Department of Computer Engineering, University of California at Santa Cruz, Santa Cruz, California, USA.
Biophys J. 2011 Mar 16;100(6):1509-16. doi: 10.1016/j.bpj.2011.01.029.
We present a novel application of active voltage control of DNA captured in a nanopore to regulate the amount of time the DNA is available to molecules in the bulk phase that bind to the DNA. In this work, the control method is used to measure hybridization between a single molecule of DNA captured in a nanopore and complementary oligonucleotides in the bulk phase. We examine the effect of oligonucleotide length on hybridization, and the effect of DNA length heterogeneity on the measurements. Using a mathematical model, we are able to deduce the binding rate of complementary oligonucleotides, even when DNA samples in experiments are affected by heterogeneity in length. We analyze the lifetime distribution of DNA duplexes that are formed in the bulk phase and then pulled against the pore by reversing the voltage. The lifetime distribution reveals several dissociation modes. It remains to be resolved whether these dissociation modes are due to DNA heterogeneity or correspond to different states of duplex DNA. The control method is unique in its ability to detect single-molecule complex assembly in the bulk phase, free from external force and with a broad (millisecond-to-second) temporal range.
我们提出了一种新的应用，即通过主动电压控制在纳米孔中捕获的 DNA，来调节 DNA 与在体相中结合到 DNA 的分子相互作用的时间。在这项工作中，该控制方法用于测量在纳米孔中捕获的单个 DNA 分子与在体相中的互补寡核苷酸之间的杂交。我们研究了寡核苷酸长度对杂交的影响，以及 DNA 长度异质性对测量的影响。通过数学模型，我们能够推导出互补寡核苷酸的结合速率，即使在实验中 DNA 样本受到长度异质性的影响时也是如此。我们分析了在体相形成的 DNA 双链的寿命分布，然后通过反转电压将其从孔中拉出。寿命分布揭示了几种解离模式。仍有待解决的是，这些解离模式是由于 DNA 异质性还是对应于双链 DNA 的不同状态。该控制方法独特之处在于它能够在不受外力影响的情况下，在毫秒到秒的宽时间范围内检测到体相中单分子复合物的组装。
