Generating nuclear pore complex mimics with DNA origami

用 DNA 折纸生成核孔复合体模拟物

基本信息

  • 批准号:
    8621296
  • 负责人:
  • 金额:
    $ 24.98万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2014
  • 资助国家:
    美国
  • 起止时间:
    2014-06-01 至 2016-05-31
  • 项目状态:
    已结题

项目摘要

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.
总结 核孔复合物(NPC)是分子分选机,其确保核孔复合物的适当区室化。 所有真核生物的细胞核和细胞质内容物。离子、代谢物和小分子自由通过 而大分子(> 40 kD)的转运受阻。同时与 由于这种屏障功能,NPC对一组高移动的核运输具有显著的选择性 受体(NTR); NTR结合携带信号的货物分子,并促进其通过 届全国人大这些选择性的运输事件是非常迅速的,一个NPC可以运输1000个 分子/秒。人大如何建立这种有选择性和有效的运输系统是不完全的 阐明并代表了对我们理解细胞区室化的根本挑战。 此外,了解NPC的功能对于设计改善 越来越多的人类疾病包括癌症、心脏异常、神经变性疾病 病毒感染,以及当NPC功能受到干扰时导致的发育缺陷。最后,在其 核心,NPC是一个有效的分子分选机-定义运输机制将导致 开发模拟其性质的合成材料,用于蛋白质纯化、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NTR和富含苯丙氨酸-甘氨酸的NPC蛋白亚类(nups)之间的相互作用 (FG)个氨基酸残基然而,NTR和FG-nup之间的测量亲和力太强,无法支持 观察到的体内转运率导致一个悖论;我们(和其他人)认为,这一悖论反映了 在其天然环境之外检查个体FG-nup的局限性,其中存在其他 具有不同性质的FG-nups、它们的化学计量以及它们在圆柱形通道内的限制 累积地有助于难以在体外重现的合作行为。通过利用我们 凭借DNA折纸的专业知识,我们有能力制造称为NuPODs(核孔复合体)的结构 由DNA组织),其模拟NPC的尺寸并含有限定的FG-nup组合物, 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,直接测试如何 空间定位和FG密度影响FG-nups的协同行为及其与 具体NTR。在目标2中,我们将在纳米孔上放置我们的NuPODs,并研究独特的组合 的FG-nups建立了对不同大小的惰性大分子的渗透屏障。这些研究将开放 用于制造NPC模拟物的门,所述NPC模拟物完全概括NPC的传输特性。此外,本发明还 我们对NPC底层设计的理解将使我们能够生成具有规定的NuPOD 选择性/渗透性特性。

项目成果

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Chenxiang Lin其他文献

Chenxiang Lin的其他文献

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{{ truncateString('Chenxiang Lin', 18)}}的其他基金

DNA nanotechnology enabled high-precision membrane engineering
DNA 纳米技术实现高精度膜工程
  • 批准号:
    10622748
  • 财政年份:
    2023
  • 资助金额:
    $ 24.98万
  • 项目类别:
A multiscale approach for elucidating nuclear entry mechanisms of HIV-1 capsid
阐明 HIV-1 衣壳核进入机制的多尺度方法
  • 批准号:
    10793845
  • 财政年份:
    2021
  • 资助金额:
    $ 24.98万
  • 项目类别:
A multiscale approach for elucidating nuclear entry mechanisms of HIV-1 capsid
阐明 HIV-1 衣壳核进入机制的多尺度方法
  • 批准号:
    10490880
  • 财政年份:
    2021
  • 资助金额:
    $ 24.98万
  • 项目类别:
Fluid shear stress mechanotransduction at endothelial cell-cell junctions
内皮细胞-细胞连接处的流体剪切应力机械转导
  • 批准号:
    10688712
  • 财政年份:
    2021
  • 资助金额:
    $ 24.98万
  • 项目类别:
A multiscale approach for elucidating nuclear entry mechanisms of HIV-1 capsid
阐明 HIV-1 衣壳核进入机制的多尺度方法
  • 批准号:
    10676822
  • 财政年份:
    2021
  • 资助金额:
    $ 24.98万
  • 项目类别:
A multiscale approach for elucidating nuclear entry mechanisms of HIV-1 capsid
阐明 HIV-1 衣壳核进入机制的多尺度方法
  • 批准号:
    10794022
  • 财政年份:
    2021
  • 资助金额:
    $ 24.98万
  • 项目类别:
Fluid shear stress mechanotransduction at endothelial cell-cell junctions
内皮细胞-细胞连接处的流体剪切应力机械转导
  • 批准号:
    10322398
  • 财政年份:
    2021
  • 资助金额:
    $ 24.98万
  • 项目类别:
A multiscale approach for elucidating nuclear entry mechanisms of HIV-1 capsid
阐明 HIV-1 衣壳核进入机制的多尺度方法
  • 批准号:
    10402986
  • 财政年份:
    2021
  • 资助金额:
    $ 24.98万
  • 项目类别:
Fluid shear stress mechanotransduction at endothelial cell-cell junctions
内皮细胞-细胞连接处的流体剪切应力机械转导
  • 批准号:
    10559534
  • 财政年份:
    2021
  • 资助金额:
    $ 24.98万
  • 项目类别:
A Nanomechanical Toolkit to Guide Membrane Structure and Dynamics
指导膜结构和动力学的纳米机械工具包
  • 批准号:
    10378021
  • 财政年份:
    2019
  • 资助金额:
    $ 24.98万
  • 项目类别:
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