Single molecule FRET spectroscopy
Single molecule FRET (Förster Resonance Energy Transfer) is a powerful method for measuring inter- and intra-molecular distances at the nanometer scale. We use smFRET to monitor the helicase activity of the eukaryotic initiation factor 4A during RNA unwinding. In the presence of a small accessory factor eIF4H, the helicase unwinds short RNA duplexes upon ATP hydrolysis. But unlike most DNA helices, eIF4A does not translocate along the strand, and instead after unwinding it let’s off one of the strand allowing it to re-zip. This results in a repeated, ATP-driven, unzipping - rezipping kinetics that can be directly observed and quantified by smFRET, by either tagging the RNA molecules with a donor-acceptor pair, or by conjugation of the helices itself (eIF4A) with a FRET pair.
Live videos of the Donor and Acceptor channels of surface immobilized RNA molecules during repeated unwinding-rewinding by eIF4A.
Sm-FRET of immobilized RNA molecules
Surface immobilization of RNA molecules enables long-time visualization of single-molecules in vitro. The molecules are illuminated by Total Internal Reflection (TIR) field allowing imaging of tens of molecules simultaneously. The experiment is performed in a custom-made microfluidic chamber that permits tight control of the buffer conditions and temperature, as well as performing abrupt changes in buffers to observe a time-dependent system response.
