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