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Time-Resolved FRET and FLIM of Four-way DNA Junctions

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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.

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Acknowledgments

This work was carried out as part of a DTI funded Beacon project. We also gratefully acknowledge the participation of Edinburgh Instruments Ltd in the work using the Fluorescence Lifetime Plate Reader and in particular Dr. Reynald Hurteaux for his assistance.

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Authors and Affiliations

  1. School of Physics, The University of Edinburgh, Room 4606 James Clerk Maxwell Building, Mayfield Road, Edinburgh, EH9 3JZ, UK

    C. P. Mountford & J. Crain

  2. Collaborative Optical Spectroscopy Micromanipulation and Imaging Centre (COSMIC), The University of Edinburgh, Mayfield Road, Edinburgh, EH9 3JZ, UK

    C. P. Mountford & J. Crain

  3. School of Chemistry, The University of Edinburgh, King’s Buildings, West Mains Road, Edinburgh, EH9 3JJ, UK

    A. R. Mount & S. A. G. Evans

  4. The Scottish Centre for Genomic Technology and Informatics, The University of Edinburgh, The Chancellor’s Building, 49 Little France Crescent, Edinburgh, EH16 4SB, Scotland, UK

    T.-J. Su, P. Dickinson, A. H. Buck, C. J. Campbell, J. S. Beattie & P. Ghazal

  5. Institute of Integrated Micro and Nano Systems, Scottish Microelectronics Centre, School of Engineering and Electronics, University of Edinburgh, Edinburgh, EH9 3JF, UK

    J. G. Terry & A. J. Walton

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  1. C. P. Mountford
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  2. A. R. Mount
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  3. S. A. G. Evans
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  4. T.-J. Su
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  5. P. Dickinson
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  6. A. H. Buck
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  7. C. J. Campbell
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  8. J. G. Terry
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  9. J. S. Beattie
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  10. A. J. Walton
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  11. P. Ghazal
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  12. J. Crain
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Mountford, C.P., Mount, A.R., Evans, S.A.G. et al. Time-Resolved FRET and FLIM of Four-way DNA Junctions. J Fluoresc 16, 839–845 (2006). https://doi.org/10.1007/s10895-006-0125-5

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  • DOI: https://doi.org/10.1007/s10895-006-0125-5

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