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.

中文翻译：

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独立式 DNA 折纸超晶格促进膜囊泡的冷冻电镜可视化

冷冻电子显微镜（cryo-EM）方法的技术突破为细胞外囊泡（EV）和合成脂质体-蛋白质组装体的高度详细的结构表征开辟了新的视角。这些囊泡在接近天然水合状态下的溶液中的结构特征对于破译细胞间通讯以及改善 EV 作为诊断标记物和作为疾病治疗中的药物载体的应用非常重要。然而，制备具有低囊泡异质性、低浓度以及化学反应或组装过程的动力学控制的多孔碳冷冻电镜网格的困难限制了冷冻电镜在电动汽车研究中的使用。我们报告了一种简单的膜囊泡冷冻电镜样品制备方法，该方法通过使用独立的 DNA 亲和超晶格覆盖多孔碳冷冻电镜网格来帮助规避这些限制。我们的方法使用 DNA 折纸自组装成溶液稳定且微米大小的有序分子模板，其中结构和功能特性可以得到合理控制。我们设计了具有胆固醇结合位点的模板来特异性捕获膜囊泡。这种 DNA-胆固醇亲和晶格 (DCAL) 的优点包括 (1) 在低浓度下局部富集人工和生物囊泡，以及 (2) 从条件化细胞培养物的预纯化沉淀中分离异质细胞来源的膜囊泡（外泌体）网格上的介质。