Characterization of the conformational changes of a tripartite molecular beacon

We have investigated the kinetics of the conformational changes of a new type of molecular beacons called tripartite molecular beacons. The rate constants corresponding to the opening and closing of the beacons have been obtained from fluorescence correlation spectroscopy experiments. We found that both rate constants are larger for the tripartite molecular beacon than for the corresponding molecular beacon. This paper outlines the importance of using very low excitation intensities for this type of measurements, and of considering the fact that the beacon still emits a residual background fluorescence in its closed form. We also report on the exploration of several strategies to improve the precision of the measurements by increasing the characteristic time associated with the diffusion of the beacons so that it would not be confused with the relaxation time associated with the conformational changes.