Probing RNA Metal Binding with Specifically Labeled RNA

金属在帮助塑造RNA复杂的三维结构和 控制着它对所有生命形式都至关重要的许多功能。我们发现~（15）N NMR是一种很好的非- 干扰方法，特别是敏感的金属的影响，是特定的网站结合到 特定的RNA基序通过15 N NMR监测金属滴定15 N特异性标记的寡核苷酸 可以提供关于RNA金属相互作用的独特信息。我们已经证明了特定的相互作用 其中Mg 2+在串联GA对上，K+在串联GU对上。我们的长期目标是制定详细的 理解RNAHig和相互作用。该提案的重点是RNA* 金属相互作用。的 下面描述的实验将使我们能够收集有关金属结合到几个小的 RNA基序，以及稍大的核酶片段。这里所述的具体目标将集中于 在合成各种15 N特异性标记的RNA时，它们在金属存在下的15 N NMR，如 以及其他结构和功能研究。许多人类疾病都涉及RNA的功能障碍， 的mRNA，其衰变途径，细菌生物膜的致病作用。更深入地了解 它们的行动方式对公共卫生至关重要。 1.三个系列特异性标记的含金属双链体的合成及~（15）N NMR研究 结合位点，以确定其基本特征。 2.几种特异性标记剪接体和Ⅱ类内含子的合成及~（15）N NMR研究 片段来探索对mRNA剪接过程至关重要的金属相互作用。 3.通过使用两个固体支持物的新方法合成特异性标记的加帽RNA。 4.通过15 N NMR多标记的加帽RNA和多标记的Ap 4A来探测嘌呤金属结合和 to identify识别the specific特定phosphate磷酸盐oxygen氧原子that also也interact相互作用with metals金属. 5.我们将合成特异性标记的c-di-GMP形式，以通过NMR（1H，13 C，31 P， 15 N）溶液中存在的金属依赖性络合物的完整结构;将使用15 N化学位移变化 监测核心的金属相互作用;并将研究结构之间的平衡， 金属和浓度，包括Mg 2 */K* 混合物的滴定和多胺的添加。

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.