Fine-tuning of highly bright benzo[c,d]indole-oxazolopyridine cyanine dye for nucleolar RNA imaging in living cells

Here we report on fine-tuning of highly bright fluorogenic cyanine dye, benzo[c,d]indole-oxazolo[5,4-c]pyridine (BIOP: λ_em = 570 nm, Φ_free = 0.00038, Φ_bound = 0.52), recently developed by our group for nucleolar RNA imaging in living cells. We tuned an emission maximum to the longer wavelength by replacing oxazolopyridine unit with its isomer. The resulting probe with oxazolo[4,5-b]pyridine, named BIOP [4,5-b], exhibited a significant off-on signaling ability for RNA (λ_em = 580 nm, Φ_free = 0.002, Φ_bound = 0.46) that almost compared with BIOP. BIOP [4,5-b] was applicable to live-cell imaging, where wash-free protocol was available. Importantly, the slight change in spectral features would be expected to minimize the false signal from BIOP [4,5-b] in co-staining experiments with typical green-emissive dyes. In addition, thanks to the unique spectral shape that is typical of cyanine dyes, we demonstrate that yellow-emissive BIOP [4,5-b] also did work as a pseudo red-emissive dye (λ_em > 600 nm) for live-cell RNA imaging, for which we just switch filter set to typical one for real red-emissive dyes (Ex 560/40 nm; Em 645/75 nm).