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Nucleic acid aptamer binding to novel coronavirus (SARS-CoV-2) nucleocapsid protein and use thereof

A nucleic acid aptamer, nucleocapsid protein technology, applied in the direction of viruses/phages, viruses, viral peptides, etc., can solve the problems of renal failure, rapid spread, and imperfect detection methods for new coronaviruses, and achieves small molecular weight, Chemically stable, easy to store and label effects

Active Publication Date: 2021-07-23
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The new coronavirus can be transmitted from person to person. Compared with SARS coronavirus, SARS-CoV-2 has a lower fatality rate, but spreads faster. People infected with SARS-CoV-2 will develop acute and severe respiratory diseases, accompanied by fever, cough, shortness of breath and shortness of breath. Difficult, renal failure and death in severe cases, currently no effective treatment
The detection method of the new coronavirus is not perfect at present, it needs to rely on specific instruments, special laboratories and professional technicians, and the detection process is complicated, it cannot meet the needs of timely detection, and there is a certain rate of missed detection

Method used

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  • Nucleic acid aptamer binding to novel coronavirus (SARS-CoV-2) nucleocapsid protein and use thereof
  • Nucleic acid aptamer binding to novel coronavirus (SARS-CoV-2) nucleocapsid protein and use thereof
  • Nucleic acid aptamer binding to novel coronavirus (SARS-CoV-2) nucleocapsid protein and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Screening of ssDNA nucleic acid aptamers specifically binding to novel coronavirus (SARS-CoV-2) nucleocapsid protein

[0046] 1. Synthesize the random single-stranded DNA library and primers shown in the following sequences:

[0047] Random ssDNA library:

[0048] SEQ ID NO: 5: 5'-GTTCGTGGTGTGCTGGATGTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTGACACATCCAGCAGCACGA-3'

[0049] Among them, 36 "N"s represent a sequence formed by connecting 36 arbitrary nucleotide bases. The library was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0050] The primer information is shown in Table 1 and was synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0051] Table 1 Primers and their sequences

[0052]

[0053] Among them, S in the primer name represents the forward primer, A in the primer name represents the reverse primer, 19 A in the sequence represent a polyA tail composed of 19 adenine nucleotides (A), and "Spacer 18" represents an 18-atom Hexaethy...

Embodiment 2

[0076] Embodiment 2: Surface plasmon resonance (SPR) detects affinity and specificity detection of SARS-CoV-2 nucleocapsid protein nucleic acid aptamer and SARS-CoV-2 nucleocapsid protein

[0077] 1. Dilute the nucleic acid aptamers SARS-CoV-2-N15, SARS-CoV-2-N48, SARS-CoV-2-N58, and SARS-CoV-2-N61 of general biosynthesis with DPBS buffer respectively into 500nM.

[0078] 2. Coupling the SARS-CoV-2 nucleocapsid protein to the second channel on the surface of the CM5 chip: first wash the chip with 50mM NaOH, inject 20 μl of the sample at a flow rate of 10 μl / min, and then use an equal volume of EDC (1-( 3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; 0.4M aqueous solution) and NHS (N-hydroxysuccinimide; 0.1M aqueous solution) were mixed and injected into 50 μl activated chip , with a flow rate of 5 μl / min. The SARS-CoV-2 nucleocapsid protein was diluted with 10 mM sodium acetate at pH 5.5 to a final concentration of 50 μg / mL and then injected. The injection volume w...

Embodiment 3

[0085] Example 3 shows the binding of the four nucleic acid aptamers screened in Example 1 of the present invention to the binding site of the N protein using surface plasmon resonance (SPR)

[0086] Using surface plasmon resonance (SPR), using the chip of Example 2, the four nucleic acid aptamers of general biosynthesis were diluted to 500nM respectively, and SARS-CoV-2-N48 was injected for 2 minutes, and then SARS-CoV- 2-N58 injection for 2 minutes, dissociation for 2 minutes. Such as Figure 4 As shown, it can be seen that the signals of the two nucleic acid aptamer samples were significantly improved, which can explain that SARS-CoV-2-N48 and SARS-CoV-2-N58 bind to different sites of the N protein. The same experimental method verified that SARS-CoV-2-N48 binds to SARS-CoV-2-N61 at different sites on the N protein, and SARS-CoV-2-N15 binds to SARS-CoV-2-N58 on the N protein SARS-CoV-2-N15 and SARS-CoV-2-N61 bind to different sites of the N protein. SARS-CoV-2-N48 and SA...

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Abstract

The invention relates to a nucleic acid aptamer binding to novel coronavirus (SARS-CoV-2) nucleocapsid protein and use thereof. The nucleic acid aptamer specifically comprises or consists of a nucleotide sequence shown in any one of SEQ ID NO: 1-4. The nucleic acid aptamer and the derivative thereof have the advantages of being rapid in chemical synthesis, small in molecular weight, stable, easy to store and label and the like, and can be used for detection, diagnosis, imaging, treatment and the like.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a nucleic acid adapter shown in SEQ ID NO: 1-4 that can be used to bind the nucleocapsid protein of SARS-CoV-2 (the nucleocapsid protein of SARS-CoV-2) Ligands and their uses. Background technique [0002] Coronavirus (Coronavirus) is a positive-sense single-stranded RNA virus with a mantle. It is a pathogen that infects humans and vertebrates and can cause a variety of acute and chronic diseases. So far, there are 7 kinds of coronaviruses that can cause human disease, including SARS coronavirus and MERS coronavirus. [0003] On January 12, 2020, the World Health Organization officially named the novel coronavirus that caused the pneumonia outbreak "2019 Novel Coronavirus (2019-nCoV)". The Committee on Taxonomy of Viruses, ICTV) announced that the official classification of the new coronavirus (2019-nCoV) is named severe acute respiratory syndrome coronavirus 2 (severe acut...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C07K14/00C07K14/165G01N33/569A61K31/7088A61P31/14
CPCC12N15/115C07K14/003C07K14/005G01N33/56983A61K31/7088A61P31/14C12N2310/16C12N2310/315C12N2770/20022G01N2469/10
Inventor 罗昭锋方晓娜张海燕欧惠超张立云周宏敏
Owner UNIV OF SCI & TECH OF CHINA
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