High-throughput synthesis and selection of DNA G-quadruplex optical probes

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Specific guanine-rich sequences of DNA can fold into four-stranded structures known as G-quadruplexes (G4s), which are thought to be involved in a range of essential biological functions in healthy cells and are implicated in diseases. However, their detection in live biological samples remains very challenging. Recently, we have suggested a novel way of monitoring G4 formation in live cells, based on detection of the fluorescence lifetime of small optical probes [Nature Commun 2015, 6, 8178]. Despite this successful proof of concept study, our probes require further modification and optimization, in order to achieve high contrast signal upon binding to G4 DNA. Multiple possible variations in the probe structure, as well as the variations in DNA topology and concentration, mean that testing of new probes will be time consuming and laborious if performed using standard synthetic procedures and fluorescence lifetime analysis. Here we propose a strategy for high throughput synthesis and analysis of a large number of derivatives of thiazole orange (TO), a molecule which showed significant promise as a G4 DNA sensor in our proof of concept studies. This project will develop a novel methodology for high throughput synthesis and statistical testing of results in a fully automated way, allowing us to select ideal optical probes for G4 that are not accessible through standard routes within the timeframe of a PhD.Keywords: DNA optical probes, high throughput synthesis and analysis, fluorescence lifetime, molecular rotors, automation, data-driven optimisation