Advances in fluorescence correlation spectroscopy with applications to studies of macromolecular interactions and folding of RNA hairpins

This work is dedicated to developing theory and applications of fluctuation spectroscopy, an experimental technique that allows one to measure and analyze signal fluctuations that are due to single molecules. By averaging many fluctuations, it is possible to obtain information about distributions of molecular properties of the ensemble without disturbing the equilibrium. Our work continues development of widely popular fluorescence correlation spectroscopy. Towards this goal, we develop full theory of two-point correlations in a light detection experiment and revive application of high-order fluorescence correlation spectroscopy in biochemistry. Our theoretical findings lead to simplified data analysis that efficiently captures all important molecular properties: brightness per molecule, diffusion coefficient of the molecules, and relaxation rates of chemical reactions. We are also able to obtain theoretical expressions that describe the signal-to-noise ratio in high-order correlation experiments. The developed methodology is especially useful in investigating irreversible chemical reactions as we show by quantitatively describing photobleaching in the excitation volume.;We demonstrate that these theoretical findings aid biochemical research by characterizing interactions of polypyrimidine tract binding protein (human isoform 1, domains 3 and 4) with a 75-nucleotide long RNA (a part of GABA A receptor gamma2 pre-mRNA). The combination of filter-binding, dual-channel (32P and fluorescence) detection in electrophoretic mobility shift assay, and high-order fluorescence correlation analysis allows us to measure stoichiometry of intermediate complexes as well as binding constants.;As the second example we study folding of RNA hairpins using high sensitivity detection based on the design used in molecular beacons. We investigate the effect of salt and loop composition on dynamics and establish, through a combination of steady state fluorescence and absorption spectroscopy, fluorescence correlation spectroscopy, and brightness analysis, that folding is not a two-state process. We present evidence for two phases in folding and show that the faster phase is accelerated by single strand stacking in the hairpin loop.