1,10-Phenanthroline-cuprous complex, (OP)2Cu+, with hydrogen peroxide as a coreactant, is an artificial nuclease which cleaves DNA and RNA by an oxidative pathway at physiological pH's and temperatures. Since the reaction proceeds via a nucleic acid-(OP)2Cu+ complex, the nuclease is dependent on the three-dimensional structure of the nucleic acid. For example, A-DNA is cleaved at 1/7th the rate of B-DNA; single stranded and Z-DNA's are resistant to cleavage. Since (OP)2Cu+ is small and structurally compact, its nuclease activity should be governed by the local DNA conformation and not any interaction with bases distal to the cleavage site. Therefore, (OP)2Cu+ may provide a unique method to map the local variations in DNA structure that may be functionally important in the expression of genetic information. The first goal of this grant is to identify the structural determinants which control DNA scission by studying the digestion patterns of oligonucleotides of known three dimensional structure. Then, the cleavage of different bacterial operons will determine if common structural features are detected by (OP)2Cu+. Conformational changes introduced in these regions by mutations and ligand binding will be analyzed. The scope of the artificial nuclease activity will be enhanced by attaching 1,10-phenanthroline to specific ligands of DNA and converting these carriers into site specific nucleases. For example, OP, bound to antibodies directed against Z-DNA, should specifically nick this left-handed structure. Finally, the efficiency of the intracellular action of this artificial nuclease will be explored with the goals of examining DNA structure in situ and designing cell-type specific cytotoxic agents.
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