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Quantifying force-dependent and zero-force DNA intercalation by single-molecule stretching

Nature Methods volume 4, pages 517–522 (2007)Cite this article

Abstract

We used single DNA molecule stretching to investigate DNA intercalation by ethidium and three ruthenium complexes. By measuring ligand-induced DNA elongation at different ligand concentrations, we determined the binding constant and site size as a function of force. Both quantities depend strongly on force and, in the limit of zero force, converge to the known bulk solution values, when available. This approach allowed us to distinguish the intercalative mode of ligand binding from other binding modes and allowed characterization of intercalation with binding constants ranging over almost six orders of magnitude, including ligands that do not intercalate under experimentally accessible solution conditions. As ligand concentration increased, the DNA stretching curves saturated at the maximum amount of ligand intercalation. The results showed that the applied force partially relieves normal intercalation constraints. We also characterized the flexibility of intercalator-saturated dsDNA for the first time.

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Figure 1: DNA-intercalator phase diagrams.
Figure 2: Dependence of ethidium binding on force.
Figure 3: Force-extension curves in the absence of intercalator, at saturated binding for Ru(phen)2dppz2+, Ru(phen)32+ and ethidium.

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Acknowledgements

We acknowledge funding from the US National Science Foundation (MCB0238190), US National Institutes of Health (GM072462) and American Chemical Society Petroleum Research Fund.

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Authors and Affiliations

  1. Department of Physics, Northeastern University, Boston, 02115, Massachusetts, USA

    Ioana D Vladescu, Micah J McCauley & Mark C Williams

  2. Department of Chemistry, Mount Holyoke College, South Hadley, 01075, Massachusetts, USA

    Megan E Nuñez

  3. Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, 55455, Minnesota, USA

    Ioulia Rouzina

  4. Center for Interdisciplinary Research on Complex Systems, Northeastern University, Boston, 02115, Massachusetts, USA

    Mark C Williams

Authors
  1. Ioana D Vladescu
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  2. Micah J McCauley
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  3. Megan E Nuñez
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  4. Ioulia Rouzina
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Contributions

I.D.V. performed most experiments. M.J.M. maintained the instrument, labeled DNA and performed some experiments. M.E.N. provided ruthenium compounds. I.D.V., I.R., M.E.N. and M.C.W. designed the research. I.R. developed the theory. I.D.V., M.E.N., I.R. and M.C.W. wrote the paper.

Corresponding authors

Correspondence to Ioulia Rouzina or Mark C Williams.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Drug effect on hysteresis behavior. (PDF 92 kb)

Supplementary Fig. 2

Dependence of Ru(phen)2dppz2+ binding on force. (PDF 81 kb)

Supplementary Fig. 3

Dependence of Ru(phen)32+ binding on force. (PDF 77 kb)

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Vladescu, I., McCauley, M., Nuñez, M. et al. Quantifying force-dependent and zero-force DNA intercalation by single-molecule stretching. Nat Methods 4, 517–522 (2007). https://doi.org/10.1038/nmeth1044

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