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Argonaute proteins repress gene expression and defend against foreign nucleic acids using short RNAs or DNAs to specify the correct target RNA or DNA sequence. We have developed single-molecule methods to analyze target binding and cleavage mediated by the Argonaute:guide complex, RISC. We find that both eukaryotic and prokaryotic Argonaute proteins reshape the fundamental properties of RNA:RNA, RNA:DNA, and DNA:DNA hybridization—a small RNA or DNA bound to Argonaute as a guide no longer follows the well-established rules by which oligonucleotides find, bind, and dissociate from complementary nucleic acid sequences. Argonautes distinguish substrates from targets with similar complementarity. Mouse AGO2, for example, binds tighter to miRNA targets than its RNAi cleavage product, even though the cleaved product contains more base pairs. By re-writing the rules for nucleic acid hybridization, Argonautes allow oligonucleotides to serve as specificity determinants with thermodynamic and kinetic properties more typical of RNA-binding proteins than of RNA or DNA., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Small interfering RNAs (siRNAs) direct Argonaute proteins, the core components of the RNA-induced silencing complex (RISC), to cleave complementary target RNAs. Here, we describe a method to purify active RISC containing a single, unique small RNA guide sequence. We begin by capturing RISC using a complementary 2'-O-methyl oligonucleotide tethered to beads. Unlike other methods that capture RISC but do not allow its recovery, our strategy purifies active, soluble RISC in good yield. The method takes advantage of the finding that RISC partially paired to a target through its siRNA guide dissociates more than 300 times faster than a fully paired siRNA in RISC. We use this strategy to purify fly Ago1- and Ago2-RISC, as well as mouse AGO2-RISC. The method can discriminate among RISCs programmed with different guide strands, making it possible to deplete and recover specific RISC populations. Endogenous microRNA:Argonaute complexes can also be purified from cell lysates. Our method scales readily and takes less than a day to complete.

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) guide Argonaute proteins to silence mRNA expression. Argonaute binding alters the properties of an RNA guide, creating functional domains. We show that the domains established by Argonaute-the anchor, seed, central, 3' supplementary, and tail regions-have distinct biochemical properties that explain the differences between how animal miRNAs and siRNAs bind their targets. Extensive complementarity between an siRNA and its target slows the rate at which fly Argonaute2 (Ago2) binds to and dissociates from the target. Highlighting its role in antiviral defense, fly Ago2 dissociates so slowly from extensively complementary target RNAs that essentially every fully paired target is cleaved. Conversely, mouse AGO2, which mainly mediates miRNA-directed repression, dissociates rapidly and with similar rates for fully paired and seed-matched targets. Our data narrow the range of biochemically reasonable models for how Argonaute-bound siRNAs and miRNAs find, bind, and regulate their targets., (Copyright © 2012 Elsevier Inc. All rights reserved.)