Nature Methods
- 4, 517 - 522 (2007)
Published online: 29 April 2007; | doi:10.1038/nmeth1044
Quantifying force-dependent and zero-force DNA intercalation by single-molecule stretchingIoana D Vladescu1, Micah J McCauley1, Megan E Nuñez2, Ioulia Rouzina3 & Mark C Williams1, 41
Department of Physics, Northeastern University, Boston, Massachusetts 02115, USA. 2
Department of Chemistry, Mount Holyoke College, South Hadley, Massachusetts 01075, USA. 3
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA. 4
Center for Interdisciplinary Research on Complex Systems, Northeastern University, Boston, Massachusetts 02115, USA.
Correspondence should be addressed to Mark C Williams mark@neu.edu or Ioulia Rouzina rouzi002@umn.edu 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.
MORE ARTICLES LIKE THIS These links to content published by NPG are automatically generated.
|
