title: Time-resolved FRET and FLIM of four-way DNA junctions.
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.
authors.affiliation: School of Physics, The University of Edinburgh, Room 4606 James Clerk Maxwell Building, Mayfield Road, Edinburgh, EH9 3JZ, UK.
authors.forename: C P
authors.initials: CP
authors.lastname: Mountford
authors.forename: A R
authors.initials: AR
authors.lastname: Mount
authors.forename: S A G
authors.initials: SA
authors.lastname: Evans
authors.forename: T-J
authors.initials: TJ
authors.lastname: Su
authors.forename: P
authors.initials: P
authors.lastname: Dickinson
authors.forename: A H
authors.initials: AH
authors.lastname: Buck
authors.forename: C J
authors.initials: CJ
authors.lastname: Campbell
authors.forename: J G
authors.initials: JG
authors.lastname: Terry
authors.forename: J S
authors.initials: JS
authors.lastname: Beattie
authors.forename: A J
authors.initials: AJ
authors.lastname: Walton
authors.forename: P
authors.initials: P
authors.lastname: Ghazal
authors.forename: J
authors.initials: J
authors.lastname: Crain
mesh_terms: D045566:DNA, Cruciform / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D009690:Nucleic Acid Conformation
chemical_list: D045566:DNA, Cruciform