Probing RNA Metal Binding with Specifically Labeled RNA

描述(申请人提供)：金属在帮助塑造RNA错综复杂的三维结构和控制其对所有生命形式至关重要的许多功能方面发挥着关键作用。我们发现15N核磁共振是一种很好的非扰动方法，它对特定位置与某些RNA基序结合的金属的影响特别敏感。用15N核磁共振监测的金属滴定15N标记的寡核苷酸可以提供关于RNA与金属相互作用的独特信息。我们已经证明了在串联GA对上与Mg2+以及在串联Gu对上与K+的特定相互作用。我们的长期目标是对RNA-配体相互作用有一个详细的了解。这项提议的重点是RNA-金属相互作用。下面描述的实验将使我们能够收集关于金属与几个小的RNA基序以及一些较大的核酶片段结合的准确信息。这里描述的具体目标将集中在合成各种特殊标记的15N RNA，它们在金属存在下的15N核磁共振，以及其他结构和功能研究。许多人类疾病涉及RNA功能障碍，从mRNA的剪接，到它的衰退途径，再到细菌生物膜的致病效应。更深入地了解它们的行动模式对公共卫生至关重要。1.合成了三个含有金属结合位点的小分子标记双链，并用15N核磁共振对其进行了研究，确定了它们的基本特征。2.合成并利用~(15)N核磁共振仪对剪接体和第II类内含子片段进行研究，以探索在mRNA剪接过程中起关键作用的金属相互作用。3.用两种固体载体合成特异性标记的帽状RNA。4.用~(15)N核磁共振多标记封端RNA和多标记Ap4A研究嘌呤与金属的结合，并鉴定与金属相互作用的特异性磷酸氧原子。5.我们将合成c-di-GMP的特殊标记形式，通过核磁共振(1H，13C，31P和15N)确定溶液中存在的金属依赖络合物的完整结构；将使用15N化学位移变化来监测核心的金属相互作用；并将研究结构之间的平衡作为金属和浓度的函数，包括镁/钾混合物的滴定和多胺的添加。

DESCRIPTION (provided by applicant): Metals play a crucial role in helping to shape the intricate three-dimensional structure of RNA and to control its many functions that are key to all forms of life. We have found that 15N NMR is an excellent non-perturbing method that is particularly sensitive to the effects of metals that are site-specifically bound to certain RNA motifs. Titration of 15N specifically labeled oligonucleotides with metals monitored by 15N NMR can provide unique information about RNA-metal interactions. We have demonstrated specific interactions with Mg2+ at a tandem GA pair and K+ at a tandem GU pair. Our long term goal is to develop a detailed understanding of RNA-ligand interactions. This proposal is focused on RNA-metal interactions. The experiments described below will allow us to gather precise information about metal binding to several small RNA motifs, as well as to somewhat larger ribozyme fragments. The specific aims described here will focus on the synthesis of a variety of 15N specifically labeled RNAs, their 15N NMR in the presence of metals, as well as other structural and functional studies. Many human diseases involve RNA malfunction, from splicing of mRNA, to its decay pathways, to the pathogenic effects of bacterial biofilms. A deeper understanding of their modes of action is critically important for public health. 1. Synthesize and study by 15N NMR three series of small specifically labeled duplexes containing metal binding sites to identify their essential features. 2. Synthesize and study by 15N NMR several specifically labeled spliceosome and group II intron fragments to explore metal interactions that are critical for the mRNA splicing process. 3. Synthesize specifically labeled capped RNA by a novel approach using two solid supports. 4. Study by 15N NMR multilabeled capped RNAs and multilabeled Ap4A to probe purine metal binding and to identify the specific phosphate oxygen atoms that also interact with metals. 5. We will synthesize specifically labeled versions of c-di-GMP to determine by NMR (1H, 13C, 31P, and 15N) full structures of the metal dependent complexes present in solution; will use 15N chemical shift changes to monitor metal interactions at the core; and will study the equilibrium among the structures as a function of metal and concentration, including titrations ofMg2+/K+ mixtures and addition of polyamines.