Controlling Topology of a Telomeric G-quadruplex DNA With a Chemical Cross-link

DNA G-quadruplexes (G4s) are noncanonical structures formed in guanine-rich sequences. Within the human genome, they are nonrandomly distributed and influence DNA replication, gene expression, and genome maintenance. Numerous proteins involved in these processes have been identified as G4-binding proteins. However, the interaction of proteins with G4s in the context of double-stranded DNA in vitro has been difficult to study due to the transient nature of G4s in the presence of complementary DNA. To overcome this challenge, introducing internal covalent cross-links between distant nucleotides within the DNA sequence may promote pre-folding of G4 structures, thereby shifting the thermodynamic equilibrium toward G4-formation. We used a Cu(I)-catalyzed azide-alkyne cycloaddition to create a cross-link between 2'-O-propargylguanosine and N⁶-azidoethyl-2'-deoxyadenosine in the DNA telomeric sequence (TAG₃T)₂. A cross-link between G3 and A8 reinforced the parallel G4 topology that was stable in the presence of complementary DNA. Moreover, even in the presence of its complementary strand, this cross-linked G4 recruited the parent native DNA (TAG₃T)₂ to form a hybrid G4. These results suggest that cross-linking provides a useful tool for stabilizing noncanonical DNA structures in the presence of complementary strands, enabling their study within the context of genomic DNA.