Figure 4.

Variation of FRET efficiency between L13-sCy3 and L13-sCy5 terminally attached to DNA as a function of helix length. E_FRET was measured for each duplex species as phospholipid vesicle-encapsulated single molecules. The E_FRET values are plotted (solid circles) as a function of helix length, with estimated errors. The data have been simulated (solid line) using a model in which 50% of the molecules had one or both fluorophores unstacked (κ² = 2/3) whereas the remaining 50% were stacked. The best fit was obtained where there was no mean rotation of the fluorophores relative to the terminal basepair. However, lateral rotation of the fluorophores was permitted about the mean, with a half-width of 20°. Standard B-form geometry of the DNA helix was used, with 10.5 bp/turn and a helical rise of 3.6 Å/bp step. These calculations were based upon a measured value of the spectral overlap integral J(λ) = 5.4 × 10⁻¹³ M⁻¹ cm³, refractive index n = 1.33, and a quantum yield for L13-sCy3 of ϕ_D = 0.35. The data (open circles) and simulation (broken line) for a duplex series with L3-Cy3 and L3-Cy5 are plotted, taken from our earlier study (12). In this simulation the mean position of each fluorophore is rotated by 31° relative to the terminal basepair, accounting for the phase difference between the two curves.