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Updated: Jul 19, 2026

Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1
Published on: September 18, 2019
Time-resolved FRET and FLIM of four-way DNA junctions
C P Mountford1, A R Mount, S A G Evans
1School of Physics, The University of Edinburgh, Room 4606 James Clerk Maxwell Building, Mayfield Road, Edinburgh, EH9 3JZ, UK.
Abstract:
Conformational transitions in a 4-way DNA junction when titrated with ionic solutions are studied using time-resolved fluorescence resonance energy transfer. Parameters characterising the transition in terms of critical ion concentration (c1/2) and the Hill coefficient for ion binding are obtained by fitting a simple two-state model using steady-state spectra. Data obtained from a fluorescence lifetime plate reader and analysed by fitting a single exponential to donor fluorescence lifetime decays are shown to be in good agreement with the parameters obtained from steady-state measurements. Fluorescence lifetimes, however, offer advantages, particularly in being independent of fluorophore concentration, output intensity, inhomogeneity in the excitation source and output wavelength. We demonstrate preliminary FRET-FLIM images of DNA junction solutions obtained using a picosecond gated CCD which are in agreement with results from a fluorescence lifetime plate reader. The results suggest that time-resolved FRET-FLIM is sensitive to subtle structural changes and may be useful in assays based on 4-way DNA junctions.
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