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

## TL;DR

This paper explores how modifying the 5′ cap of mRNA can improve its stability, reduce immune response, and enhance translation for better therapeutic applications.

## Contribution

The paper introduces both natural and engineered 5′ cap modifications as tools to optimize mRNA properties and study mRNA function.

## Key findings

- Natural 5′ cap modifications influence mRNA translation, stability, and immunogenicity.
- Engineered non-natural 5′ cap modifications can enhance mRNA properties and provide reactive handles for functional studies.
- 5′ cap modifications can be introduced using enzymes or chemical synthesis methods.

## Abstract

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.

5′ Cap modifications can be introduced via enzymes or chemical synthesis. Natural and non‐natural 5′ cap structures affect mRNA properties, including translation (T), stability (S), and/or immunogenicity (I).© 2026 WILEY‐VCH GmbH