Combinatorial selection and binding of phosphorothioate aptamers targeting human NF-κB RelA(p65) and p50
Combinatorial selection and binding of phosphorothioate aptamers targeting human NF-κB RelA(p65) and p50
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DOI：
10.1021/bi020220k
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发表时间：
2002-07-30
期刊：
影响因子：
2.9
通讯作者：
Gorenstein, DG
中科院分区：
文献类型：
--
作者：
King, DJ;Bassett, SE;Gorenstein, DG
先前，我们报道了靶向转录因子NF-IL 6的硫代磷酸酯适体或“硫代适体”的体外组合选择。使用相同的方法和纯化的重组人NF-κ B蛋白RelA-（p65）和p50，已经选择了双链体硫代适体，其表现出与双链体22-mer结合位点IgkappaB的高亲和力、竞争性结合。RelA（p65）和p50同源二聚体的结合能进行了研究，使用定量电泳迁移率变动分析或EMSA。作为竞争性适体结合的参考系统，确定称为Igkappa的双链体22聚体磷酰基结合位点以1：1的化学计量和亲和力结合每个p65和p50同源二聚体，通过全局分析确定，对于p65，K-d = 4.8 +/- 0.2 nM，对于p50，K-d = 0.8 +/- 0.2 nM。开发了竞争性NF-κ B/Igkappa结合的全局分析工具，并用于测量由p65和p50选择的硫代适体的亲和力。竞争结果表明，硫代适体与已知的启动子元件IgkappaB结合并竞争相同的NF-κ B位点（对于p65选择的适体，Kd = 78.9 +/-1.9 nM，对于p50选择的硫代适体，Kd = 19.6 +/-1.3 nM）。用p50的定性凝胶位移结合实验也证明，增强的亲和力和特异性的性质可以归因于硫的存在。总的来说，这些结果证明了硫代适体体外组合选择技术作为产生特异性高亲和力蛋白质配体的方法的可行性。
Previously, we reported the in vitro combinatorial selection of phosphorothioate aptamers or "thioaptamers" targeting the transcription factor NF-IL6. Using the same approach and purified recombinant human NF-kappaB proteins RelA-(p65) and p50, duplex thioaptamers have been selected that demonstrate high-affinity, competitive binding with the duplex 22-mer binding site, IgkappaB. Binding energetics of RelA(p65) and p50 homodimers were studied using a quantitative electrophoretic mobility shift assay or EMSA. As a reference system for competitive aptamer binding, the duplex 22-mer phosphoryl binding site known as Igkappa was determined to bind each p65 and p50 homodimer with a 1: 1 stoichiometry and with affinities, determined by global analysis, K-d = 4.8 +/- 0.2 nM for p65 and K-d = 0.8 +/- 0.2 nM for p50. A global analysis tool for competitive NF-kappaB/Igkappa binding was developed and utilized to measure the affinity of thioaptamers selected by both p65 and p50. The competition results indicate that the thioaptamers bind and compete for the same NF-kappaB site as the known promoter element IgkappaB (K-d = 78.9 +/- 1.9 nM for a p65-selected aptamer and 19.6 +/- 1.3 nM for a p50-selected thioaptamer). Qualitative gel shift binding experiments with p50 also demonstrate that the nature of enhanced affinity and specificity can be attributed to the presence of sulfur. Collectively, these results demonstrate the feasibility of the thioaptamer in vitro combinatorial selection technology as a method for producing specific, high-affinity ligands to proteins.