robinson-et-al-2023-cellular-visualization-of-g-quadruplex-rna-via-fluorescence-lifetime-imaging-microscopy (1).pdf (5.87 MB)
Cellular visualization of G-quadruplex RNA via fluorescence- lifetime imaging microscopy.
journal contributionposted on 2024-01-11, 12:41 authored by Jenna Robinson, Stine G Stenspil, Karolina Maleckaite, Molly Bartlett, Marco Di Antonio, Ramon Vilar, Marina K Kuimova
Over the past decade, appreciation of the roles of G-quadruplex (G4) structures in cellular regulation and maintenance has 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 in a dense and structurally diverse cellular environment is challenging. The use of fluorescent 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 G4s 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, independent of the local probe concentration. Furthermore, TOR-G4 primarily colocalizes with RNA in the cytoplasm and nucleoli of cells, making it the first lifetime-based probe validated for exploring the emerging roles of RNA G4s in cellulo.