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.

合成并研究了基于吖啶橙 (AO)发色团的新型同源二聚体染料。将溶液中游离态和与双链 DNA (dsDNA) 结合时测得的光物理性质与广泛使用的核酸标记染料SYBR Green I ( SG ) 和YOYO-1 进行比较。所有研究的染料在与 dsDNA 结合后显示出可忽略不计的固有荧光和强烈的荧光增强，量子产率为 0.45-0.55。与SG和YOYO-1相反, 所得染料对高温、光和氧化剂表现出更好的抵抗力。研究了所有研究染料在实时定量聚合酶链反应 (qPCR) 中的抑制作用和对 DNA 解链温度的影响。在qPCR实验中，由两个 AO 部分组成的二聚体染料通过包含 2,5-或 2,6-二取代五或六元杂环片段的中性接头偶联，在更广泛的染料浓度范围内（即0.75–1.75 微米）。此外，这些染料在使用浓度远高于 qPCR 工作浓度时是无毒的。不抑制 qPCR 的强荧光信号以及促进特异性扩增产物的形成使这些染料适用于高分辨率熔解曲线分析和广泛浓度的常规 qPCR。