Single molecule biophysics and fluorescence correlation spectroscopy

Abstract

This dissertation focuses on applications of ultra-sensitive fluorescence. It mainly contains two parts: applying single molecule Foerster resonance energy transfer (SM-FRET) in studies of dynamics and topology of looping constructs formed from DNA-protein interaction and developing a novel fluorescence correlation spectroscopy (FCS) approach to measure binding between drug molecules and different vesicles. Foerster resonance energy transfer (FRET) is a distance dependent phenomenon that can be used to detect and quantify biochemical conformations and interactions in complex samples. With ensemble measurements, it is impossible to resolve the information for dynamic heterogeneous systems without averaging the result. SM-FRET, achieved by using confocal microscopy, measures the signal from a single molecule at a time, thus eliminates ensemble averaging. In this case, SM-FRET was used to evaluate conformational dynamics in a model of negative gene regulation. Details will be discussed in chapter 1, 3 and 4. In FCS measurements, the fluctuations of fluorescence intensity of the sample is correlated to determine information from the processes that cause the fluctuations such as molecules diffusing in and out of a laser focal observation volume and intersystem crossing. In this dissertation, intensity fluctuations caused by intersystem crossing and diffusion are utilized to develop a new FCS approach. This FCS approach can further be used to determine the binding of fluorophores to larger structures. Details will be discussed in chapter 2, 5, 6 and 7.