Förster Resonance Energy Transfer (FRET) is commonly used as a spectroscopic ruler for nanometer scale distance measurements. DNA photonic wires are of interest for potential applications in synthetic photosynthesis, biological sensing, and photonics applications. Many of these applications require understanding how to engineer highly efficient energy transfer.
We have combined steady state absorption, fluorescence, ultrafast transient absorption, time-resolved photoluminescence, and single-particle (sp-)FRET measurements to examine energy transfer between Cy3 and Cy5 attached to a DNA duplex as a function of dye separation distance. The combination of these techniques allow for inhomogeneities in ensemble measurements and the contribution of direct absorption by the Cy5 acceptor molecule to be accounted for. Each cyanine dye was rigidly attached to the DNA backbone. Anisotropy measurements and increased fluorescence quantum yields confirm rigid immobile dyes. This reduces the uncertainty in dye separation as well as in the orientational factor, κ2.
Energy transfer from the donor to the acceptor dye is characterized by donor emission quenching and simultaneous acceptor emission sensitization. Time-resolved and single-particle measurements are found to be more reliable for dye separations less than the Förster distance. For time-resolved measurements, we see a fast decay in donor emission and a concomitant rise in acceptor emission. We explore energy transfer over distances that are shorter than the dye molecules and thus are outside of the Förster limit. Spectral shifts in steady state absorption and acceptor fluorescence quenching indicate interaction between the donor and acceptor when in close proximity. We observe, for the smallest separation distances, picosecond energy transfer with near unity efficiency. Single distributions in efficiency are observed via spFRET measurements and transient absorption shows no signs of charge transfer. Structural models were used to clarify the distances between dyes. Deviations from Förster theory are discussed.
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