Alternating-laser Excitation (ALEX) Implemented on a Hybrid Magnetic Tweezers Single Molecule Förster Resonance Energy Transfer Instrument

Single-molecule spectroscopy has emerged as a technique to study the mechanisms of complex biological systems. Single molecule Förster resonance energy transfer (smFRET) is a powerful method for monitoring nanometer scale motions within biological macromolecules, measured as the efficiency of energy transfer between a donor and acceptor chromophore. Magnetic tweezers (MT) is a second single molecule technique that permits the application of tension and torques to individual DNA molecules. Single molecule measurements typically employ either a fluorescence-based approach such as smFRET or a micromanipulation technique such as MT; however, each technique has its own limitations. Recently, the integration of FRET and MT (MT-FRET) has become possible, permitting researchers to mechanically manipulate molecules with MT while simultaneously measuring dynamic structural properties by smFRET. For our proof of principle experiment, we are using a model hairpin DNA to validate our instrumentation. MT-FRET is used on the hairpin to monitor the unfolded versus folded state of the DNA. One of the inherent problems with MT-FRET is when FRET efficiency (E) E = 0. When the hairpin unfolds, the DNA goes outside the measurable FRET range; therefore, we need to implement a third single molecule technique, alternating-laser excitation (ALEX), which gives us the ability to differentiate between the actual physical state of the DNA, or if E=0 is due to a photophysical effect.