Monitoring structural changes in nucleic acids with single residue spatial and millisecond time resolution by...

Hydroxyl radical (.OH) footprinting provides comprehensive site-specific quantitative information about the structural changes associated with macromolecular folding, interactions and ligand binding. 'Fast Fenton' footprinting is a laboratory-based method for time-resolved .OH footprinting capable of millisecond time resolution readily applicable to DNA and RNA. This protocol utilizes inexpensive chemical reagents (H2O2, Fe(NH4)2(SO4)2, EDTA, thiourea or ethanol) and widely available quench-flow mixers to reveal transient, often short-lived, intermediate states of complex biochemical processes. We describe a protocol developed to study RNA folding that can be readily tailored to particular applications. Once familiar with quench-flow mixer operation and its calibration, nucleic acid labeling and the conduct of a dose-response experiment, a single kinetic experiment of 30 time points takes about 1 h to perform. Sample processing and separation of the .OH reaction products takes several hours. Data analysis can take 45 min to several weeks depending on the depth of analysis conducted.