Biochemical and pre-steady-state kinetic characterization of recombinant human Argonaute2

Argonaute2 (Ago2) protein is known to play a key role in RNA interference (RNAi). It is the core of the RNA induced silencing complex (RISC) and the only member of the human Ago family with slicer activity. Here we report about a detailed biochemical and pre-steady-state kinetic characterization of substrate binding and target cleavage by hAgo2. Performing fluorescence measurements using appropriately labeled RNA substrates, we were able to determine the equilibrium dissociation constants (K_d) for single and double stranded siRNA substrates. A detailed analysis of the nucleic acid binding mechanism applying pre-steady-state techniques revealed that formation of the binary guide RNA-hAgo2 complex occurs in at least three steps. While the first fast phase reflects the formation of a collision complex, a second slower phase most likely represents anchoring of the 5'-end of the guide RNA in the Mid domain binding pocked promoted by the presence of a phosphate group. A third even slower phase is indicative of a conformational change after initial positioning of the guide strand. In a next set of experiments, we are about to determine the binding equilibrium of the binary complex for a target RNA followed by time resolved binding analyses. In parallel, target cleavage properties of hAgo2 in the presence of different siRNAs are investigated. In combining our biochemical data with structural information derived from archaebacterial enzymes, we soon should be able to derive a minimal kinetic model of the hAgo2-catalysed RNA cleavage reaction.