Biomolecule conformational dynamics studies using two-beam fluorescence fluctuation spectroscopy: dynamics of DNA hairpin structure

Abstract

This dissertation describes a new fluorescence-based technique for understanding biomolecule conformational dynamics, referred to as two-beam fluorescence fluctuation spectroscopy (2bFFS). In this approach, the fluorescence fluctuations associated with biomolecule conformational changes were observed as the molecules flowed sequentially between two spatially offset, microscopic detection volumes, and analyzed simultaneously from three different vantage points—cross-correlation analysis of the two detection channels relative to each other, autocorrelation analysis of the two detection channels independently, and photon counting histogram (PCH) analysis. The cross-correlation function characterizes the hydrodynamic properties (flow rate and diffusion rate) of the molecules under study, independent of conformational fluctuations. These parameters can then be used to constrain the measured autocorrelation function, from which precise measurements of the conformational kinetics parameters are obtained. The combined PCH analysis can resolve the equilibrium distribution between different conformations of the molecules. To demonstrate these principles, dynamic equilibrium between the folded and unfolded conformations of single stranded DNA hairpin molecules was investigated. The DNA hairpins contained a polythymine loop and a five base-pair stem sequence that was end-labeled with a fluorescent dye and a quencher. Folding and unfolding of the DNA hairpin structure caused the dye fluorescence to fluctuate on the same characteristic time scale as the conformational change. In addition to unambiguously characterizing the relaxation times of the process, the analysis revealed non-exponential relaxation kinetics and DNA size-dependent folding times characteristic of dynamic heterogeneity in the DNA hairpin-forming mechanism. Analysis of the equilibrium distribution suggests a three-state mechanism for the reaction, involving a rapid equilibrium between open and a stable intermediate form of the DNA hairpin. The final, closed form of the DNA hairpin is suggested to be stable on a much longer time scale than that of the present FFS measurements.