Measurement of in vivo RISC activity using an in vitro non-radioactive RISC assay

Argonaute 2 (Ago2) is the core catalytic enzyme of RISC (RNA-induced silencing complex) that mediates RNA cleavage by RNA-interference (RNAi). RNAi is an important gene regulatory mechanism, that depends on small interfering (si)RNAs. The minimal components of RISC are members of the Argonaute family of proteins plus small RNA guides. So far, Ago2 activity was assessed using 32P-cap-labelled in vitro synthesized mRNA targets and ATP as substrates. Cleavage products are detected after purification and separation of reaction mixtures by denaturing Urea-PAGE followed by autoradiography. This assay is time consuming, difficult to handle and only semiquantitative. Recently, we have reported the establishment of a non-radioactive RISC assay in vitro. GST-Ago2 was purified to near homogeneity from E.Coli and incubated with an 180bp or 723bp luciferase mRNA target. Cleavage reaction was started by addition of phosphorylated single stranded complementary RNA oligonucleotides and resulted in complete cleavage of the target RNA. Purified reaction mixtures were separated and visualized using RNA Chips on an Agilent 2100 Bioanalyzer instrument. Here we report, that after optimization with respect to specificity and efficacy it is also possible to use the established in vitro RISC assay to measuring activity of endogenous Ago2 isolated from cells. Thus, the non-radioactive Ago2 assay can be used to identify inhibitors or activators of Ago2 in cell-based assay systems.