Zhang Li, Xie Guoming, Qing Min
Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, P.R. China.
Chongqing Research Center for Pharmaceutical Engineering, College of Pharmacy, Chongqing Medical University, Chongqing 400016, P.R. China.
Anal Chem. 2025 Jul 22;97(28):15384-15392. doi: 10.1021/acs.analchem.5c02307. Epub 2025 Jul 10.
The inherent programmability of DNA, dictated by Watson-Crick base-pairing and sequence diversity, renders it a versatile biomolecule for nanoscale engineering. Toehold-mediated strand displacement (TMSD) enhances DNA strand exchange by over 4 orders of magnitude, underpinning applications in molecular circuits, biosensing, and nanodevices. However, its efficiency is constrained by the instability of the toehold duplexes and the reversibility of the initial binding step. Here, we introduce the click chemistry-accelerated toehold exchange (CCATE) reaction, which integrates TMSD with strain-promoted azide-alkyne cycloaddition (SPAAC) to irreversibly stabilize toehold binding and substantially accelerate strand displacement kinetics. By site-specifically conjugating azide and dibenzocyclooctyne (DBCO) near the toehold region, CCATE achieves a ∼1000-fold rate enhancement over conventional TMSD, particularly for short toeholds and mismatched substrates. This catalyst-free, highly orthogonal approach enables the rapid construction of DNA logic gates and amplification circuits, broadening the scope of DNA nanotechnology in biosensing, diagnostics, and molecular computing.
由沃森-克里克碱基配对和序列多样性所决定的DNA固有的可编程性，使其成为纳米级工程中一种多功能的生物分子。通过引入链置换（TMSD），DNA链交换增强了4个数量级以上，为分子电路、生物传感和纳米器件的应用奠定了基础。然而，其效率受到链置换双链体的不稳定性和初始结合步骤的可逆性的限制。在此，我们引入了点击化学加速链置换交换（CCATE）反应，该反应将TMSD与应变促进的叠氮化物-炔烃环加成（SPAAC）相结合，以不可逆地稳定链置换结合并显著加速链置换动力学。通过在链置换区域附近位点特异性地连接叠氮化物和二苯并环辛炔（DBCO），CCATE比传统的TMSD实现了约1000倍的速率提高，特别是对于短链置换和错配底物。这种无催化剂、高度正交的方法能够快速构建DNA逻辑门和放大电路，拓宽了DNA纳米技术在生物传感、诊断和分子计算中的应用范围。
