Novel dimeric dyes based on the acridine orange chromophore: Synthesis, characterization and application in real-time PCR

Novel homodimeric dyes based on the acridine orange (AO) chromophore were synthesized and investigated. The photophysical properties measured free in solution and on binding to double-stranded DNA (dsDNA) were compared to those of widely used nucleic acid labelling dyes, namely SYBR Green I (SG) and YOYO-1. All studied dyes demonstrated negligible intrinsic fluorescence and strong fluorescence enhancement upon binding to dsDNA with quantum yields 0.45–0.55. Contrary to SG and YOYO-1, the obtained dyes showed better resistance to high temperatures, light and oxidizing reagents. The inhibitory effect and the effect on DNA melting temperature in a real-time quantitative polymerase chain reaction (qPCR) for all studied dyes were investigated. In qPCR experiments, dimeric dyes composed of two AO moieties coupled by neutral linkers comprising 2,5- or 2,6-disubstituted five- or six-membered heterocyclic fragments were less inhibitory to qPCR than SG at a broader range of dye concentrations (i.e. 0.75–1.75 μM). Moreover, these dyes are nontoxic when used at concentrations well above the qPCR working concentration. The strong fluorescent signal without inhibiting the qPCR along with the promotion of the formation of specific amplification products make these dyes suitable for high-resolution melt curve analysis and routine qPCR at a broad range of concentrations.