Fluorescent probes for quantitation of secretory protein levels in single cells

描述(由申请人提供)：分泌蛋白通常是疾病相关细胞状态变化的强有力的标记物，包括内质网应激和转移。缺乏检测活细胞中特定管腔分泌蛋白的技术是一个重大差距。 在细胞成像工具中。我们的目标是开发和交付能够检测活细胞群体中诊断分泌蛋白表达差异的高度敏感的记者。如果不这样做，我们将结合三种现有的技术来创建一种新的成像工具，STABS(秘书靶向适体信标)。更具体地说，通过将修饰的细菌毒素与核酸酶稳定的适体信标结合起来，针对分泌蛋白质，如血管内皮生长因子或UPR诱导的内质网蛋白质Ero1和ERdj4，我们的目标是产生试剂，当直接添加到细胞或组织中时，将进入分泌途径并报告这些蛋白质的存在。理论上，多达四种不同的荧光染料可以与独特的适配子配对来报告四种不同的分泌蛋白的表达。我们预计该探针技术将对组织样本的基础研究和快速临床分析具有实用价值。 公共卫生相关性：分泌蛋白通常是与疾病相关的细胞状态变化的强有力的标志，包括内质网应激和转移。拟议的工作旨在开发新的工具来直接检测体内蛋白质水平的存在或变化 天然的、未受干扰的活细胞的分泌途径。我们预计该探针技术将对组织样本的基础研究和快速临床分析具有实用价值。

DESCRIPTION (provided by applicant): Secretory proteins are often robust markers of changes in disease-relevant cellular states including ER stress and metastasis. The absence of technologies for detecting specific luminal secretory proteins in live cells represents a major gap in cell imaging tools. Our goal is to develop and deliver highly sensitive reporters that can detec differences in the expression of diagnostic secretory proteins within a population of live cells. T do this, we will combine three existing technologies to create a new class of imaging tools, STABs (Secretory Targeting Aptamer Beacons). More specifically, by combining modified bacterial toxins with nuclease stabilized aptamer beacons specific for secreted proteins such as VEGF or the UPR-induced endoplasmic reticulum proteins Ero1 and ERdj4, we aim to generate reagents which, when added directly to cells or tissues, will enter the secretory pathway and report the presence of these proteins. Theoretically, up to four distinct fluorescent dyes can be paired with unique aptamers to report on the expression of four different secretory proteins. We envision the probe technology will have utility for basic research and rapid clinical analysis of tissue samples. PUBLIC HEALTH RELEVANCE: Secretory proteins are often robust markers of changes in disease-relevant cellular states including ER stress and metastasis. The proposed work seeks to develop new tools to directly detect the presence of or changes in the levels of proteins within the secretory pathway of na¿ve, unperturbed live cells. We envision the probe technology will have utility for basic research and rapid clinical analysis of tissue samples.