Published online 19 August 2004
Nucleic Acids Research, Vol. 32 No. 15 © Oxford University Press 2004; all rights reserved
Semi-automated, single-band peak-fitting analysis of hydroxyl radical nucleic acid footprint autoradiograms for the quantitative analysis of transitions
1 Department of Biochemistry and 2 Department of Physiology & Biophysics and Center for Synchrotron Biosciences, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
* To whom correspondence should be addressed. Tel: +1 718 430 3179; Fax: +1 718 430 8565; Email: brenowit{at}aecom.yu.edu
Received July 2, 2004; Revised and Accepted August 5, 2004
Hydroxyl radical footprinting can probe the solvent accessibility of the ribose moiety of the individual nucleotides of DNA and RNA. Semi-automated analytical tools are presented for the quantitative analyses of nucleic acid footprint transitions in which processes such as folding or ligand binding are followed as a function of time or ligand concentration. Efficient quantitation of the intensities of the electrophoretic bands comprising the footprinting reaction products is achieved by fitting a series of Lorentzian curves to line profiles obtained from gels utilizing sequentially relaxed constraints consistent with electrophoretic mobility. An automated process of data ‘standardization’ has been developed that corrects for differences in the loading amounts in the electrophoresis. This process enhances the accuracy of the derived transitions and makes generating them easier. Together with visualization of the processed footprinting in false-color two-dimensional maps, DNA and RNA footprinting data can be accurately, precisely and efficiently processed allowing transitions to be objectively and comprehensively analyzed. The utility of this new analysis approach is illustrated by its application to the ion-meditated folding of a large RNA molecule.