Dual Focus Fluorescence Cross-Correlation Spectroscopy For The Investigation Of Biomolecule Folding And Binding In Flowing Liquids

Fluorescence correlation spectroscopy measures the time constants for rate processes giving rise to fluorescence intensity fluctuations observed from one or more microscopic sub-volumes of a nanomolar aqueous solution containing the biomolecules of interest. In our approach, the analyte molecules flow through an electrophoresis capillary under the influence of pressure driven flow or electrophoretic flow and are probed by two spatially offset probe volumes in such a way that the molecules flow sequentially from one probe volume to the next. Fluorescence fluctuations are analyzed from each probe volume independently using autocorrelation analysis, and from the two spatially offset probe volumes using cross-correlation analysis. In this way, we are able to resolve fluorescence fluctuation time constants arising from diffusion, flow, triplet blinking, and conformational fluctuations. Conformational fluctuations are monitored by quenching and unquenching of dye-quencher molecules attached to DNA or RNA hairpin structures and probe the folding and unfolding kinetics of the hairpins. In this presentation, we will discuss recent results that show how base stacking within the loop region of the DNA and RNA hairpins alters the kinetics and thermodynamic stability of the hairpins. We will also discuss binding and unbinding of counterions to individual nucleotides as they flow through the capillary under the influence of an applied electric field. Emphasis will be placed on how the desired information can be extracted using our unique approach to fluorescence correlation spectroscopy.