A DNA Polymerase Stop Assay for Characterization of G-Quadruplex Formation and Identification of G-Quadruplex-Interactive Compounds

Guanine-rich DNA sequences are able to spontaneously fold into G-quadruplex structures in the presence of certain metal cations. In the human genome, the majority of DNA G-quadruplexes form at the telomeres and regulatory regions of cancer-related genes. The formation of these structures is implicated in nuclear processes involving DNA, including transcription, DNA replication, and DNA repair. In the past few decades, small molecules which can stabilize these structures have been shown to suppress the telomere extension and to inhibit oncogene transcription. Therefore, DNA G-quadruplexes are thought to be attractive targets for new anticancer therapies. In this chapter, we describe step by step a DNA polymerase extension method for the characterization of G-quadruplex formation and identification of G-quadruplex-interactive compounds. This method is based on the principle that DNA polymerase is incapable to resolve G-quadruplex structure and pauses at 3′-end of the G-quadruplex forming region when it transverses to the 5′-end of the template. Results from the DNA polymerase stop assay can provide the basis for further studies aimed at elucidating the major G-quadruplexes formed by sequences consisting of more than four runs of contiguous guanines, as well as the specificity of G-quadruplex-interactive molecules in binding different G-quadruplex topologies.