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.

The stability and characteristics of a DNA Holliday junction switch.

A Holliday junction (HJ) consists of four DNA double helices, with a branch point discontinuity at the intersection of the component strands. At low ionic strength, the HJ adopts an open conformation, with four widely spaced arms, primarily due to strong electrostatic repulsion between the phosphate groups on the backbones. At high ionic strength, screening of this repulsion induces a switch to a more compact (closed) junction conformation. Fluorescent labelling with dyes placed on the HJ arms allows this conformational switch to be detected optically using fluorescence resonance energy transfer (FRET), producing a sensitive fluorescent output of the switch state. This paper presents a systematic and quantitative survey of the switch characteristics of such a labelled HJ. A short HJ (arm length 8 bp) is shown to be prone to dissociation at low switching ion concentration, whereas an HJ of arm length 12 bp is shown to be stable over all switching ion concentrations studied. The switching characteristics of this HJ have been systematically and quantitatively studied for a variety of switching ions, by measuring the required ion concentration, the sharpness of the switching transition and the fluorescent output intensity of the open and closed states. This stable HJ is shown to have favourable switch characteristics for a number of inorganic switching ions, making it a promising candidate for use in nanoscale biomolecular switch devices.