Molecular Design of Fluorogenic Probes for Targeting rRNA: Indicator in FID Assay and Dye for Imaging of Nucleolar RNA in Living Cells

Here we report on fluorogenic indicators in the FID (fluorescence indicator displacement) assay for targeting the bacterial ribosomal RNA (Bac-rRNA) acceptor site (A-site). First, commercially available TO-PRO-3, a thiazole orange (TO) analogue with a trimethine bridge, was found to function as a deep-red fluorescent indicator with a high binding selectivity to the internal loop of the Bac-rRNA A-site (λ_em = 663 nm, K_d = 1.1±0.12 μM; pH 7.0, 20°C). TO-PRO-3 is the first example of deep-red indicators in the FID assay, which enables the assessment of the A-site binding capability of various test compounds including blue- and green-emitting compounds. Next, we developed a new class of small molecules based on the trimethylated napthyridine for internal loop binding, followed by conjugation with a triplex-forming PNA (peptide nucleic acid) containing TO base surrogate. The resulting small molecule-PNA oligomer conjugate, named the SPOC probe, is presently the strongest binding non-aminoglycosidic ligand to the Bac-rRNA A-site with a light-up signaling function (λ_em = 537 nm, K_d = 190 ±72 nM; pH 7.0, 25°C). Importantly, this is the first demonstration of small molecule-PNA conjugation to overcome the pH limitation of triplex-forming PNAs. Finally, we describe an asymmetric cyanine analogue (benzo[c,d]indole-quinoline), named BIQ as a novel RNA-selective dye with an off-on signaling ability in the deep-red spectral range (I /10 = 105-fold, λ_em = 657 nm; pH 7.0, 25°C). BIQ was applicable to the fluorescence imaging of nucleolar RNA in living cells. Also, BIQ functioned as a base surrogate in triplex-forming PNA probes. Taken together, this work hopes to contribute to more innovative probe designs for various RNA targets.