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 1-7 tandem G-quadruplex (GQ) structures. Using 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 [4n]G-Tracts, (12, 16, 20...), demonstrate the broadest accessibility patterns where the PNA 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 double stranded telomere is most accessible. We interpret these results as the folding frustration being higher in [4n]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 telomere 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 double stranded telomere) is established. Significance StatementThe ends of eukaryotic linear chromosomes are capped by telomeres which terminate with a single-stranded overhang. Telomeric overhangs fold into compact structures, called G-quadruplex, that inhibit access to these critical genomic sites. We report single molecule measurements and computational modeling studies probing the accessibility of a set of human telomeric overhangs that covers a significant portion of the physiologically relevant length scale. We observe novel accessibility patterns which have a well-defined periodicity and show that certain regions are significantly more accessible than others. These accessibility patterns also suggest the underlying folding frustration of G-quadruplexes depends on telomere length. These patterns have significant implications for regulating the access of DNA processing enzymes and DNA binding proteins that can target telomeric overhangs.