The fluorescence properties and binding mechanism of SYTOX green, a bright, low photo-damage DNA intercalating agent

Eur Biophys J. 2015 Jul;44(5):337-48. doi: 10.1007/s00249-015-1027-8. Epub 2015 May 31.


DNA intercalators are widely used in cancer therapeutics, to probe protein-DNA interactions and to investigate the statistical-mechanical properties of DNA. Here, we employ single-molecule fluorescence microscopy, magnetic tweezers, and ensemble-binding assays to investigate the fluorescence properties and binding mechanism of SYTOX green, a DNA labeling dye previously used for staining dead cells and becoming of common use for single-molecule methodologies. Specifically, we show that SYTOX green presents several advantages with respect to other dyes: (1) binds DNA rapidly and with high affinity; (2) has a good signal-to-noise ratio even at low concentrations; (3) exhibits a low photobleaching rate; and (4) induces lower light-induced DNA degradation. Finally, we show that SYTOX green is a DNA intercalator that binds DNA cooperatively with a binding site of 3.5 bp, increasing the DNA length upon binding by 43%, while not affecting its mechanical properties.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA / chemistry
  • DNA / drug effects*
  • DNA Damage
  • Fluorescence
  • Fluorescent Dyes / chemistry*
  • Fluorescent Dyes / pharmacology
  • Mutagens / chemistry*
  • Mutagens / pharmacology
  • Organic Chemicals / chemistry
  • Organic Chemicals / pharmacology
  • Ultraviolet Rays


  • Fluorescent Dyes
  • Mutagens
  • Organic Chemicals
  • SYTOX Green
  • DNA