Double-stranded phosphorothioate oligonucleotide modulation of gene expression.
Double-stranded phosphorothioate oligonucleotide modulation of gene expression.
复制标题
双链硫代磷酸酯寡核苷酸调节基因表达。
DOI：
10.1111/j.1749-6632.1994.tb21722.x
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发表时间：
1994
影响因子：
5.2
通讯作者：
Aronow,BJ
中科院分区：
文献类型：
--
作者：
Ess,KC;Hutton,JJ;Aronow,BJ
抑制显性作用癌基因的表达可以阻止或逆转肿瘤生长或增加对常规治疗的敏感性。在某些情况下，靶向细胞类型特异性顺式调节区以竞争性结合控制癌基因表达的反式激活因子可能是一种非常有效的方法。我们利用修饰的双链（DS）寡核苷酸与核心增强子元件竞争反式激活因子。硫代磷酸酯寡核苷酸，如Bielinska等人所述，因为与磷酸二酯寡核苷酸相比，它们对核酸酶具有抗性。这些特性也使硫代磷酸酯作为ss反义试剂产生了很大的兴趣。作为拮抗细胞类型特异性增强子的模型，我们尝试拮抗人腺苷脱氨酶（ADA）基因T细胞增强子。本实验室先前的工作表明，能够驱动高水平组织特异性转基因小鼠表达的调控元件位于ADA格尔的第一内含子中。DNA酶I超敏位点形成，瞬时转染实验中的缺失研究和足迹分析已经描绘了一个200个碱基对的增强子区域，该区域由一个关键的28个碱基对的核心区域（RC）组成，其活性高度依赖于侧翼顺式元件。在用构建体5 ′-ADA CAT# 87转染后，将对应于核心增强子序列的DS硫代磷酸酯核苷酸加入奥尔特4 T细胞中（图1）。在48小时收获通常表达高水平ADA的Molt 4细胞，并定量CAT活性。来自一个代表性实验的结果示于图2中。RC和APRC（RC扩展到包括相邻的AP-1位点）寡核苷酸能够以浓度依赖性方式抑制报告基因表达。这两个序列都能够作为活性核心增强子聚合时，阻碍。相比之下，突变的寡核苷酸R1和R7既不能抑制报告基因表达，也不能在转染的Molt-4细胞中作为聚合的核心增强子发挥作用。这% s工作是su国立研究院o！Gealth Grant HD 19919（JJH）. &通讯地址：布鲁斯J. Aronow博士，De
Inhibiting the expression of dominantly acting oncogenes could halt or reverse tumor growth or confer increased susceptibility to conventional therapies. Targeting cell-type specific cis-regulatory regions for competitive binding of transactivators that control the expression of an oncogene may under some circumstances be a highly effective approach. We have utilized modified doublestranded (DS) oligonucleotides to compete with a core enhancer element for trans-activating factors. Phosphorothioate oligonucleotides, as described by Bielinska et d.,'were chosen because of their resistance to nucleases as compared with phosphodiester oligonucleotides. These properties have also generated much interest in phosphorothioates as ss antisense agents. As a model for antagonizing a cell type-specific enhancer, we have attempted to antagonize the human adenosine deaminase (ADA) gene T-cell enhancer. Previous work by this laboratory has shown that regulatory elements capable of driving high-level tissue-specific transgenic mouse expression reside in the first intron of the ADA ger~ e.~.~ DNAse I hypersensitivity site formation, deletional studies in transient transfection experiments, and footprinting assays have delineated a 200-base pair enhancer region composed of a critical 28-base pair core region (RC) whose activity is highly dependent on flanking cis elements. DS phosphorothioate nucleotides corresponding to core enhancer sequences were added to M olt 4 T-cells after transfection with construct 5'-ADA CAT# 87 (FIG. 1). Molt4 cells, which normally express high levels of ADA, were harvested at 48 hours, and the CAT activity was quantitated. The results from one representative experiment are shown in FIGURE 2. RC and APRC (RC extended to include an adjacent AP-1 site) oligonucleotides were able to inhibit reporter expression in a concentration-dependent manner. Both of these sequences were able to hction when polymerized as an active core enhancer. In contrast, mutated oligonucleotides R1 and R7 failed both to inhibit reporter expression and to function as a polymerized core enhancer in transfected Molt-4 cells. This% s work was su National Institutes o! gealth Grant HD 19919 (JJH). &Address for correspondence: Bruce J. Aronow, Ph. D., De