Electron Microscopy Mapping of Oligopurine Tracts in Duplex DNA by Peptide Nucleic Acid Targeting

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 1994 Dec 11

PMID 7816609

Citations 9

Authors

V V Demidov

D I Cherny

A V Kurakin

M V Yavnilovich

V A Malkov

M D Frank-Kamenetskii

S H Sonnichsen

P E Nielsen

Affiliations

Soon will be listed here.

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.

Citing Articles

Effects of hypoxanthine substitution in peptide nucleic acids targeting KRAS2 oncogenic mRNA molecules: theory and experiment.

Sanders J, Wampole M, Chen C, Sethi D, Singh A, Dupradeau F J Phys Chem B. 2013; 117(39):11584-95.

PMID: 23972113 PMC: 3946533. DOI: 10.1021/jp4064966.

Labeling DNA for single-molecule experiments: methods of labeling internal specific sequences on double-stranded DNA.

Zohar H, Muller S Nanoscale. 2011; 3(8):3027-39.

PMID: 21734993 PMC: 3322637. DOI: 10.1039/c1nr10280j.

Peptide nucleic acids as tools for single-molecule sequence detection and manipulation.

Zohar H, Hetherington C, Bustamante C, Muller S Nano Lett. 2010; 10(11):4697-701.

PMID: 20923183 PMC: 3322611. DOI: 10.1021/nl102986v.

Specific versus nonspecific binding of cationic PNAs to duplex DNA.

Abibi A, Protozanova E, Demidov V, Frank-Kamenetskii M Biophys J. 2004; 86(5):3070-8.

PMID: 15111421 PMC: 1304173. DOI: 10.1016/S0006-3495(04)74356-8.

Analysis of various sequence-specific triplexes by electron and atomic force microscopies.

Cherny D, Fourcade A, Svinarchuk F, Nielsen P, Malvy C, Delain E Biophys J. 1998; 74(2 Pt 1):1015-23.

PMID: 9533714 PMC: 1302582. DOI: 10.1016/S0006-3495(98)74026-3.

References

Dubochet J, DUCOMMUN M, ZOLLINGER M, Kellenberger E . A new preparation method for dark-field electron microscopy of biomacromolecules. J Ultrastruct Res. 1971; 35(1):147-67. DOI: 10.1016/s0022-5320(71)80148-x. View

Nielsen P, Egholm M, Berg R, Buchardt O . Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science. 1991; 254(5037):1497-500. DOI: 10.1126/science.1962210. View

Hanvey J, Peffer N, Bisi J, Thomson S, Cadilla R, Josey J . Antisense and antigene properties of peptide nucleic acids. Science. 1992; 258(5087):1481-5. DOI: 10.1126/science.1279811. View

Nielsen P, Egholm M, Berg R, Buchardt O . Sequence specific inhibition of DNA restriction enzyme cleavage by PNA. Nucleic Acids Res. 1993; 21(2):197-200. PMC: 309092. DOI: 10.1093/nar/21.2.197. View

Cherny D, Belotserkovskii B, Frank-Kamenetskii M, Egholm M, Buchardt O, Berg R . DNA unwinding upon strand-displacement binding of a thymine-substituted polyamide to double-stranded DNA. Proc Natl Acad Sci U S A. 1993; 90(5):1667-70. PMC: 45940. DOI: 10.1073/pnas.90.5.1667. View

Nielsen P, Egholm M, Buchardt O . Peptide nucleic acid (PNA). A DNA mimic with a peptide backbone. Bioconjug Chem. 1994; 5(1):3-7. DOI: 10.1021/bc00025a001. View

Nielsen P, Egholm M, Berg R, Buchardt O . Peptide nucleic acids (PNAs): potential antisense and anti-gene agents. Anticancer Drug Des. 1993; 8(1):53-63. View

Demidov V, Frank-Kamenetskii M, Egholm M, Buchardt O, Nielsen P . Sequence selective double strand DNA cleavage by peptide nucleic acid (PNA) targeting using nuclease S1. Nucleic Acids Res. 1993; 21(9):2103-7. PMC: 309471. DOI: 10.1093/nar/21.9.2103. View

Revet B, Sena E, Zarling D . Homologous DNA targeting with RecA protein-coated short DNA probes and electron microscope mapping on linear duplex molecules. J Mol Biol. 1993; 232(3):779-91. DOI: 10.1006/jmbi.1993.1431. View

10.

Peffer N, Hanvey J, Bisi J, Thomson S, Hassman C, Noble S . Strand-invasion of duplex DNA by peptide nucleic acid oligomers. Proc Natl Acad Sci U S A. 1993; 90(22):10648-52. PMC: 47834. DOI: 10.1073/pnas.90.22.10648. View

11.

Cherny D, Malkov V, Volodin A, Frank-Kamenetskii M . Electron microscopy visualization of oligonucleotide binding to duplex DNA via triplex formation. J Mol Biol. 1993; 230(2):379-83. DOI: 10.1006/jmbi.1993.1154. View