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Tethered Particle Motion

This single molecule method allows us to monitor changes in conformation and length of DNA molecules. On one end, the DNA is rigidly attached to a glass substrate, on the other end it is bound to a small glass microsphere (a bead). Custom written software allows us to monitor the Brownian motion of the bead as a function of time. If the length of the DNA molecule changes, the radius of gyration of the bead will change, giving a readout in real time of the DNA configuration.


In this case our DNA molecule has two binding sites for Lac repressor protein which were located 300bp away from each other. This protein has two binding heads, which allow it to bind the two operators simultaneously, and hence loop the DNA between them.

When Lac repressor is added to the solution, the Brownian motion of the bead oscillates between two states. By taking a histogram of the radius of gyration over time, the existance of these two states can be readily seen..

Our temporal resolution allows us not only to discern the mere existence of two states, but also the kinetic rates of transition between them. We can also determine the free energy change of lac repressor binding and loop formation by studying the steady state probability of each state. Performing this experiment many times with varying operator distances to understand sequence dependent looping and repressor binding is a subject of great interest in the Phillips Group.


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