Photophysical properties of fluorescent DNA-dyes bound to single- and double-stranded DNA in aqueous buffered solution

The absorption and fluorescence spectra, fluorescence quantum yields, lifet imes and time-resolved fluorescence spectra are reported for nine different fluorescent DNA-dyes. The work was initiated in search of a quantitative m ethod to detect the ratio of single-to-double stranded DNA (ssDNA/dsDNA) in solution based on the photophysics of dye-DNA complexes; the result is a c omprehensive study providing a vast amount of information for users of DNA stains. The dyes examined were the bisbenzimide or indole-derived stains (H oechst 33342, Hoechst 33258 and 4',6-diamidino-2-phenylindole), phenanthrid inium stains (ethidium bromide and propidium iodide) and cyanine dyes (Pico Green, YOYO-1 iodide, SYBR Green I and SYBR Gold). All were evaluated under the same experimental conditions in terms of ionic strength, pH and dye-DN A ratio. Among the photophysical properties evaluated only fluorescence lif etimes for the cyanine stilbene dyes allowed a convenient differentiation b etween ssDNA and dsDNA. The bisbenzimide dyes showed multiexponential decay s when bound to either form of DNA, making lifetime-based analysis cumberso me with inherent errors. These dyes also presented biexponential decay when free in aqueous buffered solutions at different pH. A mechanism for their deactivation is proposed based on two different conformers decaying with di fferent kinetics. The phenanthridinium dyes showed monoexponential decays w ith ssDNA and dsDNA, but there was no discrimination between them. High dye -DNA ratios (e.g. 1:1) resulted in multiexponential decays for cyanine dyes , resulting from energy transfer or self-quenching deactivation. Shifts in both absorption and fluorescence maxima for both ssDNA and dsDNA DNA-cyanin e dye complexes were small. Broadening of dye-ssDNA absorption and fluoresc ence bands for the cyanine dyes relative to dye-dsDNA bands was detected an d attributed to higher degrees of rotational freedom in the former.