Emerging accessibility patterns in long telomeric overhangs

We present single-molecule experimental and computational modeling studies investigating the accessibility of human telomeric overhangs of physiologically relevant lengths. We studied 25 different overhangs that contain 4-28 repeats of GGGTTA (G-Tract) sequence and accommodate one to seven tandem G-quadruplex (GQ) structures. Using the FRET-PAINT method, we probed the distribution of accessible sites via a short imager strand, which is complementary to a G-Tract and transiently binds to available sites. We report accessibility patterns that periodically change with overhang length and interpret these patterns in terms of the underlying folding landscape and folding frustration. Overhangs that have [41-J]G-Tracts, (12, 16, 20 ...) demonstrate the broadest accessibility patterns where the peptide nucleic acid probe accesses G-Tracts throughout the overhang. On the other hand, constructs with [4n+2]G-Tracts, (14, 18, 22 ...) have narrower patterns where the neighborhood of the junction between single- and doublestranded telomeres is most accessible. We interpret these results as the folding frustration being higher in [41-J]G-Tract constructs compared to [4n+2]G-Tract constructs. We also developed a computational model that tests the consistency of different folding stabilities and cooperativities between neighboring GQs with the observed accessibility patterns. Our experimental and computational studies suggest the neighborhood of the junction between single- and double-stranded telomeres is least stable and most accessible, which is significant as this is a potential site where the connection between POT1/TPP1 (bound to single-stranded telomere) and other shelterin proteins (localized on doublestranded telomere) is established.

Automated summary

In this study, the accessibility and folding characteristics of human telomeric overhangs of physiologically relevant lengths were investigated through experimental and computational modeling approaches. The results revealed a correlation between the accessibility patterns and the folding landscape of the overhangs, with periodic changes observed with varying overhang lengths and different G-Tract repeat sequences.