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Competition-Enhanced Ligand Selection to Identify DNA Aptamers
ACS Combinatorial Science Pub Date : 2018-09-06 00:00:00 , DOI: 10.1021/acscombsci.8b00048 Maeling J. N. Tapp , Joseph M. Slocik 1 , Patrick B. Dennis 1 , Rajesh R. Naik 1 , Valeria T. Milam
ACS Combinatorial Science Pub Date : 2018-09-06 00:00:00 , DOI: 10.1021/acscombsci.8b00048 Maeling J. N. Tapp , Joseph M. Slocik 1 , Patrick B. Dennis 1 , Rajesh R. Naik 1 , Valeria T. Milam
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Competition-enhanced ligand screening (CompELS) was employed to rapidly screen through large DNA libraries to identify single-stranded, oligonucleotide-based ligands called aptamers that bind to a nonbiological target. This previously unreported aptamer screening approach involves the repeated introduction of unenriched random sequence populations during the biopanning process, but avoids iterative elution and polymerase chain reaction (PCR) amplification steps inherent to traditional SELEX (systematic evolution of ligands by exponential enrichment) screening. In this study, 25 aptamers were identified against a gold surface via CompELS and evaluated to identify patterns in primary structures and predicted secondary structures. Following a final one-round competition experiment with the 25 identified aptamers, one particular aptamer sequence (1N) emerged as the most competitive adsorbate species for the gold substrate. Binding analysis indicated at least an order of magnitude difference in the binding affinity of 1N (Kd = 5.6 × 10–10 M) compared to five other high affinity aptamer candidates (Kd = 10–8–10–9 M) from identical secondary structure families. Collectively, these studies introduce a rapid, reliable screening and ranking platform along with a classification scheme well-suited for identifying and characterizing aptamers for nonbiological as well as biological targets.
中文翻译：
竞争增强配体选择以鉴定DNA适体
使用竞争增强的配体筛选（CompELS）快速筛选大型DNA文库，以鉴定称为非适体的单链，基于寡核苷酸的配体，该配体与非生物靶标结合。这种先前未报道的适体筛选方法涉及在生物淘选过程中重复引入未富集的随机序列种群，但避免了传统SELEX（通过指数富集的配体的系统进化）筛选固有的迭代洗脱和聚合酶链反应（PCR）扩增步骤。在这项研究中，通过CompELS在金表面鉴定了25个适体，并对其进行了评估，以鉴定一级结构和预测的二级结构中的模式。在对25个已识别的适体进行最后的一轮竞争实验后，一种特定的适体序列（1N）成为金底物最具竞争力的吸附物。结合分析表明，与相同的其他五种高亲和力适体候选物（K d = 10 –8 –10 –9 M）相比，1N的结合亲和力（K d = 5.6×10 –10 M）至少有一个数量级差异。二级结构族。这些研究共同引入了一个快速，可靠的筛选和分级平台，以及一个分类方案，非常适合识别和表征非生物学和生物学靶标的适体。
更新日期：2018-09-06
中文翻译：
竞争增强配体选择以鉴定DNA适体
使用竞争增强的配体筛选（CompELS）快速筛选大型DNA文库，以鉴定称为非适体的单链，基于寡核苷酸的配体，该配体与非生物靶标结合。这种先前未报道的适体筛选方法涉及在生物淘选过程中重复引入未富集的随机序列种群，但避免了传统SELEX（通过指数富集的配体的系统进化）筛选固有的迭代洗脱和聚合酶链反应（PCR）扩增步骤。在这项研究中，通过CompELS在金表面鉴定了25个适体，并对其进行了评估，以鉴定一级结构和预测的二级结构中的模式。在对25个已识别的适体进行最后的一轮竞争实验后，一种特定的适体序列（1N）成为金底物最具竞争力的吸附物。结合分析表明，与相同的其他五种高亲和力适体候选物（K d = 10 –8 –10 –9 M）相比，1N的结合亲和力（K d = 5.6×10 –10 M）至少有一个数量级差异。二级结构族。这些研究共同引入了一个快速，可靠的筛选和分级平台，以及一个分类方案，非常适合识别和表征非生物学和生物学靶标的适体。
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