A Fluorescence-Based Assay for Ribonuclease A Activity

Abstract A sensitive assay for ribonuclease A activity based on the relief of fluorescence quenching within a defined oligomeric substrate (5′ fluorescein-AAAArUAAAA-3′-rhodamine) is described. The substrate can be produced using an automated nucleic acid synthesizer and commercially available reagents. Together with a nonfluorescent cosubstrate (5′-dimethoxytrityl-AAAArUAAAA), the compound can be used to determine kinetic constants for the first step (transphosphorylation) of the ribonuclease-catalyzed reaction. These measurements should be useful for structure-based analyses of ribonuclease activity since a crystal structure has been determined for a closely analogous enzyme–inhibitor complex.