Genetically Encodable Nanoparticle Tags for Combined Fluorescence and Tomographic

描述(申请人提供)：生物结构的大小介于单个大分子和细胞器之间(几纳米到100纳米)，不容易用现有的技术进行研究。因此，虽然大量的RNA和蛋白质序列和结构正在被编目，但这些结构的量化细胞背景是滞后的。理解这一背景将具有巨大的价值，因为它将使我们能够知道单个大分子的集合是如何组装成形成活细胞的动态机器的。一旦这些相互作用被揭示，细胞及其疾病状态的新图景肯定会浮出水面。该项目的长期目标是开发新的生物分子标记方法，该方法将能够使用细胞内荧光成像、电子断层扫描(ET)和电子能量损失谱(EELS)相结合的方式构建细胞中RNA和蛋白质的3D图谱。标签将由RNA或肽序列组成，这些序列经过改造后可催化形成无机纳米颗粒(“材料、核酶和酶”)。一旦在细胞中以RNA串联体和蛋白质嵌合体的形式进行遗传编码，这些序列将能够催化在活细胞内仅在感兴趣的位置形成无机纳米颗粒(直径4 nm-10 nm)。长期目标将通过确定一个独特的可克隆材料、核酶和酶的文库来实现。它们产生的纳米粒子将具有5种预期特性中的一种或多种：(1)可在活细胞中合成的纳米粒子；(2)可在细胞中通过玻璃化和冷丙酮冷冻取代制备的纳米粒子；(3)发光纳米粒子；(4)形状或大小不同的纳米粒子；以及(5)可使用电子发射光谱分辨的具有不同组成的纳米粒子。总的来说，这些标签将实现一种高度多元化的方法来成像细胞生物分子，可以想象的是，可以在一张断层图像中可视化数十到数百个生物分子。一系列明确的目标展示了如何创建和验证拟议的纳米颗粒标签库。其目的是：(1)通过生物分子体外选择方法分离材料酶和核酶，并根据一套特定的化学标准进行体外筛选，以确定它们在体内发挥作用的可能性；(2)使用满足Aim 1标准的序列在模型细菌系统中创建RNA连接体和多肽嵌合体，并在体外和体内测试它们用作纳米标签的能力；最后(3)通过用ET同时定位两个蛋白质来展示基因编码纳米标签的多重作用能力。公共卫生相关性：该项目的长期目标是生成一个完整的细胞内RNA和蛋白质空间排列的3D地图。这一目标将通过实施细胞成像的新概念来实现，在细胞成像中，无机纳米颗粒被用来标记体内的生物分子，以便用3D电子断层扫描进行可视化。绘制这些多组分的生物分子相互作用图将提供前所未有的细胞生物化学以及区分正常过程和疾病过程的离散分子变化的一瞥。

DESCRIPTION (provided by applicant): Biological structures whose sizes lie between those of individual macromolecules and cellular organelles (a few nm to 100 nm) are not readily studied with existing technologies. Thus, while vast inventories of RNA and protein sequences and structures are being catalogued, a quantitative cellular context for these structures is lagging. Understanding this context would have great value, because it would allow us to know how collections of individual macromolecules assemble into the dynamic machines that form a living cell. Once these interactions are revealed, a new picture of the cell and its disease states is sure to emerge. The long-term goal of this project is to develop new biomolecule tagging methodologies that will enable the construction of 3D maps of RNA and proteins in cells using a combination of intracellular fluorescence imaging, electron tomography (ET) and electron energy loss spectroscopy (EELS). The tags will consist of RNA or peptide sequences that are engineered to catalyze the formation of inorganic nanoparticles ("materials ribozymes and enzymes"). Once genetically encoded in cells as RNA concatemers and protein chimeras, these sequences will be able to catalyze the formation of inorganic nanoparticles (4 nm - 10 nm diameter) inside living cells exclusively at the site of interest. The long-term goal will be implemented by identifying a library of unique clonable materials ribozymes and enzymes. The nanoparticles they generate will have one or more of 5 desired properties: (1) Nanoparticles that can be synthesized in live cells; (2) Nanoparticles that can be synthesized in cells that have been prepared by vitrification and freeze-substitution in cold acetone; (3) Luminescent nanoparticles; (4) Shape or size distinct nanoparticles; and (5) Nanoparticles with distinct compositions that can be resolved using EELS. Collectively these tags will enable a highly multiplexed approach to imaging cellular biomolecules, conceivably allowing the visualization of tens to hundreds of biomolecules in a single tomogram. A series of well-defined aims demonstrate how the proposed library of nanoparticle tags may be created and validated. The aims are to: (1) isolate, through biomolecule in vitro selection methods, materials enzymes and ribozymes and screen them in vitro against a specific set of chemical criteria to determine their likelihood of functioning in vivo; (2) Use the sequences that satisfy the criteria from aim 1 to create RNA concatemers and peptide chimeras in a model bacterial system, and test them in vitro and in vivo for their ability to be used as nanoparticle tags; and finally (3) Demonstrate the multiplexing capabilities of genetically encoded nanoparticle tags by localizing two proteins simultaneously with ET. Public Health Relevance: The long-range goal of this project is to generate a complete 3D map of the spatial arrangement of RNA and proteins in a cell. This goal will be accomplished through the implementation of a new concept in cellular imaging, in which inorganic nanoparticles are used to tag biomolecules in vivo for visualization with 3D electron tomography. Mapping these multi-component biomolecule interactions will provide an unprecedented glimpse of cellular biochemistry and the discrete molecular changes that differentiate normal processes from disease processes.