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A Ligation-Triggered DNAzyme Cascade for Amplified Fluorescence Detection of Biological Small Molecules with Zero-Background Signal

化学 级联 荧光 结扎 脱氧核酶 信号(编程语言) 生物物理学 DNA 分子生物学 生物化学 色谱法 物理 光学 计算机科学 生物 程序设计语言
作者
Limin Lu,Xiaobing Zhang,Rongmei Kong,Bin Yang,Weihong Tan
出处
期刊:Journal of the American Chemical Society [American Chemical Society]
卷期号:133 (30): 11686-11691 被引量:222
标识
DOI:10.1021/ja203693b
摘要

Many types of fluorescent sensing systems have been reported for biological small molecules. Particularly, several methods have been developed for the recognition of ATP or NAD+, but they only show moderate sensitivity, and they cannot discriminate either ATP or NAD+ from their respective analogues. We have addressed these limitations and report here a dual strategy which combines split DNAzyme-based background reduction with catalytic and molecular beacon (CAMB)-based amplified detection to develop a ligation-triggered DNAzyme cascade, resulting in ultrahigh sensitivity. First, the 8-17 DNAzyme is split into two separate oligonucleotide fragments as the building blocks for the DNA ligation reaction, thereby providing a zero-background signal to improve overall sensitivity. Next, a CAMB strategy is further employed for amplified signal detection achieved through cycling and regenerating the DNAzyme to realize the true enzymatic multiple turnover (one enzyme catalyzes the cleavage of several substrates) of catalytic beacons. This combination of zero-background signal and signal amplification significantly improves the sensitivity of the sensing systems, resulting in detection limits of 100 and 50 pM for ATP and NAD+, respectively, much lower than those of previously reported biosensors. Moreover, by taking advantage of the highly specific biomolecule-dependence of the DNA ligation reaction, the developed DNAzyme cascades show significantly high selectivity toward the target cofactor (ATP or NAD+), and the target biological small molecule can be distinguished from its analogues. Therefore, as a new and universal platform for the design of DNA ligation reaction-based sensing systems, this novel ligation-triggered DNAzyme cascade method may find a broad spectrum of applications in both environmental and biomedical fields.
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