Tetrazine as a general phototrigger to turn on fluorophores

Abstract

Light-activated fluorescence affords a powerful tool for monitoring subcellular structures and dynamics with enhanced temporal and spatial control of the fluorescence signal. Here, we demonstrate a general and straightforward strategy for using a tetrazine phototrigger to design photoactivatable fluorophores that emit across the visible spectrum. Tetrazine is known to efficiently quench the fluorescence of various fluorophores via a mechanism referred to as through-bond energy transfer. Upon light irradiation, restricted tetrazine moieties undergo a photolysis reaction that generates two nitriles and molecular nitrogen, thus restoring the fluorescence of fluorophores. Significantly, we find that this strategy can be successfully translated and generalized to a wide range of fluorophore scaffolds. Based on these results, we have used this mechanism to design photoactivatable fluorophores targeting cellular organelles and proteins. Compared to widely used phototriggers (e.g., o-nitrobenzyl and nitrophenethyl groups), this study affords a new photoactivation mechanism, in which the quencher is photodecomposed to restore the fluorescence upon light irradiation. Because of the exclusive use of tetrazine as a photoquencher in the design of fluorogenic probes, we anticipate that our current study will significantly facilitate the development of novel photoactivatable fluorophores.

Fig. 1. Schematic representation of fluorescence turn-on of tetrazine-fluorophore derivatives using the tetrazine phototrigger.

Fig. 2. (A) Reaction scheme of light photolysis of Tz-BODIPY 1 and the ¹H NMR spectra of Tz-BODIPY 1 obtained at the indicated light irradiation times using a 254 nm handheld UV light. (B) Fluorescent emission spectral change of Tz-BODIPY 1 after light activation. (C) Change of fluorescence intensity at 509 nm during light irradiation using a 254 nm handheld UV light.

Fig. 3. (A) Photoactivation of Tz-BODIPY derivatives in A431 cells using 405 nm laser activation. Confocal images were obtained before and after 405 nm light activation of Tz-BODIPY derivatives. Scale bar = 10 μm. (B) Photoactivation of organelle-targeting Tz-BODIPY probes in A431 cells. A431 cells were incubated with Tz-BODIPY-TPP 9, Tz-BODIPY-MOR 7, or Tz-BODIPY-Ts 8, and the corresponding commercial organelle-targeting dye, followed by photoactivation using a 405 nm laser. Commercial MitoView™ 633, ER-Tracker™ Blue-White DPX, and LysoView™ 633 were used as markers for mitochondria, endoplasmic reticulum, and lysosomes, respectively. Scale bar = 10 μm.

Fig. 4. Synthetic route to organelle-targeting Tz-BODIPYs.

Fig. 5. PALM imaging of histone proteins using the Tz-BODIPY-Halo probe. (A) Site-specific labeling of the protein of interest (POI) with the Tz-BODIPY-Halo probe for fluorescence imaging. (B) Widefield image and corresponding PALM images of H2B labeled with H2B-HaloTag in CHO–K1 cells. (C) Histogram plot of the localization accuracy of PALM images in (B). Scale bar = 1 μm.