A Programmable DNA Origami Platform to Organize SNAREs for Membrane Fusion.
A Programmable DNA Origami Platform to Organize SNAREs for Membrane Fusion.
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DOI：
10.1021/jacs.5b13107
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发表时间：
2016-04-06
影响因子：
15
通讯作者：
Rothman JE
中科院分区：
文献类型：
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作者：
Xu W;Nathwani B;Lin C;Wang J;Karatekin E;Pincet F;Shih W;Rothman JE
SNARE复合物是膜融合的核心分子机制，是驱动细胞间和细胞内通信和运输的基本过程。仍有争议的问题之一是SNAREs是否以及如何合作。在这里，我们展示了使用自组装dna纳米结构环来模板大小均匀的小单层囊泡，其中包含预定的最大数量的外部SNAREs，以研究膜融合过程。我们还将脂质偶联的互补ssDNA作为系链结合到囊泡和靶膜中，这样可以绕过融合反应的限速对接步骤，并允许在SNARE密度低至每个囊泡一对的情况下直接观察单个膜融合事件。通过该平台，我们在单事件水平上证实，将模板suv与支持的脂质双层（SBL）对接后，1-2对SNAREs足以驱动快速的脂质混合。该平台的模块化和可编程性使其易于适用于研究涉及辅助蛋白的更复杂系统。
SNARE complexes are the core molecular machinery of membrane fusion, a fundamental process that drives inter- and intra- cellular communication and trafficking. One of the questions that remain controversial has been whether and how SNAREs cooperate. Here we show the use of self-assembled DNA-nanostructure rings to template uniform-sized small unilamellar vesicles containing predetermined maximal number of externally facing SNAREs to study the membrane-fusion process. We also incorporated lipid-conjugated complementary ssDNA as tethers into vesicle and target membranes, which enabled bypass of the rate-limiting docking step of fusion reactions and allowed direct observation of individual membrane-fusion events at SNARE densities as low as one pair per vesicle. With this platform, we confirmed at the single event level that after docking of the templated-SUVs to supported lipid bilayers (SBL), 1–2 pairs of SNAREs are sufficient to drive fast lipid mixing. Modularity and programmability of this platform makes it readily amenable to studying more complicated systems where auxiliary proteins are involved.