Cellular RNA labeling strategies based on bioorthogonal chemical reactions are much less developed in comparison to glycan, protein and DNA due to its inherent instability and lack of effective methods to introduce bioorthogonal reactive functionalities (e.g. azide) into RNA. Here we report the development of a simple and modular posttranscriptional chemical labeling and imaging technique for RNA by using a novel toolbox comprised of azide-modified UTP analogs. These analogs facilitate the enzymatic incorporation of azide groups into RNA, which can be posttranscriptionally labeled with a variety of probes by click and Staudinger reactions. Importantly, we show for the first time the specific incorporation of azide groups into cellular RNA by endogenous RNA polymerases, which enabled the imaging of newly transcribing RNA in fixed and in live cells by click reactions. This labeling method is practical and provides a new platform to study RNA in vitro and in cells.

Dynamic interplay between peptide synthesis and membrane assembly would have been crucial for the emergence of protocells on the prebiotic Earth. However, the effect of membrane-forming amphiphiles on peptide synthesis,...

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Direct incorporation of azide groups into RNA oligonucleotides by in vitro transcription reactions in the presence of a new azide-modified UTP analogue, and subsequent posttranscriptional chemical labeling of azide-modified oligoribonucleotide transcripts by click and Staudinger reactions are described. This postsynthetic labeling protocol is robust and modular, and offers an alternative access to RNA labeled with biophysical probes.

Numerous biophysical tools based on fluorescence have been developed to advance the understanding of RNA–nucleic acid, RNA–protein, and RNA–small molecule inter-actions. In this regard, fluorescent ribonucleoside analogues that are sensitive to their local environment provide sensitive probes for investigating RNA structure, dynamics, and recognition. Most of these analogues closely resemble the native ribonucleosides with respect to their overall dimension and have the ability to form canonical Watson–Crick (WC) base pairs. Therefore, it is possible to place these probes near the point of interaction in a target nucleic acid with minimum structural perturbations and gain insight into the intricacies of conformational changes taking place in and around the interaction site. Here, we provide a concise background on the development and recent advances in the applications of base-modified fluorescent ribonucleoside analogue probes. We first present various base-modified fluorescent ribonucleoside analogues, their photophysical properties, and methods to incorporate these analogues into oligoribonucleotides. We then discuss the established spectroscopic techniques, which make use of the fluorescence properties of these emissive ribonucleoside analogues. Finally, we present the applications of base-modified fluorescent ribonucleoside analogues used as probes incorporated into oligoribonucleotides in investigating RNA structures and functions.