Monitoring structural changes in nucleic acids with single residue spatial and millisecond time resolution by quantitative hydroxyl radical footprinting
Monitoring structural changes in nucleic acids with single residue spatial and millisecond time resolution by quantitative hydroxyl radical footprinting
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DOI：
10.1038/nprot.2007.533
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Brenowitz, Michael
中科院分区：
文献类型：
--
作者：
Shcherbakova, Inna;Brenowitz, Michael
羟基自由基（中心点OH）足迹提供了全面的网站特定的定量信息与大分子折叠，相互作用和配体结合的结构变化。“快速芬顿”足迹法是一种基于实验室的时间分辨方法。OH足迹能够毫秒时间分辨率容易适用于DNA和RNA。该方案利用廉价的化学试剂（H2O2、Fe（NH4）（2）（SO4）（2）、EDTA、硫脲或乙醇）和广泛使用的猝灭流混合器来揭示复杂生化过程的瞬态（通常是短暂的）中间状态。我们描述了一个协议，研究RNA折叠，可以很容易地定制特定的应用程序。一旦熟悉淬灭流混合器操作及其校准、核酸标记和剂量响应实验的进行，30个时间点的单个动力学实验需要约1小时来执行。样品的处理和分离。OH反应产物需要几个小时。数据分析可能需要45分钟到数周，具体取决于分析的深度。
Hydroxyl radical (center dot OH) footprinting provides comprehensive site-specific quantitative information about the structural changes associated with macromolecular folding, interactions and ligand binding. 'Fast Fenton' footprinting is a laboratory-based method for time-resolved. OH footprinting capable of millisecond time resolution readily applicable to DNA and RNA. This protocol utilizes inexpensive chemical reagents (H2O2, Fe(NH4)(2)(SO4)(2), EDTA, thiourea or ethanol) and widely available quench-flow mixers to reveal transient, often short-lived, intermediate states of complex biochemical processes. We describe a protocol developed to study RNA folding that can be readily tailored to particular applications. Once familiar with quench-flow mixer operation and its calibration, nucleic acid labeling and the conduct of a dose-response experiment, a single kinetic experiment of 30 time points takes about 1 h to perform. Sample processing and separation of the. OH reaction products takes several hours. Data analysis can take 45 min to several weeks depending on the depth of analysis conducted.