Classical thiazole orange and its regioisomer as fluorogenic probes for nucleolar RNA imaging in living cells

In contrast to well-established DNA-selective dyes for live cell imaging, RNA-selective dyes have been less developed owing to the challenges of making small molecules have RNA selectivity over DNA. Two kinds of dyes are now commercially available for nucleolar RNA imaging in cells, but these two dyes do not apply to living cells and have limited use in fixed and permeabilized cells. Herein, we report on thiazole orange (TO), a well-known nucleic acid stain, as a promising fluorogenic dye for nucleolar RNA imaging in living cells. TO shows clear response selectivity for RNA over DNA with a significant light-up property upon binding to RNA (lambda(em) = 532 nm, I/I-0 = 580-fold, and phi(bound)/phi(free) = 380) and is even applicable to wash-free imaging of living cells. More interestingly, 2TO, a regioisomer of TO in which the benzothiazole unit is connected to position 2 in the quinoline ring, performs much better (lambda(em) = 532 nm, I/I-0 = 430-fold, phi(bound)/phi(free) = 1200), having superior selectivity for RNA in both solution and living cells. The comparison with TO derivatives carrying different substituents at N1 of the quinoline ring reveals that the slight change in the TO framework significantly affects RNA selectivity, photostability and membrane permeability.

Automated summary

Compared to DNA-selective dyes, RNA-selective dyes are less developed due to the challenges of achieving RNA selectivity. This study introduces thiazole orange (TO) as a promising fluorogenic dye for live cell imaging of nucleolar RNA. The regioisomer 2TO shows even better selectivity and performance, with significant improvement in brightness and selectivity for RNA in both solution and living cells, compared to TO derivatives carrying different substituents.