Department of Materials Science and Engineering, University of Sheffield, Sheffield, UK.
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Phys Chem Chem Phys. 2024 Oct 17;26(40):25798-25807. doi: 10.1039/d4cp01795a.
Kinetoplast DNA is a complex nanoscale network, naturally assembled from thousands of interconnected DNA circles within the mitochondrion of certain parasites. Despite the relevance of this molecule to parasitology and the recent discovery of tuneable mechanics, its topology remains highly contested. Here we present a multiscale analysis into the structure of kDNA using a combination of high-resolution atomic force microscopy and custom-designed image analysis protocols. By capturing a notably large set of high-resolution images, we are able to look beyond individual kDNA variations and quantify population properties throughout several length scales. Within the sample, geometric fluctuations of area and mean curvature are observed, corresponding with previous measurements. These translate to localised variations in density, with a sample-wide decrease in DNA density from the outer rim of the molecule to the centre and an increase in pore size. Nodes were investigated in a single molecule study, and their estimated connectivity significantly exceeded mean valence, with a high dependence on their position in the network. While node separation was approximately half the minicircle circumference, it followed a strong bimodal distribution, suggesting more complex underlying behaviour. Finally, upon selective digestion of the network, breakdown of the fibril-cap heterogeneity was observed, with molecules expanding less upon immobilisation on the mica surface. Additionally, preferential digestion was seen in localised areas of the network, increasing pore size disproportionately. Overall, the combination of high-resolution AFM and single molecule image analysis provides a promising method to the continued investigation of complex nanoscale structures. These findings support the ongoing characterisation of kDNA topology to aid understanding of its biological and mechanical phenomena.
动基体 DNA 是一种复杂的纳米级网络，由某些寄生虫线粒体中数千个相互连接的 DNA 环自然组装而成。尽管该分子与寄生虫学有关，并且最近发现了可调节的力学特性，但它的拓扑结构仍然存在很大争议。在这里，我们使用高分辨率原子力显微镜和定制的图像分析协议的组合，对 kDNA 的结构进行了多尺度分析。通过捕获一组数量显著增加的高分辨率图像，我们能够超越单个 kDNA 变化，在几个长度尺度上量化种群特性。在样本中，观察到面积和平均曲率的几何波动，与以前的测量结果相对应。这转化为密度的局部变化，从分子的外边缘到中心，DNA 密度呈整体下降趋势，而孔径增大。在单个分子研究中研究了节点，它们的估计连接度显著超过平均价数，并且高度依赖于它们在网络中的位置。虽然节点分离大约是微环周长的一半，但它遵循强烈的双峰分布，表明存在更复杂的潜在行为。最后，在选择性消化网络后，观察到原纤维帽异质性的破坏，分子在固定在云母表面上时膨胀较少。此外，在网络的局部区域观察到优先消化，不成比例地增加了孔径。总之，高分辨率 AFM 和单分子图像分析的结合为复杂纳米结构的持续研究提供了一种很有前途的方法。这些发现支持对 kDNA 拓扑结构的持续表征，以帮助理解其生物学和力学现象。
