Two-beam fluorescence cross-correlation spectroscopy in an electrophoretic mobility shift assay.

Two-beam fluorescence cross-correlation spectroscopy (FCCS) was used to resolve the bound and unbound fractions of fluorescently labeled single-stranded DNA (ssDNA) in a ssDNA-protein complex as the analyte solution flowed continuously through an electrophoresis capillary. Cross-correlation of the single molecule fluorescence from two spatially separate excitation laser beams resulted in cross-correlation functions that consisted of well-resolved peaks characteristic of the different electrophoretic flow velocities of the bound and unbound ssDNA. This decoupled the molecular parameters of the bound and unbound ssDNA used to model the cross-correlation function, which enabled the relative concentrations to be determined without prior knowledge of the pure-component cross-correlation functions, as would be required in an analogous autocorrelation analysis. The relative concentrations of the bound and unbound ssDNA were determined by two-beam FCCS within 2-6% precision, even for samples that contained as little as 5% unbound ssDNA, and were consistent with the results obtained by capillary electrophoresis (CE) separation of the same samples. Data sufficient to obtain these results was acquired in 10-15 s per sample. Fluorescently labeled poly(dT)39 complexed with the single stranded DNA binding protein of Escherichia coli served as the model system. The measured dissociation constant of 2.5+/-0.9 nM agreed with the literature value for this complex within experimental error. The CE/two-beam FCCS experiment described here is part of a family of techniques that use single molecule fluorescence detection to resolve different components in an electrophoresis system. Advantages of these methods relative to separations-based CE include enhanced sensitivity, the potential for higher speed analyses, elimination of the sample plug injection step, and the ability to carry out the analysis in shorter flow channels.