Tuning phenolic hemicyanines for ratiometric DNA sensing and live cell nuclear bioimaging applications

Abstract

Activatable fluorescent probes with turn-on emission in response to a biological target can reduce the background signal and improve the detection limit needed for biosensor and bioimaging resolution. Turn-on probes with dual emission (ratiometric probes) have further advantages, as they have self-calibration ability for more reliable detection. Herein, we demonstrate the tunability of the phenolic hemicyanine (HC) scaffold for both DNA biosensing and bioimaging applications using the quinine-binding DNA aptamer (MN4) as a host-guest biosensor platform and live ovarian cancer cells for nuclear imaging. The DNA aptamer MN4 contains a three-way junction (3WJ) consisting of a hydrophobic branch point connecting three double-stranded stems. Phenolic HCs bearing electron-withdrawing halogen substituents (FPhOBtz and Cl 2 PhOBTz) bind to the 3WJ of MN4 as the phenolate with strong turn-on emission. Subsequent displacement of the phenolate by the quinine target causes a turn-off emission response. In contrast, the parent phenolic HC (PhOBtz) and those bearing electron-donating groups (Me 2 PhOBtz and (MeO) 2 PhOBtz) exhibit dual (phenol and phenolate) fluorescence upon MN4 binding. Phenol excitation generates phenolate emission by an excited-state proton transfer (ESPT) process with DNA components serving as the proton acceptor. Quinine displacement of PhOBtz from the 3WJ of MN4 affords a turn-on ratiometric response with preferential light-up of phenol emission. The ability of the phenolic HCs to serve as cellular nuclear stains further highlights the tunability of the phenolic HC probes with the Cl 2 PhOBtz analog displaying superior staining ability. The simplicity and tunability of phenolic HCs make them attractive fluorescent probes for DNA sensing and bioimaging applications.