Guieysse A L, Praseuth D, Grigoriev M, Harel-Bellan A, Hélène C
Laboratoire de Biophysique, INSERM U.201, CNRS URA 481, Paris, France.
Nucleic Acids Res. 1996 Nov 1;24(21):4210-6. doi: 10.1093/nar/24.21.4210.
Triple helix-forming oligonucleotides covalently linked to psoralen can be specifically cross-linked to both strands of DNA at the triplex-duplex junction following UV irradiation. We have previously shown that a 15mer psoralen-oligonucleotide conjugate forming a triple helix on the promoter of the alpha subunit gene of the interleukin-2 receptor inhibits transcription of reporter plasmids transfected into living cells after irradiation. In the present work, we directly demonstrate covalent triple helix formation at the target site inside cells. A primer extension assay using Taq polymerase was developed to quantitate the DNA which had reacted with the psoralen of the triple helix-forming oligonucleotide. Photoaddition of the psoralen at the DNA target site was demonstrated, not only when the preformed triplex was electroporated inside cells, but also when the oligonucleotide was added to the culture medium after plasmid electroporation and before irradiation of the cells.
与补骨脂素共价连接的三链螺旋形成寡核苷酸在紫外线照射后可在三链体 - 双链体交界处与DNA的两条链特异性交联。我们之前已经表明，一种在白细胞介素 - 2受体α亚基基因启动子上形成三链螺旋的15聚体补骨脂素 - 寡核苷酸缀合物在照射后可抑制转染到活细胞中的报告质粒的转录。在本研究中，我们直接证明了在细胞内的靶位点形成了共价三链螺旋。开发了一种使用Taq聚合酶的引物延伸测定法来定量与三链螺旋形成寡核苷酸的补骨脂素发生反应的DNA。不仅当预先形成的三链体通过电穿孔导入细胞内时，而且当在质粒电穿孔后且在细胞照射前将寡核苷酸添加到培养基中时，都证明了补骨脂素在DNA靶位点的光加成。
