Grigoriev M, Praseuth D, Guieysse A L, Robin P, Thuong N T, Hélène C, Harel-Bellan A
Laboratoire de Biologie des Tumeurs Humaines, Centre National de la Recherche Scientifique, Villejuif, France.
Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3501-5. doi: 10.1073/pnas.90.8.3501.
Synthetic oligodeoxynucleotides represent promising tools for gene inhibition in live systems. Triple helix-forming oligonucleotides, which bind to double-stranded DNA, are of special interest since they are targeted to the gene itself rather than to its mRNA product, as in the antisense strategy. Triple helix-forming oligonucleotides can be coupled to DNA-modifying agents and used to introduce modifications in the DNA target in a highly sequence-specific manner. We have recently designed psoralen-oligonucleotide conjugates, which, upon binding to double-stranded DNA sequences via triple helix formation, may be cross-linked in vitro to both strands of the DNA following UV irradiation. A psoralen-oligonucleotide conjugate was targeted to the promoter of the alpha subunit of the interleukin 2 receptor (IL-2R alpha) gene. The triple helix site overlaps the binding site for the transcription factor NF-kappa B, which activates transcription from the IL-2R alpha promoter. After UV irradiation, the oligonucleotide conjugate becomes cross-linked to the target site and inhibits transcription of reporter plasmids transfected in live cells. Inhibition is observed when UV-induced cross-linking occurs both in vitro (before transfection) and in vivo (after transfection). We directly demonstrate that this inhibitory effect is due to triple helix formation at the target site, since a mutant of the promoter, to which oligonucleotide binding was inhibited, was not affected by the psoralen-oligonucleotide conjugate after UV irradiation. In addition, we demonstrate that site-specific cross-linking upstream of the promoter has no effect on transcription.
合成寡脱氧核苷酸是在活细胞系统中进行基因抑制的有前景的工具。与双链DNA结合的三链螺旋形成寡核苷酸特别受关注，因为与反义策略不同，它们靶向的是基因本身而非其mRNA产物。三链螺旋形成寡核苷酸可与DNA修饰剂偶联，并以高度序列特异性的方式用于在DNA靶标中引入修饰。我们最近设计了补骨脂素 - 寡核苷酸缀合物，其通过三链螺旋形成与双链DNA序列结合后，在紫外线照射下可在体外与DNA的两条链交联。一种补骨脂素 - 寡核苷酸缀合物靶向白细胞介素2受体（IL-2Rα）基因α亚基的启动子。三链螺旋位点与转录因子NF-κB的结合位点重叠，NF-κB可激活IL-2Rα启动子的转录。紫外线照射后，寡核苷酸缀合物与靶位点交联并抑制在活细胞中瞬时转染的报告质粒的转录。当紫外线诱导的交联在体外（转染前）和体内（转染后）均发生时，可观察到抑制作用。我们直接证明这种抑制作用是由于在靶位点形成了三链螺旋，因为寡核苷酸结合被抑制的启动子突变体在紫外线照射后不受补骨脂素 - 寡核苷酸缀合物的影响。此外，我们证明在启动子上游的位点特异性交联对转录没有影响。
