Cellular visualization of G-quadruplex RNA via fluorescence lifetime imaging microscopy

Abstract

Over the last decade, appreciation of the potential roles of G-quadruplex (G4) structures in cellular regulation and maintenance have rapidly grown, making the establishment of robust methods to visualize G4s increasingly important. Fluorescent probes are commonly used for G4 detection in vitro, however, achieving sufficient selectivity to detect G4s with confidence in a dense and structurally diverse cellular environment is challenging. The use of fluorescence probes for G4 detection is further complicated by variations of probe uptake into cells, which may affect fluorescence intensity independently of G4 abundance. In this work, we report an alternative small-molecule approach to visualize G-quadruplexes that does not rely on fluorescence intensity switch-on and thus, does not require the use of molecules with exclusive G4 binding selectivity. Specifically, we have developed a novel thiazole orange derivative, TOR-G4, that exhibits a unique fluorescence lifetime when bound to G4s compared to other structures, allowing G4 binding to be sensitively distinguished from non-G4 binding, independently of local probe concentration. Furthermore, TOR-G4 primarily co-localizes with RNA in the cytoplasm and nucleoli of cells, making it the first fluorescence lifetime-based probe validated for exploring the emerging roles of RNA G-quadruplexes in cellulo.

Near-Infrared Fluorescent Switch-On Probe for Guanine-Quadruplex Imaging with Extremely Large Stokes Shift

“…Considering the large fluorescence response of QCy­(BnBT) 3 with the addition of RNA G4 sequences (Figure S8), it can be concluded that QCy­(BnBT) 3 might stain both DNA G4 and RNA G4 in cells with NIR fluorescence. Since similar fluorescence microscopic images were also reported in the case of the other fluorescent small molecular probes, ,, our results are reliable.…”

“…The Stokes shift of the fluorescent G4 probe, QCy­(BnBT) 3 , reached ca. 220 nm, which is one of the largest Stokes shifts not only among the reported G4 binding fluorescent probes − but also among the reported fluorophores. − , The highly selective NIR fluorescence response of QCy­(BnBT) 3 upon the binding with G4 nucleic acids was observed both in the case of the test tube and in the cells, suggesting that the highly selective G4 imaging in the cells can be realized by using QCy­(BnBT) 3 . Since the highly selective ligand for G4 nucleic acids is one of the most important developmental targets in the field of chemical biology , and drug discovery, , our strategy described here would contribute greatly to researchers working in these fields.…”

Structure-Informed Design of an Ultra Bright RNA-activated Fluorophore

“…6 Fluorogenic aptamers have been used in diverse cellular imaging studies, including multi-color RNA imaging and single-molecule spectroscopy. 10,11,12,13,14 In addition to their use as imaging agents, uorogenic aptamers have been leveraged to create sensors. 15,16 The fact that these aptamers are genetically encodable, coupled with exibility in choicd of excitation-emission wavelengths makes them powerful tools for diverse applications.…”