Hampel K J, Burke J M
Markey Center for Molecular Genetics, University of Vermont, Burlington, Vermont 05405, USA.
Methods. 2001 Mar;23(3):233-9. doi: 10.1006/meth.2000.1134.
Chemical footprinting methods have been used extensively to probe the structures of biologically important RNAs at nucleotide resolution. One of these methods, hydroxyl-radical footprinting, has recently been employed to study the kinetics of RNA folding. Hydroxyl radicals can be generated by a number of different methods, including Fe(II)-EDTA complexes, synchrotron radiation, and peroxynitrous acid disproportionation. The latter two methods have been used for kinetic studies of RNA folding. We have taken advantage of rapid hydroxyl-radical generation by Fe(II)-EDTA-hydrogen peroxide solutions to develop a benchtop method to study folding kinetics of RNA complexes. This technique can be performed using commercially available chemicals, and can be used to accurately define RNA folding rate constants slower than 6 min(-1). Here we report the method and an example of time-resolved footprinting on the hairpin ribozyme, a small endoribonuclease and RNA ligase.
化学足迹法已被广泛用于在核苷酸分辨率下探测具有生物学重要性的RNA的结构。其中一种方法，即羟自由基足迹法，最近已被用于研究RNA折叠动力学。羟自由基可以通过多种不同方法产生，包括Fe(II)-EDTA复合物、同步辐射和过氧亚硝酸歧化反应。后两种方法已用于RNA折叠的动力学研究。我们利用Fe(II)-EDTA-过氧化氢溶液快速产生羟自由基的特性，开发了一种台式方法来研究RNA复合物的折叠动力学。该技术可以使用市售化学品进行，并且可用于准确确定慢于6 min⁻¹的RNA折叠速率常数。在此，我们报告该方法以及对发夹状核酶（一种小型内切核糖核酸酶和RNA连接酶）进行时间分辨足迹分析的一个实例。
