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. 1994 Dec 11;22(24):5218–5222. doi: 10.1093/nar/22.24.5218

Electron microscopy mapping of oligopurine tracts in duplex DNA by peptide nucleic acid targeting.

V V Demidov 1, D I Cherny 1, A V Kurakin 1, M V Yavnilovich 1, V A Malkov 1, M D Frank-Kamenetskii 1, S H Sönnichsen 1, P E Nielsen 1
PMCID: PMC332063  PMID: 7816609

Abstract

Biotinylated homopyrimidine decamer peptide nucleic acids (PNAs) are shown to form sequence-specific and stable complexes with complementary oligopurine targets in linear double-stranded DNA. The noncovalent complexes are visualized by electron microscopy (EM) without chemical fixation using streptavidin as an EM marker. The triplex stoichiometry of the PNA-DNA complexes (two PNA molecules presumably binding by Watson-Crick and Hoogsteen pairing with one of the strands of the duplex DNA) is indicated by the appearance of two streptavidin 'beads' per target site in some micrographs, and is also supported by the formation of two retardation bands in a gel shift assay. Quantitative analysis of the positions of the streptavidin 'beads' revealed that under optimized conditions PNA-DNA complexes are preferably formed with the fully complementary target. An increase in either the PNA concentration or the incubation time leads to binding at sites containing one or two mismatches. Our results demonstrate that biotinylated PNAs can be used for EM mapping of short targets in duplex DNA.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Cherny D. I., Malkov V. A., Volodin A. A., Frank-Kamenetskii M. D. Electron microscopy visualization of oligonucleotide binding to duplex DNA via triplex formation. J Mol Biol. 1993 Mar 20;230(2):379–383. doi: 10.1006/jmbi.1993.1154. [DOI] [PubMed] [Google Scholar]
  2. Cherny D. Y., Belotserkovskii B. P., Frank-Kamenetskii M. D., Egholm M., Buchardt O., Berg R. H., Nielsen P. E. DNA unwinding upon strand-displacement binding of a thymine-substituted polyamide to double-stranded DNA. Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1667–1670. doi: 10.1073/pnas.90.5.1667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Demidov V., Frank-Kamenetskii M. D., Egholm M., Buchardt O., Nielsen P. E. Sequence selective double strand DNA cleavage by peptide nucleic acid (PNA) targeting using nuclease S1. Nucleic Acids Res. 1993 May 11;21(9):2103–2107. doi: 10.1093/nar/21.9.2103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dubochet J., Ducommun M., Zollinger M., Kellenberger E. A new preparation method for dark-field electron microscopy of biomacromolecules. J Ultrastruct Res. 1971 Apr;35(1):147–167. doi: 10.1016/s0022-5320(71)80148-x. [DOI] [PubMed] [Google Scholar]
  5. Hanvey J. C., Peffer N. J., Bisi J. E., Thomson S. A., Cadilla R., Josey J. A., Ricca D. J., Hassman C. F., Bonham M. A., Au K. G. Antisense and antigene properties of peptide nucleic acids. Science. 1992 Nov 27;258(5087):1481–1485. doi: 10.1126/science.1279811. [DOI] [PubMed] [Google Scholar]
  6. Nielsen P. E., Egholm M., Berg R. H., Buchardt O. Peptide nucleic acids (PNAs): potential antisense and anti-gene agents. Anticancer Drug Des. 1993 Feb;8(1):53–63. [PubMed] [Google Scholar]
  7. Nielsen P. E., Egholm M., Berg R. H., Buchardt O. Sequence specific inhibition of DNA restriction enzyme cleavage by PNA. Nucleic Acids Res. 1993 Jan 25;21(2):197–200. doi: 10.1093/nar/21.2.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Nielsen P. E., Egholm M., Berg R. H., Buchardt O. Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science. 1991 Dec 6;254(5037):1497–1500. doi: 10.1126/science.1962210. [DOI] [PubMed] [Google Scholar]
  9. Nielsen P. E., Egholm M., Buchardt O. Peptide nucleic acid (PNA). A DNA mimic with a peptide backbone. Bioconjug Chem. 1994 Jan-Feb;5(1):3–7. doi: 10.1021/bc00025a001. [DOI] [PubMed] [Google Scholar]
  10. Peffer N. J., Hanvey J. C., Bisi J. E., Thomson S. A., Hassman C. F., Noble S. A., Babiss L. E. Strand-invasion of duplex DNA by peptide nucleic acid oligomers. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10648–10652. doi: 10.1073/pnas.90.22.10648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Révet B. M., Sena E. P., Zarling D. A. Homologous DNA targeting with RecA protein-coated short DNA probes and electron microscope mapping on linear duplex molecules. J Mol Biol. 1993 Aug 5;232(3):779–791. doi: 10.1006/jmbi.1993.1431. [DOI] [PubMed] [Google Scholar]

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