监测RNA结构平衡的变动情况“过氧化”和“氧化”羟基自由基足
Summary
本协议描述了如何量化的镁（二）依赖RNA的三级结构的形成由两个羟基自由基足迹方法。
Abstract
RNA分子在生物学中发挥的重要作用。除了传递遗传信息，RNA折叠成独特的三级结构，实现特定的生物学作用，作为调节剂，粘结剂或催化剂。有关大专院校的联系形成的信息是必须了解的RNA分子的功能。羟基自由基（•OH）是独特的结构由于其反应活性高，体积小的核酸探针。，作为一个足迹探头使用时，羟基自由基地图的溶剂可及表面的DNA和RNA的磷酸骨干如单核苷酸分辨率罚款。羟基自由基足可以用来识别内分子间的接触面，例如在DNA -蛋白质和RNA -蛋白质复合物的核苷酸。 3平衡和动力学4转换可以由羟基自由基足迹作为一个soluti功能进行变量或时间，分别。的足迹的一个主要特点是有限的接触探头（例如，“单次击中动力学”）中的每个核苷酸的聚合物均匀采样的结果。5
在这个视频文章中，我们使用的四膜虫核酶的小四至小六的域名，说明RNA样品制备和一个镁（II）介导的折叠等温线的决心。我们描述了使用众所周知的羟基自由基足迹协议，要求H 2 O 2（我们称之为“过氧化”协议）和一个有价值的，但并不广为人知，替代使用自然溶解O 2的（我们称之为“氧化“协议）。提交的数据压缩，转换和分析程序概述。
Protocol
1。足迹试剂的制备准备了10倍的反应缓冲液含有钠cacodylate 100毫米，1毫米EDTA和1个M氯化钾。调整pH值至7.4。使用0.2μm的醋酸纤维过滤设备（NALGENE）过滤器的缓冲区。注：不吸管的RNA直接到10倍缓冲区。 准备如表1所示，每个反应的滴定反应混合。滴定混合量（1X缓冲和镁（二）所需的浓度）应为90μL，然后加入1X缓冲的RNA加入10μl。 准备一个核糖核酸酶T1消化缓冲液6.63M尿素，柠…
Discussion
羟基自由基足迹是一个有价值的工具，以评估核酸的溶剂可及表面面积。定性和定量的形成三级结构14，可以遵循作为一个功能参数，如离子种类和浓度，pH值，温度，结合蛋白或折叠的共同因素。引人注目的一条直线前进和廉价的协议，以及由此产生的溶剂可及单核苷酸水平上的折叠信息的结合，使得这种方法非常有吸引力的。传统的•OH足迹反应是增加H 2 O 2，我们称为“过氧?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作是支持的国家卫生RO1 – GM085130研究所和国家科学基金会MCB0929394的资助。我们感谢她的盛情款待，让我们在她的实验室电影马里昂施密特博士。
Materials
| Name | Company | Cat# |
|---|---|---|
| Sodium Cacodylate (Caution! Toxic) | Sigma | C4945-25g |
| EDTA (0.5 M) | Ambion | AM9260G |
| DEPC treated water | Ambion | AM9915G |
| Sodium Acetate (3 M) | Ambion | AM9740 |
| MgCl2 (1 M) | Ambion | AM9530G |
| Urea | Ambion | AM9902 |
| Sodium Citrate | Sigma | W302600 |
| tRNA | Sigma | R-7876 |
| Sodium-L-ascorbate | Sigma | A7631-25g |
| Fe(NH4)2(SO4)2 . 6 H2O | Sigma | F1543-500g |
| RNase T1 | Fermentas | EN0541 |
| Hydrogen Peroxide (30%) | Sigma | 349887 |
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Tags
MonitoringEquilibrium ChangesRNA StructurePeroxidativeOxidativeHydroxyl Radical FootprintingRNA MoleculesGenetic InformationTertiary StructuresBiologic RoleRegulatorBinderCatalystNucleic AcidsPhosphodiester BackboneSingle Nucleotide ResolutionIntermolecular Contact SurfaceEquilibrium TransitionsKinetic TransitionsSolution VariableTimeProbe ExposurePolymer SamplingVideo ArticleSample PreparationMg(II)-mediated Folding Isotherms
Citer Cet Article
Bachu, R., Padlan, F. S., Rouhanifard, S., Brenowitz, M., Schlatterer, J. C. Monitoring Equilibrium Changes in RNA Structure by ‘Peroxidative’ and ‘Oxidative’ Hydroxyl Radical Footprinting. J. Vis. Exp. (56), e3244, doi:10.3791/3244 (2011).