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bioRxiv · 10.1101/2023.05.07.539224

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

Robinson, J.·Stenspil, S. G.·Maleckaite, K.·Di Antonio, M.·Vilar, R.·Kuimova, M.
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Abstract

Over the last decade, appreciation of the 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 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 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, independently of local probe concentration. Furthermore, TOR-G4 primarily co-localizes 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/539224v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@95c5faorg.highwire.dtl.DTLVardef@f8a897org.highwire.dtl.DTLVardef@1047f23org.highwire.dtl.DTLVardef@36e9ec_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Robinson, J., Stenspil, S. G., Maleckaite, K., Di Antonio, M., Vilar, R., Kuimova, M.. 2023-05-08. Cellular visualization of G-quadruplex RNA via fluorescence lifetime imaging microscopy. https://doi.org/10.1101/2023.05.07.539224

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