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In Vitro Transcription of Long RNA Containing Modified Nucleosides

  • Norbert Pardi
  • Hiromi Muramatsu
  • Drew Weissman
  • Katalin Karikó
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 969)

Abstract

The in vitro synthesis of long RNA can be accomplished using phage RNA polymerase and template DNA. However, the in vitro synthesized RNA, unlike those transcribed in vivo in cells, lacks nucleoside modifications. Introducing modified nucleosides into in vitro transcripts is important because they reduce the potential of RNA to activate RNA sensors [1–6] and translation of such nucleoside-modified RNA is increased in cell lines, primary cells, and after in vivo delivery [1, 3, 7–10]. Here, we describe the in vitro synthesis of nucleoside-modified RNA with enhanced translational capacity and reduced ability to activate immune sensors.

Key words

messenger RNA Immunogenicity Modified nucleoside Pseudouridine 5-methylcytidine Capping In vitro transcription 

Notes

Acknowledgments

These studies were funded by the National Institutes of Health (grant numbers HL87688 to K.K. and AI050484, AI090788, and DE019059 to D.W).

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Norbert Pardi
    • 1
  • Hiromi Muramatsu
    • 2
  • Drew Weissman
    • 1
  • Katalin Karikó
    • 2
  1. 1.Department of MedicineUniversity of PennsylvaniaPhiladelphiaUSA
  2. 2.Department of NeurosurgeryUniversity of PennsylvaniaPhiladelphiaUSA

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