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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Winter J, Jung S, Keller S, Gregory RI, Diederichs S (2009) Many roads to maturity: microRNA biogenesis pathways and their regulation. Nat Cell Biol 11:228–234
Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E et al (2002) Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A 99:15524–15529
Chin LJ, Ratner E, Leng S, Zhai R, Nallur S, Babar I et al (2008) A SNP in a let-7 microRNA complementary site in the KRAS 3’ untranslated region increases non-small cell lung cancer risk. Cancer Res 68:8535–8540
Metzler M, Wilda M, Busch K, Viehmann S, Borkhardt A (2004) High expression of precursor microRNA-155/BIC RNA in children with Burkitt lymphoma. Genes Chromosomes Cancer 39:167–169
Jay C, Nemunaitis J, Chen P, Fulgham P, Tong AW (2007) miRNA profiling for diagnosis and prognosis of human cancer. DNA Cell Biol 26:293–300
Pena JT, Sohn-Lee C, Rouhanifard SH, Ludwig J, Hafner M, Mihailovic A et al (2009) miRNA in situ hybridization in formaldehyde and EDC-fixed tissues. Nat Methods 6:139–141
Politz JC, Zhang F, Pederson T (2006) MicroRNA-206 colocalizes with ribosome-rich regions in both the nucleolus and cytoplasm of rat myogenic cells. Proc Natl Acad Sci U S A 103:18957–18962
Lu J, Tsourkas A (2011) Quantification of miRNA abundance in single cells using locked nucleic acid-FISH and enzyme-labeled fluorescence. Methods Mol Biol 680:77–88
Silverman AP, Kool ET (2006) Detecting RNA and DNA with templated chemical reactions. Chem Rev 106:3775–3789
Grossmann TN, Strohbach A, Seitz O (2008) Achieving turnover in DNA-templated reactions. Chembiochem 9:2185–2192
Franzini RM, Kool ET (2009) Efficient nucleic acid detection by templated reductive quencher release. J Am Chem Soc 131:16021–16023
Franzini RM, Kool ET (2011) Two successive reactions on a DNA template: a strategy for improving background fluorescence and specificity in nucleic acid detection. Chemistry 17:2168–2175
Sando S, Kool ET (2002) Imaging of RNA in bacteria with self-ligating quenched probes. J Am Chem Soc 124:9686–9687
Silverman AP, Baron EJ, Kool ET (2006) RNA-templated chemistry in cells: discrimination of Escherichia, Shigella and Salmonella bacterial strains with a new two-color FRET strategy. Chembiochem 7:1890–1894
Silverman AP, Kool ET (2005) Quenched autoligation probes allow discrimination of live bacterial species by single nucleotide differences in rRNA. Nucleic Acids Res 33:4978–4986
Abe H, Kool ET (2006) Flow cytometric detection of specific RNAs in native human cells with quenched autoligating FRET probes. Proc Natl Acad Sci U S A 103:263–268
Pianowski Z, Gorska K, Oswald L, Merten CA, Winssinger N (2009) Imaging of mRNA in live cells using nucleic acid-templated reduction of azidorhodamine probes. J Am Chem Soc 131:6492–6497
Furukawa K, Abe H, Hibino K, Sako Y, Tsuneda S, Ito Y (2009) Reduction-triggered fluorescent amplification probe for the detection of endogenous RNAs in living human cells. Bioconjug Chem 20:1026–1036
Abe H, Wang J, Furukawa K, Oki K, Uda M, Tsuneda S et al (2008) A reduction-triggered fluorescence probe for sensing nucleic acids. Bioconjug Chem 19:1219–1226
Franzini RM, Kool ET (2008) 7-Azidomethoxy-coumarins as profluorophores for templated nucleic acid detection. Chembiochem 9:2981–2988
Zhou P, Wang M, Du L, Fisher GW, Waggoner A, Ly DH (2003) Novel binding and efficient cellular uptake of guanidine-based peptide nucleic acids (GPNA). J Am Chem Soc 125:6878–6879
Gorska K, Keklikoglou I, Tschulena I, Winssingera N (2011) Rapid fluorescence imaging of miRNAs in human cells using templated Staudinger reaction. Chem Sci 2:1969–1975
Egholm M, Buchardt O, Christensen L, Behrens C, Freier SM, Driver DA et al (1993) PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature 365:566–568
Nielsen PE (2004) PNA technology. Mol Biotechnol 26:233–248
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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Gorska, K., Winssinger, N. (2014). Rapid miRNA Imaging in Cells Using Fluorogenic Templated Staudinger Reaction Between PNA-Based Probes. In: Nielsen, P., Appella, D. (eds) Peptide Nucleic Acids. Methods in Molecular Biology, vol 1050. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-553-8_15
Download citation
DOI: https://doi.org/10.1007/978-1-62703-553-8_15
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-552-1
Online ISBN: 978-1-62703-553-8
eBook Packages: Springer Protocols