A Programmable, DNA-Exclusively-Guided Argonaute DNase and Its Higher Cleavage Specificity Achieved by 5'-Hydroxylated Guide

Overview

Journal Biomolecules

Publisher MDPI

Specialties Biochemistry
Molecular Biology

Date 2022 Oct 27

PMID 36291549

Authors

Shichao Sun

Dejin Xu

Lin Zhu

Bei Hu

Zhen Huang

Affiliations

Soon will be listed here.

Abstract

Argonaute proteins exist widely in eukaryotes and prokaryotes, and they are of great potential for molecular cloning, nucleic acid detection, DNA assembly, and gene editing. However, their overall properties are not satisfactory and hinder their broad applications. Herein, we investigated a prokaryotic Argonaute nuclease from a mesophilic bacterium (CdAgo) and explored its overall properties, especially with 5'-hydroxylated (5'-OH) guides. We found that CdAgo can exclusively use single-stranded DNA (ssDNA) as guide to cleave ssDNA and plasmid targets. Further, we found the length of the efficient guide is narrower for the 5'-OH guide (17-20 nt) than for the 5'-phosphorylated guide (5'-P, 14-21 nt). Furthermore, we discovered that the 5'-OH guides can generally offer stronger mismatch discrimination than the 5'-P ones. The 5'-OH guides offer the narrower length range, higher mismatch discrimination and more accurate cleavage than the 5'-P guides. Therefore, 5'-OH-guide-directed CdAgo has great potential in biological and biomedical applications.

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