Photoactivatable mRNA 5′ Cap Analogs for RNA‐Protein Crosslinking

Abstract Chemical modification of messenger RNA (mRNA) has paved the way for advancing mRNA‐based therapeutics. The intricate process of mRNA translation in eukaryotes is orchestrated by numerous proteins involved in complex interaction networks. Many of them bind specifically to a unique structure at the mRNA 5′‐end, called 5′‐cap. Depending on the 5′‐terminal sequence and its methylation pattern, different proteins may be involved in the translation initiation and regulation, but a deeper understanding of these mechanisms requires specialized molecular tools to identify natural binders of mRNA 5′‐end variants. Here, a series of 8 new synthetic 5′‐cap analogs that allow the preparation of RNA molecules with photoreactive tags using a standard in vitro transcription reaction are reported. Two photoreactive tags and four different modification sites are selected to minimize potential interference with cap‐protein contacts and to provide complementary properties regarding crosslinking chemistry and molecular interactions. The tailored modification strategy allows for the generation of specific crosslinks with model cap‐binding proteins, such as eIF4E and Dcp2. The usefulness of the photoreactive cap analogs is also demonstrated for identifying the cap‐binding subunit in a multi‐protein complex, which makes them perfect candidates for further development of photoaffinity labeling probes to study more complex mRNA‐related processes.