Experimental Determination of the Forster Critical Distance

Forster resonance energy transfer (FRET) can be used as a spectroscopic ruler to measure nanometer-scale distances. The recovery of inter-dye distance depends on a calibration factor known as the Forster critical distance (R0). This distance is currently estimated based on measurements of the quantum yield of the donor dye, the overlap integral between the donor and acceptor dyes, and assumptions about the index of refraction and the relative orientation of the donor and acceptor dye molecules. Here, we report a method to experimentally measure R0, using B-DNA as a structural reference. Fifteen donor (Cy3)-labeled oligonucleotides were generated, by placing donor-labeled Thymidines at positions 11, 14, ⋯, 39. A single complementary strand was synthesized with acceptor (AlexaFluor647) at position 10. The strands were annealed, producing dsDNA consisting of a 30 base pair (bp) ruler with a 10 bp cap on each end. For each freely diffusing construct, the mean transfer efficiency (TE) was measured by single-pair FRET (sp-FRET) and ensemble (en-FRET). The TE's as a function of bp were fit to a reduced representation model of B-DNA that provided the absolute inter-dye distances. The reduced model was formulated based on an atomistic model of dye-labeled B-DNA, R0 was recovered from the fit. We repeated our approach using three different donor/acceptor pairs, each with a different R0.