Reaction of a Biscationic Distamycin-Ellipticine Hybrid Ligand with DNA. Mode and Sequence Specificity of Binding

Abstract

Molecular modeling of complexes between the octanucleotide d(CGATATCG)2 and either a monocationic or biscationic distamycin-ellipticine hybrid molecule predicted that the extra positive charge on the latter conjugate ligand should ensure tight fitting into the minor groove of the duplex without affecting intercalation of the ellipticine chromophore. To test this prediction, we have synthesized a biscationic compound Distel (2+) and investigated its interaction with DNA using various optical and gel electrophoresis techniques. Viscosity, fluorescence lifetime, and circular and linear dichroism measurements bear out the validity 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 ellipticine ring intercalated nearby. Linear dichroism experiments with a range of polynucleotides indicate that, unlike its monocationic homologue, the biscationic ligand engages in bidentate binding to AT sequences but not to GC sequences. Footprinting studies employing DNase I and methidiumpropyl-EDTA.FeII 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 with Distel (2+) are 4- to 10-fold lower than those required to produce comparable DNase I footprints with distamycin alone. Also, in accord with the energy-minimized model of the hybrid-oligonucleotide complex, chemical probing experiments using diethyl pyrocarbonate and osmium tetroxide reveal that the hybrid causes significant distortion of the DNA helix, explicable in terms of bending of the duplex toward the minor groove, which greatly enhances the reactivity toward probes in the major groove of the DNA. The experimental results help to identify the determinant factors, predominantly steric and electrostatic interactions, which shape the DNA-binding reaction. Thus, molecular modeling has correctly predicted the DNA-binding properties of a doubly charged ligand and shown that appending an auxiliary basic group onto the distamycin moiety was the right way to proceed in order to convert a nonspecific conjugate into a highly specific DNA reader.

The DNA Intercalating Alkaloid Cryptolepine Interferes with Topoisomerase II and Inhibits Primarily DNA Synthesis in B16 Melanoma Cells

“…Such biphasic evolution of the CD signal of the cryptolepine with increasing P / D ratios is characteristic of an excitonic coupling and fully consistent with an intercalative mode of binding to DNA. A similar variation of molar dichroism with the P / D ratio has been reported with other intercalating agents such as ethidium bromide (), acridine orange (), and ellipticine derivatives ( , ). The low value of the maximum Δε (Δε max = 7) also favors the intercalation mode ().…”

The Cell’s Nucleolus: an Emerging Target for Chemotherapeutic Intervention

“…Biophysical and biochemical experiments were performed to confirm the proposed hybrid binding mode of Distel, which distorts the structure of B-form DNA significantly. [52] Fluorescence microscopy was used to detect Distel and ellipticine in treated cells. [53] Both the conjugate and the intercalator itself preferentially accumulated in the nuclei of HeLa cells.…”

Sequence-Specific DNA Minor Groove Binders. Design and Synthesis of Netropsin and Distamycin Analogues

“…The calculation proved to be correct. Indeed, the substitution of the terminal formamido group of Distel(1+) for an aminopropionamido group charged at neutral pH [Distel(2+) (83)] was the correct way to proceed to convert a nonspecific conjugate into a highly AT-specific DNA reader (280). In contrast to Distel(1+), the interaction of Distel(2+) with DNA seems to be driven as much by the distamycin moiety as by the ellipticine residue.…”