Formation and Characterization of PNA-Containing Heteroquadruplexes

  • Bruce A. Armitage
Part of the Methods in Molecular Biology book series (MIMB, volume 1050)


The guanine quadruplex is a secondary structure formed by DNA and RNA that has been implicated in regulation of gene expression and maintenance of genome stability. Guanine-rich PNA oligomers can invade DNA or RNA quadruplex targets to form heteroquadruplex structures. Affinities in the low nanomolar range are routinely observed, making PNAs among the tightest binding of all quadruplex-targeted agents. Although inherently more promiscuous than heteroduplex formation based on Watson–Crick pairing, selectivity of heteroquadruplex formation can be improved through rational design of the sequence and backbone structure of the PNA. This chapter presents design rules and methods for characterizing PNA–DNA/RNA heteroquadruplexes.

Key words

Homologous hybridization Heteroquadruplex formation Backbone-modified PNA UV melting curves Surface plasmon resonance 


  1. 1.
    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 365:566–568PubMedCrossRefGoogle Scholar
  2. 2.
    Gellert M, Lipsett MN, Davies DR (1962) Helix formation by guanylic acid. Proc Natl Acad Sci U S A 48:2013–2018PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Neidle S, Balasubramanian S (eds) (2006) Quadruplex nucleic acids. Royal Society of Chemistry, Cambridge, UKGoogle Scholar
  4. 4.
    Amato J, Oliviero G, De Pauw E, Gabelica V (2009) Hybridization of short complementary PNAs to G-quadruplex forming oligonucleotides: an electrospray mass spectrometry study. Biopolymers 91:244–255PubMedCrossRefGoogle Scholar
  5. 5.
    Datta B, Armitage BA (2001) Hybridization of PNA to structured DNA targets: quadruplex invasion and the overhang effect. J Am Chem Soc 123:9612–9619PubMedCrossRefGoogle Scholar
  6. 6.
    Green JJ, Ying L, Klenerman D, Balasubramanian S (2003) Kinetics of unfolding the human telomeric DNA quadruplex using a PNA trap. J Am Chem Soc 125:3763–3767PubMedCrossRefGoogle Scholar
  7. 7.
    Marin VL, Armitage BA (2005) RNA guanine quadruplex invasion by complementary and homologous PNA probes. J Am Chem Soc 127:8032–8033PubMedCrossRefGoogle Scholar
  8. 8.
    Marin VL, Armitage BA (2006) Hybridization of complementary and homologous peptide nucleic acid oligomers to a guanine quadruplex-forming RNA. Biochemistry 45:1745–1754PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Datta B, Schmitt C, Armitage BA (2003) Formation of a PNA2-DNA2 hybrid quadruplex. J Am Chem Soc 125:4111–4118PubMedCrossRefGoogle Scholar
  10. 10.
    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 46:10433–10443PubMedCrossRefGoogle Scholar
  11. 11.
    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 131:18415–18424PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    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(30):8524–8526. doi: 10.1039/C1031CC12805A CrossRefGoogle Scholar
  13. 13.
    Sun D, Hurley LH (2010) Biochemical techniques for the characterization of G-quadruplex structures: EMSA, DMS footprinting, and DNA polymerase stop assay. Methods Mol Biol 608:65–79PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Kumari S, Bugaut A, Huppert J, Balasubramanian S (2007) An RNA G-quadruplex in the 5' UTR of the NRAS proto-oncogene modulates translation. Nat Chem Biol 3:218–221PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Siddiqui-Jain A, Grand CL, Bearss DJ, Hurley LH (2002) Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription. Proc Natl Acad Sci U S A 99:11593–11598PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Hamilton SE, Simmons CG, Kathiriya IS, Corey DR (1999) Cellular delivery of peptide nucleic acids and inhibition of human telomerase. Chem Biol 6:343–351PubMedCrossRefGoogle Scholar
  17. 17.
    Koppelus U, Nielsen PE (2003) Cellular delivery of peptide nucleic acid (PNA). Adv Drug Deliv Rev 55:267–280CrossRefGoogle Scholar
  18. 18.
    Sahu B, Chenna V, Lathrop KL, Thomas SM, Zon G, Livak JK, Ly DH (2009) Synthesis of conformationally preorganized and cell-permeable guanidine-based γ-peptide nucleic acids (γGPNAs). J Org Chem 74:1509–1516PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    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 125:6878–6879PubMedCrossRefGoogle Scholar
  20. 20.
    Paul A, Sengupta P, Krishnan Y, Ladame S (2008) Combining G-quadruplex targeting motifs on a single peptide nucleic acid scaffold: a hybrid (3 + 1) PNA-DNA bimolecular quadruplex. Chemistry 14:8682–8689PubMedCrossRefGoogle Scholar
  21. 21.
    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 3:175–183CrossRefGoogle Scholar
  22. 22.
    Koch T (2004) PNA synthesis by Boc chemistry. In: Nielsen PE (ed) Peptide nucleic acids: protocols and applications, 2nd edn. Horizon Bioscience, Norfolk, pp 37–60Google Scholar
  23. 23.
    Mergny J-L, Phan A-T, Lacroix L (1998) Following G-quartet formation by UV-spectroscopy. FEBS Lett 435:74–78PubMedCrossRefGoogle Scholar
  24. 24.
    Marky LA, Breslauer KJ (1987) Calculating thermodynamic data for transitions of any molecularity from equilibrium melting curves. Biopolymers 26:1601–1620PubMedCrossRefGoogle Scholar
  25. 25.
    Job P (1928) Recherches sur la formation de complexes minéraux en solution, et sur leur stabilité. Ann Chim 9:113–203Google Scholar
  26. 26.
    Svanvik N, Westman G, Wang D, Kubista M (2000) Light-up probes: thiazole orange-conjugated peptide nucleic acid for detection of target nucleic acid in homogeneous solution. Anal Biochem 281:26–35PubMedCrossRefGoogle Scholar
  27. 27.
    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. doi: 10.1021/jo200482d PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Bruce A. Armitage
    • 1
  1. 1.Department of Chemistry, Center for Nucleic Acids Science and TechnologyCarnegie Mellon UniversityPittsburghUSA

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