Fluorescence-Force Spectroscopy Maps Two-Dimensional Reaction Landscape of the Holliday Junction

Author:

Hohng Sungchul12345,Zhou Ruobo12345,Nahas Michelle K.12345,Yu Jin12345,Schulten Klaus12345,Lilley David M. J.12345,Ha Taekjip12345

Affiliation:

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.

Abstract

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.

Publisher

American Association for the Advancement of Science (AAAS)

Subject

Multidisciplinary

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