A telomere carrying repetitive sequences ends with a single-stranded overhang. The G-rich overhang could fold back and bind in the major groove of its upstream duplex, forming an antiparallel triplex structure. The telomeric triplex has been proposed to function in protecting chromosome ends. However, we lack strategies to mechanically probe the dynamics of a telomeric triplex. Here, we show that the topological dynamics of a telomeric triplex involves 3′ overhang binding at the ds/ssDNA junction inferred by DNA mechanics. Assisted by click chemistry and branched polymerase chain reaction, we developed a rescue-rope-strategy for mechanically manipulating an artificial telomeric DNA with a free end. Using single-molecule magnetic tweezers, we identified a rarely forming (5%) telomeric triplex which pauses at an intermediate state upon unzipping the Watson–Crick paired duplex. Our findings revealed that a mechanically stable triplex formed in a telomeric DNA can resist a force of 20 pN for a few seconds in a physiological buffer. We also demonstrated that the rescue-rope-strategy assisted mechanical manipulation can directly rupture the interactions between the third strand and its targeting duplex in a DNA triplex. Our single-molecule rescue-rope-strategy will serve as a general tool to investigate telomere dynamics and further develop triplex-based biotechnologies.

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Comprehensive SummaryRecognition of CpG dinucleotide DNA in epigenetic information flow plays a pivotal role for cellular differentiation and development. The TET3 CXXC domain binds to CpG DNA, serving a basic epigenetic information reading mechanism. During the selective recognition of a CpG motif by a CXXC domain from crowded binding sites in a gene sequence, the protein‐DNA interactions are beyond CpG dinucleotide. However, the selective binding dynamics of CpG within a long DNA context by epigenetic enzymes have been rarely exploited, which is hard for ensemble methods to probe. Here, we used single‐molecule magnetic tweezers to quantitatively examine the dynamics of TET3's CXXC domain on a Hoxa9 promoter DNA. Our single‐molecule binding profile revealed that CXXC‐DNA interactions involve both CpG motifs and their flanking sequences. The residence time of TET3 CXXC differs by about 1000 times in five distinguished CpG clusters in the context of a CpG island. Moreover, we performed multi‐state hidden Markov modeling analysis on the zipping/unzipping dynamics of a CpG hairpin, discovering TET3 CXXC's preference on CpG motifs regarding the –2 to +2 flanking bases. Our results shed light on the selective binding dynamics of a CXXC on a gene sequence, facilitating studies on epigenetic information reading mechanisms.