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Time-resolved FRET and FLIM of four-way DNA junctions

Mountford, C. P., Mount, A. R., Evans, S. A. G., Su, T.-J., Dickinson, P., Buck, A. H., Campbell, C. J., Terry, J. G., Beattie, J. S., Walton, A. J., Ghazal, P. ORCID: and Crain, J. 2006. Time-resolved FRET and FLIM of four-way DNA junctions. Journal of Fluorescence 16 (6) , pp. 839-845. 10.1007/s10895-006-0125-5

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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 (c 1/2) 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.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Medicine
Publisher: Springer Verlag (Germany)
ISSN: 1053-0509
Date of Acceptance: 28 August 2006
Last Modified: 23 Oct 2022 14:20

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