The goal of this research program is the design and application of transition metal complexes to target specific sites on DNA and RNA. Such designs form the basis for novel chemotherapeutics targeted to nucleic acids with predictable sequence -specificities and high affinities as well as providing new tools for biotechnology. We have focused on the construction of octahedral complexes of rhodium (III) containing the phenanthrenequinone diimine (phi) ligand. These complexes bind duplex DNA primarily through metallointercalation in the major groove, with site- selectivities being governed by non-covalent interaction of the ancillary ligands with site-selectivities being governed by non-covalent interaction of the ancillary ligands with the nucleic acid. With photoactivation, the complexes promote direct DNA strand scission at the site of intercalation, and this photocleavage serves to mark the site of binding. We intend (i) the construction of a new metallointercalators with predictable site- specificities and affinities, comparable to those of DNA-binding proteins, (ii) the detailed characterization of the structural basis for these site- specificities, (iii) the application of metal complexes to probe RNA and DNA structures, and (iv) their application to examine the site-specific inhibition of nucleic acid processes. Derivatives of Rh(en)2phi3+ will be prepared to pose an array of functional groups on the ancillary ligand matched to donors and acceptors in the DNA major groove. Using both shape- selection and direct readout, derivatives of delta-Rh(phen)2phi3+, functionalized on the periphery will be prepared to target 6 base pair DNA sites. In exploiting the sequence-dependent flexibilities of DNA, derivatives of delta-Rh(phen)2phi3+ will be prepared to target 5'-XTATAX-3' sequences. Two dimerization strategies, metal activated chelation and covalent dimerization through a disulfide linkage, will be explored to expand the length of sequences targeted (8-10 base pairs). Mixed ligand complexes containing three different chelating ligands, athe intercalator, dimerization ligand, and primary recognition ligand, will also be constructed. Binding site selectivities will be determined on DNA fragments and oligonucleotides based upon photoactivated cleavage. Base substitutions and sequence-specific unwinding assays will be used to probe specific interactions. Detailed structural information will be obtained in high resolution NMR experiments of rhodium-oligonucleotide complexes and using x-ray crystallography on iridium analogues. In applying metal complexes to probe nucleic acid structure, the hammerhead and Anabaena RNA ribozymes, HIV TAR RNA, and well-defined DNA mismatches will be examined. Importantly, with the development of complexes differing in levels of site- specificity, charge and solubility characteristics, as well as in rates of dissociation, we intend to test the efficacy of metal complexes in functioning to inhibit nucleic acid processes. Metal complexes will be examined as site-specific inhibitors of DNA and RNA polymerase. Analogous experiments will then be conducted in bacterial cells, where cellular uptake, photoactivated DNA cleavage, and site-specific inhibition of RNA synthesis will be assayed. This program, from design to functional application, represents an almost unique opportunity to develop systematically develop rational strategies for site-specific nucleic acid targeting by small molecules.
Showing the most recent 10 out of 71 publications
