DNA G-quadruplex detection system employing a protein fibril ligand

Thioflavin T (ThT), a typical probe of protein fibrils, also binds human telomeric G-quadruplexes with high specificity for other DNA structures. Upon binding, ThT fluoresces brightly, making it a promising G-quadruplex probe. Because ThT was originally used for detecting protein fibrils, false-positive detection and imaging of DNA G-quadruplexes is possible in the presence of proteins, such as in living cells. We therefore developed a system for the specific discrimination of DNA G-quadruplexes from protein fibrils through measurement of fluorescence resonance energy transfer (FRET) from ThT to a DNA duplex probe. We designed a complementary DNA (cDNA) that hybridizes with the single-stranded region near the DNA G-quadruplex–forming region. Hexidium iodide (HI), a typical DNA duplex fluorescent intercalator, was utilized as an acceptor for FRET from ThT. FRET occurred only when ThT bound the G-quadruplex region and HI bound the duplex region of the cDNA. Fluorescence analysis showed that this system can detect DNA G-quadruplexes even in the presence of protein fibrils.