DNA Beyond the Double Helix: Interplay and Targeting of G-Quadruplexes and i-Motifs

The transient formation of non-canonical structures, beyond the canonical double helix DNA, has been well documented and is implicated in a myriad of gene regulatory mechanisms, including telomere maintenance and cellular homeostasis. Genome portions enriched in guanine content are prone to fold transiently in G-quadruplexes. G-quadruplexes (G4s) are non-canonical tetra helical structures derived from stacking at least two G-tetrads coordinated by monovalent cations. G-tetrad is a planar square of four guanines interacting with each other through Hoogsteen base pairs. In most cases, the complementary strand of this genomic portion contains the sequence requirement for the formation of i-Motifs (iM), a non-canonical structure held together by the intercalation of hemi-protonated C+:C base pairs. While G-quadruplexes have been extensively investigated, the research about i-Motifs has received less attention, mainly due to the acidic pH requirement to fold in vitro. However, their existence within the cell was proved in 2017. Genome-wide analysis revealed that these structures can coexist in the same genomic sites and are enriched at most oncogene promoters, attracting interest as potential targets for therapeutic applications. The possibility of dual G4/iM targeting represents a possible approach to obtain cooperative therapeutic effects. We initially screened the interaction of common G4-ligands with a resolved minimal i-Motif structure and identified TmPyP4 as the most promising compound. NMR spectroscopy revealed a specific iM structural element involved in TmPyP4 binding, providing insights for dual G4/iM binder design. The impact on gene regulation obtained with ligands depends on the biological role played by G4 and iM within the promoter of a given gene. Most studies assume that these structures form in different stages of the cell cycle and exert opposite biological roles, in line with single-molecule data showing that steric hindrance impedes their simultaneous formation. Nevertheless, recent reports indicate possible overlapping roles in gene regulation. Given these controversial findings, we investigated the dynamic formation of both G4 and iM from a structural perspective, exploring whether they can form simultaneously or are mutually exclusive. To achieve this goal, we designed a double-stranded construct to simulate the genomic context, where two lateral duplexes flank a constrained central G/C-rich region. By combining spectroscopic and calorimetric data, supported by the design of mutated constructs, we characterized the thermodynamic profile of our system, providing strong findings indicating the concomitant formation of both G-quadruplex and i-Motif structures within our double-stranded construct. Furthermore, ongoing studies are addressing whether different G4 topologies may be accommodated within a duplex environment. Currently, our data suggest that both telomeric hybrid-type G4 and a parallel G4 derived from the cKit promoter region can fold once inserted within our constrained system. Our results suggest a possible interplay between G4 and iM structures, which may contribute to the fine-tuning of complex biological mechanisms. To gain structural insight into the system and explore potential G4/iM crosstalk, we employed SAXS analysis. Preliminary SAXS data are consistent with our spectroscopic findings, and further studies are in progress to refine a model describing the most probable relative orientation between the tetraplexes. Ultimately, we established a protocol to covalently link the 5’/3’ ends of our constructs to obtain circular molecules that remain closed after double-strand dissociation. In this way, we obtained constructs in which lateral duplexes unfold through a concentration-independent mechanism, making them suitable for single-molecule techniques operating under highly diluted conditions.