Enzymatic and Physical Studies on the Triplex dTn∙dAn∙rUn

The triplex dTn∙dAn∙rUn was studied as a model system both enzymatically and physically with a view that a rational approach for attempting to isolate triplexes from in vivo situations might emerge. The triplex was characterized by mixing curves and by its equilibrium buoyant density. In 5 mM Na phosphate, pH 7.3, for KCl concentrations below 0.4 M the triplex dissociated into dTn∙dAn + rUn, dissociation being complete at about 0.3 M KCl. If MgCl2 replaced KCl a strongly cooperative dissociation occurred at 1 mM MgCl2. Whereas with alkaline titration of dTn∙dAn∙rUn, rUn was dissociated first followed by dTn∙dAn, acidic titration resulted in the whole triplex dissociating together. Both transcription and replication of dTn∙dAn were strongly inhibited by formation of a triplex. The DNA and RNA moieties in the triplex are somewhat protected against DNase I and pancreatic RNase degradation, when compared with duplex dTn∙dAn or rUn. Spermine binds equally well to dTn∙dAn and dTn∙dAn∙rUn which is consistent with spermine binding in the minor groove and RNA in the major groove of DNA.