Division of Materials Science, Faculty of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8573, Japan.
ACS Nano. 2020 Mar 24;14(3):3477-3489. doi: 10.1021/acsnano.9b09581. Epub 2020 Feb 18.
We designed and prepared a single-legged DNA walker that relies on the creation of a simple diffusion-limited nanointerface on a gold nanoparticle (DNA/PEG-GNP) track co-modified with fluorescence-labeled hairpin DNA and poly(ethylene glycol) (PEG) containing a positively charged amino group at one end. The movement of our single-legged DNA walker is driven by an enzyme-free DNA circuit mechanism through cascading toehold mediated DNA displacement reactions (TMDRs) using fuel hairpin DNAs. The acceleration of TMDRs was observed for the DNA/PEG-GNP track through electrostatic interaction between the positively charged track and negatively charged DNAs, resulting in the acceleration of the DNA circuit and amplification of the fluorescence signal. Furthermore, the DNA/PEG-GNP track allowed autonomous and persistent movement of a walker DNA strand on the same GNP track, because the intraparticle DNA circuit occurred preferentially by preventing diffusion of the negatively charged free walker DNA strand from near the positively charged tracks into solution through electrostatic interaction. Based on comparative study of kinetics of TMDRs and DNA walking behaviors, it is to be noted that the DNA/PEG-GNP track showed the fastest DNA circuit reaction (walking rate) and the largest number of steps taken by the walker DNA strand compared to other GNP tracks with varying nanointerfaces that differ in terms of their type of charges (no and negative charges), density of positive charges, and number of hairpin DNAs per GNP track. These facts reveal that the positive charges on the GNP track play an important role in the acceleration of the DNA circuit, as well as the successful walking motion of the single-legged DNA strand. By using the fluorescence signal amplification functions, our single-legged DNA walker could be applied directly and successfully to enzyme-free miRNA-detection systems. The miRNA-detection system provided higher discrimination of other mismatched miRNAs and higher sensitivity (the lowest LOD: 4.0 pM) when compared to other miRNA-detection systems based on other GNP tracks without positive charges. Unlike existing single-legged DNA walkers, our single-legged DNA walkers do not require complex processes, such as immobilization of the walker DNA strand on the tracks and precise adjustment of the sequence of walker DNA. Therefore, our strategy, based on the creation of diffusion-limited nanointerfaces, has enormous potential for the applications of single-legged DNA walkers to biosensors, bioimaging, and computing.
我们设计并制备了一种单腿 DNA walker，它依赖于在金纳米颗粒（DNA/PEG-GNP）轨道上创建一个简单的扩散受限纳米界面，该轨道通过荧光标记的发夹 DNA 和聚乙二醇（PEG）进行共修饰，PEG 的一端含有正电荷的氨基。我们的单腿 DNA walker 的运动是通过无酶 DNA 电路机制驱动的，该机制通过级联的 toehold 介导的 DNA 置换反应（TMDR）来实现，使用燃料发夹 DNA。通过带正电荷的轨道与带负电荷的 DNA 之间的静电相互作用，观察到 DNA/PEG-GNP 轨道上 TMDR 的加速，从而加速 DNA 电路并放大荧光信号。此外，DNA/PEG-GNP 轨道允许 walker DNA 链在相同的 GNP 轨道上自主且持续地运动，因为通过静电相互作用阻止带负电荷的游离 walker DNA 链从带正电荷的轨道附近扩散到溶液中，从而优先发生颗粒内 DNA 电路。基于 TMDR 动力学和 DNA 行走行为的比较研究，值得注意的是，与具有不同纳米界面的其他 GNP 轨道相比，DNA/PEG-GNP 轨道表现出最快的 DNA 电路反应（行走速度）和 walker DNA 链迈出的最大步数，这些纳米界面的电荷类型（无电荷和负电荷）、正电荷密度和每个 GNP 轨道上的发夹 DNA 数量不同。这些事实表明，GNP 轨道上的正电荷在加速 DNA 电路以及单腿 DNA 链的成功行走运动中发挥了重要作用。通过使用荧光信号放大功能，我们的单腿 DNA walker 可以直接成功应用于无酶 miRNA 检测系统。与其他基于无正电荷 GNP 轨道的 miRNA 检测系统相比，该 miRNA 检测系统提供了更高的其他错配 miRNA 的区分度和更高的灵敏度（最低检测限：4.0 pM）。与现有的单腿 DNA walker 不同，我们的单腿 DNA walker 不需要复杂的过程，例如将 walker DNA 链固定在轨道上以及精确调整 walker DNA 的序列。因此，我们的策略基于创建扩散受限纳米界面，具有将单腿 DNA walker 应用于生物传感器、生物成像和计算的巨大潜力。
