It has been >20 years since the formation of G-quadruplex (G4) secondary structures in gene promoters was first linked to the regulation of gene expression. Since then, the development of small molecules to selectively target G4s and their cellular application have contributed to an improved understanding of how G4s regulate transcription. One model that arose from this work placed these non-canonical DNA structures as repressors of transcription by preventing polymerase processivity. Although a considerable number of studies have recently provided sufficient evidence to reconsider this simplistic model, there is still a misrepresentation of G4s as transcriptional roadblocks. In this review, we will challenge this model depicting G4s as simple ‘off switches’ for gene expression by articulating how their formation has the potential to alter gene expression at many different levels, acting as a key regulatory element perturbing the nature of epigenetic marks and chromatin architecture.

G-Quadruplex DNA structures have attracted increasing attention due to their biological roles and potential as targets for the development of new drugs. While most guanine-rich sequences in the genome have the potential to form monomeric G-quadruplexes, certain sequences have enough guanine-tracks to give rise to multimeric quadruplexes. One of these sequences is the human telomere where tandem repeats of TTAGGG can lead to the formation of two or more adjacent G-quadruplexes. Herein we report on the modular synthesis via click chemistry of dimeric metal-salphen complexes (with NiII and PtII) bridged by either polyether or peptide linkers. We show by circular dichroism (CD) spectroscopy that they generally have higher selectivity for dimeric vs monomeric G-quadruplexes. The emissive properties of the PtII-salphen dimeric complexes have been used to study their interactions with monomeric and dimeric G-quadruplexes in vitro as well as to study their cellular uptake and localization.

Regulation of the epigenome is critical for healthy cell function but can become disrupted with age, leading to aberrant epigenetic profiles including altered DNA methylation. Recent studies have indicated that DNA methylation homeostasis can be compromised by the formation of DNA secondary structures known as G-quadruplexes (G4s), which form in guanine-rich regions of the genome. G4s can be recognised and bound by certain methylation-regulating enzymes, and in turn perturb the surrounding methylation architecture. However, the effect G4 formation has on DNA methylation at critical epigenetic sites remains elusive and poorly explored. In this work, we investigate the association between G4 sequences and prominent DNA methylation sites, termed ‘ageing clocks’, that act as bona fide dysregulated regions in aged and cancerous cells. Using a combination of in vitro (G4-seq) and in cellulo (BG4-ChIP) G4 distribution maps, we show that ageing clocks sites are significantly enriched with G4-forming sequences. The observed enrichment also varies across species and cell lines, being least significant in healthy cells and more pronounced in tumorigenic cells. Overall, our results suggest a biological significance of G4s in the realm of DNA methylation, which may be important for further deciphering the driving forces of diseases characterised by epigenetic abnormality, including ageing.

Over the last decade, appreciation of the potential roles of G-quadruplex (G4) structures in cellular regulation and maintenance have rapidly grown, making the establishment of robust methods to visualize G4s increasingly important. Fluorescent probes are commonly used for G4 detection in vitro, however, achieving sufficient selectivity to detect G4s with confidence in a dense and structurally diverse cellular environment is challenging. The use of fluorescence probes for G4 detection is further complicated by variations of probe uptake into cells, which may affect fluorescence intensity independently of G4 abundance. In this work, we report an alternative small-molecule approach to visualize G-quadruplexes that does not rely on fluorescence intensity switch-on and thus, does not require the use of molecules with exclusive G4 binding selectivity. Specifically, we have developed a novel thiazole orange derivative, TOR-G4, that exhibits a unique fluorescence lifetime when bound to G4s compared to other structures, allowing G4 binding to be sensitively distinguished from non-G4 binding, independently of local probe concentration. Furthermore, TOR-G4 primarily co-localizes with RNA in the cytoplasm and nucleoli of cells, making it the first fluorescence lifetime-based probe validated for exploring the emerging roles of RNA G-quadruplexes in cellulo.