Department of Bioengineering, California Institute of Technology, Pasadena, CA, USA.
Department of Neuroscience and Howard Hughes Medical Institute, University of Wisconsin-Madison, 1111 Highland Avenue, Madison, WI, 53705, USA.
Nat Commun. 2023 Mar 20;14(1):1532. doi: 10.1038/s41467-023-36996-x.
Interactions between membrane proteins are essential for cell survival but are often poorly understood. Even the biologically functional ratio of components within a multi-subunit membrane complex-the native stoichiometry-is difficult to establish. Here we demonstrate digital nanoreactors that can control interactions between lipid-bound molecular receptors along three key dimensions: stoichiometric, spatial, and temporal. Each nanoreactor is based on a DNA origami ring, which both templates the synthesis of a liposome and provides tethering sites for DNA-based receptors (modelling membrane proteins). Receptors are released into the liposomal membrane using strand displacement and a DNA logic gate measures receptor heterodimer formation. High-efficiency tethering of receptors enables the kinetics of receptors in 1:1 and 2:2 absolute stoichiometries to be observed by bulk fluorescence, which in principle is generalizable to any ratio. Similar single-molecule-in-bulk experiments using DNA-linked membrane proteins could determine native stoichiometry and the kinetics of membrane protein interactions for applications ranging from signalling research to drug discovery.
膜蛋白之间的相互作用对细胞存活至关重要，但通常人们对此了解甚少。即使是多亚基膜复合物中各组成部分的生物学功能比值（即天然计量比）也难以确定。在这里，我们展示了数字纳米反应器，它可以沿着三个关键维度控制脂质结合分子受体之间的相互作用：计量比、空间和时间。每个纳米反应器都是基于一个 DNA 折纸环，该环既能模板合成脂质体，又能为基于 DNA 的受体（模拟膜蛋白）提供系链位点。通过链置换将受体释放到脂质体膜中，然后 DNA 逻辑门测量受体异二聚体的形成。高效的受体系链使得可以通过体相荧光来观察 1:1 和 2:2 绝对计量比的受体动力学，从原则上讲，这可以推广到任何比例。使用 DNA 连接的膜蛋白进行类似的单分子在体实验，可以确定天然计量比和膜蛋白相互作用的动力学，这在从信号研究到药物发现的各种应用中都具有重要意义。
