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Site-Specific Dual-Color Labeling of Long RNAs.
Journal article

Site-Specific Dual-Color Labeling of Long RNAs.

  • Zhao M Department of Chemistry, University of Zurich, Zurich, Switzerland.
  • Börner R Department of Chemistry, University of Zurich, Zurich, Switzerland.
  • Sigel RKO Department of Chemistry, University of Zurich, Zurich, Switzerland.
  • Freisinger E Department of Chemistry, University of Zurich, Zurich, Switzerland. freisinger@chem.uzh.ch.
  • 2020-01-01
Published in:
  • Methods in molecular biology (Clifton, N.J.). - 2020
Long RNAs
Orthogonal chemistry
Riboswitch
Single-molecule fluorescence resonance energy transfer (smFRET)
Site-specific labeling
English 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.
Language
  • English
Open access status
closed
Identifiers
Persistent URL
https://sonar.ch/global/documents/298689
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