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

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

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Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5048, Centre de Biochimie Structurale, 29 rue de Navacelles, 34090, Montpellier, France.


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.

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