Nesrine Aissaoui, Allan Mills, Josephine Lai-Kee-Him, Nicolas Triomphe, Quentin Cece, Christine Doucet, Anne Bonhoure, Michel Vidal, Yonggang Ke* and Gaetan Bellot*,
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引用次数: 0
摘要
低温电子显微镜(cryo-EM)方法的技术突破为细胞外囊泡(EVs)和合成脂质体-蛋白质组装体的高精细结构表征开辟了新的前景。在溶液中,这些囊泡几乎处于原生水合状态,其结构特征对于破译细胞间通讯、改善细胞外囊泡在诊断中作为标记物和疾病治疗中作为药物载体的应用具有重要意义。然而,在低浓度、化学反应或组装过程的动力学控制下制备具有低囊泡异质性的孔状碳冷冻电镜网格存在困难,这限制了冷冻电镜在 EV 研究中的应用。我们报告了一种直接的膜囊泡低温电子显微镜样品制备方法,该方法使用独立的 DNA 亲和超晶格覆盖孔状碳低温电子显微镜网格,有助于规避这些限制。我们的方法利用 DNA 折纸自组装成溶液稳定、微米大小的有序分子模板,其中的结构和功能特性均可合理控制。我们设计的模板具有胆固醇结合位点,可以特异性地捕获膜囊泡。这种 DNA 胆固醇亲和格(DCAL)的优点包括:(1) 以低浓度局部富集人工和生物囊泡,(2) 从网格上的细胞培养条件培养基预纯化颗粒中分离异质细胞衍生膜囊泡(外泌体)。
Free-Standing DNA Origami Superlattice to Facilitate Cryo-EM Visualization of Membrane Vesicles
Technological breakthroughs in cryo-electron microscopy (cryo-EM) methods open new perspectives for highly detailed structural characterizations of extracellular vesicles (EVs) and synthetic liposome–protein assemblies. Structural characterizations of these vesicles in solution under a nearly native hydrated state are of great importance to decipher cell-to-cell communication and to improve EVs’ application as markers in diagnosis and as drug carriers in disease therapy. However, difficulties in preparing holey carbon cryo-EM grids with low vesicle heterogeneities, at low concentration and with kinetic control of the chemical reactions or assembly processes, have limited cryo-EM use in the EV study. We report a straightforward membrane vesicle cryo-EM sample preparation method that assists in circumventing these limitations by using a free-standing DNA-affinity superlattice for covering holey carbon cryo-EM grids. Our approach uses DNA origami to self-assemble to a solution-stable and micrometer-sized ordered molecular template in which structure and functional properties can be rationally controlled. We engineered the template with cholesterol-binding sites to specifically trap membrane vesicles. The advantages of this DNA–cholesterol-affinity lattice (DCAL) include (1) local enrichment of artificial and biological vesicles at low concentration and (2) isolation of heterogeneous cell-derived membrane vesicles (exosomes) from a prepurified pellet of cell culture conditioned medium on the grid.
期刊介绍:
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