Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy
Peter A. Summers, Benjamin W. Lewis, Jorge González‐García, Rosa María Porreca, Aaron H. M. Lim, Paolo Cadinu, Nerea Martín‐Pintado, David J. Mann, Joshua B. Edel, Jean‐Baptiste Vannier, Marina K. Kuimova, Ramón Vilar
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.