STRUCTURAL INFERENCE OF NATIVE AND PARTIALLY FOLDED RNA BY CONTACT MAPPING

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 核酶、核糖开关和许多其他功能RNA分子的生物学行为严重依赖于它们的三级折叠和采样多种功能状态的能力。其中大多数状态的构象异质性和部分折叠的性质使得用高分辨率结构方法来表征它们变得困难甚至难以解决。在这里，我们介绍了一种快速推断大RNA分子在其天然和非天然溶液状态下的三级螺旋排列的方法。多重羟基自由基(.oh)裂解分析(MOHCA)通过随机掺入自由基裂解剂，然后进行双向凝胶电泳，实现了对大量接触残基的高通量检测。我们通过在亚螺旋分辨率下重现一个研究得很好的模型RNA的未折叠和自然状态来验证这项技术，该模型RNA是四膜虫核酶的P4-P6结构域。然后，我们将MOHCA应用于最近发现的P4-P6 RNA的第三状态，该状态由高浓度的单价盐稳定，其偏序排除了传统结构确定技术的可能性。紧凑的非天然RNA状态的三维肖像揭示了天然三级接触的有序子集，与蛋白质的动态但在其他方面相似的熔融球状状态形成对比。由于MOHCA几乎适用于任何溶液状态，我们预计MOHCA将成为阐明大RNA分子和RNA/蛋白质复合体的许多功能结构的强大工具。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The biological behaviors of ribozymes, riboswitches, and numerous other functional RNA molecules are critically dependent on their tertiary folding and their ability to sample multiple functional states. The conformational heterogeneity and partially folded nature of most of these states has rendered their characterization by high-resolution structural approaches difficult or even intractable. Here we introduce a method to rapidly infer the tertiary helical arrangements of large RNA molecules in their native and non-native solution states. Multiplexed hydroxyl radical (.OH) cleavage analysis (MOHCA) enables the high-throughput detection of numerous pairs of contacting residues via random incorporation of radical cleavage agents followed by two-dimensional gel electrophoresis. We validated this technology by recapitulating the unfolded and native states of a well studied model RNA, the P4-P6 domain of the Tetrahymena ribozyme, at subhelical resolution. We then applied MOHCA to a recently discovered third state of the P4-P6 RNA that is stabilized by high concentrations of monovalent salt and whose partial order precludes conventional techniques for structure determination. The three-dimensional portrait of a compact, non-native RNA state reveals a well ordered subset of native tertiary contacts, in contrast to the dynamic but otherwise similar molten globule states of proteins. With its applicability to nearly any solution state, we expect MOHCA to be a powerful tool for illuminating the many functional structures of large RNA molecules and RNA/protein complexes.