REACTION OF A BISCATIONIC DISTAMYCIN-ELLIPTICINE HYBRID LIGAND WITH DNA - MODE AND SEQUENCE SPECIFICITY OF BINDING

Molecular modeling of complexes between the octanucleotide d(CGATATCG) (2) and either a monocationic or biscationic distamycin-ellipticine hy brid molecule predicted that the extra positive charge on the latter c onjugate Ligand should ensure tight fitting into the minor groove of t he duplex without affecting intercalation of the ellipticine chromopho re. To test this prediction, we have synthesized a biscationic compoun d Distel (2+) and investigated its interaction with DNA using various optical and gel electrophoresis techniques. Viscosity, fluorescence Li fetime, and circular and linear dichroism measurements bear out the va lidity of the calculations and show that Distel (2+) does indeed come to lie with its distamycin moiety in the minor groove of DNA and its e llipticine ring intercalated nearby. Linear dichroism experiments with a range of polynucleotides indicate that, unlike its monocationic hom ologue, the biscationic ligand engages in bidentate binding to AT sequ ences but not to GC sequences. Footprinting studies employing DNase I and methidiumpropyl-EDTA.Fe-II as DNA cleaving agents reveal that the biscationic hybrid is notably selective for AT-rich sequences in DNA. The concentrations required to detect a clear footprint at AT sites wi th Distel (2+) are 4- to 10-fold lower than those required to produce comparable DNase I footprints with distamycin alone. Also, in accord w ith the energy-minimized model of the hybrid-oligonucleotide complex, chemical probing experiments using diethyl pyrocarbonate and osmium te troxide reveal that the hybrid causes significant distortion of the DN A helix, explicable in terms of bending of the duplex toward the minor groove, which greatly enhances the reactivity toward probes in the ma jor groove of the DNA. The experimental results help to identify the d eterminant factors, predominantly steric and electrostatic interaction s, which shape the DNA-binding reaction. Thus, molecular modeling has correctly predicted the DNA-binding properties of a doubly charged lig and and shown that appending an auxiliary basic group onto the distamy cin moiety was the right way to proceed in order to convert a nonspeci fic conjugate into a highly specific DNA reader.