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

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

Abstract

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 

Notes

Acknowledgements

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.).

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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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