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

Mahipal Ganji, Sung Hyun Kim, Jaco Van Der Torre, Elio Abbondanzieri, Cees Dekker

Research output: Contribution to journalArticleScientificpeer-review

21 Citations (Scopus)
131 Downloads (Pure)

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.

Original languageEnglish
Pages (from-to)4699-4707
Number of pages9
JournalNano Letters: a journal dedicated to nanoscience and nanotechnology
Volume16
Issue number7
DOIs
Publication statusPublished - 13 Jul 2016

Keywords

  • DNA supercoiling
  • fluorescence
  • intercalation
  • plectoneme
  • single-molecule

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