FLUORESCENT AND LARGE METAL CLUSTER COMBINATION PROBES

描述（改编自申请人摘要）：此快速通道 应用围绕着新探头的开发联合收割机 多个荧光团和大的金属簇络合物。 这些独特 开发电子和荧光两用探针 显微镜 提出了几种新的探头， I相：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'， 2）Fab'抗体缀合的纳米金簇（1.4nm），其 将通过星状树枝状聚合物连接到多个荧光团， 为了消除与先前的荧光猝灭有关的问题， 尝试开发荧光金颗粒; 3）荧光纳米金 和更大的簇，其结合镍（II）螯合物， 将多组氨酸序列工程化到带His标签的重组蛋白中 4）Fab'抗绿色荧光蛋白（GFP） 与纳米金和更大的金属簇结合的抗体用于检测 在细胞中表达的GFP嵌合蛋白;和5）荧光纳米金 和更大的簇，其与dUTP和dATP偶联用于酶促反应， 掺入DNA并用于原位杂交。 在初始 阶段，将通过以下方法评价抗IgG抗体偶联探针： 免疫印迹连续稀释的IgG（用银增强）和作为 用于表面红血免疫荧光标记的二级试剂 细胞抗原 还将使用荧光和金属簇Fab 作为第二探针检测单克隆抗体标记的snRNP 并通过共聚焦和电子显微镜进行评价。 荧光金属 将测试簇DNA探针检测前mRNA的能力 c-fos的转录本和与当前应用相比的结果。 抗 GFP定向探针将针对几种GFP嵌合蛋白进行测试 预期具有不同的细胞内定位模式。 第二阶段将成功应用新试剂， 通过荧光和电子显微镜研究三个系统：（i） 核RNA加工位点的结构和功能;（ii） 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结构， 用显微注射双探针标记的DNA结合蛋白研究 活细胞中的动态过程。 拟定商业应用： 这些产品在研究界具有很好的应用潜力， 特别是用于高分辨率电子显微镜观察， 定义分子组织的能力。 探测器的小尺寸将 除非在高放大率下，否则限制有用性。 然而，共价 将探针附着到初级试剂上， 标记通常是用胶体标记常规标记中的问题， 黄金 开发供用户标记自己的试剂盒的潜力 含有硫醇或胺的产品是特别有吸引力的特征 的提案。

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.