3 to15 Covalent Gold for High Resolution Electron Microscopy

描述（由申请人提供）：将制备用制剂稳定化的3-15 nm胶体金试剂，所述制剂包含用于稳定金纳米颗粒的疏水螯合硫醇结构域和提供表面性质和缀合反应的广泛选择的亲水表面基团。本文提出了分别在活细胞和保存良好的组织中进行体内和体外金标记后的电子显微镜靶点定位方法。将使用可克隆的表达标签，其中黄色和绿色荧光蛋白（分别为YFP和GFP）分别与六聚组氨酸标签（His-标签）和lac阻遏物组合表达。然后，荧光蛋白、His标签和lac阻遏物将被生物相容性的、无毒的共价键合的金纳米颗粒靶向，所述金纳米颗粒用抗体或氨三乙酸（NTA）-Ni（II）功能化，所述氨三乙酸-Ni（II）是一种螯合物，其以与抗原-抗体结合相当的强度和特异性结合His标签。在第一阶段，10和15 nm金-NTA-Ni（II）标签的使用将通过金表面的修饰进行优化，以获得用于切片标记的最高可能的相容性和稳定性，而与抗lacI或抗GFP抗体共价连接的3和5 nm金将使用最近开发的显微注射技术进行优化，以用于体内金标记。最后，将研究金尺寸、保护壳厚度和蛋白质荧光之间的关系，以确定金颗粒是否产生荧光猝灭或增强，并且如果需要，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，而不需要通过银或金进行信号增强，并且将使得能够对细胞靶进行相关的光学和电子显微镜检查。公共卫生相关性：该项目将为细胞、分子和结构生物学的研究人员提供方法和简单、通用的试剂，用于与细胞中表达的有色荧光蛋白融合的lac阻遏物或聚组氨酸标签以及其他模型（如斑马鱼）平行进行的电子显微镜标记。这还将使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成为可能，为任何含有基因工程融合标签（例如荧光融合蛋白、His标签和lac操纵子重复序列）的系统提供分子水平的结构信息，以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。

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.