RESUMEN
Small molecular weight probes that can show a fluorescence signaling response upon binding to RNAs are promising for RNA imaging in living cells. Live-cell RNA imaging probes that can achieve a large light-up ability (>100-fold) and high Φbound value for RNA (>0.50) have been rarely reported to date. Here, benzo[c,d]indole-oxazolopyridine (BIOP), an unsymmetrical monomethine cyanine analogue, was newly developed as a bright and large light-up probe for imaging of nucleolar RNA in living cells. BIOP served as a yellow-emissive probe (λem = 570 nm) and exhibited a significant light-up response upon RNA binding (770-fold) with a high Φbound value (0.52). We demonstrated the advantages of BIOP over a commercially available RNA-staining probe, SYTO RNA select, for robust and sensitive RNA sensing by a systematic comparison of fluorescent properties for RNA. In addition, BIOP was found to possess high membrane permeability and low cytotoxicity in living cells. The examination of live-cell imaging revealed that BIOP exhibited emission in the nucleolus upon binding to nucleolar RNA much stronger than that of SYTO RNA select. Furthermore, BIOP facilitated the highly sensitive imaging of nucleolar RNA, in which 50 nM BIOP can stain nucleolar RNA in living cells with a 20 min incubation.
RESUMEN
RNA-binding small probes with deep-red emission are promising for RNA analysis in biological media without suffering from background fluorescence. Here benzo[c,d]indole-quinoline (BIQ), an asymmetric monomethine cyanine analogue, was newly developed as a novel RNA-selective probe with light-up signaling ability in the deep-red spectral range. BIQ features a significant light-up response (105-fold) with an emission maximum at 657 nm as well as improved photostability over the commercially available RNA-selective probe, SYTO RNA select. BIQ was successfully applied to the fluorescence imaging of nucleolar RNAs in living cells with negligible cytotoxicity. Furthermore, we found the useful ability of BIQ as a base surrogate integrated in peptide nucleic acid (PNA) oligonucleotides for RNA sequence analysis. BIQ base surrogate functioned as a deep-red light-up base surrogate in forced intercalation (FIT) and triplex-forming FIT (tFIT) systems for the sequence-selective detection of single-stranded and double-stranded RNAs, respectively.