Two new concepts to measure fluorescence resonance energy transfer via fluorescence correlation spectroscopy: Theory and experimental realizations

In this study, we demonstrate two new concepts, using fluorescence correlat ion spectroscopy (FCS), to characterize fluorescence resonance energy trans fer (FRET). The two approaches were tested experimentally by measuring a se ries of double-stranded DNA molecules, with different numbers of base-pairs separating the donor (Alexa488) and acceptor (Cy5) fluorophores. In the fi rst approach, FRET efficiencies are determined from the detected acceptor f luorescence rate per molecule. Here, the unique possibility with FCS to det ermine the mean number of molecules within the detection volume is exploite d, making a concentration calibration superfluous. The second approach take s advantage of FRET-dependent fluorescence fluctuations of photophysical or igin, in particular fluctuations generated by trans-cis isomerization of th e acceptor dye. The rate of interchange,between the trans and cis states is proportional to the excitation rate and can be conveniently measured by FC S. Under FRET-mediated excitation, this rate can be used as a direct measur e of the FRET efficiency. The measured isomerization rate depends only on t he fluctuations in the acceptor fluorescence, and is not affected by donor, fluorescence cross-talk, background, dye labeling efficiencies, or by the concentration of molecules under study. The measured FRET efficiencies are well in agreement with a structural model of DNA. Furthermore, additional s tructural information is obtained from simulations of the measured fraction of acceptor dyes being in a nonfluorescent cis conformation, from which di fferences in the position and orientation of the trans and cis form of the acceptor dye can be predicted.