FRET from Multiple Pathways in Fluorophore-Labeled DNA

Because of their ease of design and assembly, DNA scaffolds provide a valuable means for organizing fluorophores into complex light harvesting antennae. However, as the size and complexity of the DNA–fluorophore network grows, it can be difficult to fully understand energy transfer properties because of the large number of dipolar interactions between fluorophores. Here, we investigate simple DNA–fluorophore networks that represent elements of the more complex networks and provide insight into the Förster Resonance Energy Transfer (FRET) processes in the presence of multiple pathways. These FRET networks consist of up to two Cy3 donor fluorophores and two Cy3.5 acceptor fluorophores that are linked to a rigid dual-rail DNA scaffold with short interfluorophore separation corresponding to 10 DNA base pairs (∼34 Å). This configuration results in five FRET pathways: four hetero-FRET and one homo-FRET pathway. The FRET properties are characterized using a combination of steady-state and time-resolved spectroscopy and understood using Förster theory. We show that the multiple FRET pathways lead to an increase in FRET efficiency, in part because homo-FRET between donor fluorophores provides access to parallel pathways to the acceptor and thereby compensates for low FRET efficiency channels caused by a static transition dipole distribution. More generally, the results show that multiple pathways may be used in the design of artificial light harvesting devices to compensate for inhomogeneities and nonideal ensemble effects that degrade FRET efficiency.