Kennedy Scott D
Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, NY, 14642, USA.
Methods Mol Biol. 2016;1490:253-64. doi: 10.1007/978-1-4939-6433-8_16.
Knowledge of RNA secondary structure is often sufficient to identify relationships between the structure of RNA and processing pathways, and the design of therapeutics. Nuclear magnetic resonance (NMR) can identify types of nucleotide base pairs and the sequence, thus limiting possible secondary structures. Because NMR experiments, like chemical mapping, are performed in solution, not in single crystals, experiments can be initiated as soon as the biomolecule is expressed and purified. This chapter summarizes NMR methods that permit rapid identification of RNA secondary structure, information that can be used as supplements to chemical mapping, and/or as preliminary steps required for 3D structure determination. The primary aim is to provide guidelines to enable a researcher with minimal knowledge of NMR to quickly extract secondary structure information from basic datasets. Instrumental and sample considerations that can maximize data quality are discussed along with some details for optimal data acquisition and processing parameters. Approaches for identifying base pair types in both unlabeled and isotopically labeled RNA are covered. Common problems, such as missing signals and overlaps, and approaches to address them are considered. Programs under development for merging NMR data with structure prediction algorithms are briefly discussed.
了解RNA二级结构通常足以确定RNA结构与加工途径以及治疗方法设计之间的关系。核磁共振（NMR）可以识别核苷酸碱基对的类型和序列，从而限制可能的二级结构。由于NMR实验与化学图谱分析一样，是在溶液中而非单晶中进行的，因此一旦生物分子表达并纯化，即可开始实验。本章总结了NMR方法，这些方法可快速识别RNA二级结构，这些信息可作为化学图谱分析的补充，和/或作为确定三维结构所需的初步步骤。主要目的是提供指导方针，使对NMR了解最少的研究人员能够从基本数据集中快速提取二级结构信息。讨论了可使数据质量最大化的仪器和样品注意事项，以及最佳数据采集和处理参数的一些细节。涵盖了识别未标记和同位素标记RNA中碱基对类型的方法。考虑了常见问题，如信号缺失和重叠，以及解决这些问题的方法。简要讨论了正在开发的将NMR数据与结构预测算法合并的程序。
