Abstract
The use of highly reactive chemical species to probe the structure and dynamics of nucleic acids is greatly simplified by software that enables rapid quantification of the gel images that result from these experiments. Semiautomated footprinting analysis (SAFA) allows a user to quickly and reproducibly quantify a chemical footprinting gel image through a series of steps that rectify, assign, and integrate the relative band intensities. The output of this procedure is raw band intensities that report on the relative reactivity of each nucleotide with the chemical probe. We describe here how to obtain these raw band intensities using SAFA and the subsequent normalization and analysis procedures required to process these data. In particular, we focus on analyzing time-resolved hydroxyl radical ((•)OH) data, which we use to monitor the kinetics of folding of a large RNA (the L-21 T. thermophila group I intron). Exposing the RNA to bursts of (•)OH radicals at specific time points during the folding process monitors the time progress of the reaction. Specifically, we identify protected (nucleotides that become inaccessible to the (•)OH radical probe when folded) and invariant (nucleotides with constant accessibility to the (•)OH probe) residues that we use for monitoring and normalization of the data. With this analysis, we obtain time-progress curves from which we determine kinetic rates of folding. We also report on a data visualization tool implemented in SAFA that allows users to map data onto a secondary structure diagram.