Related Experiment Video
Updated: Jul 23, 2025

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
Published on: March 25, 2020
A programmable pAgo nuclease with RNA target-cleavage specificity from the mesophilic bacterium Verrucomicrobia
Qi Liu1, Wanping Chen1,2, Yue Zhang1
1State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, China.
Abstract:
Argonaute (Ago) proteins are conserved programmable nucleases present in eukaryotes and prokaryotes and provide defense against mobile genetic elements. Almost all characterized pAgos prefer to cleave DNA targets. Here, we describe a novel pAgo from Verrucomicrobia bacterium (VbAgo) that can specifically cleave RNA targets rather than DNA targets at 37°C and function as a multiple-turnover enzyme showing prominent catalytic capacity. VbAgo utilizes DNA guides (gDNAs) to cleave RNA targets at the canonical cleavage site. Meanwhile, the cleavage activity is remarkably strengthened at low concentrations of NaCl. In addition, VbAgo presents a weak tolerance for mismatches between gDNAs and RNA targets, and single-nucleotide mismatches at positions 11‒12 and dinucleotide mismatches at positions 3‒15 dramatically reduce target cleavage. Moreover, VbAgo can efficiently cleave highly structured RNA targets at 37°C. These properties of VbAgo broaden our understanding of Ago proteins and expand the pAgo-based RNA manipulation toolbox.
Related Concept Videos
Ribozymes
Ribozymes can...
Conservative Site-specific Recombination and Phase Variation
The recognition sites for Cre recombinase called LoxP...
Bacterial RNA Polymerase
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
CRISPR and crRNAs
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...

