Applications of Fluorescence Correlation Spectroscopy to the Study of Nucleic Acid Conformational Dynamics

Abstract

Publisher Summary This chapter provides an overview of the fluorescence correlation spectroscopy (FCS) technique, and focuses on its applications to the investigation of conformational dynamics of nucleic acids. FCS is a technique based on the measurement of the spontaneous fluctuations of the fluorescence signal of a small number of molecules. Fluorescence fluctuations are typically measured in an optically restricted submicron observation volume and then analyzed statistically to reveal kinetic information about the processes that lead to these fluctuations. Such processes include concentration fluctuations via molecular diffusion, chemical reactions, photophysical processes, and so on. To obtain dynamic or kinetic information from FCS experiments, the experimentally acquired auto- and cross-correlation functions have to be compared with the decays predicted by the appropriate physical models. The simplest FCS experiment involves measuring the intensity fluctuations of a fluorescent particle diffusing freely in three dimensions. For FCS to be useful in the study of conformational dynamics, the intensity of fluorescence of the probe has to be influenced by the conformation of the biopolymer being studied. The most common approach is to label the biopolymer with a fluorophore–quencher or donor–acceptor FRET pair, so that the efficiency of emission of the fluorophore is a direct measure of the distance between the two tags.