| Popis: |
Fluorescence-correlation-spectroscopy (FCS) is a powerful tool to gain information about dynamics of bio-molecules. However, the key problem is to extract the rates hidden in the FCS data by fitting the data to a meaningful model. A number of different fitting approaches have been described in recent years but the extraction of relevant information till now was still limited by numerous experimental problems and the fact that the set of starting parameter values chosen could often predefine the result. We establish a new way to globally analyze FCS data based on Bayesian inference to overcome these issues. Moreover, the influence of other remaining experimental error sources, e.g. photo-physics, is excluded by additional means. Using this approach in combination with the results from single molecule burst analysis, we investigate the kinetics of DNA hairpins labeled with a variety of different fluorescent probes as a function of salt concentration. We find that dye selection influences the rates of hairpin opening and closing as well as the equilibrium constant of the transition. Thus, great caution has to be used when utilizing dye molecules as reporters for the kinetics of dynamic macromolecular structures. |
