Wei Li, Shuaijing Wang, Haotian Zong, Jiayue Li, Yi Zhou, Zhenguang Wang
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引用次数: 0
摘要
尿嘧啶-DNA 糖基化酶(UDG)在清除受损的尿嘧啶碱基,从而维护遗传稳定性和完整性方面发挥着至关重要的作用。为了检测 UDG 的活性,我们设计了一种以酶为动力的无标记 DNA 步行器。最初,我们设计了一种无标记 DNA 轨道,其中包含一个金纳米粒子(AuNP)、多个发夹结构和各种摆臂,用于行走机构。发夹结构经过精心设计,包含一个 G-四联序列,能够产生无标记荧光信号。在没有 UDG 的情况下,摆臂保持惰性,但一旦引入 UDG,摆臂就会被激活,从而启动由酶驱动的行走过程,并产生显著的解离 G-四联序列。通过将选择性荧光染料整合到设计中,实现了增强的无标记荧光响应。所提出的 DNA 步行器是一种直接、无标记的 UDG 检测方法,灵敏度极高,检测限为 0.00004 U/mL。以尿嘧啶糖基化酶抑制剂(UGI)为抑制模型,进行了抑制剂检测,精度令人满意。此外,还成功完成了单细胞水平的细胞 UDG 分析。因此,所开发的 DNA 步行器是一种无标记、选择性和灵敏的 UDG 活性评估工具,在疾病诊断、抑制剂筛选和生物医学研究方面具有巨大的应用潜力。
Enzyme-Powered, Label-Free DNA Walker for Uracil-DNA Glycosylase Detection at Single-Cell Level.
Uracil-DNA glycosylase (UDG) plays a crucial role in the removal of damaged uracil bases, thereby upholding genetic stability and integrity. An enzyme-powered, label-free DNA walker was devised for UDG activity detection. Initially, a label-free DNA track, incorporating a gold nanoparticle (AuNP), multiple hairpin structures, and various swing arms, was engineered for walking mechanism. The hairpin structure was meticulously crafted to include a G-quadruplex sequence, enabling the generation of a label-free fluorescence signal. The swing arm remained inert in the absence of UDG, but became activated upon the introduction of UDG, thereby initiating the enzyme-powered walking process and generating significant dissociative G-quadruplex sequences. By integrating a selective fluorescent dye into the design, an enhanced label-free fluorescence response was achieved. The proposed DNA walker presented a direct and label-free approach for UDG detection, demonstrating exceptional sensitivity with a detection limit of 0.00004 U/mL. Using the uracil glycosylase inhibitor (UGI) as an inhibitory model, inhibitor assay was conducted with satisfactory precision. Furthermore, successful analysis of cellular UDG at the single-cell level was accomplished. Consequently, the developed DNA walker serves as a label-free, selective, and sensitive tool for UDG activity assessment, showing great potential for applications in disease diagnosis, inhibitor screening, and biomedical investigations.
期刊介绍:
Chemistry—An Asian Journal is an international high-impact journal for chemistry in its broadest sense. The journal covers all aspects of chemistry from biochemistry through organic and inorganic chemistry to physical chemistry, including interdisciplinary topics.
Chemistry—An Asian Journal publishes Full Papers, Communications, and Focus Reviews.
A professional editorial team headed by Dr. Theresa Kueckmann and an Editorial Board (headed by Professor Susumu Kitagawa) ensure the highest quality of the peer-review process, the contents and the production of the journal.
Chemistry—An Asian Journal is published on behalf of the Asian Chemical Editorial Society (ACES), an association of numerous Asian chemical societies, and supported by the Gesellschaft Deutscher Chemiker (GDCh, German Chemical Society), ChemPubSoc Europe, and the Federation of Asian Chemical Societies (FACS).