# Enhancing cap-independent translation of linear mRNA

## TL;DR

This paper introduces a new method to improve cap-independent mRNA translation, making it more efficient and stable for potential therapeutic use.

## Contribution

A novel 5′-end modification strategy using click chemistry enhances mRNA stability and protein output for cap-independent translation.

## Key findings

- The new modification significantly improves mRNA stability and protein production.
- The method does not trigger an immune response, making it suitable for therapeutic applications.
- The approach allows detailed study of transfection and translation processes in cells.

## Abstract

While cap-dependent translation remains the primary focus in mRNA-based therapeutics, cap-independent translation holds promise for targeting diseases ranging from cancer to neurodegeneration. However, cap-independently translated mRNAs are unstable, produce less protein than capped mRNAs, and current methods for their improvement are imperfect. Here, we propose the use of in vitro transcription priming with azido-modified dinucleotide primer and post-transcriptional modification utilising click chemistry to improve the properties of cap-independently translated mRNAs. Our results demonstrate a significant enhancement in mRNA stability and protein output without eliciting immunogenicity. Moreover, we show how the mRNA 5′-end modification strategy can be used to investigate transfection and cap-independent translation processes in cells overcoming burdens associated with previous methods. Together, our findings support cap-independent translation as a viable alternative to the established cap-dependent process and provide tools for further exploration and enhancement of this modality.

Cap-independently translated mRNAs could expand the scope of mRNA therapies, but yield limited protein, and existing enhancement strategies are imperfect. Here, the authors install a 5′-end modification that both boosts efficiency and facilitates in-depth characterisation of these mRNAs.