Figure 8.

Isothermal FRET assay. (A) Schematics of the experiment: classic FRET melting assay is carried out monitoring the donor fluorescence (F) upon heating of the NA that disrupts the fluorescence donor (F)/quencher (Q) interaction (top). In our modified isothermal assay, the disruption of the F/Q interaction occurs at r.t., upon hybridization with the conjugate (bottom). (A, B) Isothermal FRET assay results obtained for 5′-FAM and 3′-TAMRA labeled LTR-III+IV (2.5 × 10⁻⁷ M) mixed with increasing concentrations (1–8×) of unlabeled competitor, (B) LTR-III+IV or (C) hTel, and a constant amount (1 × 10⁻⁶ M) of NDI-PNA 6 or PNA 7. ΔF% is calculated as (ΔF₁/ΔF₂) × 100, where ΔF₁ is the difference between the fluorescence of the labeled NA in the presence of both NDI-PNA 6 or PNA 7 and competitor, and the basal fluorescence of the NA alone, while ΔF₂ is the difference in fluorescence measured without competitor.