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: 7916851
• 负责人: DAN L. FELDHEIM
• 金额: $ 46.47万
• 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/7916851
• 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）：在单个大分子和细胞细胞器的大小之间的生物结构（几nm至100 nm）不容易使用现有技术研究。因此，尽管正在对RNA和蛋白质序列和结构的大量清单进行分类，但这些结构的定量细胞环境仍在滞后。了解这种情况将具有很大的价值，因为它可以使我们知道如何将单个大分子的集合组装到形成活细胞的动态机器中。一旦揭示了这些相互作用，细胞及其疾病状态的新图片肯定就会出现。该项目的长期目标是开发新的生物分子标记方法，该方法将使用细胞内荧光成像，电子层析成像（ET）和电子能量损失光谱谱（EELS）的组合在细胞中构建RNA和蛋白质的3D地图。标签将由RNA或肽序列组成，这些RNA或肽序列设计为催化无机纳米颗粒的形成（“材料核酶和酶”）。一旦将细胞中的遗传编码作为RNA辅导剂和蛋白质嵌合体编码，这些序列将能够催化活细胞内部的无机纳米颗粒（4 nm -10 nm直径）的形成。长期目标将通过确定独特的可克隆材料核酶和酶的库来实现。他们生成的纳米颗粒将具有5个所需特性中的一个或多个：（1）可以在活细胞中合成的纳米颗粒； （2）可以在冷丙酮中通过玻璃化和冻结固定制备的细胞中合成的纳米颗粒； （3）发光的纳米颗粒； （4）形状或大小不同的纳米颗粒； （5）具有不同组成的纳米颗粒，可以使用鳗鱼解决。这些标签共同使高度多路复用的方法用于成像细胞生物分子，可以想象，可以在单个断层图中可视化数十个至数百个生物分子。一系列定义明确的目的表明了如何创建和验证提出的纳米颗粒标签库。目的是：（1）通过体外选择方法的生物分子隔离，材料酶和核酶，并根据一组特定的化学标准在体外进行筛查，以确定它们在体内功能的可能性； （2）使用满足AIM 1标准的序列在模型细菌系统中创建RNA串联和肽嵌合体，并在体外和体内对其进行测试，以便将其用作纳米颗粒标签；最后（3）通过与ET同时定位两种蛋白质，证明了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：该项目的远距离目标是生成一个完整的3D图，以在单元格中RNA和蛋白质的空间排列。该目标将通过在细胞成像中实现新概念来实现，其中无机纳米颗粒用于在体内标记生物分子以使用3D电子断层扫描可视化。映射这些多组分的生物分子相互作用将提供前所未有的细胞生物化学的瞥见，以及将正常过程与疾病过程区分开的离散分子变化。