Parallel Single-Molecule Study of DNA Repressor Kinetics

In a bottom up approach to understanding molecular motors, a synthetic protein-based molecular motor, the “tumbleweed”, is being designed and constructed [1]. This design uses three ligand dependent DNA repressor proteins to rectify diffusive motion of the construct along a DNA track. These proteins are MetJQ44K, TrpR, and DtxR. To predict the behavior of this artificial motor one needs to understand the binding and unbinding kinetics of the repressor proteins at a single-molecule level. An assay, similar to tethered particle motions assays [2], is used to measure the unbinding rates of these three DNA repressor proteins. In this assay the repressor is immobilized to a surface in a microchamber. Long DNA with the correct recognition sequence for one of the repressors is attached to a streptavidin-coated microsphere. As the DNA-microsphere construct diffuses through the microchamber it will sometimes bind to the repressor protein. Using brightfield microscopy and a CCD camera the diffusive motion of the microsphere can be characterized and bound and unbound states can be differentiated. On the order of ten microspheres can be easily visualized at one time allowing single-molecule measurements to be done in parallel. The resulting kinetic measurements are compared to bulk binding kinetics measured in a QCM device.[1] EHC Bromley et al. 2009. The Tumbleweed: Towards a synthetic protein motor. HFSP Journal. 3:204-212.[2] F Vanzi et al. 2006. Lac Repressor hinge flexibility and DNA looping: single molecule kinetics by tethered particle motion. Nuc. Acids. Res. 34:3409-3420.