Monitoring the Intercalation of Acridine Orange (AO) Molecules into 6-Methyl isoxanthopterin (6-MI)-Labeled DNA by Circular Dichroism and Single-Molecule FRET

Abstract

Intercalation into duplex DNA occurs when a planar and aromatic small molecule ligand binds between adjacent base pairs of duplex DNA, forming a sandwich-like structure. This insertion significantly perturbs the double-stranded DNA structure. As a result, such intercalation may also perturb DNA-structure-dependent biological processes, such as DNA transcription, replication and repair. Based on such functional perturbations, extensive studies have been performed that focus on the development of intercalative drugs with possible clinical potential. Among the intercalators that have been examined, acridine derivatives have been much investigated. However, more work is required to develop effective drugs with the desired selectivities and reduced side effects. In an attempt to provide more incisive molecular approaches to understanding intercalation mechanisms, we have shown that 6-methyl isoxanthopterin (6-MI) and acridine orange (AO) can act as a donor-acceptor Forster Resonance Energy Transfer (FRET) pair. 6-MI is a fluorescent guanine analogue that can serve as a local reporter of DNA conformational changes, while acridine orange is a well-studied DNA intercalator. We show how these two optical probes can be paired to monitor local AO-DNA intercalation dynamics with multi-millisecond time resolution. Applying single-molecule FRET and linear dichroism methods developed in our group, we are using these approaches to study the roles of intermediate conformational states during the intercalation process.