Developing optical probes for cellular imaging of G-quadruplexes.

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G-quadruplex (G4) DNA is a non-canonical structure that forms in guanine-rich regions of the genome. There is increasing evidence showing that G-quadruplexes have important biological functions, and therefore molecular tools to visualise these structures are important. We propose to use small molecule probes to carry out live cell imaging studies to detect G4s that currently are simply not possible. We will first perform imaging of G4s formed in the telomeric region of chromosomes using Fluorescence Lifetime Imaging Microscopy (FLIM). Given high stability of our probes over time and the bright emission of the probes, we should be able to detect the chromosome tips even with diffraction limited resolution. Once we are able to visualize G4s in the telomeric region (which would be the first time this is done with a small molecule rather than an antibody), we will explore a number of cellular processes. We will first investigate the ability of increasing amounts of well-established G-quadruplex binders (e.g. pyridostatine, telomestatine and metal salphens) to displace the probe from the telomeric regions. This will provide unique insights into the binding dynamics of small molecules to G-quadruplexes in live cells and eventually could be adapted as a technique to screen non-fluorescent molecules that target G-quadruplexes in telomeres as potential anticancer drugs. Next, we will use our probes to study in cellulo, the interaction and function of DNA helicases on G-quadruplexes biology. In vitro studies have proposed that some histone chaperones including ATRX have the ability to bind G-quadruplexes. However, it is unclear if this factor recognises and act at G-quadruplexes in vivo and during the cell cycle. In this project we will attempt to visualise the role of ATRX in G4 maintenance directly, for the first time.