A fluorescence correlation spectroscopy-based enzyme assay for human Dicer

Abstract

Here, we present an in vitro assay based on fluorescence correlation spectroscopy (FCS), which allows investigation of the kinetic behaviour of human Dicer. The assay is based on the different mobilities of substrate and product. The change of substrate mobility was independent of the choice of the fluorescence label, allowing exclusion of non-specific photophysical artefacts. Dicer and RNase III cleavage led to different product diffusion times. Single-stranded RNA did not change its mobility after cleavage by both double-strand-specific RNases. In agreement with the literature, the RNase activity of Dicer could be inhibited by substituting Ca²⁺ for Mg²⁺. In a defined system of two diffusion species of similar label and mobility differences, such as substrate and product, the linearity of the assay could be proven. An FCS-based enzyme assay is proposed, which allows monitoring of Dicer activity with high specificity in vitro.