3 to15 Covalent Gold for High Resolution Electron Microscopy

描述（由申请人提供）：可共价连接的3-15 nm金试剂将被制备稳定的配方，其中包含用于稳定金纳米颗粒的疏水螯合硫醇结构域，以及提供广泛选择的表面性质和共轭反应的亲水性表面基团。提出了在活细胞和保存良好的组织中分别进行活体金标和离体金标的电镜定位方法。使用克隆表达标签，分别与六组氨酸标签（His-tag）和lac抑制因子联合表达黄色和绿色荧光蛋白（分别为YFP和GFP）。荧光蛋白、his标签和lac抑制因子将被生物相容性、无毒共价可连接的金纳米颗粒靶向，这些纳米颗粒被抗体或硝酸三乙酸(NTA)-Ni（II）功能化，这是一种与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，10和15纳米金- nta - ni （II）标签的使用将通过修饰金表面来优化，以获得最高的相容性和稳定性，用于分段标记；而3和5纳米金共价连接抗laci或抗gfp抗体，将使用最新开发的显微注射技术来优化体内金标记。最后，研究金的大小、保护壳的厚度与蛋白质荧光之间的关系，以确定金颗粒是否会使荧光猝灭或增强，并在必要时调整荧光种类与标记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下直接显示，而不需要银或金的信号增强，并且将使细胞目标的相关光学和电子显微镜成为可能。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的通用试剂，用于与紫胶抑制因子或与细胞和斑马鱼等其他模型中表达的彩色荧光蛋白融合的多组氨酸标签并行进行电子显微镜标记。这也将使动态细胞生命过程和疾病过程的研究在大分子分辨率的活细胞，提供结构信息在分子水平上的任何系统，其中的基因工程融合标签，如荧光融合蛋白，his标签和lac操作重复，产生基本见解的各种系统。这将用于活细胞的电子显微镜定位和高分辨率相关的金标。

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.