It has been over a century since Ivar Bang first noted the unusual self-aggregating property of the guanylic acid-forming gel at high concentrations [1]. Gellert and colleagues later elucidated that this property is due to the formation of a four-stranded structure stabilized by hydrogen bonding between guanine moieties based on X-ray diffraction of guanylic acid fibers [2]. The observation gained biological attention when the telomeric region of DNA showed a stable self-associated structure under denaturing in vitro conditions, later confirmed to be a G-quadruplex (G4) structure [3], [4], [5], [6], [7].

With growing interest in the biological relevance of DNA G4 structures, early algorithms (G≥3 N1–7 G≥3 N1–7 G≥3 N1–7 G≥3) predicted over 300,000 potential G4 forming sequences in the human genome [8], [9]. Experimental approaches combining polymerase stop assays and next-generation sequencing revealed over 700,000 potential G4-forming structures in the human genome [10]. More than half of the potential G4 structures formed were found to have some bulge in the G-tract or long loops that did not fit in the previous algorithm, now termed potential non-canonical G4 structures. To account for both potential canonical and non-canonical G4 formations, Bedrat et al. developed a new G4 predicting algorithm called G4 hunter, which takes into account the G-richness and G-skewness to improve predictions [11]. In addition, machine learning approaches have been underway to predict potential G4 structures better [12]. Approaches like chromatin immunoprecipitation technique further revealed that G4 structures were predominately found to occur in the nucleosome depleted region (NDR) associated within promoter regions [13], [14]. Various in vitro experiments under biomimetic conditions have suggested the formation, stability, and dynamics of G4 structure inside cells, as discussed in the review.

Meanwhile, G4 structures were first visualized within telomeric regions of Stylonychia lemnae macronuclei, followed by the evidence of G4 structures in human cell and tissue [15], [16], [17], [18]. The study revealed that the G4 is a dynamic structure whose formation is cell cycle-dependent [16], [19]. Accumulating evidence indicated that G4 structures regulate diverse biological processes, including replication, transcription, translation, telomere biology, DNA repair, the cGAS–STING pathway, epigenetic regulation, neurobiology, and stem cell biology [20], [21], [22], [23], [24], [25], [26], [27], [28], [29]. This review discusses various approaches to modulate these regulatory systems by targeting the stability of G4 structures, utilizing small molecules, oligonucleotides, and peptides. While RNA G4 structures also mediate important biological phenomena [22], this review we will be focusing primarily on the DNA G4s.

In summary, we discuss different canonical and non-canonical conformations/topology of G4, biomimetic in vitro methods to probe formation, stability, dynamics, and approaches to alter G4 stability to modulate gene expression. We further discuss DNA G4s in the genome and the role of G4 structure in transcription, replication, and DNA repair. Finally, we explore the possibility and promise of DNA G4s in therapeutics.