Tethered Particle Motion (TPM) experiments are a simple, yet insightful, technique for single-molecule studies. By tracking the motion of a tiny microsphere attached to a strand of DNA, events such as the formation of protein-mediated DNA loops can be observed.Using TPM on designed hyperstable loop-forming DNA constructs, we have found that LacI-mediated DNA loops form and break down on time scales of the order of minutes. This is in stark constrast to measurements in competition assays that report loop lifetimes of days for these constructs. We are currently investigating possible explanations for the LacI loop formation process that resolves these seemingly contradictory observations.
In TPM a microsphere is attached to one end of a strand of DNA, while the other end is affixed to a microscope coverslip. The image on the left shows the unlooped state of the DNA, while the image on the right is illustrative of the looped state. The image on the right Radial trace of the motion of a tethered microsphere showing clear jumps between looped and unlooped states of the DNA.