PNA-ligand conjugates enable site-selective and directional targeting of adjacent G-quadruplexes

G-quadruplexes (G4s) are non-canonical DNA structures enriched in gene promoters and involved in transcriptional regulation, making them attractive drug targets, albeit difficult to target individually with high specificity due to their structural similarity. In the human c-KIT promoter, three adjacent G4 motifs (c-KIT1, c-KIT*, and c-KIT2) modulate oncogene expression, yet their individual contributions remain poorly understood, posing a significant challenge for selective targeting and underscoring the need for tools capable of discriminating between closely spaced G4s. Here, we report a dual-recognition strategy that combines the sequence specificity of peptide nucleic acids (PNAs) with the stabilizing properties of a G4 ligand to achieve site-selective and programmable targeting of neighboring G4s. Sequence-specific base pairing enables the PNA probe to recognize the flanking region between the c-KIT1 and c-KIT* motifs, positioning the ligand in proximity to a defined G4 structure and enabling directional and selective stabilization. Ligand conjugation at the N- or C-terminus of the PNA directs selective targeting of individual G4 units, while dual conjugation enables their simultaneous stabilization. This approach provides positional control not accessible with conventional small-molecule ligands and establishes a modular platform for targeting G4s within regions containing closely spaced motifs. A dual luciferase assay revealed that targeted G4 stabilization within the c-KIT promoter leads to distinct transcriptional outcomes in cells, indicating distinct regulatory roles of the c-KIT1 and c-KIT* motifs. This PNA-ligand platform enables site-specific recognition and discrimination between adjacent G4s and provides a general framework for selective chemical intervention in G4-regulated oncogenes.