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J Biotechnol. 2002 Jan;82(3):211-31.

Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data.

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Physikalisch-Chemishes Institut, Universität Heidelberg, Germany.


We studied the fluorescence resonance energy transfer (FRET) efficiency of different donor-acceptor labeled model DNA systems in aqueous solution from ensemble measurements and at the single molecule level. The donor dyes: tetramethylrhodamine (TMR); rhodamine 6G (R6G); and a carbocyanine dye (Cy3) were covalently attached to the 5'-end of a 40-mer model oligonucleotide. The acceptor dyes, a carbocyanine dye (Cy5), and a rhodamine derivative (JA133) were attached at modified thymidine bases in the complementary DNA strand with donor-acceptor distances of 5, 15, 25 and 35 DNA-bases, respectively. Anisotropy measurements demonstrate that none of the dyes can be observed as a free rotor; especially in the 5-bp constructs the dyes exhibit relatively high anisotropy values. Nevertheless, the dyes change their conformation with respect to the oligonucleotide on a slower time scale in the millisecond range. This results in a dynamic inhomogeneous distribution of donor/acceptor (D/A) distances and orientations. FRET efficiencies have been calculated from donor and acceptor fluorescence intensity as well as from time-resolved fluorescence measurements of the donor fluorescence decay. Dependent on the D/A pair and distance, additional strong fluorescence quenching of the donor is observed, which simulates lower FRET efficiencies at short distances and higher efficiencies at longer distances. On the other hand, spFRET measurements revealed subpopulations that exhibit the expected FRET efficiency, even at short D/A distances. In addition, the measured acceptor fluorescence intensities and lifetimes also partly show fluorescence quenching effects independent of the excitation wavelength, i.e. either directly excited or via FRET. These effects strongly depend on the D/A distance and the dyes used, respectively. The obtained data demonstrate that besides dimerization at short D/A distances, an electron transfer process between the acceptor Cy5 and rhodamine donors has to be taken into account. To explain deviations from FRET theory even at larger D/A distances, we suggest that the pi-stack of the DNA double helix mediates electron transfer from the donor to the acceptor, even over distances as long as 35 base pairs. Our data show that FRET experiments at the single molecule level are rather suited to resolve fluorescent subpopulations in heterogeneous mixture, information about strongly quenched subpopulations gets lost.

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