Skip to main content

In Vitro Transcription of Long RNA Containing Modified Nucleosides

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

This is a preview of subscription content, access via your institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-62703-260-5_2
  • Chapter length: 14 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   139.00
Price excludes VAT (USA)
  • ISBN: 978-1-62703-260-5
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   179.00
Price excludes VAT (USA)
Hardcover Book
USD   219.99
Price excludes VAT (USA)
Fig. 1.
Fig. 2.

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Anderson BR, Muramatsu H, Jha BK, Silverman RH, Weissman D, Kariko K (2011) Nucleoside modifications in RNA limit activation of 2′-5′-oligoadenylate synthetase and increase resistance to cleavage by RNase L. Nucleic Acids Res 39:9329–9338

    PubMed  CrossRef  CAS  Google Scholar 

  2. Nallagatla SR, Bevilacqua PC (2008) Nucleoside modifications modulate activation of the protein kinase PKR in an RNA structure-specific manner. RNA 14:1201–1213

    PubMed  CrossRef  CAS  Google Scholar 

  3. Kariko K, Muramatsu H, Welsh FA, Ludwig J, Kato H, Akira S, Weissman D (2008) Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol Ther 16:1833–1840

    PubMed  CrossRef  CAS  Google Scholar 

  4. Kariko K, Weissman D (2007) Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development. Curr Opin Drug Discov Devel 10:523–532

    PubMed  CAS  Google Scholar 

  5. Hornung V, Ellegast J, Kim S, Brzozka K, Jung A, Kato H, Poeck H, Akira S, Conzelmann KK, Schlee M, Endres S, Hartmann G (2006) 5′-Triphosphate RNA is the ligand for RIG-I. Science 314:994–997

    PubMed  CrossRef  Google Scholar 

  6. Kariko K, Buckstein M, Ni H, Weissman D (2005) Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 23:165–175

    PubMed  CrossRef  CAS  Google Scholar 

  7. Kariko K, Muramatsu H, Ludwig J, Weissman D (2011) Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nucleic Acids Res 39:e142

    PubMed  CrossRef  CAS  Google Scholar 

  8. Anderson BR, Muramatsu H, Nallagatla SR, Bevilacqua PC, Sansing LH, Weissman D, Kariko K (2010) Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation. Nucleic Acids Res 38:5884–5892

    PubMed  CrossRef  CAS  Google Scholar 

  9. Kormann MS, Hasenpusch G, Aneja MK, Nica G, Flemmer AW, Herber-Jonat S, Huppmann M, Mays LE, Illenyi M, Schams A, Griese M, Bittmann I, Handgretinger R, Hartl D, Rosenecker J, Rudolph C (2011) Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nat Biotechnol 29:154–157

    PubMed  CrossRef  CAS  Google Scholar 

  10. Kariko K, Muramatsu H, Keller JM, Weissman D (2012) Increased erythropoiesis in mice injected with sub-microgram quantities of pseudouridine-containing mRNA encoding erythropoietin. Mol Ther 20:948–953

    Google Scholar 

  11. Graham FL, van der Eb AJ (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:456–467

    PubMed  CrossRef  CAS  Google Scholar 

  12. Dubes GR, Wegrzyn RJ (1978) Rapid ephemeral cell sensitization as the mechanism of histone-induced and protamine-induced enhancement of transfection by poliovirus RNA. Protoplasma 96:209–223

    PubMed  CrossRef  CAS  Google Scholar 

  13. Krieg PA, Melton DA (1984) Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res 12:7057–7070

    PubMed  CrossRef  CAS  Google Scholar 

  14. Jirikowski GF, Sanna PP, Maciejewski-Lenoir D, Bloom FE (1992) Reversal of diabetes insipidus in Brattleboro rats: intrahypothalamic injection of vasopressin mRNA. Science 255:996–998

    PubMed  CrossRef  CAS  Google Scholar 

  15. Weide B, Pascolo S, Scheel B, Derhovanessian E, Pflugfelder A, Eigentler TK, Pawelec G, Hoerr I, Rammensee HG, Garbe C (2009) Direct injection of protamine-protected mRNA: results of a phase 1/2 vaccination trial in metastatic melanoma patients. J Immunother 32:498–507

    PubMed  CrossRef  CAS  Google Scholar 

  16. Weide B, Carralot JP, Reese A, Scheel B, Eigentler TK, Hoerr I, Rammensee HG, Garbe C, Pascolo S (2008) Results of the first phase I/II clinical vaccination trial with direct injection of mRNA. J Immunother 31:180–188

    PubMed  CrossRef  CAS  Google Scholar 

  17. Pascolo S (2006) Vaccination with messenger RNA. Methods Mol Med 127:23–40

    PubMed  CAS  Google Scholar 

  18. Anderson BR, Muramatsu H, Jha BK, Silverman RH, Weissman D, Kariko K (2011) Nucleoside modifications in RNA limit activation of 2′-5′ oligoadenylate synthetase and increase resistance to cleavage by RNase L. Nucleic Acids Res 39(21):9329–9338

    PubMed  CrossRef  CAS  Google Scholar 

  19. Weissman D, Ni H, Scales D, Dude A, Capodici J, McGibney K, Abdool A, Isaacs SN, Cannon G, Kariko K (2000) HIV gag mRNA transfection of dendritic cells (DC) delivers encoded antigen to MHC class I and II molecules, causes DC maturation, and induces a potent human in vitro primary immune response. J Immunol 165:4710–4717

    PubMed  CAS  Google Scholar 

  20. Angel M, Yanik MF (2010) Innate immune suppression enables frequent transfection with RNA encoding reprogramming proteins. PLoS One 5:e11756

    PubMed  CrossRef  Google Scholar 

  21. Yakubov E, Rechavi G, Rozenblatt S, Givol D (2010) Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors. Biochem Biophys Res Commun 394:189–193

    PubMed  CrossRef  CAS  Google Scholar 

  22. Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, Ebina W, Mandal PK, Smith ZD, Meissner A, Daley GQ, Brack AS, Collins JJ, Cowan C, Schlaeger TM, Rossi DJ (2010) Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7(5):618–630

    PubMed  CrossRef  CAS  Google Scholar 

  23. Zeenko V, Gallie DR (2005) Cap-independent translation of tobacco etch virus is conferred by an RNA pseudoknot in the 5′-leader. J Biol Chem 280:26813–26824

    PubMed  CrossRef  CAS  Google Scholar 

  24. Gallie DR (2001) Cap-independent translation conferred by the 5′ leader of tobacco etch virus is eukaryotic initiation factor 4G dependent. J Virol 75:12141–12152

    PubMed  CrossRef  CAS  Google Scholar 

  25. Niepel M, Gallie DR (1999) Identification and characterization of the functional elements within the tobacco etch virus 5′ leader required for cap-independent translation. J Virol 73:9080–9088

    PubMed  CAS  Google Scholar 

  26. Gallie DR, Tanguay RL, Leathers V (1995) The tobacco etch viral 5′ leader and poly(A) tail are functionally synergistic regulators of translation. Gene 165:233–238

    PubMed  CrossRef  CAS  Google Scholar 

  27. Kariko K, Kuo A, Barnathan E (1999) Overexpression of urokinase receptor in mammalian cells following administration of the in vitro transcribed encoding mRNA. Gene Ther 6:1092–1100

    PubMed  CrossRef  CAS  Google Scholar 

  28. Ensinger MJ, Martin SA, Paoletti E, Moss B (1975) Modification of the 5′-terminus of mRNA by soluble guanylyl and methyl transferases from vaccinia virus. Proc Natl Acad Sci USA 72:2525–2529

    PubMed  CrossRef  CAS  Google Scholar 

  29. Padilla R, Sousa R (2002) A Y639F/H784A T7 RNA polymerase double mutant displays superior properties for synthesizing RNAs with non-canonical NTPs. Nucleic Acids Res 30:e138

    PubMed  CrossRef  Google Scholar 

  30. Kato Y, Minakawa N, Komatsu Y, Kamiya H, Ogawa N, Harashima H, Matsuda A (2005) New NTP analogs: the synthesis of 4′-thioUTP and 4′-thioCTP and their utility for SELEX. Nucleic Acids Res 33:2942–2951

    PubMed  CrossRef  CAS  Google Scholar 

  31. Srivatsan SG, Tor Y (2009) Enzymatic incorporation of emissive pyrimidine ribonucleotides. Chem Asian J 4:419–427

    PubMed  CrossRef  CAS  Google Scholar 

  32. Aurup H, Siebert A, Benseler F, Williams D, Eckstein F (1994) Translation of 2′-modified mRNA in vitro and in vivo. Nucleic Acids Res 22:4963–4968

    PubMed  CrossRef  CAS  Google Scholar 

  33. Kormann MS, Hasenpusch G, Aneja MK, Nica G, Flemmer AW, Herber-Jonat S, Huppmann M, Mays LE, Illenyi M, Schams A, Griese M, Bittmann I, Handgretinger R, Hartl D, Rosenecker J, Rudolph C (2011) Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nat Biotechnol 29:154–157

    PubMed  CrossRef  CAS  Google Scholar 

  34. Schenborn ET, Mierendorf RC Jr (1985) A novel transcription property of SP6 and T7 RNA polymerases: dependence on template structure. Nucleic Acids Res 13:6223–6236

    PubMed  CrossRef  CAS  Google Scholar 

  35. Sastry SS, Ross BM (1997) Nuclease activity of T7 RNA polymerase and the heterogeneity of transcription elongation complexes. J Biol Chem 272:8644–8652

    PubMed  CrossRef  CAS  Google Scholar 

Download references

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).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Katalin Karikó .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2013 Springer Science+Business Media New York

About this protocol

Cite this protocol

Pardi, N., Muramatsu, H., Weissman, D., Karikó, K. (2013). In Vitro Transcription of Long RNA Containing Modified Nucleosides. In: Rabinovich, P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation. Methods in Molecular Biology, vol 969. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-260-5_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-260-5_2

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-259-9

  • Online ISBN: 978-1-62703-260-5

  • eBook Packages: Springer Protocols