Triplex targets in the human rhodopsin gene.
Triplex targets in the human rhodopsin gene.
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人类视紫红质基因中的三链体靶点。
DOI：
10.1021/bi980525s
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Vasquez,KM
中科院分区：
文献类型：
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作者：
Perkins,BD;Wilson,JH;Wensel,TG;Vasquez,KM
我们已经探索了三链体技术在人类视紫红质基因中的应用，该基因编码与遗传性疾病常染色体显性视网膜色素变性（ADRP）有关的G蛋白连接受体。我们的研究结果支持的假设，大多数人类基因含有高亲和力的三链体位点，并进一步完善了规则的识别和成功的三链体形成的寡核苷酸（TFO），这些网站的目标。使用计算机搜索长度为15个核苷酸且超过80%嘌呤的位点，我们在视紫红质基因中发现了143个不同的位点，并且在其他几个人类基因中发现了相当数量的位点。通过应用更严格的标准，我们在视紫红质基因中选择了17个潜在的靶位点，用质粒结合试验筛选它们，发现8个以亚微摩尔亲和力（Kd= 10-9−10- 7 M）结合TFO。我们比较了每个位点的嘌呤（GA）和混合（GT）TFO，发现GA-TFO始终以更高的亲和力结合，并且对靶链中的嘧啶中断不太敏感。高G含量有利于高亲和力结合;只有G含量>54%的位点与Kd ≤ 10- 8 M的TFO结合。
We have explored the application of triplex technology to the human rhodopsin gene, which encodes a G-protein-linked receptor involved in the genetic disorder autosomal dominant retinitis pigmentosa (ADRP). Our results support the hypothesis that most human genes contain high-affinity triplex sites and further refine the rules governing identification and successful targeting of triplex-forming oligonucleotides (TFOs) to these sites. Using a computer search for sites 15 nucleotides in length and greater than 80% purine, we found 143 distinct sites in the rhodopsin gene and comparable numbers of sites in several other human genes. By applying more stringent criteria, we selected 17 potential target sites in the rhodopsin gene, screened them with a plasmid binding assay, and found 8 that bound TFOs with submicromolar affinity (Kd= 10-9−10-7M). We compared purine (GA) and mixed (GT) TFOs at each site, and found that GA-TFOs consistently bound with higher affinity, and were less sensitive to pyrimidine interruptions in the target strand. High G-content favored high-affinity binding; only sites with >54% G-content bound TFOs withKd≤ 10-8M.