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Chemical Dual End-Labeling of Large Ribozymes

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DNAzymes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2439))

Abstract

Fast and efficient site-specific labeling of long RNAs is one of the main bottlenecks limiting distance measurements by means of Förster resonance energy transfer (FRET) or electron paramagnetic resonance (EPR) spectroscopy. Here, we present an optimized protocol for dual end-labeling with different fluorophores at the same time meeting the restrictions of highly labile and degradation-sensitive RNAs. We describe in detail the dual-labeling of a catalytically active wild-type group II intron as a typical representative of long functional RNAs. The modular procedure chemically activates the 5′-phosphate and the 3′-ribose for bioconjugation with a pair of fluorophores, as shown herein, or with spin labels. The mild reaction conditions preserve the structural and functional integrity of the biomacromolecule and results in covalent, dual-labeled RNA in its pre-catalytic state in yields suitable for both ensemble and single-molecule FRET experiments.

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Acknowledgements

Financial support from the Swiss National Science Foundation [200020_192153 to RKOS], the UZH Forschungskredit [FK-20-081 to EA], the University of Zurich, and the Graduate School of Chemical and Molecular Sciences Zurich (CMSZH) is gratefully acknowledged.

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Authors and Affiliations

  1. Department of Chemistry, University of Zurich, Zurich, Switzerland

    Esra Ahunbay, Fabio D. Steffen, Susann Zelger-Paulus & Roland K. O. Sigel

Authors
  1. Esra Ahunbay
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  2. Fabio D. Steffen
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  3. Susann Zelger-Paulus
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  4. Roland K. O. Sigel
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Corresponding authors

Correspondence to Susann Zelger-Paulus or Roland K. O. Sigel .

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Editors and Affiliations

  1. Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany

    Gerhard Steger

  2. Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany

    Hannah Rosenbach

  3. Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany

    Ingrid Span

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Ahunbay, E., Steffen, F.D., Zelger-Paulus, S., Sigel, R.K.O. (2022). Chemical Dual End-Labeling of Large Ribozymes. In: Steger, G., Rosenbach, H., Span, I. (eds) DNAzymes. Methods in Molecular Biology, vol 2439. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2047-2_13

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  • DOI: https://doi.org/10.1007/978-1-0716-2047-2_13

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2046-5

  • Online ISBN: 978-1-0716-2047-2

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