Department of Chemistry and the Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
ACS Nano. 2012 Jul 24;6(7):6272-83. doi: 10.1021/nn301709n. Epub 2012 Jun 18.
We present a test case example of a detailed single-molecule fluorescence study of one of the most sophisticated and complex DNA devices introduced to date, a recently published autonomous bipedal DNA motor. We used the diffusion-based single-molecule Förster resonance energy transfer technique, coupled to alternating laser excitation (sm-FRET-ALEX), to monitor the motor assembly and operation. The study included verification of the formation of the correct structures, and of the correct motor operation, determination of the formation and stepping reaction yields, and identification of side products. Finally, the mechanisms of the motor assembly and operation were elucidated by measuring the reaction kinetics profile of track-walker binding and of lifting of the walker's leg upon fuel addition. The profiles revealed a fast phase, in which about half of the reaction was completed, followed by a slow phase which adds somewhat to the yield, reflecting the incomplete motor assembly and operation identified in the equilibrium experiments. Although further study is needed to fully understand the reasons for the incomplete assembly and operation, this work demonstrates that single-molecule fluorescence, based on its ability to provide detailed in situ structural dynamics information, inaccessible for traditional methods, constitutes an excellent tool for chaperoning the development of DNA-based technology.
我们提出了一个详细的单分子荧光研究的测试案例，该研究针对的是迄今为止引入的最复杂和最先进的 DNA 设备之一，即最近发表的自主双足 DNA 马达。我们使用基于扩散的单分子Förster 共振能量转移技术（sm-FRET-ALEX）来监测马达的组装和运作。该研究包括验证正确结构的形成，以及正确的马达操作，确定形成和步进反应的产率，并鉴定副产物。最后，通过测量轨道行走者结合的反应动力学曲线和燃料添加时行走者腿部抬起的反应动力学曲线，阐明了马达的组装和操作机制。这些曲线揭示了一个快速相，其中大约一半的反应已经完成，然后是一个缓慢相，略微增加了产率，这反映了在平衡实验中发现的不完全马达组装和操作。尽管需要进一步的研究来完全理解不完全组装和操作的原因，但这项工作表明，基于其提供传统方法无法获得的详细原位结构动力学信息的单分子荧光，是为 DNA 技术的发展提供指导的极好工具。
