Dual-Color Fluorescence Switch-On Probe for Imaging G‑Quadruplex and Double-Stranded DNA in Living Cells

A tripodal quinone-cyanine dye having one donor and three acceptors, that is, one quinone and three N-methylbenzothiazolium moieties, QCy­(MeBT)₃, was synthesized by simple Knoevenagel condensation between 2-hydroxybenzene-1,3,5-tricarbaldehyde and N-methyl-2-methylbenzothiazolium iodide. The 700 nm (λ_ex, 570 nm) and 600 nm (λ_ex, 470 nm) fluorescence emission of QCy­(MeBT)₃ was significantly and individually enhanced with the addition of G-quadruplex (G4) DNA and double-stranded DNA (dsDNA), respectively. The results of docking simulations and the response against the viscosity change revealed that the dual-fluorescence response was caused by the difference in the binding mode of QCy­(MeBT)₃ depending on the DNA structure. The results of fluorescence microscopy imaging experiments using QCy­(MeBT)₃ suggested that G4 DNAs and dsDNAs in the cell nucleus can be imaged with near-infrared (NIR, 700 nm) and red (600 nm) fluorescence emissions. Furthermore, pyridostatin-induced G4 formation in the living cells can be imaged with NIR fluorescence. The results indicated that QCy­(MeBT)₃ has huge potential to be a NIR-fluorescent molecular probe for analyzing the structural dynamics of nucleic acids in living cells with a normal fluorescence microscope.