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 Gquadruplexes (G4). Increasing evidence suggests that these G4 structures form in vivo with a crucial role in cellular processes, however, their direct observation in live cells remains a challenge. Here we unequivocally 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 have developed a new FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4, which can be applied to a wide range of drug candidates. We demonstrate that DAOTA-M2 can be used to study G4 stability in live cells. Disruption of FancJ DNA helicase activity increases G4 lifetime, directly establishing for the first time its biological activity in mammalian cells. Figure 1. In vitro fluorescence-lifetime of DAOTA-M2 bound to different DNA topologies. (a) Chemical structures of the DNA binders under study in this work. (b) Time resolved fluorescence decays of DAOTA-M2 (2 µM, black trace) and following the subsequent additions of dsDNA (CT-DNA, 20 µM, green trace) and then G4 (c-Myc, 4 µM, red trace). (c) Variation of the average lifetime (τw) of DAOTA-M2 in the presence of different G4 (red dots), ss/dsDNA (green dots) and ss/dsRNA (blue dots), adapted from reference 30 . (d) Fluorescence-lifetimes of DAOTA-M2 (2 µM) in aqueous buffer, buffered Xenopus egg extract (33 µL egg extract + 12 µL aqueous buffer), and in buffered cell extract supplemented with G4 (4 µM c-Myc) and dsDNA (44 µM ds26). Both measurements remained constant over 0.5 hr incubation at 21 °C. (e) In a mixture of DAOTA-M2 (2 µM), dsDNA (CT-DNA, 20 µM) and G4 (c-Myc, 4 µM), increasing amounts of PDS (5.3. 7.9. 9.9 and 14.4 µM, black traces) displaces DAOTA-M2 from a G4 to dsDNA environment. (f) Variation of τw in a mixture of DAOTA-M2 (2 µM), dsDNA (CT-DNA, 20 µM), and G4 (c-Myc, 4 µM), with increasing concentrations of G4 binders PDS (black dots) and Ni-salphen (orange dots), and a non-G4 binder DAPI (purple dots). See Figure S2 for example decays. (g) Variation of τw in a mixture of DAOTA-M2 (2 µM) and G4 (c-Myc, 4 µM), with increasing concentrations of Zn (grey dots), VO (purple dots), Ni (orange dots) and Cu (green dots) salphens. Error bars are ± standard deviation of three repeats. In less stated otherwise, all experiments in 10 mM lithium cacodylate buffer (pH 7.3) with 100 mM KCl.

The Genome Stability Maintenance DNA Helicase DDX11 and Its Role in Cancer

“…Besides, the DNA strands forming a G4 structure can run parallel or anti-parallel or have a mixed arrangement. Although several studies have revealed that G4 structures may impact many genome metabolism pathways, including gene transcription regulation, DNA replication and telomere maintenance, their physiological relevance has been disputed, and only very recently has their existence in live cells been robustly proved using innovative single-molecule real-time imaging technique based on G4-specific fluorescent probe [ 21 , 22 ]. However, it is well known that G4 is an important source of replication stress, especially in highly proliferating cells cancer cells, because they represent a roadblock for the advancing replication machineries.…”

G‐Quartets, 4‐Way Junctions and Triple Helices but Not DNA Duplexes: Planarization of Twisted Push–Pull Flipper Probes by Surface Recognition Rather Than Physical Compression

“…The most promising DNA motif for chemical flipper planarization were G‐quartets 4 ( Figure 3 ,A ) [33–46] . G‐Quartets have important functions in biology, from telomeres to gene expression.…”

“…To elaborate on flipper planarization, cMyc 4p and human telomers hTel22 4a were selected as single‐stranded parallel and antiparallel G‐quartets, respectively ( Figure 4 ,A and 4,B ) [39–42,46] . The sequence of the triple helix 6 was chosen because it is one of the few examples with characterized intercalators ( Figure 4 ,C ) [46,67] .…”

Role of the DDX11 DNA Helicase in Warsaw Breakage Syndrome Etiology

“…While formation and stability of these peculiar DNA structures has been analyzed in vitro by many different sophisticated biophysical techniques, their physiological relevance has been a matter of debate. Only very recently a robust evidence in support of their existence and dynamic formation in living cells has derived from innovative single-molecule real-time imaging technique based on G4-specific fluorescent probe [ 29 , 30 ]. Several studies revealed that G4 structures might impact many aspects of the genome metabolism, including gene transcription regulation, replication origin definition and activation, DNA replication and telomere maintenance [ 31 ].…”