Yang Yantao, Chen Mingming, Krueger Christopher J, Tsourkas Andrew, Chen Antony K
Department of Biomedical Engineering, College of Engineering, Peking University, No. 5 Yiheyuan Road, Haidian District, Beijing, 100871, China.
Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China.
Methods Mol Biol. 2018;1649:231-242. doi: 10.1007/978-1-4939-7213-5_15.
Molecular beacons (MBs), a class of oligonucleotide-based probes, have enabled researchers to study various RNA molecules in their native live-cell contexts. However, it is also increasingly recognized that, when delivered into cells, MBs have the tendency to be sequestered into the nucleus where they may generate false positive signals. In an attempt to overcome this issue, MBs have been synthesized with chemically modified oligonucleotide backbones to confer greater biostability. Alternatively, strategies have been developed to minimize nuclear entry. In the latter approach, we have combined functional elements of MBs with functional elements of siRNAs that facilitate nuclear export to create a new RNA imaging platform called ratiometric bimolecular beacons (RBMBs). We showed that RBMBs exhibited long-term cytoplasmic retention, and hence a marginal level of false positive signals in living cells. Subsequent studies demonstrated that RBMBs could sensitively and accurately quantify mRNA transcripts engineered to contain multiple tandem repeats of an MB target sequence at the single-molecule level. In this chapter, we describe the synthesis of RBMBs and their applications for absolute quantification and tracking of single mRNA transcripts in cells.
分子信标（MBs）是一类基于寡核苷酸的探针，使研究人员能够在天然活细胞环境中研究各种RNA分子。然而，人们也越来越认识到，当将分子信标导入细胞时，它们往往会被隔离到细胞核中，在那里可能产生假阳性信号。为了克服这个问题，人们合成了具有化学修饰寡核苷酸骨架的分子信标，以赋予其更高的生物稳定性。或者，已经开发出策略来尽量减少进入细胞核。在后一种方法中，我们将分子信标的功能元件与促进核输出的小干扰RNA（siRNA）的功能元件相结合，创建了一个名为比率双分子信标（RBMBs）的新RNA成像平台。我们表明，RBMBs在活细胞中表现出长期的细胞质滞留，因此假阳性信号水平较低。随后的研究表明，RBMBs能够在单分子水平上灵敏且准确地定量工程化包含多个MB靶序列串联重复的mRNA转录本。在本章中，我们描述了RBMBs的合成及其在细胞中对单个mRNA转录本进行绝对定量和追踪的应用。
