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Methods Mol Biol. 2013;969:55-72. doi: 10.1007/978-1-62703-260-5_4.

Synthetic mRNAs with superior translation and stability properties.

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1
Department 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.

PMID:
23296927
DOI:
10.1007/978-1-62703-260-5_4
[Indexed for MEDLINE]
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