Fluorescence lifetime imaging of G-quadruplex RNA dynamics in Alzheimer's disease using a novel nucleic acid-sensitive probe

Abstract

Real-time monitoring of G-quadruplex (G4) RNA dynamics is vital for understanding their biological roles in the progression and treatment of Alzheimer's disease. However, this remains challenging due to two key difficulties. First, current fluorescent probes lack sufficient selectivity for G4 RNA over G4 DNA and other non-G4 secondary structures in live-cell competitive environments. Second, fluorescence intensity-based imaging cannot detect subtle changes in G4 RNA because of variations in fluorophore uptake and photobleaching. Herein, we report a novel thiazole orange derivative (TOGR) for fluorescence lifetime imaging of G4 RNA in living cells. Structural modifications of thiazole orange enhance RNA-binding affinity and G4 selectivity. TOGR exhibits a unique fluorescence lifetime when bound to G4 structures, enabling sensitive detection of G4 formation independent of local probe concentration via FLIM. FLIM imaging reveals that TOGR primarily colocalizes with RNA in the cytoplasm and nucleoli. Due to its preferential RNA-binding affinity in competitive cellular environments, TOGR enables selective monitoring of G4 RNA dynamics, facilitating the exploration of novel roles of G4 RNA in cells without interference from G4 DNA. Importantly, the dynamic behavior of G4 RNA during Alzheimer's disease pathology and the effects of glucocorticoids on G4 RNA dynamics were successfully revealed using this lifetime-sensitive and RNA-selective imaging probe. This research not only paves the way for advanced probe design for detailed G4 RNA imaging but also lays the foundation for exploring G4 RNA-related pathological mechanisms in Alzheimer's disease.