Strong temperature effects on the fidelity of target DNA recognition by a thermophilic pAgo nuclease

Prokaryotic Argonaute (pAgo) proteins are programmable nucleases with great promise in genetic engineering and biotechnology. Previous studies identified several DNA-targeting pAgo nucleases from mesophilic and thermophilic prokaryotic species that are active in various temperature ranges. However, the effects of temperature on the specificity of target recognition and cleavage by pAgos have not been studied. Here, we describe a thermostable pAgo nuclease from the thermophilic bacterium Thermobrachium celere, TceAgo. We show that TceAgo preferentially uses 50-phosphorylated small DNA guides and can perform specific cleavage of both single-stranded and double-stranded DNA substrates in a wide range of temperatures. Single-nucleotide mismatches between guide and target molecules differently change the reaction efficiency depending on the mismatch position, with the fidelity of target recognition greatly increased at elevated temperatures. Thus, TceAgo can serve as a tool to allow specific detection and cleavage of DNA targets in a temperature-dependent manner. The results demonstrate that the specificity of programmable nucleases can be strongly affected by the reaction conditions, which should be taken into account when using these nucleases in various in vitro and in vivo applications. (c) 2023 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

Automated summary

This study describes a thermostable pAgo nuclease, TceAgo, from the thermophilic bacterium Thermobrachium celere, which can perform specific cleavage of both single-stranded and double-stranded DNA substrates in a wide range of temperatures. The fidelity of target recognition is greatly increased at elevated temperatures. These findings highlight the importance of considering reaction conditions when using programmable nucleases in different in vitro and in vivo applications.