Fluorescence-Force Spectroscopy Maps Two-Dimensional Reaction Landscape of the Holliday Junction
Sungchul Hohng,1,2*
Ruobo Zhou,1
Michelle K. Nahas,1
Jin Yu,1,4
Klaus Schulten,1,3,4
David M. J. Lilley,5
Taekjip Ha1,2,3,4
Despite the recent advances in single-molecule manipulation techniques, purely mechanical approaches cannot detect subtle conformational changes in the biologically important regime of weak forces. We developed a hybrid scheme combining force and fluorescence that allowed us to examine the effect of subpiconewton forces on the nanometer scale motion of the Holliday junction (HJ) at 100-hertz bandwidth. The HJ is an exquisitely sensitive force sensor whose force response is amplified with an increase in its arm lengths, demonstrating a lever-arm effect at the nanometer-length scale. Mechanical interrogation of the HJ in three different directions helped elucidate the structures of the transient species populated during its conformational changes. This method of mapping two-dimensional reaction landscapes at low forces is readily applicable to other nucleic acid systems and their interactions with proteins and enzymes.
1 Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
2 Howard Hughes Medical Institute, Urbana, IL 61801, USA.
3 Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
4 Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
5 Cancer Research UK Nucleic Acid Structure Research Group, MSI/WTB Complex, University of Dundee, Dundee DD1 5EH, UK.
* Present address: Department of Physics and Astronomy, Seoul National University, Seoul 151-742, Korea.
To whom correspondence should be addressed. E-mail: tjha{at}uiuc.edgu
