Dynamic Structures of Large and Flexible RNAs

描述(由申请人提供)：非编码RNA(NcRNAs)已经成为细胞机制的丰富关键元素，越来越多地成为药物发现努力的目标。NcRNAs的高分辨率三维结构测定为在原子水平上理解其功能机制和实施基于结构的药物发现方法提供了基础。然而，RNA的独特特性仍然给用X射线结晶学和核磁共振光谱进行高分辨率结构测定带来了巨大的挑战。许多ncRNA的全球结构高度灵活，因此可以抵抗结晶。许多ncRNA的分子量超过了核磁共振应用的极限(100个核苷酸)。这使得有必要从更大的背景中去除适合用X射线和核磁共振表征的单个RNA结构域。这种分而治之的策略经常受到质疑，因为有实验表明，结构域具有重叠的功能，并有可能相互关联形成更高级别的结构。相比之下，计算和实验方法的进步使人们能够确定越来越大和复杂的RNA的二级结构。最近，我们发现拓扑约束提供了RNA二级结构与3D全局和动态适应之间缺失的链接。在这个建议中，我们建议开发新的计算方法，利用这些新建立的拓扑约束来定义仅基于二级结构的RNA结构的全局特征。通过将这些全局限制与足迹数据和一种新的用于识别三级基序的核磁共振化学位移指纹策略相结合，我们建议开发一种新的范式来确定大型柔性RNA的三维结构组织。该方法将通过确定鸟嘌呤敏感核糖开关适配子结构域在其稳定配体结合形式下的3D构象来验证，并随后用于表征该结构域在其无配体形式下更灵活的构象。结果将被用来检验这样的假设，即在唯一的三向连接中编码的拓扑约束，而不是长距离的环-环三级相互作用，定义了适配子结构域的全局结构，从而产生了为自适应配体结合而优化的空间调谐动力学。 公共卫生相关性：拟议的研究将测试开发一种新的计算核磁共振方法的可行性，以确定在溶液条件下非常大的RNA的结构。这种方法不需要结晶或尖锐的核磁共振谱，可以应用于大到1000个核苷酸的RNA结构，并有望比传统方法提高一到两个数量级的产量。这种确定RNA结构的新范式将把几种目前由于X射线和核磁共振的限制而不可能进行的研究模式带入应用领域。

DESCRIPTION (provided by applicant): Non-coding RNAs (ncRNAs) have emerged as abundant critical elements of the cellular machinery that are increasingly being targeted in drug discovery efforts. High-resolution 3D structure determination of ncRNAs provides the basis for understanding their functional mechanisms at the atomic level and for implementing structure-based approaches for drug discovery. However, RNA's unique characteristics continue to pose significant challenges to high-resolution structure determination by X-ray crystallography and NMR spectroscopy. The global structures of many ncRNAs are highly flexible and can therefore resist crystallization. Many ncRNAs have molecular weights that exceed the NMR limit of application (<100 nucleotides). This has made it necessary to excise individual RNA domains, suitable for characterization by X-ray and NMR, from their much larger context. This 'divide and conquer strategy' is often called into question by experiments showing that domains have overlapping functions and potentially associate to form higher order structures. By contrast, advances in computational and experimental methods are allowing determination of secondary structures for increasingly large and complex RNAs. Recently, we showed that topological constraints provide the missing link between RNA secondary structure and 3D global and dynamic adaptation. In this proposal, we propose to develop new computational methods that exploit these newly founded topological constraints to define global features of RNA structure based on secondary structure alone. By combining these global constraints with footprinting data and a new NMR chemical shift fingerprinting strategy for identifying tertiary motifs, we propose to develop a new paradigm for determining the 3D structural organization of large flexible RNAs. The methodology will be validated by determining the 3D conformation of the guanine sensing riboswitch aptamer domain in its stable ligand bound form and subsequently used to characterize the more flexible conformation of the domain in its ligand free form. Results will be used to test the hypothesis that topological constraints encoded in the unique three-way junction, and not long range loop-loop tertiary interactions, define the global structure of the aptamer domain giving rise to spatially tuned dynamics that are optimized for adaptive ligand binding. PUBLIC HEALTH RELEVANCE: The proposed research will test the feasibility of developing a novel computational NMR method for determining structures of very large RNAs under solution conditions. The method obviates the need for crystallization or sharp NMR spectra, can be applied to RNA structures as large as 1000 nucleotides, and is expected to increase throughput over conventional methods by one-to-two orders of magnitude. This new paradigm for RNA structure determination will bring into the realm of application several modes of investigation that are currently impossible due to limitations in X-ray and NMR.