Mayfield C, Miller D
Department of Medicine, University of Alabama at Birmingham.
Nucleic Acids Res. 1994 May 25;22(10):1909-16. doi: 10.1093/nar/22.10.1909.
A region of the human Ha-ras promoter (-8 to -28) which contains two of the three Sp1 binding sites essential for transcriptional activity forms a sequence specific oligonucleotide-directed pur*pur:pyr triple helix. The relative binding of oligonucleotides containing different substitutions, including an abasic propanediol linker, over three potentially destabilizing C:G interruptions in the otherwise poly G:poly C target was examined. DNase I footprint titrations reveal that substitution of the positively charged abasic propanediol linker results in approximately ten fold greater binding than cytosine substitution which in turn provides greater sequence specific binding than substitution of a guanine in the third strand oligonucleotide over the C:G interruptions. Protein binding assays demonstrate that triplex formation by the linker substituted oligomer (HR21Xap) is less effective in inhibiting Sp1 binding than the cytosine substituted oligomer (HR21ap) both to the target sequence as well as an upstream sequence. As an indication of the effect of linker substitution and targeting consensus Sp1 sites on triplex specificity, the relative ability of the Ha-ras promoter targeted oligonucleotides to interact with non-target Sp1 sequences within the Ha-ras promoter as well as in the DHFR promoter and HIV-1 LTR was also investigated. At concentrations which afford complete DNase I protection of the target sequence, HR21ap does not bind to the non-target sequences while HR21Xap interacts weakly only at a distal site in the DHFR promoter. Also, HR21ap as well as HR21Xap are specific in their inhibition of Sp1 binding. These results suggest that the propanediol linker is able to skip over interruptions in a target sequence thereby stabilizing triplex but, slightly compromises sequence specificity and the ability to inhibit Sp1 binding to the Ha-ras promoter.
人类Ha-ras启动子的一个区域（-8至-28）包含转录活性所必需的三个Sp1结合位点中的两个，形成了一种序列特异性的寡核苷酸定向嘌呤*嘌呤：嘧啶三链螺旋。研究了含有不同取代基的寡核苷酸（包括无碱基丙二醇连接体）在原本为聚G：聚C靶标的三个潜在不稳定C：G间断处的相对结合情况。DNase I足迹滴定显示，带正电荷的无碱基丙二醇连接体的取代导致的结合比胞嘧啶取代大约强十倍，而胞嘧啶取代又比第三链寡核苷酸中鸟嘌呤在C：G间断处的取代提供更高的序列特异性结合。蛋白质结合分析表明，连接体取代的寡聚物（HR21Xap）形成三链体对Sp1结合的抑制作用不如胞嘧啶取代的寡聚物（HR21ap）对靶序列以及上游序列的抑制作用有效。作为连接体取代和靶向共有Sp1位点对三链体特异性影响的一个指标，还研究了靶向Ha-ras启动子的寡核苷酸与Ha-ras启动子内以及二氢叶酸还原酶（DHFR）启动子和HIV-1长末端重复序列（LTR）中非靶Sp1序列相互作用的相对能力。在能使靶序列获得完全DNase I保护的浓度下，HR21ap不与非靶序列结合，而HR21Xap仅在DHFR启动子的一个远端位点有弱相互作用。此外，HR21ap以及HR21Xap在抑制Sp1结合方面具有特异性。这些结果表明，丙二醇连接体能够跳过靶序列中的间断从而稳定三链体，但略微损害了序列特异性以及抑制Sp1与Ha-ras启动子结合的能力。
