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

Seiichi Nishizawa, En Ting Tabitha Lee, Yukina Yoshino, Sayaka Yajima, Masafumi Rokugawa, Yusuke Sato

Research output: Contribution to journalArticlepeer-review


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 (λem = 663 nm, Kd = 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 (λem = 537 nm, Kd = 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, λem = 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.

Original languageEnglish
Pages (from-to)703-714
Number of pages12
Issue number12
Publication statusPublished - 2021


  • Bacterial A-site
  • Fluorescence imaging
  • Fluorescence indicator displacement assay
  • Molecular probe
  • Nucleolar RNA
  • Small molecule
  • Triplex-forming peptide nucleic acid

ASJC Scopus subject areas

  • Analytical Chemistry


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