Fast Fenton footprinting: a laboratory-based method for the time-resolved analysis of DNA, RNA and proteins

‘Footprinting’ describes assays in which ligand binding or structure formation protects polymers such as nucleic acids and proteins from either cleavage or modification; footprinting allows the accessibility of individual residues to be mapped in solution. Equilibrium and time-dependent footprinting links site-specific structural information with thermodynamic and kinetic transitions. The hydroxyl radical (·OH) is a particularly valuable footprinting probe by virtue of it being among the most reactive of chemical oxidants; it reports the solvent accessibility of reactive sites on macromolecules with as fine as a single residue resolution. A novel method of millisecond time-resolved ·OH footprinting has been developed based on the Fenton reaction, Fe(II) + H(2)O(2) → Fe(III) + ·OH + OH(−). This method can be implemented in laboratories using widely available three-syringe quench flow mixers and inexpensive reagents to study local changes in the solvent accessibility of DNA, RNA and proteins associated with their biological function.