Exploring Trianguleniums as Fluorescence Lifetime-based Probes for G-Quadruplex DNA

Session

Using FLIM and FCS to Determine Interactions and Dynamics

Authors

Miss Tigerlily Bradford (1), Dr Ben Lewis (1), Professor Marina Kuimova (1), Professor Ramon Vilar (1), Dr Jean-Baptiste Vannier (2)

Affiliations

1. Imperial College London
2. MRC London

Keywords

fluorescence, lifetime, G-quadruplex, DNA, ATRX, probes

Abstract text

G-quadruplex DNA, formed by Hoogsteen bonded guanine tetrads, is a non-canonical structure which is thought to impact crucial biological processes, such as gene expression and  regulation, specifically, DNA replication  in guanine-rich sequences like promoter regions of genes and at telomeres. While several methods exist to monitor nuclear G4 content, reliably visualising this structure in live cells is still widely considered a challenge.¹ 

For example, the “gold standard” antibody probes require cells to be fixed and, as a result, may not accurately reflect the biological environment of a live cell nuclei. Other probes rely on switch-on in fluorescence intensity upon G4 binding and hence are influenced by probe concentration. Fluorescence lifetime is concentration independent, and so does not have the same limitations as optical probes that depend on fluorescent intensity alone. ² Our group has studied one such fluorescence lifetime probe, a triangulenium-based dye termed DAOTA-M₂.³ We have shown that the lifetime of this probe changes when bound to different DNA topologies due to the conformational changes affecting the PET quenching process in vitro. ³  

We demonstrated that DAOTA-M₂ successfully localises in the nucleus of live cells. We used fluorescence lifetime imaging microscopy (FLIM), to assess the specificity of binding of DAOTA-M₂ to G4s in live cells by incubating the cells with a strong competitive G4 binder. The displacement of DAOTA-M₂ from G4s resulted in an expected decrease in lifetime observed from cells. This strong dependence of DAOTA-M₂ lifetime on the concentration of G4s available for binding was then utilized to monitor different concentrations of G4 between distinct cell lines. HeLa is a telomerase positive cell line characterised by endogenous production of the protein ATRX. ATRX is understood to indirectly unwind G4 structures,⁴ expected to result in a shorter lifetime of DAOTA-M₂, as compared to that of an ALT (alternative lengthening of telomeres) cell line, such as U2OS, which has no or little ATRX. However, when U2OS cells are forced to express ATRX under the inducible system established by Clynes et al., 2015 using a doxycycline treatment, the production of ATRX protein  in U2OS cells facilitates the unwinding of G4s. We were able to monitor the probe's lifetime variation depending on ATRX dependent G4 concentration in HeLa and U2OS cell lines, upon induction or deletion of ATRX genes, using FLIM of DAOTA-M₂, indicating that DAOTA-M₂ can detected a quantitative change in G4 character in live-cell nuclei. 

References

1.           Rhodes D, Lipps HJ. G-quadruplexes and their regulatory roles in biology. Nucleic Acids Res. 2015;43(18). doi:10.1093/nar/gkv862

2.           Hänsel-Hertsch R, di Antonio M, Balasubramanian S. DNA G-quadruplexes in the human genome: detection, functions and therapeutic potential. Nat Rev Mol Cell Biol. 2017;18(5). doi:10.1038/nrm.2017.3

3.           Shivalingam A, Vyšniauskas A, Albrecht T, White AJP, Kuimova MK, Vilar R. Trianguleniums as Optical Probes for G-Quadruplexes: A Photophysical, Electrochemical, and Computational Study. Chemistry - A European Journal. 2016;22(12). doi:10.1002/chem.201504099

4.           Clynes D, Jelinska C, Xella B, et al. Suppression of the alternative lengthening of telomere pathway by the chromatin remodelling factor ATRX. Nat Commun. 2015;6(1). doi:10.1038/ncomms8538