Nile Blue: a versatile scaffold for modulating DNA-interacting properties

Nile Blue (NB) is a red-emitting phenoxazine dye with affinity for DNA G-quadruplexes (G4s), yet its low fluorescence quantum yield in aqueous media and limited selectivity restrict biological applications. We synthesized three NB derivatives bearing hydroxy (5a), amino (5b), or carboxyl (5c) substituents. These derivatives exhibit blue-shifted absorption/emission maxima (approximately 40 nm) relative to NB, enhanced quantum yields (19-27% vs. 3% in aqueous buffer), and up to a 4-fold increase in brightness. Their interactions with parallel, hybrid, and antiparallel DNA G4s and a DNA hairpin were systematically investigated. The neutral 5a and positively charged 5b bound G4s presumably via stacking, resulting in hypochromism, red-shifted absorbance maxima, and fluorescence quenching. In turn, the negatively charged 5c showed negligible binding. Circular dichroism melting experiments revealed that 5a stabilizes only parallel G4s, whereas 5b stabilizes all G4 topologies, with neither compound affecting the DNA hairpin. Microscale thermophoresis demonstrated that 5b binds parallel G4s with submicromolar affinity, exhibiting up to 100- and 1000-fold selectivity over other G4 topologies and the DNA hairpin, respectively. Leveraging the efficient quenching of 5b by G4s, we suggested a red-fluorescent Nile Blue Displacement Assay (NBDA) for profiling G4 ligands, which provided dissociation constants comparable to those obtained in the standard thiazole orange-based displacement assay but with the advantage of monitoring emission enhancement in the red region. In cytotoxicity tests, 5b inhibited the proliferation of lung adenocarcinoma A549 cells with an IC₅₀ value comparable to that of the G4 ligand pyridostatin and a superior selectivity index vs. non-cancerous HEK293T cells. Due to its enhanced photophysical properties and selective cytotoxicity, 5b is a promising lead for further studies as an antitumor and/or theranostic agent.