Impact of Molecular Crowding on Accessibility of Telomeric Overhangs Forming Multiple G-Quadruplexes.

Molecular crowding─a defining feature of the cellular environment─affects folding kinetics, conformation, and stability of G-quadruplex (GQ) structures. However, its influence on the overall architecture and accessibility of telomeric overhangs containing multiple GQs remains largely unexplored. In this study, we employed single-molecule FRET and FRET-PAINT to address this question. We examined the accessibility of telomeric overhangs, capable of forming 1-6 GQs, to a short complementary peptide nucleic acid (PNA) imager probe in the presence of 200 and 6000 Da polyethylene glycol (PEG) molecules (PEG-200 and PEG-6000). We observed a progressive compaction and architectural condensation of the overhang as PEG concentration increased. At 30% concentration, this compaction was accompanied by approximately 3-fold and 8-fold reduction in probe accessibility in PEG-200 and PEG-6000, respectively. These findings offer new insights into how the crowded cellular environment may compact telomeric overhangs and modulate their structural and functional properties.