Design and Properties of Ligand-Conjugated Guanine Oligonucleotides for Recovery of Mutated G-Quadruplexes

Abstract

The formation of a guanine quadruplex DNA structure (G4) is known to repress the expression of certain cancer-related genes. Consequently, a mutated G4 sequence can affect quadruplex formation and induce cancer progression. In this study, we developed an oligonucleotide derivative consisting of a ligand-containing guanine tract that replaces the mutated G4 guanine tract at the promoter of the vascular endothelial growth factor (VEGF) gene. A ligand moiety consisting of three types of polyaromatic hydrocarbons, pyrene, anthracene, and perylene, was attached to either the 3′ or 5′ end of the guanine tract. Each of the ligand-conjugated guanine tracts, with the exception of anthracene derivatives, combined with other intact guanine tracts to form an intermolecular G4 on the mutated VEGF promoter. This intermolecular G4, exhibiting parallel topology and high thermal stability, enabled VEGF G4 formation to be recovered from the mutated sequence. Stability of the intramolecular G4 increased with the size of the conjugated ligand. However, suppression of intermolecular G4 replication was uniquely dependent on whether the ligand was attached to the 3′ or 5′ end of the guanine tract. These results indicate that binding to either the top or bottom guanine quartet affects unfolding kinetics due to polarization in DNA polymerase processivity. Our findings provide a novel strategy for recovering G4 formation in case of damage, and fine-tuning processes such as replication and transcription.

Chemical Modulation of DNA Replication along G-Quadruplex Based on Topology-Dependent Ligand Binding

“…A number of G4 ligands, including porphyrin and phthalocyanine, that interact with G-tetrads through stacking interactions have been characterized. , Since the G-tetrad is a common feature of all G4 topologies, these ligands cannot be used to target a particular G4 conformation. Alternatively, specific small ligands that discriminate G4 topologies have been reported, which may have the capacity to interact directly with G4 loops and grooves. , Further, these ligands have been used for sequence- or topology-dependent G4 sensing via their fluorescence, suggesting the application in topology-dependent control of replication by G4 ligands. , Additionally, we have recently demonstrated the topology-control of human vascular endothelial growth factor ( VEGF ) G4. , We found that it contains an oxidized guanine in one G-tract, which forms an unstable hybrid G4, and the replication stall did not occur. The ligand-conjugated guanine tract could be replaced with a G-tract of the VEGF G4 due to the unstable hybrid G4, which would help in replication process recovery, and regenerated parallel G4 to recover the replication stall.…”

“…23,24 Additionally, we have recently demonstrated the topology-control of human vascular endothelial growth factor (VEGF) G4. 25,26 We found that it contains an oxidized guanine in one G-tract, which forms an unstable hybrid G4, and the replication stall did not occur. The ligand-conjugated guanine tract could be replaced with a G-tract of the VEGF G4 due to the unstable hybrid G4, which would help in replication process recovery, and regenerated parallel G4 to recover the replication stall.…”