Probing RNA Metal Binding with Specifically Labeled RNA

金属在帮助形成复杂的RNA三维结构和 控制它的许多功能，这些功能是所有生命形式的关键。我们发现15N核磁共振是一种很好的非核磁共振 一种对特定部位结合的金属的影响特别敏感的扰动方法 某些RNA基序。~(15)N核磁共振监测金属滴定~(15)N标记寡核苷酸 可以提供关于RNA金属相互作用的独特信息。我们已经演示了特定的相互作用 其中在串联GA对处具有镁离子，并且在串联GU对处具有K+。我们的长期目标是制定一个详细的 了解RNAHigand相互作用。这项提议的重点是RNA*金属相互作用。这个 下面描述的实验将允许我们收集关于金属结合到几个小的 RNA基序，以及更大的核酶片段。这里描述的具体目标将集中在 关于各种特殊标记的~(15)N-RNA的合成及其在金属存在下的~(15)N-核磁共振，AS 以及其他结构和功能研究。许多人类疾病都与RNA功能障碍有关，这是由于剪接 到其腐烂途径，到细菌生物膜的致病作用。更深入地了解 他们的行动模式对公共卫生至关重要。 1.三个系列含金属特殊标记小分子双链的合成及~(15)N核磁共振研究 结合位点以确定其基本特征。 2.几种特异标记剪接体和第二组内含子的合成及~(15)N核磁共振研究 片段，以探索在mRNA剪接过程中至关重要的金属相互作用。 3.用两种固体载体合成特异性标记的帽状RNA。 4.~(15)N核磁共振多标记封端RNA和多标记Ap4A对嘌呤金属结合的研究 以确定也与金属相互作用的特定磷酸盐氧原子。 5.我们将合成特殊标记的FC-di-GMP版本，以通过核磁共振(1H，13C，31P，和 15N)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 监测核心的金属相互作用；并将研究作为以下函数的结构之间的平衡 金属和浓度，包括滴定的镁*/钾*混合物和添加的多胺。

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 RNAHigandinteractions. 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 NMRmultilabeled capped RNAs and multilabeledAp4A to probe purine metal binding and to identify the specificphosphate oxygen atoms that also interact with metals. 5. We will synthesize specifically labeled versions ofc-di-GMP to determine by NMR (1H, 13C, 31P, and 15N) full structures of the metal dependent complexes present in solution; will use15Nchemical 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.