17] Probing of DNA structure in cells with osmium tetroxide-2,2'-bipyridine

Abstract

Publisher Summary This chapter discusses the probing of DNA structure in cells using osmium tetroxide-2,2´-bipyridine (Os-bipy). The hypersensitivity of local open DNA structures toward Os-bipy is exploited for analysis in bacterial cells. Cells are treated with a millimolar concentration of Os-bipy for a short time, the excess reagent is removed, plasmid DNA is isolated by the boiling method, and osmium binding sites are determined. Direct probing of DNA in cells with Os-bipy at neutral pH showed no triplex formation in the homopurine–homopyrimidine segment of the intracellular pEJ4 plasmid. If E. coli cells were preincubated and treated with Os-bipy at pH 4.5 or 5.0, a modification pattern characteristic of H triplexes was obtained. Shifting the intracellular superhelical density to more negative values resulted in a stronger site-specific modification. In another experiment, attempt was made to search for triplex DNA in cells with the Os-bipy probe. The presence of triplex H-DNA in vitro is manifested by a strong modification in the center of the (C-T) n sequence and a weaker modification at the triplex boundary. To probe the homopurine strand in the triplex structure in vitro , diethyl pyrocarbonate or glyoxal was applied. So far no reports on the application of these or other single-strand-selective probes reacting with purines to studies of DNA structure in cells have been published. Thus, information about triplex structure in cells obtained by direct chemical probing is limited to the pyrimidine strand.