A fluorescence correlation spectroscopy-based enzyme assay for human dicer

Abstract

We used fluorescence correlation spectroscopy (FCS) to establish an in vitro assay to investigate RNase activity of human Dicer (Werner et al., Biol Chem 393(3):187-193). FCS allows investigating the interactions of different particles due to their differing diffusion mobility, provided that one of the interacting partners exhibit a fluorescence label. In our case we used a fluorophore-labeled double-stranded RNA (dsRNA) as substrate to monitor Dicer activity. The dsRNA was cleaved by the enzyme resulting in a five-nucleotide-short single-stranded RNA (ssRNA) fragment carrying the fluorophore, which could be distinguished from the substrate and unlabeled second product by FCS. Furthermore, we refer to additional (control) experiments to confirm obtained data. © 2014 Springer Science+Business Media, New York.