Zhou Cun Yu, Alexander Seth C, Devaraj Neal K
Department of Chemistry and Biochemistry , University of California , 9500 Gilman Dr La Jolla , San Diego , CA 92093 , USA . Email:
Chem Sci. 2017 Oct 1;8(10):7169-7173. doi: 10.1039/c7sc03150e. Epub 2017 Aug 29.
Investigating the many roles RNA plays in cellular regulation and function has increased demand for tools to explore RNA tracking and localization within cells. Our recently reported RNA-TAG (transglycosylation at guanine) approach uses an RNA-modifying enzyme, tRNA-guanine transglycosylase (TGT), to accomplish covalent labeling of an RNA of interest with fluorescent tracking agents in a highly selective and efficient manner. Unfortunately, labeling by this method currently suffers from a high nonspecific fluorescent background and is currently unsuitable for imaging RNA within complex cellular environments. Herein we report the design and synthesis of novel fluorogenic thiazole orange probes that significantly lower nonspecific binding and background fluorescence and, as a result, provide up to a 100-fold fluorescence intensity increase after labeling. Using these fluorogenic labeling agents, we were able to image mRNA expressed in Chinese Hamster Ovary cells in a wash-free manner.
对RNA在细胞调控和功能中所起的多种作用进行研究，增加了对探索RNA在细胞内追踪和定位工具的需求。我们最近报道的RNA-TAG（鸟嘌呤转糖基化）方法使用一种RNA修饰酶，即tRNA-鸟嘌呤转糖基化酶（TGT），以高度选择性和高效的方式用荧光追踪剂对感兴趣的RNA进行共价标记。不幸的是，目前这种方法的标记存在高非特异性荧光背景，目前不适用于在复杂细胞环境中对RNA进行成像。在此，我们报告了新型荧光噻唑橙探针的设计与合成，这些探针显著降低了非特异性结合和背景荧光，结果在标记后荧光强度增加了100倍。使用这些荧光标记剂，我们能够以无需洗涤的方式对中国仓鼠卵巢细胞中表达的mRNA进行成像。
