DNA structures alternative to the double helix have emerged as key features of the genome for the understanding of genetics and diseases. In particular, G-quadruplexes (G4s), four-stranded structures formed in guanine-rich sequences of cellular chromatin, are implicated in transcription, replication and genome stability. G4s may also present new opportunities for targeting in anti-cancer therapeutic interventions with small molecules. In this thesis, I expand the G4-profiling toolkit to better understand the biological roles of G4s, and potentially offer practical insights for G4-targeting drug development.

First, quantitative G4-chromatin immunoprecipitation with sequencing (qG4-ChIP-seq), a method for mapping and comparing G4 landscapes between samples, was used to study G4s in cell lines and patient-derived tumour xenografts from different breast cancer subtypes. Differentially enriched G4s in each cancer model were associated with copy number aberrations and single-nucleotide variants, as well as common breast cancer driver regions, suggesting a link between cancer genome instability and G4 structure formation.

Subsequently, to increase the versatility of G4 profiling, I developed G4-Cleavage Under Targets and Tagmentation (G4-CUT&Tag), a more efficient method to profile G4s with higher signal-to-noise ratio and 100-fold lower cellular input than G4-ChIP-seq. Further pushing the detection limit, I optimised G4-CUT&Tag for the first mapping of G4s at single-cell resolution. I demonstrated that individual cell identity can be discerned within a mixed cellular population based solely on single-cell G4 profiles. This result demonstrates that G4 signatures in individual cells relate to the fundamental identity of a cell. Next, I developed single-nuclei G4&RNA-seq, a multiomic method to simultaneously profile G4s and poly(A)-tailed RNA within the same single nucleus. Preliminary data provides proof-of-principle to directly associate G4 formation at individual loci with their transcriptional output within individual cells. Using this approach, I then showed its potential applications in discerning G4 landscapes in different cellular states with reference to cell cycle transcriptomic data within a mixed cell population. My work now enables future genomic investigations on cell-to-cell variation of a DNA secondary structure relative to other chromatin features that were previously not possible.

Overall, this thesis demonstrates advancements in G4-profiling methodologies and enables a high resolution and multi-dimensional exploration of the incidence of G4s and their functions.