Accessibility Patterns and Folding Frustration in Long Telomeric Overhangs

We present single molecule experimental and computational modeling studies investigating accessibility and folding landscape of human telomeric overhangs that contain 4-28 repeats of GGGTTA (G-Tract) sequence. These sequences reach physiologically relevant lengths and can accommodate 1-7 G-quadruplex (GQ) structures. Using FRET-PAINT, we probed the distribution of telomeric sites that are accessible external agents via a short imager strand that transiently binds to G-Tracts that are not folded into GQ. We report accessibility patterns that periodically change with overhang length and interpret these patterns in terms of their implications for folding frustration. Overhangs that have multiples of four G-Tracts (12, 16, 20…), demonstrate maximum folding frustration, while those with two additional G-Tracts, (14, 18, 22…), have minimal frustration. We also developed a computational model that suggests positive folding cooperativity and lower folding stability at 5´-end, compared to other regions, are required for persistence of such folding frustration and accessibility patterns.