Ing N H, Beekman J M, Kessler D J, Murphy M, Jayaraman K, Zendegui J G, Hogan M E, O'Malley B W, Tsai M J
Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030.
Nucleic Acids Res. 1993 Jun 25;21(12):2789-96. doi: 10.1093/nar/21.12.2789.
Oligonucleotides provide novel reagents for inhibition of gene expression because of their high affinity binding to specific nucleotide sequences. We describe a 38 base, single-stranded DNA that forms a triple helix or 'triplex' on progesterone response elements of a target gene. This triplex-forming oligonucleotide binds with a Kd = 100 nM at 37 degrees C and physiological pH, and blocks binding of progesterone receptors to the target. Furthermore, it completely inhibited progesterone receptor-dependent transcription in vitro. To approach in vivo conditions, triplex-forming oligonucleotides were tested in cell transfection studies. The derivation of the oligonucleotides with cholesterol enhanced their cellular uptake and nuclear concentration by at least four-fold. The cholesterol-derivatized triplex-forming oligonucleotide specifically inhibited transcription of the PRE-containing reporter gene in cells when applied to the medium at micromolar concentrations. This is the first demonstration of steroid-responsive gene inhibition by triplex formation and joins the growing body of evidence indicating that oligonucleotides have therapeutic potential.
由于寡核苷酸能与特定核苷酸序列高亲和力结合，因此可提供用于抑制基因表达的新型试剂。我们描述了一种38个碱基的单链DNA，它能在靶基因的孕酮反应元件上形成三螺旋或“三链体”。这种形成三链体的寡核苷酸在37℃和生理pH条件下以Kd = 100 nM的亲和力结合，并阻断孕酮受体与靶标的结合。此外，它在体外完全抑制了孕酮受体依赖性转录。为了接近体内条件，在细胞转染研究中对形成三链体的寡核苷酸进行了测试。带有胆固醇的寡核苷酸衍生物使它们的细胞摄取和核内浓度提高了至少四倍。当以微摩尔浓度应用于培养基时，胆固醇衍生的形成三链体的寡核苷酸特异性抑制细胞中含PRE的报告基因的转录。这是通过形成三链体抑制类固醇反应性基因的首次证明，并加入了越来越多的证据表明寡核苷酸具有治疗潜力。
