Lowering Entropic Barriers in Triplex DNA Switches Facilitating Biomedical Applications at Physiological pH

Triplex DNA switches are attractive allosteric tools for engineering smart nanodevices, but their poor triplex‐forming capacity at physiological conditions limited the practical applications. To address this challenge, we proposed a low‐entropy barrier design to facilitate triplex formation by introducing a hairpin duplex linker into the triplex motif, and the resulting triplex switch was termed as CTNS ds . Compared to the conventional clamp‐like triplex switch, CTNS ds increased the triplex‐forming ratio from 30 % to 91 % at pH 7.4 and stabilized the triple‐helix structure in FBS and cell lysate. CTNS ds was also less sensitive to free‐energy disturbances, such as lengthening linkers or mismatches in the triple‐helix stem. The CTNS ds design was utilized to reversibly isolate CTCs from whole blood, achieving high capture efficiencies (>86 %) at pH 7.4 and release efficiencies (>80 %) at pH 8.0. Our approach broadens the potential applications of DNA switches‐based switchable nanodevices, showing great promise in biosensing and biomedicine.