Novel combinatorial selection of phosphorothioate oligonucleotide aptamers
Novel combinatorial selection of phosphorothioate oligonucleotide aptamers
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DOI：
10.1021/bi981780f
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发表时间：
1998-11-24
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;Ventura, DA;Gorenstein, DG
描述了一种新的组合方法，用于构建和筛选增强的核酸酶抗性硫代磷酸酯DNA“诱饵”或“适体”。“已经选择了与人IL 6（NF-IL 6）的核因子紧密结合的适体，这是一种碱性亮氨酸拉链转录因子，参与诱导急性期反应和细胞因子基因启动子对炎症的反应。在PCR扩增中使用随机组合选择方法和dNTP（α S），我们可以选择在结合靶NF-IL 6方面具有最高特异性的特定硫代取代试剂。使用22个核苷酸长的双链体随机文库，选择纳摩尔结合、特异性22聚体硫代磷酸骨架取代序列（仅在dA位置）。这些显示了与正常磷酸骨架CCAAT/增强子结合蛋白识别NF-IL 6的位点不同的共有序列。与结合1个蛋白质二聚体/双链体的野生型10聚体序列不同，这些22聚体硫代磷酸适体以2个二聚体/双链体的化学计量结合。
A novel combinatorial approach is described for construction and screening of enhanced nuclease-resistant phosphorothioate DNA "decoys" or "aptamers." Aptamers have been selected that bind tightly to the nuclear factor for human IL6 (NF-IL6), a basic leucine zipper transcription factor involved in the induction of acute-phase responsive and cytokine gene promoters in response to inflammation. Using a random combinatorial selection approach and dNTP(alpha S)'s in the PCR amplification, we can select specific thio-substituted agents which have the highest specificity in binding to target NF-IL6. Using a 22-nucleotide-long duplex random library, nanomolar binding, specific 22-mer thiophosphate backbone substitution sequences (at dA positions only) were selected. These show a different consensus sequence than normal phosphate backbone CCAAT/enhancer binding protein recognition sites for NF-IL6. Unlike the wild-type 10-mer sequences, which bind 1 protein dimer/duplex, these 22-mer thiophosphate aptamers bind with a stoichiometry of 2 dimers/duplex.