Molecular machines constructed by three-dimensional (3-D) DNA walker have emerged as a hot topic in applications such as novel biosensors, cargo delivery platforms and intracellular imaging. Herein, we first propose a lame DNA walker that can randomly and autonomously move on microsphere-based 3-D track. The stochastic lame walker has a long leg mainly responsible for persistent movement and a short leg cutting substrates rapidly. Its motion is propelled by a nicking endonuclease cleavage of hybridized DNA tracks. Kinetic and persistent study show that the lame DNA walker enables reaction equilibrium at 30 min, need a cleat domain of at least 14 nucleotides and can persistently move on 3-D tracks with an average rate of 6.467 × 10⁻¹¹ M s⁻¹. We also demonstrate that the lame walker can be used to detect target DNA in the detection range of 10 pM–5 nM with high specificity by toehold exchange mechanism. This work will further expand the performance of 3-D DNA walkers and substantially contributes to the improved understanding of DNA walking systems.

由三维（3-D）DNA Walker构建的分子机器已成为诸如新型生物传感器，货物运输平台和细胞内成像等应用中的热门话题。在这里，我们首先提出了一个la脚的DNA步行者，它可以在基于微球的3-D轨道上随机自主地运动。随机la足助行器的腿长，主要用于持续运动，而腿部短，可以快速切割基材。它的运动是由杂交DNA轨道的切口内切核酸酶切割推动的。动力学和持续性研究表明，me脚的DNA沃克分子能够在30分钟内实现反应平衡，需要至少14个核苷酸的楔形结构域，并且能够以平均6.467×10 ^-11  M s ^-1的速率在3-D轨道上持续移动。我们还证明了me足助行器可以通过脚趾交换机制以高特异性检测10 pM-5 nM检测范围内的靶DNA。这项工作将进一步扩展3-D DNA Walker的性能，并为增进对DNA Walking系统的理解做出巨大贡献。