DNA and RNA molecules can adopt a variety of noncanonical conformations including hairpin structures. These hairpins are presumed to play an integral role in replication, transcription, and genetic recombination, though new information regarding their biological functions continues to be learned through kinetics and thermodynamics studies.
Our group is interested in investigating the full range of hairpin conformational states and probing the mechanism of hairpin folding, which was once thought to be a two-state mechanism but is now recognized as a three-state mechanism. Using higher-order FCS as well as dual-beam FCS in combination with stopped flow kinetic measurements, our work has not only validated a three-state folding model, but also demonstrated that external salt concentrations and temperature influence the folding kinetics and the free energy landscape.
To conduct FCS studies, single-stranded DNA is labeled with a fluorophore and fluorescence quencher, thereby enabling measurements of the fluorescence intensity fluctuations characteristic of the transitions between the folded, intermediate, and unfolded conformational states.