title: Intercalation-Based Single-Molecule Fluorescence Assay To Study DNA Supercoil Dynamics.
abstract: DNA supercoiling crucially affects cellular processes such as DNA replication, gene expression, and chromatin organization. However, mechanistic understanding of DNA supercoiling and the related DNA-processing enzymes has remained limited, mainly due to the lack of convenient experimental tools to probe these phenomena. Here, we report a novel high-throughput single-molecule assay for real-time visualization of supercoiled DNA molecules, named ISD (Intercalation-induced Supercoiling of DNA). We use an intercalating dye to induce supercoiling of surface-attached DNA molecules as well as to visualize coiled-loop structures (i.e., plectonemes) formed on DNA. The technique is solely based on epifluorescence microscopy and requires no mechanical manipulation of the DNA molecules. This new assay allows to track positions and sizes of individual plectonemes and characterize their position-dependent dynamics such as nucleation, termination, and diffusion. We describe the ISD technique and demonstrate its potential by establishing that plectonemes are pinned to a local 10-nucleotide long mispaired sequence along a double-stranded DNA molecule.
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology , Van der Maasweg 9, Delft, 2629HZ, The Netherlands.
authors.forename: Mahipal
authors.initials: M
authors.lastname: Ganji
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology , Van der Maasweg 9, Delft, 2629HZ, The Netherlands.
authors.forename: Sung Hyun
authors.initials: SH
authors.lastname: Kim
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology , Van der Maasweg 9, Delft, 2629HZ, The Netherlands.
authors.forename: Jaco
authors.initials: J
authors.lastname: van der Torre
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology , Van der Maasweg 9, Delft, 2629HZ, The Netherlands.
authors.forename: Elio
authors.initials: E
authors.lastname: Abbondanzieri
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology , Van der Maasweg 9, Delft, 2629HZ, The Netherlands.
authors.forename: Cees
authors.initials: C
authors.lastname: Dekker
mesh_terms: D004278:DNA, Superhelical / Q000737:chemistry*; D004058:Diffusion; D005453:Fluorescence; D009690:Nucleic Acid Conformation
chemical_list: D004278:DNA, Superhelical
keywords: DNA supercoiling; fluorescence; intercalation; plectoneme; single-molecule