Single-Molecule Analysis for RISC Assembly and Target Cleavage
RNA-induced silencing complex (RISC) is a small RNA–protein complex that mediates silencing of complementary target RNAs. Biochemistry has been successfully used to characterize the molecular mechanism of RISC assembly and function for nearly two decades. However, further dissection of intermediate states during the reactions has been warranted to fill in the gaps in our understanding of RNA silencing mechanisms. Single-molecule analysis with total internal reflection fluorescence (TIRF) microscopy is a powerful imaging-based approach to interrogate complex formation and dynamics at the individual molecule level with high sensitivity. Combining this technique with our recently established in vitro reconstitution system of fly Ago2-RISC, we have developed a single-molecule observation system for RISC assembly. In this chapter, we summarize the detailed protocol for single-molecule analysis of chaperone-assisted assembly of fly Ago2-RISC as well as its target cleavage reaction.
Key wordsSingle-molecule imaging RNA interference Argonaute RISC Small interfering RNA Total internal reflection fluorescence microscopy
We thank S. Iwasaki for constructing vectors for protein expression and establish the in vitro reconstitution system of fly Ago2-RISC assembly. This work was supported in part by Grants-in-Aid for Scientific Research on Innovative Areas (‘Functional machinery for non-coding RNAs’ 21115002 and ‘Non-coding RNA neo-taxonomy’ 26113007) (to H.T. and Y.T.), and Grants-in-Aid for challenging Exploratory Research (24657115 and 26650047) (to H.M.S.) from The Ministry of Education, Culture, Sports, Science and Technology in Japan.