Life Science Division, Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
RNA. 2010 Mar;16(3):638-46. doi: 10.1261/rna.1946310. Epub 2010 Jan 27.
Defining the shape, conformation, or assembly state of an RNA in solution often requires multiple investigative tools ranging from nucleotide analog interference mapping to X-ray crystallography. A key addition to this toolbox is small-angle X-ray scattering (SAXS). SAXS provides direct structural information regarding the size, shape, and flexibility of the particle in solution and has proven powerful for analyses of RNA structures with minimal requirements for sample concentration and volumes. In principle, SAXS can provide reliable data on small and large RNA molecules. In practice, SAXS investigations of RNA samples can show inconsistencies that suggest limitations in the SAXS experimental analyses or problems with the samples. Here, we show through investigations on the SAM-I riboswitch, the Group I intron P4-P6 domain, 30S ribosomal subunit from Sulfolobus solfataricus (30S), brome mosaic virus tRNA-like structure (BMV TLS), Thermotoga maritima asd lysine riboswitch, the recombinant tRNA(val), and yeast tRNA(phe) that many problems with SAXS experiments on RNA samples derive from heterogeneity of the folded RNA. Furthermore, we propose and test a general approach to reducing these sample limitations for accurate SAXS analyses of RNA. Together our method and results show that SAXS with synchrotron radiation has great potential to provide accurate RNA shapes, conformations, and assembly states in solution that inform RNA biological functions in fundamental ways.
定义 RNA 在溶液中的形状、构象或组装状态通常需要多种研究工具，包括核苷酸类似物干扰作图到 X 射线晶体学。这个工具包的一个关键补充是小角 X 射线散射（SAXS）。SAXS 提供了关于溶液中颗粒的大小、形状和灵活性的直接结构信息，并且已被证明对于最小化样品浓度和体积要求的 RNA 结构分析非常有效。原则上，SAXS 可以提供关于小 RNA 分子和大 RNA 分子的可靠数据。实际上，RNA 样品的 SAXS 研究可能会显示出不一致性，这表明 SAXS 实验分析存在局限性或样品存在问题。在这里，我们通过对 SAM-I 核糖开关、I 组内含子 P4-P6 结构域、来自 Sulfolobus solfataricus 的 30S 核糖体亚基 (30S)、木薯花叶病毒 tRNA 样结构 (BMV TLS)、Thermotoga maritima asd 赖氨酸核糖开关、重组 tRNA(val)和酵母 tRNA(phe)的研究表明，许多 RNA 样品 SAXS 实验中的问题源于折叠 RNA 的异质性。此外，我们提出并测试了一种通用方法，以减少这些样品限制，从而实现 RNA 的准确 SAXS 分析。我们的方法和结果表明，具有同步辐射的 SAXS 具有很大的潜力，可以提供溶液中 RNA 的准确形状、构象和组装状态，从而以基本的方式告知 RNA 的生物学功能。
