Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
The Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332, USA.
Int J Mol Sci. 2023 Feb 6;24(4):3222. doi: 10.3390/ijms24043222.
This study analyzed the nuclease- and serum-driven degradation of millimeter-scale, circular DNA-histone mesostructures (DHMs). DHMs are bioengineered chromatin meshes of defined DNA and histone compositions designed as minimal mimetics of physiological extracellular chromatin structures, such as neutrophil extracellular traps (NETs). Taking advantage of the defined circular shape of the DHMs, an automated time-lapse imaging and image analysis method was developed and used to track DHM degradation and shape changes over time. DHMs were degraded well by 10 U/mL concentrations of deoxyribonuclease I (DNase I) but not by the same level of micrococcal nuclease (MNase), whereas NETs were degraded well by both nucleases. These comparative observations suggest that DHMs have a less accessible chromatin structure compared to NETs. DHMs were degraded by normal human serum, although at a slower rate than NETs. Interestingly, time-lapse images of DHMs revealed qualitative differences in the serum-mediated degradation process compared to that mediated by DNase I. Importantly, despite their reduced susceptibility to degradation and compositional simplicity, the DHMs mimicked NETs in being degraded to a greater extent by normal donor serum compared to serum from a lupus patient with high disease activity. These methods and insights are envisioned to guide the future development and expanded use of DHMs, beyond the previously reported antibacterial and immunostimulatory analyses, to extracellular chromatin-related pathophysiological and diagnostic studies.
本研究分析了毫米级圆形 DNA-组蛋白介观结构（DHM）在核酸酶和血清作用下的降解情况。DHM 是一种经过生物工程设计的、具有特定 DNA 和组蛋白组成的染色质介观结构，旨在模拟生理细胞外染色质结构，如中性粒细胞胞外诱捕网（NETs）。利用 DHM 的规则圆形形状，开发了一种自动化的延时成像和图像分析方法，并用于跟踪 DHM 随时间的降解和形状变化。10U/mL 浓度的脱氧核糖核酸酶 I（DNase I）可有效降解 DHM，但相同浓度的微球菌核酸酶（MNase）则不能；而 NETs 可被这两种核酸酶有效降解。这些对比观察结果表明，DHM 的染色质结构比 NETs 更不易接近。DHM 可被正常人类血清降解，尽管降解速度比 NETs 慢。有趣的是，DHM 的延时图像显示，与 DNase I 介导的降解过程相比，血清介导的降解过程存在定性差异。重要的是，尽管 DHM 降解的敏感性较低且组成简单，但与狼疮患者高疾病活动期的血清相比，DHM 更易被正常供体血清降解，这与 NETs 的情况相似。这些方法和见解有望指导 DHM 的进一步开发和广泛应用，超越之前报道的抗菌和免疫刺激分析，以进行细胞外染色质相关的病理生理和诊断研究。
