Characterization and application of novel high-temperature Argonaute protein

A technology of reaction and reaction system, applied in the field of molecular biology and biology, can solve the problems of cumbersome steps and high detection cost

Active Publication Date: 2021-07-13
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006]However, some current detection methods using nucleic acid tool en...

Method used

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  • Characterization and application of novel high-temperature Argonaute protein
  • Characterization and application of novel high-temperature Argonaute protein
  • Characterization and application of novel high-temperature Argonaute protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0229] Embodiment 1: Obtaining of TeAgo gene sequence

[0230]In the database, the amino acid sequence of known PfAgo is searched for similarity, and some amino acid sequences with high sequence consistency are selected, analyzed by MEGA software, and a homologous evolution tree is constructed, and TeAgo is selected as a candidate enzyme, wherein TeAgo and The sequence similarity of the characterized PfAgo reaches 33.02%. The amino acid sequence of TeAgo (WP_050002102.1) and the corresponding gene sequence (NZ_CP008887.1) encoding the protein were obtained. After the gene sequence was synthesized by codon optimization, it was cloned into pET28a expression vector.

Embodiment 2

[0231] Example 2: TeAgo protein heterologous expression and purification

[0232] The above-mentioned TeAgo-pET28a prokaryotic expression plasmid was introduced into E.coli BL21(DE3) to obtain a TeAgo-pET28a / E.coli BL21(DE3) prokaryotic expression strain. The expression strain E.coliBL21(DE3) containing the recombinant plasmid TeAgo-pET28a was inoculated in LB medium containing 50 μg / mL kanamycin, and cultured on a shaker at 37°C and 220 rpm to OD 600 Between 0.6-0.8, add IPTG with a final concentration of 0.4-0.6mM, and continue culturing on a shaker at 200rpm at 18°C ​​for 16-20h to induce the expression of TeAgo protein. Collect the cells by centrifugation, resuspend the cells in a resuspension buffer (containing 20mM Tris-HCl, pH around 8.0, 500mM NaCl), then crush the cells by high pressure, and centrifuge to obtain the supernatant. Using Ni-NTA column to affinity purify the protein, the eluate is concentrated by ultrafiltration, desalted and other steps to obtain the pu...

Embodiment 3

[0234] Example 3: TeAgo Shearing Activity Determination

[0235] Design fluorescently modified 45nt single-stranded DNA, RNA target nucleic acid and four complementary 16nt DNA and RNA guide strands, and send them to the company for synthesis.

[0236] DNA target nucleic acid sequence (SEQ ID NO:21):

[0237] 5'-FAM-CGCAGCATGTCAAGATCACAGATTTTGGGCTGGCCAAACTGCTGG-3'

[0238] RNA target nucleic acid sequence (SEQ ID NO:22):

[0239] 5’-FAM-CGCAGCAUGUCAAGAUCACAGAUUUUGGGCUGGCCAAACUGCUGG-3’

[0240] gDNA sequence (SEQ ID NO: 23):

[0241] 5'-HO / P-TAGTTTGGCCAGCCCA-3'

[0242] gRNA sequence (SEQ ID NO:24):

[0243] 5'-HO / P-UAGUUUGGCCAGCCCA-3'

[0244] Prepare the reaction buffer (containing 15mM Tris-HCl pH8.0, 250mM NaCl), and add MnCl with a final concentration of 0.5mM in the reaction buffer 2 , 400nM TeAgo, 2μM synthetic gDNA or gRNA and 0.8μM 5' fluorescently modified sequence complementary single-stranded DNA or RNA target nucleic acid, react at 95°C for 15min, af...

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Abstract

The invention provides characterization and application of a novel high-temperature Argonaute protein. Specifically, the invention provides a nucleic acid cleavage system, which is characterized in that the nucleic acid cleavage system comprises: (a) guide DNA (gDNA); (b) a programmable endonuclease Argonaute (Ago); and (c) optionally a reporter nucleic acid wherein if the reporter nucleic acid is cleaved, the cleavage can be detected. In addition, the invention also provides a system and a method for enriching and detecting low-abundance target nucleic acid based on the programmable endonuclease Ago provided by the invention. The detection system and method provided by the invention can specifically and effectively enrich and efficiently detect low-abundance nucleic acid, and the programmable endonuclease Ago provided by the invention has strong gene manipulation potential.

Description

technical field [0001] The invention belongs to the field of molecular biology and biotechnology, and specifically relates to the characterization and application of a novel high-temperature Argonaute protein. Background technique [0002] The Argonaute (Ago) protein was first mentioned in a study describing a mutant of Arabidopsis thaliana. Ago proteins are key players in the eukaryotic RNA interference (RNAi) pathway that regulate gene expression post-transcriptionally, thereby defending against host-invading RNA viruses and preserving genome integrity. The reported Ago proteins are mainly divided into two types: eukaryotic Ago (eAgo) and prokaryotic Ago (pAgo). [0003] Prokaryotic Ago can bind to single-stranded guide DNA or RNA, and catalyze the cleavage of target DNA or RNA that is complementary to guide. Different from the CRISPR / Cas system, prokaryotic Ago does not need a PAM sequence when cutting the target nucleic acid chain. It can bind to the guide (DNA or RNA)...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/6806C12Q1/6851C12N15/70C12N15/55C12N9/22
CPCC12N15/10C12Q1/6806C12Q1/6851C12N15/70C12N9/22C12N2800/22C12Q2521/327C12Q2525/161C12Q2531/113C12Q2561/101C12Q2545/114
Inventor 冯雁孙莹璎郭翔陆慧李忠磊
Owner SHANGHAI JIAO TONG UNIV
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