Measuring DNA–Protein Binding Affinity on a Single Molecule Using Optical Tweezers
DNA–protein interactions may be observed on single molecules with a variety of techniques. However, quantifying the binding affinity is difficult and often requires many (∼100) individual events to characterize the interaction. We use a single λ DNA molecule that provides a lattice of binding sites for proteins. Extending and relaxing the tethered molecule reversibly melts DNA, allowing it to be converted between double-stranded (ds) and single-stranded (ss) forms. By monitoring changes in the properties of the DNA as a function of added protein concentration and fitting to binding models, the DNA–protein interaction may be characterized and quantified. As an example, the high mobility group protein HMGB1(box A + B) is observed to stabilize dsDNA. Measuring the strength of this effect allows us to determine the equilibrium association constant for HMGB1(box A + B) binding to dsDNA.
Key wordsSingle molecule Optical tweezers Force spectroscopy DNA binding DNA melting
This work was supported by NIH (GM75965) and NSF (MCB-02381890). L. James Maher and Jeff Zimmerman are thanked for purified samples of HMGB2(box A + B). Additionally, the authors would like to thank Karin Musier-Forsyth and Penny J. Beuning for technical assistance and advice with the DNA labeling protocol.
- 9.van Mameren, J., Gross, P., Farge, G., Hooijman, P., Modesti, M., Falkenberg, M., Wuite, G., and Peterman, E. (2009) Unraveling the structure of DNA during overs-tretching using multicolor, single-molecule fluorescence imaging, Proc Natl Acad Sci USA, 10.1073/PNAS.0904322106.Google Scholar
- 13.Cruceanu, M., Urbaneja, M. A., Hixson, C. V., Johnson, D. G., Datta, S. A., Fivash, M. J., Stephen, A. G., Fisher, R. J., Gorelick, R. J., Casas-Finet, J. R., Rein, A., Rouzina, I., and Williams, M. C. (2006) Nucleic acid binding and chaperone properties of HIV-1 Gag and nucleocapsid proteins, Nucleic Acids Res 34, 593–605.CrossRefGoogle Scholar