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首页> 美国卫生研究院文献>Nucleic Acids Research >Construction and selection of bead-bound combinatorial oligonucleoside phosphorothioate and phosphorodithioate aptamer libraries designed for rapid PCR-based sequencing
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Construction and selection of bead-bound combinatorial oligonucleoside phosphorothioate and phosphorodithioate aptamer libraries designed for rapid PCR-based sequencing

机译：珠结合的组合寡核苷酸硫代磷酸酯和二硫代磷酸酯适体文库的构建和选择用于基于PCR的快速测序

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Chemically synthesized combinatorial libraries of unmodified or modified nucleic acids have not previously been used in methods to rapidly select oligonucleotides binding to target biomolecules such as proteins. Phosphorothioate oligonucleotides (S-ODNs) or phosphorodithioate oligonucleotides (S2-ODNs) with sulfurs replacing one or both of the non-bridging phosphate oxygens bind to proteins more tightly than unmodified oligonucleotides and have the potential to be used as diagnostic reagents and therapeutics. We have applied a split synthesis methodology to create one-bead one-S-ODN and one-bead one-S2-ODN libraries. Binding and selection of specific beads to the transcription factor NF-κB p50/p50 protein were demonstrated. Sequencing both the nucleic acid bases and the positions of any 3′-O-thioate/dithioate linkages was carried out by using a novel PCR-based identification tag of the selected beads. This approach allows us to rapidly and conveniently identify S-ODNs or S2-ODNs that bind to proteins.
机译：未经修饰或修饰的核酸的化学合成组合文库先前尚未用于快速选择与目标生物分子（例如蛋白质）结合的寡核苷酸的方法。与未修饰的寡核苷酸相比，硫取代了一个或两个非桥连的磷酸氧的硫代磷酸酯寡核苷酸（S-ODN）或二硫代磷酸酯寡核苷酸（S2-ODN）与蛋白质的结合更紧密，并且具有用作诊断试剂和治疗剂的潜力。我们已应用拆分合成方法来创建一个单珠一-S-ODN和一个单珠一-S2-ODN库。证明了特异性珠与转录因子NF-κBp50 / p50蛋白的结合和选择。通过使用所选珠子的新颖的基于PCR的识别标签对核酸碱基和任何3'-O-硫代酸酯/二硫代酸酯键的位置进行测序。这种方法使我们能够快速方便地识别与蛋白质结合的S-ODN或S2-ODN。

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