We will use 15N NMR of 15N/13C specifically labeled RNA fragments to identify and evaluate metal binding sites that involve base nitrogens. The major focus will be the RNA systems described below, for which we will prepare specifically labeled fragments to study binding of a wide range of metal cations. 1. Several small motifs containing various combinations of GU and GA pairs, some with other non-Watson-Crick pairs and/or bulged nucleosides, have been implicated as specific metal binding sites. The relatively small size of these isolated motifs makes them particularly suited to multiple experiments with different metal ions. 2. The hammerhead and hairpin are relatively small ribozymes (40 - 50 nt) that are accessible by chemical synthesis. They require metal cations to function, and show much promise for therapeutic and diagnostic applications. 3. The group I and II introns are much larger (400 - 600 nt) and require metal cations to form active tertiary structures as well as for catalysis. We will synthesize and study these systems in subunits of increasing size, with the ultimate goal of ligating our labeled fragments to much longer RNA. 4. Ribosomal RNA plays a central role in protein synthesis in all living systems, and requires metal cations to do so. A particular focus will be three-helix junctions which often play essential roles by forming the necessary ribosomal architecture and binding to metals and proteins.
