Identification of the Target Binding Site of Ethanolamine-Binding Aptamers and Its Exploitation for Ethanolamine Detection
Identification of the Target Binding Site of Ethanolamine-Binding Aptamers and Its Exploitation for Ethanolamine Detection
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DOI：
10.1021/ac5034819
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发表时间：
2015-01-06
影响因子：
7.4
通讯作者：
Scheper, Thomas
中科院分区：
文献类型：
--
作者：
Heilkenbrinker, Alexandra;Reinemann, Christine;Scheper, Thomas
核酸适体是一种很有前途的小分子识别元件，可用于小分子的灵敏和特异性检测。我们之前已经选择了乙醇胺的ssDNA适体，乙醇胺是迄今为止最小的适体靶标之一。这里介绍的工作集中在确定的适体结构内的结合区域和开发的一个基于适体的检测乙醇胺的测定。适体的序列分析导致鉴定出富含G的共有序列，其能够以典型的两层或三层G-四重体结构折叠。逐步截短的适体变体的实验表明，共有序列是负责的，足以结合到目标。基于对适体结合位点的了解，我们开发了一种基于适体的微阵列检测方法，该方法依赖于乙醇胺和与共有序列互补的寡核苷酸之间的竞争。乙醇胺和荧光标记的互补寡核苷酸的竞争性结合导致荧光强度依赖于乙醇胺浓度，检测限为10 pM。该方法能够检测小分子，而无需对分析物进行任何标记。竞争性测定可能被转移到其他适体，从而提供了一个有前途的系统，用于基于适体的检测不同的小分子。
Aptamers are promising recognition elements for sensitive and specific detection of small molecules. We have previously selected ssDNA aptamers for ethanolamine, one of the smallest aptamer targets so far. The work presented here focuses on the determination of the binding region within the aptamer structure and its exploitation for the development of an aptamer-based assay for detection of ethanolamine. Sequence analysis of the aptamers resulted in the identification of a G-rich consensus sequence, which was able to fold in a typical two- or three-layered G-quartet structure. Experiments with stepwise truncated variants of the aptamers revealed that the consensus sequence is responsible and sufficient for binding to the target. On the basis of the knowledge of the aptamers binding site, we developed an aptamer-based microarray assay relying on competition between ethanolamine and an oligonucleotide complementary to the consensus sequence. Competitive binding of ethanolamine and fluorescently labeled complementary oligonucleotides resulted in fluorescence intensities dependent on ethanolamine concentration with a limit of detection of 10 pM. This method enables detection of small molecules without any labeling of analytes. The competitive assay could potentially be transferred to other aptamers and thus provides a promising system for aptamer-based detection of diverse small molecules.