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

C. P. Mountford, A. R. Mount, S. A. G. Evans, T. -J. Su, P. Dickinson, A. H. Buck, C. J. Campbell, J. G. Terry, J. S. Beattie, A. J. Walton, P. Ghazal, J. Crain, Colin Campbell

Research output: Contribution to journalArticlepeer-review

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 (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.

Original languageEnglish
Pages (from-to)839-845
Number of pages7
JournalJournal of fluorescence
Volume16
Issue number6
DOIs
Publication statusPublished - Nov 2006

Keywords

  • nucleic acids
  • fluorescence
  • energy transfer
  • imaging
  • RESONANCE ENERGY-TRANSFER
  • HOLLIDAY JUNCTIONS
  • MODEL

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