WhenR  >  0.8R₀: fluorescence anisotropy, non-additive intensity, and cluster size

Abstract

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 , however, these methods break down when R> 0.8R . Fused trimeric DNA clusters labelled with fluorescein were engineered to provide inter-fluorophore distances from 0.7 to 1.6 R/R 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 and is applicable to cases where the anisotropy of a cluster of N fluorophores is not well approximated by r/N.

Lipid Headgroup Charge Controls Melittin Oligomerization in Membranes: Implications in Membrane Lysis

“…Standalone measurements of fluorescence anisotropy in membranes suffer from ambiguity of interpretation since additional factors (such as differential lipid composition, membrane localization of fluorophores, and changes in values of fluorescence lifetime) could lead to observed changes in fluorescence anisotropy. Anisotropy enhancement on photobleaching ,, represents a robust analytical method to quantify the oligomeric size of membrane-bound peptides because artifactual interferences in observed changes in fluorescence anisotropy could be screened out during data analysis (see later). The perturbative nature of the photobleaching assays motivated us to utilize NBD-labeled melittin for our experiments, in spite of the presence of an intrinsically fluorescent tryptophan residue (Trp-19) in this peptide.…”

Pursuing excitonic energy transfer with programmable DNA-based optical breadboards

“…As noted above, this can result in overestimating homoFRET values if not properly taken into account. 327,363,364 Using a different assembly approach, the Asanuma Group focused on closely examining homoFRET with base-stacking dyes such as perylene and pyrene. Exploiting DNA's modularity they conceived an ingenious approach of using heteroFRET as a means of looking at homoFRET efficiency.…”

“…As noted above, this can result in overestimating homoFRET values if not properly taken into account. 327,363,364…”

“…As the anisotropy of the dyes depends on the rotational freedom of the entire system, if there is considerable size or rigidity changes in the system upon creation of the homoFRET section this must be taken into account when trying to quantify subsequent ET. 327 Once the appropriate anisotropy values are obtained, the ET efficiency can be determined by the following equations in a manner akin to heteroFRET. 324 The homoFRET rate is given by ω (equivalent to the FRET rate in eqn (1) ), which in combination with τ , the fluorescence lifetime, can, in turn, provide the homoFRET efficiency ( E hF ).…”