Genetically Encodable Nanoparticle Tags for Combined Fluorescence and Tomographic

用于组合荧光和断层扫描的基因可编码纳米颗粒标签

• 批准号: 7694357
• 负责人: DAN L. FELDHEIM
• 金额: $ 46.09万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7694357
• 关键词: Acetone; Address; Amino Acid Sequence; Bacterial Model; Biochemical Process; Biochemistry; Biological; Blinking; Caliber; Cataloging; Catalogs; Catalytic RNA; Cell Cycle; Cells; Chemicals; Chimera organism; Chimeric Proteins; Collection; Cytoskeletal Proteins; Disease; Dreams; Electron energy loss spectroscopy; Engineering; Enzymes; Equipment and supply inventories; Escherichia coli; Fluorescence; Fluorescence Microscopy; Freeze Substitution; Goals; Growth; Hand; Imagery; In Vitro; Individual; Label; Libraries; Life; Likelihood Functions; Maps; Metals; Methodology; Methods; Molecular; Nature; Noise; Organelles; Oxides; Peptide Sequence Determination; Peptides; Process; Property; Proteins; Proteomics; RNA; RNA Sequences; Research; Ribosomes; Semiconductors; Series; Shapes; Signal Transduction; Signal Transduction Pathway; Site; Spectrum Analysis; Structure; System; Technology; Testing; Tomogram; Tubulin; Viral; Visual; analog; catalyst; cellular imaging; electron tomography; fluorescence imaging; germanium oxide; in vivo; insight; interest; luminescence; macromolecule; metal oxide; nanoparticle; particle; protein aminoacid sequence; public health relevance; quantum; reconstruction; stoichiometry

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.

描述（由申请人提供）：其大小介于单个大分子和细胞器（几nm至100 nm）之间的生物结构不易用现有技术进行研究。因此，虽然大量的RNA和蛋白质序列和结构正在被编目，但这些结构的定量细胞背景却很落后。理解这一背景将具有巨大的价值，因为它将使我们了解单个大分子的集合如何组装成形成活细胞的动态机器。一旦这些相互作用被揭示，细胞及其疾病状态的新图像肯定会出现。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）和电子能量损失光谱（EELS）的组合构建细胞中RNA和蛋白质的3D图谱。这些标签将由RNA或肽序列组成，这些序列被改造成催化无机纳米颗粒（“材料核酶和酶”）的形成。一旦在细胞中作为RNA多联体和蛋白质嵌合体进行遗传编码，这些序列将能够催化活细胞内仅在感兴趣的位点形成无机纳米颗粒（4 nm - 10 nm直径）。长期目标将通过确定一个独特的可克隆材料核酶和酶库来实现。它们产生的纳米颗粒将具有5种所需性质中的一种或多种：（1）可以在活细胞中合成的纳米颗粒;（2）可以在已经通过在冷丙酮中玻璃化和冷冻置换制备的细胞中合成的纳米颗粒;（3）发光纳米颗粒;（4）形状或尺寸不同的纳米颗粒;（5）可以在细胞中合成的纳米颗粒;（6）可以在细胞中合成的纳米颗粒;（7）可以在细胞中合成的纳米颗粒;（8）可以在细胞中合成的纳米颗粒;（9）可以在细胞中合成的纳米颗粒;（10）可以在细胞中合成的纳米颗粒;（11）可以在细胞中合成的纳米颗粒;（12）可以在细胞中合成的纳米颗粒;（13）可以在细胞中合成的纳米颗粒;（14）可以在细胞中合成的纳米颗粒;（15）可以在细胞中合成的纳米颗粒。和（5）具有不同组成的纳米颗粒，其可以使用EELS解析。这些标签将共同实现对细胞生物分子进行成像的高度多路复用的方法，可以想象地允许在单个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所提出的纳米颗粒标签库。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，并针对一组特定的化学标准在体外筛选它们，以确定它们在体内发挥功能的可能性;（2）使用满足目标1的标准的序列在模型细菌系统中产生RNA多联体和肽嵌合体，并在体外和体内测试它们用作纳米颗粒标签的能力;以及最后（3）通过用ET同时定位两种蛋白质来证明遗传编码的纳米颗粒标签的多路复用能力。公共卫生相关性：该项目的长期目标是生成细胞中RNA和蛋白质空间排列的完整3D图。这一目标将通过实施细胞成像的新概念来实现，其中无机纳米颗粒用于标记体内生物分子，以便通过3D电子断层扫描进行可视化。绘制这些多组分生物分子相互作用将提供细胞生物化学和区分正常过程与疾病过程的离散分子变化的前所未有的一瞥。