title: 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.
authors.affiliation: Small Biosystems Lab, Departament de Física Fonamental, Facultat de Física, Universitat de Barcelona, 08028 Barcelona, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina, Instituto de Salud Carlos III, 28029 Madrid, Spain.
authors.forename: Joan
authors.initials: J
authors.lastname: Camunas-Soler
authors.affiliation: Small Biosystems Lab, Departament de Física Fonamental, Facultat de Física, Universitat de Barcelona, 08028 Barcelona, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina, Instituto de Salud Carlos III, 28029 Madrid, Spain.
authors.forename: Maria
authors.initials: M
authors.lastname: Manosas
authors.affiliation: Small Biosystems Lab, Departament de Física Fonamental, Facultat de Física, Universitat de Barcelona, 08028 Barcelona, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina, Instituto de Salud Carlos III, 28029 Madrid, Spain.
authors.forename: Silvia
authors.initials: S
authors.lastname: Frutos
authors.affiliation: Institute for Research in Biomedicine (IRB Barcelona), Barcelona Science Park, Baldiri Reixac 10-12, 08028 Barcelona, Spain.
authors.forename: Judit
authors.initials: J
authors.lastname: Tulla-Puche
authors.affiliation: Institute for Research in Biomedicine (IRB Barcelona), Barcelona Science Park, Baldiri Reixac 10-12, 08028 Barcelona, Spain.
authors.forename: Fernando
authors.initials: F
authors.lastname: Albericio
authors.affiliation: Small Biosystems Lab, Departament de Física Fonamental, Facultat de Física, Universitat de Barcelona, 08028 Barcelona, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina, Instituto de Salud Carlos III, 28029 Madrid, Spain fritort@gmail.com.
authors.forename: Felix
authors.initials: F
authors.lastname: Ritort
mesh_terms: D000465:Algorithms; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018983:DNA Footprinting / Q000379:methods*; D047630:Depsipeptides / Q000737:chemistry / Q000378:metabolism*; D004548:Elasticity; D007364:Intercalating Agents / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D052898:Optical Tweezers; D011485:Protein Binding; D013816:Thermodynamics; D013997:Time Factors
chemical_list: D047630:Depsipeptides; D007364:Intercalating Agents; D008024:Ligands; C109005:thiocoraline; D004247:DNA