Li Yuqing, Liu Biwu, Huang Zhicheng, Liu Juewen
Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo Waterloo Ontario N2L 3G1 Canada
Chem Sci. 2020 Feb 10;11(10):2735-2743. doi: 10.1039/d0sc00086h.
The DNA aptamer for adenosine and ATP has been used as a model system for developing analytical biosensors. For practical reasons, it is important to distinguish adenosine from ATP, although this has yet to be achieved despite extensive efforts made on selection of new aptamers. We herein report a strategy of excising an adenine nucleotide from the backbone of a one-site adenosine aptamer, and the adenine-excised aptamer allowed highly specific binding of adenosine. Cognate analytes including AMP, ATP, guanosine, cytidine, uridine, and theophylline all failed to bind to the engineered aptamer according to the SYBR Green I (SGI) fluorescence spectroscopy and isothermal titration calorimetry (ITC) results. Our A-excised aptamer has two binding sites: the original aptamer binding site in the loop and the newly created one due to base excision from the DNA backbone. ITC demonstrated that the A-excised aptamer strand can bind to two adenosine molecules, with a of 14.8 ± 2.1 μM at 10 °C and entropy-driven binding. Since the wild-type aptamer cannot discriminate adenosine from AMP and ATP, we attributed this improved specificity to the excised site. Further study showed that these two sites worked cooperatively. Finally, the A-excised aptamer was tested in diluted fetal bovine serum and showed a limit of detection of 46.7 μM adenosine. This work provides a facile, cost-effective, and non-SELEX method to engineer existing aptamers for new features and better applications.
用于腺苷和三磷酸腺苷（ATP）的DNA适配体已被用作开发分析生物传感器的模型系统。出于实际原因，区分腺苷和ATP很重要，尽管在选择新的适配体方面已付出巨大努力，但这一目标尚未实现。我们在此报告了一种从单位点腺苷适配体主链上切除腺嘌呤核苷酸的策略，切除腺嘌呤后的适配体对腺苷具有高度特异性结合能力。根据SYBR Green I（SGI）荧光光谱和等温滴定量热法（ITC）的结果，包括一磷酸腺苷（AMP）、ATP、鸟苷、胞苷、尿苷和茶碱在内的同源分析物均无法与工程化适配体结合。我们切除腺嘌呤后的适配体有两个结合位点：环中的原始适配体结合位点和由于从DNA主链上切除碱基而新产生的结合位点。ITC表明，切除腺嘌呤后的适配体链可以结合两个腺苷分子，在10℃时解离常数为14.8±2.1μM，结合由熵驱动。由于野生型适配体无法区分腺苷与AMP和ATP，我们将这种提高的特异性归因于切除的位点。进一步研究表明，这两个位点协同工作。最后，在稀释的胎牛血清中对切除腺嘌呤后的适配体进行了测试，其对腺苷的检测限为46.7μM。这项工作提供了一种简便、经济高效且无需指数富集的配体系统进化技术（SELEX）的方法，用于改造现有适配体以获得新特性和更好的应用。
