Demonstration of ligand decoration, and ligand-induced perturbation, of G-quadruplexes in a plasmid using atomic force microscopy.

Abstract:: G-Quadruplexes are nucleic acid secondary structures consisting of a planar arrangement of four guanine residues. Potential G-quadruplex-forming sequences are widely distributed throughout the genome. Significantly, they are present in telomeres and are enriched in gene promoters and first introns, raising the possibility that perturbation of G-quadruplex stability might have therapeutic potential, for example in the treatment of cancer. Ligands that interact selectively with G-quadruplexes include both proteins and small molecules, although the interactions between ligands and their G-quadruplex targets have been monitored using indirect methods. In addition, the G-quadruplex targets have often been short DNA fragments. Here, we have used atomic force microscopy imaging to examine directly at the single-molecule level the interaction of ligands with G-quadruplexes generated during transcription of a plasmid containing a G-rich insert. We show that the structures produced during transcription are decorated specifically by the single-chain antibody HF1 and by the nuclear protein PARP-1, both of which are known to recognize G-quadruplexes. Our results provide clear structural evidence of G-quadruplex formation in a transcription-dependent case and demonstrate directly how small-molecule stabilizers and destabilizers can manipulate these structures in a biochemically functional system.

Authors:: I Mela, R Kranaster, RM Henderson, S Balasubramanian, JM Edwardson

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