Dynamics Of Human Telomeric G-Quadruplex Probed By Single Molecule Fluorescence-Force Spectroscopy

G-quadruplexes (GQs) are constituted by stacks of G-quartets formed in guanine rich sequences in nucleic acids. Among its many functions, GQ plays a pivotal role in regulating telomere length in cells. Mechanistic understanding of dynamics of GQ is key to understanding its biological functions. Integrated single molecule force-fluorescence spectroscopy has been used herein to study the dynamics of human telomeric GQs under tension. GQ unfolding at physiological K+ concentration showed three features: a significant population resisted perturbation by force (∼35 pN) and remained in the folded state, while unfolding culminated into either a partially or completely unfolded state. Complete unfolding of GQs occurred stochastically within a wide spectrum of forces between 3 and 35 pN, even within the same molecule across different stretching cycles. Correspondingly, although GQs exhibited diverse refolding behavior, most of the refolding occurred within 5-10 pN. Such diversity in the forces required for complete unfolding of GQs was also observed with Na+. Replacement of a single guanine precluded GQ formation at lower metal ion concentrations (up to 100 mM) and only partially folded GQ-like species were induced which unfolded/refolded at lower forces with no evident hysteresis. At high metal ion concentrations (1M of Na+/K+) GQ with single point mutation showed an unfolding/refolding behavior redolent of GQ, suggesting formation of GQ-like species. The understanding of different levels of stability in human telomeric GQ can be leveraged into developing anti-cancer drugs.