Herein, a novel self-powered 3D DNA walking machine triggered by triple-helix molecular switch (THMS) has been proposed for microRNA-21 (miRNA-21) imaging in living cells. Impressively, the 3D DNA walking machine was constructed with swing arm and THMS co-functionalized gold nanoparticles, THMS could precisely customize as a multifunctional structure with variable configuration. Once the 3D DNA walking machine was delivered into living cells, THMS was promptly disassembled due to the formation of DNA duplex (miRNA-21/TAMRA-DNA2), thus the 3D DNA walking machine was immediately triggered on AuNPs. Meanwhile, FAM-DNA1 quickly self-hybridized into hairpin and exposed the Mg²⁺ ribonucleoside adenosine (rA) recognition site in the loop region. Sequentially, swing arm hybridized with hairpin FAM-DNA1 and cleaved it with the assistance of Mg²⁺. Finally, numerous FAM-labeled segments were released from AuNPs and further paired with the spare part of TAMRA-DNA2 in the miRNA-21/TAMRA-DNA2 duplex, resulting in the two fluorescent dyes approaching each other, which induced the efficient FRET from FAM to TAMRA, thereby turned the system into “ON” state for signal amplification. It is noteworthy that the ingenious design of the self-powered 3D DNA walking machine avoids the external addition of fuel DNA strands or protein enzymes, which enables the autonomous motion of the 3D DNA walking machine in complex cellular microenvironment, greatly simplifies the experimental steps and improves the efficiency of the walking machine. Moreover, this assay will provide a novel signal amplification strategy for evaluating intracellular miRNA levels.

本文提出了一种由三螺旋分子开关 (THMS) 触发的新型自供电 3D DNA 步行机，用于活细胞中的 microRNA-21 (miRNA-21) 成像。令人印象深刻的是，3D DNA 步行机由摆臂和 THMS 共功能化金纳米粒子构成，THMS 可以精确地定制为具有可变配置的多功能结构。一旦 3D DNA 步行机被输送到活细胞中，由于 DNA 双链体（miRNA-21/TAMRA-DNA2）的形成，THMS 被迅速分解，因此 3D DNA 步行机立即在 AuNPs 上触发。同时，FAM-DNA1迅速自杂交成发夹，暴露出Mg ²⁺环区的核糖核苷腺苷 (rA) 识别位点。随后，摆臂与发夹 FAM-DNA1 杂交并在 Mg ^{2+的帮助下将其切割}. 最后，大量 FAM 标记的片段从 AuNPs 中释放出来，并进一步与 miRNA-21/TAMRA-DNA2 双链体中的 TAMRA-DNA2 的备用部分配对，导致两种荧光染料相互接近，从而诱导 FAM 的有效 FRET到 TAMRA，从而使系统进入“ON”状态以进行信号放大。值得注意的是，自供电3D DNA步行机的巧妙设计避免了外部添加燃料DNA链或蛋白质酶，使得3D DNA步行机能够在复杂的细胞微环境中自主运动，大大简化了实验步骤和提高了步行机的效率。此外，该测定将为评估细胞内 miRNA 水平提供一种新的信号放大策略。