Single-molecule kinetics and footprinting of DNA bis-intercalation: the paradigmatic case of Thiocoraline.

Joan Camunas-Soler,Maria Manosas,Fernando Albericio,Felix Ritort,Silvia Frutos,Judit Tulla-Puche

doi:10.1093/nar/gkv087

Abstract

DNA bis-intercalators are widely used in molecular biology with applications ranging from DNA imaging to anticancer pharmacology. Two fundamental aspects of these ligands are the lifetime of the bis-intercalated complexes and their sequence selectivity. Here, we perform single-molecule optical tweezers experiments with the peptide Thiocoraline showing, for the first time, that bis-intercalation is driven by a very slow off-rate that steeply decreases with applied force. This feature reveals the existence of a long-lived (minutes) mono-intercalated intermediate that contributes to the extremely long lifetime of the complex (hours). We further exploit this particularly slow kinetics to determine the thermodynamics of binding and persistence length of bis-intercalated DNA for a given fraction of bound ligand, a measurement inaccessible in previous studies of faster intercalating agents. We also develop a novel single-molecule footprinting technique based on DNA unzipping and determine the preferred binding sites of Thiocoraline with one base-pair resolution. This fast and radiolabelling-free footprinting technique provides direct access to the binding sites of small ligands to nucleic acids without the need of cleavage agents. Overall, our results provide new insights into the binding pathway of bis-intercalators and the reported selectivity might be of relevance for this and other anticancer drugs interfering with DNA replication and transcription in carcinogenic cell lines.

Highlights

Small DNA binders have become powerful tools in biomedicine due to their ability to interact with nucleic acids at specific sites and modulate key processes such as repair, transcription and replication [1,2,3,4]
DNA stretching experiments performed at varying pulling speeds show a hysteresis that increases with decreasing pulling speed, whereas hysteresis is not observed at the fastest speed of 1.5 ␮m/s (Figure 2a, red, blue, green and Supplementary Movie SV1)
The observation of hysteresis in DNA pulling curves was previously reported for the bisintercalator dye YOYO-1 [23], and is caused by the fact that the affinity constant and kinetic rates are force-dependent

Summary

Introduction

Small DNA binders have become powerful tools in biomedicine due to their ability to interact with nucleic acids at specific sites and modulate key processes such as repair, transcription and replication [1,2,3,4]. DNA intercalators and bis-intercalators have received continued interest during the last decades due to their particular properties that range from being excellent DNA staining probes (e.g. ethidium, YOYO) to effective anti-proliferative drugs (e.g. doxorubicin, echinomycin) [4,5]. From a structural point of view, intercalators are small planar compounds that bind nucleic acids by sliding between two adjacent base pairs and bringing them apart. This process unwinds the DNA double helix and increases its contour length by ∼3.4 Aper intercalator [6]. The compound is able to ‘clamp’ DNA by sandwiching two consecutive base pairs between its aromatic rings, distorting and elongating DNA a length twice that of a mono-intercalator

Methods

Results

Conclusion