acid, has an overall complementarity of 77.8% to the target nucleic acid, and therefore falls within the scope of the present invention.
  • the complementary percentage of the 5'-end fixed sequence to the 3'-end fixed sequence can be routinely determined using BLAST programs (basic local alignment search tool) and PowerBLAST programs known in the art.
  • gLuc sequence and Tet sequence are exogenous artificial sequences that do not belong to the human genome, which have good targeting capabilities and can avoid unnecessary side effects caused by targeting endogenous genomic sites in cells.
  • linker refers to a short segment of a nucleic acid sequence containing two or more identical or different nucleotides, wherein the nucleotides are selected from adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U).
  • promoter is a DNA sequence that RNA polymerase recognizes, binds to, and initiates transcription. It contains conserved sequences required for specific binding of RNA polymerase and transcription initiation, most of which are located upstream of the transcription start point of the structural gene, and the promoter itself is not transcribed.
  • Basic promoter (basal promoter, minimal promoter) is a promoter consisting only of all the basic elements required for transcription initiation (e.g., TATA-box and/or start codon). In the presence of appropriate transcription factors, the basal promoter functions to transcribe.
  • the first promoter is selected from U6, 7SK, H1.
  • the second promoter is EF1a.
  • the selection marker comprises a basic promoter selected from mini-promoter-1 (SEQ ID NO:28), mini-promoter-2 (SEQ ID NO:27), mini-TK promoter (SEQ ID NO:29), mini-CMV promoter (SEQ ID NO:30) and Crystallin basal promoter (SEQ ID NO:31).
  • the CRISPR/Cas system is derived from the adaptive immune system of bacteria and archaea, which protect themselves from invasion by foreign viruses and plasmid nucleic acids.
  • the CRISPR sequence can transcribe and process non-coding RNA, namely CRISPR RNA, crRNA, which can directly act on DNA instead of RNA.
  • Guide RNA gRNA
  • sgRNA small guide RNA
  • It acts in a post-transcriptional modification process called RNA editing in kinetoplastids. It is also a small non-coding RNA.
  • CRISPR-associated protein is a nuclease that can use the RNA corresponding to the spacer in the CRISPR sequence to guide, identify and cut a specific DNA chain complementary to its sequence.
  • Deactivated Cas refers to a Cas protein that loses nuclease activity through point mutations but retains binding enzyme activity.
  • the dCas protein can be fused with a transcription repression domain (TRD) or a transcription activation domain (TAD), and the dCas protein can be fused with effector proteins, including transcription activators, repressors, and epigenetic regulators, respectively, to achieve effective gene-specific CRISPR-mediated activation (CRISPRa), interference (CRISPRi), and epigenomic modification.
  • CRISPRa CRISPR-mediated activation
  • CRISPRi interference
  • epigenomic modification e.g., the dCas protein is dCas9.
  • the dCas9 protein is dCasMINI-V4.
  • the CRISPR/Cas9 system is guided by crRNAs and causes double-strand breaks in DNA. There are some small RNAs tracrRNAs near the CRISPR sequence, which complement and bind to the repetitive sequence. In the presence of the Cas9 factor, they are recognized and sheared by RNase III to produce mature crRNA. In 2012, Jennifer et al. connected tracrRNA and crRNA into a single-stranded guide RNA chimera, and used the Streptococcus pyogenes CRISPR/Cas system (spCas9 system) to achieve the first in vitro double-stranded cutting of the targeted DNA by the guide RNA-guided Cas9 protein.
  • spCas9 system Streptococcus pyogenes CRISPR/Cas system
  • the CRISPR/Cas9 system specifically binds to the targeted DNA sequence through base complementary pairing of sgRNA, guiding the Cas9 protein with nuclease cutting activity to recognize and act on the target sequence containing the 3' end PAM sequence (NGG) complementary to the sgRNA and achieve specific double-strand breaks. Subsequently, the endogenous DNA repair mechanism of the cell is activated, which can achieve non-homologous end joining and homology-mediated DNA double-strand repair, thereby generating corresponding gene editing events.
  • the nuclease domain of Cas9 (for example, H840A mutation in HNH domain and D10A mutation in RuvC domain) is mutated to generate nuclease-deficient "dCas9".
  • This "blunted” and “dead” Cas9 loses the function of cutting DNA, but can still target and bind DNA with the same precision under the guidance of gRNA.
  • dCas9 can recruit effectors (repressor and activator domains) to promoter regions, regulatory regions or coding regions to precisely regulate any gene without causing DNA damage.
  • CasMINI is a variant version of Un1Cas12f1, which is smaller than Cas9 and belongs to a new generation of smaller gene editing systems.
  • dCasMINI-V4 refers to the 4th version of dCasMINI, which only has the function of targeting and binding to the target gene, but no cutting function.
  • SARS-CoV-2 novel coronavirus
  • Delta and Omicron are particularly noteworthy due to their extremely high infectiousness.
  • the new coronavirus SARS-CoV-2 belongs to the ⁇ species of the Coronaviridae family and is an enveloped, single-stranded positive-sense RNA virus.
  • the SARS-CoV-2 genome contains a 5' cap and a 3' PolyA tail structure, encoding 29 proteins, including 25 non-structural proteins and auxiliary proteins and 4 structural proteins.
  • Non-structural proteins play a key role in viral RNA replication and immune evasion, while auxiliary proteins perform multiple functions such as assisting viral infection, survival, and propagation in host cells; structural proteins are responsible for viral assembly and the formation of mature viral particles.
  • the replicase gene which is about two-thirds of the length of the SARS-CoV-2 genome near the 5' end, encodes two open reading frames (ORF), ORF1a and ORF1b.
  • ORF open reading frames
  • ORF1a ORF1a
  • ORF1b ORF1b
  • the gene in the first third of its length encodes several ORFs, of which four encode coronavirus structural proteins, namely spike glycoprotein (S), membrane protein (M), envelope protein (E) and nucleocapsid protein (N), and the other ORFs encode several auxiliary proteins.
  • the SARS-CoV-2 virus S protein belongs to the type I viral fusion protein, which contains two functional subunits S1 and S2.
  • the coronavirus enters the host cell mediated by the S protein.
  • the S1 subunit is responsible for binding to the host cell receptor, and the S2 subunit is responsible for the fusion of the viral membrane and the cell membrane.
  • the receptor binding domain (RBD) of the S1 subunit is responsible for binding to the angiotensin converting enzyme 2 (ACE2) receptor of the host cell.
  • ACE2 angiotensin converting enzyme 2
  • the S1 protein or the RBD region of the S1 protein is often used as a therapeutic target for blocking the binding of the virus to the host receptor hACE2, and is an important target for the prevention and treatment of COVID-19 vaccines, neutralizing antibody research and development, and SARS-COV-2 clinical diagnosis.
  • the nucleic acid aptamer screened by the RNA nucleic acid aptamer screening system can effectively and sensitively detect the S protein or the RBD region of the S protein, and can effectively block the binding of RBD to hACE2.
  • Nucleic acid aptamers are a class of short, single-stranded DNA (ssDNA) or RNA oligonucleotide molecules that fold to form complex and unique three-dimensional structures, binding to target protein molecules with high specificity and good affinity. Compared with ssDNA nucleic acid aptamers, RNA nucleic acid aptamers are more flexible because they can form complex spatial structures.
  • Aptamers usually have certain specific three-dimensional structures due to their tendency to form complementary base pairs. They can fold to form different secondary structures such as stem, loop, bulge, pseudoknot, G-quadruplex, kissing hairpin, etc. The collection of these secondary structures can then form a unique three-dimensional spatial conformation, thereby specifically recognizing the relevant target molecule.
  • the interactions between these three-dimensional spatial conformations such as hydrophobic and electrostatic interactions, hydrogen bonds, van der Waals forces, structural complementarity, and base stacking, are crucial to the binding affinity and specific recognition of aptamers. Similar to the antibody-antigen binding mode, the interactions between specific three-dimensional structures It can drive the formation of nucleic acid aptamer-target molecule complex.
  • an RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus wherein the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, and the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:7.
  • the random region comprises a nucleic acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:7.
  • the SARS-CoV-2 virus is a SARS-CoV-2 wild type, Delta or Omicron BA.1 or BA.2 virus strain. Based on its broad-spectrum activity, it is speculated that the RNA nucleic acid aptamer of the present invention against the S1 protein of the SARS-CoV-2 virus is also applicable to SARS-CoV-2 variants that appear thereafter and in the future.
  • the fixed sequence at the 5' end of the RNA aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:8.
  • the fixed sequence at the 3' end of the RNA aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:9.
  • the fixed sequence at the 5' end of the RNA aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:8, and the fixed sequence at the 3' end of the RNA aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:9.
  • nucleic acid sequences having at least about 70%, or alternatively at least about 75%, or alternatively at least about 80%, or alternatively at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively at least about 97% sequence identity with any of the sequences shown in SEQ ID NO:1 to SEQ ID NO:9 are considered to be within the scope of the present invention.
  • nucleic acid aptamers can help improve the stability of nucleic acid molecules, extend their half-life, and reduce their immunogenicity.
  • RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus
  • the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, and the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 7, and the chemical modification is selected from one or more modifications of fluorine modification, methoxy modification, polyethylene glycol (PEG) polymer modification, and cholesterol-PEG polymer modification.
  • PEG polyethylene glycol
  • the PEG polymer is selected from a hexamer of small molecular weight to a polymer of 40kD high molecular weight.
  • the chemical modification is selected from one or more of fluorine modification, methoxy modification, cholesterol-PEG6 compound modification, cholesterol-PEG24 compound modification, PEG 40kDa compound modification, and cholesterol-PEG 40kDa compound modification.
  • the random region comprises a nucleic acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 7.
  • the fixed sequence at the 5' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 8.
  • the fixed sequence at the 3' end of the RNA aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 9.
  • the fixed sequence at the 5' end of the RNA aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 8
  • the fixed sequence at the 3' end of the RNA aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 9.
  • the SARS-CoV-2 virus is a SARS-CoV-2 wild type, Delta or Omicron BA.1 or BA.2 virus strain.
  • the chemical modification is a fluorine modification, wherein the hydroxyl group at the 2' position of the pyrimidine ribose of the chemically modified nucleic acid aptamer sequence is replaced by fluorine.
  • the chemical modification is methoxy modification, wherein the hydroxyl group at the 2' position of the purine ribose of the chemically modified nucleic acid aptamer sequence is replaced by a methoxy group.
  • the chemical modification is fluorine and methoxy modification, wherein the hydroxyl group at the 2' position of the pyrimidine ribose of the chemically modified nucleic acid aptamer sequence is replaced by fluorine, and the hydroxyl group at the 2' position of the purine ribose is replaced by a methoxy group.
  • the chemical modification is modification by fluorine, methoxy and cholesterol-PEG6 compound, wherein the hydroxyl group at the 2' position of the pyrimidine ribose of the chemically modified nucleic acid aptamer sequence is replaced by fluorine, the hydroxyl group at the 2' position of the purine ribose is replaced by methoxy, and the cholesterol-PEG6 compound is attached to the 5' end of the nucleic acid aptamer sequence.
  • the chemical modification is modification by fluorine, methoxy and cholesterol-PEG24 compounds, wherein the hydroxyl group at the 2' position of the pyrimidine ribose of the chemically modified nucleic acid aptamer sequence is replaced by fluorine, the hydroxyl group at the 2' position of the purine ribose is replaced by methoxy, and the cholesterol-PEG24 compound is attached to the 5' end of the nucleic acid aptamer sequence.
  • the chemical modification is modification by fluorine, methoxy and PEG 40kDa compound, wherein the hydroxyl group at the 2' position of the pyrimidine ribose of the chemically modified nucleic acid aptamer sequence is replaced by fluorine, the hydroxyl group at the 2' position of the purine ribose is replaced by methoxy, and the PEG 40kDa compound is attached to the 5' end of the nucleic acid aptamer sequence.
  • the chemical modification is modification by fluorine, methoxy and cholesterol-PEG 40kDa compound, wherein the hydroxyl group at the 2' position of the pyrimidine ribose of the chemically modified nucleic acid aptamer sequence is replaced by fluorine, the hydroxyl group at the 2' position of the purine ribose is replaced by methoxy, and the cholesterol-PEG 40kDa compound is attached to the 5' end of the nucleic acid aptamer sequence.
  • Homology refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. "Unrelated" or “non-homologous" sequences are not homologous to the present invention. One of the inventive sequences shares less than 40% identity, but preferably less than 25% identity.
  • a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" to another sequence, meaning that when aligned, that percentage of bases (or amino acids) are the same when comparing the two sequences.
  • a certain percentage e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%
  • sequence identity e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%
  • Viral vectors can carry genetic material into cells by using the molecular mechanism of viruses to transmit their genome into other cells for infection. Viral vectors can also be called vectors, vector virus particles or vector particles. Examples of viral vectors include, but are not limited to: retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, vaccinia viruses, baculoviruses or lentiviruses.
  • Retroviral vectors may be derived or capable of being derived from any suitable retrovirus.
  • retroviruses include, but are not limited to, murine leukemia virus (MLV), human T-cell leukemia virus (HTLV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelocytoma virus-29 (MC29), and avian erythrocytosis virus (AEV).
  • MMV murine leukemia virus
  • HTLV human T-cell leukemia virus
  • MMTV mouse mammary tumor virus
  • RSV Rous sarcoma virus
  • Fujinami sarcoma virus FuSV
  • Adenoviruses are double-stranded, linear DNA viruses that do not replicate via an RNA intermediate.
  • Adenoviruses are double-stranded, DNA, non-enveloped viruses that are able to transduce a wide range of cell types of human and non-human origin in vivo, ex vivo, and in vitro.
  • Adeno-associated virus also known as adeno-associated virus, belongs to the genus Dependoviridae of the family Parvoviridae. It is the simplest single-stranded DNA defective virus discovered so far. Recombinant AAV vectors have been successfully used for in vitro, ex vivo and in vivo transduction of marker genes and genes involved in human diseases. Certain AAV vectors have been developed that can effectively bind large payloads (up to 8-9kb).
  • Herpes simplex virus is an enveloped, double-stranded DNA virus that naturally infects neurons. It can accommodate large segments of exogenous DNA and has been adopted as a vector for gene delivery to neurons. The use of HSV in therapeutic procedures requires that the strains be attenuated so that they cannot establish a lytic cycle.
  • the viral vector of the present invention can be a vaccinia virus vector, such as MVA or NYVAC. It should be understood that portions of the viral genome can remain intact after insertion of the recombinant gene. This means that the viral vector can retain the concept of the ability to infect cells and subsequently express additional genes that support its replication and may promote the lysis and death of infected cells.
  • Lentiviruses are part of a larger group of retroviruses. They can be divided into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV), the etiological agent of human autoimmune deficiency syndrome (AIDS), and simian immunodeficiency virus (SIV).
  • the non-primate lentivirus group includes the prototype "lentivirus" visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), and bovine immunodeficiency virus (BIV).
  • Non-viral vectors utilize the physicochemical properties of non-viral carrier materials to mediate gene transfer. Any suitable non-viral vector can be used to introduce nucleic acid aptamers into the cells of a subject. Examples of non-viral vectors include, but are not limited to, plasmids, liposomes, inorganic nanoparticles, and exosomes.
  • Plasmids are small circular DNA molecules. They are the most commonly used and simplest vectors in genetic engineering. They must include three parts: genetic marker gene, replication region, and target gene. Plasmids can be found in all bacterial groups. They are self-replicating DNA molecules outside the bacterial chromosome. Examples of plasmid vectors include, but are not limited to: Escherichia coli plasmid vectors, Bacillus subtilis plasmid vectors, yeast plasmid vectors, Agrobacterium plasmid vectors, and cyanobacterial plasmid vectors.
  • Liposomes are liposomes (hollow) made of lecithin and ceramide, etc., with a bilayer structure, which is an artificial membrane.
  • the composition of liposomes is usually a combination of phospholipids (especially phospholipids with high phase transition temperature), usually combined with steroids (especially cholesterol). Other phospholipids or other lipids can also be used.
  • the physical properties of liposomes depend on the presence of pH, ionic strength and divalent cations.
  • the transduction efficiency of liposomes can be increased by using dioleoylphosphatidylethanolamine during transduction. High-efficiency liposomes are commercially available. Examples of liposomes include, but are not limited to, neutral liposomes, negatively charged liposomes and positively charged liposomes.
  • Inorganic nanoparticles mainly play a role in treating diseases by transporting drugs or biomolecules into organisms through cell membranes.
  • Examples of inorganic nanoparticles used in gene delivery include but are not limited to silicon, iron oxides, carbon nanotubes, calcium phosphate, metal nanoparticles, quantum dots, etc.
  • Exosomes are small extracellular vesicles with an average diameter of 50-150nm. They serve as a means of intercellular communication. Typically, they consist of structural proteins and selected proteins, miRNAs, mRNAs, and long non-coding RNAs. RNA contains short nucleotide sequences that are recognized by proteins, which transport them to the cytoplasm and package them into exosomes. Exosomes transport payloads from one cell to another. After entering the recipient cell, the exosome payload is released into the cytoplasm.
  • Drugs are substances used to prevent, treat and diagnose diseases.
  • drugs refer to any chemical substance that can affect the physiological functions of body organs and cell metabolic activities.
  • RNA nucleic acid adaptor comprising an S1 protein that specifically binds to a SARS-CoV-2 virus.
  • the RNA aptamer sequence comprises a random region in the middle and fixed sequences at both ends, and the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:7.
  • the random region comprises a nucleic acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:7.
  • the fixed sequence at the 5' end of the RNA aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:8.
  • the fixed sequence at the 3' end of the RNA aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:9. In some embodiments, the fixed sequence at the 5' end of the RNA aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:8, and the fixed sequence at the 3' end of the RNA aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:9.
  • the RNA aptamer is chemically modified, and the chemical modification is selected from one or more of fluorine modification, methoxy modification, polyethylene glycol (PEG) polymer modification, and cholesterol-PEG polymer modification, wherein the PEG polymer can be selected from a small molecular weight hexamer to a 40kD high molecular weight polymer.
  • the drug further comprises a viral vector or a non-viral vector, wherein the vector comprises or carries the RNA aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus.
  • the viral vector is a lentivirus.
  • the non-viral vector is a plasmid.
  • the SARS-CoV-2 virus is a SARS-CoV-2 wild type, Delta or Omicron BA.1 or BA.2 virus strain.
  • “Pharmaceutical composition” refers to a pharmaceutical preparation for human use.
  • the pharmaceutical composition comprises a drug of the present invention and a suitable preparation of a carrier, a stabilizer and/or an excipient.
  • One aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a drug of the present invention and a pharmaceutically acceptable excipient
  • the drug comprises an RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus
  • the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, and the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 7.
  • the random region comprises a nucleic acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 7.
  • the fixed sequence at the 5' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 8. In some embodiments, the fixed sequence at the 3' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 9. In some embodiments, the fixed sequence at the 5' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 8, and the fixed sequence at the 3' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 9.
  • the RNA aptamer is chemically modified, and the chemical modification is selected from one or more of fluorine modification, methoxy modification, polyethylene glycol (PEG) polymer modification, and cholesterol-PEG polymer modification, wherein the PEG polymer can be selected from a small molecular weight hexamer to a 40kD high molecular weight polymer.
  • the drug further comprises a viral vector or a non-viral vector, wherein the vector comprises Or carrying the RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus.
  • the viral vector is a lentivirus.
  • the non-viral vector is a plasmid.
  • the SARS-CoV-2 virus is a SARS-CoV-2 wild type, Delta or Omicron BA.1 or BA.2 virus strain.
  • a pharmaceutical composition or sterile composition the drug is mixed with a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient for example, lyophilized powders, slurries, aqueous solutions or suspensions.
  • compositions are well known in the art.
  • pharmaceutically acceptable excipients include materials that allow the active ingredient of the composition to remain biologically active when combined with the ingredient and do not cause a destructive reaction with the subject's immune system. These may include stabilizers, preservatives, salt or sugar complexes or crystals, etc.
  • “Pharmaceutically acceptable” refers to molecules and ingredients that do not produce allergic reactions or similar undesirable reactions when administered to the human body. It is known in the art how to prepare aqueous compositions containing as active ingredients. Typically, these compositions are prepared as injections or sprays, such as liquid solutions or suspensions; they can also be prepared in a solid form suitable for being formulated into a solution or suspension before injection or spraying.
  • the drugs or pharmaceutical compositions of the present invention may be used alone or in combination with each other. Accordingly, the present invention provides a pharmaceutical kit to facilitate the above-mentioned combination therapy, which comprises two or more drugs or pharmaceutical compositions of the present invention that exist independently. In some embodiments, subjects sometimes use two or more drugs or pharmaceutical compositions of the present invention simultaneously. In some embodiments, subjects sometimes use two or more drugs or pharmaceutical compositions of the present invention separately.
  • One aspect of the present invention provides a method for screening RNA aptamers for a target protein, comprising the following steps: (1) providing a screening cell, wherein the screening cell has a guide RNA, a targeting sequence, a selection marker, a fusion protein and a dCas protein; wherein the guide RNA contains a recognition sequence and a random library of aptamers of a predetermined length; the targeting sequence is paired with the recognition sequence of the guide RNA; the selection marker is located downstream of the targeting sequence and includes a basic promoter and a selection marker gene; the fusion protein comprises the target protein and a transcriptional activation element, and the target protein binds to the RNA aptamer in the random library of aptamers, resulting in the binding of the transcriptional activation element to the selection marker; the dCas protein specifically recognizes the targeting sequence under the guidance of the guide RNA; (2) screening the screening cells using the selection marker gene; (3) collecting the screening cells expressing the selection marker gene; (4) lysing the screening
  • step (4) further comprises analyzing and verifying the obtained aptamer sequence.
  • the dCas protein is a dCas9 protein or a dUn1Cas12f1 protein.
  • the basic promoter is selected from mini-promoter-1 (SEQ ID NO: 28), mini-promoter-2 (SEQ ID NO: 27), mini-TK promoter (SEQ ID NO: 29), mini-CMV promoter (SEQ ID NO: 30) and Crystallin basal promoter (SEQ ID NO: 31).
  • the target protein is derived from a virus, bacteria, fungus or animal. In some embodiments, the target protein is derived from humans.
  • the target protein is green fluorescent protein or the RBD region derived from the SARS-CoV-2 spike protein.
  • the screening cell is a human cell HEK293T or a prokaryotic cell Escherichia coli E. coli.
  • the screening cell is obtained by transfecting one or more plasmid vectors into prokaryotic cells, the plasmid vector comprising one or more selected from guide RNA; targeting sequence and selection marker; nucleic acid sequence encoding fusion protein; and nucleic acid sequence encoding dCas protein, wherein the guide RNA contains a recognition sequence and a random library of nucleic acid aptamers of predetermined length, the targeting sequence is paired with the recognition sequence of the guide RNA, the selection marker is located downstream of the targeting sequence and includes a basic promoter and a selection marker gene, the fusion protein comprises the target protein and a transcriptional activation element, the target protein binds to the RNA nucleic acid aptamer in the random library of nucleic acid aptamers, resulting in the binding of the transcriptional activation element to the selection marker, and the dCas protein specifically recognizes the targeting sequence under the guidance of the guide RNA.
  • the guide RNA contains a recognition sequence and a random library of nucle
  • the screening cell is obtained by transfecting a plasmid vector into a prokaryotic cell, the plasmid vector comprising the guide RNA, the targeting sequence, the selection marker, and the nucleic acid sequence encoding the fusion protein and the dCas protein.
  • the prokaryotic cell is an Escherichia coli cell.
  • the method further comprises step (5): subcloning the nucleic acid aptamer sequence obtained in step (4) into a plasmid vector, and repeating steps (2) to (4).
  • the screening cell is provided by the following steps: (1.1) providing a cell expressing the dCas protein, the dCas protein specifically recognizes the targeting sequence under the guidance of the guide RNA, the cell also comprising the targeting sequence and the selection marker, the targeting sequence is paired with the recognition sequence of the guide RNA, the selection marker is located downstream of the targeting sequence and includes a basic promoter and a selection marker gene; (1.2) providing a viral expression vector encoding a guide RNA and a fusion protein, the guide RNA contains a recognition sequence and a random library of nucleic acid aptamers of a predetermined length, the fusion protein comprises the target protein and a transcriptional activation element, the target protein binds to the RNA nucleic acid aptamer in the random library of nucleic acid aptamers, resulting in the binding of the transcriptional activation element to the selection marker; (1.3) packaging the viral expression vector, and then using the packaged viral expression vector to infect the cell.
  • the method further comprises step (5): subcloning the nucleic acid aptamer sequence obtained in step (4) into the viral expression vector in step (1.2), and then repeating steps (1.3) and steps (2) to (4).
  • step (5) is further repeated 1 to 3 times.
  • an MOI of 3-5 is used for virus infection
  • an MOI of 0.1-0.3 is used for virus infection.
  • antibiotics are used to detect the viral titer of the packaged viral expression vector in step (1.3).
  • the viral expression vector is a lentiviral expression vector.
  • the cell is a HEK293T cell.
  • RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus
  • the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, and the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:7.
  • the random region comprises a nucleic acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:7.
  • the fixed sequence at the 5' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:8. In some embodiments, the fixed sequence at the 3' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:9. In some embodiments, the fixed sequence at the 5' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:8, and the fixed sequence at the 3' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:9.
  • the RNA aptamer is chemically modified, and the chemical modification is selected from one or more of fluorine modification, methoxy modification, polyethylene glycol (PEG) polymer modification, and cholesterol-PEG polymer modification, wherein the PEG polymer can be selected from a small molecular weight hexamer to a 40kD high molecular weight polymer.
  • the SARS-CoV-2 virus is a SARS-CoV-2 wild type, Delta or Omicron BA.1 or BA.2 virus strain.
  • Another aspect of the present invention provides a method for detecting SARS-CoV-2 virus, the method comprising contacting a sample to be detected with an RNA nucleic acid aptamer of the present invention that specifically binds to the S1 protein of the SARS-CoV-2 virus, wherein the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, and the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 7. In some embodiments, the random region comprises a nucleic acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 7.
  • the fixed sequence at the 5' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 8. In some embodiments, the fixed sequence at the 3' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 9. In some embodiments, the fixed sequence at the 5' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 8, and the fixed sequence at the 3' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 9.
  • the RNA aptamer is chemically modified, and the chemical modification is selected from one or more of fluorine modification, methoxy modification, polyethylene glycol (PEG) polymer modification, and cholesterol-PEG polymer modification, wherein the PEG polymer can be selected from a small molecular weight hexamer to a 40kD high molecular weight polymer.
  • the SARS-CoV-2 virus is a SARS-CoV-2 wild type, Delta or Omicron BA.1 or BA.2 virus strain.
  • the present invention provides an RNA that specifically binds to the S1 protein of the SARS-CoV-2 virus of the present invention.
  • the use of nucleic acid aptamers in the preparation of a detection reagent or kit for detecting SARS-CoV-2 virus wherein the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, and the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:7.
  • the random region comprises a nucleic acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:7.
  • the fixed sequence at the 5' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:8. In some embodiments, the fixed sequence at the 3' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:9. In some embodiments, the fixed sequence at the 5' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:8, and the fixed sequence at the 3' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:9.
  • the RNA aptamer is chemically modified, and the chemical modification is selected from one or more of fluorine modification, methoxy modification, polyethylene glycol (PEG) polymer modification, and cholesterol-PEG polymer modification, wherein the PEG polymer can be selected from a small molecular weight hexamer to a 40kD high molecular weight polymer.
  • the SARS-CoV-2 virus is a SARS-CoV-2 wild type, Delta or Omicron BA.1 or BA.2 virus strain.
  • Another aspect of the present invention provides a method for neutralizing SARS-CoV-2 virus in a subject, the method comprising administering an effective amount of the drug of the present invention to a subject in need thereof, wherein the drug comprises an RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus, the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, and the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 7. In some embodiments, the random region comprises a nucleic acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 7.
  • the fixed sequence at the 5' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 8. In some embodiments, the fixed sequence at the 3' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 9. In some embodiments, the fixed sequence at the 5' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 8, and the fixed sequence at the 3' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 9.
  • the RNA aptamer is chemically modified, and the chemical modification is selected from one or more of fluorine modification, methoxy modification, polyethylene glycol (PEG) polymer modification, and cholesterol-PEG polymer modification, wherein the PEG polymer can be selected from a small molecular weight hexamer to a 40kD high molecular weight polymer.
  • the drug further comprises a viral vector or a non-viral vector, wherein the vector comprises or carries the RNA aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus.
  • the viral vector is a lentivirus.
  • the non-viral vector is a plasmid.
  • the SARS-CoV-2 virus is a SARS-CoV-2 wild type, Delta or Omicron BA.1 or BA.2 virus strain.
  • the present invention provides the use of the drug of the present invention in the preparation of a drug for neutralizing the SARS-CoV-2 virus in a subject, wherein the drug comprises an RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus, and the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, and the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:7. In some embodiments, the random region comprises a nucleic acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:7.
  • the fixed sequence at the 5' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:8. In some embodiments, the fixed sequence at the 3' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:9. In some embodiments, the fixed sequence at the 5' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:8, and the fixed sequence at the 3' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:9.
  • the RNA nucleic acid aptamer is chemically modified, and the chemical modification is selected from one or more of fluorine modification, methoxy modification, polyethylene glycol (PEG) polymer modification, and cholesterol-PEG polymer modification, wherein the PEG polymer can be selected from a small molecular weight hexamer to a 40kD high molecular weight polymer.
  • the drug further comprises a viral vector or a non-viral vector, wherein the vector comprises or carries the RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus.
  • the viral vector is a lentivirus.
  • the non-viral vector is a plasmid.
  • the SARS-CoV-2 virus is a SARS-CoV-2 wild type, Delta or Omicron BA.1 or BA.2 virus strain.
  • Another aspect of the present invention provides a method for treating or preventing SARS-CoV-2 virus infection, the method comprising administering an effective amount of the drug of the present invention to a subject in need thereof, wherein the drug comprises an RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus, the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, and the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 7. In some embodiments, the random region comprises a nucleic acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 7.
  • the fixed sequence at the 5' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 8. In some embodiments, the fixed sequence at the 3' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 9. In some embodiments, the fixed sequence at the 5' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 8, and the fixed sequence at the 3' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 9.
  • the RNA aptamer is chemically modified, and the chemical modification is selected from one or more of fluorine modification, methoxy modification, polyethylene glycol (PEG) polymer modification, and cholesterol-PEG polymer modification, wherein the PEG polymer can be selected from a small molecular weight hexamer to a 40kD high molecular weight polymer.
  • the drug further Comprising a viral vector or a non-viral vector, wherein the vector comprises or carries the RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus.
  • the viral vector is a lentivirus.
  • the non-viral vector is a plasmid.
  • the SARS-CoV-2 virus is a SARS-CoV-2 wild type, Delta or Omicron BA.1 or BA.2 virus strain.
  • the present invention provides the use of the drug of the present invention in the preparation of a drug for treating or preventing SARS-CoV-2 virus infection in a subject, wherein the drug comprises an RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus, and the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, and the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:7. In some embodiments, the random region comprises a nucleic acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:7.
  • the fixed sequence at the 5' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:8. In some embodiments, the fixed sequence at the 3' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:9. In some embodiments, the fixed sequence at the 5' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 8, and the fixed sequence at the 3' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 9.
  • the RNA nucleic acid aptamer is chemically modified, and the chemical modification is selected from one or more of fluorine modification, methoxy modification, polyethylene glycol (PEG) polymer modification, and cholesterol-PEG polymer modification, wherein the PEG polymer can be selected from a small molecular weight hexamer to a 40kD high molecular weight polymer.
  • the drug further comprises a viral vector or a non-viral vector, wherein the vector comprises or carries the RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus.
  • the drug further comprises a viral vector or a non-viral vector, wherein the vector comprises or carries the RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus.
  • the viral vector is a lentivirus.
  • the non-viral vector is a plasmid.
  • the SARS-CoV-2 virus is a SARS-CoV-2 wild-type, Delta or Omicron BA.1 or BA.2 strain.
  • Another aspect of the present invention provides a method for neutralizing SARS-CoV-2 virus in a subject, the method comprising administering an effective amount of the pharmaceutical composition of the present invention to a subject in need thereof, wherein the pharmaceutical composition comprises the drug of the present invention and a pharmaceutically acceptable excipient, the drug comprising an RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus, the RNA nucleic acid aptamer sequence comprising a random region in the middle and fixed sequences at both ends, the fixed sequences at both ends being complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:7.
  • the random region comprises a nucleic acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:7.
  • the fixed sequence at the 5' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:8.
  • the fixed sequence at the 3' end of the RNA nucleic acid aptamer sequence comprises A nucleic acid sequence as shown in SEQ ID NO:9.
  • the fixed sequence at the 5' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:8, and the fixed sequence at the 3' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:9.
  • the RNA nucleic acid aptamer is chemically modified, and the chemical modification is selected from one or more of fluorine modification, methoxy modification, polyethylene glycol (PEG) polymer modification, and cholesterol-PEG polymer modification, wherein the PEG polymer can be selected from a small molecular weight hexamer to a 40kD high molecular weight polymer.
  • the drug further comprises a viral vector or a non-viral vector, wherein the vector comprises or carries the RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus.
  • the viral vector is a lentivirus.
  • the non-viral vector is a plasmid.
  • the SARS-CoV-2 virus is a SARS-CoV-2 wild type, Delta or Omicron BA.1 or BA.2 virus strain.
  • the present invention provides the use of the pharmaceutical composition of the present invention in the preparation of a drug for neutralizing SARS-CoV-2 virus, wherein the pharmaceutical composition comprises the drug of the present invention and a pharmaceutically acceptable excipient, the drug comprises an RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus, the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, and the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 7.
  • the random region comprises a nucleic acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 7.
  • the fixed sequence at the 5' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 8.
  • the fixed sequence at the 3' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 9.
  • the fixed sequence at the 5' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:8, and the fixed sequence at the 3' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:9.
  • the RNA nucleic acid aptamer is chemically modified, and the chemical modification is selected from one or more of fluorine modification, methoxy modification, polyethylene glycol (PEG) polymer modification, and cholesterol-PEG polymer modification, wherein the PEG polymer can be selected from a small molecular weight hexamer to a 40kD high molecular weight polymer.
  • the drug further comprises a viral vector or a non-viral vector, wherein the vector comprises or carries the RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus.
  • the viral vector is a lentivirus.
  • the non-viral vector is a plasmid.
  • the SARS-CoV-2 virus is a SARS-CoV-2 wild type, Delta or Omicron BA.1 or BA.2 virus strain.
  • Another aspect of the present invention provides a method for treating or preventing SARS-CoV-2 virus infection in a subject, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition of the present invention, wherein the pharmaceutical composition comprises a drug of the present invention and a pharmaceutically acceptable excipient, the drug comprising an S1 protein that specifically binds to the SARS-CoV-2 virus.
  • the present invention relates to an RNA aptamer of the present invention, wherein the RNA aptamer sequence comprises a random region in the middle and fixed sequences at both ends, and the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:7.
  • the random region comprises a nucleic acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:7.
  • the fixed sequence at the 5' end of the RNA aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:8.
  • the fixed sequence at the 3' end of the RNA aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:9.
  • the drug further comprises a viral vector or a non-viral vector, wherein the vector comprises or carries the RNA aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus.
  • the fixed sequence at the 5' end of the RNA aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:8, and the fixed sequence at the 3' end of the RNA aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO:9.
  • the RNA aptamer is chemically modified, and the chemical modification is selected from one or more of fluorine modification, methoxy modification, polyethylene glycol (PEG) polymer modification, and cholesterol-PEG polymer modification, wherein the PEG polymer can be selected from a small molecular weight hexamer to a 40kD high molecular weight polymer.
  • the drug further comprises a viral vector or a non-viral vector, wherein the vector comprises or carries the RNA aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus.
  • the viral vector is a lentivirus.
  • the non-viral vector is a plasmid.
  • the SARS-CoV-2 virus is a SARS-CoV-2 wild type, Delta or Omicron BA.1 or BA.2 virus strain.
  • the present invention provides the use of the pharmaceutical composition of the present invention in the preparation of a drug for treating or preventing SARS-CoV-2 virus infection in a subject, wherein the pharmaceutical composition comprises the drug of the present invention and a pharmaceutically acceptable excipient, the drug comprises an RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus, the RNA nucleic acid aptamer sequence comprises a random region in the middle and fixed sequences at both ends, and the fixed sequences at both ends are complementary, wherein the random region comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 7.
  • the random region comprises a nucleic acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 7.
  • the fixed sequence at the 5' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 8.
  • the fixed sequence at the 3' end of the RNA nucleic acid aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 9.
  • the drug further comprises a viral vector or a non-viral vector, wherein the vector comprises or carries the RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus.
  • the fixed sequence at the 5' end of the RNA aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 8
  • the fixed sequence at the 3' end of the RNA aptamer sequence comprises a nucleic acid sequence as shown in SEQ ID NO: 9.
  • the RNA aptamer is chemically modified, and the chemical modification is selected from One or more of fluorine modification, methoxy modification, polyethylene glycol (PEG) polymer modification and cholesterol-PEG polymer modification, wherein the PEG polymer can be selected from a small molecular weight hexamer to a 40kD high molecular weight polymer.
  • the drug further comprises a viral vector or a non-viral vector, wherein the vector comprises or carries the RNA nucleic acid aptamer that specifically binds to the S1 protein of the SARS-CoV-2 virus.
  • the viral vector is a lentivirus.
  • the non-viral vector is a plasmid.
  • the SARS-CoV-2 virus is a SARS-CoV-2 wild type, Delta or Omicron BA.1 or BA.2 virus strain.
  • Suitable routes of administration include parenteral administration (e.g., intramuscular, intravenous or subcutaneous administration), oral administration, nasal spray and aerosol inhalation administration.
  • the drugs or pharmaceutical compositions of the methods of the present invention can be administered in a variety of conventional ways, such as administration via endotracheal intubation, oral ingestion, inhalation, topical application or transdermal, subcutaneous, intraperitoneal, parenteral, intraarterial or intravenous injection.
  • people can administer the RNA nucleic acid aptamers, drugs or pharmaceutical compositions of the present invention in a targeted drug delivery system.
  • the RNA nucleic acid aptamers, drugs or pharmaceutical compositions of the present invention are administered via intravenous injection.
  • the appropriate dosage is determined by the clinician, for example, using parameters or factors known or suspected in the art to affect treatment or expected to affect treatment.
  • the initial dose is slightly lower than the optimal dose and then increased by small amounts until the desired or optimal effect relative to any adverse side effects is achieved.
  • Important diagnostic measurements include measuring, for example, inflammatory symptoms or the levels of inflammatory cytokines produced.
  • the medicament or pharmaceutical composition of the present invention is administered by continuous administration or by administration at certain intervals (e.g., one day, one week, or 1-7 times per week).
  • the dosage can be provided by endotracheal intubation, intravenous, subcutaneous, intraperitoneal, transdermal, topical, oral, nasal, rectal, intramuscular, intracerebral or intraspinal.
  • the preferred dosage regimen is a regimen that includes the maximum dose or administration frequency that avoids significant undesirable side effects.
  • the HEK293T cell line used in this article was purchased from the American Type Culture Collection (ATCC).
  • HEK293T was cultured in DMEM (Dulbecco's Modified Eagle Medium) containing 100U/ml Penicillin/Streptomycin and 10% fetal bovine serum (FBS).
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • 293T/dCas9 is a monoclonal cell line obtained by stably integrating the dCas9 expression element by lentivirus in the wild-type HEK293T cell line.
  • the CRISmers screening cell line dCas9/9 ⁇ gLuc-puro cell line is a monoclonal cell line obtained by further stably integrating the 9 ⁇ gLuc-puro reporter gene element (9 ⁇ gLuc-minPro-puro) by lentivirus in the 293T/dCas9 cell line.
  • the HEK293T/hACE2 cell line is a polyclonal cell line obtained by stably integrating the hACE2 expression element by lentivirus in the wild-type HEK293T cell line. All cells were mycoplasma-free and cultured in The cells were incubated in a cell culture incubator at 37°C and containing 5% CO 2 .
  • the construction of expression vectors mainly includes the preparation of linearized vectors, the preparation of inserted fragments, the connection between vectors and fragments, the transformation of plasmids with E. coli competent cells, the identification of E. coli clones by colony PCR, plasmid extraction, sequencing and identification of the obtained plasmids, etc.
  • the lentiviral vector backbones used in this paper are all from Addgene.
  • HEK293T cells are a common and conventional cell line that is easy to operate and is widely used in retroviral production, gene expression, and protein expression.
  • the cultivation of this cell line mainly includes cell recovery, cell maintenance culture, cell passaging, cell freezing, and other operations well known to those skilled in the art.
  • transfection can be performed in 96-well plates, 24-well plates, 6-well plates, and 10 cm dishes.
  • the luciferase reporter system was used to verify the affinity of nucleic acid aptamers, which was mostly performed in 96-well plates, and lentiviral packaging was mostly performed in 6-well plates or 10 cm dishes.
  • Adhesive transfection is suitable for transfection of conventional cells such as HEK293T; suspension transfection can expand the contact between the transfection complex and the cells, further improve the transfection efficiency, and is suitable for experiments that are difficult to transfect or require high transfection efficiency.
  • the following is an example of 96-well plate adherent transfection:
  • Seeding cells one day before transfection Coat the area of the 96-well plate to be seeded with 0.1% gelatin in advance, take HEK293T cells with fast growth rate and good growth status or transfected cells and seed them into the 96-well plate at a cell seeding volume of 2.5 ⁇ 104 . It is best to keep the cell confluence above 60% on the day of transfection (test the experimental effect after 48 hours, no need to be too dense).
  • This article established 293T/dCas9 stable monoclonal cell lines, dCas9/9 ⁇ gLuc-puro stable monoclonal cell lines, and HEK293T/hACE2 stable polyclonal cell lines.
  • the establishment process mainly includes the pre-planting of HEK293T cells, lentiviral packaging, lentiviral collection, preparation of cells to be infected, lentiviral infection, cell line screening and identification, etc.
  • Monoclonal cell lines also need to be selected for identification after cell line screening, while polyclonal cell lines do not need to be selected separately for monoclonal cell lines.
  • HEK293T cells Seeding of HEK293T cells: One day before transfection, HEK293T cells with fast growth rate and good cell status were seeded at a cell seeding volume of 1 ⁇ 10 6 into a 6-well plate coated with 0.1% gelatin in advance. It is best to keep the cell confluence above 80% on the day of transfection.
  • Lentivirus packaging The lentivirus packaging process is consistent with the non-liposome transient transfection process. There are three plasmids involved in transfection: pRRL lentivirus vector plasmid containing the target gene expression frame, pCMV-VSV-G plasmid expressing the lentivirus envelope protein VSVG, and psPAX2 plasmid expressing the lentivirus packaging proteins GAG and POL. The mass ratio of the three plasmids is 10:9:1.
  • Lentivirus collection Collect the cell culture supernatant 48 hours after transfection, and then slowly add fresh culture medium along the wall and continue culturing for 24 hours. Filter the collected virus liquid with a 0.45 ⁇ m filter and store it in a 4°C refrigerator. Collect the cell culture supernatant again 72 hours after transfection, filter it with a 0.45 ⁇ m filter and mix it with the virus liquid collected 48 hours ago. It can be packaged and stored at -80°C. If it is used within a short period of time, it can be temporarily stored in a 4°C refrigerator. Gun tips and their Its experimental equipment needs to be placed in a special virus waste collection bag for centralized treatment after high temperature and high pressure.
  • Virus infection is the same as transient cell transfection.
  • Adherent infection or suspension infection can also be used. The principles are the same. Taking adherent infection as an example, one day before infection, cells to be infected with fast growth rate and good cell status are inoculated at a cell inoculation rate of 1x10 5 into a 24-well plate coated with 0.1% gelatin in advance, and one well of cells is reserved as a blank control group for virus infection.
  • Lentivirus infection Pipette the culture medium in the 24-well plate along the wall, add the virus solution containing 8 ⁇ g/ml Polybrene (co-infection agent), and fix the culture volume at 250 ⁇ l. Gently shake and place in the incubator for culture. After 8-12 hours, aspirate the virus solution along the wall, gently add fresh culture medium along the wall, and continue to culture in the incubator. The blank control group is not infected with lentivirus, and other operations are the same. After the lentivirus infects the cells, the target gene will be integrated into the host cell genome. The target gene often contains resistance genes, which are used for cell screening.
  • the dCas9/9 ⁇ gLuc-puro cell line is a monoclonal cell line obtained by further stably integrating the 9 ⁇ gLuc-puro reporter gene element into the 293T/dCas9 cell line using lentivirus.
  • dCas9/9 ⁇ gLuc-puro cell line When identifying the dCas9/9 ⁇ gLuc-puro cell line, different monoclonal clones were independently infected with two groups of lentiviruses, one group was lenti-hU6-gLuc sgRNA-1.2 GFP apt-EF1a-NLS-GFP-VPH (PC); the other group was lenti-hU6-gLuc sgRNA-1.2blank-EF1a-NLS-GFP-VPH (NC). Each monoclonal cell line was infected with the virus group.
  • PC lenti-hU6-gLuc sgRNA-1.2 GFP apt-EF1a-NLS-GFP-VPH
  • NC lenti-hU6-gLuc sgRNA-1.2blank-EF1a-NLS-GFP-VPH
  • luciferase reporter assay was used to validate the CRISmers system and to screen the affinity of nucleic acid aptamers selected in the subsequent secondary identification.
  • CRISmers system validation The steps are consistent with the non-lipid transient transfection steps of the 96-well plate mentioned above.
  • the following plasmids (based on the 293T/dCas9 stable transfection cell line) were transiently transfected in the corresponding wells of the 96-well plate using PEI transfection reagent: p1 ⁇ gLuc target firefly luciferase or p9 ⁇ gLuc target firefly luciferase, phU6-gLuc sgRNA-1.1 GFP aptamer or phU6-gLuc sgRNA-1.2 GFP aptamer, pGFP-VPH and pCMV-renilla luciferase.
  • the mass of pCMV-renilla luciferase plasmid is 20ng, and the molar ratio of the remaining three plasmids is 1:1:1. 6-8h after transfection, replace the 10% FBS/DMEM fresh culture medium.
  • the blank expression vector phU6-gLuc sgRNA-1.2 BsmBI-BsmBI-polyT-EF1a-NLS-linker-EcoRI-RBD-EcoRI-linker-VP64-P65-HSF1-NLS-linker-P2A-linker-Zeo-WPRE must be constructed first, and the nucleic acid aptamer sequence is inserted into the gLuc sgRNA stem loop2 (1.2) backbone region using the BsmBI restriction endonuclease before transfection experiments.
  • the constructed expression vectors can be directly used in transfection experiments after rapid plasmid extraction, without the need to obtain high-purity plasmids on a large scale. Replace the 10% FBS/DMEM fresh culture medium 6-8h after transfection.
  • Luciferase activity detection Luciferase activity was detected using the dual-luciferase reporter kit from Vigorous Company 48 hours after transfection.
  • CRISmers system verification Cell lysis: 48 hours after transfection, carefully aspirate the culture medium in the 96-well plate, use a dispenser to add 30 ⁇ l 1universal lysis buffer to each well of cells, and incubate at room temperature for 5-10 minutes on a micro-oscillator or low-speed shaker. After the cells are completely lysed, use a dispenser to take 20 ⁇ l of the lysed cell sample to a new corresponding 96-well plate. Detection of firefly luciferase activity: Use a dispenser to take 20 ⁇ l of Fassay Reagent to the bottom of the well, gently tap the wall of the well 3-5 times to mix, and immediately place it in a microplate reader for chemiluminescence detection.
  • the luminescence value recorded at this time is the activity reading of firefly luciferase.
  • Detection of Renilla luciferase activity Use a dispenser to take 20 ⁇ l of Rassay Reagent to the bottom of the tube, gently tap the wall of the tube 3-5 times to mix. Mix well for 5 times and immediately put it into the microplate reader for chemiluminescence detection. At this time, the luminescence value is recorded as the activity reading of Renilla luciferase.
  • the target protein expression reading frame of interest for screening was inserted into the lentiviral blank expression vector with the help of restriction endonuclease EcoRI, and then the synthetic 20bp random oligonucleotide library was inserted into the expression vector using restriction endonuclease BsmBI to form a 20bp oligonucleotide screening library for the target protein.
  • the construction of the library is mainly divided into PCR amplification of the random synthetic oligonucleotide library, enzyme digestion of the lentiviral vector, Gibson assembly, library electroporation, library lentiviral packaging and titer determination.
  • the synthesized oligonucleotide library has a total of 100bp, with the middle 20bp being an oligonucleotide random sequence (A, T, C, G), and the 5' end and 3' being 40bp nucleotide linker sequences homologous to the expression vector, namely AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGGCCAnnnnnnnnnnnnnnnnnnnnnnnnnCTGCAGGGCCAAGTGGCACCGAGTCGGTGCTTTTTATCGA (SEQ ID NO: 10).
  • oligo-Primer F TATGTTTAAGAGCTAGAAATAGCAAGTTAAAATAAGGCTA (SEQ ID NO: 11)
  • oligo-Primer R CCATCTTTGCAAAGCTTATATCGATAAAAAGCACCGACTC (SEQ ID NO: 12).
  • the QIAquick PCR purification kit for purification and recovery according to the instructions. If the sample concentration is low after purification, multiple parallel amplification reactions can be added to ensure sufficient samples when the library is connected.
  • the sample can be placed in a 4°C refrigerator for short-term storage, and a -20°C or -80°C refrigerator for long-term storage to prevent degradation.
  • the lentiviral expression vector inserted into the SARS-CoV-2 S protein RBD expression reading frame was digested and dephosphorylated with the BsmBI restriction endonuclease. After the reaction, 2 ⁇ l of the sample was subjected to a 1% agarose gel electrophoresis experiment to verify the digestion efficiency, and 1 ⁇ l of the original vector without digestion was taken for control. Compared with the spiral original vector, the vector after digestion is linear, and the band position is slightly higher than the spiral vector. After verification, the QIAquick gel recovery kit was used for gel cutting and purification according to the instructions.
  • the vector In order to remove impurities such as salts that may be introduced in the digestion reaction, improve the subsequent Gibson ligation efficiency, and concentrate the concentration of the purified vector, the vector needs to be precipitated with isopropanol.
  • the reaction system is shown in the following table.
  • Multiple parallel Gibson ligation reactions can be performed simultaneously to ensure sufficient samples during library electrotransfer.
  • a blank control group needs to be set up during ligation, and the volume of random library amplified by PCR is replaced with an equal volume of deionized water to determine the background effect of the ligation reaction and calculate the library coverage.
  • the concentration of the ligated library after concentration and purification needs to be precipitated with isopropanol in the reaction system.
  • the above reaction mixture is mixed by a vortex shaker and placed at room temperature for 15 minutes, and centrifuged at 12000rpm for 15 minutes.
  • Electroporation Set the electroporation parameters according to the instructions. Take out the electroporation competent cells from the -80°C freezer and place them on compacted ice. When the cells are just thawed, add 2 ⁇ l of the above ligation product, gently blow and mix 2-3 times, then quickly transfer the mixture to the pre-cooled electroporation cup and cover the cup lid. Wipe the moisture outside the electroporation cup with a towel and quickly remove the excess water. Quickly place the electroporation cup in the electroporation tank for electroporation. After electroporation, quickly add 1 ml of preheated SOC medium to the electroporation cup and culture at 37°C, 220 rpm for 1 hour. During electroporation, avoid bubbles and wipe off the water on the cup wall as much as possible to ensure that the electroporation action is smooth and fast.
  • Counting Count the bacterial plates to evaluate the electroporation efficiency and library coverage. Before the final library electroporation, a preliminary experiment should be conducted to estimate the background effect of the constructed library, electroporation efficiency, etc. Since there are too many initial sequences in the first round (4 20 ), the existing library construction technology cannot cover all sequences and can only harvest as many clones as possible. Starting from the second round of library construction, the existing library construction technology can cover all sequences and achieve at least 500-fold coverage.
  • Bacteria collection After evaluating the number of bacterial colonies harvested from this electroporation, use a small medicine spoon or bacterial scraper to collect the bacteria in a clean bench. After the collection is completed, place the bacteria in a shaker at 37°C and 220 rpm for 2-3 hours.
  • the lentiviral packaging process of the library is basically the same as the lentiviral packaging process in the establishment of the above-mentioned lentiviral-mediated stable cell line. Due to the large demand for library lentivirus, the packaging process is carried out simultaneously in multiple 10cm dishes. The lentiviral packaging process is consistent with non-liposome transient transfection. There are three plasmids involved in transfection: pRRL lentiviral vector plasmid containing lentiviral random library, pCMV-VSV-G plasmid expressing lentiviral envelope protein VSVG, and psPAX2 plasmid expressing lentiviral packaging proteins GAG and POL. The mass ratio of the three plasmids is 10:9:1.
  • the Zeocin resistance gene of the library vector was used to determine the virus titer.
  • the dCas9/9 ⁇ gLuc-puro cells in good condition were inoculated into 12-well plates, with 3 ⁇ 10 6 cells in each well (defined as the "starting cell number"). Subsequently, 0 ⁇ l, 25 ⁇ l, 50 ⁇ l, 100 ⁇ l, 200 ⁇ l and 400 ⁇ l library lentivirus were added for suspension centrifugation infection (8 ⁇ g/ml polybrene was added to ensure infection efficiency). After centrifugation at 37°C and 2400rpm for 2h, the cells were placed in a 37°C cell culture incubator and cultured for 12-14h.
  • the infected cells were inoculated into the corresponding new 12-well plates at a density of 1 ⁇ 10 4 , and a culture medium containing 500 ⁇ g/ml zeocin antibiotics was added.
  • the infected virus without antibiotics group and the uninfected virus with antibiotics group were set as controls. After 5-6 days of continuous antibiotic screening, all the cells in the group not infected with the virus and adding antibiotics died. When the cell confluence of the group infected with the virus without adding antibiotics reached 80%-90%, the cells were digested and counted.
  • the number of cells in each group infected with the virus and adding antibiotics is defined as the "number of live cells", and the number of cells in each group infected with the virus without adding antibiotics is defined as the "number of control cells”.
  • Titer (IFU/ml) (number of live cells ⁇ starting number of cells) / (number of control cells ⁇ number of milliliters of virus volume).
  • Lentivirus library screening was based on the monoclonal cell line #4-53 dCas9/9 ⁇ gLuc-puro.
  • the virus screening process was basically the same as the lentivirus screening in the process of establishing the above-mentioned lentivirus-mediated stable cell line. Since the library screening requires a large amount of cells, the screening process was carried out in multiple 10cm dishes.
  • a medium (3-5) MOI was used for virus infection to ensure that most functional sequences were covered and screened, and to reduce the workload of screening.
  • a low (0.1-0.3) MOI was used for virus infection to ensure that most cells received only one nucleic acid aptamer sequence.
  • the cells were passaged, the cells were maintained at a density of 30%-40%, and the culture medium was replaced with a 2 ⁇ g/ml puro antibiotic culture medium for screening, and fresh resistance culture medium was replaced every 2-3 days. After continuous screening for about 10 days, clones grown from single cells can be seen. All clones were picked with a stereoscope and lysed with cell genome PCR lysis buffer. The lysis process was incubated at 50°C for 1 hour and 95°C for 15 minutes. 1 ⁇ l of lysis buffer was taken for direct genome PCR amplification.
  • the genome PCR amplification system, amplification procedure (annealing, extension, final extension for 20 cycles), amplification primers, agarose gel electrophoresis experimental verification after amplification, and purity were determined.
  • the chemical recovery was basically the same as the PCR amplification process of the above oligonucleotide random library.
  • the deep sequencing of the samples was performed by GeneVision.
  • the main evaluation contents are the number of output reads and the quality of base sequencing (Q30).
  • nucleic acid aptamers When performing deep sequencing data analysis, first filter out the nucleic acid aptamer sequences that do not match the homologous sequence at both ends, then remove the adapter sequence and homologous sequence at both ends of the nucleic acid aptamer to obtain several nucleic acid aptamer sequences. In order to identify high-affinity sequences, we selected nucleic acid aptamers for the second round of luciferase reporter experiment screening based on two indicators.
  • each independent sequence was sorted from high to low according to the frequency of occurrence, and the top 0.1% high-frequency independent sequences were selected for verification in the second round of screening; on the other hand, the enrichment index of independent sequences between the two rounds of screening was calculated and sorted (the enrichment index refers to the ratio of the number of repetitions of the same independent sequence in the two rounds), and the top 15 high-frequency enrichment index sequences were selected for verification in the second round of screening.
  • the packaging of SARS-CoV-2 pseudovirus is basically the same as the aforementioned lentivirus packaging process.
  • Pseudovirus packaging involves a total of 5 plasmids: pCMV-firefly luciferase, pSpike or pDelta or pOmicron, pTAT, pRev and pGAGpol.
  • pseudovirus packaging Before pseudovirus packaging, take 1 ml of 10% FBS/DMEM fresh medium to replace the original medium in the 6-well plate.
  • the pseudovirus packaging process is the non-liposomal transient transfection process of plasmids.
  • the following 5 plasmids are co-transfected: 2 ⁇ g pCMV-firefly luciferase, 0.4 ⁇ g pSpike or 0.4 ⁇ g pDelta or 0.4 ⁇ g pOmicron, 0.2 ⁇ g pTAT, 0.2 ⁇ g pRev and 0.2 ⁇ g pGAGpol.
  • Pseudovirus collection After 48 hours of cell culture, carefully aspirate the cell supernatant with a syringe and filter it with a 0.45 ⁇ m filter column. Pseudoviruses of the same type can be mixed together for storage. Pseudoviruses at 48 hours can be temporarily stored in a 4°C refrigerator. After 48 hours of pseudovirus collection, carefully add 2 ml of 10% FBS/DMEM fresh culture medium along the wall and continue to culture for 24 hours, that is, a total of 72 hours. After 72 hours, also carefully aspirate the cell supernatant with a syringe and filter it with a 0.45 ⁇ m filter column.
  • Pseudoviruses collected at 48 hours and 72 hours can be mixed together for storage. Pseudoviruses are divided and frozen at -80°C. During pseudovirus collection The pipette tips and other experimental equipment that come into contact with the pseudovirus should be collected independently and processed after sterilization.
  • Blocking Use a vacuum suction pump to suck away the incubation solution, use a pistol to suck up the washing buffer to clean the wells, and then suck away the washing buffer along the wall. Repeat this process three times. Try to dry the liquid at the bottom of the well and avoid touching the bottom of the well during the aspiration process. After washing, use a pistol to suck up the blocking solution and incubate at 37°C for 30 minutes. After blocking is completed, repeat the washing process three times. (Washing buffer: PBS + 0.05% (vol/vol) Tween-20, PH7.4; blocking solution: washing buffer + 2% (weight/vol) BSA).
  • Streptavidin-HRP incubation Add 0.1 ⁇ g/ml Streptavidin-HRP prepared with sterile water and incubate at room temperature for 30 min. Then use a vacuum pump to carefully suck away the incubation solution along the wall, use a spray gun to suck up the washing buffer to clean the wells, and then suck away the washing buffer along the wall. Repeat this three times, try to dry the liquid at the bottom of the well, avoid touching the bottom of the well, and be sure to replace the gun tip.
  • Substrate incubation Add 200 ⁇ l of substrate and incubate at room temperature for 20 min. The wells with strong signals will turn blue.
  • Stop reaction Add 50 ⁇ l of stop solution and incubate at room temperature for 2-5 min. The color of the wells with strong signals will change from blue to yellow.
  • Virus plating One day before the test, add pseudovirus particles of different titers (TCID50) in a total volume of 100 ⁇ l to a 96-well ELISA plate with a high binding surface, seal with a disposable plastic film and place in a 4°C refrigerator for overnight incubation. The remaining operations are consistent with the aforementioned "protein-based ELONA experiment". It should be noted that the gun tips and other experimental equipment that have been in contact with pseudoviruses need to be placed in a special virus waste collection bag for centralized treatment after high temperature and high liquid. The pseudovirus titer detection is carried out according to the quantitative detection method published by Youchun Wang et al.
  • the experiment was performed according to the instructions provided by the Chemiluminescent EMSA kit (Biyuntian).
  • the simple operation is as follows: a fixed amount of biotinylated aptamer (1 pmol) was incubated with different ratios of SARS-CoV-2 wild-type or mutant Omicron BA.2 RBD protein (1:0, 1:1, 1:10, 1:20, 1:50) in EMSA binding buffer at room temperature for 30 minutes.
  • biotinylated aptamer (1 pmol) was incubated with SARS-CoV-2 RBD protein at a ratio of 1:25, and cold probes (non-biotinylated aptamers, 1:1, 1:2, 1:100, 1:200) in different ratios with biotinylated aptamers were incubated in EMSA binding buffer at room temperature for 30 minutes.
  • biotinylated aptamer (1 pmol) was incubated with the SARS-CoV-2 mutant Omicron BA.2 RBD protein at a ratio of 1:50, and cold probes (non-biotinylated aptamers, 1:1, 1:2, 1:100, 1:200) at different ratios with the biotinylated aptamer in EMSA binding buffer at room temperature for 30 minutes. After incubation, the incubated samples were mixed with the loading buffer and electrophoresed with 4% polyacrylamide gel at 100V for 1 hour, and the samples were transferred to N+ nylon membranes. After electrophoretic transfer, crosslinking was performed with a handheld UV lamp for 5-10 minutes. The biotinylated aptamers were then detected by chemiluminescence.
  • RNA aptamers #2-1-18 and #5-2-15 were tested for the inhibitory effects of RNA aptamers #2-1-18 and #5-2-15 on the above two strains of SARS-CoV-2 Delta strain, SARS-CoV-2 Omicron BA.1 strain, and SARS-CoV-2 Omicron BA.2 strain.
  • the virus-related experimental process was carried out in the biological safety level-3 laboratory (BSL-3) of Shenzhen Center for Disease Control and Prevention, and the operation process was strictly carried out in accordance with the BSL-3 laboratory operation regulations.
  • the strain used for SARS-CoV-2 Delta is SARS-CoV-2 B.1.617.2-Shenzhen Center for Disease Control and Prevention isolate (SZ/03/2022/SARS-CoV-2(D)); the strain used for SARS-CoV-2 Omicorn BA.1 is SARS-CoV-2 B.1.1.529 BA.1 Shenzhen Center for Disease Control and Prevention isolate (SZ/08/2022/SARS-CoV-2(O)).
  • the strain used for SARS-CoV-2 Omicorn BA.2 is SARS-CoV-2 B.1.1.529 BA.2 Shenzhen Center for Disease Control and Prevention isolate SARS-CoV-2/shenzhen/13/2022 (Omicron BA.2).
  • virus diluents Use 2% FBS/DMEM to dilute the SARS-CoV-2 Delta strain with a titer of 1 ⁇ 10 6 TCID50 and the SARS-CoV-2 Omicron strain with a titer of 1 ⁇ 10 6 TCID50 to 50 TCID50, respectively, and label them as virus diluents Mix-D and Mix-O.
  • 600nM Take 3.6 ⁇ l of 100 ⁇ M sample storage stock solution + 596.4 ⁇ l of the above virus dilution Mix-D;
  • 200nM Take 200 ⁇ l of 600nM sample + 400 ⁇ l of the above virus dilution Mix-D;
  • 22.22nM Take 200 ⁇ l of 66.67nM sample + 400 ⁇ l of the above virus dilution Mix-D;
  • 0.27nM Take 200 ⁇ l of 0.82nM sample + 400 ⁇ l of the above virus dilution Mix-D;
  • 0.09nM Take 200 ⁇ l of 0.27nM sample + 400 ⁇ l of the above virus dilution Mix-D;
  • 0 nM 400 ⁇ l of the above virus dilution Mix-D.
  • 600nM Take 3.6 ⁇ l of 100 ⁇ M sample storage stock solution + 596.4 ⁇ l of the above virus dilution Mix-O;
  • 200nM Take 200 ⁇ l of 600nM sample + 400 ⁇ l of the above virus dilution Mix-O;
  • 22.22nM Take 200 ⁇ l of 66.67nM sample + 400 ⁇ l of the above virus dilution Mix-O;
  • 0.27nM Take 200 ⁇ l of 0.82nM sample + 400 ⁇ l of the above virus dilution Mix-O;
  • 0.09nM Take 200 ⁇ l of 0.27nM sample + 400 ⁇ l of the above virus dilution Mix-O;
  • 0 nM 400 ⁇ l of the above virus dilution Mix-O.
  • sample-virus incubation mixture diluted in multiple ratios was incubated in a cell culture incubator at 37°C and 5% CO2 for 1 hour.
  • RNA volume of each sample is 100 ⁇ l, which needs to be aliquoted and stored to avoid repeated freezing and thawing during subsequent use. After collecting the cell supernatant from each well, add 100 ⁇ l of 2% FBS/DMEM culture medium.
  • RNA volume of each sample is 100 ⁇ l, which needs to be aliquoted and stored to avoid repeated freezing and thawing during subsequent use.
  • RNA extraction in this experiment was mainly for SARS-CoV-2 Delta strain, Omicron BA.1 and Omicron BA.2 strain infection, and the cell supernatant was collected at different time points for RNA extraction.
  • the RNA extraction of the cell supernatant was batch extracted by Roche automated nucleic acid extraction instrument MagNA Pure 96 system in the new crown nucleic acid testing laboratory.
  • MagNA Pure 96 system in the new crown nucleic acid testing laboratory.
  • the cell supernatant contains viruses, and virus inactivation reagents must be added and disinfected with alcohol when taken out of the BSL-3 laboratory. All operations in the new crown nucleic acid testing laboratory should also be carried out in accordance with regulations, and experimental waste should also be handled as required.
  • the extracted RNA needs to be subjected to a one-step RT-qPCR experiment to identify the expression of the virus in order to measure the neutralizing inhibitory effect of nucleic acid drugs on the virus.
  • the fluorescence channel settings are: ORF1ab detection target: FAM reporter; N detection target: VIC/HEX.
  • Reagent preparation During the experiment, RNase-free pipette tips, centrifuge tubes, etc. must be used, and the operation must be performed in a biosafety cabinet. Take out the aliquoted RNA samples from the -80°C refrigerator and thaw them on ice. Prepare the reaction system according to the fluorescent PCR reaction system described in the preparation of the standard curve above.
  • the virus-related experimental process was carried out in the biosafety level 3 laboratory of Guangzhou Customs Technology Center, and the operation process was strictly carried out in accordance with the BSL-3 laboratory operation regulations.
  • mice were gently anesthetized with isoflurane and 1 x 10 5 FFU of Omicron BA.2 live virus was administered intranasally. After 24/48 hours, the mice were anesthetized, and the lung tissues were observed and collected. The lungs were transferred to PBS and homogenized, and the clear supernatant was harvested, RNA was extracted, and RT-qPCR was performed to quantify viral RNA. At the same time, the virus titer per gram of lung tissue was determined using the FRNT method in VERO E6 cells.
  • mice without specific pathogens were gently anesthetized with isoflurane and 1 x 10 5 FFU of live Omicron BA.2 virus was administered intranasally. After 3 hours, the mice were gently anesthetized with isoflurane and the modified nucleic acid aptamer (Chol-PEG40K-5-2-15-2'-FO) dissolved in sterile enzyme-free water was administered intranasally, 25 ⁇ l per nostril. After 24/48 hours, the mice were anesthetized, the lung tissue was observed visually and the lung tissue was collected.
  • the modified nucleic acid aptamer Chol-PEG40K-5-2-15-2'-FO
  • the lungs were transferred to PBS and homogenized, the clear supernatant was harvested, RNA was extracted and RT-qPCR was performed to quantify the viral RNA.
  • the virus titer per gram of lung tissue was determined using the FRNT method in VERO E6 cells.
  • amino acid or nucleic acid sequences described in the present invention are provided in the table below.
  • the nucleic acid aptamer screening system of the present invention utilizes the guiding effect of sgRNA and the targeting effect of dCas9 to anchor the complex of dCas9 and sgRNA to the sgRNA target sequence upstream of the basic promoter (mini-promoter-2 (SEQ ID NO: 27)), and at the same time, the target protein of interest (X-protein) is fused and expressed with the transcription activation element.
  • the basic promoter mini-promoter-2 (SEQ ID NO: 27)
  • the transcription activation element fused with the X-protein can be recruited to the sgRNA target sequence, thereby activating the expression of the selection marker gene downstream of mini-promoter-2.
  • the expression of the downstream selection marker gene is an indirect reflection of the affinity binding ability of the nucleic acid aptamer to the target protein.
  • loop regions can tolerate the addition of nucleic acid aptamers to enhance the recruitment of Cas9 and related effector element complexes.
  • the sgRNA backbone can allow large fragments to be inserted without affecting the activity of dCas9.
  • the screening process is shown in Figure 1B.
  • the synthesized oligonucleotide random sequence is cloned into the loop region of the sgRNA backbone of the customized lentiviral vector in the manner of sgRNA library construction, thereby forming an initial library containing the targeting exogenous artificial sgRNA sequence and the oligonucleotide random sequence attached to its backbone region.
  • the customized lentiviral vector should already contain an independent reading frame for co-expression of X-protein and transcriptional activation elements, and it is easy to molecularly clone and replace the X-protein.
  • the library is packaged with lentivirus and infected with lentivirus based on a customized universal screening reporter cell line.
  • the universal screening reporter cell line should have integrated the selection reporter system (sgRNA target sequence-mini-promoter-2-selection marker gene), and the reporter system is sensitive to selection pressure. Due to the integration characteristics of the lentivirus, the DNA sequence encoding the oligonucleotide random sequence can be integrated into the genome of the screening reporter cell line.
  • the transcriptional activation element co-expressed with X-protein can be recruited to the sgRNA targeting sequence, that is, the upstream sequence of the screening reporter system.
  • the reporter system in the screening cell line begins to respond.
  • the selection reporter system in the corresponding cell works. The stronger the affinity of the aptamer to X-protein, the more co-expressed transcriptional activation elements are recruited, the stronger the activation of the downstream selection marker gene, and the stronger the feedback of the screening reporter cell line when facing the screening pressure.
  • the genomic information of the enriched cells is obtained, the affinity aptamer sequence inserted in the loop region of the sgRNA backbone is amplified, and the sequence information is analyzed by next-generation sequencing (NGS) ( Figure 1B), and the aptamer sequence with affinity to the target protein can be obtained.
  • NGS next-generation sequencing
  • Verification was performed using the luciferase reporter system, and the luciferase reporter gene was located downstream of mini-promoter-2.
  • the guiding effect of sgRNA and the targeting effect of dCas9 anchor the complex of dCas9 and sgRNA to the sgRNA target sequence. If the aptamer in the sgRNA backbone can recognize the target protein with affinity, the CRISPRa (transcription activation element) co-expressed with the target protein can be recruited to the sgRNA target sequence, and then the transcription activation element acts on mini-promoter-2, thereby activating the expression of the reporter gene luciferase (Figure 2B).
  • the expression of the downstream luciferase reporter gene depends on the efficiency of the aptamer in recruiting the target protein-transcription activation element complex, converting the affinity of the aptamer and the target protein into the expression ability of the luciferase reporter gene.
  • Green Fluorescent Protein and the canonical GFP aptamer identified by the SELEX screening system were used to verify that the affinity of RNA aptamers with target proteins can be used to recruit transcriptional activators and activate the expression of luciferase reporter genes in cells.
  • the canonical GFP aptamer was inserted into the tetra loop region of the sgRNA backbone (sgRNA 1.1) or stem loop 2 region (sgRNA 1.2) (Figure 2A), and GFP protein was fused with transcriptional activation elements VP64, P65 and HSF1 (also referred to as VPH) ( Figure 2B). Then, a luciferase reporter plasmid containing 1 copy of the gLuc targeting sequence was constructed (1 ⁇ gLuc-minPro-luciferase).
  • the 293T/dCas9 cell line stably expressing dCas9 was co-transfected with a total of 3 plasmids expressing (1) sgRNA1.1 structure or sgRNA1.2 structure, (2) GFP-VPH, and (3) 1 ⁇ gLuc-minPro-luciferase.
  • the luciferase expression signal was detected 48 hours later.
  • a luciferase reporter plasmid containing 9 copies of the gLuc targeting sequence (9 ⁇ gLuc-minPro-luciferase) was constructed to explore whether the system of the present invention has an additive effect.
  • the 293T/dCas9 cell line stably expressing dCas9 was co-transfected with a total of 3 plasmids expressing (1) sgRNA1.1 structure or sgRNA1.2 structure, (2) GFP-VPH, (3) 1 ⁇ gLuc-minPro-luciferase or 9 ⁇ gLuc-minPro-luciferase.
  • the luciferase expression signal was detected after 48 hours.
  • the monoclonal cell line with a small number of surviving clones after infection with the NC virus component as a monoclonal cell line with low background activity
  • the monoclonal cell line with a large number of surviving clones after infection with the PC virus component as a monoclonal cell line that is sensitive to the screening system and has a strong survival ability
  • the monoclonal cell line with a high PC/NC ratio as a monoclonal cell line with good overall activity.
  • RNA aptamers screening system based on the CRIS PR/Cas9 system, which was named CRISmers.
  • CRISmers screening system can complete the quantitative conversion of aptamer-protein affinity recognition in cells, maximally maintain the natural structural state of the target protein, and at the same time, maximize the authenticity of the environment in which the aptamer will play a role in the future; on the other hand, multiple rounds of screening and high-concentration antibiotic screening in each round avoid background noise caused by the presence of dead cells, and also improve the stringency of screening.
  • lentiviral vector namely phU6-gLuc sgRNA-1.2 BsmBI-BsmBI-polyT-EF1a-NLS-linker-EcoRI-EcoRI-linker-VP64-P65-HSF1-NLS-linker-P2A-linker-Zeo-WPRE, as shown in Figure 12.
  • BsmBI a nucleic acid aptamer insertion site in the gLuc sgRNA-1.2 structural region
  • EcoRI an insertion site for the target protein to be screened
  • Fusion expression of the target protein to be screened and the transcription activation element VPH This lentiviral vector only requires one step of molecular cloning to replace the target protein of interest to be screened, and only one library construction is required to obtain an oligonucleotide random sequence library for the target protein of interest to be screened.
  • the universal screening monoclonal cell line #4-53 dCas9/9 ⁇ gLuc-puro constructed in the above Example 3 was used.
  • the workflow of a screening cycle can be divided into the following seven steps: First, based on the universal lentiviral expression vector, the target protein of interest is customized to be inserted with the help of EcoRI restriction endonuclease to form an intermediate vector. Synthesize an oligonucleotide random sequence library, and clone the random sequence library into the gLuc sgRNA backbone stem loop2 region of the intermediate vector with the help of BsmBI restriction endonuclease, thus completing the library construction.
  • oligonucleotide random sequence library containing the specific target protein for screening into lentiviral particles, and use Zeocin antibiotic to detect the viral titer of the lentiviral particles, thus completing the library lentiviral packaging.
  • these lentiviral particles will be in a certain The above #4-53 dCas9/9 ⁇ gLuc-puro universal screening reporter monoclonal cell line was infected with an MOI of .
  • 2 ⁇ g/ml puromycin selection pressure is applied to the cells infected with the lentiviral library.
  • the surviving cells are collected.
  • the cells are lysed, the DNA sequence containing the nucleic acid aptamer region is specifically amplified, and the obtained sequence is analyzed and verified by deep sequencing.
  • the selected nucleic acid aptamer sequences are secondary screened and verified using the luciferase reporter system.
  • the nucleic acid aptamer sequence amplified in the previous round is subcloned into the original universal lentiviral expression vector to start the next round of screening.
  • the sublibrary was infected with the #4-53 9 ⁇ gLuc-puro universal screening reporter monoclonal cell line at an MOI of about 0.1-0.2 to ensure that most cells only enter a virus particle containing 20 oligonucleotide sequences.
  • RNA aptamers for the RBD region of the SARS-CoV-2 S protein. Since the length of aptamers is mostly concentrated in 20-40 bases, we synthesized a 20-base oligonucleotide random sequence library. Existing molecular experimental techniques cannot obtain all sequence possibilities, that is, 4 20 differential sequences. We expanded the labor as much as possible during the construction of the library to obtain more oligonucleotide sequences. In the screening, we independently screened 2 parallel samples and infected cells with a larger lentiviral infection multiplicity in the initial round of screening to maximize the representativeness of aptamers in infected cells.
  • the obtained sequences were sorted in two ways: first, all independent sequences were arranged according to the number of repetitions, and the top 0.1% high-frequency sequences were selected for verification; second, the enrichment index of the sequence (the ratio of the number of repetitions of each independent sequence in two adjacent rounds) was calculated and sorted, and the top 15 sequences with high enrichment index in each round were selected for verification.
  • the RNA aptamers selected by the above two methods we first used the luciferase reporter system for the second screening after library screening.
  • the selected sequence was synthesized and constructed into the S1 protein RBD expression vector using BsmBI restriction endonuclease, namely phU6-gLuc sgRNA-1.2-BsmBI-BsmBI-polyT-EF1a-NLS-linker-S1 RBD-linker-VP64-P65-HSF1-NLS-linker-P2A-linker-Zeo-WPRE, and a total of three plasmids including 9 ⁇ gLuc sgRNA-gaussia luciferase and dCas9 expression elements were co-transfected into 293T cells to verify whether the selected RNA nucleic acid aptamer can recruit transcriptional activation elements and activate the expression of Gaussia luciferase by virtue of its affinity recognition with RBD.
  • BsmBI restriction endonuclease namely phU6-gLuc sgRNA-1.2-BsmBI-BsmBI-polyT-
  • nucleic acid aptamers that showed high and significant luciferase activation signals in the verification are #2-1-17, #2-1-18, #2-2-24, #3-1-31, #3-2-3, #4-1-20 and #5-2-15, and their sequences are listed in Table 8 above.
  • FIG. 8B shows the structures of aptamer sequence #2-1-18 and sequence #5-2-15 in sgRNA.
  • Figure 8C shows the secondary structure and free energy value of RNA aptamer predicted by Mfold webserver software.
  • Sequence #2-1-18 and sequence #5-2-15 were selected for subsequent functional verification.
  • ELONA enzyme-linked oligonucleotide assay
  • RNA aptamers were used 100 nM of aptamers against different concentrations of RBD recombinant protein in ELONA reaction to examine the detection limit of RNA aptamers.
  • PD1 programmed cell death protein 1
  • NSP7-8 non-structural proteins 7 and 8
  • random RNA sequences of the same length were used as negative controls.
  • the two aptamers targeting the wild-type spike protein RBD recombinant protein of SARS-CoV-2 showed good affinity and specificity, that is, the RBD recombinant protein at a concentration as low as 0.1 ⁇ g/ml could be detected.
  • the two aptamers targeting the SARS-CoV-2 concern mutant Delta spike protein RBD recombinant protein showed good affinity and specificity.
  • the two aptamers targeting the SARS-CoV-2 concern mutant Omicron BA.1 spike protein RBD recombinant protein showed good affinity and specificity.
  • the two aptamers targeting the SARS-CoV-2 concern mutant Omicron BA.2 spike protein RBD recombinant protein showed good affinity and specificity.
  • both nucleic acid aptamers showed good affinity and specificity, and could detect four pseudoviruses with a pseudovirus concentration titer as low as 1 TCID50/100 ⁇ l (50% tissue culture infectious dose, TCID50).
  • the results of the ELONA experiment based on the RBD recombinant protein are shown in Figure 10.
  • the fixed sequences at the ends are complementary and convenient for sequence synthesis and amplification, and the affinity activity of RNA aptamers for target proteins is significantly improved.
  • the random sequence may form different secondary structures with some bases of the fixed sequences at both ends, thereby affecting the formation of RNA aptamers. Therefore, in a preferred embodiment, the complete sequence of RNA aptamers includes the effective sequence obtained by the intermediate screening and the fixed sequences at both ends.
  • both nucleic acid aptamers can maintain binding activity at 4°C, 25°C and 37°C, indicating that they still have potential application advantages in a wider working temperature range.
  • RNA aptamers against SARS-CoV-2 concern variant live virus beads Delta (B.1.617.2) and Omicron BA.1 (B.1.1.529 BA.1) in the P3 laboratory.
  • the detection scheme is shown in Figure 12A. Because #5-2-15 showed better binding activity in multiple previous verifications, we first performed the following verification on #5-2-15.
  • RNA aptamers Before infecting VERO cells with live SARS-CoV-2 virus, we incubated different concentrations of RNA aptamers with live SARS-CoV-2 virus, and then incubated the RNA aptamer-live virus mixture with VERO cells. Then, on the second and fourth days after virus infection, the cell supernatant was collected and viral RNA was extracted, and the viral copy number was analyzed by RT-qPCR.
  • RNA aptamer #5-2-15 showed good dose-dependent inhibitory activity against two SARS-CoV-2 mutant live virus beads at two different time points.
  • the neutralization activity was detected 2 days after virus infection.
  • the IC50 value (half maximal inhibitory concentration) of #5-2-15 against SARS-CoV-2 Delta was 26.25ng ⁇ ml -1
  • the IC50 value for SARS-CoV-2 Omicron BA.1 was 0.4628ng ⁇ ml -1
  • the neutralization activity was detected 4 days after virus infection.
  • the IC50 value of #5-2-15 against SARS-CoV-2 Delta was 19.91 ng ⁇ ml -1
  • the IC50 value against SARS-CoV-2 Omicron BA.1 was 0.6006 ng ⁇ ml -1 .
  • nucleic acid aptamers helps to improve the stability of nucleic acid molecules, prolong their half-life, and reduce their immunogenicity.
  • the aptamers co-modified with fluorination and methoxyl groups have stronger binding activity than unmodified or other aptamers modified with fluorination or methoxyl groups alone.
  • the aptamers modified with 2'-F-O showed stronger binding activity than the unmodified aptamers, and the negative control had no binding activity.
  • the modified aptamers showed good dose-dependent binding activity against the RBD recombinant proteins of the two viruses, and when the unbiotinylated aptamers (Cold-apt) were added at the same time, they showed good dose-dependent competitive binding activity.
  • the results show that the Chol-PEG6-#5-2-15-2'-FO nucleic acid aptamer has virus prevention and treatment effects in animals 24 and 48 hours after SARS-CoV-2 Omicron BA.2 virus infection. Chol-PEG6-#5-2-15-2'-FO has better preventive and therapeutic effects on live virus infection for 48 hours and 24 hours.
  • the affinity binding ability of the nucleic acid aptamer with the target protein will be converted into the expression ability of the kanamycin reporter gene, that is, the survival ability of Escherichia coli when kanamycin is applied to Escherichia coli.
  • the results show that when the CRISmers system is transformed into Escherichia coli, the affinity of the #5-2-15 nucleic acid aptamer with the SARS-CoV-2 RBD target protein is converted into a resistance to kanamycin, supporting the growth of Escherichia coli in LB agar plates containing 100 ⁇ g ml -1 kanamycin.
  • the affinity of the #2-1-18 nucleic acid aptamer with the SARS-CoV-2 RBD target protein is converted into a resistance to kanamycin, supporting the growth of Escherichia coli in LB agar plates containing 75 ⁇ g ml -1 kanamycin. It shows that the CRISmers system of the present invention can be used to screen cells in different hosts.
  • the CRISmers system screening component into a vector, and used the second restriction endonuclease EcoRI to insert the target protein of interest into the vector for screening.
  • the first restriction endonuclease BsmBI was used to insert the random sequence library into the vector in the same way as the CRISPR sgRNA library was constructed to form a final screening library for screening.
  • the final screening library was transferred into the Escherichia coli screening host by electroporation, and after ampicillin enrichment and kanamycin screening, the Escherichia coli that survived ampicillin and kanamycin resistance were collected, and the plasmids in the Escherichia coli were extracted and the downstream sequencing and verification process was performed.

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Abstract

一种基于CRISPR/Cas技术针对靶蛋白筛选RNA核酸适配体的系统，系统包含：（i）向导RNA，其含有识别序列和预定长度的核酸适配体随机文库；（ii）靶向序列，其与向导RNA的识别序列配对；（iii）选择标记，其位于靶向序列下游，包括基本启动子和选择标记基因；（iv）融合蛋白，其包含靶蛋白和转录激活元件，靶蛋白与核酸适配体随机文库中的RNA核酸适配体结合导致转录激活元件与选择标记的结合；（v）dCas蛋白，其在所导RNA引导下特异性识别靶向序列，以及（vi）筛选细胞。还提供了利用该系统筛选针对靶蛋白的RNA核酸适配体的方法，以及针对SARS-CoV-2病毒的S1蛋白筛选到的RNA核酸适配体及其应用。

Description

筛选RNA核酸适配体的系统及方法 技术领域
本发明涉及筛选RNA核酸适配体的系统及方法，尤其涉及基于CRISPR/Cas技术的筛选RNA核酸适配体的系统及方法。本发明还涉及一种RNA核酸适配体，具体涉及一种用于检测或中和SARS-CoV-2病毒的RNA核酸适配体及其应用。
背景技术
1990年以来，指数富集配基的系统进化技术(Systematic evolution of ligands by exponential enrichment，SELEX)是筛选ssDNA或RNA核酸适配体的金标准方法。其过程主要包括SELEX技术方法的选择，寡核苷酸随机序列文库的合成与建立，靶标分子的获得，靶标分子与核酸文库的孵育、结合、洗脱、回收、扩增等反复选择性循环过程，最终特异的亲和序列即核酸适配体将会占据剩余文库序列种类的主体而最终被鉴定获得，后续再进行功能验证及化学修饰等后续探索性工作。最终的筛选产物对筛选环境即实验条件如筛选文库与靶标分子比例、缓冲液成分、离子强度、pH值、孵育温度、孵育时间以及靶标分子固有属性等要求十分敏感严苛，这些筛选变量叠加会产生特定的选择严格性，从而影响获得核酸适配体的真实亲和力和有效性。
基于纯化蛋白的SELEX筛选方式可能无法模拟靶标蛋白的天然结构状态；基于细胞的SELEX筛选其靶标往往集中在细胞表面，且可用于筛选的靶标仅占比较小的群体；基于动物的SELEX筛选成本高、缺乏特异性和通量，并且可能因在体内筛选环境变得复杂。
目前，具有良好应用前景的核酸适配体数量还远不能满足实际需求，核酸适配体的有效筛选方法仍有可发展的空间。
核酸适配体可应用于生物医学领域的不同场景，主要包括药物治疗、生物传感器技术、分子成像与体外诊断技术、生物标记与生物发现技术、药物递送技术以及抗病毒和疫苗的基因治疗技术。核酸适配体可偶联、标记及化学修饰的特性使其更易于与其他新技术如内源核酸分析、微流体细胞分离、流式细胞术、纳米颗粒等结合，用于提高诊断效果，促进临床研究。
随着新型冠状病毒疫情的爆发，各国科研机构和制药公司积极推进新冠病毒的药物筛选和疫苗研发。有必要提供更多的预防和/或治疗新冠肺炎的药物和方法。
发明内容
本发明的一方面提供一种针对靶蛋白筛选RNA核酸适配体的系统，其包含：(i)向 导RNA，含有识别序列和预定长度的核酸适配体随机文库；(ii)靶向序列，其与所述向导RNA的识别序列配对；(iii)选择标记，位于所述靶向序列下游，包括基本启动子和选择标记基因；(iv)融合蛋白，其包含所述靶蛋白和转录激活元件，所述靶蛋白与所述核酸适配体随机文库中的RNA核酸适配体结合导致所述转录激活元件与所述选择标记的结合；(v)dCas蛋白，其在所述向导RNA引导下特异性识别靶向序列；以及(vi)筛选细胞。在一些实施方案中，所述核酸适配体随机文库由长度为8至60个碱基、105至1036数量级的单链寡核苷酸随机序列组成。在一些实施方案中，核酸适配体随机文库插入到向导RNA骨架的loop区。在一些实施方案中，所述靶向序列是外源和/或人工序列。在一些实施方案中，所述靶向序列包含复数个拷贝的gLuc序列或Tet序列。在一些实施方案中，所述选择标记基因是荧光素酶基因、荧光蛋白基因或抗生素抗性基因。在一些实施方案中，所述荧光素酶基因选自萤火虫荧光素酶(Firefly luciferase)基因、海肾荧光素酶(Renilla luciferase)基因和长腹水蚤荧光素酶(Gaussia Luciferase)基因。在一些实施方案中，所述抗生素抗性基因是嘌呤霉素(Puromycin)抗性基因或卡那霉素(Kanamycin)抗性基因。在一些实施方案中，所述荧光蛋白基因是绿色荧光蛋白基因或红色荧光蛋白基因。在一些实施方案中，所述转录激活元件包含多个转录激活因子。在一些实施方案中，所述转录激活元件包含VP64、P65和HSF1。在一些实施方案中，其中所述基本启动子选自mini-promoter-1(SEQ ID NO:28)、mini-promoter-2(SEQ ID NO:27)、mini-TK promoter(SEQ ID NO:29)、mini-CMV promoter(SEQ ID NO:30)和Crystallin basal promoter(SEQ ID NO:31)。在一些实施方案中，所述dCas蛋白是dCas9蛋白或dUn1Cas12f1蛋白。在一些实施方案中，所述靶蛋白来源于病毒、细菌、真菌或动物。在一些实施方案中，所述靶蛋白来源于人类。在一些实施方案中，所述靶蛋白是绿色荧光蛋白或来源于SARS-CoV-2的S1蛋白的RBD区域。在一些实施方案中，所述筛选细胞是人源细胞HEK293T或原核细胞大肠杆菌E.coli。
本发明的另一方面提供一种针对靶蛋白筛选RNA核酸适配体的系统，其包含：病毒表达载体，所述病毒表达载体包含向导RNA和编码融合蛋白的核酸序列，其中所述向导RNA含有识别序列和预定长度的核酸适配体随机文库，所述融合蛋白包含所述靶蛋白和转录激活元件，所述靶蛋白与所述核酸适配体随机文库的结合导致所述转录激活元件与所述选择标记的结合；以及表达所述dCas蛋白的筛选细胞，其包含靶向序列和选择标记，其中所述靶向序列与所述向导RNA的识别序列配对，所述选择标记位于所述靶向序列下游且包括基本启动子和选择标记基因。在一些实施方案中，所述病毒表达载体是慢病毒表达载体。在一些实施方案中，所述慢病毒表达载体进一步包含可操作地连接至所述向导RNA的第一启 动子和可操作地连接至所述融合蛋白的编码序列的第二启动子。在一些实施方案中，所述核酸适配体随机文库通过第一限制性内切酶插入到向导RNA骨架。在一些实施方案中，所述靶蛋白的编码序列通过第二限制性内切酶可操作地连接至所述第二启动子。在一些实施方案中，所述慢病毒表达载体包含位于向导RNA下游的polyT。在一些实施方案中，所述慢病毒表达载体包含核定位序列(NLS)。在一些实施方案中，所述NLS包括在第二启动子下游的第一NLS和在所述融合蛋白的编码序列的下游的第二NLS。在一些实施方案中，所述慢病毒表达载体包含位于第二NLS下游的P2A、抗生素抗性基因和转录后调控元件。在一些实施方案中，所述P2A通过连接子分别与上游的第二NLS和下游的抗生素抗性基因连接，所述靶蛋白的编码序列通过连接子分别与上游的第一NLS和下游的转录激活因子的编码序列连接。
本发明的另一方面提供一种针对靶蛋白筛选RNA核酸适配体的系统，其包含：一种或多种质粒载体，所述质粒载体包含选自所述向导RNA；所述靶向序列和所述选择标记；编码所述融合蛋白的核酸序列；以及编码所述dCas蛋白的核酸序列中的一个或多个，其中所述向导RNA含有识别序列和预定长度的核酸适配体随机文库，所述靶向序列与所述向导RNA的识别序列配对，所述选择标记位于所述靶向序列下游且包括基本启动子和选择标记基因，所述融合蛋白包含所述靶蛋白和转录激活元件，所述靶蛋白与所述核酸适配体随机文库中的RNA核酸适配体结合导致所述转录激活元件与所述选择标记的结合，所述dCas蛋白在所述向导RNA引导下特异性识别靶向序列；以及筛选细胞。在一些实施方案中，所述系统包含一种质粒载体，所述质粒载体包含所述向导RNA、所述靶向序列、所述选择标记、以及编码所述融合蛋白和所述dCas蛋白的核酸序列。在一些实施方案中，所述筛选细胞是原核细胞。
本发明的另一方面提供一种筛选针对靶蛋白的RNA核酸适配体的方法，其包含以下步骤：(1)提供筛选细胞，所述筛选细胞具有向导RNA、靶向序列、选择标记、融合蛋白和dCas蛋白；其中所述向导RNA含有识别序列和预定长度的核酸适配体随机文库；所述靶向序列与所述向导RNA的识别序列配对；所述选择标记位于所述靶向序列下游且包括基本启动子和选择标记基因；所述融合蛋白包含所述靶蛋白和转录激活元件，所述靶蛋白与所述核酸适配体随机文库中的RNA核酸适配体结合导致所述转录激活元件与所述选择标记的结合；所述dCas蛋白在所述向导RNA引导下特异性识别靶向序列；(2)利用选择标记基因对筛选细胞进行筛选；(3)收集表达所述选择标记基因的筛选细胞；(4)裂解筛选细胞，对含有核酸适配体区域的序列，进行特异性扩增，通过测序获得所述核酸适配体的序列信息。在 一些实施方案中，步骤(4)进一步包含对获得的核酸适配体序列进行分析与验证。在一些实施方案中，所述筛选细胞是通过将一种或多种质粒载体转染至原核细胞中得到的，所述质粒载体包含选自所述向导RNA；所述靶向序列和所述选择标记；编码所述融合蛋白的核酸序列；以及编码所述dCas蛋白的核酸序列中的一个或多个。在一些实施方案中，所述原核细胞是大肠杆菌细胞。在一些实施方案中，所述筛选细胞通过以下具体步骤提供：(1.1)提供一种表达所述dcas蛋白的细胞，所述细胞还包含所述靶向序列和所述选择标记；(1.2)提供一种编码所述向导RNA和所述融合蛋白的病毒表达载体；(1.3)对所述病毒表达载体进行包装；然后使用包装后的病毒表达载体感染所述细胞。在一些实施方案中，所述病毒表达载体是慢病毒表达载体。
本发明的另一方面提供一种特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。
本发明的另一个方面提供一种检测SARS-CoV-2病毒的检测试剂或试剂盒，其包含特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。
本发明的另一方面提供一种检测SARS-CoV-2病毒的方法，所述方法包含使待检测样品与本发明的特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体接触，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。
本发明的另一个方面提供本发明的特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体在制备检测SARS-CoV-2病毒的检测试剂或试剂盒中的应用，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。
本发明的另一方面提供一种包含特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体的药物，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述药物进一步包含病毒载体或非病毒载体，其中所述载体包含或携带所述特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。
本发明的另一个方面提供一种在对象中中和SARS-CoV-2病毒的方法，所述方法包含 向有需要的对象施用有效量的本发明的药物，其中所述药物包含特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述药物进一步包含病毒载体或非病毒载体，其中所述载体包含或携带所述特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。
本发明的另一方面提供本发明的药物在制备用于在对象中中和SARS-CoV-2病毒的药物中的应用，其中所述药物包含特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述药物进一步包含病毒载体或非病毒载体，其中所述载体包含或携带所述特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。
本发明的另一个方面提供一种治疗或预防SARS-CoV-2病毒感染的方法，所述方法包含向有需要的对象施用有效量的本发明的药物，其中所述药物包含特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述药物进一步包含病毒载体或非病毒载体，其中所述载体包含或携带所述特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。
本发明的另一个方面提供本发明的药物在制备用于在对象中治疗或预防SARS-CoV-2病毒感染的药物中的应用，其中所述药物包含特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述药物进一步包含病毒载体或非病毒载体，其中所述载体包含或携带所述特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。
本发明的另一方面提供一种药物组合物，其包含本发明的药物和药学上可接受的赋形剂，其中所述药物包含特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述药物进一步包含病毒载体或非病毒载体，其中所述载体包含或携带所述特异性 结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。
本发明的另一个方面提供一种在对象中中和SARS-CoV-2病毒的方法，所述方法包含向有需要的对象施用有效量的本发明的药物组合物，其中所述药物组合物包含本发明的药物和药学上可接受的赋形剂，所述药物包含特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述药物进一步包含病毒载体或非病毒载体，其中所述载体包含或携带所述特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。
本发明的另一个方面提供本发明的药物组合物在制备用于中和SARS-CoV-2病毒的药物中的应用，其中所述药物组合物包含本发明的药物和药学上可接受的赋形剂，所述药物包含特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述药物进一步包含病毒载体或非病毒载体，其中所述载体包含或携带所述特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。
本发明的另一个方面提供一种在对象中治疗或预防SARS-CoV-2病毒感染的方法，所述方法包含向有需要的对象施用有效量的本发明的药物组合物，其中所述药物组合物包含本发明的药物和药学上可接受的赋形剂，所述药物包含特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述药物进一步包含病毒载体或非病毒载体，其中所述载体包含或携带所述特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。
本发明的另一个方面提供本发明的药物组合物在制备用于在对象中治疗或预防SARS-CoV-2病毒感染的药物中的应用，其中所述药物组合物包含本发明的药物和药学上可接受的赋形剂，所述药物包含特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述药物进一步包含病毒载体或非病毒载体，其中所述载体包含或携带所述特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。
本发明的另一方面提供一种在对象中中和SARS-CoV-2病毒的药品套装，所述药品套 装包含独立存在的两种或多种本发明的药物或药物组合物。
本发明的另一方面提供一种在对象中治疗或预防SARS-CoV-2病毒感染的药品套装，所述药品套装包含独立存在的两种或多种本发明的药物或药物组合物。
附图说明
图1：基于CRISPR/Cas9的核酸适配体筛选系统。(A)RNA核酸适配体筛选系统的分子装置示意图。(B)RNA核酸适配体筛选系统筛选RNA核酸适配体的过程。其中，核酸适配体标注为红色；感兴趣的靶蛋白(X-protein)标注为橙色；转录激活结构域标注为绿色；mini-promoter标注为蓝色，选择标记基因标注为粉红色。
图2：验证核酸适配体筛选系统。(A)GFP aptamer分别被插入到sgRNA骨架的tetra loop区(sgRNA 1.1)或stem loop 2区(sgRNA 1.2)。(B)用于检测GFP靶蛋白与其RNA核酸适配体亲和力的荧光素酶报告系统模式图。(C)基于B中的荧光素酶报告体系，使用1个拷贝的gLuc sgRNA靶序列来检测系统灵敏度(1×gLuc)。(D)基于B中的荧光素酶报告体系，使用9个拷贝的gLuc sgRNA靶序列来检测系统灵敏度(9×gLuc)。(E)在#4-53 dCas9/9×gLuc-Puro单克隆细胞系中分别递送表达sgRNA 1.2-GFP aptamer或sgRNA1.2-随机aptamer序列、GFP-VPH元件的慢病毒，经由嘌呤霉素筛选后对存活克隆的数量进行定量统计。PC/NC比率在数据图顶端显示，嘌呤霉素使用浓度在数据图底部显示。Mock表示瞬时转染质粒对照；UR表示随机序列对照；NC表示递送表达sgRNA 1.2-随机aptamer序列以及GFP-VPH慢病毒组分的阴性实验组；PC表示递送表达sgRNA 1.2-GFP aptamer序列以及GFP-VPH慢病毒组分的阳性实验组；数据来自三个生物学重复的平均值±标准差。*代表P<0.05，**P<0.01，***代表P<0.001，双尾t检验。
图3：用于检测GFP靶蛋白与GFP RNA核酸适配体亲和力的嘌呤霉素报告系统的示意图与dCas9/9×gLuc-puro单克隆细胞系的筛选、鉴定流程。
图4：通过量化存活克隆数来鉴定dCas9/9×gLuc-puro单克隆细胞系的性能。嘌呤霉素所使用的三个不同浓度在图中展示。NC表示阴性对照；PC表示阳性对照。数据来自三个生物学重复的平均值±标准差。
图5：基于CRISPR/Cas9的CRISmers RNA核酸适配体筛选系统示意图。
图6：针对SARS-CoV-2野生型刺突蛋白RBD区域进行CRISmers筛选后利用深度测序的序列分析结果。R：筛选轮数，Filtered sequences：过滤掉一些错误序列后的测序序列，Unique sequences：单一/独特序列。
图7：基于荧光素酶报告系统的二次筛选结果。第二轮、第三轮、第四轮以及第五轮 筛选中获取的序列经深度测序并分析后获得的前0.1％高频序列和排名前15条高富集指数序列经荧光素酶报告系统进行二次筛选。实验共进行了2个独立的CRISmers筛选(结果分别为A和B)。数据来自三个生物学重复的平均值±标准差。
图8：CRISmers系统靶向SARS-CoV-2野生型刺突蛋白RBD区域筛选获得的活性序列。(A)经荧光素酶报告系统二次筛选后对图7中高激活序列进行平行比较的结果。(B)在荧光素酶平行比较实验中(图7A)激活倍数最高的两条核酸适配体序列#2-1-18和#5-2-15。(C)利用Mfold webserver软件对这两条核酸适配体的二级结构以及自由能进行分析。数据来自三个生物学重复的平均值±标准差。
图9：核酸适配体对SARS-CoV-2的检测能力。(A)酶联寡核苷酸测定方法的示意图。(B)在250ng的SARS-CoV-2野生型刺突蛋白RBD固定浓度下检测核酸适配体的剂量依赖活性。乱序RNA为阴性对照。(C，G)使用100nM核酸适配体测试对野生型SARS-CoV-2刺突蛋白RBD蛋白的检测极限，重组PD-1蛋白和SARS-CoV-2 NSP7-8复合蛋白作为阴性对照用于检测核酸适配体的检测特异性。(D，H)使用100nM核酸适配体测试对SARS-CoV-2突变型刺突蛋白Delta RBD蛋白的检测极限，重组PD-1蛋白和SARS-CoV-2 NSP7-8复合蛋白作为阴性对照用于检测核酸适配体的检测特异性。(E，I)使用100nM核酸适配体测试对SARS-CoV-2突变型刺突蛋白Omicron BA.1 RBD蛋白的检测极限，重组PD-1蛋白和SARS-CoV-2 NSP7-8复合蛋白作为阴性对照用于检测核酸适配体的检测特异性。(F，J)使用100nM核酸适配体测试对SARS-CoV-2突变型刺突蛋白Omicron BA.2 RBD蛋白的检测极限，重组PD-1蛋白和SARS-CoV-2 NSP7-8复合蛋白作为阴性对照用于检测核酸适配体的检测特异性。(K，L，M，N)分别使用100nM核酸适配体测试SARS-CoV-2野生型刺突蛋白假病毒(K)，SARS-CoV-2突变型Delta刺突蛋白假病毒(L)，SARS-CoV-2突变型Omicron BA.1刺突蛋白假病毒(M)以及SARS-CoV-2突变型Omicron BA.2刺突蛋白假病毒(N)的检测极限。乱序RNA为阴性对照。TCID50：半数组织培养感染剂量。数据来自三个生物学重复的平均值±标准差。*代表P<0.05，**代表P<0.01，***代表P<0.001，****代表P<0.0001，双尾t检验，n.s.代表差异不显著。
图10：两端固定序列对RNA核酸适配体活性的影响探究(ELONA)。利用ELONA检测经筛选获得的中间序列#Core-2-1-18和#Core-5-2-15与结合两端固定序列的完整序列#2-1-18、#5-2-15针对RBD蛋白的结合活性。乱序RNA为阴性对照。数据来自三个生物学重复的平均值±标准差。**代表P<0.01，****代表P<0.0001，双尾t检验，n.s.代表差异不显著。
图11：核酸适配体在不同温度下的结合活性测定。利用ELONA检测核酸适配体对RBD蛋白在4℃，25℃和37℃的结合活性。乱序RNA为阴性对照。数据来自三个生物学重复的平均值±标准差。***代表P<0.001，****代表P<0.0001，双尾t检验。
图12：核酸适配体(#5-2-15)对SARS-CoV-2变异活病毒珠的中和活性检测。(A)针对SARS-CoV-2活病毒中和实验的示意图。病毒感染2天后核酸适配体#5-2-15中和SARS-CoV-2 Delta(B)和Omicron BA.1(C)变异病毒珠，相对病毒RNA拷贝数的定量结果。病毒感染4天后核酸适配体#5-2-15中和SARS-CoV-2 Delta(D)和Omicron BA.1(E)变异病毒珠，相对病毒RNA拷贝数的定量结果。阴性对照读值被用来做100％标准化计算。实验样品分别为病毒感染2天和4天后收集的细胞上清。IC50在图中被标注。数据来自三个生物学重复的平均值±标准差。
图13：核酸适配体(#2-1-18)对SARS-CoV-2变异活病毒珠Omicron BA.1的中和活性检测结果。病毒感染2天和4天后，分别收集细胞培养上清，核酸适配体中和SARS-CoV-2 Omicron BA.1变异病毒珠后2天(A)和4天(B)，相对病毒RNA拷贝数的定量结果。阴性对照读值被用来做100％标准化计算。实验样品为病毒感染2天后收集的细胞上清。IC50在图中被标注。数据来自三个生物学重复的平均值±标准差。
图14：修饰后的核酸适配体结合活性与特异性验证。(A，B)利用ELONA方法检测未修饰和修饰后核酸适配体对SARS-CoV-2变异病毒株Omicron BA.2刺突蛋白RBD重组蛋白的结合活性验证。核酸适配体浓度分别为100nM(A)和1000nM(B)。Native:未经修饰的RNA核酸适配体；2’-F:核酸序列嘧啶核糖2’位置羟基进行氟化修饰；2’-O:核酸序列嘌呤核糖2’位置羟基进行甲氧基修饰；2’-F-O:核酸序列嘧啶2’位置进行氟化修饰，嘌呤2’位置进行甲氧基修饰。(C)凝胶阻滞迁移率检测实验EMSA示意图。(D)利用凝胶阻滞迁移率检测实验平行检测未修饰和修饰后核酸适配体对SARS-CoV-2刺突蛋白RBD重组蛋白的结合活性。scrambled RNA:无义乱序RNA，scrambled RNA-2’-F-O:2’-F-O修饰的无义乱序RNA，(E，F)利用凝胶阻滞迁移率检测实验分别检测修饰后核酸适配体针对SARS-CoV-2野生型病毒株刺突蛋白RBD重组蛋白(E)与SARS-CoV-2变异病毒株Omicron BA.2刺突蛋白RBD重组蛋白的剂量依赖结合活性与剂量依赖特异竞争性结合活性(F)。乱序RNA作为阴性对照。Biotin-apt：生物素化的核酸适配体；Cold-apt：未经修饰的核酸适配体。数据来自三个生物学重复的平均值±标准差。**P<0.01,***P<0.001,****P<0.0001,双尾t检验，n.s.,无显著差异。
图15：氟化甲氧基修饰的核酸适配体(#2-1-18-2’-F-O)对SARS-CoV-2变异活病 毒珠Omicron BA.1的中和活性检测结果。核酸适配体中和SARS-CoV-2 Omicron BA.1变异病毒珠后2天(A)和4天(B)，相对病毒RNA拷贝数的定量结果。阴性对照读值被用来做100％标准化计算。实验样品为病毒感染2天和4天后收集的细胞上清。IC50在图中被标注。数据来自三个生物学重复的平均值±标准差。
图16：氟化甲氧基修饰的核酸适配体(#5-2-15-2’-F-O)对SARS-CoV-2变异活病毒珠Omicron BA.1的中和活性检测结果。核酸适配体中和SARS-CoV-2 Omicron BA.1变异病毒珠后2天(A)和4天(B)，相对病毒RNA拷贝数的定量结果。阴性对照读值被用来做100％标准化计算。实验样品为病毒感染2天和4天后收集的细胞上清。IC50在图中被标注。数据来自三个生物学重复的平均值±标准差。
图17：氟化甲氧基修饰的核酸适配体(#2-1-18-2’-F-O和#5-2-15-2’-F-O)对SARS-CoV-2变异活病毒珠Omicron BA.2的中和活性检测结果。#2-1-18-2’-F-O(A)和#5-2-15-2’-F-O(B)核酸适配体中和SARS-CoV-2 Omicron BA.2变异病毒珠后，相对病毒RNA拷贝数的定量结果。阴性对照读值被用来做100％标准化计算。实验样品为病毒感染2天后收集的细胞上清。IC50在图中被标注。数据来自三个生物学重复的平均值±标准差。
图18：5’端附加40kDa PEG的氟化甲氧基修饰的核酸适配体(PEG40K-2-1-18-2’-F-O和PEG40K-5-2-15-2’-F-O)对SARS-CoV-2变异活病毒珠Omicron BA.2的中和活性检测结果。PEG40K-2-1-18-2’-F-O(A)和PEG40K-5-2-15-2’-F-O(B)核酸适配体中和SARS-CoV-2 Omicron BA.2变异病毒珠后，相对病毒RNA拷贝数的定量结果。阴性对照读值被用来做100％标准化计算。实验样品为病毒感染2天后收集的细胞上清。IC50在图中被标注。数据来自三个生物学重复的平均值±标准差。
图19：5’端附加胆固醇-PEG6的氟化甲氧基修饰的核酸适配体(chol-PEG6-5-2-15-2’-F-O)对SARS-CoV-2变异活病毒珠Omicron BA.2的中和活性检测结果。chol-PEG6-5-2-15-2’-F-O核酸适配体中和SARS-CoV-2 Omicron BA.2变异病毒珠后，相对病毒RNA拷贝数的定量结果。阴性对照读值被用来做100％标准化计算。实验样品为病毒感染2天后收集的细胞上清。IC50在图中被标注。数据来自三个生物学重复的平均值±标准差。
图20：5’端附加胆固醇-PEG24的氟化甲氧基修饰的核酸适配体(chol-PEG24-5-2-15-2’-F-O)对SARS-CoV-2变异活病毒珠Omicron BA.2的中和活性检测结果。chol-PEG24-5-2-15-2’-F-O核酸适配体中和SARS-CoV-2 Omicron BA.2变异病毒珠后，相对病毒RNA拷贝数的定量结果。阴性对照读值被用来做100％标准化计算。实验样品为病毒感染2天后收集的细胞上清。IC50在图中被标注。数据来自三个生物学重复的平均值±标准差。
图21：5’端附加胆固醇-40kDa PEG的氟化甲氧基修饰的核酸适配体(chol-PEG40K-5-2-15-2’-F-O)对SARS-CoV-2变异活病毒珠Omicron BA.2的中和活性检测结果。chol-PEG40K-5-2-15-2’-F-O核酸适配体中和SARS-CoV-2 Omicron BA.2变异病毒珠后，相对病毒RNA拷贝数的定量结果。阴性对照读值被用来做100％标准化计算。实验样品为病毒感染2天后收集的细胞上清。IC50在图中被标注。数据来自三个生物学重复的平均值±标准差。
图22：胆固醇对SARS-CoV-2变异活病毒珠Omicron BA.2的中和活性检测结果。胆固醇作用SARS-CoV-2 Omicron BA.2变异病毒珠后，相对病毒RNA拷贝数的定量结果。阴性对照读值被用来做100％标准化计算。实验样品为病毒感染2天后收集的细胞上清。IC50在图中被标注。数据来自三个生物学重复的平均值±标准差。
图23：40kDa PEG对SARS-CoV-2变异活病毒珠Omicron BA.2的中和活性检测结果。40kDa PEG作用SARS-CoV-2 Omicron BA.2变异病毒珠后，相对病毒RNA拷贝数的定量结果。阴性对照读值被用来做100％标准化计算。实验样品为病毒感染2天后收集的细胞上清。IC50在图中被标注。数据来自三个生物学重复的平均值±标准差。
图24：胆固醇-40kDa PEG对SARS-CoV-2变异活病毒珠Omicron BA.2的中和活性检测结果。胆固醇-40kDa PEG作用SARS-CoV-2 Omicron BA.2变异病毒珠后，相对病毒RNA拷贝数的定量结果。阴性对照读值被用来做100％标准化计算。实验样品为病毒感染2天后收集的细胞上清。IC50在图中被标注。数据来自三个生物学重复的平均值±标准差。
图25：关于Chol-PEG40K-5-2-15-2’-F-O的动物验证实验。(A)为检测Chol-PEG40K-5-2-15-2’-F-O在体内针对SARS-CoV-2活病毒变异株Omicron BA.2预防作用与治疗作用的动物实验示意图。(B)RT-qPCR实验检测预防实验中肺组织Omicron BA.2病毒RNA相对拷贝数。(C)FRNT实验检测预防实验中肺组织中Omicron BA.2病毒RNA滴度。(D)RT-qPCR实验检测治疗效果实验中肺组织Omicron BA.2病毒RNA相对拷贝数。(E)FRNT实验检测治疗效果实验中肺组织Omicron BA.2病毒RNA滴度。FRNT：活病毒灶减少中和实验。阴性对照读值被用来做100％标准化计算。数据来自三个生物学重复的平均值±标准差。**P<0.01，***代表P<0.001，****代表P<0.0001，双尾t检验。
图26：关于Chol-PEG6-5-2-15-2’-F-O的动物验证实验。(A)RT-qPCR实验检测预防实验中肺组织Omicron BA.2病毒RNA相对拷贝数。(B)FRNT实验检测预防实验中肺组织中Omicron BA.2病毒RNA滴度。(C)RT-qPCR实验检测治疗效果实验中肺组织Omicron BA.2病毒RNA相对拷贝数。(D)FRNT实验检测治疗效果实验中肺组织Omicron BA.2病 毒RNA滴度。FRNT：活病毒灶减少中和实验。阴性对照读值被用来做100％标准化计算。数据来自三个生物学重复的平均值±标准差。*P<0.05，**P<0.01，***代表P<0.001，n.s.,无显著差异，双尾t检验。
图27：CRISmers系统在不同CRISPR/Cas系统中的验证。以现目前体积较小版本的dCasMINI-V4(UniCas12f1 CRISPR/Cas系统的进化版本)用于替换CRISmers系统中dCas9以检测CRISmers系统的可迁移性的实验示意图(A)。aptamer序列被插入dCasMINI-V4系统sgRNA骨架的loop2位置。萤火虫荧光素酶报告实验用以检测激活效果(B)。乱序RNA核酸适配体作为阴性对照。数据来自三个生物学重复的平均值±标准差。***代表P<0.001，****代表P<0.0001，双尾t检验。
图28：CRISmers系统使用不同选择标志物的效果验证。将CRISmers系统中原嘌呤霉素报告基因替换为绿色荧光蛋白报告基因用于报告实验的示意图(A)。核酸适配体与靶蛋白的亲和结合能力将转化为绿色荧光蛋白的表达能力。流式细胞术检测实验中由核酸适配体#2-1-18、#5-2-15介导的绿色荧光蛋白激活的效率(B)。cell：空细胞组；NC：阴性对照组；GFP％：GFP阳性细胞所占百分比。
图29：CRISmers系统在不同宿主细胞中的效果验证。将CRISmers系统中原嘌呤霉素报告基因替换为大肠杆菌卡纳霉素报告基因，并转化至大肠杆菌中检测激活实验的示意图(A)。CRISmers系统在大肠杆菌宿主中的应用效果(B)。
图30：可定制化的用于真核细胞HEK293T-dCas9细胞系筛选的慢病毒载体(phU6-gLuc sgRNA-1.2 BsmBI-BsmBI-polyT-EF1a-NLS-linker-EcoRI-EcoRI-linker-VP64-P65-HSF1-NLS-linker-P2A-linker-Zeo-WPRE)的示意图。
图31：可定制化的用于原核细胞大肠杆菌筛选的质粒载体(phU6-gLuc sgRNA-1.2BsmBI-BsmBI-polyT-EF1a-NLS-linker-EcoRI-X-protein-EcoRI-linker-VP64-P65-HSF1-NLS-linker-P2A-dCas9-WPRE-SV40promoter-Zeo)的示意图。
具体实施方式
定义
术语“一”或“一个”实体是指该实体中的一个或多个；例如，“外泌体”被理解为表示一个或多个外泌体。因此，术语“一个”、“一个或多个”和“至少一个”可以在本文中互换使用。
“对象”或“患者”、“个体”是指任何期望进行诊断、预后或治疗的对象，特别是哺乳动物对象。哺乳动物包括人、家畜、农畜、动物园动物、竞技动物或宠物，例如狗、猫、猪、兔、大鼠、小鼠、马、牛、奶牛等。本文所称的对象优选是人。
在本发明中，术语“治疗”是指疗法上的以及预防性的措施，其阻止或减缓对象发生不期望的生理学改变或病症，例如病毒感染、发热、干咳、乏力等上呼吸道和消化道症状。有利或期望的临床效果包括，但不限于，病毒的减少或消灭、症状的缓解、疾病程度的降低、疾病状态的稳定化(即不恶化)、疾病进展的延迟或减缓、疾病状态的减轻或缓和以及疾病的部分或全部治愈，而不论上述效果是否可检测到。“治疗”也可指与不治疗相比生存期延长。需要治疗的对象包括已患有该疾病或病症的对象，以及有可能患有该疾病或病症的对象，或要预防该疾病或病症的对象。
本文所用的术语“有治疗需要的患者”或“有治疗需要的对象”包括因施用本发明用于例如检测、诊断和/或治疗用途的多肽或其组合物而受益的对象，如哺乳动物对象。
如本文中所使用的，术语“治疗有效量”或“有效量”是指当将本发明的药物或药物组合物单独给予或与另外的治疗剂联合给予细胞、组织或受治疗者时，其有效防止或减缓待治疗的疾病或病症的量。治疗有效剂量进一步指所述化合物足以导致症状减缓的量，所述减缓症状例如为治疗、治愈、防止或减缓相关医学状态，或提高对所述病征的治疗率、治愈率、防止率或减缓率。当施用给个体单独给予的活性成分时，治疗有效量是指该单独的成分。当施用组合时，治疗有效量是指产生治疗效果的活性成分的联合的量，而不论其是联合给予、连续给予还是同时给予。治疗有效量将减轻症状通常至少10％；通常至少20％；优选至少约30％；更优选至少40％和最优选至少50％。
在本发明中，“约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，在本领域每一次实行中“约”可意味着在1内或超过1的标准差。或者，“约”或“基本上包含”可意味着至多20％的范围。此外，对于生物学系统或过程而言，该术语可意味着至多一个数量级或数值的至多5倍。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“基本上包含”的含义应该假定为在该具体值的可接受误差范围内。
如本文所使用的，“互补的”是指寡聚化合物的两个核碱基之间精确配对的能力。例如，如果寡核苷酸(寡聚化合物)的某个位置处的核碱基能够与靶核酸的某个位置处的核碱基形成氢键，则所述靶核酸是DNA、RNA或寡核苷酸分子，然后，寡核苷酸和靶核酸之间氢键的位置被认为是互补位置。当每个分子中足够数量的互补位置被能够彼此结合成氢键的核碱基占据时，寡核苷酸和另外的DNA、RNA或寡核苷酸分子彼此互补。因此，“互补的”是用于表示足够数量的核碱基足够程度的精确配对或互补性，使得在寡核苷酸和靶核酸之间稳定的和特异性的结合的术语。
应理解，在本领域中核酸适配体两端的固定序列不需要100％互补即可特异性杂交形成“茎”结构。此外，寡核苷酸可以在一个或多个区段上杂交，使得插入或相邻区段不参与杂交事件(例如环结构或发夹结构)。优选本发明的核酸适配体两端的固定序列具有至少70％、或至少75％、或至少80％、或至少85％的序列互补性，更优选地是它们包含至少90％的序列互补性，甚至更优选地包含至少95％或至少99％的序列互补性。例如，5’端固定序列的20个核碱基中的18个与3’端固定序列互补将代表90％的互补性。在该实施例中，剩余的非互补核碱基可以与互补的核碱基成簇或散布，并且不需要彼此邻接或与互补的核碱基邻接。因此，长度为18个核碱基的反义寡聚体具有4个非互补核碱基，其侧翼为与靶核酸完全互补的两个区域，与靶核酸具有77.8％的总体互补性，因此属于本发明的范围。可以使用本领域已知的BLAST程序(基础的局部比对搜索工具)和PowerBLAST程序常规地确定5’端固定序列与3’端固定序列的互补百分比。
如本文中所使用的，“gLuc序列”和“Tet序列”是外源人造的不属于人类基因组的序列，其具有较好的可靶向能力，并且可避免因靶向细胞内源基因组位点而引发不必要的副作用。
如本文所使用的，术语“连接子”是指含有两个或更多个相同的或不同的核苷酸的核酸序列的短片段，其中所述核苷酸选自腺嘌呤(A)、鸟嘌呤(G)、胞嘧啶(C)、胸腺嘧啶(T)和尿嘧啶(U)。
如本文所使用的，术语“启动子”是RNA聚合酶识别、结合和开始转录的一段DNA序列，它含有RNA聚合酶特异性结合和转录起始所需的保守序列，多数位于结构基因转录起始点的上游，启动子本身不被转录。“基本启动子(basal promoter,minimal promoter)”是仅仅由所有的转录起始所需的基础元件(例如TATA-框和/或起始密码子)组成的一种启动子。存在适当的转录因子时，基本启动子发挥作用以进行转录。在一些实施方案中，第一启动子选自U6、7SK、H1。在一些实施方案中，第二启动子是EF1a。在一些实施方案中，选择标记包含的基本启动子选自mini-promoter-1(SEQ ID NO:28)、mini-promoter-2(SEQ ID NO:27)、mini-TK promoter(SEQ ID NO:29)、mini-CMV promoter(SEQ ID NO:30)和Crystallin basal promoter(SEQ ID NO:31)。
CRISPR/Cas
CRISPR/Cas系统源自细菌和古细菌的适应性免疫系统，它们通过该套系统保护自己免受外来病毒和质粒核酸的入侵。其中的CRISPR序列可转录并可加工非编码RNA，即CRISPRRNA,crRNA，其可直接作用于DNA而非RNA。向导RNA(guide RNA，gRNA)， 也称为小向导RNA(small guide RNA，sgRNA)。是作用于动质体(kinetoplastid)体内一种称为RNA编辑(RNA editing)的后转录修饰过程中。也是一种小型非编码RNA。CRISPR相关蛋白(简称Cas蛋白)是一种核酸内切酶，可以利用CRISPR序列中间隔序列(spacer)对应的RNA指引，识别并且切割特定与其序列互补的DNA链。失活的Cas(Deactivated Cas，dCas)是指通过点突变失去核酸酶活性但保留结合酶活性的Cas蛋白。dCas蛋白可与转录抑制结构域(TRD)或转录激活结构域(TAD)融合，dCas蛋白可以与效应蛋白融合，包括转录激活子、抑制子和表观调节子，分别实现有效的基因特异性CRISPR介导的激活(CRISPRa)、干扰(CRISPRi)和表观基因组修饰。在一些实施方案中，dCas蛋白是dCas9。在一些实施方案中，dCas9蛋白是dCasMINI-V4。
CRISPR/Cas9系统是由crRNAs指引并在DNA中造成双链断裂。在CRISPR序列附近存在一些小RNA tracrRNA，其与重复序列互补结合，在Cas9因子存在前提下经过RNA酶III识别与剪切，产生成熟的crRNA。2012年Jennifer等人将tracrRNA和crRNA连接成为单链向导RNA嵌合体，并借助化脓链球菌CRISPR/Cas系统(spCas9系统)首次在体外实现向导RNA引导Cas9蛋白完成对靶向DNA的双链切割。CRISPR/Cas9系统通过sgRNA与靶向DNA序列以碱基互补配对的方式进行特异性结合，引导具有核酸酶切割活性的Cas9蛋白识别并作用于与sgRNA互补的含有3‘端PAM序列(NGG)的靶序列并实现特异性的双链断裂，随后细胞内源的DNA修复机制启动，可实现非同源末端连接和同源介导的DNA双链修复，从而产生相应的基因编辑事件。
Cas9的核酸酶结构域(例如，在HNH结构域中产生H840A突变和在RuvC结构域中产生D10A突变)进行突变，以产生核酸酶缺陷的“dCas9”。这种“钝化”和“死亡”Cas9失去切割DNA的功能，但在gRNA的指导下仍能以相同的精确度靶向和结合DNA。dCas9可以招募效应器(阻遏蛋白和激活剂结构域)到启动子区域、调控区域或编码区域，对任何基因进行精确定点调控而不造成DNA损伤。
CasMINI是Un1Cas12f1的变体版本，其比Cas9尺寸更小，属于新一代体积更小的基因编辑系统。在本发明中，我们采用CasMINI的“dead”版本dUn1Cas12f1(dCasMINI-V4，V4指dCasMINI的第4版本)，即仅具有靶向结合靶基因的功能，无切割功能。
SARS-CoV-2 S1蛋白RBD区域
近年来，全球相继暴发由新型冠状病毒(Severe cute respiratory syndrome coronavirus 2,SARS-CoV-2)引发的新型冠状病毒肺炎(COVID-19)，下文简称新冠肺炎，严重威胁全人类的健康和生命安全，随着疫情的不断发展，除了最开始出现的SARS-CoV-2病毒野生型之 外，SARS-CoV-2病毒已经进化出一系列不同的新冠病毒变异株，其中以Delta、Omicron因传染性极强特点尤引人关注。
新型冠状病毒SARS-CoV-2属于冠状病毒科β种属成员，是有被膜、单链正义RNA病毒。SARS-CoV-2基因组包含5’端加帽和3’端加PolyA尾结构，编码29种蛋白，包括25种非结构蛋白与辅助蛋白以及4种结构蛋白。非结构蛋白在病毒RNA复制和免疫逃避过程中发挥着关键作用，辅助蛋白执行辅助病毒感染、存活和在宿主细胞中传播等多种功能；结构蛋白负责病毒组装并形成成熟的病毒颗粒。新冠病毒基因组靠近5’端约三分之二长度复制酶基因编码两个开放阅读框(Open reading frame，ORF)，ORF1a和ORF1b。近3’端三分之一长度基因编码几个ORFs，其中4个ORFs编码冠状病毒结构蛋白，分别是刺突糖蛋白(spike glycoprotein，S)、膜蛋白(membrane protein，M)、包膜蛋白(envelope protein，E)和衣壳蛋白(nucleocapsid protein，N)，其他ORFs编码几种辅助蛋白。
SARS-CoV-2病毒S蛋白属于I型病毒融合蛋白，包含两个功能性亚基S1和S2。冠状病毒由S蛋白介导从而进入宿主细胞，S1亚基负责与宿主细胞受体结合，S2亚基负责病毒膜与细胞膜融合，其中S1亚基的受体结合结构域(receptor-binding domain，RBD)负责与宿主细胞的血管紧张素转换酶2(angiotensin converting enzyme 2，ACE2)受体结合。因此S1蛋白或S1蛋白RBD区域常被作为阻断病毒与宿主受体hACE2结合的治疗性靶点，是预防与治疗COVID-19的疫苗、中和性抗体研发以及SARS-COV-2临床诊断的重要靶点。在本发明中，使用RNA核酸适配体筛选系统筛选的核酸适配体能有效灵敏检测S蛋白或S蛋白RBD区域、能有效阻断RBD与hACE2结合。
核酸适配体
核酸适配体(Nucleic acid aptamers)是一类短的、单链DNA(single-stranded DNA,ssDNA)或RNA寡核苷酸分子，它们通过折叠形成复杂且独特的空间三维结构，以较高特异性和较好亲和性结合靶标蛋白分子。相比ssDNA核酸适配体，RNA核酸适配体因可形成复杂的空间结构而更灵活。
核酸适配体由于其倾向于形成互补碱基对而通常具有某些特定的三维结构。它们可以折叠形成如茎(stem)、环(loop)、凸起(bulge)、赝结(pseudoknot)、G-四链体(G-quadruplex)、香吻发卡(kissing hairpin)等不同的二级结构。随后这些二级结构的集合可以形成独特的三维空间构象，从而对相关靶标分子进行特异性识别。这些三维空间构象间的相互作用如疏水和静电相互作用、氢键、范德华力、结构互补性以及碱基堆积对核酸适配体结合亲和力与特异性识别力至关重要。与抗体-抗原结合方式类似，特定三维结构间的相互作 用可驱动核酸适配体-靶标分子复合物的形成。
在本发明的一方面，提供了一种特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述随机区域包含如SEQ ID NO:2或SEQ ID NO:7所示的核酸序列。在一些实施方案中，SARS-CoV-2病毒是SARS-CoV-2野生型、Delta或Omicron BA.1或BA.2病毒株。根据其广谱活性，推测本发明的针对SARS-CoV-2病毒的S1蛋白的RNA核酸适配体也适用于此后出现以及未来出现的SARS-CoV-2变异株。
经实验验证发现两端的固定序列有助于序列合成和扩增，并且对于RNA核酸适配体针对靶蛋白亲和力活性有明显提升。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列，并且所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。
此外，与SEQ ID NO:1至SEQ ID NO:9所示序列中的任一个序列具有至少约70％、或替代地至少约75％、或替代地至少约80％、或替代地至少约85％、或替代地至少约90％、或者替代地至少约95％、或替代地至少约97％的序列同一性的核酸序列被认为在本发明的范围内。
经修饰的RNA核酸适配体
核酸适配体的化学修饰有助于提高核酸分子的稳定性，延长半衰期，降低其免疫原性等。
在本发明的一方面，提供了一种特异性结合SARS-CoV-2病毒的S1蛋白的经化学修饰的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列，所述化学修饰选自氟修饰、甲氧基修饰、聚乙二醇(PEG)多聚体修饰和胆固醇-PEG多聚体修饰中的一个或多个修饰。在一些实施方案中，所述PEG多聚体是选自小分子量的六聚体至40kD高分子量的多聚体。在一些实施方案中，所述化学修饰选自氟修饰、甲氧基修饰、胆固醇-PEG6化合物修饰、胆固醇-PEG24化合物修饰、PEG 40kDa化合物修饰和胆固醇-PEG 40kDa化合物修饰中的一个或多个。在一些实施方案中，所述随机区域包含如SEQ ID NO:2或SEQ ID NO:7所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列。在一些实 施方案中，所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列，并且所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，SARS-CoV-2病毒是SARS-CoV-2野生型、Delta或Omicron BA.1或BA.2病毒株。
在一些实施方案中，所述化学修饰是经氟修饰，其中所述经化学修饰的核酸适配体序列的嘧啶核糖2’位置的羟基替换为氟。
在一些实施方案中，所述经化学修饰是经甲氧基修饰，其中所述经化学修饰的核酸适配体序列的嘌呤核糖2’位置的羟基替换为甲氧基。
在一些实施方案中，所述化学修饰是经氟和甲氧基修饰，其中所述经化学修饰的核酸适配体序列的嘧啶核糖2’位置的羟基替换为氟，嘌呤核糖2’位置的羟基替换为甲氧基。
在一些实施方案中，所述化学修饰是经氟、甲氧基和胆固醇-PEG6化合物修饰，其中所述经化学修饰的核酸适配体序列的嘧啶核糖2’位置的羟基替换为氟，嘌呤核糖2’位置的羟基替换为甲氧基，以及核酸适配体序列的5’端附加胆固醇-PEG6化合物。
在一些实施方案中，所述化学修饰是经氟、甲氧基和胆固醇-PEG24化合物修饰，其中所述经化学修饰的核酸适配体序列的嘧啶核糖2’位置的羟基替换为氟，嘌呤核糖2’位置的羟基替换为甲氧基，以及核酸适配体序列的5’端附加胆固醇-PEG24化合物。
在一些实施方案中，所述化学修饰是经氟、甲氧基和PEG 40kDa化合物修饰，其中所述经化学修饰的核酸适配体序列的嘧啶核糖2’位置的羟基替换为氟，嘌呤核糖2’位置的羟基替换为甲氧基，以及核酸适配体序列的5’端附加PEG 40kDa化合物。
在一些实施方案中，所述化学修饰是经氟、甲氧基和胆固醇-PEG 40kDa化合物修饰，其中所述经化学修饰的核酸适配体序列的嘧啶核糖2’位置的羟基替换为氟，嘌呤核糖2’位置的羟基替换为甲氧基，以及核酸适配体序列的5’端附加胆固醇-PEG 40kDa化合物。
同一性
“同源性”或“同一性”或“相似性”是指两个肽之间或两个核酸分子之间的序列相似性。同源性能够通过比较每个序列中的位置来确定，该序列可以为了比较的目的而被比对。当比较的序列中的位置被相同的碱基或氨基酸占据时，那么分子在该位置是同源的。序列间同源性的程度是序列共有的匹配或同源位置的数量的函数。“无关的”或“非同源的”序列与本 发明的序列之一共享小于40％的同一性，但优选地小于25％的同一性。
多核苷酸或多核苷酸区域(或者多肽或多肽区域)与另一个序列具有一定百分比(例如60％、65％、70％、75％、80％、85％、90％、95％、98％或者99％)的“序列同一性”，意味着当比对时，在比较两个序列时该百分比的碱基(或氨基酸)是相同的。这种比对和百分比同源性或序列同一性可以使用本领域已知的软件程序来确定。
病毒载体
病毒载体可将遗传物质带入细胞，原理是利用病毒具有传送其基因组进入其他细胞，进行感染的分子机制。病毒载体也可以称为载体、载体病毒粒子或载体粒子。病毒载体的实例包括但不限于：逆转录病毒、腺病毒、腺相关病毒、单纯性疱疹病毒、牛痘病毒、杆状病毒或慢病毒。
逆转录病毒载体可以使衍生自或能够衍生自任何适合的逆转录病毒。大量的不同的逆转录病毒已经被鉴定。实施例包括但不限于：鼠白血病病毒(MLV)、人T-细胞白血病病毒(HTLV)、小鼠乳腺肿瘤病毒(MMTV)、劳氏肉瘤病毒(RSV)、Fujinami肉瘤病毒(FuSV)、莫洛尼鼠白血病病毒(Mo MLV)、FBR鼠骨肉瘤病毒(FBR MSV)、莫洛尼鼠肉瘤病毒(Mo-MSV)、Abelson鼠白血病病毒(A-MLV)、禽骨髓细胞瘤病毒-29(MC29)和禽红细胞增多症病毒(AEV)。
腺病毒是双链的线性DNA病毒，其不通过RNA中间体复制。腺病毒是双链的DNA无包膜病毒，其能够在体内、离体和体外转导大范围的人和非人来源的细胞类型。
腺相关病毒(adeno-associated virus，AAV)，也称腺伴随病毒，属于微小病毒科依赖病毒属，是目前发现的一类结构最简单的单链DNA 缺陷型病毒。重组的AAV载体已成功地被用于标记基因和涉及人类疾病的基因的体外、离体和体内的转导。已经开发了某些AAV载体，其可以有效地结合大的有效载荷(高达8-9kb)。
单纯性疱疹病毒(HSV)是包膜的双链DNA病毒，其天然地感染神经元。它可以容纳外源DNA的大区段，并且已经被采用为用于对神经元的基因递送的载体。在治疗过程中HSV的使用需要使毒株减毒，从而它们不能建立裂解周期。
本发明的病毒载体可以是牛痘病毒载体，例如MVA或NYVAC。应当理解的是，在重组基因的插入之后病毒基因组的部分可以保持完整。这意味着病毒载体可以保留感染细胞并随后表达额外的基因的能力的概念，该额外的基因支持其复制并可能促进被感染细胞的裂解和死亡。
慢病毒是更大群体的逆转录病毒的一部分。可以分为灵长类动物和非灵长类动物群 体。灵长类慢病毒的例子包括但不限于：人免疫缺陷病毒(HIV)、人自身免疫缺陷综合症(AIDS)的病原体以及猿猴免疫缺陷病毒(SIV)。非灵长类动物慢病毒群体包括原型“慢病毒”visna/maedi病毒(VMV)，以及相关的山羊关节炎-脑炎病毒(CAEV)、马传染性贫血病毒(EIAV)、猫免疫缺陷病毒(FIV)和牛免疫缺陷病毒(BIV)。
非病毒载体
非病毒载体是利用非病毒的载体材料的物化性质来介导基因的转移。任何合适的非病毒载体可被用于将核酸适配体引入对象的细胞中。非病毒载体的实施例包括但不限于，质粒、脂质体、无机纳米粒子和外泌体。
质粒是小型环状DNA分子，在基因工程中作为最常用，最简单的载体，必须包括三部分：遗传标记基因，复制区，目的基因。质粒在所有的细菌类群中都可发现，它们是独立于细菌染色体外自我复制的DNA分子。质粒载体的例子包括但不限于：大肠杆菌质粒载体、枯草杆菌质粒载体、酵母质粒载体、农杆菌质粒载体和蓝细菌质粒载体。
脂质体由卵磷脂和神经酰胺等制得的脂质体(空心)，具有的双分子层结构，是一种人工膜。脂质体的组成通常是磷脂(特别是高相转变温度的磷脂)的组合，通常与类固醇(尤其是胆固醇)组合。也可以使用其他磷脂或其他脂质。脂质体的物理特性取决于pH、离子强度和二价阳离子的存在。通过在转导期间通过使用二油酰磷脂酰乙醇胺可以增加脂质体的转导效率。高效脂质体可商购获得。脂质体的例子包括但不限于：中性脂质体、负电荷脂质体和正电荷脂质体。
无机纳米粒子主要通过穿过细胞膜将药物或生物分子转运到生物体中而起到治疗疾病的作用。应用于基因转运的无机纳米粒子的例子包括但不限于硅、铁氧化物、碳纳米管、磷酸钙、金属纳米粒子、量子点等。
外泌体是平均直径为50-150nm的小的细胞外囊泡。它们作为细胞间通讯的手段。通常，它们由结构蛋白以及所选的蛋白质、miRNA、mRNA和长非编码RNA组成。RNA包含被蛋白质识别的短核苷酸序列，所述蛋白质将其运输至细胞质中并将其包装进外泌体中。外泌体将有效负载从一个细胞运输至另一个细胞。在进入受体细胞后，外泌体有效负载被释放到细胞质中。
药物组合物和药品套装
“药物”是用以预防、治疗及诊断疾病的物质。理论上，药物是指凡能影响机体器官生理功能及细胞代谢活动的化学物质都属于药物的范畴。
发明的一方面提供一种包含特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适 配体的药物，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述随机区域包含如SEQ ID NO:2或SEQ ID NO:7所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列，并且所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体经化学修饰，所述化学修饰选自氟修饰、甲氧基修饰、聚乙二醇(PEG)多聚体修饰和胆固醇-PEG多聚体修饰中的一个或多个，其中PEG多聚体可以选自小分子量的六聚体至40kD高分子量的多聚体。在一些实施方案中，所述药物进一步包含病毒载体或非病毒载体，其中所述载体包含或携带所述特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。在一些实施方案中，所述病毒载体是慢病毒。在一些实施方案中，所述非病毒载体是质粒。在一些实施方案中，SARS-CoV-2病毒是SARS-CoV-2野生型、Delta或Omicron BA.1或BA.2病毒株。
“药物组合物”是指用于人的药物制剂。该药物组合物包含本发明的药物以及载体、稳定剂和/或赋形剂的合适制剂。
本发明的一方面提供一种药物组合物，其包含本发明的药物和药学上可接受的赋形剂，其中所述药物包含特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述随机区域包含如SEQ ID NO:2或SEQ ID NO:7所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列，并且所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体经化学修饰，所述化学修饰选自氟修饰、甲氧基修饰、聚乙二醇(PEG)多聚体修饰和胆固醇-PEG多聚体修饰中的一个或多个，其中PEG多聚体可以选自小分子量的六聚体至40kD高分子量的多聚体。在一些实施方案中，所述药物进一步包含病毒载体或非病毒载体，其中所述载体包含 或携带所述特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。在一些实施方案中，所述病毒载体是慢病毒。在一些实施方案中，所述非病毒载体是质粒。在一些实施方案中，SARS-CoV-2病毒是SARS-CoV-2野生型、Delta或Omicron BA.1或BA.2病毒株。
为了制备药物组合物或无菌组合物，让药物与可药用载体或赋形剂混合。可通过与生理学上可接受的载体、赋形剂或稳定剂混合，来制备呈例如冻干粉、浆液、水溶液或混悬剂形式的治疗及诊断药物的制剂。
药学上可接受的赋形剂是本领域熟知的。如本文所用，“药学上可接受的赋形剂”包括当与组合物的活性成分组合时允许该成分保持生物活性并且不会引起与对象的免疫系统的破坏性反应的材料。这些可以包括稳定剂、防腐剂、盐或糖配合物或晶体等。“药学上可接受的”是指当施用至人体时不会产生过敏反应或类似的不期望的反应的分子和成分。本领域已知如何制备包含作为活性组分的水性组合物。通常，这些组合物被制备成注射剂或喷雾剂，例如液态溶液或悬浮液；也可以制备成适于在注射或喷雾之前配制成溶液或悬浮液的固体形式。
本发明的药物或药物组合物可以单独使用或彼此联合使用。相应地，本发明提供一种药品套装以便于进行上述联合疗法，其包含独立存在的两种或多种本发明的药物或药物组合物。在一些实施方式中，对象有时会同时施用两种或多种本发明的药物或药物组合物。在一些实施方式中，对象有时会分别施用两种或多种本发明的药物或药物组合物。
筛选方法、治疗和/或预防方法
本发明的一个方面提供一种筛选针对靶蛋白的RNA核酸适配体的方法，其包含以下步骤：(1)提供筛选细胞，所述筛选细胞具有向导RNA、靶向序列、选择标记、融合蛋白和dCas蛋白；其中所述向导RNA含有识别序列和预定长度的核酸适配体随机文库；所述靶向序列与所述向导RNA的识别序列配对；所述选择标记位于所述靶向序列下游且包括基本启动子和选择标记基因；所述融合蛋白包含所述靶蛋白和转录激活元件，所述靶蛋白与所述核酸适配体随机文库中的RNA核酸适配体结合导致所述转录激活元件与所述选择标记的结合；所述dCas蛋白在所述向导RNA引导下特异性识别靶向序列；(2)利用选择标记基因对筛选细胞进行筛选；(3)收集表达所述选择标记基因的筛选细胞；(4)裂解筛选细胞，对含有核酸适配体区域的序列，进行特异性扩增，通过测序获得所述核酸适配体的序列信息。在一些实施方案中，步骤(4)进一步包含对获得的核酸适配体序列进行分析与验证。在一些实施方案中，所述dCas蛋白是dCas9蛋白或dUn1Cas12f1蛋白。在一些实施方案中，所述基本启动子选自mini-promoter-1(SEQ ID NO:28)、mini-promoter-2(SEQ ID NO:27)、mini-TK  promoter(SEQ ID NO:29)、mini-CMV promoter(SEQ ID NO:30)和Crystallin basal promoter(SEQ ID NO:31)。在一些实施方案中，所述靶蛋白来源于病毒、细菌、真菌或动物。在一些实施方案中，所述靶蛋白来源于人类。在一些实施方案中，所述靶蛋白是绿色荧光蛋白或来源于SARS-CoV-2刺突蛋白的RBD区域。在一些实施方案中，所述筛选细胞是人源细胞HEK293T或原核细胞大肠杆菌E.coli。
在一些实施方案中，所述筛选细胞是通过将一种或多种质粒载体转染至原核细胞中得到的，所述质粒载体包含选自向导RNA；靶向序列和选择标记；编码融合蛋白的核酸序列；以及编码dCas蛋白的核酸序列中的一个或多个，其中所述向导RNA含有识别序列和预定长度的核酸适配体随机文库，所述靶向序列与所述向导RNA的识别序列配对，所述选择标记位于所述靶向序列下游且包括基本启动子和选择标记基因，所述融合蛋白包含所述靶蛋白和转录激活元件，所述靶蛋白与所述核酸适配体随机文库中的RNA核酸适配体结合导致所述转录激活元件与所述选择标记的结合，所述dCas蛋白在所述向导RNA引导下特异性识别靶向序列。在一些实施方案中，所述筛选细胞是通过将一种质粒载体转染至原核细胞中得到的，所述质粒载体包含所述向导RNA、所述靶向序列、所述选择标记、以及编码所述融合蛋白和所述dCas蛋白的核酸序列。在一些实施方案中，所述原核细胞是大肠杆菌细胞。在一些实施方案中，所述方法进一步包含步骤(5)：将步骤(4)中得到的核酸适配体序列亚克隆到质粒载体中，重复步骤(2)至步骤(4)。
在一些实施方案中，所述筛选细胞通过以下步骤提供：(1.1)提供一种表达所述dCas蛋白的细胞，所述dCas蛋白在向导RNA引导下特异性识别靶向序列，所述细胞还包含所述靶向序列和所述选择标记，所述靶向序列与所述向导RNA的识别序列配对，所述选择标记位于所述靶向序列下游且包括基本启动子和选择标记基因；(1.2)提供一种编码向导RNA和融合蛋白的病毒表达载体，所述向导RNA含有识别序列和预定长度的核酸适配体随机文库，所述融合蛋白包含所述靶蛋白和转录激活元件，所述靶蛋白与所述核酸适配体随机文库中的RNA核酸适配体结合导致所述转录激活元件与所述选择标记的结合；(1.3)对所述病毒表达载体进行包装，然后使用包装后的病毒表达载体感染所述细胞。在一些实施方案中，所述方法进一步包含步骤(5)：将步骤(4)中得到的核酸适配体序列亚克隆至步骤(1.2)中的病毒表达载体，然后重复步骤(1.3)和步骤(2)至步骤(4)。在一些实施方案中，步骤(5)进一步被重复1至3次。在一些实施方案中，第一次执行步骤(1.3)时，采用3-5的MOI进行病毒感染，再次执行步骤(1.3)时，采用0.1-0.3的MOI进行病毒感染。在一些实施方案中，在步骤(1.3)中利用抗生素检测包装后的病毒表达载体的病毒滴度。在一 些实施方案中，所述病毒表达载体是慢病毒表达载体。在一些实施方案中，所述细胞是HEK293T细胞。
本发明的另一方面提供一种检测SARS-CoV-2病毒的检测试剂或试剂盒，其包含特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述随机区域包含如SEQ ID NO:2或SEQ ID NO:7所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列，并且所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体经化学修饰，所述化学修饰选自氟修饰、甲氧基修饰、聚乙二醇(PEG)多聚体修饰和胆固醇-PEG多聚体修饰中的一个或多个，其中PEG多聚体可以选自小分子量的六聚体至40kD高分子量的多聚体。在一些实施方案中，SARS-CoV-2病毒是SARS-CoV-2野生型、Delta或Omicron BA.1或BA.2病毒株。
本发明的另一方面提供一种检测SARS-CoV-2病毒的方法，所述方法包含使待检测样品与本发明的特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体接触，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述随机区域包含如SEQ ID NO:2或SEQ ID NO:7所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列，并且所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体经化学修饰，所述化学修饰选自氟修饰、甲氧基修饰、聚乙二醇(PEG)多聚体修饰和胆固醇-PEG多聚体修饰修饰中的一个或多个，其中PEG多聚体可以选自小分子量的六聚体至40kD高分子量的多聚体。在一些实施方案中，SARS-CoV-2病毒是SARS-CoV-2野生型、Delta或Omicron BA.1或BA.2病毒株。
进一步地，本发明提供了本发明的特异性结合SARS-CoV-2病毒的S1蛋白的RNA 核酸适配体在制备检测SARS-CoV-2病毒的检测试剂或试剂盒中的应用，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述随机区域包含如SEQ ID NO:2或SEQ ID NO:7所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列，并且所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体经化学修饰，所述化学修饰选自氟修饰、甲氧基修饰、聚乙二醇(PEG)多聚体修饰和胆固醇-PEG多聚体修饰中的一个或多个，其中PEG多聚体可以选自小分子量的六聚体至40kD高分子量的多聚体。在一些实施方案中，SARS-CoV-2病毒是SARS-CoV-2野生型、Delta或Omicron BA.1或BA.2病毒株。
本发明的另一个方面提供一种在对象中中和SARS-CoV-2病毒的方法，所述方法包含向有需要的对象施用有效量的本发明的药物，其中所述药物包含特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述随机区域包含如SEQ ID NO:2或SEQ ID NO:7所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列，并且所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体经化学修饰，所述化学修饰选自氟修饰、甲氧基修饰、聚乙二醇(PEG)多聚体修饰和胆固醇-PEG多聚体修饰中的一个或多个，其中PEG多聚体可以选自小分子量的六聚体至40kD高分子量的多聚体。在一些实施方案中，所述药物进一步包含病毒载体或非病毒载体，其中所述载体包含或携带所述特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。在一些实施方案中，所述病毒载体是慢病毒。在一些实施方案中，所述非病毒载体是质粒。在一些实施方案中，SARS-CoV-2病毒是SARS-CoV-2野生型、Delta或Omicron BA.1或BA.2病毒株。
进一步地，本发明提供了本发明的药物在制备用于在对象中中和SARS-CoV-2病毒的药物中的应用，其中所述药物包含特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述随机区域包含如SEQ ID NO:2或SEQ ID NO:7所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列，并且所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体经化学修饰，所述化学修饰选自氟修饰、甲氧基修饰、聚乙二醇(PEG)多聚体修饰和胆固醇-PEG多聚体修饰中的一个或多个，其中PEG多聚体可以选自小分子量的六聚体至40kD高分子量的多聚体。在一些实施方案中，所述药物进一步包含病毒载体或非病毒载体，其中所述载体包含或携带所述特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。在一些实施方案中，所述病毒载体是慢病毒。在一些实施方案中，所述非病毒载体是质粒。在一些实施方案中，SARS-CoV-2病毒是SARS-CoV-2野生型、Delta或Omicron BA.1或BA.2病毒株。
本发明的另一个方面提供一种治疗或预防SARS-CoV-2病毒感染的方法，所述方法包含向有需要的对象施用有效量的本发明的药物，其中所述药物包含特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述随机区域包含如SEQ ID NO:2或SEQ ID NO:7所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列，并且所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体经化学修饰，所述化学修饰选自氟修饰、甲氧基修饰、聚乙二醇(PEG)多聚体修饰和胆固醇-PEG多聚体修饰中的一个或多个，其中PEG多聚体可以选自小分子量的六聚体至40kD高分子量的多聚体。在一些实施方案中，所述药物进一步 包含病毒载体或非病毒载体，其中所述载体包含或携带所述特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。在一些实施方案中，所述病毒载体是慢病毒。在一些实施方案中，所述非病毒载体是质粒。在一些实施方案中，SARS-CoV-2病毒是SARS-CoV-2野生型、Delta或Omicron BA.1或BA.2病毒株。
进一步地，本发明提供本了发明的药物在制备用于在对象中治疗或预防SARS-CoV-2病毒感染的药物中的应用，其中所述药物包含特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述随机区域包含如SEQ ID NO:2或SEQ ID NO:7所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列，并且所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体经化学修饰，所述化学修饰选自氟修饰、甲氧基修饰、聚乙二醇(PEG)多聚体修饰和胆固醇-PEG多聚体修饰中的一个或多个，其中PEG多聚体可以选自小分子量的六聚体至40kD高分子量的多聚体。在一些实施方案中，所述药物进一步包含病毒载体或非病毒载体，其中所述载体包含或携带所述特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。在一些实施方案中，所述药物进一步包含病毒载体或非病毒载体，其中所述载体包含或携带所述特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。在一些实施方案中，所述病毒载体是慢病毒。在一些实施方案中，所述非病毒载体是质粒。在一些实施方案中，SARS-CoV-2病毒是SARS-CoV-2野生型、Delta或Omicron BA.1或BA.2病毒株。
本发明的另一个方面提供一种在对象中中和SARS-CoV-2病毒的方法，所述方法包含向有需要的对象施用有效量的本发明的药物组合物，其中所述药物组合物包含本发明的药物和药学上可接受的赋形剂，所述药物包含特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述随机区域包含如SEQ ID NO:2或SEQ ID NO:7所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的3’端的固定序列包含 如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列，并且所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体经化学修饰，所述化学修饰选自氟修饰、甲氧基修饰、聚乙二醇(PEG)多聚体修饰和胆固醇-PEG多聚体修饰中的一个或多个，其中PEG多聚体可以选自小分子量的六聚体至40kD高分子量的多聚体。在一些实施方案中，所述药物进一步包含病毒载体或非病毒载体，其中所述载体包含或携带所述特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。在一些实施方案中，所述病毒载体是慢病毒。在一些实施方案中，所述非病毒载体是质粒。在一些实施方案中，SARS-CoV-2病毒是SARS-CoV-2野生型、Delta或Omicron BA.1或BA.2病毒株。
进一步地，本发明提供了本发明的药物组合物在制备用于中和SARS-CoV-2病毒的药物中的应用，其中所述药物组合物包含本发明的药物和药学上可接受的赋形剂，所述药物包含特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述随机区域包含如SEQ ID NO:2或SEQ ID NO:7所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列，并且所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体经化学修饰，所述化学修饰选自氟修饰、甲氧基修饰、聚乙二醇(PEG)多聚体修饰和胆固醇-PEG多聚体修饰中的一个或多个，其中PEG多聚体可以选自小分子量的六聚体至40kD高分子量的多聚体。在一些实施方案中，所述药物进一步包含病毒载体或非病毒载体，其中所述载体包含或携带所述特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。在一些实施方案中，所述病毒载体是慢病毒。在一些实施方案中，所述非病毒载体是质粒。在一些实施方案中，SARS-CoV-2病毒是SARS-CoV-2野生型、Delta或Omicron BA.1或BA.2病毒株。
本发明的另一方面提供一种在对象中治疗或预防SARS-CoV-2病毒引感染的方法，所述方法包含向有需要的对象施用有效量的本发明的药物组合物，其中所述药物组合物包含本发明的药物和药学上可接受的赋形剂，所述药物包含特异性结合SARS-CoV-2病毒的S1蛋 白的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述随机区域包含如SEQ ID NO:2或SEQ ID NO:7所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述药物进一步包含病毒载体或非病毒载体，其中所述载体包含或携带所述特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列，并且所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体经化学修饰，所述化学修饰选自氟修饰、甲氧基修饰、聚乙二醇(PEG)多聚体修饰和胆固醇-PEG多聚体修饰中的一个或多个，其中PEG多聚体可以选自小分子量的六聚体至40kD高分子量的多聚体。在一些实施方案中，所述药物进一步包含病毒载体或非病毒载体，其中所述载体包含或携带所述特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。在一些实施方案中，所述病毒载体是慢病毒。在一些实施方案中，所述非病毒载体是质粒。在一些实施方案中，SARS-CoV-2病毒是SARS-CoV-2野生型、Delta或Omicron BA.1或BA.2病毒株。
进一步地，本发明提供了本发明的药物组合物在制备用于在对象中治疗或预防SARS-CoV-2病毒感染的药物中的应用，其中所述药物组合物包含本发明的药物和药学上可接受的赋形剂，所述药物包含特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。在一些实施方案中，所述随机区域包含如SEQ ID NO:2或SEQ ID NO:7所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列。在一些实施方案中，所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述药物进一步包含病毒载体或非病毒载体，其中所述载体包含或携带所述特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。在一些实施方案中，所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列，并且所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。在一些实施方案中，所述RNA核酸适配体经化学修饰，所述化学修饰选自 氟修饰、甲氧基修饰、聚乙二醇(PEG)多聚体修饰和胆固醇-PEG多聚体修饰中的一个或多个，其中PEG多聚体可以选自小分子量的六聚体至40kD高分子量的多聚体。在一些实施方案中，所述药物进一步包含病毒载体或非病毒载体，其中所述载体包含或携带所述特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。在一些实施方案中，所述病毒载体是慢病毒。在一些实施方案中，所述非病毒载体是质粒。在一些实施方案中，SARS-CoV-2病毒是SARS-CoV-2野生型、Delta或Omicron BA.1或BA.2病毒株。
合适的给药途径包括胃肠外给药(例如肌内、静脉内或皮下给药)，口服，鼻喷以及雾化吸入等给药。可按多种常规方式给予本发明方法的药物或药物组合物，这些方法例如有经气管插管给予、经口摄取、吸入、局部施用或经皮肤、皮下、腹膜内、胃肠外、动脉内或静脉内注射。此外，人们可以在靶向药物递送系统中给予本发明的RNA核酸适配体、药物或药物组合物。在一些实施方案中，经静脉内注射给予本发明的RNA核酸适配体、药物或药物组合物。
由临床医生例如用本领域已知或怀疑影响治疗或预期影响治疗的参数或因子来测定合适的剂量。通常，开始剂量比最佳剂量稍低，此后少量增加直到达到相对于任何不良副作用所要的或最佳的作用效果。重要的诊断测量包括测量例如炎性症状或所产生的炎性细胞因子的水平。
通过连续给药或通过以一定间隔(例如一天、一周或每周1-7次)给药来施用本发明的药物或药物组合物。可通过气管插管、静脉内、皮下、腹膜内、经皮肤、局部、经口、经鼻、经直肠、肌内、大脑内或脊柱内来提供剂量。优选剂量方案是包括避免显著的不合乎需要的副作用的最大剂量或给药频率的方案。
实验细胞系和实验方法
实验细胞系
本文中使用的HEK293T细胞系购买于美国模式培养物集存库(American Type Culture Collection，ATCC)。HEK293T在含有100U/ml Penicillin/Streptomycin，10％胎牛血清(Fetal Bovine Serum，FBS)的DMEM(Dulbecco’s Modified Eagle Medium)培养基中培养。293T/dCas9是通过在野生型HEK293T细胞系中通过慢病毒稳定整合dCas9表达元件得到的单克隆细胞系。CRISmers筛选细胞系dCas9/9×gLuc-puro细胞系是通过在293T/dCas9细胞系中通过慢病毒进一步稳定整合9×gLuc-puro报告基因元件(9×gLuc-minPro-puro)得到的单克隆细胞系。HEK293T/hACE2细胞系是通过在野生型HEK293T细胞系中通过慢病毒稳定整合hACE2表达元件得到的多克隆细胞系。所有的细胞均不含有支原体，培养在 含5％CO2、37℃的细胞培养箱中。
表达载体构建
本文所有表达载体构建方法参考《分子克隆实验指南(第四版)(套装上下册)》。表达载体的构建主要包括线性化载体的制备、插入片段的制备、载体与片段的连接处理、借助大肠杆菌感受态细胞的质粒转化处理、大肠杆菌克隆的菌落PCR鉴定处理、质粒提取、对所获质粒的测序与鉴定处理等过程。本论文中所使用的慢病毒载体骨架均来自addgene。
高纯度质粒提取
为了获得大量高纯度高质量的质粒，我们使用碱裂解法进行质粒提取。相比快速质粒提取，高纯度质粒提取可逐步去除内毒素、RNA、蛋白质和有机溶剂等杂质。经高纯度提取的质粒可用于表达载体构建、多种细胞实验等。
HEK293T细胞的培养
HEK293T细胞是一株较为常见且常规的细胞系，易于操作，被广泛用于逆病毒生产、基因表达和蛋白表达等。该细胞系的培养主要包括细胞复苏、细胞维持培养、细胞传代以及细胞冻存等本领域技术人员熟知的操作。
HEK293T细胞的非脂质体瞬时转染
进行瞬时转染的细胞应保证较好的细胞状态且一定的细胞汇合度。根据实验目的的不同，转染可在96孔板、24孔板、6孔板以及10cm皿中进行。本文中利用荧光素酶报告系统验证核酸适配体亲和性时多在96孔板中进行，慢病毒包装多在6孔板或10cm皿中进行。
不同的培养孔底面积所对应的培养基体积、转染试剂与DNA用量不同，具体如下表所示(仅列举与本文实验相关且常用的信息，数据可能存在细微主观差异)
表1 HEK293T细胞非脂质体瞬时转染相关试剂用量
转染时可根据实验目的的不同选择贴壁转染或悬浮转染，贴壁转染适用于常规细胞如HEK293T的转染；悬浮转染可扩大转染复合体与细胞的接触，进一步提高转染效率，适用于难转染、对转染效率要求高的实验。下面以96孔板贴壁转染为例进行举例说明：
(1)转染前一天种植细胞：提前用0.1％明胶包被待种植的96孔板区域，取用生长速度快，生长状态好的HEK293T细胞或带转染的细胞按2.5×104的细胞接种量将其接种于96孔板中，保持转染当天细胞汇合度在60％以上为宜(48h后检测实验效果，无需太密)。
(2)转染当天：使用10μl无血清培养基稀释200ng DNA并均匀混合(多种DNA需按一定的比例配置，总量不变)；使用10μl无血清培养基稀释转染试剂PEI并均匀混合，室温下静置孵育5min；将稀释后的转染试剂逐滴加入稀释后的DNA种并均匀混合，室温下静置孵育20min；取20μl转染试剂与DNA复合物加至含有细胞和培养基的孔中，呈井字形晃匀，置于37℃，5％CO2细胞培养箱中培养。6h后更换新鲜培养基后继续置于培养箱中培养。24孔板、6孔板以及10cm皿的转染时，在加入转染试剂与DNA复合物前，可对细胞进行换液处理，更换二分之一培养体积的10％FBS/DMEM培养基。
(3)转染后48h：48h后可对细胞进行相关检测或其他处理。
慢病毒介导的稳转细胞系的建立
本文共建立了293T/dCas9稳转单克隆细胞系、dCas9/9×gLuc-puro稳转单克隆细胞系以及HEK293T/hACE2稳转多克隆细胞系，建立流程主要包括HEK293T细胞的提前种植、慢病毒包装、慢病毒收集、待感染细胞准备、慢病毒感染、细胞系筛选与鉴定等过程。单克隆细胞系还需在细胞系筛选后挑取单克隆细胞系进行鉴定，多克隆细胞系无需进行单克隆细胞系的单独挑选。
(1)HEK293T细胞的种植：转染前一天将生长速度快、细胞状态良好的HEK293T细胞按1×106的细胞接种量接种至提前用0.1％明胶包被的6孔板中，保持转染当天细胞汇合度在80％以上为宜。
(2)慢病毒包装：慢病毒包装过程与非脂质体瞬时转染过程一致，涉及转染的质粒共有3个：含有目的基因表达框的pRRL慢病毒载体质粒、表达慢病毒包膜蛋白VSVG的pCMV-VSV-G质粒以及表达慢病毒包装蛋白GAG、POL的psPAX2质粒，三种质粒质量比为10：9：1。使用50μl无血清培养基稀释DNA并均匀混合；使用50μl无血清培养基稀释转染试剂PEI并均匀混合，室温下静置孵育5min；将稀释后的转染试剂逐滴加入稀释后的DNA并均匀混合，室温下静置孵育20min；孵育期间，给待转染的6孔板细胞更换10％FBS/DMEM新鲜培养基1ml；孵育时间结束后取100μl转染试剂与DNA复合物加至含有细胞和培养基的孔中，呈井字形晃匀，置于37℃，5％CO2细胞培养箱中培养。6-8h后更换新鲜培养基后继续置于培养箱中培养。
(3)慢病毒收集：转染后48h收集细胞培养上清，然后再沿壁缓慢加入新鲜培养基继续培养24h。收集的病毒液用0.45μm滤器过滤，过滤后的病毒液暂放4℃冰箱保存。转染后72h再次收集细胞培养上清，用0.45μm滤器过滤后与48h收集的病毒液混合，可分装后置于-80℃保存。若短时间内使用，则可暂放4℃冰箱保存。接触过病毒液的枪头及其 它实验器械需高温高压后放入专用病毒废弃收集袋中集中处理。
(4)待感染细胞准备：病毒感染与细胞瞬时转染一致，同样可采用贴壁感染或悬浮感染，原理一致。以贴壁感染为例，在感染前一天，将生长速度快、细胞状态良好的待感染细胞按1x105的细胞接种量接种至提前用0.1％明胶包被的24孔板中，预留一个孔细胞作为病毒感染的空白对照组。
(5)慢病毒感染：沿壁吸走24孔板中的培养基，加入含8μg/ml Polybrene(助感染剂)的病毒液，并将培养体积固定在250μl，轻轻晃匀后置于培养箱中培养。8-12h之后，沿壁吸走病毒液，轻轻沿壁加入新鲜培养基，置于培养箱中继续培养。空白对照组不感染慢病毒，其他操作一致。慢病毒感染细胞后，目的基因会整合至宿主细胞基因组，目的基因中多含有抗性基因，用于细胞的筛选。
(6)单克隆细胞系筛选：病毒感染48h后，细胞密度较大，需传代至较低密度并用含有相应抗生素浓度的培养基替换原培养基进行培养，每隔两天更换一次含抗生素的培养基。不同抗生素作用机理不同，且不同细胞对抗生素的敏感程度不同，对应的抗性筛选时间也不同，实验前需对抗生素使用浓度、作用时间等进行预实验分析。空白对照组由于未感染慢病毒缺少抗性基因表达，会随着筛选时间延长而完全死亡。以空白对照组细胞完全死亡为界，观察病毒感染组是否有细胞存活。
(7)单克隆细胞系挑取：一般在抗生素培养10-12天后，感染成功的细胞会长成紧密的小团克隆样细胞，在体式镜下使用200μl移液枪枪头轻刮/吸取细胞团块，后置于24孔板中培养。应尽可能挑取分散的、边缘光滑的、无融合克隆形成的、生长状态较好的单克隆细胞团块。挑取数目根据慢病毒感染效率而定，一般以20-40个为宜。多克隆细胞系的建立无需挑取单克隆。
(8)稳转细胞系的鉴定：挑取至24孔板的单克隆细胞团块长至一定数目时，一部分用于冻存，另一部分用于鉴定。细胞系鉴定的方法取决于递送的目的基因的特征，如插入片段序列、荧光基因、蛋白标签、蛋白功能性质等，可对应地通过基因型鉴定/Sanger测序、流式细胞术、Western blot、酶标仪等进行评估。
(9)CRISmers筛选细胞系的获得与鉴定：dCas9/9×gLuc-puro细胞系是通过在293T/dCas9细胞系中利用慢病毒进一步稳定整合9×gLuc-puro报告基因元件得到的单克隆细胞系。dCas9/9×gLuc-puro细胞系鉴定时分别对挑取的不同单克隆独立感染2组慢病毒，一组为lenti-hU6-gLuc sgRNA-1.2 GFP apt-EF1a-NLS-GFP-VPH(PC)；另一组为lenti-hU6-gLuc sgRNA-1.2blank-EF1a-NLS-GFP-VPH(NC)。每一个挑取的单克隆细胞系在感染病毒组 分48h后施加不同浓度的嘌呤霉素(0.5μg/ml，1μg/ml，2μg/ml)，随后定量各单克隆细胞系经抗生素筛选后存活克隆的个数。PC/NC比率越高即单克隆细胞系活性、敏感性更好。同时附加关注NC组病毒组分感染后存活克隆个数，个数越低即本底效应越低。
荧光素酶报告实验
本文中使用荧光素酶报告实验来验证CRISmers系统，以及筛选后期二次鉴定挑选的核酸适配体亲和力。
(1)HEK293T细胞的转染：
(a)CRISmers系统验证：步骤与前文提到的96孔板的非脂质体瞬时转染步骤一致，使用PEI转染试剂在96孔板对应的孔中瞬时转染以下质粒(基于293T/dCas9稳转细胞系)：p1×gLuc target firefly luciferase或p9×gLuc target firefly luciferase，phU6-gLuc sgRNA-1.1 GFP aptamer或phU6-gLuc sgRNA-1.2 GFP aptamer、pGFP-VPH以及pCMV-renilla luciferase。其中pCMV-renilla luciferase质粒质量为20ng，剩余3个质粒摩尔比为1：1：1。转染后6-8h更换10％FBS/DMEM新鲜培养基。
(b)鉴定核酸适配体的亲和力：与前文提到的96孔板的非脂质体瞬时转染步骤一致，使用PEI转染试剂在96孔板对应的孔中瞬时转染以下质粒(基于293T细胞系)：phPGK-dCas9，p9×gLuc target gaussia luciferase，phU6-gLuc sgRNA-1.2 X aptamer-EF1a-NLS-RBD of S1-VPH，3个质粒摩尔比为1：1：1。在鉴定时需先对空白表达载体phU6-gLuc sgRNA-1.2 BsmBI-BsmBI-polyT-EF1a-NLS-linker-EcoRI-RBD-EcoRI-linker-VP64-P65-HSF1-NLS-linker-P2A-linker-Zeo-WPRE进行分子构建，利用BsmBI限制性内切酶将核酸适配体序列插入gLuc sgRNA stem loop2(1.2)骨架区域再进行转染实验。因需平行比较的核酸适配体数量较多，构建的各表达载体经质粒快速小提后可直接用于转染实验，无需大规模获得高纯度质粒。转染后6-8h更换10％FBS/DMEM新鲜培养基。
(2)荧光素酶活性检测：转染48h后使用Vigorous公司的双荧光素酶报告试剂盒进行荧光素酶活性检测。
(a)CRISmers系统验证：裂解细胞：转染后48h，小心吸走96孔板中的培养基，用排枪在每孔细胞中加入30μl 1universal lysis buffer，在微型震荡仪或低速摇床上孵育室温孵育5-10min。细胞完全裂解后用排枪取20μl裂解细胞样品至新的对应的96孔板中。萤火虫荧光素酶活性的检测：用排枪取20μl Fassay Reagent至孔底部，轻轻敲击孔壁3-5次混匀，立即放入酶标仪中进行化学发光检测。此时记录发光值为萤火虫荧光素酶的活性读值。海肾荧光素酶活性的检测：用排枪取20μl Rassay Reagent至管底部，轻轻敲击管壁3- 5次混匀，立即放入酶标仪中进行化学发光检测。此时记录发光值为海肾荧光素酶的活性读值。
(b)鉴定核酸适配体的亲和力：转染后48h，用排枪吸取20μl 96孔板中的细胞培养上清至新的黑孔96孔板中，用排枪吸取20μl Rassay Reagent加至对应的每孔底部，轻轻敲击孔壁3-5次混匀，立即放入酶标仪中进行化学发光检测。此时记录发光值为长腹水蚤荧光素酶的活性读值。由于二次筛选对挑选出的核酸适配体进行鉴定时，数量较多，故改为Gaussia可分泌至细胞上清的荧光素酶，便于检测。
(3)荧光素酶活性分析：
(a)CRISmers系统验证：数据以萤火虫荧光素酶活性/海肾荧光素酶活性计算得出，海肾荧光素酶为实验内参。然后将实验所有数据除以对照组平均值，进行数据标准化处理。
(b)鉴定核酸适配体的亲和力：数据以实验所有数据除以对照组平均值，进行数据标准化处理。
核酸适配体寡核苷酸随机序列文库构建
在本文中，为了实验可操作性与便捷性，我们构建了一个空白表达载体，即phU6-gLuc sgRNA-1.2 BsmBI-BsmBI-polyT-EF1a-NLS-linker-EcoRI-EcoRI-linker-VP64-P65-HSF1-NLS-linker-P2A-linker-Zeo-WPRE。首先借助限制性内切酶EcoRI将感兴趣的用于筛选的靶蛋白表达阅读框插入慢病毒空白表达载体，随后利用限制性内切酶BsmBI将合成的20bp随机寡核苷酸文库插入表达载体，形成针对靶蛋白的20bp寡核苷酸筛选文库。文库的构建工作主要分为寡核苷酸随机合成文库的PCR扩增、慢病毒载体的酶切处理、Gibson组装、文库电转化、文库慢病毒包装与滴度测定等。
(1)寡核苷酸随机文库的PCR扩增
合成的寡核苷酸文库共有100bp，中间20bp为寡核苷酸随机序列(A，T，C，G)，5’端与3’各为与表达载体同源的40bp核苷酸接头序列，即AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGGCCAnnnnnnnnnnnnnnnnnnnnCTGCAGGGCCAAGTGGCACCGAGTCGGTGCTTTTTATCGA(SEQ ID NO:10)。首先，将合成的寡核苷酸随机文库干粉以12000rpm，离心5min，将附着在管壁上的粉末离至管底，避免样品损失，浓度不精确。随后用无菌水将干粉溶解至1μg/μl，再稀释至0.1ng/μl，置于冰上待用。为确保样品扩增的保真性和覆盖度，寡核苷酸随机文库的PCR反应体系与反应程序与一般的PCR反应不同，具体如下表所示：
表2 oligo文库PCR扩增体系

表3 oligo文库PCR扩增程序
Oligo文库PCR扩增引物序列为(5’至3’)：oligo-Primer F：TATGTTTAAGAGCTAGAAATAGCAAGTTAAAATAAGGCTA(SEQ ID NO:11)，oligo-Primer R：CCATCTTTGCAAAGCTTATATCGATAAAAAGCACCGACTC(SEQ ID NO:12)。扩增反应完成后，取2μl样品进行琼脂糖凝胶电泳实验验证产物大小与特异性，目的片段大小为140bp，以无非特异性条带为宜。跑胶时，由于条带较小应使用2％琼脂糖凝胶便于区分。验证后使用QIAquick PCR纯化试剂盒根据说明书进行纯化回收。若纯化后样品浓度较低，可增设多个平行扩增反应以确保文库连接时样品充分。样品可置于4℃冰箱短暂保存，长期保存需置于-20℃或-80℃冰箱，防止降解。
(2)慢病毒载体的酶切处理
将已插入SARS-CoV-2 S蛋白RBD表达阅读框的慢病毒表达载体用BsmBI限制性内切酶进行酶切与去磷酸化反应。反应后取2μl样品进行1％琼脂糖凝胶电泳实验验证酶切效率，同时取1μl未酶切的原载体进行对照。与螺旋型原载体相比，酶切后的载体呈现线性，且条带位置略高于螺旋型载体。验证后使用QIAquick胶回收试剂盒根据说明书进行切胶纯化。为去除酶切反应中可能引入的盐等杂质，提高后续Gibson连接效率，浓缩纯化后载体浓度，需对载体进行异丙醇沉淀，反应体系如下表所示。
表4异丙醇沉淀反应体系
将上述反应体系经涡旋震荡仪混匀后室温放置15min，12000rpm，离心15min，此时离心管管底出现圆形蓝色沉淀。弃上清，用预冷的-20℃ 80％(vol/vol)乙醇清洗2次，离心并去除残余乙醇，置于空气中干燥5min。后视沉淀多少加入不同体积的dH2O并置于55℃金属浴中溶解沉淀。若纯化后样品浓度较低，可多个异丙醇沉淀反应可混合后按比例进行。样品可置于4℃冰箱短暂保存，长期保存需置于-20℃或-80℃冰箱，防止降解。
(3)Gibson组装
按如下表所示配置Gibson反应体系，并置于PCR仪中50℃孵育1h。
表5 Gibson组装反应体系
可同时进行多个平行Gibson连接反应，以保证文库电转时样品充分。连接时需设置空白对照组，用去离子水等体积替换PCR扩增的随机文库体积，以判断连接反应的本底效应，计算文库覆盖度。为去除Gibson反应中可能引入的盐等杂质，提高文库后续电转效率，浓缩纯化后连接文库浓度，需对反应体系进行异丙醇沉淀。将上述反应混合物经涡旋震荡仪混匀后室温放置15min，12000rpm，离心15min，此时离心管管底出现圆形蓝色沉淀。弃上清，用预冷的-20℃ 80％(vol/vol)乙醇清洗2次，离心并去除残余乙醇，置于空气中干燥5min。后视沉淀多少加入不同体积的dH2O并置于55℃金属浴中溶解沉淀。若纯化后样品浓度较低，可多个异丙醇沉淀反应可混合后按比例进行。样品可置于4℃冰箱短暂保存，长期保存需置于-20℃或-80℃冰箱，防止降解。最终加水溶解时应保证最终连接产物浓度为100-150ng/μl范围，以保证电转效率。
(4)文库电转
(a)电转杯预冷：将电转杯(0.1cm)插入碎冰中，压实冰面，静置待用。
(b)SOC培养基预热：将无抗生素的SOC培养基放置于37℃烘箱中，预热待用。
(c)电转：根据电转仪使用说明设置电转仪电击参数。从-80℃冰箱取出电转感受态细胞并置于压实的冰上，待细胞刚化冻时，加入2μl上述连接产物，轻轻吹打混匀2-3次后迅速将混合物转移至预冷的电转杯中，盖上电转杯杯盖。用毛巾擦拭电转杯外部水分，迅 速将电转杯置于电转槽中电转。电转完成后，迅速往电转杯中加入1ml预热的SOC培养基，37℃，220rpm震荡培养1h。电转过程中，避免产生气泡，尽量擦干杯壁水分，保证电转动作一气呵成，迅速敏捷。
(d)涂板：将上述培养菌液以每培养皿250μl的体积均匀涂布，后置于37℃培养箱中过夜培养。
(e)计数：对菌板进行计数，评估电转效率和文库覆盖度。在最终文库电转前，应进行预实验预估本次构建文库的本底效应，电转效率等。由于第一轮初始序列太多(420)，现有的文库构建技术无法覆盖全部序列，仅能尽可能多的收获克隆数。从第二轮文库构建开始，现有的文库构建技术已能覆盖全部序列，并实现至少500倍覆盖度。
(f)集菌：在评估完本次电转收获的细菌克隆数后，在洁净工作台中使用小药勺或细菌刮刀进行集菌工作。集菌完成后置于37℃，220rpm摇床中培养2-3h。
(g)高纯度质粒提取：扩菌完成后，收集菌体，进行高纯度质粒提取。将提取后的质粒放于-80℃冰箱，便于长期保存。
核酸适配体寡核苷酸随机序列文库的慢病毒包装与慢病毒滴度测定
(1)文库慢病毒包装
文库慢病毒包装过程与上述慢病毒介导的稳转细胞系建立过程中慢病毒包装过程基本一致。由于文库慢病毒需求量较大，故包装过程在多个10cm皿中同步进行。慢病毒包装过程与非脂质体瞬时转染一致，涉及转染的质粒共有3个：含有慢病毒随机文库的pRRL慢病毒载体质粒、表达慢病毒包膜蛋白VSVG的pCMV-VSV-G质粒以及表达慢病毒包装蛋白GAG、POL的psPAX2质粒，三种质粒质量比为10：9：1。
(a)第一天：提前用0.1％明胶包被10cm皿，取生长速度快，生长状态良好的HEK293T细胞按8×106的细胞接种量将其接种于10cm皿，保持转染当天细胞汇合度在80％以上为宜。
(b)第二天：使用500μl无血清培养基稀释DNA并均匀混合；使用500μl无血清培养基稀释转染试剂PEI并均匀混合，室温下静置孵育5min；将稀释后的转染试剂逐滴加入稀释后的DNA并均匀混合，室温下静置孵育20min；孵育间隔给10cm皿中待转染的细胞更换10％FBS/DMEM新鲜培养基5ml。孵育时间结束后，取1000μl转染试剂与DNA复合物加至含有细胞和培养基的孔中，呈井字形晃匀，置于37℃，5％CO2细胞培养箱中培养。6-8h后更换新鲜培养基后继续置于培养箱中培养。
(c)慢病毒收集：转染后48h收集细胞培养上清，然后再沿壁缓慢加入新鲜培养基 继续培养24h。收集后的病毒液用0.45μm滤器过滤，过滤后的病毒液暂放4℃冰箱保存。转染后72h再次收集细胞培养上清，过滤后与48h收集的病毒液混合，可分装后置于-80℃保存。若短时间内使用，则可暂放4℃冰箱保存。接触过病毒液的枪头及其它实验器械需高温高液后放入专用病毒废弃收集袋中集中处理。
(2)病毒滴度测定
在病毒滴定时，采用文库载体自带的Zeocin抗性基因进行病毒滴度的测定。将状态良好的dCas9/9×gLuc-puro细胞接种至12孔板中，每孔接种3×106个细胞(定义为“起始细胞数”)。随后分别加入0μl、25μl、50μl、100μl、200μl以及400μl文库慢病毒进行悬浮离心感染(需加入8μg/ml polybrene保证感染效率)。37℃，2400rpm离心2h后，置于37℃细胞培养箱中培养12-14h。感染48h后将感染的细胞以1×104密度接种至对应的新的12孔板中，加入含有500μg/ml zeocin抗生素的培养基。此外需设置感染病毒不加抗生素组和未感染病毒加抗生素组作为对照。抗生素持续筛选5-6天后，未感染病毒加抗生素组全部死亡，感染病毒不加抗生素组细胞汇合度达到80％-90％时，对细胞进行消化并计数。此处定义病毒感染加抗生素的各组细胞数为“活细胞数”，感染病毒不加抗生素组的各组细胞数为“对照细胞数”。按照病毒滴度计算公式进行慢病毒滴度计算：滴度(IFU/ml)＝(活细胞数×起始细胞数)/(对照细胞数×毫升病毒体积数)。最后求平均值，得到最终慢病毒滴度。需注意的是，若某个实验组细胞存活比例大于50％，此组读值需舍弃，不参与平均滴度计算。
核酸适配体寡核苷酸随机序列文库的慢病毒筛选
慢病毒文库筛选基于单克隆细胞系#4-53 dCas9/9×gLuc-puro进行。病毒筛选过程与上述慢病毒介导的稳转细胞系建立过程中慢病毒筛选基本一致。由于文库筛选对细胞量需求量较大，故筛选过程在多个10cm皿中进行。在初始轮文库感染筛选时，采用中等(3-5)的MOI进行病毒感染，目的是确保大多数的功能序列被覆盖、筛选，且减小筛选的工作量。在随后几轮亚文库筛选时，采用低(0.1-0.3)的MOI进行病毒感染，以确保大多数细胞仅接收1个核酸适配体序列。病毒感染48h后，细胞进行传代处理，将细胞维持在30％-40％的密度下，并将培养基更换含有2μg/ml的puro抗生素培养基进行筛选，每隔2-3天更换一次新鲜的抗性培养基。持续筛选10天左右，可见由单个细胞长成的克隆团块。用体式镜挑取所有克隆，用细胞基因组PCR裂解液裂解细胞，裂解过程为50℃孵育1h，95℃孵育15min。取1μl裂解液直接进行基因组PCR扩增。基因组PCR扩增体系、扩增程序(退火、延伸、终延伸为20个循环)、扩增引物、扩增完成后的琼脂糖凝胶电泳实验验证以及纯 化回收与上述寡核苷酸随机文库的PCR扩增过程基本一致。样品深度测序交由金维智公司进行。
深度测序数据分析
获得每轮深度测序结果时，首先评估本轮测序质量是否符合预期，主要评估内容为产出reads数和碱基测序质量(Q30)。
进行深度测序数据分析时，首先过滤掉两端与同源序列不匹配的核酸适配体序列，随后去除核酸适配体两端的接头序列与同源序列，获得若干核酸适配体序列。为鉴定高亲和性序列，我们以两方面的指标挑选用于第二轮荧光素酶报告实验筛选的核酸适配体。一方面以每个独立序列按出现频率由高至低进行排序，二轮筛选时选择前0.1％高频独立序列进行验证；另一方面计算在两轮筛选间独立序列的富集指数并加以排序(富集指数指在两轮中同一独立序列的重复数之比)，二轮筛选时选择前15条高频富集指数序列进行验证。
SARS-CoV-2假病毒包装实验
SARS-CoV-2假病毒的包装与前述慢病毒包装过程基本一致。假病毒包装共涉及5个质粒：pCMV-firefly luciferase，pSpike或pDelta或pOmicron，pTAT，pRev以及pGAGpol。
(1)假病毒包装前一天：提前用0.1％明胶包被6孔板，使接种后的细胞更易于贴附。选择生长速度快、状态良好的HEK293T细胞，将其以1×106的细胞密度接种至6孔板中，置于37℃，5％CO2的细胞培养箱中过夜培养。
(2)假病毒包装：假病毒包装前，取1ml 10％FBS/DMEM新鲜培养基更换6孔板中原培养基。假病毒包装过程即为质粒的非脂质体瞬时转染过程，共转染以下5个质粒：2μg pCMV-firefly luciferase，0.4μg pSpike或0.4μg pDelta或0.4μg pOmicron，0.2μg pTAT，0.2μg pRev以及0.2μg pGAGpol。用200μl无血清DMEM稀释共转染的5质粒；用200μl无血清DMEM稀释9μl转染试剂PEI，混匀后室温静置5min；5min后将稀释后的转染试剂PEI逐滴加入稀释后的质粒混合物，混匀后室温静置20min。孵育时间结束后，将400μl质粒与转染试剂的混合物逐滴沿壁加入，呈井字形晃匀后放入37℃，5％CO2细胞培养箱孵育6-8h。6-8h后更换10％FBS/DMEM新鲜培养基。
(3)假病毒收集：细胞培养48h后用注射器小心吸取细胞上清，用0.45μm过滤柱过滤，同一种假病毒可以混在一起进行保存。48h的假病毒可以暂放4℃冰箱保存。48h假病毒收集后，沿壁小心加入2ml 10％FBS/DMEM新鲜培养基，继续培养24h即共培养72h。72h后同样用注射器小心吸取细胞上清，用0.45μm过滤柱过滤。48h和72h时间点收集的假病毒可以混在一起进行保存。假病毒分装后置于-80℃冻存。假病毒收集过程中 的枪头与其他与假病毒接触的实验器具应独立收集并灭菌后处理。
基于蛋白的ELONA实验
在本文中，我们借助了ELONA实验检测筛选到的核酸适配体的亲和力及检测潜能。分别进行了基于蛋白的ELONA实验和基于假病毒的ELONA实验。
(1)铺板：检测前一天，将溶于无菌水的不同浓度的蛋白以100μl总体积加至高结合表面的96孔酶标板中，用一次性塑料膜密封后置于4℃冰箱过夜孵育。(解离常数Kd值检测实验中，铺板RBD蛋白浓度为250ng，蛋白敏感性检测实验中，铺板RBD蛋白浓度分别为0.001μg、0.01μg、0.1μg、0.2μg以及0.4μg)。
(2)封闭：用真空吸液泵吸走孵育液，用排枪吸取清洗缓冲液对铺板孔进行清洗，再沿壁吸走清洗缓冲液，如此重复3次，尽量吸干孔底液体，吸液过程中避免触及孔底。清洗后，用排枪吸取封闭液并置于37℃孵育30min。封闭完成后仍重复3次清洗工作。(清洗缓冲液：PBS+0.05％(vol/vol)Tween-20，PH7.4；封闭液：清洗缓冲液+2％(weight/vol)BSA)。
(3)核酸适配体孵育：加入用灭菌的无酶水稀释不同浓度的生物素修饰核酸适配体后，置于不同温度如4℃、室温23℃以及37℃孵育1h。用真空泵小心沿壁吸走孵育液，用排枪吸取清洗缓冲液对铺板孔进行清洗，再沿壁吸走清洗缓冲液，如此重复3次，尽量吸干孔底液体，避免触及孔底，注意替换枪头。
(4)Streptavidin-HRP孵育：加入用灭菌水配置的0.1μg/ml Streptavidin-HRP室温孵育30min。随后用真空泵小心沿壁吸走孵育液，用排枪吸取清洗缓冲液对铺板孔进行清洗，再沿壁吸走清洗缓冲液，如此重复3次，尽量吸干孔底液体，避免触及孔底，注意替换枪头。
(5)底物孵育：加入200μl底物室温孵育20min，可观察到信号强的孔出现蓝色。
(6)终止反应：加入50μl终止液室温孵育2-5min，可观察到信号强的孔颜色由蓝色变为黄色。
(7)信号检测：利用酶标仪检测OD450nm吸光度，OD450nm读值越高，反映核酸适配体与蛋白的亲和结合越强。
(8)数据分析：本论文涉及的解离常数Kd值计算为Y＝BmaxX/(Kd+X)，Y代表OD450的平均值，Bmax代表OD450的最大值，X代表生物素修饰的核酸适配体的对应浓度，由GraphPad Prism软件自动分析得出。
基于假病毒的ELONA实验
病毒铺板：检测前一天，将不同滴度(TCID50)的假病毒粒子以100μl总体积加至高结合表面的96孔酶标板中，用一次性塑料膜密封后置于4℃冰箱过夜孵育。剩余操作与前述“基于蛋白的ELONA实验”一致。需注意的是，接触过假病毒的枪头及其它实验器械需高温高液后放入专用病毒废弃收集袋中集中处理。假病毒滴度检测参照Youchun Wang等团队发表的定量检测方法进行检测。
凝胶阻滞迁移率检测实验
实验根据Chemiluminescent EMSA试剂盒(碧云天)提供的操作说明书进行操作。简单操作如下：固定用量的生物素化核酸适配体(1pmol)与不同比例的SARS-CoV-2野生型或变异突变型Omicron BA.2 RBD蛋白(1：0，1：1，1：10，1：20，1：50)在EMSA结合缓冲液中于室温孵育30分钟。对应地，固定用量的生物素化核酸适配体(1pmol)与SARS-CoV-2 RBD蛋白以1：25的比例，同时加以与生物素化核酸适配体不同比例的冷探针(非生物素化核酸适配体，1：1，1：2，1：100，1：200)在EMSA结合缓冲液中于室温孵育30分钟。固定用量的生物素化核酸适配体(1pmol)与SARS-CoV-2变异突变型Omicron BA.2 RBD蛋白以1：50的比例，同时加以与生物素化核酸适配体不同比例的冷探针(非生物素化核酸适配体，1：1，1：2，1：100，1：200)在EMSA结合缓冲液中于室温孵育30分钟。孵育后，将孵育样品与上样缓冲液混合，并用4％聚丙烯酰胺凝胶在100V电压下电泳1小时，并将样品转移至N+尼龙膜上。电泳转移后，用手持紫外灯交联5-10分钟。随后通过化学发光检测生物素化标记的核酸适配体。
SARS-CoV-2真病毒中和实验
为检测筛选获得的RNA核酸适配体针对SARS-CoV-2病毒的抑制效率，结合现下SARS-CoV-2的流行情况，我们针对SARS-CoV-2 Delta毒株、SARS-CoV-2 Omicron BA.1毒株、SARS-CoV-2 Omicron BA.2毒株验证了RNA核酸适配体#2-1-18与#5-2-15对上述两株病毒的抑制效果。与病毒相关的实验过程在深圳市疾病预防控制中心生物安全三级实验室(Biological safety level-3 laboratory,BSL-3)进行，操作过程严格按照BSL-3实验室操作规定进行。SARS-CoV-2 Delta毒株所使用株系为SARS-CoV-2 B.1.617.2-深圳市疾病预防控制中心分离株(SZ/09/2022/SARS-CoV-2(D))；SARS-CoV-2 Omicorn BA.1毒株所使用株系为SARS-CoV-2 B.1.1.529 BA.1深圳市疾病预防控制中心分离株(SZ/08/2022/SARS-CoV-2(O))。SARS-CoV-2 Omicorn BA.2毒株所使用株系为SARS-CoV-2 B.1.1.529 BA.2深圳市疾病预防控制中心分离株SARS-CoV-2/shenzhen/13/2022(Omicron BA.2)。
(1)细胞准备。实验前一天在P2实验室将1.5×104的VERO细胞接种至96孔板中，培养基为10％FBS/DMEM。
(2)配置病毒稀释液。用2％FBS/DMEM分别将滴度为1×106TCID50的SARS-CoV-2 Delta毒株和滴度为1×106TCID50的SARS-CoV-2 Omicron毒株稀释为50 TCID50，标注为病毒稀释液Mix-D和Mix-O。
(3)配置样品-病毒孵育混合物：
(a)核酸样品-SARS-CoV-2 Delta毒株混合物
600nM:取100μM样品储存母液3.6μl+596.4μl上述病毒稀释液Mix-D；
200nM：取600nM样品200μl+400μl上述病毒稀释液Mix-D；
66.67nM：取200nM样品200μl+400μl上述病毒稀释液Mix-D；
22.22nM：取66.67nM样品200μl+400μl上述病毒稀释液Mix-D；
7.41nM：取22.22nM样品200μl+400μl上述病毒稀释液Mix-D；
2.47nM：取7.41nM样品200μl+400μl上述病毒稀释液Mix-D；
0.82nM：取2.47nM样品200μl+400μl上述病毒稀释液Mix-D；
0.27nM：取0.82nM样品200μl+400μl上述病毒稀释液Mix-D；
0.09nM：取0.27nM样品200μl+400μl上述病毒稀释液Mix-D；
0nM：400μl上述病毒稀释液Mix-D。
(b)核酸样品-SARS-CoV-2 Omicron毒株混合物
600nM:取100μM样品储存母液3.6μl+596.4μl上述病毒稀释液Mix-O；
200nM：取600nM样品200μl+400μl上述病毒稀释液Mix-O；
66.67nM：取200nM样品200μl+400μl上述病毒稀释液Mix-O；
22.22nM：取66.67nM样品200μl+400μl上述病毒稀释液Mix-O；
7.41nM：取22.22nM样品200μl+400μl上述病毒稀释液Mix-O；
2.47nM：取7.41nM样品200μl+400μl上述病毒稀释液Mix-O；
0.82nM：取2.47nM样品200μl+400μl上述病毒稀释液Mix-O；
0.27nM：取0.82nM样品200μl+400μl上述病毒稀释液Mix-O；
0.09nM：取0.27nM样品200μl+400μl上述病毒稀释液Mix-O；
0nM：400μl上述病毒稀释液Mix-O。
(4)样品-病毒孵育混合物孵育。将上述倍比稀释的样品-病毒孵育混合物在37℃，5％CO2细胞培养箱孵育1h。
(5)加药与孵育：吸走96孔板细胞培养基液体，将不同浓度的样品-病毒孵育混合物加至对应标记的96孔板中，每孔100μl，在37℃，5％CO2细胞培养箱孵育2h。2h后每孔更换120μl 2％FBS/DMEM培养基，在37℃，5％CO2细胞培养箱孵育48h。
(6)感染2天后上清核酸提取：48h后取100μl细胞上清于1.5ml管中，再加入200μl病毒灭活剂(试剂盒中的binding buffer)，混匀孵育10min后带至新冠核酸检测实验室，利用Roche自动化核酸提取仪进行样本的RNA提取。提取后每样本RNA体积为100μl，需进行分装保存，避免后续使用时反复冻融。每孔收完细胞上清后再补足100μl 2％FBS/DMEM培养基。
(7)感染5天后上清核酸提取：感染5天后再取100μl细胞上清于1.5ml管中，再加入200μl病毒灭活剂(试剂盒中的binding buffer)，混匀孵育10min以上带至新冠核酸检测实验室，利用Roche自动化核酸提取仪进行样本的RNA提取。提取后每样本RNA体积为100μl，需进行分装保存，避免后续使用时反复冻融。
RNA提取
本次实验的RNA提取主要为SARS-CoV-2 Delta毒株、Omicron BA.1以及Omicron BA.2毒株感染后，收集不同时间点的细胞上清进行RNA的提取。细胞上清的RNA提在新冠核酸检测实验室由Roche自动化核酸提取仪MagNA Pure 96 system进行批量提取。细胞上清中含病毒，带出BSL-3实验室需加入病毒灭活试剂并用酒精消毒处理。在新冠核酸检测实验室所有操作也应按规定进行，实验垃圾也应按要求处理。
一步法RT-qPCR实验
对提取的RNA需进行一步法RT-qPCR实验鉴定病毒的表达，以此衡量核酸药物对病毒的中和抑制作用。
(1)标准曲线制作
(a)溶解标准品：将购买的规格为5×105拷贝数/支的新型冠状病毒核酸N基因标准品干粉离心，12000rpm，离心2min。后加入500μl DEPC水溶解，使之浓度为1×106拷贝数/ml。
(b)稀释标准品：用DEPC水对标准品进行稀释，配置1000copies/μl、200copies/μl、40copies/μl、8copies/μl以及1.6copies/μl等不同浓度的标准品稀释液。实验过程中需使用无RNA酶的枪头、离心管等，操作在生物安全柜中操作。
(c)试剂配置：实验过程中需使用无RNA酶的枪头、离心管等，操作在生物安全柜中操作。按下表配置荧光PCR反应体系：
表6荧光PCR反应体系
(d)荧光定量PCR仪扩增：按下表设置荧光PCR反应程序：
表7荧光PCR扩增程序
荧光通道设置为：ORF1ab检测靶标：FAM reporter；N检测靶标：VIC/HEX。
(e)标准曲线制作：荧光定量PCR运行完后，点击分析，导出实验数据。将对应浓度下的CT值列入Excel表中，此时浓度应转化为对数值Log。选中数据，点击插入，选择“散点图”，选中散点图中数据点，单击右键，选择“添加趋势线”，设置趋势线格式，添加“显示公式”与“显示R平方值”，设置好坐标，标题，图例等。若R平方值大于0.99，该组实验数据可用。本次实验R2＝0.9975，得出的线性为：Y＝-3.7149X+37.122，X为样品浓度的对数值(拷贝数/μl)，Y为对应样品的实验CT值。
(2)实验样品测定
(a)反应试剂配置：实验过程中需使用无RNA酶的枪头、离心管等，操作在生物安全柜中操作。从-80℃冰箱取出分装的RNA样品，并置于冰上融化。按上述标准曲线制备中所述的荧光PCR反应体系配制反应体系。
(b)荧光定量PCR仪扩增：按上述扩增程序设置反应程序。因标准曲线制作时使用的标准品为N基因，故主要侧重于N检测靶标的数据收集。
(c)实验数据处理：荧光定量PCR运行完后，点击分析，导出实验数据。将对应样本的CT值导入上述公式，获得对应的样品浓度，乘以体积即得所测样品的病毒拷贝数。相对拷贝数＝实验组/对照组平均值*100％。
动物实验
与病毒相关的实验过程在广州海关技术中心生物安全三级实验室进行，操作过程严格按照BSL-3实验室操作规定进行。文献报道SARS-CoV-2 Omicron BA.2活病毒变异株可以感染野生型BALB/c小鼠。针对预防型验证实验：无特定病原5-6周BALB/c小鼠用异氟醚轻轻麻醉小鼠后，滴鼻给药溶于无菌无酶水以1.6mg/kg修饰后的核酸适配体(Chol-PEG40K-5-2-15-2’-F-O)，每边鼻孔25μl。滴鼻给药3小时后，用异氟醚轻轻麻醉小鼠后，给小鼠滴鼻给入1 x 105FFU的Omicron BA.2活病毒。24/48小时后，对小鼠进行麻醉，对肺脏组织进行肉眼观察并采集肺组织。将肺移入PBS并匀浆，收获澄清的上清液，提取RNA并进行RT-qPCR以定量病毒RNA。同时，在VERO E6细胞中测定使用FRNT方法测定每克肺组织中的病毒滴度。针对治疗型验证实验：无特定病原5-6周BALB/c小鼠用异氟醚轻轻麻醉小鼠后，给小鼠滴鼻给入1 x 105FFU的Omicron BA.2活病毒。3小时后用异氟醚轻轻麻醉小鼠并滴鼻给药溶于无菌无酶水以1.6mg/kg修饰后的核酸适配体(Chol-PEG40K-5-2-15-2’-F-O)，每边鼻孔25μl。24/48小时后，对小鼠进行麻醉，对肺脏组织进行肉眼观察并采集肺组织。将肺移入PBS并匀浆，收获澄清的上清液，提取RNA并进行RT-qPCR以定量病毒RNA。同时，在VERO E6细胞中测定使用FRNT方法测定每克肺组织中的病毒滴度。
统计方法和数据分析
本文所有数据均来自三次以上重复实验，并以平均值±标准差进行展示。我们利用Microsoft Excel 2021软件进行数据统计，利用GraphPad Prism 9进行数据处理和作图。实验采用双尾Student’t test进行显著性分析，其中*代表t<0.05，**代表t<0.01，***代表t<0.001，****代表t<0.0001，NS(not significant)代表差异不显著。
序列
本发明中描述的氨基酸或核酸序列提供于下表中。
表8本发明涉及的序列






实施例1核酸适配体筛选系统的设计
如图1A所示，本发明的核酸适配体筛选系统利用sgRNA的向导作用与dCas9的靶向作用将dCas9与sgRNA的复合物锚定在基本启动子(mini-promoter-2(SEQ ID NO:27))上游的sgRNA靶序列，同时将感兴趣的靶蛋白(X-protein)与转录激活元件融合表达。若插入在sgRNA骨架环状区的核酸适配体与游离表达的X-protein进行亲和识别，即可将与X-protein融合表达的转录激活元件招募至sgRNA靶序列，从而激活mini-promoter-2下游选择标记基因的表达。下游选择标记基因的表达即为核酸适配体与靶蛋白亲和结合能力的间接反映。之前的研究已经表明，sgRNA骨架如stem loop2和tetra loop区域大数量的碱基替换或删除不 会影响Cas9蛋白的催化功能。这些loop区域可以容忍添加核酸适配体以增强对Cas9与相关效应元件复合物的募集。此外sgRNA骨架可以允许大片段插入而不影响dCas9的活性。
筛选过程如图1B所示，将合成的寡核苷酸随机序列以sgRNA文库构建的方式克隆进定制化的慢病毒载体sgRNA骨架的环状区，从而形成了含有靶向外源人造sgRNA序列且其骨架区域附加寡核苷酸随机序列的初始文库。值得注意的是，定制化的慢病毒载体应已包含独立的X-protein与转录激活元件共表达的阅读框，且易于对X-protein进行分子克隆替换。随后，对该文库进行慢病毒包装与基于定制化通用型筛选报告细胞系的慢病毒感染。值得注意的是，通用型筛选报告细胞系应已整合选择报告系统(sgRNA靶序列-mini-promoter-2-选择标记基因)，且该报告系统对选择压力反应敏感。由于慢病毒的整合特性，编码寡核苷酸随机序列的DNA序列可以整合进筛选报告细胞系的基因组。若插入在sgRNA骨架区域的寡核苷酸随机序列中存在对X-protein的亲和性核酸适配体，即可将与X-protein共表达的转录激活元件招募至sgRNA靶向序列，即筛选报告系统的上游序列。当施加相应的报告系统筛选压力，筛选细胞系中的报告系统开始响应。当施加相应的选择标记基因筛选压力时，对应地细胞中的选择报告系统进行工作。核酸适配体与X-protein的亲和力越强，招募的共表达转录激活元件更多，对下游的选择标记基因的激活越强，筛选报告细胞系面对筛选压力时的反馈就越强。筛选完成后，获取富集细胞的基因组信息，扩增插入在sgRNA骨架环状区域的亲和性核酸适配体序列并通过二代测序(Next-Generation Sequencing,NGS)进行序列信息分析(图1B)，即可获取与靶蛋白有亲和力的核酸适配体序列。
实施例2核酸适配体筛选系统的验证
借助荧光素酶报告系统来进行验证，荧光素酶报告基因位于mini-promoter-2下游。sgRNA的向导作用与dCas9的靶向作用将dCas9与sgRNA的复合物锚定sgRNA靶序列，若sgRNA骨架中的核酸适配体可亲和识别靶蛋白，即可将与靶蛋白共表达的CRISPRa(转录激活元件)招募至sgRNA靶序列，随后转录激活元件作用于mini-promoter-2，从而激活报告基因荧光素酶的表达(图2B)。下游荧光素酶报告基因的表达取决于核酸适配体招募靶蛋白-转录激活元件复合物的效率，将核酸适配体与靶蛋白的亲和能力转化为荧光素酶报告基因的表达能力。
使用绿色荧光蛋白(Green Fluorescent Protein,GFP)和针对该蛋白利用SELEX筛选系统鉴定报道的RNA核酸适配体canonical GFP aptamer验证在细胞内可借助RNA核酸适配体与靶蛋白的亲和作用招募转录激活因子并激活荧光素酶报告基因的表达。
首先，将此canonical GFP aptamer分别插入到sgRNA骨架的tetra loop区域(sgRNA  1.1)或stem loop2区域(sgRNA 1.2)(图2A)，同时将GFP蛋白与转录激活元件VP64、P65和HSF1(也简称为VPH)融合表达(图2B)。然后构建含1个拷贝gLuc靶向序列的荧光素酶报告质粒(1×gLuc-minPro-luciferase)。随后将稳定表达dCas9的293T/dCas9细胞系与表达(1)sgRNA1.1结构或sgRNA1.2结构，(2)GFP-VPH，(3)1×gLuc-minPro-luciferase等共计3个质粒共转染。48小时后检测荧光素酶表达信号。
如图2C所示，sgRNA 1.1组和sgRNA 1.2组的报告基因的表达能力没有显著改变，说明canonical GFP aptamer插入到sgRNA骨架的tetra loop区域(sgRNA 1.1)或stem loop2区域(sgRNA 1.2)不会影响dCas9的活性。
进一步地，构建含9个拷贝gLuc靶向序列的荧光素酶报告质粒(9×gLuc-minPro-luciferase)来探索本发明的系统是否存在叠加效应。将稳定表达dCas9的293T/dCas9细胞系与表达(1)sgRNA1.1结构或sgRNA1.2结构，(2)GFP-VPH，(3)1×gLuc-minPro-luciferase或9×gLuc-minPro-luciferase等共计3个质粒共转染。48小时后检测荧光素酶表达信号。
结果如图2C和2D所示，相比单个拷贝的gLuc sgRNA靶序列(图2C)，使用9个串联重复的gLuc sgRNA靶序列时，可检测到显著的荧光素酶表达信号，这表明借助多个拷贝的gLuc靶序列可招募更多的CRISPRa转录激活元件，提高了系统测试的灵敏度(图2D)。此外，相比附加到sgRNA骨架的tetraloop区域(sgRNA 1.1)的GFP aptamer，附加到sgRNA骨架stem loop2区域(sgRNA 1.2)的GFP aptamer显示出了更显著的荧光素酶激活效果。
实施例3通用型筛选细胞系的构建
为便于收集筛选后经施加选择压力成功富集的细胞，我们将报告基因由荧光素酶替换成了嘌呤酶素抗性基因。为便于后续系统筛选的可操作性和便捷性，我们在上述293T/dCas9单克隆细胞系的基础上通过进一步递送慢病毒报告系统元件9×gLuc-minPro-puro的方式获得了一些dCas9/9×gLuc-puro的293T单克隆细胞系。
为鉴定这些单克隆细胞系的有效性与本底效应，我们仍利用前述GFP和GFP aptamer的亲和关系进行验证。这些单克隆细胞系分别进行了如下慢病毒组分感染：阳性对照组(Positive Controls，PC：gLuc sgRNA-1.2 GFP aptamer；GFP-VPH)，阴性对照组(Negative Controls，NC：gLuc sgRNA-1.2随机序列；GFP-VPH)(图3)。慢病毒感染48小时后，分别对这些单克隆细胞系的不同实验组施以不同浓度的嘌呤霉素(0.5μg/ml、1μg/ml和2μg/ml)，随着浓度的增加，代表着筛选压力不断增加。嘌呤霉素持续培养7-10天后，对每 个dCas9/9×gLuc-puro单克隆细胞系的存活克隆数量进行定量分析(图4)。
我们定义NC病毒组份感染后存活克隆数少的单克隆细胞系为本底活性较低的单克隆细胞系，PC病毒组份感染后存活克隆数多的单克隆细胞系为对筛选体系反应敏感且存活能力较强的单克隆细胞系，PC/NC比率高的单克隆细胞系为综合活性较好的单克隆细胞系。
定量结果如图4所示，相比其他浓度的嘌呤霉素，使用2μg/ml的嘌呤霉素浓度时多数单克隆细胞系显示出了较高的PC/NC比率，这表明高浓度嘌呤霉素下能过滤一些本底活性。此外，不同嘌呤霉素浓度下，克隆#4-53均显示出了较高的PC/NC比率，同时在2μg/ml的嘌呤霉素作用下，该细胞系显示出了极低的本底效应却较高的存活能力(图2E)，选择克隆#4-53为后续核酸适配体筛选的通用型筛选细胞系。
实施例4通用型核酸适配体筛选系统的构建及筛选流程
基于上述CRISPR/Cas9的RNA核酸适配体筛选系统的验证结果，我们提出了一种基于CRISPR/Cas9系统的通用型RNA aptamers筛选系统，将其命名为CRISmers。CRISmers筛选系统作为RNA核酸适配体筛选时，一方面可在细胞内完成对核酸适配体与蛋白亲和识别力的定量转化，最大限度地维持了靶点蛋白的天然结构状态，同时最大可能地保持了核酸适配体未来发挥作用时环境的真实性；另一方面，多轮的筛选以及每轮高浓度的抗生素筛选，避免了因死细胞存在带来的背景噪音，也提高了筛选的严格性。
为了便于筛选文库构建、荧光素酶报告系统验证等实验的可操作性与便捷性，我们构建了一个可定制化的慢病毒载体，即phU6-gLuc sgRNA-1.2 BsmBI-BsmBI-polyT-EF1a-NLS-linker-EcoRI-EcoRI-linker-VP64-P65-HSF1-NLS-linker-P2A-linker-Zeo-WPRE，如图12所示。具体表现为：(1)含gLuc sgRNA-1.2结构区域核酸适配体插入位点BsmBI；(2)待筛选的靶蛋白插入位点EcoRI；(3)待筛选的靶蛋白与转录激活元件VPH的融合表达。该慢病毒载体仅需一步分子克隆即可替换感兴趣的待筛选靶蛋白，仅需一次文库构建即可获得针对感兴趣的待筛选靶蛋白的寡核苷酸随机序列文库。同时为了承接文库的筛选，使用上述实施例3中构建的通用型筛选单克隆细胞系即#4-53 dCas9/9×gLuc-puro。
如图5所示，一个筛选循环的工作流程可分为以下七个步骤：首先，基于通用慢病毒表达载体借助EcoRI限制性内切酶自定义插入感兴趣的靶蛋白形成中间载体。合成寡核苷酸随机序列文库，借住BsmBI限制性内切酶将该随机序列文库克隆到中间载体的gLuc sgRNA骨架stem loop2区域，至此完成文库构建的工作。第二，将上述表达含有针对特定靶蛋白用于筛选的寡核苷酸随机序列文库包装到慢病毒颗粒中，并利用Zeocin抗生素检测慢病毒颗粒的病毒滴度，至此完成文库慢病毒包装的工作。第三，这些慢病毒颗粒将以一定 的MOI感染上述#4-53 dCas9/9×gLuc-puro通用型筛选报告单克隆细胞系。第四，随着筛选的进行，对慢病毒文库感染后的细胞施加2μg/ml的嘌呤霉素选择压力。第五，随着嘌呤霉素的筛选富集，收集存活的细胞。第六，裂解细胞，特异性扩增含有核酸适配体区域的DNA序列，通过深度测序对获取的序列进行分析与验证。对挑出来的核酸适配体序列利用荧光素酶报告系统进行二次筛选验证。第七，必要时，将前一轮扩增的核酸适配体序列亚克隆至原始的通用慢病毒表达载体来启动下一轮筛选工作。
实施例5针对SARS-CoV-2刺突蛋白受体结构域的RNA核酸适配体筛选
考虑到SARS-CoV-2诊断和治疗方法需求的迫切性，我们利用CRISmers系统靶向SARS-CoV-2 S蛋白RBD区域进行了RNA核酸适配体的筛选。在实验中，我们分别进行了两个独立的平行筛选，每个独立筛选共计五轮。在第一轮筛选中，为了以最大限度地扩大在感染细胞中核酸适配体的代表性，在gLuc sgRNA骨架stem loop2区域附加20个寡核苷酸随机序列且靶向S1蛋白RBD区域的慢病毒文库以约为3的MOI感染#4-53 9×gLuc-puro通用型筛选报告单克隆细胞系。从第二轮筛选开始，亚文库以约为0.1-0.2的MOI感染#4-53 9×gLuc-puro通用型筛选报告单克隆细胞系以确保大多数细胞仅进入一个含有20个寡核苷酸序列的病毒粒子。
利用CRISmers系统，我们针对SARS-CoV-2 S蛋白RBD区域进行了RNA核酸适配体的筛选，由于核酸适配体的长度多集中在20-40个碱基，我们合成了20个碱基的寡核苷酸随机序列文库。现有的分子实验技术无法获得所有序列可能性即420个差异序列，我们在文库的构建过程中尽可能多的扩大劳动量，获得更多的寡核苷酸序列。在筛选中，我们独立进行了2个平行样本的筛选，并在初始轮筛选时以较大的慢病毒感染复数感染细胞，最大限度地扩大在感染细胞中核酸适配体的代表性。
在经历寡核苷酸随机序列文库构建、文库慢病毒包装、文库慢病毒感染、2μg/ml的嘌呤霉素筛选、存活细胞收集、扩增序列与深度测序、亚文库构建等循环，我们共计进行了2个独立样本的五轮筛选。针对第二轮、第三轮、第四轮以及第五轮中每份独立样本的深度测序数据分析并过滤掉非特异性序列后我们发现，随着后续筛选轮数的进行，获得的核酸适配体独立序列数量逐渐减少(如下表所示)。在第四轮和第五轮筛选时，核酸适配体独立序列数量不再显著减少，这表明此时筛选富集压力已经足够，文库筛选已经饱和，不再需要进一步的亚文库筛选(图6)。
表9每轮筛选所获序列分析
接下来，我们将对挑选出的RNA核酸适配体进行基于荧光素酶报告系统的二轮筛选与验证。
两个独立筛选样本在第二轮、第三轮、第四轮和第五轮筛选后经深度测序后将获得的序列通过两种方式进行排序：第一，将所有独立序列按出现重复数进行排列，选择前0.1％高频序列进行验证；第二，计算序列的富集指数(每个独立序列相邻两轮出现的重复数比率)并排序，选择每轮富集指数高的前15条序列进行验证。经上述两种方式挑选出的RNA核酸适配体，我们首先利用荧光素酶报告系统进行文库筛选后的第二次筛选。挑选得到的序列经合成并利用BsmBI限制性内切酶构建到靶向S1蛋白RBD表达载体中，即phU6-gLuc sgRNA-1.2-BsmBI-BsmBI-polyT-EF1a-NLS-linker-S1 RBD-linker-VP64-P65-HSF1-NLS-linker-P2A-linker-Zeo-WPRE，在293T细胞中共转染9×gLuc sgRNA-gaussia luciferase与dCas9表达元件等共计3个质粒，验证挑选的RNA核酸适配体是否可以凭借其与RBD的亲和识别招募转录激活元件并激活Gaussia荧光素酶的表达。需要注意的是，此时的二次筛选我们将Firefly荧光素酶替换成了分泌型的Gaussia荧光素酶,可直接检测分泌到细胞上清的荧光素酶，在批量实验检测中简化了实验步骤，提高了实验效率。另需要注意的是，高频出现的序列可能是PCR反应的偏倚副产物，不一定表现出高富集倍数，两者并非完全对等的关系。此外，我们发现在第四轮尤其在第五轮的序列分析挑选时，一些高频序列或高富集指数序列在前序轮次筛选中已被挑出及验证，这表明第四轮至第五轮的筛选压力已经足够，与上述对深度测序后数据分析结果一致，这验证了该系统筛选结果的一致性。我们对上述挑选的RNA核酸适配体序列用荧光素酶报告系统进行了验证，结果如图7所示，第2轮筛选已经可以筛选到表现出高荧光素酶激活信号的核酸适配体。在验证中显示出荧光素酶高显著激活信号的核酸适配体有#2-1-17、#2-1-18、#2-2-24、#3-1-31、#3-2-3、#4-1-20和#5-2-15，其序列被列于上述表8中。
为保证实验结果的准确性，我们将多轮在基于荧光素酶激活实验的二次筛选实验中显示荧光素酶显著激活的序列在同一实验中进行了平行比较。如图8A所示，序列#2-1-18和 序列#5-2-15显示出了最高的荧光素酶激活信号。图8B展示了核酸适配体序列#2-1-18和序列#5-2-15在sgRNA中的结构。图8C展示了利用Mfold webserver软件预测的RNA核酸适配体的二级结构和自由能值。
实施例6 RNA核酸适配体针对SARS-CoV-2及其变体假病毒的检测能力
选择了序列#2-1-18和序列#5-2-15进行后续功能验证。首先，我们借助酶联寡核苷酸测定方法(Enzyme-linked oligonucleotide assay，ELONA)进行亲和力与特异性的测定(图9A)。
结果如图9B所示，两条RNA核酸适配体序列均表现出了与RBD重组蛋白的高亲和性结合，经过计算，RNA核酸适配体#2-1-18和#5-2-15解离常数(equilibrium association constant,Kd)值分别为16.61nM和22.65nM。
进一步，为了检测RNA核酸适配体的诊断敏感性，我们在ELONA反应中使用100nM的核酸适配体针对不同浓度的RBD重组蛋白，检查了RNA核酸适配体的检测极限。在该实验中，重组的程序性细胞死亡受体1蛋白(Programmed cell death protein 1，PD1)，SARS-CoV-2的非结构蛋白7和8蛋白复合物(Non-structural proteins 7and 8，NSP7-8)，以及同样长度的随机RNA序列作为阴性对照。
结果如图9C和图9D所示，两个核酸适配体靶向SARS-CoV-2野生型刺突蛋白RBD重组蛋白都展示出了较好的亲和性和特异性，即可检测浓度低至0.1μg/ml的RBD重组蛋白。如图9D和图9H所示，两个核酸适配体靶向SARS-CoV-2关切突变株Delta刺突蛋白RBD重组蛋白都展示出了较好的亲和性和特异性。如图9E和图9I所示，两个核酸适配体靶向SARS-CoV-2关切突变株Omicron BA.1刺突蛋白RBD重组蛋白都展示出了较好的亲和性和特异性。如图9F和图9J所示，两个核酸适配体靶向SARS-CoV-2关切突变株Omicron BA.2刺突蛋白RBD重组蛋白都展示出了较好的亲和性和特异性。
同样的，我们在ELONA反应中使用100nM的核酸适配体针对不同浓度滴度的SARS-CoV-2野生型刺突蛋白假病毒粒子(图9K)、SARS-CoV-2 Delta突变的刺突蛋白假病毒粒子(图9L)、SARS-CoV-2 Omicron BA.1突变的刺突蛋白假病毒粒子(图9M)和SARS-CoV-2 Omicron BA.2突变的刺突蛋白假病毒粒子(图9N)，检查了RNA核酸适配体的检测极限。在实验中，同样长度的随机RNA序列作为阴性对照。
结果如图9K-9N所示，两个核酸适配体都展示出了较好的亲和性和特异性，即可检测假病毒浓度滴度低至1 TCID50/100μl(50％tissue culture infective dose，TCID50)的四种假病毒。
进一步地，基于RBD重组蛋白的ELONA实验的结果如图10所示，#core-5-2-15两 端的固定序列互补且便于序列合成和扩增，并且对于RNA核酸适配体针对靶蛋白亲和力活性有明显提升。因筛选过程中，随机序列可能会与两端固定序列部分碱基形成不同的二级结构从而影响RNA核酸适配体的形成。因此，在优选的实施方案中，RNA核酸适配体完整序列包含中间筛选得到的有效序列与两端固定序列。
此外，如图11所示，在基于RBD重组蛋白的ELONA实验中，两种核酸适配体均能在4℃、25℃和37℃保持结合活性，这表明它们在较宽的工作温度范围内仍具有潜在的应用优势。
实施例7 RNA核酸适配体针对SARS-CoV-2关切变异活病毒珠的中和活性
考虑到RNA核酸适配体与RBD的结合可能会阻断SARS-CoV-2刺突蛋白与hACE2的相互作用，我们在P3实验室对#5-2-15RNA核酸适配体对SARS-CoV-2关切变异活病毒珠Delta(B.1.617.2)和Omicron BA.1(B.1.1.529 BA.1)进行了病毒中和活性检测，检测方案如图12A所示。因#5-2-15在前期多项验证中展示出了更好的结合活性，我们首先对#5-2-15进行了下述验证。
在对SARS-CoV-2活病毒感染VERO细胞前，我们分别将不同浓度的RNA核酸适配体与SARS-CoV-2活病毒进行了孵育，随后将RNA核酸适配体-活病毒混合物与VERO细胞进行孵育。随后分别在病毒感染细胞后第2天、第4天收取细胞上清并提取病毒RNA，利用RT-qPCR对病毒拷贝数进行分析。
结果表明，与前述针对假病毒的结合活性结果一致(图9H和图9I)，RNA核酸适配体#5-2-15在两个不同时间点，针对两种SARS-CoV-2关切变异活病毒珠均显示出了较好的剂量依赖抑制活性，如图12B、图12C所示，在病毒感染后2天检测中和活性，#5-2-15对SARS-CoV-2 Delta的IC50值(半抑制浓度，half maximal inhibitory concentration)为26.25ng·ml-1，对SARS-CoV-2 Omicron BA.1的IC50值为0.4628ng·ml-1。如图12D、图12E所示，在病毒感染后4天检测中和活性，#5-2-15对SARS-CoV-2 Delta的IC50值为19.91ng·ml-1，对SARS-CoV-2 Omicron BA.1的IC50值为0.6006ng·ml-1。
此外，我们对另一个备选核酸适配体#2-1-18针对Omicron BA.1的抗病毒活性进行了检测。如图13A、图13B所示，在病毒感染后2天检测中和活性，#2-1-18对SARS-CoV-2Omicron BA.1的IC50值为6.732ng·ml-1(图13A)，在病毒感染后4天检测中和活性，#2-1-18对SARS-CoV-2 Omicron BA.1的IC50值为10.23ng·ml-1(图13B)。
实施例8经修饰的RNA核酸适配体针对SARS-CoV-2关切变异活病毒珠的中和活性
核酸适配体的化学修饰有助于提高核酸分子的稳定性，延长半衰期，降低其免疫原性等，我们也对筛选得到的核酸适配体进行了化学修饰并利用ELONA实验和EMSA实验对未修饰与修饰的核酸适配体进行了结合活性与特异性的检测。
如图14A-14B所示，氟化与甲氧基共修饰的核酸适配体相比未修饰或其他单独氟化或甲氧基修饰的结合活性更强。如图14C-14D所示，相比未修饰的核酸适配体，2’-F-O修饰的核酸适配体展现了更强的结合活性，阴性对照无结合活性。如图14E-14F所示，针对两种病毒的RBD重组蛋白，修饰后的核酸适配体展现了较好的剂量依赖结合活性，同时加入未生物素化修饰的核酸适配体(Cold-apt)时，展现出了较好的剂量依赖竞争性结合活性。
跟据上述结果，我们又进一步针对氟化与甲氧基共修饰的核酸适配体体针对SARS-CoV-2 Omicron BA.1与BA.2进行了活病毒中和实验验证。结果如图15-图17所示，修饰后的核酸适配体均表现出较好的病毒中和活性。
依据上述结果，我们又进一步针对不同的PEG与胆固醇修饰后的核酸适配体针对SARS-CoV-2 Omicron BA.2进行了活病毒中和实验验证。结果如图18所示，修饰后的核酸适配体均表现出较好的病毒中和活性。#5-2-15在活病毒中和活性中展示了更好的潜能，我们进一步应用#5-2-15核酸适配体进行了更丰富的化学修饰。结果如图19-图21所示，修饰后的核酸适配体均表现出更好的病毒中和活性。
为验证核酸适配体的结合特异性，针对上述的修饰后核酸适配体，我们将化学修饰物胆固醇、PEG40K与胆固醇-PEG40K同步进行了活病毒中和活性验证。结果分别如图22-图24所示，均未见病毒抑制活性；这提示上述核酸适配体对活病毒的中和活性并非来自单独的化学修饰物胆固醇、PEG40K与胆固醇-PEG40K的作用。
实施例9经修饰的RNA核酸适配体对病毒的预防作用与治疗作用
为验证所筛选得到的核酸适配体与修饰后核酸适配体在动物体内的针对活病毒的中和活性，模拟核酸适配体对病毒的预防作用与治疗作用，结合以上结果，我们对化学修饰的核酸适配体Chol-PEG40K-#5-2-15-2’-F-O和Chol-PEG6-#5-2-15-2’-F-O进行了动物实验，验证其在动物体内针对SARS-CoV-2 Omicron BA.2活病毒的预防作用和治疗作用。
如图25所示，结果表明Chol-PEG40K-#5-2-15-2’-F-O RNA核酸适配体在动物体内针对SARS-CoV-2 Omicron BA.2病毒感染后24与48小时均具有很好的病毒预防作用与治疗作用。
如图26所示，结果表明，时Chol-PEG6-#5-2-15-2’-F-O核酸适配体在动物体内针对SARS-CoV-2 Omicron BA.2病毒感染后24与48小时均具有病毒预防作用与治疗作用。相比 病毒感染48小时，Chol-PEG6-#5-2-15-2’-F-O对活病毒感染24小时具有更好的预防与治疗作用。
实施例10 CRISmers系统的应用型探索
为拓展CRISmers系统的应用范围，我们进行了不同CRISPR/Cas系统、不同筛选选择标记物以及不同筛选宿主的嫁接替换实验。
如图27所示，将CRISmers系统中dCas9替换为dCasMINI-V4之后，核酸适配体与靶蛋白的亲和结合能力仍然能有效转化为荧光素酶的表达能力，激活荧光素酶的表达，表明本发明的CRISmers系统可以适用于不同的CRISPR/Cas系统。
如图28所示，将CRISmers系统中嘌呤霉素报告基因替换为绿色荧光蛋白报告基因之后，结果显示核酸适配体与靶蛋白的亲和结合能力仍然能有效转化为绿色荧光蛋白的表达能力，激活绿色荧光蛋白的表达。表明本发明的CRISmers系统可以使用不同的选择标志物。
如29所示，将CRISmers系统中原嘌呤霉素报告基因替换为大肠杆菌卡纳霉素报告基因并转化至大肠杆菌中之后，核酸适配体与靶蛋白的亲和结合能力将转化为卡那霉素报告基因的表达能力即对大肠杆菌施加卡那霉素时其生存能力。结果显示，CRISmers系统转化至大肠杆菌中，#5-2-15核酸适配体与SARS-CoV-2 RBD靶蛋白的亲和作用转化为卡那霉素的抗性作用，支持大肠杆菌在含100μg ml-1卡那霉素LB琼脂板中生长。#2-1-18核酸适配体与SARS-CoV-2 RBD靶蛋白的亲和作用转化为卡那霉素的抗性作用，支持大肠杆菌在含75μg ml-1卡那霉素LB琼脂板中生长。表明本发明的CRISmers系统可以适用不同的宿主筛选细胞。如图31所示，我们将CRISmers系统筛选组分原件整合于一个载体中，利用第二限制性内切酶EcoRI可以将感兴趣的待筛选靶蛋白插入载体中用于筛选，利用第一限制性内切酶BsmBI将随机序列文库按CRISPR sgRNA文库构建的方式将其插入载体中形成最终筛选文库用于筛选。将最终筛选文库利用电转化的方式转入大肠杆菌筛选宿主，经过氨苄抗生素富集、卡那霉素抗生素筛选后收集抗氨苄抗生素，抗卡那霉素抗生素存活的大肠杆菌，提取大肠杆菌中质粒并进行下游的测序与验证过程。
应该理解的是，尽管已经通过优选实施方式和任选的特征具体公开了本发明，但是本领域技术人员可以对本文所公开的本发明进行修改、改进和变化，这些修改、改进和变化被认为在本发明的范围内。在此提供的材料、方法和实施例是优选的实施方式的代表和示例性的，并且不旨在作为对本发明范围的限制。

Claims (55)

  1. 一种针对靶蛋白筛选RNA核酸适配体的系统，其包含：
    (i)向导RNA，含有识别序列和预定长度的核酸适配体随机文库；
    (ii)靶向序列，其与所述向导RNA的识别序列配对；
    (iii)选择标记，位于所述靶向序列下游，包括基本启动子和选择标记基因；
    (iv)融合蛋白，其包含所述靶蛋白和转录激活元件，所述靶蛋白与所述核酸适配体随机文库中的RNA核酸适配体结合导致所述转录激活元件与所述选择标记的结合；
    (v)dCas蛋白，其在所述向导RNA引导下特异性识别靶向序列，以及
    (vi)筛选细胞。
  2. 根据权利要求1所述的系统，其中所述核酸适配体随机文库由长度为8至60个碱基、105至1036数量级的单链寡核苷酸随机序列组成。
  3. 根据权利要求1所述的系统，所述核酸适配体随机文库插入到向导RNA骨架的loop区。
  4. 根据权利要求1所述的系统，其中所述靶向序列是外源和/或人工序列。
  5. 根据权利要求4所述的系统，其中所述靶向序列包含复数个拷贝的gLuc序列或Tet序列。
  6. 根据权利要求1所述的系统，其中所述选择标记基因是荧光素酶基因、荧光蛋白基因或抗生素抗性基因。
  7. 根据权利要求1所述的系统，其中所述转录激活元件包含多个转录激活因子。
  8. 根据权利要求7所述的系统，其中所述转录激活元件包含VP64、P65和HSF1。
  9. 根据权利要求1所述的系统，其中所述基本启动子选自mini-promoter-1(SEQ ID NO:28)、mini-promoter-2(SEQ ID NO:27)、mini-TK promoter(SEQ ID NO:29)、mini-CMV promoter(SEQ ID NO:30)和Crystallin basal promoter(SEQ ID NO:31)。
  10. 根据权利要求1所述的系统，其中所述dCas蛋白是dCas9蛋白或dUn1Cas12f1蛋白。
  11. 根据权利要求1所述的系统，其包含病毒表达载体，所述病毒表达载体包含所述向导RNA和编码所述融合蛋白的核酸序列；并且所述筛选细胞表达所述dCas蛋白且包含所述靶向序列和所述选择标记。
  12. 根据权利要求11所述的系统，其中所述病毒表达载体是慢病毒表达载体。
  13. 根据权利要求12所述的系统，其中所述慢病毒表达载体进一步包含可操作地连接至所述向导RNA的第一启动子和可操作地连接至所述融合蛋白的编码序列的第二启动子。
  14. 根据权利要求13所述的系统，其中所述核酸适配体随机文库通过第一限制性内切酶插 入到向导RNA骨架。
  15. 根据权利要求13所述的系统，其中所述靶蛋白的编码序列通过第二限制性内切酶可操作地连接至所述第二启动子。
  16. 根据权利要求15所述的系统，其中所述慢病毒表达载体包含位于向导RNA下游的polyT。
  17. 根据权利要求16所述的系统，其中所述慢病毒表达载体包含核定位序列(NLS)。
  18. 根据权利要求17所述的系统，其中所述NLS包括在第二启动子下游的第一NLS和在所述融合蛋白的编码序列的下游的第二NLS。
  19. 根据权利要求18所述的系统，其中所述慢病毒表达载体包含位于第二NLS下游的P2A、抗生素抗性基因和转录后调控元件。
  20. 根据权利要求19所述的系统，其中所述P2A通过连接子分别与上游的第二NLS和下游的抗生素抗性基因连接，所述靶蛋白的编码序列通过连接子分别与上游的第一NLS和下游的转录激活因子的编码序列连接。
  21. 根据权利要求1所述的系统，其中所述筛选细胞是真核细胞或原核细胞。
  22. 根据权利要求21所述的系统，其中所述筛选细胞是原核细胞，所述系统包含一种或多种质粒载体，所述质粒载体包含选自所述向导RNA；所述靶向序列和所述选择标记；编码所述融合蛋白的核酸序列；以及编码所述dCas蛋白的核酸序列中的一个或多个。
  23. 根据权利要求22所述的系统，其中所述系统包含一种质粒载体，所述质粒载体包含所述向导RNA、所述靶向序列、所述选择标记、以及编码所述融合蛋白和所述dCas蛋白的核酸序列。
  24. 根据权利要求1所述的系统，其中所述靶蛋白来源于病毒、细菌、真菌或动物。
  25. 根据权利要求1所述的系统，其中所述靶蛋白来源于人类。
  26. 根据权利要求1所述的系统，其中所述靶蛋白是绿色荧光蛋白或来源于SARS-CoV-2的S1蛋白的RBD区域。
  27. 一种筛选针对靶蛋白的RNA核酸适配体的方法，其包含以下步骤：
    (1)提供筛选细胞，所述筛选细胞具有向导RNA、靶向序列、选择标记、融合蛋白和dCas蛋白；其中所述向导RNA含有识别序列和预定长度的核酸适配体随机文库；所述靶向序列与所述向导RNA的识别序列配对；所述选择标记位于所述靶向序列下游且包括基本启动子和选择标记基因；所述融合蛋白包含所述靶蛋白和转录激活元件，所述靶蛋白与所述核酸适配体随机文库中的RNA核酸适配体结合导致所述转录激活元件与所述选择标记的结合；所 述dCas蛋白在所述向导RNA引导下特异性识别靶向序列；
    (2)利用选择标记基因对筛选细胞进行筛选；
    (3)收集表达所述选择标记基因的筛选细胞；
    (4)裂解筛选细胞，对含有核酸适配体区域的序列，进行特异性扩增，通过测序获得所述核酸适配体的序列信息。
  28. 根据权利要求27所述的方法，其中步骤(4)进一步包含对获得的核酸适配体序列进行分析与验证。
  29. 根据权利要求27所述的方法，其中所述筛选细胞是通过将一种或多种质粒载体转染至原核细胞中得到的，所述质粒载体包含选自所述向导RNA；所述靶向序列和所述选择标记；编码所述融合蛋白的核酸序列；以及编码所述dCas蛋白的核酸序列中的一个或多个。
  30. 根据权利要求29所述的方法，其中所述筛选细胞是通过将一种质粒载体转染至原核细胞中得到的，所述质粒载体包含所述向导RNA、所述靶向序列、所述选择标记、以及编码所述融合蛋白和所述dCas蛋白的核酸序列。
  31. 根据权利要求29所述的方法，所述原核细胞是大肠杆菌细胞。
  32. 根据权利要求27所述的方法，其中所述筛选细胞通过以下步骤提供：
    (1.1)提供一种表达所述dCas蛋白的细胞，所述细胞还包含所述靶向序列和所述选择标记；
    (1.2)提供一种编码所述向导RNA和所述融合蛋白的病毒表达载体；
    (1.3)对所述病毒表达载体进行包装；然后使用包装后的病毒表达载体感染所述细胞。
  33. 根据权利要求32所述的方法，其进一步包含步骤(5)：将步骤(4)中得到的核酸适配体序列亚克隆至步骤(1.2)中的病毒表达载体，然后重复步骤(1.3)和步骤(2)至步骤(4)。
  34. 根据权利要求33所述的方法，步骤(5)进一步被重复1至3次。
  35. 根据权利要求33或34所述的方法，其中第一次执行步骤(1.3)时，采用3-5的MOI进行病毒感染，再次执行步骤(1.3)时，采用0.1-0.3的MOI进行病毒感染。
  36. 根据权利要求33或34所述的方法，其中在步骤(1.3)中利用抗生素检测包装后的病毒表达载体的病毒滴度。
  37. 根据权利要求32所述的方法，其中所述病毒表达载体是慢病毒表达载体。
  38. 根据权利要求32所述的方法，其中所述细胞是HEK293T细胞。
  39. 根据权利要求27所述的方法，其中所述dCas蛋白是dCas9蛋白或dUn1Cas12f1蛋白。
  40. 根据权利要求27所述的方法，其中所述基本启动子选自mini-promoter-1(SEQ ID NO:28)、mini-promoter-2(SEQ ID NO:27)、mini-TK promoter(SEQ ID NO:29)、mini-CMV promoter(SEQ ID NO:30)和Crystallin basal promoter(SEQ ID NO:31)。
  41. 根据权利要求27所述的方法，其中所述靶蛋白来源于病毒、细菌、真菌或动物。
  42. 根据权利要求27所述的系统，其中所述靶蛋白来源于人类。
  43. 根据权利要求27所述的方法，其中所述靶蛋白是来源于SARS-CoV-2的S1蛋白的RBD区域。
  44. 一种特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体，所述RNA核酸适配体序列包含中间的随机区域和两端的固定序列，两端的固定序列互补，其中所述随机区域包含如SEQ ID NO:1至SEQ ID NO:7中任一个所示的核酸序列。
  45. 根据权利要求44所述的RNA核酸适配体，其中所述随机区域包含如SEQ ID NO:2或SEQ ID NO:7所示的核酸序列。
  46. 根据权利要求44所述的RNA核酸适配体，其中所述RNA核酸适配体序列的5’端的固定序列包含如SEQ ID NO:8所示的核酸序列。
  47. 根据权利要求44所述的RNA核酸适配体，所述RNA核酸适配体序列的3’端的固定序列包含如SEQ ID NO:9所示的核酸序列。
  48. 根据权利要求44所述的RNA核酸适配体，其中所述RNA核酸适配体进一步被选自氟、甲氧基、聚乙二醇(PEG)多聚体、胆固醇-PEG多聚体中的一个或多个修饰。
  49. 根据权利要求48所述的RNA核酸适配体，其中所述PEG多聚体是选自小分子量的六聚体至40 kD高分子量的多聚体。
  50. 根据权利要求44所述的RNA核酸适配体，其中所述SARS-CoV-2病毒是SARS-CoV-2野生型、Delta或Omicron BA.1、Omicron BA.2病毒株。
  51. 一种用于检测SARS-CoV-2病毒的检测试剂或试剂盒，其包含如权利要求44至50中任一项所述的RNA核酸适配体。
  52. 一种用于在对象中中和SARS-CoV-2病毒的药物，其包含如权利要求44至50中任一项所述的RNA核酸适配体。
  53. 根据权利要求52所述的药物，其进一步包含病毒载体或非病毒载体，其中所述载体包含或携带所述特异性结合SARS-CoV-2病毒的S1蛋白的RNA核酸适配体。
  54. 一种药物组合物，其包含如权利要求52或53所述的药物和药学上可接受的赋形剂。
  55. 权利要求44至50中任一项所述的RNA核酸适配体在制备用于预防或治疗SARS-CoV-2 病毒感染的药物中的应用。
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