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Updated: May 15, 2026

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells
Published on: November 13, 2014
Synthetic mRNAs with superior translation and stability properties
Ewa Grudzien-Nogalska1, Joanna Kowalska, Wei Su
1Department of Biochemistry and Molecular Biology, Lousisiana State University Health Sciences Center, Shreveport, LA, USA.
Abstract:
The translational efficiency and stability of synthetic mRNA in both cultured cells and whole animals can be improved by incorporation of modified cap structures at the 5'-end. mRNAs are synthesized in vitro by a phage RNA polymerase transcribing a plasmid containing the mRNA sequence in the presence of all four NTPs plus a cap dinucleotide. Modifications in the cap dinucleotide at the 2'- or 3'-positions of m(7)Guo, or modifications in the polyphosphate chain, can improve both translational efficiency and stability of the mRNA, thereby increasing the amount and duration of protein expression. In the context of RNA-based immunotherapy, the latter is especially important for antigen production and presentation by dendritic cells. Protocols are presented for synthesis of modified mRNAs, their introduction into cells and whole animals, and measurement of their translational efficiency and stability.
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