Studies of transition metal complex-RNA interactions using mass spectrometry

RNA is a structurally diverse macromolecule that can have a variety of tertiary conformations. This structural diversity plays a role in many important interactions between RNA and other molecules. Our group is interested in examining RNA structure at a variety of levels of complexity through biophysical and chemical methods. Chemical probing and mass spectrometry with transition metal complexes allows us to elucidate which base and sugar residues are accessible to solvent, and this in turn gives information about the RNA structure. Here we will focus on the results of RNA interactions with metal polypyridal complexes and cobalt hexammine. The use of RNases can allow for the mapping of the metal complex binding sites.; Mass spectrometry is a technique that has the potential for rapid and high-throughput characterization of biomolecule non-covalent complexes. Chapter 1 provides an introduction into the study of RNA structure, and concludes with the use of mass spectrometry to characterize RNA-ligand interactions. The experimental protocols used are described in Chapter 2. Chapter 3 explores the interactions of Co(NH3)63+ with model RNA hairpins, and we observe a discrimination of binding between RNAs that contain GU versus AC wobble pairs. In addition, we observe subtle discrimination amongst RNAs with different levels of base modification. Chapter 4 explores the use of mass spectrometry to calculate dissociation constants. There is much debate whether or not mass spectrometry results are indicative of solution-phase interactions. We explore the use of different methods for dissociation constant calculation, and these are results are compared to a solution-phase study. Chapter 5 explores the interactions of five transition metal polypyridal complexes with the RNA hairpins. These metal complexes have been used as probes of nucleic acid structure, and our results indicate that binding does not always equal cleavage. Competition experiments between the polypyridal complexes and Co(NH 3)63+ indicate they may share similar binding sites. Chapter 6 contains the conclusions and future directions of this project.