Rationally Designed\nG‑Quadruplex Selective\n“Turn-On” NIR Fluorescent Probe with Large Stokes Shift\nfor Nucleic Acid Research-Based Applications

Guanine-rich DNA/RNA sequences can form Hoogsteen bonds\nto adopt\nnoncanonical secondary structures called G-quadruplexes, and these\nhave been associated with diverse cellular processes. There has been\nconsiderable research interest in the design of G4-interacting ligands\nfor cellular probing of the G4 structure and understanding its associated\nbiological function. Most of the fluorescent G4 ligands either do\nnot have significant selectivity over other nucleic acid structures,\nhave high Stokes shift, or are not in the near-infrared (NIR) region,\nwhich limits its cellular visualization. The current work involves\nthe rational design and synthesis of NIR fluorescent probes comprising\na (Z)-1-methyl-2-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium\nscaffold. Among the designed molecules, <b>4a</b> exhibited\nfar-red fluorescence (λ<sub>max</sub> = 680 nm) with large Stokes\nshift (∼182 nm) upon selective binding to human telomeric G-quadruplexes.\nThe dye <b>4a</b> does not disturb the conformation and stability\nof G-quadruplexes, thereby making it suitable for nucleic acid research\nbased applications. Interestingly, <b>4a</b> showed remarkable\nselectivity over single- and double-stranded structures in contrast\nto a commercially available quadruplex binding probe, Thiazole orange\n(TO). The molecular docking studies indicate that <b>4a</b> binds\nat the groove region of the telomeric DNA G-quadruplex through π–π\nstacking interactions with the quinoline and amine-substituted phenyl\nring and with the phosphate backbone through anion−π\ninteractions with the benzothiazole ring. The designed molecule <b>4a</b> has interesting photophysical properties, cell permeability,\nand biocompatibility with minimal cytotoxicity. Fluorescence imaging\nstudies in live HeLa cells showed that probe <b>4a</b> binds\nto the transient population of the DNA G-quadruplex in the nucleus\nand RNA quadruplexes in the cytoplasm. In brief, G-quadruplex NIR\nfluorescent probe <b>4a</b> with a higher signal/noise ratio\nhas significant potential for cellular imaging studies and thus opens\navenues to decipher the biological pathways for better understanding\nof G-quadruplex biology.