Single Molecule FRET Microscopy of Immobilized DNA Molecules: Keeping Track of Dye Integrity and Stoichiometry

Abstract

Fluorescence Resonance Energy Transfer (FRET) at the single molecule level is a powerful technique for studying conformational changes of biomolecules. Dual color excitation schemes help sorting the single molecule data and quantifying FRET efficiencies within a single molecule. Here, we compare several data analysis methods for accurate FRET measurements and for discriminating between conformational changes in single molecules and other effects caused by dye photophysics or multimers. Single molecule FRET measurements were conducted on a series of immobilized double-stranded and G-quadruplex DNA molecules using a widefield microscope. Donor (Cy3/TMR) and acceptor (Cy5) molecules were both excited via an alternated laser excitation scheme. The double stranded DNA samples serve as molecular standards and G-quadruplex DNA structures, in this context, are interesting as they show large conformation diversity.