Measuring the Folding Transition Time of Single RNA Molecules

We describe a new, time-apertured photon correlation method for resolving the transition time between two states of RNA in folding—i.e., the time of the transition between states rather than the time spent in each state. Single molecule fluorescence resonance energy transfer and fluorescence correlation spectroscopy are used to obtain these measurements. Individual RNA molecules are labeled with fluorophores such as Cy3 and Cy5. Those molecules are then immobilized on a surface and observed for many seconds during which time the molecules spontaneously switch between two conformational states with different levels of flourescence resonance energy transfer efficiency. Single photons are counted from each fluorophore and cross correlated in a small window around a transition. The average of over 1000 cross correlations can be fit to a polynomial, which can determine transition times as short as the average photon emission interval. We applied the method to the P4–P6 domain of the Tetrahymena group I self-splicing intron to yield the folding transition time of 240 μs. The unfolding time is found to be too short to measure with this method.