Chen Antony K, Rhee Won Jong, Bao Gang, Tsourkas Andrew
Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA.
Methods Mol Biol. 2011;714:159-74. doi: 10.1007/978-1-61779-005-8_10.
Over the past decade, a variety of oligonucleotide-based probes have been developed that allow for direct visualization of RNA molecules in living cells. Of these, molecular beacons have garnered a particularly high degree of interest due to their simple yet exquisite unimolecular stem-loop design that allows for the efficient conversion of target recognition into a specific fluorescent signal. As a result of their favorable fluorescent enhancement and their high specificity, molecular beacons have been used for a wide range of applications, including the monitoring of RNA expression and localization in living cells, cancer cell detection, and the study of viral infections. In this chapter we describe a general methodology that can be followed for the imaging and analysis of RNA in living cells using molecular beacons. Several commonly employed methods for delivering molecular beacons into the cytosol are discussed including toxin-based cell membrane permeabilization, microinjection, and microporation. Strategies for acquiring ratiometric measurements are also described.
在过去十年中，已开发出多种基于寡核苷酸的探针，可直接观察活细胞中的RNA分子。其中，分子信标因其简单而精巧的单分子茎环设计而备受关注，该设计可将目标识别高效转化为特定的荧光信号。由于其良好的荧光增强效果和高特异性，分子信标已被广泛应用于多种领域，包括监测活细胞中的RNA表达和定位、癌细胞检测以及病毒感染研究。在本章中，我们描述了一种使用分子信标对活细胞中的RNA进行成像和分析的通用方法。讨论了几种将分子信标递送至细胞质的常用方法，包括基于毒素的细胞膜通透化、显微注射和微孔穿孔。还描述了获取比率测量值的策略。
