Comparative Binding of Ethidium and Three Azido Analogs to Dinucleotides: Affinity and Intercalation Geometry

Geometrical and thermodynamic information has been obtained from theoretical analysis of both visible and 1 H‐NMR spectroscopic binding isotherms of ethidium and three photoactivable derivatives (8‐azido‐ethidium, 3‐azido‐ethidium and 3,8‐diazido‐ethidium) to self‐complementary ribodinucleosides. The following results have been obtained.1 Interaction with pyrimidine(3–5′)purine sequences is well accounted for by multicomponent equilibria involving self‐association of the dyes in oligomers, formation of 1:1 and 2:1 (nucleoside:dye) complexes. This model provided evidence for intercalation of all dyes, though with weaker affinity in the case of diazido‐ethidium (2 × 10 6 M −2 vs 6 × 10 7 M −2 ). Moreover 3‐azido‐ethidium was shown to intercalate into cytidylyl(3′–5′)guanosine (CpG) with its phenyl group lying in the major groove of the minihelix. This geometry is inverted with respect to that of all other compounds. It should be emphasized that visible and 1 H‐NMR techniques independently provided similar results (intercalation, affinity constants) therefore supporting this stepwise model. 2 Interaction of all dyes with purine(3′–5′)pyrimidine sequences is not intercalative, even at low temperature (4°C), but is well described by self‐association of the dyes and formation of 1:1 (nucleoside:dye) complexes. Regarding the reversible DNA intercalation process, these studies show that 8‐azido‐ethidium is the only photoactivatable derivative which behaves exactly as ethidium. Therefore 8‐azido‐ethidium can be used as a covalent probe to investigate the DNA‐related cytotoxic effects of ethidium.