Synthetic mRNAs with Superior Translation and Stability Properties

  • Ewa Grudzien-Nogalska
  • Joanna Kowalska
  • Wei Su
  • Andreas N. Kuhn
  • Sergey V. Slepenkov
  • Edward Darzynkiewicz
  • Ugur Sahin
  • Jacek Jemielity
  • Robert E. Rhoads
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 969)

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 m7Guo, 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.

Key words

Boranophosphate Cap analogs Decapping Gene therapy Immunotherapy mRNA ­stability Nucleoporation Phosphorothioate Protein expression Translational efficiency 

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Ewa Grudzien-Nogalska
    • 1
  • Joanna Kowalska
    • 2
  • Wei Su
    • 1
  • Andreas N. Kuhn
    • 3
    • 4
  • Sergey V. Slepenkov
    • 1
  • Edward Darzynkiewicz
    • 2
  • Ugur Sahin
    • 3
    • 5
    • 4
  • Jacek Jemielity
    • 2
  • Robert E. Rhoads
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyLousisiana State University Health Sciences CenterShreveportUSA
  2. 2.Division of Biophysics, Institute of Experimental Physics, Faculty of PhysicsUniversity of WarsawWarsawPoland
  3. 3.TRON-Translational Oncology at the University Medical Center MainzMainzGermany
  4. 4.Ribological, Biontech AGMainzGermany
  5. 5.III. Medical DepartmentUniversity Medical Center of the Johannes Gutenberg-University MainzMainzGermany

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