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（包括ER应激和转移）变化的稳健标志物。缺乏检测活细胞中特定腔分泌蛋白的技术是一个主要差距 在细胞成像工具中。我们的目标是开发和提供高灵敏度的报告，可以检测在活细胞群体内的诊断分泌蛋白的表达差异。为了做到这一点，我们将联合收割机结合现有的三种技术，创造一种新的成像工具，STAB（分泌靶向适体BETAB）。更具体地说，通过将修饰的细菌毒素与对分泌蛋白如VEGF或UPR诱导的内质网蛋白Ero 1和ERdj 4具有特异性的核酸酶稳定的适体信标组合，我们的目标是产生试剂，当将其直接加入细胞或组织时，将进入分泌途径并报告这些蛋白的存在。理论上，多达四种不同的荧光染料可以与独特的适体配对，以报告四种不同分泌蛋白的表达。我们设想探针技术将用于组织样本的基础研究和快速临床分析。 公共卫生相关性：分泌蛋白通常是疾病相关细胞状态（包括ER应激和转移）变化的稳健标志物。拟议的工作旨在开发新的工具，以直接检测体内蛋白质的存在或水平的变化。 未受干扰的原始活细胞的分泌途径。我们设想探针技术将用于组织样本的基础研究和快速临床分析。

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.