Hydroxyl radical is produced by ionizing radiation and causes DNA scission by abstracting the hydrogen atoms of deoxyribose. The ability to cleave DNA in a non-sequence specific manner has made this agent an effective tool in footprinting experiments used to gain structural information about DNA, RNA, and their complexes with cellular proteins in solution. Despite the widespread use of the hydroxyl radical footprinting technique, very little information is available about the mechanism by which hydroxyl radical cleaves nucleic acids. Initial deuterium isotope effect experiments with a 19-bp DNA probe demonstrated that the greater solvent accessibility of the 5' and 4' hydrogens of the deoxyribose correlated with larger deuterium isotope effects upon reaction with hydroxyl radical. The proposed work seeks to expand these initial deuterium kinetic isotope effect experiments and investigate further the mechanism of DNA cleavage by hydroxyl radical. The mechanism of RNA cleavage will also be examined with this technique. The results of these experiments will increase the amount of information obtained from the footprinting experiments by allowing the reactivity to be interpreted at the level of the deoxyribose and ribose hydrogens instead of the entire sugar moiety. These advances will help to provide more structural details about biomolecules in solution.
