Analytical and Bioanalytical Chemistry ( IF 3.8 ) Pub Date : 2021-06-10 , DOI: 10.1007/s00216-021-03444-y Annelies Dillen 1 , Wouter Vandezande 2 , Devin Daems 3 , Jeroen Lammertyn 1
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Duplexed aptamers (DAs) are widespread aptasensor formats that simultaneously recognize and signal the concentration of target molecules. They are composed of an aptamer and aptamer complementary element (ACE) which consists of a short oligonucleotide that partially inhibits the aptamer sequence. Although the design principles to engineer DAs are straightforward, the tailored development of DAs for a particular target is currently based on trial and error due to limited knowledge of how the ACE sequence affects the final performance of DA biosensors. Therefore, we have established a thermodynamic model describing the influence of the ACE on the performance of DAs applied in equilibrium assays and demonstrated that this relationship can be described by the binding strength between the aptamer and ACE. To validate our theoretical findings, the model was applied to the 29-mer anti-thrombin aptamer as a case study, and an experimental relation between the aptamer-ACE binding strength and performance of DAs was established. The obtained results indicated that our proposed model could accurately describe the effect of the ACE sequence on the performance of the established DAs for thrombin detection, applied for equilibrium assays. Furthermore, to characterize the binding strength between the aptamer and ACEs evaluated in this work, a set of fitting equations was derived which enables thermodynamic characterization of DNA-based interactions through thermal denaturation experiments, thereby overcoming the limitations of current predictive software and chemical denaturation experiments. Altogether, this work encourages the development, characterization, and use of DAs in the field of biosensing.
Graphical abstract
中文翻译：
阐明适体互补元件对双链体适体性能的影响：热力学研究
双链适体 (DA) 是广泛使用的适体传感器格式，可同时识别目标分子的浓度并发出信号。它们由适体和适体互补元件 (ACE) 组成，ACE 由部分抑制适体序列的短寡核苷酸组成。尽管设计 DA 的设计原则很简单，但由于对 ACE 序列如何影响 DA 生物传感器的最终性能的了解有限，因此针对特定目标的 DA 的定制开发目前基于反复试验。因此，我们建立了一个热力学模型，描述了 ACE 对应用于平衡测定的 DA 性能的影响，并证明了这种关系可以通过适体和 ACE 之间的结合强度来描述。为了验证我们的理论发现，将该模型应用于29-mer抗凝血酶适体作为案例研究，建立了适体-ACE结合强度与DAs性能之间的实验关系。获得的结果表明，我们提出的模型可以准确地描述 ACE 序列对已建立的用于凝血酶检测的 DA 性能的影响，适用于平衡测定。此外，为了表征在这项工作中评估的适体和 ACE 之间的结合强度，推导出了一组拟合方程，通过热变性实验能够对基于 DNA 的相互作用进行热力学表征，从而克服当前预测软件和化学变性实验的局限性. 总之，这项工作鼓励发展、表征、
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