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Enzymatic Transfer of Photo-Cross-Linkers for RNA-Protein Photo-Cross-Linking at the mRNA 5′-Cap

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Proximity Labeling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2008))

Abstract

Photo-cross-linking moieties have proven invaluable for elucidating interactions of biomolecules. While methods for site-specific incorporation of those moieties into proteins have been developed, comparable methods for nucleic acids are lacking. Utilizing the inherent specificity of enzymes, methyltransferases (MTase) exhibiting relaxed cosubstrate specificity in combination with synthetic analogs of S-adenosyl-l-methionine (AdoMet) allow for the precise installation of reporter molecules or affinity tags in various biomolecules. In this chapter, we describe AdoMet analogs with photo-cross-linking moieties that—in combination with an MTase—are ideal for site-specific installation. The workflow for chemo-enzymatic installation of photo-cross-linking moieties at the mRNA cap based on AdoMet analogs is given in detail.

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Acknowledgments

A. R. gratefully acknowledges financial support from the Emmy Noether-Programme of the Deutsche Forschungsgemeinschaft (RE 2796/2-1) and the Fonds der Chemischen Industrie. This work was partly supported by the Deutsche Forschungsgemeinschaft, DFG EXC 1003 Cells in Motion—Cluster of Excellence, Münster, Germany. We would like to thank Prof. Birgit Dräger (University of Halle, Germany) for plasmids encoding LuxS and MTAN.

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Authors and Affiliations

  1. Westfälische Wilhelms-Universität Münster, Institute of Biochemistry, Münster, Germany

    Nils Muthmann, Fabian Muttach & Andrea Rentmeister

  2. Cells-in-Motion Cluster of Excellence (EXC 1003—CiM), University of Muenster, Münster, Germany

    Andrea Rentmeister

Authors
  1. Nils Muthmann
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  2. Fabian Muttach
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  3. Andrea Rentmeister
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Corresponding author

Correspondence to Andrea Rentmeister .

Editor information

Editors and Affiliations

  1. Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany

    Murat Sunbul  & Andres Jäschke  & 

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Muthmann, N., Muttach, F., Rentmeister, A. (2019). Enzymatic Transfer of Photo-Cross-Linkers for RNA-Protein Photo-Cross-Linking at the mRNA 5′-Cap. In: Sunbul, M., Jäschke, A. (eds) Proximity Labeling. Methods in Molecular Biology, vol 2008. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9537-0_11

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  • DOI: https://doi.org/10.1007/978-1-4939-9537-0_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9536-3

  • Online ISBN: 978-1-4939-9537-0

  • eBook Packages: Springer Protocols

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