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

ABSTRACT 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.