Single Molecule Investigation of Tandem G-Quadruplex Structures Formed by Human Telomeric Sequence

Abstract

Human telomeric overhangs consist of 100-300 nucleotide long GGGTTA repeats, which can fold into multiple tandem G-quadruplex (GQ) structures. These structures protect and stabilize the telomeres and have been shown to inhibit telomerase activity, which made them an attractive target for anti-cancer drugs. Using single molecule Forster resonance energy transfer (FRET) spectroscopy, we interrogated the accessibility of 24-144 nucleotide long human telomeric DNA molecules (surface-immobilized and donor labeled) with short PNA molecules (acceptor labeled) that are complementary to a single repetition. These telomeric overhangs contain 4-24 GGGTTA repeats and can form 1-6 GQ structures when fully folded. However, when not fully folded, certain repeats can be accessed by PNA. PNA strands bind to these open sites on the telomeric DNA for a short period of time prior to dissociation, resulting in discrete FRET bursts as observed in the implementations of the FRET-PAINT method. These bursts were analyzed in terms of their dwell times, binding frequencies, and topographic distributions. Both dwell times and binding frequencies were greater for binding to intermediate regions of telomeric DNA compared to 3′-or 5′-ends. Finally, the binding frequency per telomeric repeat decreased monotonically with increasing telomere length, indicating a length-dependent compaction of the structure. These observations provide a potential framework to understand triggering of senescence or apoptosis when telomeric overhangs become shorter than a critical length (∼50 nt) where the necessary compaction may not be possible. The higher stability of the ends is consistent with GQ structures protecting telomeres against exonuclease activity.