Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy
Copyright policy )Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy
AbstractGuanine 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 thatDAOTA-M2can be used to study G4 stability in live cells. Reduction ofFancJandRTEL1expression in mammalian cells increases theDAOTA-M2lifetime and therefore suggests an increased number of G4s in these cells, implying thatFancJandRTEL1play a role in resolving G4 structures in cellulo.
- Medical Research Council United Kingdom
- University Medical Center Utrecht Netherlands
- Oncode Institute Netherlands
- Imperial College of London - Department of Chemistry
- Utrecht University Netherlands
Intravital Microscopy/methods, 570, Indoles, RNA Helicases/genetics, Intravital Microscopy, Science, General Biochemistry,Genetics and Molecular Biology, Fanconi Anemia Complementation Group Proteins/genetics, General Physics and Astronomy, Fluorescence, Article, Cell Line, Mice, Cell Line, Tumor, Journal Article, Animals, Humans, Fluorescent Dyes/chemistry, DNA/chemistry, Microscopy, Fluorescence/methods, Fluorescent Dyes, Microscopy, Tumor, DNA Helicases/genetics, Research Support, Non-U.S. Gov't, Q, DNA Helicases, General Chemistry, DNA, Fibroblasts, 540, Fanconi Anemia Complementation Group Proteins, Molecular Imaging, Indoles/chemistry, G-Quadruplexes, Microscopy, Fluorescence, Gene Knockdown Techniques, Molecular Imaging/methods, RNA Helicases
Intravital Microscopy/methods, 570, Indoles, RNA Helicases/genetics, Intravital Microscopy, Science, General Biochemistry,Genetics and Molecular Biology, Fanconi Anemia Complementation Group Proteins/genetics, General Physics and Astronomy, Fluorescence, Article, Cell Line, Mice, Cell Line, Tumor, Journal Article, Animals, Humans, Fluorescent Dyes/chemistry, DNA/chemistry, Microscopy, Fluorescence/methods, Fluorescent Dyes, Microscopy, Tumor, DNA Helicases/genetics, Research Support, Non-U.S. Gov't, Q, DNA Helicases, General Chemistry, DNA, Fibroblasts, 540, Fanconi Anemia Complementation Group Proteins, Molecular Imaging, Indoles/chemistry, G-Quadruplexes, Microscopy, Fluorescence, Gene Knockdown Techniques, Molecular Imaging/methods, RNA Helicases
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