Dynamic Anchoring of the 3′-End of the Guide Strand Controls the Target Dissociation of Argonaute–Guide Complex

Argonaute (Ago) is the catalytic core of small RNA-based gene regulation. Despite plenty of mechanistic studies on Ago, the dynamical aspects and the mechanistic determinants of target mRNA binding and dissociation of Ago–guide strand remain unclear. Here, by using single-molecule fluorescence resonance energy transfer (FRET) assays and Thermus thermophilus Ago (TtAgo), we reveal that the 3′-end of the guide strand dynamically anchors at and releases from the PAZ domain of Ago, and that the 3′-end anchoring of the guide strand greatly accelerates the target dissociation by destabilizing the guide–target duplex. Our results indicate that the target binding/dissociation of Ago–guide is executed through the dynamic interplays among Ago, guide, and target.