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

Biotinylated homopyrlmidlne decamer peptide nucleic acids (PNAs) are shown to form sequence-specific and stable complexes with complementary oligopurine targets in linear double-stranded DNA. The non-covalent complexes are visualized by electron microscopy (EM) without chemical fixation using strept...

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Bibliographic Details
Published in:	Nucleic acids research Vol. 22; no. 24; pp. 5218 - 5222
Main Authors:	Demidov, Vadim V., Cherny, Dmitry I., Kurakin, Alexey V., Yavnilovich, Michael V., Malkov, Vladislav A., Frank-Kamenetskii, Maxim D., Sdnnichsen, Spren H., Nielsen, Peter E.
Format:	Journal Article
Language:	English
Published:	England Oxford University Press 11-12-1994
Subjects:	Bacterial Proteins Base Sequence Biotin DNA - metabolism DNA - ultrastructure DNA Probes - chemical synthesis DNA Probes - chemistry DNA Probes - metabolism Microscopy, Electron Molecular Sequence Data Nucleic Acid Conformation Nucleic Acid Hybridization Oligodeoxyribonucleotides - chemical synthesis Oligodeoxyribonucleotides - chemistry Oligodeoxyribonucleotides - metabolism Peptide Nucleic Acids Poly A - metabolism Streptavidin
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Summary:	Biotinylated homopyrlmidlne decamer peptide nucleic acids (PNAs) are shown to form sequence-specific and stable complexes with complementary oligopurine targets in linear double-stranded DNA. The non-covalent complexes are visualized by electron microscopy (EM) without chemical fixation using streptavldln 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 streptavldin ‘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 streptavldln ‘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.
Bibliography:	istex:7C7B8DE6CDD2EEB3AB3922BC6758FA9986394156 ark:/67375/HXZ-PJTNQ9MV-W Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel ArticleID:22.24.5218 ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2
ISSN:	0305-1048 1362-4962
DOI:	10.1093/nar/22.24.5218