Synthetic mRNAs with superior translation and stability properties

Abstract

The translational ef ficiency and stability of synthetic mRNA in both cultured cells and whole animals can be improved by incorporation of modi fied 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 ef ficiency 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 modi fied mRNAs, their introduction into cells and whole animals, and measurement of their translational ef ficiency and stability. © Springer Science+Business Media New York 2013.