Labeling of RNA through bioorthogonal reactions is typically split into two main-steps: The metabolic incorporation (in vivo) or enzymatic installation (in vitro) of the reporter group into RNA and the chemical reaction with tagged probes for detection.
A common approach for RNA synthesis monitoring relies on incorporation of ethynyl-labeled uridine and subsequent labelling with Azide-functionalized detection molecules (fluorescent Azides, Azides of (Desthio)Biotin or FLAG Azide)(Fig. 1A)[1,2]. This Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC) is widely applied due to its fast kinetics and high efficiency, but is hampered by cytotoxicity of in situ generated Cu(I). The amount of Cu(I) can be reduced by using stabilizing ligands.
An attractive copper-free alternative to measure de novo RNA synthesis in proliferating cells is presented by using vinyl-modified nucleosides as chemical reporter, such as 5-Vinyl-uridine (5-VU). Labeling of the resulting vinyl-functionalized RNA is achieved via Inverse-Electron Demand Diels-Alder reaction (IEDDA) that allows for subsequent introduction of either a Biotin group for purification (via Tetrazine-containing Biotinylation Reagents) or a fluorescent group for subsequent microscopic imaging (via Tetrazine-containing Fluorescent Dyes).
Recently, the marked efficiency of Alkene-Tetrazine Ligation was demonstrated by pairing the properties of 2-Vinyladenosine (2-VA) or 7-Deaza-vinyl-adenosine (7-dVA), respectively with highly reactive Pyrimidyl tetrazines (Fig. 1B).
Figure 1: A) Workflow of RNA synthesis monitoring with 5-EU using copper-catalyzed Alkyne-Azide Ligation. B) Schematic overview of RNA labeling using vinyl-functionalized nucleosides and imaging through bioorthogonal IEDDA reaction with Pyrimidyl tetrazines.
 Jao et al. (2008) Exploring RNA transcription and turnover in vivo by using click chemistry. Proc. Natl. Acad. Sci. USA 105 (41):15779.
 Darzynkiewicz et al. (2011) Cytometry of DNA Replication and RNA Synthesis: Historical Perspective and Recent Advances Based on "Click Chemistry". Cytometry A 79A:328.
 Liu et al. (2019) A Nucleoside Derivative 5‑Vinyluridine (VrU) for Imaging RNA in Cells and Animals. Bioconjugate Chem. 30:2958.
 Kubota et al. (2019) Expanding the Scope of RNA Metabolic Labeling with Vinyl Nucleosides and Inverse Electron-Demand Diels−Alder Chemistry. ACS Chem. Biol. 14:1698.