An Engineered PfAgo with Wide Catalytic Temperature Range and Substrate Spectrum.

ABSTRACT: PfAgo, a thermophilic Argonaute nuclease from Pyrococcus furiosus, is widely used in various fields due to its high DNA-guided DNA cleavage activity. However, its high-temperature-dependent cleavage activity largely restricts its applications in moderate-temperature scenarios. In this study, PfAgo is engineered for cold adaptation based on its ternary complex structure and the attributes of cold-adapted enzymes, yielding a series of variants with better performance at moderate temperatures. Among those, mPfAgo (K617G, L618G) exhibits significantly promoted cleavage activity at 37 °C and a wider catalytic temperature range of 30-95 °C. Its high-temperature cleavage activity is also greatly improved, enabling its application in DNA detection with attomolar sensitivity in the presence of Mg

SUBMITTER: Wang L 

PROVIDER: S-EPMC12362761 | biostudies-literature | 2025 Aug

REPOSITORIES: biostudies-literature