Biophysics Graduate Program, ‡Department of Physics, and §Department of Mechanical and Aerospace Engineering, The Ohio State University , Columbus, Ohio 43214, United States.
ACS Nano. 2016 Jul 26;10(7):7073-84. doi: 10.1021/acsnano.6b03218. Epub 2016 Jul 6.
The organization of eukaryotic DNA into nucleosomes and chromatin undergoes dynamic structural changes to regulate genome processing, including transcription and DNA repair. Critical chromatin rearrangements occur over a wide range of distances, including the mesoscopic length scale of tens of nanometers. However, there is a lack of methodologies that probe changes over this mesoscopic length scale within chromatin. We have designed, constructed, and implemented a DNA-based nanocaliper that probes this mesoscopic length scale. We developed an approach of integrating nucleosomes into our nanocaliper at two attachment points with over 50% efficiency. Here, we focused on attaching the two DNA ends of the nucleosome to the ends of the two nanocaliper arms, so the hinge angle is a readout of the nucleosome end-to-end distance. We demonstrate that nucleosomes integrated with 6, 26, and 51 bp linker DNA are partially unwrapped by the nanocaliper by an amount consistent with previously observed structural transitions. In contrast, the nucleosomes integrated with the longer 75 bp linker DNA remain fully wrapped. We found that the nanocaliper angle is a sensitive measure of nucleosome disassembly and can read out transcription factor (TF) binding to its target site within the nucleosome. Interestingly, the nanocaliper not only detects TF binding but also significantly increases the probability of TF occupancy at its site by partially unwrapping the nucleosome. These studies demonstrate the feasibility of using DNA nanotechnology to both detect and manipulate nucleosome structure, which provides a foundation of future mesoscale studies of nucleosome and chromatin structural dynamics.
真核生物 DNA 组织成核小体和染色质，经历动态结构变化以调节基因组处理，包括转录和 DNA 修复。关键的染色质重排发生在广泛的距离范围内，包括数十纳米的介观长度尺度。然而，缺乏探测染色质中这个介观长度尺度上变化的方法。我们设计、构建和实施了一种基于 DNA 的纳米卡尺，用于探测这个介观长度尺度。我们开发了一种将核小体整合到纳米卡尺中的方法，在两个附着点的效率超过 50%。在这里，我们专注于将核小体的两个 DNA 末端附着到纳米卡尺臂的末端，因此铰链角度是核小体末端到末端距离的读数。我们证明，用 6、26 和 51 bp 连接 DNA 整合的核小体被纳米卡尺部分解开，解开的程度与先前观察到的结构转变一致。相比之下，用较长的 75 bp 连接 DNA 整合的核小体仍然完全包裹。我们发现纳米卡尺角度是核小体解组装的敏感测量指标，可以读取转录因子 (TF) 在核小体中与其靶位点的结合。有趣的是，纳米卡尺不仅检测 TF 结合，而且通过部分解开核小体，显著增加 TF 占据其位点的概率。这些研究证明了使用 DNA 纳米技术来检测和操纵核小体结构的可行性，为未来核小体和染色质结构动力学的介观研究提供了基础。
