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

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.

DNA intercalation optimized by two-step molecular lock mechanism

“…These findings confirm a previous prediction by Bahira et al 11. that combining two-step intercalation111329 with a molecular lock mechanism57 would result in higher DNA intercalation affinity than that observed for each of these properties alone. The first intercalative step of the binuclear complex Pi has an equilibrium constant of ~80 nM, which is close to the ~100 nM equilibrium constant reported in bulk for conventional DNA intercalation by an analogous mononuclear version of Pi (which has only one of the bulky threading units, enabling the exposed benzene ring to intercalate conventionally)30.…”

“…This plot illustrates the distinct nature of each type of intercalating system in terms of dissociation rates, governed by two different regimes of DNA structural fluctuations. Based on the available equilibrium and kinetic single molecule studies of DNA intercalators5711121314, for cyanine dyes (cyan symbols), all but YOYO are conventional intercalators, while polypeptide intercalators (red), and threading intercalators (purple) are unconventional intercalators. We consider YOYO to be an unconventional intercalator because its relatively long linker must be accommodated before its second moiety is fully intercalated, resulting in overall slower intercalation relative to conventional intercalators12.…”

The intrinsically disordered linker of E. coliSSB is critical for the release from single‐stranded DNA

“…Magnetic tweezers experiments are performed with a PicoTwist magnetic tweezers instrument (www.picotwist.com). DNA hairpins (480 bp) were prepared as described elsewhere 62 and tethered between a glass surface treated with anti-digoxigenin antibody (Roche) and a 1-lm streptavidin-coated Dynal magnetic bead (Invitrogen). The DNA hairpins were mechanically stretched by capturing the bead in a magnetic trap generated by a pair of permanent magnets.…”

Gene transcription in bursting: a unified mode for realizing accuracy and stochasticity

“…(6) Low-affinity DNA binding sites, unstable protein complexes, and DNA supercoiling can play crucial roles in regulating transcription. Investigating transcriptional dynamics necessitates both live imaging methods with high resolution (Skupsky et al, 2010;Suter et al, 2011;Evans et al, 2012;Friedman, Mumm, & Gelles, 2013;Gebhardt et al, 2013;Hocine et al, 2013;Kouno et al, 2013;Lickwar, Mueller, & Lieb, 2013;Yunger et al, 2013;Sidaway-Lee et al, 2014;Annibale & Gratton, 2015;Camunas-Soler et al, 2015;Gocheva et al, 2015;Roberts et al, 2015;Rybakova et al, 2015a;Corrigan et al, 2016;Tantale et al, 2016) and quantitative computer simulations with appropriate theories and models (Skupsky et al, 2010;Suter et al, 2011;Wang et al, 2012;Maina et al, 2014;Choubey, Kondev, & Sanchez, 2015;Stefan et al, 2015;Rybakova et al, 2015a,b;Corrigan et al, 2016;Tantale et al, 2016). Specifically, integrating diverse sets of data makes it possible to present a coherent dynamic picture of gene transcription in bacteria ).…”