Emerging accessibility patterns in long telomeric overhangs

Abstract

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 [4n]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 double-stranded telomeres 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 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 double-stranded telomere) is established.

Shelterin reduces the accessibility of telomeric overhangs

“…We studied the accessibility of telomeric overhangs with 4–24 G-tracts in the absence or presence of 20 nM POT1 or shelterin using a FRET-PAINT assay (Figure 1 ) ( 32 , 42 ). The observed FRET levels are correlated with the position of the accessible G-Tracts with respect to the Cy3 donor located at the ssDNA/dsDNA junction.…”

“…Removing the dsTEL segment of the DNA constructs also eliminated the additional protection provided by shelterin. These findings suggest that tethering of POT1 to dsTEL tracts via the rest of shelterin stabilizes its binding to ssTEL and reduces the accessibility of the telomeric overhangs ( 42 ).…”

“…The compaction of ssTEL into higher-order structures via tandem GQs could potentially contribute to the robust protection of telomeres in coordination with telomere-associated proteins. Using single molecule FRET-PAINT (Förster Resonance Energy Transfer- Point Accumulation in Nanoscale Topography) ( 41 ), we recently investigated the accessibility of telomeric overhangs ( 42 ). In this assay, transient bindings of a complementary short peptide nucleic acid (PNA) strand to accessible regions of ssTEL are used to characterize the accessibility of the overhang.…”

“…In this assay, transient bindings of a complementary short peptide nucleic acid (PNA) strand to accessible regions of ssTEL are used to characterize the accessibility of the overhang. We showed that a FRET signal is detected between Cy5-PNA and Cy3-labeled telomeric DNA with up to 168 nt long overhang, presumably because ssTEL becomes highly compact upon tandem GQ formation ( 42 ). The PNA binding frequency to an overhang that contains four GGGTTA repeats (which can fold into a GQ) was an order of magnitude smaller than that of an overhang that contains a single GGGTTA repeat (cannot form a GQ), even though the 4-GGGTTA overhang contains four times as many potential binding sites ( 42 ).…”

“…We showed that a FRET signal is detected between Cy5-PNA and Cy3-labeled telomeric DNA with up to 168 nt long overhang, presumably because ssTEL becomes highly compact upon tandem GQ formation ( 42 ). The PNA binding frequency to an overhang that contains four GGGTTA repeats (which can fold into a GQ) was an order of magnitude smaller than that of an overhang that contains a single GGGTTA repeat (cannot form a GQ), even though the 4-GGGTTA overhang contains four times as many potential binding sites ( 42 ). Therefore, the majority of ssTEL tracts remain folded into GQs while PNA primarily binds to sites not protected within a GQ ( 42 ).…”