DNA-binding proteins play a crucial role in every aspect of genetic activity. In the cell, they regulate events such as transcription, packaging, rearrangement, replication and repair. Important examples include RNA polymerase, transcription factors and restriction endonucleases. It has been known for some time that these proteins interact with specific DNA sequences, but it is less clear how the protein is able to find its target site among the abundance of cellular DNA. By understanding the nature of these protein-DNA interactions, scientists hope to gain insight into the most important physiological processes of an organism.

To precisely describe the motion of individual proteins along DNA, single-molecule fluorescence spectroscopy can be used to provide information regarding the activity of a single biological molecule without the effects of ensemble averaging. Researchers have taken advantage of fluorescence resonance energy transfer (FRET), a weak dipole-dipole interaction between a donor and acceptor fluorophore, to study macromolecules. The efficiency of energy transfer scales as the inverse sixth power of the distance between fluorophores, ideal for probing interactions in the range of 10 to 75 Å. FRET is a powerful tool with high colocalization accuracy and the ability to report dynamical changes in distance or orientation between fluorophores.

Currently, we are investigating the application of single-molecule fluorescence techniques to establish the mechanism of protein translocation used by restriction endonucleases on DNA. This is done under physiological conditions and tracked spatially in real time.