Quencher-Free FluorescenceMonitoring of G‑QuadruplexFolding

Guanine-rich sequences exhibit a high degree of polymorphism and can form single-stranded, Watson–Crick duplex, and four-stranded G-quadruplex structures. These sequences have found a wide range of uses in synthetic biology applications, arising in part from their structural plasticity. High-throughput, low-cost tools for monitoring the folding and unfolding transitions of G-rich sequences would provide an enabling technology for accelerating the prototyping of synthetic biological systems and for accelerating design–build–test cycles. Here, we show that unfolding transitions of a range of G-quadruplex-forming DNA sequences can be monitored in a FRET-like format using DNA sequences that possess only a single dye label, with no quencher. These quencher-free assays can be performed at low cost, with both cost and lead times ca. 1 order of magnitude lower than FRET-labeled strands. Thus, quencher-free secondary structure monitoring promises to be a valuable tool for the testing and development of synthetic biology systems employing G-quadruplexes.