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 (PNAs) 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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Tanious FA, Nguyen B, Wilson WD (2008) Biosensor surface plasmon resonance methods for quantitative analysis of macromolecular interactions. Methods Cell Biol 84:53–77
Egholm M, Buchardt O, Christensen L, Behrens C, Freier SM, Driver DA, Berg RH, Kim SK, Nordén B, Nielsen PE (1993) PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature 365:566–568
Nielsen PE, Egholm M, Berg RH, Buchardt O (1991) Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254:1498–1500
Datta B, Schmitt C, Armitage BA (2003) Formation of a PNA2-DNA2 hybrid quadruplex. J Am Chem Soc 125:4111–4118
Jensen KK, Ørum H, Nielsen PE, Nordén B (1997) Kinetics for hybridization of peptide nucleic acids (PNA) with DNA and RNA studied with the BIAcore technique. Biochemistry 36:5072–5077
Lao AIK, Su X, Aung KMM (2009) SPR study of DNA hybridization with DNA and PNA probes under stringent conditions. Biosens Bioelectron 24:1717–1722
Joung H-A, Lee N-R, Lee SK, Ahn J, Shin YB, Choi H-S, Lee C-S, Kim S, Kim M-G (2008) High sensitivity detection of 16s rRNA using peptide nucleic acid probes and a surface plasmon resonance biosensor. Anal Chim Acta 630:168–173
Sato Y, Fujimoto K, Kawaguchi H (2003) Detection of a K-ras point mutation employing peptide nucleic acid at the surface of a SPR biosensor. Colloids Surf B Biointerfaces 27:23–31
Ananthanawat C, Vilaivan T, Hoven VP, Su X (2010) Comparison of DNA, aminoethylglycine PNA and pyrrolidinyl PNA as probes for detection of DNA hybridization using surface plasmon resonance technique. Biosens Bioelectron 25:1064–1069
Ananthanawat C, Hoven VP, Vilaivan T, Su X (2011) Surface plasmon resonance study of PNA interactions with double-stranded DNA. Biosens Bioelectron 26:1918–1923
Roy S, Tanious FA, Wilson WD, Ly DH, Armitage BA (2007) High affinity homologous peptide nucleic acid probes for targeting a quadruplex forming sequence from a MYC promoter element. Biochemistry 46:10433–10443
Lusvarghi S, Murphy CT, Roy S, Tanious FA, Sacui I, Wilson WD, Ly DH, Armitage BA (2009) Loop and backbone modifications of PNA improve G quadruplex binding selectivity. J Am Chem Soc 131:18415–18424
Roy S, Zanotti KJ, Murphy CT, Tanious FA, Wilson WD, Ly DH, Armitage BA (2011) Kinetic discrimination in recognition of DNA quadruplex targets by guanine-rich heteroquadruplex-forming PNA probes. Chem Commun 47:8524–8526
Sahu B, Sacui I, Rapireddy S, Zanotti KJ, Bahal R, Armitage BA, Ly DH (2011) Synthesis and characterization of conformationally preorganized, (R)-diethylene glycol-containing γ-peptide nucleic acids with superior hybridization properties and water solubility. J Org Chem 76:5614–5627
Christensen L, Fitzpatrick R, Gildea B, Petersen KH, Hansen HF, Koch T, Egholm M, Buchardt O, Nielsen PE, Coull J, Berg RH (1995) Solid-phase synthesis of peptide nucleic acids. J Pept Sci 3:175–183
Koch T (2004) PNA synthesis by Boc chemistry. In: Nielsen PE (ed) Peptide nucleic acids: protocols and applications, 2nd edn. Horizon Bioscience, pp 37–60
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
Armitage, B.A. (2014). Analysis of PNA Hybridization by Surface Plasmon Resonance. 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_13
Download citation
DOI: https://doi.org/10.1007/978-1-62703-553-8_13
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