High Resolution Labels for EM

描述（由申请人提供）：用于EM结构研究的金标是一种重要的工具，可用于进一步了解蛋白质，复合物和细胞成分的组织。荧光标记在光学显微镜水平上非常有用，但在高分辨率的EM研究中并不有用。随着低温电子显微镜、大阵列ccd、氦级、能量过滤器、像差校正器、层析成像和自动化的出现，电子显微镜最近取得了进步。同样，在冷冻电镜（规则网格和控制冷冻单元）的标本制备方法和更强大和有用的图像处理软件方面也取得了重大进展。标记也取得了一些技术进步，例如量子点，ReAsH和小金标的发展，例如胺和硫醇反应金簇，氟纳米金（结合金和Alexa染料）和ni - nta -纳米金。这对于识别大分子上的特定位点、复合物中的特定亚基和细胞中的成分是有用的。在此，我们建议继续开发EM的高分辨率金标，并将构建几个解决当前问题的新标签。这项工作将包括：a)金壳的专门配体的有机合成，b)稳定和表征良好的金颗粒的形成，c)功能性测试，d)减少或消除非特异性结合的工作，e)引入融合标签的分子生物学，以及e)与合作者验证和应用于生物学问题。标签将包括：1；2-15纳米金标。功能化的、高度规则的金标签将被制成更大的尺寸，以提高可视性（许多应用都需要），并提供活性金试剂、更大的ni - nta金和改进的金免疫探针。2. 金融合标签：金颗粒功能化，以结合蛋白融合标签。a.噬菌体展示标签：噬菌体展示将用于寻找与金标签结合的序列，作为蛋白质融合标签。连接的副本将增加绑定。b.商品及服务税黄金。用GST标记的蛋白质会用金标记这个位点。c. TetraCys金。金颗粒将被制造出来，专门与四半胱氨酸基因融合标签结合。d.钙调蛋白金：钙调蛋白对钙调蛋白结合域具有很高的结合亲和力，钙调蛋白金提供了另一种有用的标签标记选择。3. PhotoGold。金上具有低结合常数的分子（如肽）在结合时将被光锁定，从而可以从未结合的金中纯化并通过EM定位结合位点。ATP/GTP/AMPPNP/GMPPNP-gold用于定位ATP和GTP位点。5. DNA插层金：高度标记的DNA将使其能够在DNA-蛋白质复合物中识别。这个标签库将为高分辨率电子显微镜提供重要的新工具。鉴于目前用于高分辨率电子显微镜分子分析的合适探针的缺乏，该探针的开发工作将显著推进细胞和分子生物学领域的发展。公共卫生相关性：该项目将通过在电子显微镜水平上提供识别分子的新标签，使发现重要生物蛋白、复合物和细胞的重要新信息成为可能。这些研究的最终目的是了解生命过程，从而导致疾病的有效治疗。

DESCRIPTION (provided by applicant): Gold labels for EM structural studies are an important tool that can be used to further understand the organization of proteins, complexes and cellular components. Fluorescent labels have been incredibly useful at the light microscope level, but are not useful for higher resolution EM studies. Electron microscopes have recently advanced with the advent of cryoEM, large array CCDs, helium stages, energy filters, aberration correctors, tomography, and automation. Similarly, significant advances have been made in specimen preparation methods for cryoEM (regular grids and controlled freezing units) and more powerful and useful image processing software. Labeling has also seen some technological advances, such as the development of quantum dots, ReAsH, and small gold labels, such as amine and thiol reactive gold clusters, FluoroNanogold (combining gold and Alexa dyes) and Ni-NTA-Nanogold. These are useful for identifying specific sites on macromolecules, particular subunits in a complex, and components in cells. Here we propose to continue development of high resolution gold labels for EM. Several new labels addressing current problems will be constructed. The work will include: a) organic synthesis of specialized ligands for the gold shell, b) formation of stable and well-characterized gold particles, c) testing to demonstrate functionality, d) work to reduce or eliminate non-specific binding, e) molecular biology to introduce fusion tags, and e) validation and application to biological problems with collaborators. Label will include: 1. 2-15 nm gold labels. Functionalized, highly regular, gold labels will be made in larger sizes to improve visibility (needed for many applications) and provide reactive gold reagents, larger Ni-NTA-gold, and improved gold immunoprobes. 2. Gold Fusion-Tag Labels: Gold particles functionalized to bind to protein fusion tags. a. Phage Display Tags: Phage display will be used to find sequences that bind to gold labels for use as protein fusion tags. Concatenated copies will increase binding. b. GST gold. Proteins tagged with GST would have this site labeled with gold. c. TetraCys gold. Gold particles will be made that specifically bind to the tetra-cysteine genetic fusion tag. d. Calmodulin gold: Calmodulin has a high binding affinity for the calmodulin binding domain, and calmodulin-gold provides another useful tag labeling alternative. 3. PhotoGold. Molecules with low binding constants (e.g., peptides) on the gold will be photolocked when they bind, enabling purification from unbound gold and binding site localization by EM. 4. ATP/GTP/AMPPNP/GMPPNP-gold for localizing ATP and GTP sites. 5. DNA intercalator gold: Highly labeled DNA will enable its identification in DNA-protein complexes. This arsenal of labels will provide important new tools for high resolution electron microscopy. Given the paucity of suitable probes for high resolution molecular analysis using electron microscopy, this probe development work would significantly advance the fields of cell and molecular biology. PUBLIC HEALTH RELEVANCE: This project will enable important new information to be found about important biological proteins, complexes, and cells by providing new labels for identifying molecules at the electron microscope level. The ultimate aim of these studies is to understand life processes, which in turn, can lead to effective treatment of disease.