Intracellular RNA Labeling Technologies for the Analysis of RNA Biology^†

As a cornerstone of the central dogma of molecular biology, RNA plays vital roles in living organisms. Over the past few decades, many RNA labeling technologies have been developed to elucidate the biological function of RNA. These technologies have significantly advanced our understanding of RNA secondary structure, localization, and turnover. Additionally, taking advantage of these innovative RNA labeling approaches, plenty of tool kits have been devised for the regulation of RNA-related biological process, such as gene expression and gene editing. In this review, we primarily focus on an array of intracellular RNA labeling methods, encompassing chemical probes-based labeling, metabolic labeling, and proximity-dependent labeling. We also provide a brief overview of their applications in the research of RNA biology. Finally, the perspectives of RNA labeling are also discussed.

Key Scientists

4SU was first used in1978 as a safer way to directly examine RNA synthesis. However, the enrichment of 4SU-labeled RNA seriously relied on mercurated cellulose columns, which are toxic. In 2005, Boothroyd et al. introduced a novel covalent-modification method of 4SU based on thiol exchange reaction. Then, Salic et al. applied 5EU, which is clickable, for RNA metabolic labeling in animals. Later, Spitale et al. designed 5EC and 2’AzU, which enabled cell specific RNA metabolic labeling under the help of specific enzyme. In 2013, directly chemical RNA labeling in living cell for RNA structure mapping was realized by Chang and Kool et al. using SHAPE reagent, which was further modified with an azide handle in 2015. Weissman, Bevilacqua and Assmann et al. introduced DMS probe that targets adenine and cytosine of RNA for RNA structure mapping in 2014. Then, Chang et al. exploited psoralen for RNA duplex mapping in 2016. Later, He, Zhou and Zhang et al. described another clickable probe N₃-kethoxal targeting guanine for RNA secondary structure mapping. Besides, Chang and Ting et al. described a proximity-dependent RNA labeling method based on APEX in 2019, revealing subcellular RNA localization. Meanwhile, Zou and Wang et al. achieved proximity-dependent RNA labeling using miniSOG oxidase, which provided an alternative method to investigate the spatial transcriptome. Spitale et al. utilizing photosensitizer dibromofluorescein (DBF) to realize proximity-dependent RNA labeling and sequencing.