G-quadruplexes formed in the 3′ telomere overhang (~200 nucleotides) have shown to regulate biological functions of human telomeres. The mechanism governing the population pattern of multiple telomeric G-quadruplexes is yet to be elucidated inside the telomeric overhang in a time window shorter than thermodynamic equilibrium. Using a single-molecule force ramping assay, we quantified G-quadruplex populations in telomere overhangs in a full physiological range of 99 to 291 nucleotides. We found that G-quadruplexes randomly form in these overhangs within seconds, which leads to a population governed by a kinetic, rather than thermodynamic, folding pattern. The kinetic folding gives rise to vacant G-tracts between G-quadruplexes. By targeting these vacant G-tracts using complementary DNA fragments, we demonstrated that binding to the telomeric G-quadruplexes becomes more efficient and specific for telomestatin derivatives.

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.

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We present single molecule experimental and computational modeling studies investigating the accessibility and folding landscape of human telomeric overhangs of physiologically relevant lengths. The overhangs contain 4-28 repeats of GGGTTA (G-Tract) sequence and accommodate 1-7 tandem G-quadruplex (GQ) structures. Using FRET-PAINT, we probed the distribution of accessible sites via a short imager strand, which is complementary to a G-Tract and transiently binds to unfolded sites. We report accessibility patterns that periodically change with overhang length and provide insights about the underlying folding frustration. Overhangs that have 4n G-Tracts, (12, 16…), demonstrate maximum frustration, while those with 4n+2 G-Tracts, (14, 18…), have minimal frustration. We also developed a computational model that suggests positive folding cooperativity between neighboring GQs is required for persistence of such patterns. Our experimental and computational studies suggest lower folding stability at the junction between single and double stranded telomeric DNA, which has implications for Shelterin complex formation.

To modulate biological functions, G-quadruplexes in genome are often non-specifically targeted by small molecules. Here, specificity is increased by targeting both G-quadruplex and its flanking duplex DNA in a naturally occurring dsDNA–ssDNA telomere interface using polyamide (PA) and pyridostatin (PDS) conjugates (PA-PDS). We innovated a single-molecule assay in which dissociation constant ( K d ) of the conjugate can be separately evaluated from the binding of either PA or PDS. We found K d of 0.8 nM for PA-PDS, which is much lower than PDS ( K d ∼ 450 nM) or PA ( K d ∼ 35 nM). Functional assays further indicated that the PA-PDS conjugate stopped the replication of a DNA polymerase more efficiently than PA or PDS. Our results not only established a new method to dissect multivalent binding into actions of individual monovalent components, they also demonstrated a strong and specific G-quadruplex targeting strategy by conjugating highly specific duplex-binding molecules with potent quadruplex ligands.