Novel Amplex Red Oxidases Based on Noncanonical DNA Structures: Property Studies and Applications in MicroRNA Detection
Novel Amplex Red Oxidases Based on Noncanonical DNA Structures: Property Studies and Applications in MicroRNA Detection
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基于非典型 DNA 结构的新型 Amplex 红色氧化酶：特性研究及其在 MicroRNA 检测中的应用
DOI：
10.1021/ac402535a
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发表时间：
2014-03-18
影响因子：
7.4
通讯作者：
Zhou, Xiang
中科院分区：
文献类型：
--
作者：
Wang, Shaoru;Fu, Boshi;Zhou, Xiang
g -三重体是近年来在富g序列中发现的一种新的二级结构。然而，它的功能和生物学作用在很大程度上仍然未知。本研究首先开发了两种Amplex红色氧化酶，一种是基于较新的g -三联体结构，另一种是基于常见的g -四联体结构。采用圆二色性（CD）光谱法、CD熔化法和紫外滴定法等DNA结合分析方法测定G3低聚物的g -三聚体结构。g -三聚体和g -四聚体显著增强了血红蛋白的内在氧化活性。仅G3寡聚物或G4寡聚物的一个关键鸟嘌呤缺失就会导致Amplex Red氧化活性大幅降低。据我们所知，这是第一个报告直接使用空气作为基于DNAzyme策略产生荧光的氧化剂的病例。进一步的机理研究表明，O-2和水在现场产生H2O2，随后Amplex Red氧化为再间酚，导致荧光增强。此外，新开发的氧化酶仅使用一种生物素标记探针和一种小分子底物就能有效地用于microRNA检测。目标DNA与g -三联体或g -四联体形成序列结合，在少量miRNA存在的情况下通过核酸内切酶产生g -三联体或g -四联体，并放大Amplex红氧化产生的荧光信号。我们发现的新型Amplex红色氧化酶具有广泛的应用前景。
G-triplex has recently been identified as a new secondary structure in G-rich sequences. However, its functions and biological roles remain largely unknown. This study first developed two kinds of Amplex Red oxidases, which were based on relatively new G-triplex structure and a common G-quadruplex one. A collection of DNA binding assays including circular dichroism (CD) spectroscopy, a CD melting assay, and a UV titration study were used to determine the G-triplex structure of G3 oligomer. The low intrinsic oxidative activity of hemin was significantly enhanced using G-triplex or G-quadruplex. Only one key guanine deletion from the G3 oligomer or G4 one could result in a much decreased Amplex Red oxidation activity. To the best of our knowledge, this is the first case reporting direct use of air as the oxidant for fluorescence generation based on DNAzyme strategies. Further mechanism studies demonstrated an involvement of on-site H2O2 generation from O-2 and water and a following oxidation of Amplex Red to resorufin, causing a fluorescence enhancement. Furthermore, the newly developed oxidases have been effectively used in microRNA detection, using only one biotin-labeled probe and one small-molecule substrate. The conjugation of a target DNA to the G-triplex- or G-quadruplex-forming sequence enabled one to produce G-triplex or G-quadruplex by endonuclease in the presence of a slight amount of miRNA and amplify the signal of fluorescence from the oxidation of Amplex Red. Our findings of novel Amplex Red oxidases could potentially be used in a wide range of applications.