Effect of abasic linker substitution on triplex formation, Sp1 binding, and specificity in an oligonucleotide targeted to the human Ha-ras promoter.
Effect of abasic linker substitution on triplex formation, Sp1 binding, and specificity in an oligonucleotide targeted to the human Ha-ras promoter.
复制标题
无碱基接头取代对靶向人 Ha-ras 启动子的寡核苷酸的三链体形成、Sp1 结合和特异性的影响。
DOI：
10.1093/nar/22.10.1909
复制
发表时间：
1994
影响因子：
14.9
通讯作者：
Miller,D
中科院分区：
文献类型：
--
作者：
Mayfield,C;Miller,D
人类Ha-raspromoter（- 8至- 28）的一个区域包含转录活性必需的三个Sp1结合位点中的两个，形成序列特异性寡核苷酸导向的pur*pur:pyr三螺旋。含有不同取代的寡核苷酸的相对结合，包括一个碱性丙二醇连接，在三个潜在的不稳定的C:G中断，否则poly G:poly C目标进行了检查。dna酶I足迹滴定显示，取代带正电的碱性丙二醇连接体的结合效果比取代胞嘧啶的结合效果大大约10倍，而胞嘧啶的结合效果又比取代第三链寡核苷酸中的鸟嘌呤在C:G中断上的结合效果更好。蛋白结合实验表明，与胞嘧啶取代寡聚物（HR21ap）相比，连接物取代寡聚物（HR21ap）在抑制Sp1与靶序列和上游序列结合方面的效果更差。作为连接子替代和靶向一致Sp1位点对三联体特异性影响的指示，还研究了Ha-raspromoter靶向寡核苷酸与Ha-raspromoter、DHFR启动子和HIV-1 LTR内的非靶标Sp1序列相互作用的相对能力。在提供完全DNase I保护目标序列的浓度下，HR21ap不与非目标序列结合，而HR21Xap仅在DHFR启动子的远端位点弱相互作用。此外，HR21ap和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目标序列的中断，从而稳定三联体，但稍微损害了序列特异性和抑制Sp1与Ha-raspromoter结合的能力。
A region of the human Ha-raspromoter (−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-raspromoter targeted oligonucleotides to interact with non-target Sp1 sequences within the Ha-raspromoter 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-raspromoter.