STRUCTURAL INFERENCE OF NATIVE AND PARTIALLY FOLDED RNA BY CONTACT MAPPING

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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及其对多种功能状态进行采样的能力。大多数这些国家的构象异质性和部分折叠的性质，使其表征的高分辨率结构的方法困难，甚至棘手。在这里，我们介绍了一种方法来快速推断大RNA分子在其原生和非原生溶液状态的三级螺旋排列。多重羟基自由基（.OH）裂解分析（MOHCA）能够通过随机掺入自由基裂解剂，然后进行二维凝胶电泳，高通量检测许多对接触残基。我们验证了这种技术，通过重演展开和天然状态的一个很好的研究模型RNA，P4-P6结构域的四膜虫核酶，在亚螺旋分辨率。然后，我们将MOHCA应用于最近发现的P4-P6 RNA的第三种状态，该状态通过高浓度的单价盐稳定，并且其偏序排除了用于结构测定的常规技术。一个紧凑的，非天然的RNA状态的三维画像揭示了一个良好的本地三级接触有序的子集，相反，动态的，但在其他方面类似的熔融球状态的蛋白质。由于其适用于几乎任何溶液状态，我们希望MOHCA是一个强大的工具，用于阐明大RNA分子和RNA/蛋白质复合物的许多功能结构。

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.