Probing RNA Metal Binding with Specifically Labeled RNA

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.