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PMID: 8990164 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Accessibility of nuclear DNA to triplex-forming oligonucleotides: the integrated HIV-1 provirus as a target.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 94 ·No. 1 ·1997-01-07 ·Pages 79-84

Giovannangeli C, Diviacco S, Labrousse V, Gryaznov S, Charneau P, Helene C

Abstract

The control of gene transcription by antigene oligonucleotides rests upon the specific recognition of double-helical DNA by triplex-forming oligonucleotides. The development of the antigene strategy requires access to the targeted DNA sequence within the chromatin structure of the cell nucleus. In this sudy we have used HIV-1 chronically infected cells containing the HIV provirus as endogenous genes to demonstrate that the integrated HIV-1 proviral genome is accessible to triplex-forming oligonucleotides within cell nuclei. An oligonucleotide-psoralen conjugate targeted to the polypurine tract (PPT) of the HIV-1 proviral sequence was used as a tool to convert the noncovalent triple-helical complex into a covalent lesion on genomic DNA after UV irradiation of cells. Triplex-derived adducts were analyzed using two different methods. The photo-induced psoralen cross-link prevented cleavage of the target sequence by DraI restriction endonuclease, and the sequence-specific inhibition of cleavage was revealed and quantitated by Southern blot analysis. A quantitative analysis of cross-linking efficiency was also carried out by a competitive PCR-based assay. These two approaches allowed us to demonstrate that a triplex-forming oligonucleotide can recognize and bind specifically to a 15-bp sequence within the chromatin structure of cell nuclei.

MeSH Terms
Cell Membrane Permeability Cells, Cultured Chromatin/drug effects Cross-Linking Reagents DNA/drug effects DNA, Viral/drug effects Deoxyribonucleases, Type II Site-Specific/metabolism Ficusin/chemistry HIV-1/drug effects Oligodeoxyribonucleotides/chemistry,pharmacology Polymerase Chain Reaction Proviruses/drug effects
Chemicals
Chromatin Cross-Linking Reagents DNA, Viral Oligodeoxyribonucleotides triplex DNA DNA Deoxyribonucleases, Type II Site-Specific TTTAAA -specific type II deoxyribonucleases Ficusin
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Giovannangeli C
Institut National de la Santé et de la Recherche Médicale-Unité 201,Centre National de la Recherche Scientifique, Paris, France. [email protected]
Diviacco S
Labrousse V
Gryaznov S
Charneau P
Helene C
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.94.1.79
Email
[email protected]
Published
1997-01-07
Pages
79-84
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC19239
Subset
IM
External Links
PubMed source DOI: 10.1073/pnas.94.1.79 PMC Full Text View journal details
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