@article{dadee810b4fe45c6a902983212751968,
title = "Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy",
abstract = "Guanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4s). 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 G4s within nuclei of live and fixed cells. We present a FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4s 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 G4s in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.",
keywords = "Animals, Cell Line, Tumor, DNA/chemistry, DNA Helicases/genetics, Fanconi Anemia Complementation Group Proteins/genetics, Fibroblasts, Fluorescent Dyes/chemistry, G-Quadruplexes, Gene Knockdown Techniques, Humans, Indoles/chemistry, Intravital Microscopy/methods, Mice, Microscopy, Fluorescence/methods, Molecular Imaging/methods, RNA Helicases/genetics",
author = "Summers, {Peter A.} and Lewis, {Benjamin W.} and Jorge Gonzalez-Garcia and Porreca, {Rosa M.} and Lim, {Aaron H.M.} and Paolo Cadinu and Nerea Martin-Pintado and Mann, {David J.} and Edel, {Joshua B.} and Vannier, {Jean Baptiste} and Kuimova, {Marina K.} and Ramon Vilar",
note = "Funding Information: The Engineering and Physical Sciences Research Council (EPSRC) of the UK is thanked for financial support including a studentship to B.W.L. and P.C., and a fellowship for M.K.K. (EP/I003983/1). The Royal Society-Newton Fellowships is thanked for financial support to J.G.-G. The Singaporean government is thanked for funding a studentship to A.H.M.L. Imperial College London is thanked for support for this project via the Excellence Fund for Frontier Research. N.M.P. was supported by the European Commission and the production of the Xenopus egg extract was funded by the European Union{\textquoteright}s Horizon 2020 research and innovation program under the Marie Sk{\l}odowska-Curie grant agreement 750035 (ReXeG). Dr Marco Di Antonio is thanked for donating a sample of pyridostatin and useful discussions. Vannier lab{\textquoteright}s work is supported by the London Institute of Medical Sciences (LMS), which receives its core funding from UKRI (MRC) and by an ERC Starter Grant (637798; MetDNASecStr). Rosa Maria Porreca is funded by ERC Starter Grant (637798; MetDNASecStr). We thank the LMS/NIHR Imperial Biomedical Research Centre Flow Cytometry Facility for support. Publisher Copyright: {\textcopyright} 2021, The Author(s).",
year = "2021",
month = jan,
day = "8",
doi = "10.1038/s41467-020-20414-7",
language = "English",
volume = "12",
journal = "Nature Communications",
issn = "2041-1723",
publisher = "Nature Publishing Group",
number = "1",
}