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Rapid miRNA Imaging in Cells Using Fluorogenic Templated Staudinger Reaction Between PNA-Based Probes

  • Katarzyna Gorska
  • Nicolas Winssinger
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1050)

Abstract

Reactions templated by a specific nucleic acid sequence have emerged as an attractive strategy for nucleic acid sensing. The Staudinger reaction using an azide-quenched fluorophore and a phosphine is particularly well suited by virtue of its bioorthogonality and biocompatibility. The reaction is promoted by a complementary nucleic acid that aligns the phosphine with the azide-quenched fluorophore. Cellular RNAs can catalyze the Staudinger reaction and signal amplification can be achieved through multiple turnover of the template. Peptide nucleic acids (PNA) provide a convenient platform for the preparation of specific probes as they combine desirable hybridization properties, robust synthesis, ease of fluorophore conjugation, and high biochemical stability. Herein, we describe protocols for fast fluorescent detection of miRNAs in human cells with PNA-based probes via reductive unquenching of bis-azidorhodamine by trialkylphosphine.

Key words

Peptide nucleic acid (PNA) Imaging miRNA Templated reaction Pro-fluorophore Rhodamine Staudinger reaction 

Notes

Acknowledgments

The authors thank the granting agencies which have supported this work (European Research Council-ERC 201749, The Institut Universitaire de France, Boehringer Ingelheim Fonds). The authors thank their numerous collaborators in the various aspects of this work.

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Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Katarzyna Gorska
    • 1
  • Nicolas Winssinger
    • 1
  1. 1.Laboratoire de Chemie Organique et BioorganiqueInstitut de Science et d’Ingénierie Supramoléculaires, Université Louis PasteurStrasbourgFrance

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