Proteins and DNA constantly interact with one another and are inextricably linked by both the central dogma of molecular biology and the critical need for maintenance and faithful transfer of genomic information from mother to daughter cells. However, many important protein-DNA interactions are transient and dynamic, which makes them particularly challenging to observe and characterize. Technological advances in fluorescence microscopy, including total internal reflection fluorescence (TIRF) microscopy, have made it possible to observe these processes in real-time at the single-molecule level. These types of studies can reveal interactions and dynamics that would normally be obscured by the ensemble averaging that occurs in other approaches. Molecular processes are stochastic at the single-molecule level, and they often involve multiple intermediate steps. Therefore, careful statistical analysis of single-molecule data can provide key insights into the mechanistic details of these processes.
In this talk, I will describe some examples of how single-molecule TIRF microscopy can be used to directly observe protein-DNA interactions. I will also discuss the methods used to extract information from the data collected. And, finally, I will describe how TIRF microscopy can be used to collect single-molecule Förster Resonance Energy Transfer (FRET) data. This approach can provide even more detailed information about a broad range of protein-DNA interactions.