A FLUORESCENCE-BASED ASSAY FOR RIBONUCLEASE-A ACTIVITY

A sensitive assay for ribonuclease A activity based on the relief of f luorescence quenching within a defined oligomeric substrate (5' fluore scein-AAArAUAAAA-3'-rhodamine) is described. The substrate can be prod uced using an automated nucleic acid synthesizer and commercially avai lable reagents. Together with a nonfluorescent cosubstrate (5'-dimetho xytrityl-AAAAr-UAAAA), the compound can be used to determine kinetic c onstants 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, (C) 1998 Academic Press.