Distinct Cleavage Trajectories Mediated by DNA and RNA Substrate in Argonaute From Methanocaldococcus Fervens

Prokaryotic Argonautes (pAgos) have emerged as highly versatile, programmable nucleases, while the full exploitation of their biotechnological utility still requires a deeper understanding of their catalytic mechanisms. Here, we characterize a thermophilic Argonaute from Methanocaldococcus fervens (MfAgo), which demonstrates diverse substrate targeting and high catalytic efficiency across a broad temperature range. Notably, MfAgo exhibits exceptional accuracy during RNA cleavage, and structural analysis of MfAgo in various states throughout the process reveals a stepwise conformational rearrangement, highlighting the necessity of dimerization for the stabilization of the catalytic tetrad. In contrast, cleavage assays reveal that DNA cleavage by MfAgo is robust and independent of dimerization. Furthermore, we identified an additional nucleic acid binding pocket on the surface of MID domain, which may mediate the temperature-dependent activity. Collectively, these findings establish MfAgo as a highly versatile and promising tool for biotechnology applications.