The 5′ Cap Epitranscriptome and Beyond: Natural and Engineered 5′ Cap Modifications for Optimizing mRNA Therapeutics and Functional Studies

Eukaryotic mRNAs made by in vitro transcription have emerged as medical modalities for vaccination and protein replacement therapy. The 5' cap is an essential feature of eukaryotic mRNAs providing stability, reducing immunogenicity, and serving as starting point for translation initiation. The "cap epitranscriptome" comprises several natural 5' cap modifications that can impact mRNA interactions and fate. Manipulating this privileged structure provides a powerful handle to optimize mRNA properties and to build a toolbox for investigating and controlling mRNA-related processes. In this article, the impact of natural 5' cap modifications on mRNA translation, immunogenicity, and stability is highlighted. Then, it is shown how non-natural 5' cap modifications have been used to manipulate and optimize various mRNA properties. Finally, non-natural modifications can equip mRNA with reactive handles, which provide a toolbox for studying interactions and controlling the function of mRNAs.