Das Rhiju, Kudaravalli Madhuri, Jonikas Magdalena, Laederach Alain, Fong Robert, Schwans Jason P, Baker David, Piccirilli Joseph A, Altman Russ B, Herschlag Daniel
Departments of Biochemistry, Bioengineering, and Genetics, Stanford University, Stanford, CA 94305, USA.
Proc Natl Acad Sci U S A. 2008 Mar 18;105(11):4144-9. doi: 10.1073/pnas.0709032105. Epub 2008 Mar 5.
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.
核酶、核糖开关及众多其他功能性RNA分子的生物学行为，严重依赖于它们的三级折叠以及对多种功能状态的采样能力。大多数这些状态的构象异质性和部分折叠性质，使得通过高分辨率结构方法对其进行表征变得困难甚至难以处理。在此，我们介绍一种方法，可快速推断大RNA分子在其天然和非天然溶液状态下的三级螺旋排列。多重羟基自由基（·OH）切割分析（MOHCA）通过随机掺入自由基切割剂，随后进行二维凝胶电泳，能够高通量检测众多相互接触的残基对。我们通过在亚螺旋分辨率下重现一个经过充分研究的模型RNA（嗜热四膜虫核酶的P4-P6结构域）的未折叠和天然状态，验证了这项技术。然后，我们将MOHCA应用于最近发现的P4-P6 RNA的第三种状态，该状态由高浓度单价盐稳定，其部分有序性排除了传统的结构测定技术。一种紧凑的非天然RNA状态的三维图谱揭示了天然三级接触的一个有序子集，这与蛋白质动态但其他方面相似的熔球状态形成对比。鉴于其适用于几乎任何溶液状态，我们预计MOHCA将成为阐明大RNA分子和RNA/蛋白质复合物众多功能结构的有力工具。
