Demonstration of ligand decoration, and ligand-induced perturbation, of G-quadruplexes in a plasmid using atomic force microscopy.
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.
Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, UK.
Ioanna
I
Mela
Ramon
R
Kranaster
Robert M
RM
Henderson
Shankar
S
Balasubramanian
J Michael
JM
Edwardson
D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D054856:G-Quadruplexes / Q000187:drug effects*; D006801:Humans; D008024:Ligands; D051379:Mice; D018625:Microscopy, Atomic Force; D010848:Picolinic Acids / Q000494:pharmacology; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
D000634:Aminoquinolines; D008024:Ligands; D010848:Picolinic Acids; D057127:Single-Chain Antibodies; C567962:pyridostatin; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases