Figure 3.

Probing telomeric triplex structures using single-molecule magnetic tweezers based on the rescue-rope strategy. (A) The existence of a telomeric triplex indicated by a hopping signal upon stretching a rescue-rope DNA. In the top panels, the left black trace shows an unfolding event without hopping (Zoom in for details in the right green-framed panel). In the bottom panels, the left red trace shows an infrequent hopping event (4.2% in 788 pulling-relaxing circles, zoom in for details in the right blue-framed panel). The assays were performed at 23°C in a buffer of 20 mM HEPES (pH 7.5), 0.00315% Tween-20(v/v), 0.1 mM EDTA and 100 mM NaCl. (B) Telomeric triplex detection in force-jump assays. The profile of manipulating forces is shown in the top panel. Rescue-rope construct undergoes a sharp transition during a force-jump from 6 to 20 pN (middle panel). Force-jump manipulations capture a rare event of unfolding a triplex, indicated by the pause as an intermediate state (bottom panel). Same buffer and temperature conditions as that in (A). (C) Probabilities of pausing events measured for the telomere and its mutants in force-jump assays. The telomere construct has eight repeats of TTAGGG. The mutant of permutation of 3′ overhang indicates that the bases in ssDNA tail are permutated. The mutant of deletion of 3′ overhang has the ssDNA tail deleted. The junction mutant stands for that the fifth and sixth repeats of TTAGGG are mutated at the ds/ssDNA junction. The upstream mutant represents that the first two repeats of TTAGGG are mutated which are far from the ds/ssDNA junction. In the internal loop mutant, the second TTAGGG is mutated to be mismatched as ATGTAG/ACCCTA. AA mutant changed the motif of TTAGGG to AAAGGG. Blue lines indicate mutated bases in cartoons. The buffer and temperature conditions were the same as that in (A) except that AA mutant was examined in a buffer containing 10 mM Tris (pH 7.5), 30 mM NaCl, 10 mM MgCl₂ and 0.00315% Tween 20. (D) A possible triplex in an antiparallel conformation formed by our telomeric DNA. Vertical lines represent Watson–Crick pairs. Asterisks stand for reverse Hoogsteen pairs.