Controlling Phase separation and Higher Order Structure of Chromosomal DNA Molecules

在精胺存在下，研究了在限制性内切酶ApaLI的作用下，Lambda DNA链在折叠/未折叠状态下的生物化学特性。利用荧光显微镜在单分子水平上研究了依赖于其高阶结构的DNA链的这些特征。在低浓度的精胺下，lambda DNA呈随机卷曲的构象并允许酶消化，而在高浓度的精胺下，lambda DNA具有紧凑的折叠结构并抑制这种攻击。结合对短寡聚DNA的比较实验，我们的结果表明，高阶结构的转变导致了对酶的敏感性的开/关型切换。此外，我们还提出了一种非侵入性的方法来操作单个Mb大小的全基因组DNA分子。细胞受到渗透电击，从爆裂的细胞中释放的基因组DNA被转移到更高盐浓度的区域，使用光钳。捕获的基因组DNA呈现出从凝聚态到拉伸态的构象转变，并伴随着环境的变化。提出了光钳在大基因组DNA现场操作中的适用性，即平板实验室。

The biochemical characteristics of lambda DNA chains in folded/unfolded states upon cleavage by the restriction enzyme ApaLI were investigated in the presence of spermine. These characteristics of DNA chains depending on their higher-order structure were studied at the single-molecule level using fluorescence microscopy. With a low concentration of spermine, lambda DNA takes a random coiled conformation and allows digestion by the enzyme, while under a high concentration of spermine, lambda DNA takes a compact folded structure and inhibits such attack. Together with comparative experiments on short oligomeric DNA, our results suggest that the transition in the higher-order structure causes on/off type switching of sensitivity to the enzyme. In addition, we proposed a noninvasive methodology for manipulating single Mb-size whole-genome DNA molecules. Cells were subjected to osmotic shock and the genorne DNA released from the burst cells was transferred to a region of higher salt concentration using optical tweezers. The trapped genome DNA exhibits a conformational transition from a condensed state to an elongated state, accompanied by the change in its environment. The applicability of optical tweezers to the on-site manipulation of giant genome DNA is suggested, i.e., lab-on-a-plate.