The dynamics of forming a triplex in an artificial telomere inferred by DNA mechanics.

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.