Porumb H, Gousset H, Letellier R, Salle V, Briane D, Vassy J, Amor-Gueret M, Israël L, Taillandier E
Laboratoire de Spectroscopie Bio-moléculaire, URA CNRS 1430, Université Paris-Nord, Bobigny, France.
Cancer Res. 1996 Feb 1;56(3):515-22.
A 28-base phosphodiester triple helix-forming oligonucleotide, mostly G and A containing, targeted to a polypurine tract interrupted by a purine-pyrimidine inversion, situated upstream from the TATA box of the promoter of the human HER2 gene, was conceived by computer modeling. The "energetically best choice" was oligo 28(C), which formed the triple helix in vitro, as proved by gel retardation and Fourier transform infrared spectroscopy. When administered as a complex with lipofectin, fluorescence confocal microscopy and electrophoresis confirmed the delivery and persistence of this unprotected oligonucleotide inside MCF7 (breast cancer) cells. At a concentration of 2 microM, the oligonucleotide reduced within 6 h the HER2 mRNA level to 42% (Northern blot) but did not interfere with the transcription of a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. During the first day of administration at 0.22 microM, it lowered to 59% the HER2 protein in treated, as compared to nontreated, cells (ELISA). The effect was sequence specific when compared to that of five different negative controls, and it was target selective when compared to the expression of a related, nontargeted protein, the epidermal growth factor receptor. By day 2, the inhibitory effect was overcome by replenishment reactions.
通过计算机建模设计出一种28个碱基的磷酸二酯三链螺旋形成寡核苷酸，其主要包含G和A，靶向于人HER2基因启动子TATA框上游被嘌呤-嘧啶倒置中断的聚嘌呤序列。“能量上的最佳选择”是寡核苷酸28(C)，凝胶阻滞和傅里叶变换红外光谱证明其在体外形成了三链螺旋。当与脂质体转染试剂形成复合物给药时，荧光共聚焦显微镜和电泳证实了这种未受保护的寡核苷酸在MCF7（乳腺癌）细胞内的递送和存留。在2微摩尔浓度下，该寡核苷酸在6小时内将HER2 mRNA水平降低至42%（Northern印迹法），但不干扰管家基因甘油醛-3-磷酸脱氢酶的转录。在以0.22微摩尔浓度给药的第一天，与未处理细胞相比，处理细胞中的HER2蛋白降低至59%（酶联免疫吸附测定法）。与五个不同的阴性对照相比，该效应具有序列特异性，与相关的非靶向蛋白表皮生长因子受体的表达相比，具有靶标选择性。到第2天，补充反应克服了抑制作用。
