Characterization of DNA-protein complexes by capillary electrophoresis-single molecule fluorescence correlation spectroscopy

A high-speed capillary electrophoresis mobility shift assay (CEMSA) for det ermining the binding ratios of DNA-protein complexes in solution is demonst rated. Single molecule fluorescence correlation spectroscopy (FCS) was used to resolve the bound and unbound fluorescently labeled DNA molecules as th ey flowed continuously through a fused silica capillary under the influence of an applied electric field. Resolution of the bound and unbound complexe s was based on the difference in their electrophoretic mobilities, and was accomplished without the need to perform a chemical separation. Data suffic ient to perform the analysis was acquired in less than 10 s, compared to th e minutes that are normally needed to carry out such measurement via CE sep aration, The binding ratios were determined with 5 to 10% precision and agr eed with the results obtained by CE separation within experimental error. T he resolution of the CEMSA based FCS analysis (CEMSA-FCS) was significantly higher than for the analysis performed by conventional diffusional FCS, du e to the higher mass sensitivity of the electrophoretic mobility compared t o the translational diffusion coefficient. Fluorescently labeled 39-mer sin gle stranded DNA (ssDNA) and the single stranded binding protein (SSB) from Escherichia coli was used as the model system. The dissociation constant o f the ssDNA-SSB complex was estimated to be approximate to 2 nM based on th e CEMSA-FCS analysis.