Characterization of DNA-protein complexes by capillary electrophoresis-single molecule fluorescence correlation spectroscopy.
A high-speed capillary electrophoresis mobility shift assay (CEMSA) for determining the binding ratios of DNA-protein complexes in solution is demonstrated. Single molecule fluorescence correlation spectroscopy (FCS) was used to resolve the bound and unbound fluorescently labeled DNA molecules as they flowed continuously through a fused silica capillary under the influence of an applied electric field. Resolution of the bound and unbound complexes was based on the difference in their electrophoretic mobilities, and was accomplished without the need to perform a chemical separation. Data sufficient to perform the analysis was acquired in less than 10 s, compared to the minutes that are normally needed to carry out such measurement via CE separation. The binding ratios were determined with 5 to 10% precision and agreed with the results obtained by CE separation within experimental error. The resolution of the CEMSA based FCS analysis (CEMSA-FCS) was significantly higher than for the analysis performed by conventional diffusional FCS, due to the higher mass sensitivity of the electrophoretic mobility compared to the translational diffusion coefficient. Fluorescently labeled 39-mer single stranded DNA (ssDNA) and the single stranded binding protein (SSB) from Escherichia coli was used as the model system. The dissociation constant of the ssDNA-SSB complex was estimated to be approximately 2 nM based on the CEMSA-FCS analysis.
LeCaptain
D J
DJ
Department of Chemistry, Colorado State University, Fort Collins 80523, USA.
Michel
M A
MA
Van Orden
A
A
D004247:DNA / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000379:methods; D011485:Protein Binding; D011506:Proteins / Q000032:analysis*; D013050:Spectrometry, Fluorescence
D011506:Proteins; D004247:DNA