CN113122542B - ssDNA aptamer of 2019-nCoV S1 protein, screening method and application - Google Patents
ssDNA aptamer of 2019-nCoV S1 protein, screening method and application Download PDFInfo
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Abstract
The invention relates to a set of ssDNA (deoxyribonucleic acid) aptamer of 2019-nCoV S1 protein, a screening method and application thereof, and belongs to the technical field of targeted nucleic acid medicines. The nucleotide sequence of the aptamer is selected from SEQ ID No. 1-SEQ ID No.6 in a nucleotide sequence table. The aptamer is obtained by adopting CE-SELEX technology and through three rounds of screening under the conditions of forward screening, reverse screening and normal human serum background. The aptamer has an inhibition effect on the combination of the S1 protein and the receptor ACE2 thereof, and can be used for separating, purifying and immobilizing the 2019-nCoV S1 protein; the method is used for preparing the biological sensor; and in the preparation of reagents for clinical diagnosis and medicaments for disease treatment.
Description
Technical Field
The invention relates to a group of ssDNA (deoxyribonucleic acid) aptamer of 2019-nCoV S1 protein, a screening method and application thereof, in particular to a ssDNA aptamer with high affinity and specificity for novel coronavirus (2019-nCoV) spinous process protein 1 (S1 protein) and a screening method thereof, and application of the aptamer in inhibiting the binding of 2019-nCoV S1 protein and receptor angiotensin converting enzyme 2 (ACE 2) thereof, belonging to the technical field of targeted nucleic acid medicaments.
Background
The Aptamer (Aptamer) is single-stranded DNA (ssDNA) or RNA obtained by screening a random deoxidized oligonucleotide library through an exponential enrichment ligand systematic evolution (SELEX) technology, and has the advantages of high affinity, strong specificity, easiness in preparation and modification, no immunogenicity and the like. Through the unique three-dimensional structure of the aptamer, the aptamer is able to selectively interact with a variety of target molecules, from proteins to polypeptides and small molecules. The binding properties, production properties and non-toxicity of the aptamer make it a potential candidate for the development of clinically useful drugs. The aptamer has precise binding specificity and tissue penetration ability. The targeted therapeutic method based on the nucleic acid aptamer can be realized according to the function of the nucleic acid aptamer, and can be directly used as a therapeutic agent to block the function of the target through interaction with the target; can also be used as a targeting element to form a delivery vehicle for other therapeutic agents.
In the prior art, the screening method of the aptamer mainly comprises a magnetic bead separation method, a microfluidic chip method, a membrane separation method, an affinity chromatography method, a particle display method, a capillary electrophoresis method (CE) and the like. The CE has the advantages of high efficiency, rapidness, low experimental cost and the like, and can be used for rapid separation and complex collection of target molecule-ssDNA complex and free ssDNA in aptamer screening. The CE-SELEX screening efficiency is high, and the aptamer with high affinity and high specificity can be obtained only by 1-4 rounds.
The epidemic of the novel coronavirus (2019-nCoV) infection is spread over a plurality of countries in the world and becomes a public health threat for people worldwide. Currently, antiviral drugs for treating AIDS virus, hepatitis B Virus (HBV), hepatitis C Virus (HCV) and influenza, which have been approved, are attempted for treating 2019-nCoV, but their efficacy is still alarming. However, no targeted drug or vaccine has been approved exclusively for the treatment of 2019-nCoV, and developers of potential drugs for the treatment of 2019-nCoV are still working to seek safer and more effective treatment regimens.
Disclosure of Invention
In view of the drawbacks of the prior art, it is an object of the present invention to provide a set of ssDNA aptamers of the 2019-nCoV S1 protein, which aptamers can bind with high affinity and specificity to the 2019-nCoV S1 protein. It is a second object of the present invention to provide a set of methods for screening ssDNA aptamers to 2019-nCoV S1 protein, said method being CE-SELEX.
The invention further aims to provide an application of a group of ssDNA aptamer of 2019-nCoV S1 protein, wherein the application is to take the aptamer as a nucleic acid drug of 2019-nCoV according to the inhibition effect of the aptamer on the binding of 2019-nCoV S1 protein and a receptor ACE2 thereof, so that the problem of deficiency of the targeted inhibition type drug of 2019-nCoV is solved, and the 2019-nCoV infection inhibition type nucleic acid drug with clinical transformation potential is developed.
In order to achieve the purpose of the invention, the following technical scheme is provided.
A set of ssDNA aptamers of 2019-nCoV S1 protein, said aptamers binding with high affinity and specificity to 2019-nCoV S1 protein, the nucleotide sequence of said aptamers being selected from the nucleotide sequences set forth in sequence identifiers 1 to 6 represented by the numerical identifier <210> in the nucleotide sequence listing, namely SEQ ID No.1 to SEQ ID No.6 in the nucleotide sequence listing, specifically as follows:
SEQ IDNo.1：
5'-AGCAGCACAGAGGTCAGATGCCGCAGGCAGCTGCCATTAGTCTCTATCCGTGACGGTATGCCTATGCGTGCTACCGTGAA-3'；
SEQ IDNo.2：
5'-AGCAGCACAGAGGTCAGATGGCAGCTAAGCAGGCGGCTCACAAAACCATTCGCATGCGGCCCTATGCGTGCTACCGTGAA-3'；
SEQ IDNo.3：
5'-AGCAGCACAGAGGTCAGATGGGGAATGCTTGTGGAGATGAACACGCCATTACTGCCGTACCCTATGCGTGCTACCGTGAA-3'；
SEQ IDNo.4：
5'-AGCAGCACAGAGGTCAGATGGCGAAGCGTACCGGCTACCCAGTGACAGTCGCCGTGGGTCCCTATGCGTGCTACCGTGAA-3'；
SEQ IDNo.5：
5'-AGCAGCACAGAGGTCAGATGGCCACATTAGTCTCACCACTACCTGCGTACCTACCGCCGCCCTATGCGTGCTACCGTGAA-3'；
SEQ IDNo.6：
5'-AGCAGCACAGAGGTCAGATGCGACTTGCCTATCGGCATGACACAATCTTTTGGAGCGTAACCTATGCGTGCTACCGTGAA-3'。
the screening method of the ssDNA aptamer of the 2019-nCoV S1 protein is CE-SELEX, and comprises the following specific steps:
(1) The target protein used in the first round of screening is recombinant 2019-nCoV S1 protein (hereinafter abbreviated as recombinant S1 protein) with an Fc fragment (abbreviated as Fc fragment) tag of Ig G; mixing the fluorescence labeled original random deoxyoligonucleotide library solution with the recombinant S1 protein solution, and then incubating to obtain a mixture A; and (3) carrying out capillary zone electrophoresis on the mixture A, wherein in the electrophoresis process, migration rates of free ssDNA, recombinant S1 protein and ssDNA-recombinant S1 protein complex in capillaries are different, the free ssDNA, recombinant S1 protein and ssDNA-recombinant S1 protein complex sequentially pass through a detection window at the tail end of the capillaries, the ssDNA-recombinant S1 protein complex is collected, and after PCR amplification, gel cutting, recovery and purification, a random deoxyoligonucleotide library formed by ssDNA capable of specifically combining the S1 protein and Fc fragment is obtained and is a first round of secondary library.
(2) The second round of screening is reverse screening, and because the recombinant S1 protein contains the Fc fragment, the Fc fragment is used as a reverse screening target for the screening; mixing the first round of secondary library solution obtained in the step (1) with the Fc fragment solution, then incubating the mixed solution together to obtain a mixture B, carrying out capillary zone electrophoresis on the mixture B, sequentially passing through detection windows at the tail ends of capillaries with different migration rates of free ssDNA, fc fragment and ssDNA-Fc fragment complex in the capillaries in the electrophoresis process, collecting the free ssDNA, and obtaining a random deoxyoligonucleotide library formed by ssDNA which is only specifically combined with S1 protein after PCR amplification, gel cutting, recovery and purification, wherein the random deoxyoligonucleotide library is the second round of secondary library;
(3) The third round of screening is screening under the serum background of normal people, and the target protein used is recombinant S1 protein; incubating the second secondary stock solution obtained in the step (2) with the recombinant S1 protein solution in normal human serum diluted twenty times to obtain a mixture C, carrying out capillary zone electrophoresis on the mixture C, sequentially passing through detection windows at the tail ends of capillaries in sequence when migration rates of free ssDNA, recombinant S1 protein and ssDNA-recombinant S1 protein complex in the capillaries are different in the electrophoresis process, and collecting the ssDNA-recombinant S1 protein complex;
performing PCR amplification by taking the ssDNA-recombinant S1 protein complex obtained by the screening of the round as a template, cutting gel, recovering and purifying to obtain an amplification product, and performing high-throughput sequencing and identification, and selecting 6 sequences with highest enrichment abundance for affinity and specificity evaluation to obtain 6 fluorescent-labeled ssDNA nucleic acid aptamers: nCoV-S1-A1, nCoV-S1-A2, nCoV-S1-A3, nCoV-S1-A4, nCoV-S1-A5 and nCoV-S1-A6, wherein the aptamer is combined with the S1 protein with high affinity and specificity, and the nucleotide sequences of the aptamer are SEQ ID No. 1-6 in a nucleotide sequence table respectively.
Wherein:
preparing a random deoxyoligonucleotide library solution by dissolving a random deoxyoligonucleotide library in water; dissolving protein in water to obtain protein solution; in the third round of screening, the random deoxyoligonucleotide library and the target protein are dissolved in normal human serum diluted twenty times to prepare a solution which can be used as a stock solution; the water is water with the purity higher than that of deionized water.
Preferably, in the first round of screening, the molar concentration of the random deoxyoligonucleotide library solution is 0.2 mu mol/L, and the molar concentration of the recombinant S1 protein solution is 0.1 mu mol/L; in the second screening round, the molar concentration of the Fc fragment solution was 2. Mu. Mol/L; in the third screening round, the molar concentration of the recombinant S1 protein solution was 0.04. Mu. Mol/L.
The detection method when passing through the detection window is capillary electrophoresis-laser induced fluorescence detection.
The outlet end of the capillary tube is a negative electrode, and the inlet end of the capillary tube is a positive electrode;
the capillary used in the capillary zone electrophoresis is fused quartz capillary, the total length is 50.2cm, the effective length is 40cm, and the inner diameter is 75 μm; performing the electrophoresis by using a Beckman P/ACE MDQ capillary electrophoresis apparatus; the electrophoresis buffer solution adopts boric acid-borax solution, and the pH value is 7.8; the sample injection conditions are as follows: protein sample injection: 0.5psi,5s; the analysis conditions were: the analysis temperature is 25 ℃ and the analysis voltage is 20kV; the capillary tube adopts laser induced fluorescence (CE-LIF) detection during online reaction, and the detection conditions are as follows: excitation wavelength 488nm and emission wavelength 520nm.
In steps (1), (2) and (3):
the upstream primer for PCR amplification of ssDNA sequence in the complex is designed, the nucleotide sequence is SEQ ID No.7 in the nucleotide sequence table, and the method specifically comprises the following steps: 5'-AGCAGCACAGAGGTCAGATG-3';
the downstream primer has a nucleotide sequence of SEQ ID No.8 in a nucleotide sequence table, and specifically comprises the following components: 5'-TTCACGGTAGCACGCATAGG-3'.
The application of the ssDNA aptamer of the 2019-nCoV S1 protein is that the aptamer is used for separating, purifying and immobilizing the 2019-nCoV S1 protein, is applied to the preparation of a biosensor, and is applied to the preparation of a 2019-nCoV S1 protein detection kit and the preparation of reagents for clinical diagnosis and medicaments for disease treatment.
Preferably the use is the use of the aptamer in the preparation of a 2019-nCoV S1 protein detection kit; preferably, the application is that the aptamer is used as a nucleic acid drug of 2019-nCoV, so that the problem of deficiency of a targeted inhibition type drug of 2019-nCoV is solved, and the 2019-nCoV infection inhibition type nucleic acid drug with clinical transformation potential is developed.
Advantageous effects
1. The invention provides a group of ssDNA (deoxyribonucleic acid) aptamer of 2019-nCoV S1 protein and application thereof, provides the ssDNA aptamer with an inhibition effect on the combination of the S1 protein and a receptor ACE2 thereof, can specifically identify S1 protein and 2019-nCoV simulated virus in serum, and provides a molecular basis for the development of a 2019-nCoV rapid detection and infection screening method;
the aptamer can be used as a targeting neutralizing aptamer of 2019-nCoV mimic virus, and provides a novel nucleic acid therapeutic molecule for SARS-CoV-2 targeted therapy and infection inhibition;
the invention also constructs a simulation structure of the 2019-nCoV S1-RBD-Aptamer compound, predicts an inhibition mechanism of the screened ssDNA Aptamer on the combination of the 2019-nCoV S1 protein and ACE2 through a molecular docking technology, and can provide better theoretical guidance for the development and optimization of the 2019-nCoV infection inhibitor.
2. The invention provides a screening method of a group of ssDNA (deoxyribonucleic acid) aptamers of 2019-nCoV S1 protein, which adopts CE-SELEX to obtain the aptamers with excellent affinity and specificity for 2019-nCoV S1 protein through forward screening, reverse screening and three rounds of screening under the serum background of normal people.
Drawings
FIG. 1 shows the result of capillary zone electrophoresis analysis of the first round of screening in example 1 and gel electrophoresis of PCR amplification products.
FIG. 2 shows the result of gel electrophoresis of the PCR amplification products of the second round of screening in example 1.
FIG. 3 shows the result of gel electrophoresis of the third round of screening of the capillary zone electrophoresis pattern and the PCR amplification product in example 1.
FIG. 4 shows CZE analysis patterns and equilibrium dissociation constant calculations for a first group of blank samples of nucleic acid aptamer nCoV-S1-A1 and corresponding mixture samples in example 2.
FIG. 5 shows CZE analysis patterns and equilibrium dissociation constant calculations for a second group of aptamer nCoV-S1-A2 of example 2, a blank sample and a corresponding mixture sample.
FIG. 6 shows CZE analysis patterns and equilibrium dissociation constant calculations for a blank sample of the third group of nucleic acid aptamers nCoV-S1-A3 and corresponding mixture samples in example 2.
FIG. 7 shows CZE analysis patterns and equilibrium dissociation constant calculations for a blank sample of the fourth group of nucleic acid aptamers nCoV-S1-A4 and corresponding mixture samples in example 2.
FIG. 8 shows CZE analysis patterns and equilibrium dissociation constant calculations for a blank sample of the fifth group of nucleic acid aptamers nCoV-S1-A5 and corresponding mixture samples in example 2.
FIG. 9 shows CZE analysis patterns and equilibrium dissociation constant calculations for a blank sample of the sixth group of nucleic acid aptamers nCoV-S1-A6 and corresponding mixture samples in example 2.
FIG. 10 is a three-dimensional model of the aptamer nCoV-S1-A1 and S1 protein complex based on molecular dynamics model simulation verification in example 3.
FIG. 11 is an analysis of the interaction of aptamer nCoV-S1-A1 with RBD domain by CZE analysis in example 3.
FIG. 12 is an analysis of the interaction of aptamer nCoV-S1-A2 with RBD domain by CZE analysis in example 3.
FIG. 13 is a graph showing the inhibition of binding of the S1 protein to the ACE2 protein by aptamer nCoV-S1-A1 determined in example 4.
FIG. 14 is a graph showing the inhibition of binding of the S1 protein to the ACE2 protein by aptamer nCoV-S1-A2 determined in example 4.
FIG. 15 is a CZE analysis chart of example 5 after incubation of the aptamer nCoV-S1-A1 in combination with seven sets of concentration gradients of recombinant S1 proteins, respectively.
FIG. 16 is a CZE analysis chart of the SARS-CoV-2 simulated virus with FAM fluorescent labeled aptamer nCoV-S1-A1 assay standard added to normal human serum in example 6.
FIG. 17 is a standard curve of detection of SARS-CoV-2 mimetic virus using FAM fluorescent labeled aptamer nCoV-S1-A1 in example 6.
FIG. 18 shows the results of experiments for inhibiting luciferase expression by neutralizing SARS-CoV-2 mimetic virus infection with aptamer nCoV-S1-A1 of example 7.
FIG. 19 is a graph showing the results of a laser scanning microscope for inhibiting the expression of green fluorescent protein by neutralizing the infection of SARS-CoV-2 mimetic virus with the aptamer nCoV-S1-A1 of example 7.
Detailed Description
In order to fully illustrate the nature of the invention and the manner in which it may be practiced, examples are set forth below.
In the following examples:
FAM fluorescence labeled original random deoxyoligonucleotide library (FAM fluorescence labeled ssDNA) N40 ) The method comprises the following steps: 5'-FAM-CGTAGAATTCATGAGGACGT- (N40) -AGCTAAGCTTACCAGTGCGAT-3';
recombinant S1 protein, RBD domain, fc fragment, and SARS-CoV-2 inhibitor screening kit (SARS-CoV-2 (SARS-CoV-2) Inhibitor Screening ELISA Kit) were purchased from Beijing Yiqiao Shenzhou technologies Co.
FAM fluorescent-labeled ssDNA N40 FAM fluorescent-labeled aptamer nCoV-S1-Apt 1-nCoV-S1-A6 sequences and aptamer nCoV-S1-Apt 1-nCoV-S1-A6 sequences were purchased from Biotechnology Inc.
SARS-CoV-2 mimetic virus is purchased from Shanghai Fubai Biotechnology Co., ltd. And contains Spike glycoprotein (Spike Protein) of novel coronavirus on the surface of the envelope, and the virus encapsulates RNA sequences of Green Fluorescent Protein (GFP) and Luciferase (Luciferase). After the virus infects target cells for 48-72 hours, the infection efficiency can be judged by observing the expression of green fluorescent protein and detecting the activity of luciferase.
Normal human serum was purchased from beijing bayer di biotechnology limited.
Fused silica capillaries were purchased from ottak biotechnology limited, a development area of the handan, northland.
High throughput sequencing of the PCR products in example 1 below was accomplished by Biotechnology Inc.
Example 1
A screening method for ssDNA aptamer of 2019-nCoV S1 protein, wherein the screening method is CE-SELEX, and comprises the following specific steps:
(1) The target protein used in the first round of screening is recombinant S1 proteinThe method comprises the steps of carrying out a first treatment on the surface of the FAM fluorescence-labeled ssDNA N40 Mixing the solution with the recombinant S1 protein solution, and then incubating to obtain a mixture A; and (3) carrying out capillary zone electrophoresis on the mixture A, wherein in the electrophoresis process, migration rates of free ssDNA, recombinant S1 protein and ssDNA-recombinant S1 protein complex in capillaries are different, the free ssDNA, recombinant S1 protein and ssDNA-recombinant S1 protein complex sequentially pass through a detection window at the tail end of the capillaries, the ssDNA-recombinant S1 protein complex is collected, and after PCR amplification, gel cutting, recovery and purification, a random deoxyoligonucleotide library formed by ssDNA capable of specifically combining the S1 protein and Fc fragment is obtained and is a first round of secondary library.
(2) The second round of screening is reverse screening, and because the recombinant S1 protein contains the Fc fragment, the Fc fragment is used as a reverse screening target for the screening; mixing the first round of secondary library solution obtained in the step (1) with the Fc fragment solution, then incubating the mixed solution together to obtain a mixture B, carrying out capillary zone electrophoresis on the mixture B, sequentially passing through detection windows at the tail ends of capillaries with different migration rates of free ssDNA, fc fragment and ssDNA-Fc fragment complex in the capillaries in the electrophoresis process, collecting the free ssDNA, and obtaining a random deoxyoligonucleotide library formed by ssDNA which is only specifically combined with S1 protein after PCR amplification, gel cutting, recovery and purification, wherein the random deoxyoligonucleotide library is the second round of secondary library;
(3) The third round of screening is screening under the serum background (Human serum background) of normal people, and the target protein is recombinant S1 protein; incubating the second secondary stock solution obtained in the step (2) with the recombinant S1 protein solution in normal human serum diluted twenty times to obtain a mixture C, carrying out capillary zone electrophoresis on the mixture C, sequentially passing through detection windows at the tail ends of capillaries in sequence when migration rates of free ssDNA, recombinant S1 protein and ssDNA-recombinant S1 protein complex in the capillaries are different in the electrophoresis process, and collecting the ssDNA-recombinant S1 protein complex;
performing PCR amplification by taking the ssDNA-recombinant S1 protein complex obtained by the screening of the round as a template, cutting gel, recovering and purifying to obtain an amplification product, and performing high-throughput sequencing and identification, and selecting 6 sequences with highest enrichment abundance for affinity and specificity evaluation to obtain 6 FAM fluorescent marked ssDNA nucleic acid aptamers: the method comprises the steps of nCoV-S1-A1, nCoV-S1-A2, nCoV-S1-A3, nCoV-S1-A4, nCoV-S1-A5 and nCoV-S1-A6, wherein the aptamer is combined with S1 protein with high affinity and specificity, the nucleotide sequences of the aptamer are SEQ ID No. 1-6 in a nucleotide sequence table respectively, and the nucleotide sequences are delivered to a biological engineering Co-efficient company to synthesize the aptamer for subsequent experiments.
Wherein the purchased crystalline FAM fluorescently labeled ssDNA is used N40 Adding deionized water to prepare 1.0X10 by 8000 rpm for 5min -4 mol/L of original ssDNA N40 The solution is used as stock solution, and is cooled to 4 ℃ after 5min of metal bath at 94 ℃ and is preserved at-20 ℃; before use, the mixture is diluted to the required concentration by deionized water, and stored at 4 ℃ for a short time.
Before the S1 protein and the Fc fragment are used, deionized water is used for dilution to the required concentration, and the S1 protein solution and the Fc fragment solution are obtained and stored at 4 ℃ when being used as stock solution for short time.
During the third round of screening, a second round of secondary pool (sub-ssDNA) obtained from the second round of screening N40 ) And S1 protein solution is prepared and diluted by normal human serum diluted twenty times.
In the three rounds of screening, equal volumes of protein solution and ssDNA were taken separately N40 The solutions were mixed and incubated at 37℃for 15min.
FAM fluorescence-labeled ssDNA in the first round of screening N40 The molar concentration of the solution is 0.2 mu mol/L, and the molar concentration of the recombinant S1 protein solution is 0.1 mu mol/L; in the second screening round, the molar concentration of the Fc fragment solution was 2. Mu. Mol/L; in the third screening round, the molar concentration of the recombinant S1 protein solution was 0.04. Mu. Mol/L.
The detection method when passing through the detection window is capillary electrophoresis-laser induced fluorescence detection.
The capillary used in capillary zone electrophoresis (capillary zone electrophoresis, CZE) was a fused silica capillary having a total length of 50.2cm, an effective length of 40cm, and an inner diameter of 75 μm; feeding the mixture into Beckman P/ACE MDQ capillary electrophoresis to perform electrophoresis; 50mmol/L boric acid-borax solution is adopted as the electrophoresis buffer solution, and the pH value is 7.8; sample introduction conditions: 0.5psi,5s; the electrophoresis analysis conditions were: the analysis voltage is 20kV, the analysis temperature is 25 ℃, and the detection is carried out by adopting laser induced fluorescence (CE-LIF) under the following detection conditions: excitation wavelength 488nm, emission wavelength 520nm; the outlet end of the capillary tube is a negative electrode, and the inlet end is a positive electrode.
During electrophoresis, free ssDNA N40 Sequentially passing through detection windows at the tail end of the capillary with the protein complex, and directly collecting ssDNA in the first round of positive screening and the third round of positive screening N40 -an S1 protein complex; in the second round of reverse screening step, free ssDNA was collected N40 . The results of capillary zone electrophoresis analysis are shown in FIG. 1.
In steps (1), (2) and (3):
the upstream and downstream primers for the ssDNA sequences in the PCR amplification complex were designed:
the upstream primer is as follows: 5'-AGCAGCACAGAGGTCAGATG-3';
the downstream primer is: 5'-TTCACGGTAGCACGCATAGG-3'.
The PCR amplification conditions for the collected samples were: 95 ℃ for 30s;60 ℃ for 30s;72 ℃,30s; amplification was performed for 30 cycles with DNase/RNase-Free deionized water, 2X Taq PCR Mastermix available from Tiangen Biochemical technologies Co. Agarose gel electrophoresis conditions were: 60ml of 0.5 XTBE buffer and 01.2g of agarose, prepare agarose gel with mass fraction of 2%, and electrophorese 50min at 90V voltage of Bio-Rad electrophoresis apparatus; agarose, geneGreen nucleic acid dye, 6X DNA Loading Buffer,50bp DNA Ladder was purchased from Tiangen Biochemical technologies Co.
The capillary zone electrophoresis analysis chart of the first Round of screening (Round 1) in step (1) is shown in FIG. 1, and the lower spectral line in FIG. 1 is ssDNA N40 Is ssDNA N40 Generating a signal peak at 7.2 min; the upper line is the analysis line of the ssDNA-recombinant S1 protein complex (S1-ssDNA complex), the S1-ssDNA complex generates a signal peak at 3.2min, and the complex Collection zone (Collection) is 2.8 min-3.6 min.
The capillary zone electrophoresis analysis map of the second Round of screening (Round 2) in the step (2) is shown in FIG. 2, and the lower spectral line in FIG. 2 is the first Round of secondary library (sub-ssDNA) N40 ) Is of the analysis line, sub-ssDNA N40 Generating a signal peak at 6.3 min; the upper line is the analysis line of the ssDNA-Fc fragment complex, and the free ssDNA Collection region (Collection) is 5.9min to 6.8min.
The capillary zone electrophoresis analysis chart of the third screening (Round 3) in the step (3) is shown in fig. 3, the spectrum line in fig. 3 is the analysis spectrum line of the ssDNA-recombinant S1 protein complex (S1-ssDNA complex), the ssDNA-recombinant S1 protein complex generates a signal peak at 3.7min, and the complex collecting section (Collection) is 3.1 min-4.0 min.
The upper right panels in fig. 1 to 3 are the gel electrophoresis results of each round of screening PCR amplification products, wherein the leftmost band is a DNA marker, 50bp, 100bp, 150bp, 200bp, 250bp, 300 bp..500 bp are respectively arranged from bottom to top, the PCR amplification product band of the collected sample is arranged on the right side, the band length is consistent with the position of 80bp in the DNA marker, and the amplification product with the target length is determined.
The conditions related to electrophoresis in capillary zones (capillary zone electrophoresis, CZE) and analysis thereof in the following examples are the same as in example 1.
Example 2
6 FAM fluorescent-labeled ssDNA nucleic acid aptamers: the nCoV-S1-A1, the nCoV-S1-A2, the nCoV-S1-A3, the nCoV-S1-A4, the nCoV-S1-A5 and the nCoV-S1-A6 are prepared into six groups of nucleic acid aptamer solutions by deionized water, so that six groups of blank control samples are obtained; and then incubating the six groups of nucleic acid aptamer solutions with recombinant S1 proteins respectively to obtain six corresponding groups of mixture samples, wherein the final concentration of the nucleic acid aptamer is 0.2 mu mol/L, and the final concentration of the recombinant S1 protein is 2 mu mol/L.
Injecting the blank control sample and the corresponding mixture sample into a capillary electrophoresis apparatus for CZE analysis; the equilibrium dissociation constant calculation formula (1) is as follows:
wherein [ P ]] 0 : protein concentration; [ DNA ]] 0 : aptamer concentration; a1: free aptamer peak area;a2: protein-aptamer complex peak area; a3: dissociation region peak area.
Fig. 4 to 9 are the CZE analysis spectra and equilibrium dissociation constant calculation results of six groups of the blank control samples and the corresponding mixture samples, respectively, wherein the lower spectral line is the blank control sample, and the upper spectral line is the mixture sample. As can be seen, the aptamer forms a stable complex with the S1 protein in the mixed sample, and the equilibrium dissociation constants are 0.327+/-0.016 nmol/L, 0.313+/-0.078 nmol/L, 0.118+/-0.033 nmol/L, 28.422 +/-3.666 nmol/L, 18.829 +/-3.806 nmol/L and 85.61+/-14.219 nmol/L respectively.
Example 3
Molecular dynamics simulation is carried out on FAM fluorescent marked ssDNA aptamer nCoV-S1-A1, and the specific method is as follows:
predicting the three-dimensional structure of nCoV-S1-A1 through Mode RNA webserver and an MDWeb webserver network server, predicting the binding residues of the S1 protein and ACE2 according to a UniProt database, and predicting the complex structure between the S1 protein and the nCoV-S1-A1 aptamer molecule by using a protein-DNA hybrid docking algorithm HDOCK; binding residue analysis was done by OrppegioWeb webserver web server.
FIG. 10 is a three-dimensional model of a nucleic acid aptamer nCoV-S1-A1 and S1 protein complex based on molecular dynamics model simulation verification, and can observe that the nCoV-S1-A1 base at positions 45-71 binds to two specific main active sites of RBD domain on S1 protein; binding free energy of 12.17kcal/mol, K D The value was 0.1nmol/L. Thus, it is speculated that nCoV-S1-A1 can be used as an RBD domain with S1 protein, as a potential inhibitor of SARS-CoV-2 binding to cell surface ACE 2.
Preparing FAM fluorescence labeled nCoV-S1-A1 and FAM fluorescence labeled nCoV-S1-A1 into nucleic acid aptamer solution by deionized water to obtain two groups of blank control samples: unbounded nCoV-S1-A1 and unbounded nCoV-S1-A2; and then incubating the two groups of nucleic acid aptamer solutions with RBD domains respectively to obtain two corresponding groups of mixture samples, wherein the final concentration of the nucleic acid aptamer is 0.2 mu mol/L, and the final concentration of the RBD domain is 0.2 mu mol/L.
The blank samples and the corresponding mixture samples were injected into a capillary electrophoresis apparatus for CZE analysis, and the results are shown in fig. 11 and 12.
FIG. 11 shows interaction of aptamer nCoV-S1-A1 with RBD domain by CZE analysis, the lower line in FIG. 11 is a blank sample and the upper line is a mixture sample. It can be seen that the aptamer nCoV-S1-A1 in the mixed sample can form a stable Complex (Complex) with the S1 protein.
FIG. 12 shows interaction of aptamer nCoV-S1-A2 with RBD domain by CZE analysis, the lower line in FIG. 12 is a blank sample and the upper line is a mixture sample. It can be seen that the aptamer nCoV-S1-A2 in the mixed sample can form a stable Complex (Complex) with the S1 protein.
Example 4
The inhibition of the binding of the aptamer nCoV-S1-A1 and nCoV-S1-A2 to the S1 protein and ACE2 protein was determined by using a SARS-CoV-2 inhibitor screening kit (SARS-CoV-2 (SARS-CoV-2) Inhibitor Screening ELISAKit) according to the experimental description of the kit.
The inhibition curve of the binding of the aptamer nCoV-S1-A1 to the S1 protein and the ACE2 protein was determined, and the IC50 was 80.12nM, as shown in FIG. 13.
The inhibition curve of the binding of the aptamer nCoV-S1-A2 to the S1 protein and the ACE2 protein was determined, and the IC50 was 256.38nM, as shown in FIG. 14.
Example 5
Adding seven groups of concentration gradient recombinant S1 protein standard solutions with the same volume into normal human serum, mixing and incubating the recombinant S1 protein standard solutions with the same volume of FAM fluorescent-labeled aptamer nCoV-S1-A1 solution to obtain seven groups of samples; wherein the final concentration gradient of the recombinant S1 protein is 0. Mu. Mol/L, 0.005. Mu. Mol/L, 0.01. Mu. Mol/L, 0.02. Mu. Mol/L, 0.05. Mu. Mol/L, 0.1. Mu. Mol/L and 0.2. Mu. Mol/L; the final concentration of FAM fluorescence-labeled aptamer nCoV-S1-A1 was 0.1. Mu. Mol/L. Seven sets of samples were injected into a capillary electrophoresis apparatus for CZE analysis and the results are shown in fig. 15.
The figure shows that the nucleic acid aptamer nCoV-S1-A1 recognizes the S1 protein added in standard in normal human serum, the first line to the seventh line sequentially correspond to CZE analysis patterns after the nucleic acid aptamer nCoV-S1-A1 is mixed and incubated with 0 mu mol/L, 0.005 mu mol/L, 0.01 mu mol/L, 0.02 mu mol/L, 0.05 mu mol/L, 0.1 mu mol/L and 0.2 mu mol/L of recombinant S1 protein respectively from bottom to top, and a signal peak appears in 4.8min when the nucleic acid aptamer nCoV-S1-A1 can form a compound with the recombinant S1 protein with the concentration of 0.005 mu mol/L in serum; and with the increase of the concentration of the recombinant S1 protein, the peak surface of a complex generated by the nucleic acid aptamer nCoV-S1-A1 and the recombinant S1 protein is gradually increased, which indicates that the nucleic acid aptamer nCoV-S1-A1 can specifically recognize the recombinant S1 protein in serum.
Example 6
Nine groups of SARS-CoV-2 simulated viruses with different concentrations are respectively added into normal human serum diluted by 20 times, each group is added with FAM fluorescent-labeled aptamer nCoV-S1-A1, nine groups of samples are obtained after mixing and incubation, wherein the final concentrations of the SARS-CoV-2 simulated viruses are respectively as follows: the final concentrations of FAM fluorescence-labeled aptamer nCoV-S1-A1 were all 0.2. Mu. Mol/L, 0TU/ml, 293TU/ml, 586TU/ml, 1172TU/ml, 2344TU/ml, 4688TU/ml, 9375TU/ml, 18750TU/ml and 75000 TU/ml. Nine samples were injected into a capillary electrophoresis apparatus for CZE analysis and the results are shown in fig. 16 and 17.
FIG. 16 shows the FAM fluorescence-labeled aptamer nCoV-S1-A1 assay against SARS-CoV-2 mock virus added to normal human serum, wherein the concentration of the SARS-CoV-2 mock virus is 0TU/ml, 293TU/ml, 586TU/ml, 1172TU/ml, 2344TU/ml, 4688TU/ml, 9375TU/ml, 18750TU/ml and 75000TU/ml, respectively, corresponding to the concentration from bottom to top, and it is known that a complex of FAM fluorescence-labeled aptamer nCoV-S1-A1 and SARS-CoV-2 mock virus occurs at 2.9min (C PV-A1 ) The complex gradually increased as the number of SARS-CoV-2 mimicking viral particles increased; the nucleic acid aptamer is described as being capable of detecting SARS-CoV-2 mimetic virus in serum.
FIG. 17 is a standard curve (R) for detection of SARS-CoV-2 mimetic virus using FAM fluorescent labeled aptamer nCoV-S1-A1 2 =0.981), the detection of SARS-CoV-2 mimetic virus in serum by the nucleic acid aptamer was linear.
Example 7
SARS-CoV-2 mimics the RNA sequence of the virus that encapsulates the Green Fluorescent Protein (GFP) and luciferase, and infection efficiency can be determined by observing the expression of the green fluorescent protein and the luciferase activity; the 293T/ACE2 cell line expressing ACE2 was used to mimic pseudoviral infection.
293T/ACE2 cells were seeded in 96-well plates (1X 10) according to SARS-CoV-2 mock virus instructions 4 Cells/well), 24h later, to simulate viral infection. Frozen SARS-CoV-2 mimetic virus was removed and thawed on ice. In experiments for inhibiting SARS-CoV-2 virus infection by aptamer, 2 μl of each of six groups of different concentrations of aptamer nCoV-S1-A1 was added to a 96-well plate for 293T/ACE2 cell culture together with 2 μl of SARS-CoV-2 mimetic virus, the final concentration of the six groups of aptamer nCoV-S1-A1 being: 0. Mu.M, 0.1. Mu.M, 0.2. Mu.M, 0.5. Mu.M, 1. Mu.M and 2. Mu.M; after 6 hours, changing fresh culture medium to continue culturing; after 60 hours, luciferase activity was assayed to determine the efficiency of mimicking viral infection, and the results are shown in FIG. 18.
As shown in fig. 18, as the nucleic acid aptamer concentration increased from 0 μm to 2 μm, the luciferase activity expressed by 293T/ACE2 cell lines mimicking virus infection decreased, indicating that the aptamer inhibited infection of 293T/ACE2 cell lines by mimicking virus.
293T/ACE2 cells were seeded in 96-well plates (1X 10) according to SARS-CoV-2 mock virus instructions 4 Cells/well), 24h later, to simulate viral infection. Frozen SARS-CoV-2 mimetic virus was removed and thawed on ice. In experiments for inhibiting SARS-CoV-2 virus infection by aptamer, 2 μl of each of six groups of different concentrations of aptamer nCoV-S1-A1 was added to a 96-well plate for 293T/ACE2 cell culture together with 2 μl of SARS-CoV-2 mimetic virus, the final concentration of the six groups of aptamer nCoV-S1-A1 being: 0. Mu.M, 0.01. Mu.M, 0.05. Mu.M, 0.1. Mu.M, 0.5. Mu.M and 1.00. Mu.M; after 6 hours, changing fresh culture medium to continue culturing; after 60h, the expression of green fluorescent protein was observed under a laser scanning microscope as shown in fig. 19.
FIG. 19 is a laser scanning microscope of the aptamer nCoV-S1-A1 inhibiting the expression of green fluorescent protein by neutralizing infection with SARS-CoV-2 mock virus, showing that the aptamer inhibited infection of 293T/ACE2 cell line by mock virus with less expression of green fluorescent protein expressed by 293T/ACE2 cell line as the concentration of aptamer increased from 0. Mu.M to 1. Mu.M. Nuclei were stained with DAPI (blue); the scale bar is 25 μm.
In summary, for the S1 protein, capillary electrophoresis nucleic acid aptamer screening is carried out, high-affinity and specific nucleic acid aptamers can be obtained through three rounds of screening, and the inhibition mechanism of the ssDNA aptamers obtained through screening on the S1 protein is predicted through a molecular docking technology; the S1 protein can be specifically identified in serum, and the binding effect of the S1 protein and a receptor ACE2 is inhibited; SARS-CoV-2 mimetic virus can be specifically identified in serum and inhibit infection of cells by the mimetic virus. Is expected to be a target nucleic acid drug for inhibiting SARS-CoV-2 infection.
The foregoing is merely a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
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Claims (3)
1. A ssDNA aptamer for 2019-nCoV S1 protein, characterized in that: the nucleotide sequence of the aptamer is SEQ ID No.1 in a nucleotide sequence table, recombinant 2019-nCoV S1 protein with Fc fragment tag is used as target protein, and the aptamer is obtained by screening by a CE-SELEX method.
2. Use of ssDNA aptamer to the 2019-nCoV S1 protein of claim 1: the following uses of the aptamer for non-disease diagnostic use: separating, purifying and immobilizing 2019-nCoV S1 protein; the method is used for preparing the biological sensor; and in the preparation of reagents for clinical diagnosis and medicaments for disease treatment.
3. The use of an ssDNA aptamer to the 2019-nCoV S1 protein of claim 2, wherein: the application is the application in preparing a 2019-nCoV S1 protein detection kit and the application in preparing a 2019-nCoV targeted inhibition type drug.
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