Efficient and rational development of a new fluorescent probe specific for RNA G-quadruplex imaging in cells

The visualization of RNA G-quadruplexes in cells is critical to studying and understanding their biological functions. However, while various fluorescent probes have been developed for RNA imaging, very few RNA G-quadruplex-specific fluorescent probes have been found. To address this challenge, we reasoned that turning an RNA fluorescent probe into an RNA G-quadruplex-specific fluorescent probe by rational structural modification to improve the specificity would be an efficient new strategy. Therefore, the benzothiazole-indole-based RNA fluorescent probe BEDO-0 was selected to test this approach. Based on its sensing mechanism, we designed several candidate probes with the aim of improving their G-quadruplex binding selectivity while maintaining the specific fluorescence response. Among them, BEDO-3, which possesses an extended aromatic ring and amino side chain targeting the G-quartet and phosphate backbone, is the best candidate with the strongest G-quadruplex binding affinity and specificity. The relationships between the candidate structure and imaging performance were examined in detail. Furthermore, the practicability of using BEDO-3 as an RNA G-quadruplex-specific fluorescent probe was determined. These findings fully validate the feasibility of our proposed efficient and rational strategy, which will illuminate the development of brand-new fluorescent probes for RNA G-quadruplexes and even for other RNAs.