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.