Synthetic Capped mRNAs for Cap-Specific Photo-Cross-Linking Experiments

  • Joanna Kowalska
  • Franck MartinEmail author
  • Jacek JemielityEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1428)


The 7-methylguanosine triphosphate cap present at the 5′ ends of eukaryotic mRNAs plays numerous roles in mRNA expression and metabolism. The identification and studies on cap-binding partners can be significantly advanced using tailored chemical tools such as synthetic cap analogues or RNAs carrying modified cap structures. Here we provide protocols for the production of mRNAs specifically labeled within the 5′ cap with a nucleoside capable of being photo-activated, either 6-thioguanosine or 7-methyl-6-thioguanosine, which can be used in photo-cross-linking experiments to identify or characterize cap-binding biomolecules. We also describe a protocol for the cross-linking experiments with capped RNAs to map histone H4 cap-binding pocket.

Key words

5′ cap analogs 6-thioguanosine mRNA Transcription Photo-cross-linking Cap binding Histone mRNA 



This research was supported by grant UMO-2012/05/E/ST5/03893 from the National Science Centre (to J.J.) and grants ANR-06-BLAN-0206-01 and ANR-2011-SVSE8-025-01 from CNRS (Centre National de la Recherche Scientifique) and Agence Nationale pour la Recherche (to F.M.).


  1. 1.
    Furuichi Y, Shatkin AJ (2000) Advances in virus research, vol 55. Academic Press Inc, San Diego, pp 135–184Google Scholar
  2. 2.
    Cougot N, van Dijk E, Babajko S, Séraphin B (2004) Cap-tabolism. Trends Biochem Sci 29:436–444CrossRefPubMedGoogle Scholar
  3. 3.
    Rhoads RE (2009) eIF4E: new family members, new binding partners, new roles. J Biol Chem 284:16711–16715CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Contreras R, Cheroutre H, Degrave W, Fiers W (1982) Simple, efficient in vitro synthesis of capped RNA useful for direct expression of cloned eukaryotic genes. Nucleic Acids Res 10:6353–6362CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Grudzien-Nogalska E, Kowalska J, Su W, Kuhn A, Slepenkov S, Darzynkiewicz E, Sahin U, Jemielity J, Rhoads R (2013) In: Rabinovich PM (ed) Synthetic messenger RNA and cell metabolism modulation, vol 969. Humana Press, Totowa, pp 55–72CrossRefGoogle Scholar
  6. 6.
    Grudzien-Nogalska E, Stepinski J, Jemielity J, Zuberek J, Stolarski R, Rhoads RE, Darzynkiewicz E (2007) Synthesis of anti-reverse cap analogs (ARCAs) and their applications in mRNA translation and stability. Method Enzymol 431:203–227CrossRefGoogle Scholar
  7. 7.
    Stepinski J, Waddell C, Stolarski R, Darzynkiewicz E, Rhoads RE (2001) Synthesis and properties of mRNAs containing the novel “anti-reverse” cap analogs 7-methyl(3′-O-methyl)GpppG and 7-methyl(3′-deoxy)GpppG. RNA 7:1486–1495PubMedPubMedCentralGoogle Scholar
  8. 8.
    Peng ZH, Sharma V, Singleton SF, Gershon PD (2002) Synthesis and application of a chain-terminating dinucleotide mRNA cap analog. Org Lett 4:161–164CrossRefPubMedGoogle Scholar
  9. 9.
    Jemielity J, Fowler T, Zuberek J, Stepinski J, Lewdorowicz M, Niedzwiecka A, Stolarski R, Darzynkiewicz E, Rhoads RE (2003) Novel “anti-reverse” cap analogs with superior translational properties. RNA 9:1108–1122CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Kore AR, Shanmugasundaram M, Charles I, Vlassov AV, Barta TJ (2009) Locked nucleic acid (LNA)-modified dinucleotide mRNA Cap analogue: synthesis, enzymatic incorporation, and utilization. J Am Chem Soc 131(18):6364–6365CrossRefPubMedGoogle Scholar
  11. 11.
    Kore AR, Charles I (2010) Synthesis and evaluation of 2′-O-allyl substituted dinucleotide cap analog for mRNA translation. Bioorg Med Chem 18:8061–8065CrossRefPubMedGoogle Scholar
  12. 12.
    Kalek M, Jemielity J, Darzynkiewicz ZM, Bojarska E, Stepinski J, Stolarski R, Davis RE, Darzynkiewicz E (2006) Enzymatically stable 5′ mRNA cap analogs: Synthesis and binding studies with human DcpS decapping enzyme. Bioorg Med Chem 14:3223–3230CrossRefPubMedGoogle Scholar
  13. 13.
    Jemielity J, Kowalska J, Rydzik AM, Darzynkiewicz E (2010) Synthetic mRNA cap analogs with a modified triphosphate bridge—synthesis, applications and prospects. New J Chem 34:829–844CrossRefGoogle Scholar
  14. 14.
    Rydzik AM, Kulis M, Lukaszewicz M, Kowalska J, Zuberek J, Darzynkiewicz ZM, Darzynkiewicz E, Jemielity J (2012) Synthesis and properties of mRNA cap analogs containing imidodiphosphate moiety-fairly mimicking natural cap structure, yet resistant to enzymatic hydrolysis. Bioorg Med Chem 20(5):1699–1710CrossRefPubMedGoogle Scholar
  15. 15.
    Kowalska J, Wypijewska del Nogal A, Darzynkiewicz ZM, Buck J, Nicola C, Kuhn AN, Lukaszewicz M, Zuberek J, Strenkowska M, Ziemniak M et al (2014) Synthesis, properties, and biological activity of boranophosphate analogs of the mRNA cap: versatile tools for manipulation of therapeutically relevant cap-dependent processes. Nucleic Acids Res 42:10245–10264CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Jemielity J, Lukaszewicz M, Kowalska J, Czarnecki J, Zuberek J, Darzynkiewicz E (2012) Synthesis of biotin labelled cap analogue—incorporable into mRNA transcripts and promoting cap-dependent translation. Org Biomol Chem 10:8570–8574CrossRefPubMedGoogle Scholar
  17. 17.
    Ziemniak M, Szabelski M, Lukaszewicz M, Nowicka A, Darzynkiewicz E, Rhoads RE, Wieczorek Z, Jemielity J (2013) Synthesis and evaluation of fluorescent cap analogues for mRNA labelling. RSC Adv 3:20943–20958CrossRefGoogle Scholar
  18. 18.
    Martin SA, Paoletti E, Moss B (1975) Purification of messenger-Rna guanylyltransferase and messenger-RNA(guanine-7-)-methyltransferase from vaccinia virions. J Biol Chem 250:9322–9329PubMedGoogle Scholar
  19. 19.
    Issur M, Bougie I, Despins S, Bisaillon M (2013) Enzymatic synthesis of RNAs capped with nucleotide analogues reveals the molecular basis for substrate selectivity of RNA capping enzyme: impacts on RNA metabolism. PLoS One 8, e75310CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Thillier Y, Decroly E, Morvan F, Canard B, Vasseur J-J, Debart F (2012) Synthesis of 5′ cap-0 and cap-1 RNAs using solid-phase chemistry coupled with enzymatic methylation by human (guanine-N7)-methyl transferase. RNA 18:856–868CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Nowakowska M, Kowalska J, Martin F, d’Orchymont A, Zuberek J, Lukaszewicz M, Darzynkiewicz E, Jemielity J (2014) Cap analogs containing 6-thioguanosine—reagents for the synthesis of mRNAs selectively photo-crosslinkable with cap-binding biomolecules. Org Biomol Chem 12:4841–4847CrossRefPubMedGoogle Scholar
  22. 22.
    Martin F, Barends S, Jaeger S, Schaeffer L, Prongidi-Fix L, Eriani G (2011) Cap-assisted internal initiation of translation of histone H4. Mol Cell 41:197–209CrossRefPubMedGoogle Scholar
  23. 23.
    Meier VS, Böhni R, Schümperli D (1989) Nucleotide sequence of two mouse histone H4 genes. Nucleic Acids Res 17:795CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Division of Biophysics, Institute of Experimental Physics, Faculty of PhysicsUniversity of WarsawWarsawPoland
  2. 2.Architecture et Réactivité de l’ARN, Université de Strasbourg, CNRSInstitut de Biologie Moléculaire et CellulaireStrasbourg CEDEXFrance
  3. 3.Center of New TechnologiesUniversity of WarsawWarsawPoland

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