8] Probing DNA structure with osmium tetroxide complexes in vitro

Abstract

Publisher Summary This chapter discusses the structure of DNA by probing osmium tetroxide (Os-py) complexes. Os-py has been applied to study local supercoil-stabilized DNA structures in vitro as well as parallel stranded DNA, DNA-drug interactions, etc. In local supercoil stabilized DNA structures, it reacts site specifically with bases in the formally single-stranded cruciform loop, triplex loop, displaced half of the homopyrimidine strand in triplex DNA, and with bases at the B-Z junctions. Substituting monodentate pyridine in Os-py reagent by bidentate 2,2'- bipyridine results in more stable DNA adducts and allows working with osmium tetroxide and the ligand at equimolar concentrations. Different ligands have been tested for their ability to modify site specifically the cruciform loop and B-Z junctions in supercoiled plasmids. It has been shown the OsO 4 complexes with tetramethylethylenediamine (TEMED) or bathophenanthrolinedisulfonic acid (bpds) can site specifically modify the above-mentioned structures at millimolar and sub-millimolar concentrations. The adducts resulting from DNA treatment with Os-bipy, Os-bpds, or Os-TEMED labilize the sugar-phosphate backbone in a way similar to the Os-py adducts, and their reaction sites in the polynucleotide chain can be detected by hot piperidine cleavage and sequencing.