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

Peter A. Summers, Benjamin W. Lewis, Jorge Gonzalez-Garcia, Rosa M. Porreca, Aaron H. M. Lim, Paolo Cadinu, Nerea Martin-Pintado, David J. Mann, Joshua B. Edel, Jean Baptiste Vannier (), Marina K. Kuimova () and Ramon Vilar ()
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Peter A. Summers: Department of Chemistry, Molecular Sciencess Research Hub, White City Campus, Imperial College London
Benjamin W. Lewis: Department of Chemistry, Molecular Sciencess Research Hub, White City Campus, Imperial College London
Jorge Gonzalez-Garcia: Department of Chemistry, Molecular Sciencess Research Hub, White City Campus, Imperial College London
Rosa M. Porreca: Telomere Replication and Stability group, Medical Research Council – London Institute of Medical Sciences
Aaron H. M. Lim: Department of Chemistry, Molecular Sciencess Research Hub, White City Campus, Imperial College London
Paolo Cadinu: Department of Chemistry, Molecular Sciencess Research Hub, White City Campus, Imperial College London
Nerea Martin-Pintado: Oncode Institute, Hubrecht Institute–KNAW and University Medical Center Utrecht
David J. Mann: Department of Life Sciences, Imperial College London South Kensington
Joshua B. Edel: Department of Chemistry, Molecular Sciencess Research Hub, White City Campus, Imperial College London
Jean Baptiste Vannier: Telomere Replication and Stability group, Medical Research Council – London Institute of Medical Sciences
Marina K. Kuimova: Department of Chemistry, Molecular Sciencess Research Hub, White City Campus, Imperial College London
Ramon Vilar: Department of Chemistry, Molecular Sciencess Research Hub, White City Campus, Imperial College London

Nature Communications, 2021, vol. 12, issue 1, 1-11

Abstract: 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.

Date: 2021
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DOI: 10.1038/s41467-020-20414-7

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