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Nucleic Acids Res. 2013 Oct;41(19):e184. doi: 10.1093/nar/gkt755. Epub 2013 Aug 23.

Heterogeneous staining: a tool for studies of how fluorescent dyes affect the physical properties of DNA.

Author information

1
Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden and Department for Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.

Abstract

The commonly used fluorescent dye YOYO-1 (YOYO) has, using bulk techniques, been demonstrated to stain DNA heterogeneously at substoichiometric concentrations. We here, using nanofluidic channels and fluorescence microscopy, investigate the heterogeneous staining on the single DNA molecule level and demonstrate that the dye distribution is continuous. The equilibration of YOYO on DNA is extremely slow but can be accelerated by increasing the ionic strength and/or the temperature. Furthermore, we demonstrate how to use the heterogeneous staining as a tool for detailed and time-efficient studies of how fluorescent dyes affect the physical properties of DNA. We show that the relative increase in extension of DNA with increasing amount of YOYO bound is higher at low ionic strengths and also extrapolate the extension of native DNA. Our study reveals important information on how YOYO affects the physical properties of DNA, but it also has broader applications. First, it reveals how cationic intercalators, such as potential DNA drugs, affect DNA under strong confinement. Second, the strategy of using heterogeneous staining is of general use for single molecule studies of DNA interacting with proteins or ligands.

PMID:
23975199
PMCID:
PMC3799460
DOI:
10.1093/nar/gkt755
[Indexed for MEDLINE]
Free PMC Article

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