Novel fluorescent sensors for simple, sensitive, and specific protein detection

描述（由申请人提供）：蛋白质的检测基本上是所有生物医学研究的基础。目前检测蛋白质的策略包括蛋白质印迹和酶联免疫吸附测定等技术。然而，开发针对特定蛋白质的测定是耗时的，并且是高通量筛选开发、生物标志物表征和许多基础科学研究项目中的瓶颈。检测细胞中的蛋白质同样困难，并且通常需要通过固定然后进行免疫荧光测定来牺牲细胞。目前，还没有允许在活细胞中真实的实时监测内源性表达蛋白质水平的技术。本项目的目标是开发一种可推广的和简单的方法来检测蛋白质在体外和体内设置。我们已经开发了一类新的基于多核苷酸的传感器，我们已经证明，这些传感器允许特定分析物的荧光检测。该传感器通过在分析物结合时将原本非荧光的分子转换成荧光构型来起作用。我们建议开发一种可推广的方法，使传感器开发几乎任何蛋白质。我们描述了涉及在细胞内表达基因编码的传感器以检测细胞生长和分裂期间特定内源性蛋白质的实验。这种方法具有比目前基于荧光共振能量转移（FRET）的遗传编码探针更通用的潜力。我们还描述了一种使用这些传感器生成蛋白质传感微阵列的简单方法。在该技术中，生物样品将不需要样品处理，例如用荧光标记进行化学衍生。这种无标记的方法有可能为组织样品的蛋白质组学分析提供一种简单的方法。我们还描述了使用这些传感器在体外蛋白质定量。总之，本申请中描述的实验描述了一种多功能传感器技术，该技术将从根本上为整个生物医学研究界引入新颖且广泛有用的技术。 公共卫生相关性：检测蛋白质是生物医学研究中最基本的要求之一。然而，内源性蛋白质不能在活细胞中真实的时间检测。此外，用于生物样品中蛋白质定量的简单的无试剂技术不可用。我们已经开发出新型的基于三聚氰胺的传感器，其在暴露于特定分析物时会发出荧光。本申请描述了使用这些传感器检测和定量蛋白质的简单、灵敏和特异的方法。本申请还描述了这些传感器如何用于真实的监测细胞中的蛋白质表达，并描述了一种新的蛋白质传感微阵列技术。这些实验将为蛋白质检测带来强大的新工具，从而显著增强和扩大生物医学研究。

DESCRIPTION (provided by applicant): The detection of proteins is fundamental to essentially all biomedical research. Current strategies to detect proteins include techniques such as Western blotting and enzyme-linked immunosorbent assays. However, developing assays for specific proteins is time consuming and a bottleneck in high throughput screen development, biomarker characterization, and many basic science research projects. Detecting proteins in cells is similarly difficult, and typically requires sacrificing the cell by fixation followed by immunofluorescence assays. Currently, there are no techniques that permit the levels of endogenously expressed proteins to be monitored in real time in living cells. The goal of this project is to develop a generalizable and simple method to detect proteins in an in vitro and in vivo setting. We have developed a novel class of oligonucleotide-based sensors, and we have demonstrated that these sensors permit the fluorometric detection of specific analytes. The sensor functions by converting an otherwise nonfluorescent molecule into a fluorescent configuration upon analyte binding. We propose to develop a generalizable method that would allow sensors to be developed for virtually any protein. We describe experiments that involve expressing genetically encoded sensors within cells to detect specific endogenous proteins during cell growth and division. This approach has the potential to be vastly more versatile that current fluorescence resonance energy transfer (FRET)-based genetically encodable probes. We also describe a simple approach to generate protein sensing microarrays using these sensors. In this technique, biological samples will not require sample processing such as chemical derivatization with a fluorescent tag. This label-free approach has the potential to provide a simple methodology for proteomic analyses of tissue samples. We also describe the use of these sensors for in vitro protein quantification. Together, the experiments described in this application describe a versatile sensor technology that will introduce fundamentally novel and widely useful technologies to the entire biomedical research community. PUBLIC HEALTH RELEVANCE: Detecting proteins is one of the most fundamental requirements in biomedical research. However, endogenous proteins cannot be detected in living cells in real time. Furthermore, simple, reagentless techniques for protein quantification in biological samples are not available. We have developed novel oligonucleotide-based sensors that become fluorescent upon exposure to specific analytes. This application describes a simple, sensitive, and specific method for using these sensors for detecting and quantifying proteins. This application also describes how these sensors can be used to monitor protein expression in cells in real time, and describes a novel protein-sensing microarray technology. These experiments will result in powerful new tools for protein detection that will markedly enhance and expand biomedical research.