Cellular function and nucleic acid structure are inextricably linked in biology. Whilst DNA often
exists as a double helical structure in vivo, G-quadruplexes (G4s) - that form in DNA and RNA
regions rich in guanines - are an alternative nucleic acid structure that have attracted attention
due to their significant enrichment in cancer cells. Through diverse interactions with regulatory
proteins, G4s are thought to regulate gene expression which may allow cancer cells to rapidly
respond to external stressors such as drug treatment. In my PhD, I have taken a two-pronged
approach to map and image G4s in cancer cells, particularly in the model of drug-resistant
ovarian cancer.
The first genomics-based method aimed to link G4s to transcriptional networks in drugresistant
cancer, utilising CUT&Tag, ChIP-, ATAC- and RNA- sequencing. Through this study, I
found that G4 formation, specifically at previously unstudied non-promoter sites, was associated
with elevated expression of genes that are critical for modulating the drug response. The second
approach used to study G4s was based on the design and characterisation of small-molecule,
fluorescent probes that allow G4s to be globally visualised in different cancer cell lines. Most
fluorescence intensity probes fail to achieve sufficient G4 binding selectivity to robustly detect
G4s in a complex cellular environment. In my work, I have taken a novel approach to visualise
G4s that considers the fluorescence lifetime of a probe rather than its fluorescence intensity.
Specifically, I have developed the first fluorescence lifetime probe validated for imaging the
emerging roles of RNA G4s within cells.