RNA footprinting analysis using ion pair reverse phase liquid chromatography
RNA footprinting analysis using ion pair reverse phase liquid chromatography
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DOI：
10.1017/s1355838202012888
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发表时间：
2002-02-01
期刊：
影响因子：
4.5
通讯作者：
Hornby, DP
中科院分区：
文献类型：
--
作者：
Dickman, MJ;Conroy, MJ;Hornby, DP
羟基自由基足迹法是一种有效的技术，常用于表征蛋白质和核酸之间的三级相互作用。在通过化学或酶促反应产生核酸“梯”之后，传统上通过变性凝胶电泳分离放射性标记的产物，并通过磷光成像技术进一步定量。在这里，我们报告使用离子对反相液相色谱分析的RNA足迹反应，使用荧光标记的RNA分子的产品。这种技术提供了几个优势，现有的程序，包括快速分析，自动化，并直接定量的裂解产物，而不需要采用放射性标记。为了说明这种技术的分辨率，我们分析了从荧光标记的RNA分子产生的碱水解产物，并随后使用这种方法来定义底物链与发夹核酶对接时的溶剂可及性。
Hydroxyl radical footprinting is a powerful technique often employed in characterization of the tertiary interactions between proteins and nucleic acids. Following the generation of a nucleic acid "ladder" either by chemical or enzymatic reactions, the radiolabeled products are traditionally separated by denaturing gel electrophoresis and further quantified by phosphorimaging techniques. Here we report the use of ion pair reverse phase liquid chromatography to analyze the products of an RNA footprinting reaction using fluorescently labeled RNA molecules. This technique offers several advantages over existing procedures, including rapid analysis, automation, and direct quantification of the cleavage products without the need to employ radiolabeling. To illustrate the resolving power of this technique, we have analyzed the products of base hydrolysis, generated from a fluorescently labeled RNA molecule and have subsequently used this method to define the solvent accessibility of the substrate strand as it docks with the hairpin ribozyme.