Single Molecule FRET Analysis of DNA Binding Proteins

The complex binding dynamics between DNA and proteins are often obscured by ensemble averaging effects in conventional biochemical experiments. Single-molecule fluorescence methods are powerful tools to investigate DNA-protein interaction dynamics in real time. In this chapter, we focus on using single-molecule Förster Resonance Energy Transfer (smFRET) to probe the binding dynamics of individual proteins on single DNA molecules. We provide a detailed discussion of total internal reflection fluorescence (TIRF) instrument design, nucleic acid labeling with fluorophores, flow cell surface passivation, and data analysis methods.