Intercalation-based single-molecule fluorescence assay to study DNA supercoil dynamics

Intercalation-based single-molecule fluorescence assay to study DNA supercoil dynamics

Ganji, M. (TU Delft BN/Elio Abbondanzieri Lab)
Kim, S.H. (TU Delft BN/Cees Dekker Lab)
van der Torre, J. (TU Delft BN/Technici en Analisten)
Abbondanzieri, E. (TU Delft BN/Elio Abbondanzieri Lab)
Dekker, C. (TU Delft BN/Cees Dekker Lab)

2016-07-13

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.

DNA supercoiling
fluorescence
intercalation
plectoneme
single-molecule

http://resolver.tudelft.nl/uuid:80982261-eaac-4e2f-8003-32ed67908211

https://doi.org/10.1021/acs.nanolett.6b02213

2017-07-13

1530-6984

Nano Letters: a journal dedicated to nanoscience and nanotechnology, 16 (7), 4699-4707

Accepted Author Manuscript

Institutional Repository

journal article

© 2016 M. Ganji, S.H. Kim, J. van der Torre, E. Abbondanzieri, C. Dekker