When R  >  0.8R ₀: fluorescence anisotropy, non-additive intensity, and cluster size

Assembly and clustering feature in many biological processes and homo-FRET and fluorescence anisotropy can assist in estimating the aggregation state of a system. The distance dependence of resonance energy transfer is well described and tested. Similarly, assessment of cluster size using steady state anisotropy is well described for non-oriented systems when R  <  0.8R ₀, however, these methods break down when R  >  0.8R ₀. Fused trimeric DNA clusters labelled with fluorescein were engineered to provide inter-fluorophore distances from 0.7 to 1.6 R/R ₀ and intensity and anisotropy were measured. These constructs cover a range where anisotropy effects depend on distance. Analytical expressions were derived for fully labelled and fractionally labelled clusters and the experimental results analysed. The experimental results showed that: (1) the system underwent distance dependent quenching; (2) when incompletely labelled both doubly and triply labelled forms could be assessed to obtain distance dependent intensity factors; (3) the anisotropy behaviour of a multiply labelled cluster of a particular size depends on the behaviour of the fluorophores and their distance in a cluster. This work establishes that when emission intensity data are available the analytically useful range for investigating clusters does not have to be restricted to R  <  0.8R ₀ and is applicable to cases where the anisotropy of a cluster of N fluorophores is not well approximated by r ₁/N.