Small-molecule-based CUT&RUN for G-quadruplex DNA using a cyclic naphthalene diimide-copper complex.

G-quadruplex (G4) DNA structures are widely distributed in biologically important genomic regions and are involved in gene regulation and genome stability. However, experimental identification of G4 sites in chromatin remains challenging. Here, we report a multifunctional small-molecule ligand, cNDI-CuGGHE, that integrates selective G4 recognition and copper-mediated DNA cleavage within a single molecular scaffold. Spectroscopic analysis showed that cNDI-CuGGHE preferentially binds to various G4 structures through terminal stacking on the G-quartet plane with binding constants on the order of 10⁶ M⁻¹ and significantly stabilizes G4 DNA compared with duplex DNA. Gel electrophoresis confirmed efficient and structure-dependent DNA cleavage in the presence of sodium ascorbate and hydrogen peroxide. Genomic DNA recovered from a cNDI-CuGGHE-based CUT&RUN-like assay in HeLa cells was analyzed by quantitative PCR using primers flanking the G4-forming regions of the c-kit and c-myc promoters. The reduced amplification of G4-spanning regions was consistent with preferential DNA cleavage at G4 sites in chromatin. These results demonstrate the feasibility of using small-molecule ligands as alternatives to antibody-enzyme conjugates for G4-targeted chromatin analysis.