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RNA Chaperones pp 253-270 | Cite as

Site-Specific Dual-Color Labeling of Long RNAs

  • Meng Zhao
  • Richard Börner
  • Roland K. O. Sigel
  • Eva FreisingerEmail author
Protocol
  • 325 Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 2106)

Abstract

Labeling of large RNAs with reporting entities, e.g., fluorophores, has significant impact on RNA studies in vitro and in vivo. Here, we describe a minimally invasive RNA labeling method featuring nucleotide and position selectivity, which solves the long-standing challenge of how to achieve accurate site-specific labeling of large RNAs with a least possible influence on folding and/or function. We use a custom-designed reactive DNA strand to hybridize to the RNA and transfer the alkyne group onto the targeted adenine or cytosine. Simultaneously, the 3′-terminus of RNA is converted to a dialdehyde moiety under the experimental condition applied. The incorporated functionalities at the internal and the 3′-terminal sites can then be conjugated with reporting entities via bioorthogonal chemistry. This method is particularly valuable for, but not limited to, single-molecule fluorescence applications. We demonstrate the method on an RNA construct of 275 nucleotides, the btuB riboswitch of Escherichia coli.

Key words

Long RNAs Site-specific labeling Orthogonal chemistry Riboswitch Single-molecule fluorescence resonance energy transfer (smFRET) 

Notes

Acknowledgement

We thank Anna Zemann, Nora Grundmann, and Dr. Sofia Gallo for their valuable feedback regarding the synthesis of the RG precursor. Financial support from the Swiss National Science Foundation [to E.F. and R.K.O.S.], the European Research Council [ERC to R.K.O.S.], SystemsX.ch [to R.K.O.S.], the UZH Forschungskredit [FK-14-096, FK-15-095 to R.B.], the UZH Stiftung für wissenschaftliche Forschung [to R.K.O.S. and R.B.], the University of Zurich, and the SBFI [COST Action CM1105 to E.F. and R.K.O.S] is gratefully acknowledged. Funding for open access charge: University of Zurich.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Meng Zhao
    • 1
    • 2
  • Richard Börner
    • 1
    • 3
  • Roland K. O. Sigel
    • 1
  • Eva Freisinger
    • 1
    Email author
  1. 1.Department of ChemistryUniversity of ZurichZurichSwitzerland
  2. 2.Department of PhysicsUniversity of AlbertaEdmontonCanada
  3. 3.Laserinstitut Hochschule MittweidaUniversity of Applied Sciences MittweidaMittweidaGermany

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