Genomic DNA typically exists as a double helix stabilized by hydrogen bonding interactions between Watson-Crick base pairs. However, it is also known that DNA can adopt various other non-canonical conformations including hairpin, triplex, homo-duplex, quadruplex etc. depending on the DNA sequence and the intracellular environment. It has been hypothesised, and to an extent shown, that some of these non-canonical DNA structures are involved in various biological functions. Amongst these structures, four stranded structures known as G-quadruplexes (G4s) have received particular attention over the past two decades. However, to date, we are still lacking tools that allows us to visualizing G-quadruplexes directly in live cells. While several small-molecule probes that fluoresce upon interaction with DNA have been reported, none of them have been successful at imaging G-quadruplexes in a cellular environment. This is mainly due to the fact that they rely on changes in intensity which are not possible to track properly in a cellular environment. Recently, the host lab reported a novel strategy to image G-quadruplexes in live cells. This approach makes use of the changes in emission lifetime (rather than intensity) of optical probes upon their interaction with different topologies of DNA. Since lifetime is concentration independent, this approach can be successfully used to image G-quadruplexes in live cells. While this has proven to be a highly successful approach, it is still in its infancy since the probe developed so far has a number of limitations such as low brightness, relatively small lifetime range and low selectivity. Thus, this project was aimed to develop a new set of probes that could address all these issues and use them to image the dynamics of G-quadruplexes in live cells in real time via phosphorescence lifetime imaging microscopy (PLIM).
• Objective 1 (O1): Develop a high-throughput screening methodology to synthesise new ligands and Pt complexes with a range of substituents to tune their DNA selectivity (WP1).
• Objective 2 (O2): Develop automated processes to screen the phosphorescence of the new Pt-complexes (libraries from O1) in the absence and presence of different DNA structures (including G4s) (WP2).
• Objective 3 (O3): From O2, select the probes with the highest switch-on emission effect and selectivity for G4 DNA, and conduct detailed photophysical studies including the determination of affinity constants and the emission lifetime of the probes in the presence of different DNA structures (WP2).
• Objective 4 (O4): Select the probe(s) with the largest emission lifetime difference when bound to different DNA structures and study their cellular uptake and localisation using confocal microscopy (WP3).
• Objective 5 (O5): Following O4, conduct Phosphorescence Lifetime Imaging Phosphorescence (PLIM) with the selected probe(s) on live cells to establish the distribution of G4s. Different eukaryotic cell types (e.g. cancer vs. non-cancer) and at different stages of the cell cycle will be studied (WP4).
