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Toward measuring transition paths for RNA folding with single molecule FRET

Thursday, November 07, 2013 — Poster Session II

12:00 p.m. – 2:00 p.m.

FAES Academic Center (Upper-Level Terrace)




  • K. Truex
  • H.S. Chung
  • J. M. Louis
  • W. A. Eaton


Studying RNA folding mechanisms requires single molecule methods, since ensemble measurements give no information on the predicted heterogeneity of folding dynamics. All of the mechanistic information on how RNA folds is contained in the transition paths (TPs) – the rare trajectories when the molecule crosses the free energy barrier separating folded and unfolded states. Due to experimental challenges, TPs have not yet been observed for any molecular process. Only an average TP time (~2 µs) has been determined in one case for a protein (Chung, Louis, McHale, Eaton, Science 2012). As a prelude to measurements on more complex RNA molecules, we use single molecule FRET spectroscopy to study the kinetics and dynamics of sub-millisecond-folding DNA and RNA hairpins labeled with a fluorescent dye pair and immobilized on a glass surface with a biotin-streptavidin-biotin linkage. To extract rate coefficients, we employ a photon-by-photon maximum likelihood analysis. The next steps are to determine the average TP time (which is much shorter than the residence times), determine the TP time distribution by increasing the solvent viscosity sufficiently to slow the motion over the barrier, and ultimately obtain intra-chain distance versus time trajectories during TPs, i.e. watch individual RNA molecules fold.

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