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

Telomeric overhangs, composed of repeating GGGTTA sequences, can fold into multiple G-quadruplex (GQ) structures that are essential for maintaining genomic stability and regulating telomere length. Molecular crowding, a defining feature of the cellular environment, affects folding kinetics, conformation, and stability of individual GQs. 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 Forster Resonance Energy Transfer (smFRET) and FRET-Point Accumulation for Imaging in Nanoscale Topography (FRET-PAINT) to address this question. We examined the accessibility of telomeric overhangs capable of forming one to six GQs to a short complementary peptide nucleic acid (PNA) imager probe under molecular crowding conditions. These conditions were simulated by polyethylene glycol (PEG) molecules of two different molecular weights: 200 Da (PEG-200) and 6000 Da (PEG-6000). Our results reveal a systematic reduction in the overhang accessibility with increasing PEG concentration, showing approximately a 3-fold reduction at 30% (v/v) PEG-200 and an 8-fold reduction at 30% PEG-6000. We also observed a progressive compaction of the overhang as PEG concentration increased, suggesting molecular crowding promotes architectural condensation, thereby reducing probe accessibility. These findings offer new insights into how the crowded cellular environment may compact telomeric overhangs and modulate their structural and functional properties.