FLUORESCENT AND LARGE METAL CLUSTER COMBINATION PROBES

描述(改编自申请者的摘要)：这条快速通道 应用围绕着开发新的探针，这些探针结合了 多个荧光团和大的金属簇合物。这些独特的 将开发用于电子和荧光双重用途的探针 显微镜。在此基础上提出了几点新的探索。 第一阶段：1)大的铂和钯簇合物(1.8-3.6 nm) 其共价连接到多个荧光实体和Fab‘ 抗体；2)Fab‘抗体结合的纳米金簇(1.4 nm)，其 将通过星爆树枝状聚合物连接到多个荧光团， 要消除与以前的 尝试开发荧光金粒子；3)荧光纳米金 以及结合镍(II)络合物的较大团簇 His标记重组蛋白中的多组氨酸序列 4)Fab‘抗绿色荧光蛋白(GFP) 用于检测的与纳米金和更大的金属簇结合的抗体 在细胞中表达的GFP嵌合蛋白；以及5)荧光纳米金 和更大的簇，与dUTP和dATP偶联以用于酶 掺入DNA并用于原位杂交。在最初 阶段，抗-IgG抗体结合的探针将通过以下方式进行评估 免疫印迹连续稀释的免疫球蛋白(含银增强)和AS 表面红血球免疫荧光标记的二次试剂 细胞抗原。荧光和金属簇-FAB也将被使用 作为二次探针检测单抗标记的SnRNPs 并通过共聚焦显微镜和电子显微镜进行评价。荧光金属 将测试簇状DNA探针检测前-mRNA的能力 C-fos的转录和结果与当前应用的比较。反- GFP导向的探针将针对几种GFP嵌合蛋白进行测试 预计会有不同的细胞内定位模式。 第二阶段将在新试剂成功应用于 用荧光和电子显微镜研究三种体系：(一) 核RNA加工位点的结构和功能；(2) 平滑肌Na~+/K~+和Na~+/Ca~++泵的大分子结构 细胞；和(Iii)间期染色体结构和核构型， 微注射双探针标记DNA结合蛋白的研究 活细胞中的动态过程。 建议的商业应用： 这些产品在研究界具有极好的使用潜力， 尤其是针对高分辨率电子显微镜观察 在定义分子组织方面。探测器的小尺寸将 除非使用高倍率，否则限制使用。然而，共价键 探针与初级试剂的结合应减少非特异性 在常规胶体标记中通常存在的标记问题 金子。为用户开发自己的标签试剂盒的潜力 含有硫醇或胺的产品是一个特别吸引人的特征 这项提议。

DESCRIPTION (Adapted from applicant's abstract): This Fast Track application revolves around the development of new probes that combine multiple fluorophores and large metal cluster complexes. These unique probes are to be developed for dual use in electron and fluorescence microscopy. Several new probes are proposed for the development during Phase I: 1) large platinum and palladium cluster complexes (1.8-3.6 nm) that are covalently linked to multiple fluorescent entities and to Fab' antibodies; 2) Fab' antibody conjugated Nanogold clusters (1.4 nm) that will be linked to multiple fluorophores via starburst dendrimer polymers, to eliminate fluorescent quenching problems associated with previous attempts to develop fluorescent gold particles; 3) fluorescent Nanogold and larger clusters that incorporate nickel (II) chelate that binds polyhistidine sequences engineered into His- tagged recombinant protein expressed in cells; 4) Fab' anti-green fluorescence protein (GFP) antibodies conjugated to Nanogold and larger metal clusters for detection of GFP-chimeric proteins expressed in cells; and 5) fluorescent Nanogold and larger clusters that are coupled to dUTP and dATP for enzymatic incorporation into DNA and used for in situ hybridization. In the initial phase, anti-IgG antibody-conjugated probes would be evaluated by immunoblotting serially diluted IgG (with silver enhancement) and as secondary reagents for immunofluorescence labeling of surface red blood cell antigens. The fluorescent and metal cluster-Fab's would also be used as secondary probes to detect snRNPs labeled with monoclonal antibodies and evaluated by confocal and electron microscopy. Fluorescent metal cluster-DNA probes would be tested for ability to detect pre-mRNA transcripts of c-fos and results compared to current applications. Anti- GFP directed probes would be tested against several GFP-chimeric proteins that are expected to have distinct intracellular localization patterns. Phase II would follow with successful application of the new reagents to study three systems by fluorescence and electron microscopy: (i) structure and function of nuclear RNA processing sites; (ii) macromolecular structure of the Na+/K+ and Na+/Ca++ pumps in smooth muscle cells; and (iii) interphase chromosome structure and nuclear architecture, using microinjected dual-probe labeled DNA-binding proteins to study dynamic processes in living cells. PROPOSED COMMERCIAL APPLICATION: The products have excellent potential for use in the research community, particularly for high resolution electron microscope observations aimed at defining molecular organization. The small size of the probes will limit usefulness unless at high magnification. However, the covalent attachment of probes to primary reagents should reduce nonspecific labeling that is typically a problem in routine labeling with colloidal gold. The potential to develop kits for users to label their own products that contain thiols or amines is an especially attractive feature of the proposal.