The chemical nuclease activity of 1,10-phenanthroline-copper has led to a) the Conversion of DNA binding proteins into site specific nucleases; b) the discovery of transcription inhibitors which bind to the open- complex formed at the initiation of RNA synthesis; and c) a reagent to analyze the secondary structure/ligand binding of RNA. a) The homeodomain motif of Drosophila engrailed and the leucine zipper motif of Epstein-Barr zebra will be converted into site specific scission reagents by linking 1,10-phenanthroline to cysteines introduced by site directed mutagenesis. Binding sites for the factor for inversion stimulation (fis) protein and Trp repressor, in E. coli, and the zebra protein, in Epstein-Barr virus, will be identified by their sites of scission using a ligation mediated PCR strategy. The mechanism of scission will be investigated using deoxyribose-deuterated nucleotides to identify the initial site of oxidative attack by the chemical nuclease. Since the Trp repressor chimera cleaves its wild-type sites with 100% efficiency, a family of rare cutters based on the Trp repressor structural scaffold will be prepared by mutagenizing the DNA binding domain of Trp repressor E49C-OP. b) The open complexes formed with promoters and RNA polymerase will be used as the target for two distinct types of transcription inhibitors: i) the 2:1 2,9-dimethyl-1,10-phenanthroline-cuprous complex ((NC)2Cu+); and ii) oligonucleotides complementary to the single-stranded DNA of the open complex. i) The interaction of a tetrahedral ligand like (NC)2Cu+ to single stranded DNA either in an enzyme active site or free in solution has no precedent. Therefore, the binding of(NC)2Cu+ to both procaryotic and eucaryotic open complexes will be explored using stereochemistry, site directed mutagenesis, in vitro selection of mismatched DNAs and affinity labelling. ii) In contrast to (NC)2Cu+ which is a global transcription inhibitor, ribooligonucleotides and abiological analogs (e.g. 2-OMe oligonucleotides) are capable of gene specific inhibition and could provide a new approach for the design of antiviral and antibacterial agents at the DNA level. The length, target, and backbone structure of the most inhibitory oligonucleotides will be determined. Derivatized with reactive groups, they will serve as affinity ligands to modify neighboring components of transcription complexes. c) The scission of RNA by the 2:1 1,10-phenanthroline-cuprous complex ((OP2)Cu+)) will be investigated with RNAs which have been selected in vitro for their binding affinity for the isosteric (NC)2Cu+. The binding specificity of these RNAs for (NC.)2Cu+ and its derivatives will be determined. Cleavage of ribose-deuterated RNAs with (OP2)Cu+ will suggest chemical mechanisms for the oxidative scission reaction.
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