Hydroxyl-radical footprinting to probe equilibrium changes in RNA tertiary structure

Hydroxyl-radical footprinting utilizes the ability of a highly reactive species to nonspecifically cleave the solvent accessible regions of a nucleic acid backbone. Thus, changes in a nucleic acids structure can be probed either as a function of time or of a reagent's concentration. When combined with techniques that allow single nucleotide resolution of the resulting fragments, footprinting experiments provide richly detailed information about local changes in tertiary structure of a nucleic acid accompanying its folding or ligand binding. In this chapter, we present two protocols of equilibrium hydroxyl-radical footprinting based on peroxidative and oxidative Fenton chemistry and discuss how to adjust the Fenton reagent concentrations for a specific experimental condition. We also discuss the choice of the techniques to separate the reaction products and specifics of the data analysis for equilibrium footprinting experiments. Protocols addressing the use of peroxidative Fenton chemistry for time-resolved studies have been published [Schlatterer and Brenowitz, 2009. Methods; Shcherbakova and Brenowitz, 2008. Nat. Protoc.3(2), 288-302; Shcherbakova et al., 2006. Nucleic Acids Res.34(6), e48; Shcherbakova et al., 2007. Methods Cell Biol.84, 589-615].