Central guide-target pairing activates human Argonaute2 by uncoupling the N-PAZ and MID-PIWI lobes.

Small interfering RNAs (siRNAs) are widely used to repress gene expression. For human Argonaute2 (AGO2) to cleave target RNA, siRNAs must be almost perfectly complementary to their targets, particularly in the central region, yet the mechanistic basis for this requirement has remained unclear. Here, cryo-EM structures and biochemical analyses show that centrally mismatched targets propagate along the nucleic acid-binding channel of bilobed AGO2, whereas centrally matched targets form a duplex that pries open the tapered channel, collapsing the bilobed architecture into a duplex-bound MID-PIWI lobe and an RNA-free N-PAZ lobe. This rearrangement generates a preactive open intermediate that accommodates the extensively paired duplex. The uncoupled N-PAZ lobe must then swing back and reassemble the bilobed architecture, thereby positioning the duplex in the PIWI catalytic center and promoting target cleavage.