Accessibility of nuclear DNA to triplex-forming oligonucleotides: The integrated HIV-1 provirus as a target
Accessibility of nuclear DNA to triplex-forming oligonucleotides: The integrated HIV-1 provirus as a target
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DOI：
10.1073/pnas.94.1.79
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发表时间：
1997-01-07
影响因子：
11.1
通讯作者：
Helene, C
中科院分区：
文献类型：
--
作者：
Giovannangeli, C;Diviacco, S;Helene, C
反基因寡核苷酸对基因转录的控制依赖于形成三联体的寡核苷酸对双螺旋DNA的特异性识别。反基因策略的开发需要获得细胞核染色质结构内的目标DNA序列。在本研究中，我们使用含有HIV原病毒作为内源基因的HIV-1慢性感染细胞来证明整合的HIV-1原病毒基因组可被细胞核内的三联体形成寡核苷酸接近。使用靶向 HIV-1 前病毒序列的多嘌呤束 (PPT) 的寡核苷酸-补骨脂素缀合物作为工具，在细胞紫外线照射后将非共价三螺旋复合物转化为基因组 DNA 上的共价损伤。使用两种不同的方法分析三倍体衍生的加合物，光诱导的补骨脂素交联阻止了 DraI 限制性核酸内切酶对靶序列的切割，并且通过 Southern 印迹分析揭示并定量了切割的序列特异性抑制。还通过基于竞争性 PCR 的测定进行了交联效率的定量分析。这两种方法使我们能够证明三联体形成寡核苷酸可以识别并特异性结合细胞核染色质结构内的 15 bp 序列。
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.