Probing the Folding Dynamics of Human Telomeric G-Quadruplex with Single-Molecule FRET

Abstract

Guanine-rich sequences consisting of several tandem repeats of TTAGGG are abundant in human telomeric DNA. They have been shown to fold into unique secondary structures under physiological conditions. One of these is the G-quadruplex structure that can undergo complex folding pathways [1-3]. Single-molecule fluorescence microscopy combined with Forster resonance energy transfer (FRET) allows for probing the conformation and dynamics of individual G-quadruplex molecules without time and population averaging and thus resolving their structural heterogeneity that is otherwise hidden in ensemble experiments [4,5].Here we utilize single-molecule FRET microscopy to probe the K+-induced folding dynamics of human telomeric G-quadruplex DNA. Our single-molecule experiments identify several distinct FRET states populated along the folding of G-quadruplexes. We find that initial dynamic interconversion occurs between three states - the unfolded state and two transient folded states, followed by transition into a dominant long-lived folded G-quadruplex conformation. Additional single-molecule FRET measurements, employing specific chemically modified G-quadruplex sequences, and advanced molecular modeling enabled linking the experimentally observed FRET states to particular G-quadruplex conformations. Our results thus allow uncovering a possible folding pathway of the human telomeric G-quadruplex structures.(1) Bochman, M. L.; Paeschke, K.; Zakian, V. A. Nature Reviews Genetics 2012, 13, 770.(2) Biffi, G.; Tannahill, D.; McCafferty, J.; Balasubramanian, S. Nature Chemistry 2013, 5, 182.(3) Gray, R. D.; Trent, J. O.; Chaires, J. B. J. Mol. Biol. 2014, 426, 1629.(4) Single-Molecule Techniques - A Laboratory Manual; Selvin, P. R.; Ha, T., Eds.; Cold Spring Harbor, NY, 2008.(5) Lee, J. Y.; Okumus, B.; Kim, D. S.; Ha, T. J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 18938.