Expanding the Scope of RNA Metabolic Labeling with Vinyl Nucleosides and Inverse Electron-Demand Diels–Alder Chemistry

Optimized and stringent chemical methods to profile nascent RNA expression are still in demand. Herein, we expand the toolkit for metabolic labeling of RNA through application of inverse electron demand Diels-Alder (IEDDA) chemistry. Structural examination of metabolic enzymes guided the design and synthesis of vinyl-modified nucleosides, which we systematically tested for their ability to be installed through cellular machinery. Further, we tested these nucleosides against a panel of tetrazines to identify those which are able to react with a terminal alkene, but are stable enough for selective conjugation. The selected pairings then facilitated RNA functionalization with biotin and fluorophores. We found that this chemistry not only is amenable to preserving RNA integrity but also endows the ability to both tag and image RNA in cells. These key findings represent a significant advancement in methods to profile the nascent transcriptome using chemical approaches.