Real-Time Observation of G-Quadruplex Dynamics Using Single-Molecule FRET Microscopy

The potential importance of G-quadruplex structures was implied by the recent findings that the human POT1 disrupts G-quadruplex and stimulates the telomerase activity. A solid understanding of the range of conformations that can be adopted by guanine-rich sequences can potentially shed much light on the molecular mechanisms underlying certain human diseases related to telomeres. Furthermore, structure-based design of chemotherapeutic drugs for cancer might be realized by addressing different types of G-quadruplex structures. Using the unique capabilities of single-molecule spectroscopy, we have recently reported on the intricate dynamic structural properties of a minimal form of human telomeric DNA. Here, we present the detailed step-by-step methods for the real-time observation of G-rich DNA sequences by means of single-molecule FRET microscopy and provide the protocols for vesicle encapsulation and surface immobilization assays. Such assays provide a firm basis for future studies aimed at elucidating the interaction between telomeric DNA and telomere-associated proteins as well as the synthetic therapeutic agents that specifically stabilize certain G-quadruplex topologies.