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

被引:164
作者
Giovannangeli, C
Diviacco, S
Labrousse, V
Gryaznov, S
Charneau, P
Helene, C
机构
[1] INST PASTEUR,LAB ONCOL VIRALE,CNRS,UNITE RECH ASSOCIEE 1156,F-75724 PARIS 15,FRANCE
[2] LYNX THERAPEUT INC,HAYWARD,CA 94545
关键词
oligonucleotide-psoralen conjugate; DNA accessibility; competitive PCR; antigene oligonucleotides; HIV;
D O I
10.1073/pnas.94.1.79
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
The control of gene transcription by antigene oligonucleotides rests upon the specific recognition of double-helical DNA by tripler-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 tripler-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. Tripler-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 tripler-forming oligonucleotide can recognize and bind specifically to a 15-bp sequence within the chromatin structure of cell nuclei.
引用
收藏
页码:79 / 84
页数:6
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