3 to15 Covalent Gold for High Resolution Electron Microscopy

用于高分辨率电子显微镜的 3 至 15 共价金

• 批准号: 7672793
• 负责人: VISHWAS N JOSHI
• 金额: $ 16.45万
• 依托单位: NANOPROBES, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-15 至 2011-01-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7672793
• 关键词: Address; Adenocarcinoma; Antibodies; Antigen Targeting; Architecture; Binding; Biocompatible; Biological Preservation; Cell Nucleus; Cells; Chimeric Proteins; Chinese Hamster; Collaborations; Color; Common Epitope; Complement; Cytokeratin; Cytoplasm; DNA biosynthesis; Detection; Detergents; Development; Diffuse; Disease; Dot Immunoblotting; Drug Formulations; Electron Microscope; Electron Microscopy; Electrons; Engineering; Epitopes; Fluorescence; Gold; Gold Colloid; Green Fluorescent Proteins; Heavy Metals; Hela Cells; His-His-His-His-His-His; Histidine; Human; Illinois; Image; Imagery; Imaging technology; Insulin; Ion Exchange; Islet Cell; Islets of Langerhans; Label; Laboratories; Lac Repressors; Lead; Life; Light; Light Cell; Link; Methods; Microinjections; Microscopic; Microscopy; Modeling; Modification; Molecular; Molecular Sieve Chromatography; Morphologic artifacts; Nitrilotriacetic Acid; Noise; Nuclear Structure; Osmium Tetroxide; Ovary; Particulate; Phase; Preparation; Process; Proliferating Cell Nuclear Antigen; Protein Engineering; Proteins; Quantum Dots; Rattus; Reaction; Reagent; Recombinant Proteins; Recombinants; Research Personnel; Resolution; Sampling; Semiconductors; Signal Transduction; Silver; Site; Specificity; Spectrum Analysis; Spottings; Staining method; Stains; Sulfhydryl Compounds; Surface; Surface Properties; System; Techniques; Thick; Tissues; Transmission Electron Microscopy; Universities; Zebrafish; activating transcription factor 1; antibody conjugate; antigen antibody binding; cellular targeting; density; electron tomography; fluorescence imaging; improved; in vivo; insight; interest; light microscopy; nanoGold; nanoparticle; particle; protein complex; public health relevance; reconstruction; sample fixation; structural biology; tomography; transmission process

DESCRIPTION (provided by applicant): Covalently linkable 3-15 nm gold reagents will be prepared stabilized with formulations that incorporate a hydrophobic chelating thiol domain for stabilizing the gold nanoparticles, and hydrophilic surface groups that provide a wide choice of surface properties and conjugation reactions. Methods are proposed for electron microscopic localization of targets following in vivo and on section-ex vivo gold-labeling in living cells and in well preserved tissues respectively. Clonable expression tags will be used in which yellow and green fluorescent proteins (YFP and GFP respectively) are expressed in combination with hexahistidine tag (His-tag) and lac repressor respectively. The fluorescent protein, His-tag and lac repressor will then be targeted by biocompatible, non-toxic covalently linkable gold nanoparticles functionalized with antibodies or nitrilotriacetic acid (NTA)-Ni(II), a chelate which binds to His-tags with comparable strength and specificity to antigen-antibody binding. In phase I, the use of the 10 and 15 nm gold-NTA-Ni(II) tag will be optimized by modification of the gold surface for highest possible compatibility and stability for on-section labeling while 3 and 5 nm gold covalently linked to anti-lacI or anti-GFP antibodies will be optimized for in vivo gold labeling using recently developed microinjection technique. Finally, the relation between gold size, thickness of the protective shell and protein fluorescence will be investigated in order to determine whether gold particles give fluorescence quenching or enhancement, and the spacing between the fluorescent species and labeled expression tags adjusted, if necessary. The proposed labels can be directly visualized in the transmission electron microscope without the need for signal enhancement by silver or gold and will enable correlative light and electron microscopy of cellular targets. PUBLIC HEALTH RELEVANCE: This project will provide researchers in cell, molecular and structural biology with methods and simple, universal reagents for electron microscopy labeling conducted in parallel with lac repressor or poly-histidine tags fused with colored fluorescent protein expressed in cells, and other models like zebrafish. This will also enable the study of dynamic cellular life processes and disease processes in living cells at macromolecular resolution, providing structural information at the molecular level for any system in which genetically engineered fused tags such as, fluorescent fusion proteins, His-tags and lac operator repeats, to yield fundamental insights into a wide variety of systems. This will be used for both, electron microscopic localization in living cells, and for high resolution correlative gold labeling.

描述（由申请人提供）：可共价连接的 3-15 nm 金试剂将通过包含用于稳定金纳米颗粒的疏水性螯合硫醇结构域和提供多种表面特性和共轭反应选择的亲水性表面基团的配方来制备稳定。提出了分别在活细胞和保存完好的组织中体内和切片离体金标记后对目标进行电子显微镜定位的方法。将使用可克隆表达标签，其中黄色和绿色荧光蛋白（分别为 YFP 和 GFP）分别与六组氨酸标签（His-标签）和 lac 阻遏物结合表达。然后，荧光蛋白、His 标签和 lac 阻遏物将被生物相容性、无毒、可共价连接的金纳米颗粒靶向，该纳米颗粒由抗体或次氮基三乙酸 (NTA)-Ni(II) 功能化，次氮基三乙酸 (NTA)-Ni(II) 是一种与 His 标签结合的螯合物，其强度和特异性与抗原抗体结合相当。在第一阶段，将通过修饰金表面来优化 10 和 15 nm 金-NTA-Ni(II) 标签的使用，以获得尽可能高的切片标记兼容性和稳定性，同时将使用最近开发的显微注射技术，优化与抗 lacI 或抗 GFP 抗体共价连接的 3 和 5 nm 金标记，用于体内金标记。最后，将研究金的大小、保护壳的厚度和蛋白质荧光之间的关系，以确定金颗粒是否会产生荧光猝灭或增强，并在必要时调整荧光物质和标记的表达标签之间的间距。所提出的标记可以在透射电子显微镜中直接可视化，无需通过银或金增强信号，并且可以实现细胞靶标的相关光学和电子显微镜。公共健康相关性：该项目将为细胞、分子和结构生物学的研究人员提供电子显微镜标记的方法和简单、通用的试剂，与细胞中表达的彩色荧光蛋白融合的 lac 阻遏物或多聚组氨酸标签以及斑马鱼等其他模型并行进行。这也将使得能够以大分子分辨率研究活细胞中的动态细胞生命过程和疾病过程，为其中荧光融合蛋白、His标签和lac操纵子重复等基因工程融合标签的任何系统提供分子水平的结构信息，从而获得对各种系统的基本见解。这将用于活细胞中的电子显微镜定位和高分辨率相关金标记。