Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy

Summers, P. A.; Lewis, B.; Gonzalez-Garcia, J.; Lim, A. H. M.; Cadinu, P.; Porreca, R. M.; Martin-Pintado, N.; Mann, D.; Edel, J. B.; Vannier, J.-B.; Kuimova, M.; Vilar, R.

doi:10.1101/2020.04.01.019794

bioRxiv · 10.1101/2020.04.01.019794

Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy

Summers, P. A.·Lewis, B.·Gonzalez-Garcia, J.·Lim, A. H. M.·Cadinu, P.·Porreca, R. M.·Martin-Pintado, N.·Mann, D.·Edel, J. B.·Vannier, J.-B.·Kuimova, M.·Vilar, R.

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Abstract

Guanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4). Increasing evidence suggests that these G4 structures form in vivo and play a crucial role in cellular processes. However, their direct observation in live cells remains a challenge. Here we demonstrate that a fluorescent probe (DAOTA-M2) in conjunction with Fluorescence Lifetime Imaging Microscopy (FLIM) can identify G4 within nuclei of live and fixed cells. We present a new FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4 and apply it to a wide range of drug candidates. We also demonstrate that DAOTA-M2 can be used to study G4 stability in live cells. Reduction of FancJ and RTEL1 expression in mammalian cells increases the DAOTA-M2 lifetime and therefore suggests an increased number of G4 in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.

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Summers, P. A., Lewis, B., Gonzalez-Garcia, J., Lim, A. H. M., Cadinu, P., Porreca, R. M., Martin-Pintado, N., Mann, D., Edel, J. B., Vannier, J.-B., Kuimova, M., Vilar, R.. 2020-04-01. Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy. https://doi.org/10.1101/2020.04.01.019794

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