Structural and functional insights into the fly microRNA biogenesis factor Loquacious

Stoichiometry of Loquacious bound to Dicer using single-molecule coimmunoprecipitation (co-IP) analyses. (A) Schematic representation of the single-molecule co-IP assay. (B) Total internal reflection fluorescence (TIRF) images of Alexa647-labeled molecules (Loqs-3^AF647) pulled down via Dicer. Dicer was genetically fused to GFP, enabling immobilization on the quartz slide via a biotinylated anti-GFP antibody (scale bar: 10 µm). As a control, Loquacious was flushed over the slide without prior incubation with GFP-dmDcr1. (C) Average numbers of Loqs-3^AF647 molecules per imaging area (6800 μm²). (D) Representative single-molecule fluorescence transients of Loqs-3^AF647 molecules that exhibit one-step and two-step photobleaching. (E) Photobleaching step distribution for Loqs-3^AF647 bound to dmDcr1 indicating a 1:1 stoichiometry for the Dicer–Loquacious complex (molecule numbers accumulated from four independent experiments, a total of 1558 molecules were analyzed).