Takamoto Keiji, Chance Mark R, Brenowitz Michael
Department of Biochemistry, Center for Synchrotron Biosciences, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Nucleic Acids Res. 2004 Aug 19;32(15):E119. doi: 10.1093/nar/gnh117.
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.
羟自由基足迹法可探测DNA和RNA单个核苷酸核糖部分的溶剂可及性。本文介绍了用于核酸足迹转变定量分析的半自动分析工具，其中折叠或配体结合等过程可作为时间或配体浓度的函数进行跟踪。通过将一系列洛伦兹曲线拟合到利用与电泳迁移率一致的顺序松弛约束从凝胶获得的线轮廓上，实现了对构成足迹反应产物的电泳条带强度的有效定量。已开发出一种数据“标准化”的自动化过程，可校正电泳中上样量的差异。该过程提高了导出转变的准确性，并使其生成更容易。连同在伪彩色二维图中对处理后的足迹进行可视化，DNA和RNA足迹数据可以准确、精确和高效地处理，从而使转变能够得到客观和全面的分析。这种新分析方法的实用性通过其在大RNA分子离子介导折叠中的应用得到了说明。
