School of Environmental Science and Technology, Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), Dalian POCT Laboratory, Dalian University of Technology, Dalian, Liaoning 116024, China.
Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S4K1, Canada.
J Am Chem Soc. 2023 Feb 1;145(4):2630-2637. doi: 10.1021/jacs.2c12666. Epub 2023 Jan 19.
Functional nucleic acids (FNAs), such as DNAzymes and DNA aptamers, can be engineered into circular forms for improved performance. Circular FNAs are promising candidates for bioanalytical and biomedical applications due to their intriguing properties of enhanced biological stability and compatibility with rolling circle amplification. They are typically made from linear single-stranded (ss) DNA molecules via ligase-mediated ligation. However, it remains a great challenge to synthesize circular ssDNA molecules in high yield due to inherent side reactions where two or more of the same ssDNA molecules are ligated. Herein, we present a strategy to overcome this issue by first using in vitro selection to search from a random-sequence DNA library a ligatable DNA aptamer that binds a DNA ligase and then by engineering this aptamer into a general-purpose templating DNA scaffold to guide the ligase to execute selective intramolecular circularization. We demonstrate the broad utility of this approach via the creation of several species of circular DNA molecules, including a circular DNAzyme sensor for a bacterium and a circular DNA aptamer sensor for a protein target with excellent detection sensitivity and specificity.
功能核酸（FNAs），如 DNA 酶和 DNA 适体，可以被工程化为环状形式以提高性能。由于其增强的生物稳定性和与滚环扩增的兼容性等有趣特性，环状 FNAs 是生物分析和生物医学应用的有前途的候选物。它们通常是通过连接酶介导的连接从线性单链（ss）DNA 分子制成的。然而，由于两个或更多相同的 ssDNA 分子发生连接等固有副反应，以高产率合成环状 ssDNA 分子仍然是一个巨大的挑战。在此，我们提出了一种策略来克服这个问题，首先使用体外选择从随机序列 DNA 文库中搜索与 DNA 连接酶结合的可连接 DNA 适体，然后将该适体工程化为通用模板 DNA 支架，以引导连接酶执行选择性的分子内环化。我们通过创建几种环状 DNA 分子来证明这种方法的广泛适用性，包括用于细菌的环状 DNA 酶传感器和用于蛋白质靶标的环状 DNA 适体传感器，具有出色的检测灵敏度和特异性。
