In this application note, we demonstrate how fluorescence cross-correlation spectroscopy (FCCS) can be performed on the EI-FLEX system to characterise the formation of a ternary complex. A model DNA oligo system was used for the purposes of this application note, although FCCS is also ideal for studying other ternary complexes, such as PROTACs, molecular glues, and bispecific antibodies.
Here, direct quantification of ternary complex formation was performed using FCCS. We generated hook plots to establish the optimal concentration of the unlabelled oligo to produce the desired stoichiometry and avoid unwanted dimers. The impact of salt concentration on hybridisation kinetics was also investigated.Â
Overview of this application note:
- FCCS provides a direct quantification of ternary complexes in solution, whereby cross-correlation is only detected when all three subunits have bound
- Hook plots produced by titration of the unlabelled oligo identify the optimal concentration for ternary complex formation
- Time-resolved kinetics of ternary complex formation can be calculated using FCCS
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Figure 1 – Cross-correlation provides a direct readout of ternary complex formation
(A) Schematic of ternary complex formation of oligo 1 (green label), oligo 2 (no label) and oligo 3 (red label).
(B) Cross-correlation measured on the EI-FLEX with 20nM oligos 1 and 3 and varying concentrations of oligo 2 (coloured datapoints). The data was fitted with a diffusion model accounting for possible triplet states (black lines). All measurements were acquired in TE buffer supplemented with 150mM NaCl and 1% glycerol.
