Effects of modified nucleotides in a mRNA construct encoding SARS-CoV-2 spike protein: implications for functionality and therapeutic potential

Introduction: Advances in mRNA technology have garnered significant attention, especially due to its rapid development and versatility, positioning it as a valuable tool in vaccine and immunobiological production.Despite its potential, challenges related to stability and immunogenicity remain significant in mRNA-based therapies.To address these limitations, several strategies for improving mRNA are being explored, such as the incorporation of modified nucleotides.Objectives: To investigate the impact of structural adaptations on mRNA functionality by introducing modified nucleotides during the in vitro transcription, assessing their influence on integrity, dsRNA synthesis and protein expression.Methodology: During in vitro transcription, modified uridine (UTP1, UTP2, UTP3 and UTP4), cytidine (CTP1) and adenosine (ATP1) nucleotides were incorporated into an mRNA construct encoding the SARS-CoV-2 Spike protein fused with GFP, replacing corresponding regular nucleotides.The mRNA integrity was analyzed through capillary electrophoresis, and the dsRNA content quantified using ELISA assay.Protein expression was evaluated through flow cytometry after mRNA transfection into HEK-293T cells.Results: All samples demonstrated integrity above 80%, as assessed by capillary electrophoresis, except for UTP4, which exhibited no integrity after in vitro transcription and was not included in subsequent experiments.ELISA assay quantification of dsRNA content revealed higher levels in mRNAs containing regular nucleotides and UTP3 compared to other tested modified nucleotides.In terms of in vitro protein expression, mRNA with regular nucleotides, UTP1, UTP2 and CTP1 exhibited considerable activity, while UTP3 and ATP1 showed notably weaker responses. Conclusion:The observed differences in integrity, dsRNA content and protein expression suggest that some specific modifications may influence key aspects of mRNA processing.These findings provide insights into how nucleotide modifications impact mRNA properties and may contribute to future optimization strategies for mRNA-based applications.