Stronger together for in-cell translation: natural and unnatural base modified mRNA

The preparation of highly modified mRNAs and visualization of their cellular distribution are challenging. We report in-cell application of in vitro transcribed mRNA containing natural base modifications and site-specifically introduced artificial nucleotides. Click chemistry on mRNA allows visualization in cells with excellent signal intensities. While non-specific introduction of reporter groups often leads to loss in mRNA functionality, we combined the benefits from site-specificity in the 3 '-UTR incorporated unnatural nucleotides with the improved translation efficiency of the natural base modifications psi and 5mC. A series of experiments is described to observe, quantify and verify mRNA functionality. This approach represents a new way to visualize mRNA delivery into cells and monitor its spread on a cellular level and translation efficiency. We observed increased protein expression from this twofold chemically modified, artificial mRNA counterbalancing a reduced transfection rate. This synergetic effect can be exploited as a powerful tool for future research on mRNA therapeutics.

Automated summary

In this study, we report the in-cell application of highly modified mRNA using in vitro transcription and visualization of their distribution in cells through click chemistry. By incorporating natural base modifications and site-specifically introduced artificial nucleotides, we were able to improve the translation efficiency of mRNA while maintaining its functionality. This approach provides a new way to enhance mRNA therapeutics.