Studying Exoribonuclease Activity Using Fluorescence Anisotropy Assay

Fluorescence anisotropy is a powerful technique, widely used for investigating ligand-macromolecule binding and high-throughput screens for drugs. Here, we employ fluorescence anisotropy to quantitatively study the activity of exoribonucleases exemplified by the Xrn2 enzyme. Recording changes in the fluorescence anisotropy over time allows real-time detection of enzymatic activity and provides a framework that can be tailored to particular questions. We discuss the experimental setup, the potential substrate RNAs and highlight data analysis. We envision that this assay can be applied to study other nucleic acid-degrading enzymes and further expanded to include competition and inhibitor screens.