The combined results of in vitro protein-protein interaction detection assays and X-ray crystallography with multiple Ago2 and TNRC6B mutants indicate that all three pockets in the trp-binding region of Ago2 contribute to physical interaction with TNRC6B. Specific Trp residues in TNRC6B Ago-binding domain (ABD) have pocket preferences, suggesting that the three trp-binding pockets are not perfectly redundant with each other, however, each pocket is able to interact with multiple different trp residues in TNRC6B, thus the interaction is multivalent and the structures formed may be heterogeneous and complex. Under physiological salt concentrations, in vitro, solutions containing both Ago2 and the TNRC6B-ABD quickly became opaque (change in optical properties), indicative of the formation of massive particles. Concentrated solutions of the ABD alone (but not that of Ago2) also became turbid at temperatures below 15°C. The fluorescently labeled TNRC6B ABD and Ago2 formed liquid droplets (morphology) in a protein concentration-dependent and salt concentration-dependent manner (particle size and count) as assessed by confocal microscopy. Ago valency (finetuned with mutations of the Trp-binding pockets) had a strong effect on droplet formation (particle size and count by microscopy). In a 293 HEK cell line stably overexpressing GFP-fused TNRC6B (genetic transformation) GFP-labeled, dynamic foci formed in vivo with 0.2 to 1mm in size (particle size and count by microscopy). Transfecting cells with a plasmid encoding mCherry-fused Ago2 revealed that Ago2 co-localized with TNRC6B foci in vivo. Ago2-TNRC6B droplets selectively sequester miRNA targets from the bulk solution: when mixed with droplets containing Ago2-let7 (Ago2 loaded with the miRNA let-7) in vitro, a target RNA with eight seed-matched let-7 binding sites (8xlet7) appeared almost exclusively in the pellet fraction (co-localization). In contrast, the 8xlet7 target RNA remained in the supernatant when added to droplets formed with Ago2-miR122. Ago2 kept its endonucleolytic cleavage (termed‘‘slicing’’) activity towards target RNAs within the condensates in the presence of a small RNA guide and the required ions in vitro (enzymatic activity assay). Ago2 and TNRC6B were mixed in the presence of soluble lysate from HEK293 cells, and the resulting droplets were isolated by centrifugation. Analysis by proteomics techniques (western blot) revealed that subunits of the CCR4-NOT deadenylase complex (a known miRISC component) co-pelleted with TNRC6B, while actin, which is not a component of miRISC, did not. (PMID:29576456).