Department of Physics, NC State University , Raleigh, North Carolina 27695, USA.
Biomicrofluidics. 2014 Jun 12;8(3):034113. doi: 10.1063/1.4882775. eCollection 2014 May.
We present an analytic technique for probing protein-catalyzed transient DNA loops that is based on nanofluidic channels. In these nanochannels, DNA is forced in a linear configuration that makes loops appear as folds whose size can easily be quantified. Using this technique, we study the interaction between T4 DNA ligase and DNA. We find that T4 DNA ligase binding changes the physical characteristics of the DNApolymer, in particular persistence length and effective width. We find that the rate of DNA fold unrolling is significantly reduced when T4 DNA ligase and ATP are applied to bare DNA. Together with evidence of T4 DNA ligase bridging two different segments of DNA based on AFM imaging, we thus conclude that ligase can transiently stabilize folded DNA configurations by coordinating genetically distant DNA stretches.
我们提出了一种基于纳米通道探测蛋白催化的瞬时 DNA 环的分析技术。在这些纳米通道中，迫使 DNA 呈线性排列，使环呈现出易于定量的折叠形状。使用该技术，我们研究了 T4 DNA 连接酶与 DNA 的相互作用。我们发现，T4 DNA 连接酶的结合改变了 DNA 聚合物的物理特性，特别是持久长度和有效宽度。我们发现，当 T4 DNA 连接酶和 ATP 应用于裸 DNA 时，DNA 折叠的展开速度显著降低。结合基于 AFM 成像的 T4 DNA 连接酶桥接两条不同 DNA 片段的证据，我们因此得出结论，连接酶可以通过协调遗传上遥远的 DNA 片段来瞬时稳定折叠的 DNA 构象。
