Evaluating the Relationship between FRET Changes and Distance Changes Using DNA Length and Restriction Enzyme Specificity

FRET (Förster resonance energy transfer) involves the transfer of energy from an excited donor fluorophore to an acceptor molecule in a manner that is dependent on the distance between the two. A biochemistry laboratory experiment is described that teaches students how to use FRET to evaluate distance changes in biological molecules. Students measured the apparent FRET between donor and acceptor fluorophores located on the ends of several DNAs of unknown lengths, enabling them to order the DNAs according to size. In addition, students investigated site-specific DNA cleavage by restriction endonucleases, using loss of apparent FRET to determine which enzyme cut sites were present in each of the DNAs. After completing this experiment, students understood the inverse relationship between changes in FRET and changes in distance, and understood how changes in FRET could be used to monitor a conformational change in a molecule. As an extension to the experiment, a tutorial is included that uses the same DNAs to illustrate the ability of single-molecule FRET measurements to resolve heterogeneity in a sample, which cannot be done via more traditional ensemble measurements.