DNA three-way junctions (3WJs) consist of a Y-shaped hydrophobic branch point connecting three double-stranded stems and are viewed as druggable targets for cancer treatment. They are also important building blocks for the construction of DNA nanostructures and serve as recognition elements for DNA aptasensors for a wide variety of diagnostic applications. However, visible fluorescent light-up probes for specific staining of DNA 3WJs are currently lacking. Herein, we report that a merocyanine containing the N-methylbenzothiazolium (Btz) acceptor vinyl linked to a 2-fluorophenolic (FPhO) donor (FPhOBtz) serves as a universal fluorescent turn-on dye for DNA 3WJs. Our evidence is based on a multifaceted approach to define the specificity and affinity of FPhOBtz for 3WJ DNA aptamers; the cocaine binding aptamer MN4, the cholic acid binding aptamer (CABA), and four steroid aptamers (DOGS.1, DISS.1, BES.1, DCAS.1). FPhOBtz exhibits impressive turn-on (up to 730-fold) fluorescence at 580 nm upon aptamer binding with low micromolar affinity. Direct FPhOBtz displacement from the 3WJ binding domain through competitive alkaloid and steroid binding provides immediate fluorescent read out for host–guest detection strategies in human blood serum in the low micromolar regime. Our results present the first visible light-up fluorescent probe for DNA 3WJ detection strategies.

中文翻译：

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用于 DNA 三通接头的可见荧光发光探针提供了宿主-客体生物传感应用

DNA 三向连接 (3WJ) 由连接三个双链茎的 Y 形疏水分支点组成，被视为癌症治疗的药物靶点。它们也是构建 DNA 纳米结构的重要组成部分，并作为 DNA 适体传感器的识别元件，用于各种诊断应用。然而，目前缺乏用于 DNA 3WJ 特异性染色的可见荧光发光探针。在此，我们报告了一种含有N与 2-氟酚 (FPhO) 供体 (FPhOBtz) 相连的-甲基苯并噻唑 (Btz) 受体乙烯基可用作 DNA 3WJ 的通用荧光开启染料。我们的证据基于多方面的方法来定义 FPhOBtz 对 3WJ DNA 适配体的特异性和亲和力；可卡因结合适体 MN4、胆酸结合适体 (CABA) 和四种类固醇适体 (DOGS.1、DISS.1、BES.1、DCAS.1)。FPhOBtz 在以低微摩尔亲和力结合适体时在 580 nm 处表现出令人印象深刻的开启（高达 730 倍）荧光。通过竞争性生物碱和类固醇结合从 3WJ 结合结构域直接置换 FPhOBtz 为低微摩尔水平的人血清中的主客体检测策略提供即时荧光读数。