title: Kinetic Analysis of Target RNA Binding and Slicing by Human Argonaute 2 Protein.
abstract: Analyzing the mechanisms of Argonaute-mediated gene silencing is essential to the understanding of RNA interference (RNAi). RNAi is a process to regulate gene expression on a posttranscriptional level. Directed by single-stranded small RNA guides, Argonaute 2 binds complementary target RNAs, and if the guide displays full complementarity to the targeted sequence, Argonaute 2 slices the bound target RNA. This on the one hand is an important mechanism to regulate gene expression in the cell and on the other hand represents a powerful tool to interfere with harmful gene expression levels. Here, we present techniques to kinetically characterize recombinant Argonaute 2-mediated guide and target binding as well as target RNA slicing. We focus on fluorescence-based steady-state and in particular pre-steady-state techniques to unravel mechanistic details. Furthermore, we describe a cleavage assay to analyze Argonaute 2-mediated slicing using radioactively labeled target strands.
authors.affiliation: Institute of Molecular Medicine, Universitätsklinikum Schleswig-Holstein, University of Lübeck, Ratzeburger Allee 160, 23538, Lübeck, Germany.|Department of Microbiology, Single Molecule Biochemistry Lab, Universität Regensburg, Regensburg, Germany.
authors.forename: Sarah
authors.initials: S
authors.lastname: Willkomm
authors.affiliation: Institute of Molecular Medicine, Universitätsklinikum Schleswig-Holstein, University of Lübeck, Ratzeburger Allee 160, 23538, Lübeck, Germany. restle@imm.uni-luebeck.de.
authors.forename: Tobias
authors.initials: T
authors.lastname: Restle
mesh_terms: D060565:Argonaute Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*; D020868:Gene Silencing; D006801:Humans; D007700:Kinetics; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000235:genetics*; D000067776:RNA Recognition Motif Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000235:genetics; D034743:RNA-Induced Silencing Complex / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; D035683:MicroRNAs; D000067776:RNA Recognition Motif Proteins; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex
keywords: Fluorescence; Human Argonaute 2; Kinetics; Pre-steady state; RNA interference; RNAi; Steady state; Stopped flow; microRNA; siRNA