Selective molecular probes for G-quadruplex and G-quadruplex–duplex junctions: Cyanine dye interactions and structural insight

There is growing interest in hybrid DNA structures, particularly G-quadruplex-duplex junctions, as potential ligand binding sites. In this work, we investigate the interaction of two cyanine dyes (R9 and 3b), which differ in hydrophilicity, with various DNA structures, including duplex DNA, parallel and antiparallel G-quadruplexes, and a G-quadruplex-duplex junction. We employed molecular spectroscopic techniques (UV-visible absorption, circular dichroism, fluorescence), nuclear magnetic resonance (NMR) spectroscopy, multivariate analysis, and molecular docking studies. UV-visible absorption and circular dichroism provided insights into dye aggregation and the overall interaction modes, while NMR and docking yielded structural details. The results reveal that the two dyes exhibit distinct interactions with the studied DNA structures due to their differing behaviors in aqueous solution. The less polar 3b dye is aggregation-prone and prefers π-stacking with the terminal tetrads in the parallel G-quadruplex structure, leading to strong induced circular dichroism in the visible region and fluorescence responses toward parallel G-quadruplex. The more polar R9 dye forms defined non-stacking complexes, especially at structured duplex or G-quadruplex-duplex junction sites, showing selective fluorescence enhancement. NMR and molecular docking support the idea that R9 binds selectively and without aggregation, while 3b binds strongly and non-specifically, often resulting in precipitation.