Probing juxtaposed G-quadruplex and hairpin motifs using a responsive nucleoside probe: a unique scaffold for chemotherapy

Paucity of efficient probes and small molecule ligands that can distinguish different G-quadruplex (GQ) topologies poses challenges not only in understanding their basic structure but also in targeting an individual GQ form from others. Alternatively, G-rich sequences that harbour unique chimeric structural motifs (e.g., GQ-duplex or GQ-hairpin junctions) are perceived as new therapeutic hotspots. In this context, the epidermal growth factor receptor (EGFR) gene, implicated in many cancers, contains a 30 nucleotide G-rich segment in the promoter region, which adopts in vitro two unique architectures each composed of a GQ topology (parallel and hybrid-type) juxtaposed with a hairpin domain. Here, we report the use of a novel dual-app probe, C5-trifluoromethyl benzofuran-modified 2 '-deoxyuridine (TFBF-dU), in the systematic analysis of EGFR GQs and their interaction with small molecules by fluorescence and F-19 NMR techniques. Notably, distinct fluorescence and F-19 NMR signals exhibited by the probe enabled the quantification of the relative population of random, parallel and hybrid-type GQ structures under different conditions, which could not be obtained by conventional CD and H-1 NMR techniques. Using the fluorescence component, we quantified ligand binding properties of GQs, whereas the F-19 label enabled the assessment of ligand-induced changes in GQ dynamics. Studies also revealed that mutations in the hairpin domain affected GQ formation and stability, which was further functionally verified in polymerase stop assay. We anticipate that these findings and useful properties of the nucleoside probe could be utilized in designing and evaluating binders that jointly target both GQ and hairpin domains for enhanced selectivity and druggability.

Automated summary

The lack of efficient probes and small molecule ligands that can differentiate different G-quadruplex (GQ) topologies presents challenges in understanding their structure and targeting specific GQ forms. However, G-rich sequences with unique chimeric structural motifs have been identified as potential therapeutic targets. In this study, a novel dual-app probe was used to analyze the EGFR GQs and their interaction with small molecules. The probe exhibited distinct fluorescence and NMR signals, enabling the quantification of different GQ structures and their ligand binding properties. The findings could be utilized in designing binders that target both GQ and hairpin domains for enhanced selectivity and druggability.