Zhang Zijie, Oni Olatunji, Liu Juewen
Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
Nucleic Acids Res. 2017 Jul 27;45(13):7593-7601. doi: 10.1093/nar/gkx517.
The DNA aptamer for adenosine (also for AMP and ATP) is a highly conserved sequence that has recurred in a few selections. It it a widely used model aptamer for biosensor development, and its nuclear magnetic resonance structure shows that each aptamer binds two AMP molecules. In this work, each binding site was individually removed by rational sequence design, while the remaining site still retained a similar binding affinity and specificity as confirmed by isothermal titration calorimetry. The thermodynamic parameters of binding are presented, and its biochemical implications are discussed. The number of binding sites can also be increased, and up to four sites are introduced in a single DNA sequence. Finally, the different sequences are made into fluorescent biosensors based on the structure-switching signaling aptamer design. The one-site aptamer has 3.8-fold higher sensitivity at lower adenosine concentration with a limit of detection of 9.1 μM adenosine, but weaker fluorescence signal at higher adenosine concentrations, consistent with a moderate cooperativity in the original aptamer. This work has offered insights into a classic aptamer for the relationship between the number of binding sites and sensitivity, and a shorter aptamer for improved biosensor design.
用于腺苷（也适用于AMP和ATP）的DNA适配体是一种高度保守的序列，在几次筛选中反复出现。它是生物传感器开发中广泛使用的模型适配体，其核磁共振结构表明每个适配体结合两个AMP分子。在这项工作中，通过合理的序列设计分别去除了每个结合位点，同时通过等温滴定量热法证实，剩余的位点仍保留相似的结合亲和力和特异性。给出了结合的热力学参数，并讨论了其生化意义。结合位点的数量也可以增加，在单个DNA序列中最多可引入四个位点。最后，基于结构转换信号适配体设计将不同序列制成荧光生物传感器。单位点适配体在较低腺苷浓度下灵敏度高3.8倍，腺苷检测限为9.1 μM，但在较高腺苷浓度下荧光信号较弱，这与原始适配体中的适度协同性一致。这项工作为经典适配体中结合位点数量与灵敏度之间的关系提供了见解，并为改进生物传感器设计提供了更短的适配体。
