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Group of SsDNA aptamers of 2019-nCoV S1 protein, screening method and applications

A 2019-ncovs1, screening method technology, applied in recombinant DNA technology, DNA/RNA fragments, biochemical equipment and methods, etc., can solve problems such as efficacy concerns, and achieve the effect of excellent affinity and specificity

Active Publication Date: 2021-07-16
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, approved antiviral drugs for HIV, hepatitis B virus (HBV), hepatitis C virus (HCV) and influenza are being tried against 2019-nCoV, but their efficacy remains a concern
However, no targeted drugs or vaccines have been approved specifically to treat 2019-nCoV, and developers of potential drugs to treat 2019-nCoV remain committed to finding safer and more effective treatment options

Method used

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  • Group of SsDNA aptamers of 2019-nCoV S1 protein, screening method and applications
  • Group of SsDNA aptamers of 2019-nCoV S1 protein, screening method and applications
  • Group of SsDNA aptamers of 2019-nCoV S1 protein, screening method and applications

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] A screening method for ssDNA aptamers of 2019-nCoV S1 protein, the screening method is CE-SELEX, the specific steps are as follows:

[0072] (1) The target protein used in the first round of screening is recombinant S1 protein; FAM fluorescently labeled ssDNA N40 The solution was mixed with the recombinant S1 protein solution and incubated together to obtain mixture A; the mixture A was subjected to capillary zone electrophoresis, during the electrophoresis process, the migration rates of free ssDNA, recombinant S1 protein and ssDNA-recombinant S1 protein complex in the capillary were different, successively Sequentially pass through the detection window at the end of the capillary to collect ssDNA-recombinant S1 protein complexes, and after PCR amplification, gel cutting, recovery and purification, a random deoxyoligonucleotide library formed by ssDNA that can specifically bind to S1 protein and Fc fragments is obtained. For the first round of secondary library.

[00...

Embodiment 2

[0095] Six FAM fluorescently labeled ssDNA aptamers: nCoV-S1-A1, nCoV-S1-A2, nCoV-S1-A3, nCoV-S1-A4, nCoV-S1-A5 and nCoV-S1-A6 were used The ionized water was configured as six groups of nucleic acid aptamer solutions to obtain six groups of blank control samples; then the six groups of nucleic acid aptamer solutions were incubated with the recombinant S1 protein respectively to obtain six groups of corresponding mixture samples, wherein the The final concentration of the nucleic acid aptamer was 0.2 μmol / L, and the final concentration of the recombinant S1 protein was 2 μmol / L.

[0096] The blank control sample and its corresponding mixture sample are injected into a capillary electrophoresis instrument for CZE analysis; the equilibrium dissociation constant calculation formula (1) is as follows:

[0097]

[0098] Among them, [P] 0 : protein concentration; [DNA] 0 : aptamer concentration; A1: peak area of ​​free aptamer; A2: peak area of ​​protein-aptamer complex; A3: pe...

Embodiment 3

[0101] Carry out molecular dynamics simulation on FAM fluorescently labeled ssDNA nucleic acid aptamer nCoV-S1-A1, the specific method is as follows:

[0102] The three-dimensional structure of nCoV-S1-A1 was predicted through the Mode RNA webserver and MDWeb webserver, and the binding residues between the S1 protein and ACE2 were predicted according to the UniProt database. The protein-DNA hybrid docking algorithm HDOCK was used to predict the relationship between the S1 protein and nCoV-S1 - Complex structure between A1 aptamer molecules; binding residue analysis is done by OrppegioWeb webserver network server.

[0103] Figure 10 It is a three-dimensional model of nucleic acid aptamer nCoV-S1-A1 and S1 protein complex based on molecular dynamics model simulation verification. Specific primary active site binding; binding free energy 12.17 kcal / mol, K D The value is 0.1 nmol / L. Therefore, it can be speculated that nCoV-S1-A1 can be used as a potential inhibitor of SARS-Co...

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Abstract

The invention relates to a group of ssDNA aptamers of 2019-nCoV S1 protein, a screening method and applications, and belongs to the technical field of targeted nucleic acid drugs. The nucleotide sequences of the aptamers are selected from SEQ ID No.1-SEQ ID No.6 in a nucleotide sequence table. The aptamers are obtained by adopting a CE-SELEX technology and through three rounds of screening under the background of forward screening, reverse screening and normal human sera. The aptamers have an inhibition effect on binding of S1 protein and a receptor ACE2 of the S1 protein, and can be used for separating, purifying and immobilizing the 2019-nCoV S1 protein. The aptamers are used for preparing a biological sensor, and preparing reagents for clinical diagnosis and drugs for disease treatment.

Description

technical field [0001] The present invention relates to a group of ssDNA aptamers, screening methods and applications of 2019-nCoV S1 protein, in particular, relates to novel coronavirus (2019-nCoV) spike protein 1 (S1 protein) with high affinity and specificity The ssDNA nucleic acid aptamer and its screening method, and the application of the aptamer to the inhibition of the binding of the 2019-nCoV S1 protein to its receptor angiotensin-converting enzyme 2 (ACE2), belong to the technical field of targeted nucleic acid drugs . Background technique [0002] Nucleic acid aptamer (Aptamer) is a single-stranded DNA (ssDNA) or RNA screened from a random deoxyoligonucleotide library through the exponential enrichment ligand system evolution (SELEX) technology, which has high affinity, strong specificity, It has the advantages of easy preparation and modification, and no immunogenicity. Through the unique three-dimensional structure of aptamers, aptamers can selectively interac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/569
CPCC12N15/115G01N33/56983C12N2310/16G01N2333/165Y02A50/30
Inventor 屈锋黄渊余杨歌
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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