Target Recognition Mechanism of <i>Thermus thermophilus</i> Argonaute at the Supplementary Site

<i>Thermus thermophilus</i> Argonaute (<i>Tt</i>Ago) utilizes a guide DNA strand to cleave foreign DNA, defending the bacteria against invasive genetic elements and thus offering potential as a gene-editing tool. However, the underlying mechanism for target recognition remains underexplored. For example, the necessity of guide-target complementarity at the supplementary site (positions g13-16) for target cleavage has been debated for years. Here, using multiple transition pathways generated by atomistic molecular dynamics simulations, we identified three stages in this process: tail release, base pairing, and final refinement. The tail release leads to full exposure of the guide DNA (gDNA) to solvents, thereby positioning base-pairing between gDNA and target DNA (tDNA) as the principal force driving recognition. Consequently, all rate-determining steps are situated within the base-pairing stage. Detailed examination indicates that π-π stacking between the nucleobases, the extrusion of bases, and mismatches significantly influence these rate-limiting stages. Our results also suggest that base dislocations are less disruptive factors than π-π stacking for tDNA recognition in the supplementary site.