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Measuring DNA–Protein Binding Affinity on a Single Molecule Using Optical Tweezers

  • Micah J. McCauley
  • Mark C. WilliamsEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 749)

Abstract

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 words

Single molecule Optical tweezers Force spectroscopy DNA binding DNA melting 

Notes

Acknowledgments

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.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of PhysicsNortheastern UniversityBostonUSA

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