Bead size effects on protein-mediated DNA looping in tethered-particle motion experiments

Tethered particle motion (TPM) has become an important tool for single-molecule studies of biomolecules; however, concerns remain that the method may alter the dynamics of the biophysical process under study. We investigate the effect of the attached microsphere on an illustrative biological example: the formation and breakdown of protein-mediated DNA loops in the lac repressor system. By comparing data from a conventional TPM experiment with 800 nm polystyrene beads and dark-field TPM using 50 nm Au nanoparticles, we found that the lifetimes of the looped and unlooped states are only weakly modified, less than two-fold, by the presence of the large bead. This is consistent with our expectation of weak excluded-volume effects and hydrodynamic surface interactions from the cover glass and microsphere.