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.

Guanine‐rich sequences of DNA are known to readily fold into tetra‐stranded helical structures known as G‐quadruplexes (G4). Due to their biological relevance, G4s are potential anticancer drug targets and therefore there is significant interest in molecules with high affinity for these structures. Most G4 binders are polyaromatic planar compounds which π–π stack on the G4′s guanine tetrad. However, many of these compounds are not very selective since they can also intercalate into duplex DNA. Herein we report a new class of binder based on an octahedral cobalt(III) complex that binds to G4 via a different mode involving hydrogen bonding, electrostatic interactions and π–π stacking. We show that this new compound binds selectivity to G4 over duplex DNA (particularly to the G‐rich sequence of the c‐myc promoter). This new octahedral complex also has the ability to template the formation of G4 DNA from the unfolded sequence. Finally, we show that upon binding to G4, the complex prevents helicase Pif1‐p from unfolding the c‐myc G4 structure.

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.