Small‐Molecule Activation of mRNA Translation by Click‐to‐Release Reaction in Cells

mRNA is an emerging medical modality, however, approaches to control its activity lack behind other biologics. Bioorthogonal click-to-release reactions enable breaking chemical bonds at high reaction rates even in living cells to release a functionally active biomolecule ("uncaging"). We developed a 5' cap modified with a trans-cyclooctene (TCO-cap) that reacts with hydroxyaryl-tetrazines to efficiently release the native cap 0. This strategy is compatible with in vitro transcription and facilitates HPLC-based purification of the resulting TCO-capped mRNA, circumventing the need to digest uncapped mRNA produced in the process. Using eGFP- and luciferase-mRNAs in mammalian cells, we show that TCO-capped mRNAs are translationally muted and can be activated for translation by addition of cell-permeable, non-toxic sulfonamide-modified hydroxyphenyl-tetrazines. This work presents a new approach for small-molecule-induced translation in eukaryotes with potential to be applicable to any mRNA.