The nuclease activity of 1,10-phenanthroline-Cu (OP-Cu) nicks RNA and DNA by oxidative attack on the ribose moiety. The free coordination complex is useful for investigating nucleic acid structure/function relationships. Linked to carrier ligands, OP-Cu allows synthesis of nucleases with designed specificities. Targeted nuclease activities for genomic mapping will be developed which involve a) genetically engineered derivatives of the bacterial repressor cro protein; and b) R-loop formation using RNAs modified with 1,10-phenanthroline. Both methods will be capable of scission at any chosen marker gene sequence. The synthesis of gene specific inhibitors of transcription will be achieved by linking OP-Cu to oligonucleotides a) complementary to single stranded regions of transcription initiation sites; or b) capable of formal, triple helices with polypurine/polypyrimidine tracts which flank eucaryotic genes. The chemical mechanism and secondary structure specificity of the RNase activity of OP-Cu will be determined. Along with light-activated uranyl acetate, its potential as a footprinting reagent for RNA-protein interactions will be explored. Triple helix formation between double-stranded RNA and deoxyoligonucleotides chemically derivatized with OP-Cu will be investigated. By analyzing the scission pattern of the target RNA, the stringency, polarity, and stability of triple helix formation will be determined. This interaction provides a new approach for the design of antisense reagents to block gene expression at the level of RNA.
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