A Fluorescence-Based G-Quadruplex DNA Cleavage Assay

摘要

We describe a fluorescence-based G-quadruplex DNA cleavage assay based on intramolecular G-quadruplex-forming 5',3'-dual labeled oligonucleotides. By utilizing the difference in fluorescence emission spectra between an intact and cleaved quadruplex, the extent of G-quadruplex cleavage can be determined for a range of agents including hydroxyl radical, photo-activated porphyrin TMPyP4, and the oxo-metalloporphyrin Mn(=O)TMPyP4. This assay has been adapted to 96-well format for rapid screening, and the cleavage reaction samples can be subsequently directly analyzed by PAGE with fluorescence visualization. Agents that selectively cleave G-quadruplexes may serve as valuable probes for elucidating the formation and resolution of these structures in cells or as therapeutic agents. This assay should aid in identifying G-quadruplex cleavage agents for these various applications.