An Engineered PfAgo with Wide Catalytic Temperature Range and Substrate Spectrum

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 Mg²⁺. Additionally, mPfAgo shows versatile cleavage activities, including DNA cleavage guided by 5'OH-gDNA, 5'P-gDNA, or 5'COOH-gDNA, as well as RNA cleavage with 5'OH-gDNA, 5'P-gDNA, 5'P-gRNA, or 5'COOH-gDNA as guides. Further analysis through far-UV CD spectra and DSF indicates that mPfAgo has a more flexible structure than wild-type PfAgo. Furthermore, this established strategy is applied to engineer TtdAgo, likewise obtaining its variants with enhanced moderate-temperature activity and expanded substrate spectrum. In summary, this work provides a novel method for the rational design of thermophilic Agos, thereby greatly expanding their application scopes.