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G-quadruplex (G4) structures are non-canonical DNA and RNA conformations formed in guanine-rich regions that play roles in gene regulation, genome stability, and RNA processing. However, targeting the approximately 700,000 G4s in the human genome with high specificity remains challenging due to their structural similarities. Despite their biological significance, this inability to selectively study or manipulate individual G4s presents a significant barrier to understanding their distinct roles in human cells and complicates efforts to dissect their contributions to cellular processes.

To address this limitation, we developed a strategy based on click chemistry to covalently link short single-stranded oligonucleotides (Os) to G4 ligands (GLs). This approach combines the stabilising properties of G4 ligands with the sequence specificity of guide oligonucleotides to create G4-ligand-oligonucleotide (GL-O) conjugates. The oligonucleotide forms double-stranded DNA (dsDNA) with the flanking region of the target G4, ensuring selective binding and stabilisation of the desired G4 structure. Through biophysical and biochemical assays, we demonstrated that this approach enables the selective stabilisation of individual target G4s, highlighting its utility for studying specific G4 structures.

In refining the GL-O platform, we systematically evaluated various linker configurations. This work demonstrated that longer and more flexible linkers enhance the adaptability of GL-O conjugates, allowing efficient targeting of G4s with varying distances between the G4-forming region and the complementary oligonucleotide binding sequence. This insight is particularly valuable for addressing steric hindrances and expanding the range of targetable G4 structures.

Additionally, we explored the broader principles of G4 ligand design by focusing on dispersion forces and electrostatic interactions. Synthesising heterocyclic G4 ligands and studying their interactions with G4s showed that dispersion components in arene-arene interactions and electron-deficient electrostatics are central to achieving high-affinity binding and stabilisation. These findings enhance the GL-O approach by providing a framework to fine-tune the stabilisation effect of the GL-Os, potentially reducing off-target effects.

In parallel, we pursued a separate project that examined G4 structures within human mitochondrial DNA (mtDNA), aiming to elucidate their roles in cellular function. Human mtDNA contains regions that have been predicted to form G4 structures in silico. We mapped these mtDNA G4s using high-resolution techniques and demonstrated their formation in vivo. Stabilisation or replication stalling increases their formation, potentially contributing to mitochondrial dysfunction and genomic instability in disease. 

Together, these findings advance our understanding of G4 biology, from selective targeting strategies to the unique dynamics of mitochondrial G4s, offering valuable insights into the biological roles of G4s in maintaining genome stability and regulating cellular processes.