Generating nuclear pore complex mimics with DNA origami

摘要 核孔复合体(NPC)是一种分子分选机，可确保正确划分 所有真核生物的核内和胞质内的含量。离子、代谢物和小分子自由通过 而更大的(40kD)大分子的移位则受到阻碍。同时与 这种屏障功能，NPC对一组高流动性的核运输表现出显著的选择性 受体(NTRs)；NTRs结合承载信号的货物分子，并通过 全国人大。这些选择性传输事件非常迅速，单个NPC可以传输1000个 分子/秒。全国人大如何建立这种选择性和高效的交通系统并不完全 阐明并代表了对我们对细胞区隔的理解的根本挑战。 此外，了解全国人民代表大会的职能对于制定战略以改善 越来越多的人类疾病，包括癌症、心脏异常、神经退行性疾病、 病毒感染，以及当鼻咽癌功能受到干扰时导致的发育缺陷。最后，在ITS 核心，NPC是一台高效的分子分选机-定义传输机制将导致 开发模拟其特性的合成材料用于蛋白质提纯、生物技术和 制药应用，包括生物反应器。NPC选择性和快速转运的分子基础 是NTRs与富含苯丙氨酸-甘氨酸的NPC蛋白(NUP)之间的相互作用 (Fg)氨基酸残基。然而，NTR和FG-NUP之间测量的亲和力太强，无法支持 观察到的体内转运速率导致了一个悖论；我们(和其他人)认为这个悖论反映了 在其原生环境之外检查单个FG-nup的限制，在那里存在其他 具有不同性质的FG-nup、它们的化学计量比以及它们在圆柱形通道中的限制 累积形成了一种在体外很难概括的合作行为。通过利用我们的 在DNA-折纸方面的专业知识我们有能力制造出称为NuPODS(核孔复合体)的结构 由DNA组织)，模拟NPC的尺寸，并包含FG-nup的定义成分， 在空间上是精确排列的。在目标1中，我们将使用这些NuPOD作为绑定支持，以直接测试 空间位置和FG密度影响FG-NUP的协同行为及其结合动力学 特定的非关税壁垒。在目标2中，我们将把我们的NuPOD固定在纳米孔上，并研究独特的组合 对于不同大小的惰性大分子，FG-NUP建立了渗透屏障。这些研究将开启 制造鼻咽癌仿制品的大门完全概括了鼻咽癌的运输特性。此外， 我们对NPC底层设计的理解将使我们能够生成符合规定的NuPOD 选择性/渗透性特性。

SUMMARY Nuclear pore complexes (NPCs) are molecular sorting machines that ensure proper compartmentalization of nuclear and cytoplasmic contents in all eukaryotes. Ions, metabolites and small macromolecules pass freely across the NPC, whereas the translocation of larger (>40kD) macromolecules is impeded. Simultaneously with this barrier function, the NPC exhibits remarkable selectivity towards a group of highly mobile nuclear transport receptors (NTRs); NTRs bind signal-bearing cargo molecules and facilitate their import/export through the NPC. These selective transport events are extremely rapid and an individual NPC can transport 1000 molecules/second. How the NPC establishes this selective and efficient transport system is not completely elucidated and represents a fundamental challenge to our understanding of cellular compartmentalization. Moreover, understanding the function of the NPC will be critical for designing strategies to ameliorate a growing number of human pathologies including cancers, heart abnormalities, neurodegenerative diseases, viral infection, in addition to developmental defects that result when NPC function is perturbed. Lastly, at its core, the NPC is an efficient molecular sorting machine - defining the mechanism of transport will lead to the development of synthetic materials that mimic its properties for protein purification, biotechnology and pharmaceutical applications, including bioreactors. The molecular basis for NPC selectivity and rapid transport are interactions between NTRs and a subset of NPC proteins (nups) that are rich in phenylalanine-glycine (FG) amino acid residues. However, measured affinities between NTRs and FG-nups are too strong to support observed in vivo transport rates leading to a paradox; we (and others) suggest that this paradox reflects the limitations of examining individual FG-nups outside of their native environment, where the presence of other FG-nups with different properties, their stoichiometry, and their confinement within a cylindrical channel cumulatively contribute to a cooperative behavior that is difficult to recapitulate in vitro. By leveraging our expertise in DNA-origami we have the ability to fabricate structures termed NuPODs (Nuclear Pore Complex Organized by DNA) that mimic the dimensions of the NPC and contain defined compositions of FG-nups that are precisely spatially arranged. In Aim 1, we will use these NuPODs as binding supports to directly test how spatial-positioning and FG-density impact the cooperative behavior of FG-nups and their binding kinetics to specific NTRs. In Aim 2, we will immobilize our NuPODs on nanopores and examine how unique combinations of FG-nups establish a permeability barrier to inert macromolecules of varying sizes. These studies will open the door for the fabrication of NPC-mimics that fully recapitulate the transport properties of the NPC. Further, our understanding of the NPCs underlying design will allow us to generate NuPODs with prescribed selectivity/permeability characteristics.