SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
Measuring the Folding Transition Time of Single RNA Molecules
Tae-Hee Lee 1*, Lisa J Lapidus 1, Wei Zhao 1, Kevin J Travers 1, Daniel Herschlag 1 and Steven Chu 1
1 Stanford University
* To whom correspondence should be addressed. E-mail: leeth{at}stanford.edu.
Submitted on August 7, 2006
Revised on October 10, 2006
Accepted on 18 January 2007
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Abstract |
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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 FRET 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 microseconds. The unfolding time is found to be too short to measure with this method.
Key Words:
FCS, FRET, Folding, RNA, Single Molecule
