Specific regulation of gene expression via modulation of G-quadruplex stability using reduction-sensitive azobenzene ligands

The G-quadruplex (G4) DNA structures are a noncanonical nucleic acid secondary structure involved in numerous fundamental biological processes, including replication and transcription. Accordingly, regulating the folding/unfolding of G-quadruplexes in genes is important for understanding the studies of G4-associated biological processes. Here, we propose a reduction-sensitive G4 binder for regulation of replication and transcription of G4-containing DNA, thereby enabling specific control over G4-associated biological processes. Biophysical studies, combined with molecular dynamics simulations, displays the reduction-sensitive azobenzene ligands target binding to G4 structure in template DNA, while dissociate the G4 structure by the reductive cleavage of the ligand. In vitro polymerase assays revealed that azobenzene ligands stalled the replication and transcription of template DNA at G4, and the inhibitory effects were significantly weakened on polymerase processivity with incubation of ligands at high concentration of GSH. Finally, we integrated the G4-forming sequence and G4-negative control into a synthetic reporter gene, respectively. We observed effective suppression in expression of G4-containing gene by adding the ligands compared to the corresponding G4-negative control in a way, and effective restoration of gene expression induced by their reductive states of ligands. Our findings highlight the potential of endogenous-responsive G4 binders in advancing specific regulation of biological process in a G4-dependent manner, but may also represent a promising strategy as a future therapeutic modality.