Mechanical Unfolding of Human Telomere G-Quadruplex DNA Probed by Integrated Fluorescence and Magnetic Tweezers Spectroscopy

Abstract

Telomeres are specialized chromatin structures that protect chromosome ends from nucleolytic processing by DNA repair machinery. The foundation of human telomere structure is a long array of tandem DNA sequences (TTAGGG), which can fold into a class of secondary structures known as G-quadruplexes (GQ). Previous studies revealed that GQs are highly polymorphic and a variety of topologically distinct forms may coexist under a single folding condition1-3. Single molecule Förster resonance energy transfer (smFRET) experiments demonstrated the dynamic nature of GQ structure, and suggested that inter-conversion between topologically distinct GQ folds proceeds through an obligatory transient intermediate4. To further characterize this GQ folding intermediate we developed employed an integrated fluorescence and magnetic tweezers spectroscopy technique, which permits the application of a wide range of stretching forces (0.1-50 picoNewtons) to individual GQ folds, together with simultaneous detection of GQ folding and unfolding through smFRET. Here, we present our investigation of the Na+-induced anti-parallel GQ conformation. Analysis of the force-dependent rate constants for the GQ folding and unfolding reactions provided an estimate of the position of transition state for GQ unfolding along the DNA stretching coordinate. The results suggest that telomere GQ is sensitive to mechanical force; only small perturbations can disrupt the entire structure. Furthermore, by comparing the GQ unfolded state with a single-stranded polyT DNA we show the unfolded GQ exhibits a significantly compacted non-native conformation reminiscent of the protein molten globule. 1. Ambrus, D., et al., Nucleic Acids Research 2006, 34, 2723-2735. 2. Luu, KN., et al., J Am Chem Soc 2006, 128, 9963-9970. 3. Gray, RD., et al., J Phys Chem B 2009, 113, 2676-2683. 4. Lee, JY., et al., Proc Natl Acad Sci U S A 2005, 102, 18938-18943.