title: Rapid and specific purification of Argonaute-small RNA complexes from crude cell lysates.
abstract: 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.
authors.affiliation: Howard Hughes Medical Institute and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
authors.forename: C Fabián
authors.initials: CF
authors.lastname: Flores-Jasso
authors.forename: William E
authors.initials: WE
authors.lastname: Salomon
authors.forename: Phillip D
authors.initials: PD
authors.lastname: Zamore
mesh_terms: D000818:Animals; D060565:Argonaute Proteins / Q000302:isolation & purification* / Q000378:metabolism; D020029:Base Pairing; D015152:Blotting, Northern; D004330:Drosophila / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000302:isolation & purification / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D035683:MicroRNAs / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D034622:RNA Interference; D018075:RNA, Complementary / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012680:Sensitivity and Specificity; D013997:Time Factors; D058727:RNA, Small Untranslated
chemical_list: C489654:AGO1 protein, Drosophila; C433807:AGO2 protein, Drosophila; C526735:Ago2 protein, mouse; D060565:Argonaute Proteins; D029721:Drosophila Proteins; D035683:MicroRNAs; D018075:RNA, Complementary; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; D058727:RNA, Small Untranslated