Chemical biology studies have demonstrated that G-quadruplex (G4) DNA is a potential target for tumor diagnosis and therapy. The development of fluorescent ligands targeting G4 structures is expected to advance the fields of precision medicine and molecular diagnostics. With the aim of improving the selectivity of G4 ligands, a new red fluorescent ligand DEAST was synthesized under mild conditions through modification of an existing benzothiazole scaffold. The ligand DEAST exhibited a large Stokes shift of voer 80 nm. The absorption and fluorescence experiments indicated that the ligand displayed a fast and sensitive "turn-on" fluorescence response specifically triggered by G4 DNA, which allowed it to be clearly distinguished from other nucleic acid structures, achieving a detection limit of 2.8 nM for G-quadruplex DNA (bcl-2). Notably, substitution of the methyl groups on the aniline moiety with ethyl groups enhanced the fluorescence sensitivity of the ligand toward G4. In addition, cell viability assays revealed that DEAST exhibited selective inhibition of tumor cell proliferation, suggesting its potential for development as a targeted anticancer therapeutic. Confocal fluorescence imaging indicated that DEAST preferentially accumulated in the mitochondria of live A549 cells. Urea-denaturation experiments further confirmed that the target site of DEAST in A549 cells was mitochondria G4 DNA. Collectively, this study will provide valuable insights for the structural optimization of fluorescent ligands, thereby promoting drug discovery and tumor diagnosis targeting G4.

Key words: G-quadruplex DNA, Fluorescent ligand, Cancer cells, Live cell imaging