Characterization of argonaute nucleases from mesophilic bacteria Pseudobutyrivibrio ruminis

Mesophilic Argonautes (Agos) from microbial resources have received significant attention due to their potential applications in genome editing and molecular diagnostics. This study characterizes a novel Ago from Pseudobutyrivibrio ruminis ( Pr Ago), which can cleave single-stranded DNA using guide...

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Bibliographic Details
Published in:Bioresources and bioprocessing 2024-10, Vol.11 (1), p.94-13, Article 94
Main Authors: Xu, Xiaoyi, Yang, Hao, Dong, Huarong, Li, Xiao, Liu, Qian, Feng, Yan
Format: Article
Language:English
Subjects:
Biochemical Engineering
Biotechnology
Chemistry
Chemistry and Materials Science
Cleavage
Deoxyribonucleic acid
Discovery and engineering of novel enzymes for synthesis of new moleculars
DNA
DNA cleavage
Endonuclease
Environmental Engineering/Biotechnology
Enzymes
Industrial and Production Engineering
Magnesium
Manganese ions
Mesophilic Argonaute
Mesophilic bacteria
Microorganisms
Molecular dynamics
Nucleic acids
Nucleotides
pH effects
Prokaryotic Argonaute
Pseudobutyrivibrio ruminis Argonaute
Single-stranded DNA
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Summary:Mesophilic Argonautes (Agos) from microbial resources have received significant attention due to their potential applications in genome editing and molecular diagnostics. This study characterizes a novel Ago from Pseudobutyrivibrio ruminis ( Pr Ago), which can cleave single-stranded DNA using guide DNA (gDNA). Pr Ago, functioning as a multi-turnover enzyme, effectively cleaves DNA using 5′-phosphate gDNA, 14–30 nucleotides in length, in the presence of both Mn 2+ and Mg 2+ ions. Pr Ago demonstrates DNA cleavage activity over a broad pH range (pH 4–12), with optimal activity at pH 11. As a mesophilic enzyme, Pr Ago cleaves efficiently DNA at temperatures ranging from 25 to 65 °C, particularly at 65 °C. Pr Ago does not show strong preferences for the 5′-nucleotide in gDNA. It shows high tolerance for single-base mismatches, except at positions 13 and 15 of gDNA. Continuous double-nucleotide mismatches at positions 10–16 of gDNA significantly reduce cleavage activity. Furthermore, Pr Ago mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at 65 °C. Additionally, molecular dynamic simulations suggest that interactions between the PAZ domain and different nucleic acids strongly influence cleavage efficiency. These findings expand our understanding of Protokaryotic Agos and their potential applications in biotechnology. Graphical Abstract
ISSN:2197-4365
2197-4365
DOI:10.1186/s40643-024-00797-x