Mapping the two-dimensional reaction landscape of Holliday junction via dynamic force-fluorescence spectroscopy

Sungchul Hohng, Ruobo Zhou, Michelle K. Nahas, David M. J. Lilley & Taekjip Ha, Department of Physics, University of Illinois at Urbana-Champaign

As mechanical parameters are being recognized as important determinants of many biological processes, single-molecule manipulation techniques are continually breaking new records in their sensitivity. However, biologically more relevant regime of high spatial resolution under weak applied forces has been out of reach. A hybrid instrument combining optical tweezers and single molecule FRET allowed us to probe the effect of 0.5-2 pN forces on the 2-3 nm scale motion of the four-way DNA (Holliday) junction with 100 Hz bandwidth, a currently unachievable task for purely mechanical methods. Since the force effect depends on its direction and on the length of the pulled arms, the intrinsic reaction coordinates must be two-dimensional and angular. Our analysis further elucidates the structure of the transition states during the Holliday junction’s conformational exchanges and shows that the Holliday junction is an exquisitely sensitive force sensor.