Label-free detection of nucleic acids by turn-on and turn-off G-quadruplex-mediated fluorescence
Label-free detection of nucleic acids by turn-on and turn-off G-quadruplex-mediated fluorescence
复制标题
通过打开和关闭 G-四链体介导的荧光对核酸进行无标记检测
DOI：
10.1007/s00216-011-4669-0
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发表时间：
2011-03-01
影响因子：
4.3
通讯作者：
Wang, Jin
中科院分区：
文献类型：
--
作者：
Ren, Jiangtao;Qin, Haixia;Wang, Jin
在这项研究中，我们使用了两种荧光探针，四（二异丙基胍）-锌-酞菁（Zn-DIGP）和N-甲基中卟啉IX（NMM），来监测“分裂”G-四联体探针与任意“目标”DNA杂交时的重组。根据这种方法，每个分裂探针被设计成含有一半的G-四链体形成序列，该序列与与靶DNA互补的可变序列融合。混合各个组分后，碱基配对相互作用和 G-四链体片段重新组装会产生双链体-四链体三向连接，该连接可以以 G-四链体特异性方式与荧光染料结合。所得复合物的总体荧光强度取决于双链体区域中正确碱基配对相互作用的形成以及荧光探针的确切身份。与缺乏任何“目标”DNA 的样品相比，添加目标 DNA 后，含有 Zn-DIGP 的样品的荧光强度较低，而含有 NMM 的样品的荧光强度较高。因此，基于 Zn-DIGP 的生物传感器被称为“关闭”，而含有 NMM 的生物传感器被定义为“开启”。这两种生物传感器都可以检测目标 DNA，检测限在纳摩尔范围内，并且可以区分不匹配的目标 DNA 和完全匹配的目标 DNA。与之前基于血红素结合分裂 G-四链体探针的过氧化物酶活性的生物传感器相比，荧光染料的使用消除了对不稳定传感成分（H2O2、血红素和 ABTS）的需要。我们的方法直接、易于实施，并且与生物体液中特定 DNA 序列的检测完全兼容。拥有两种不同类型的探针在应用研究中非常有价值，因为 Zn-DIGP 被发现与含有血清和尿液的样品兼容，而 NMM 与尿液兼容，但与含有血清的样品不兼容。
In this study we have used two fluorescent probes, tetrakis(diisopropylguanidino)-zinc-phthalocyanine (Zn-DIGP) and N-methylmesoporphyrin IX (NMM), to monitor the reassembly of "split" G-quadruplex probes on hybridization with an arbitrary "target" DNA. According to this approach, each split probe is designed to contain half of a G-quadruplex-forming sequence fused to a variable sequence that is complementary to the target DNA. Upon mixing the individual components, both base-pairing interactions and G-quadruplex fragment reassembly result in a duplex-quadruplex three-way junction that can bind to fluorescent dyes in a G-quadruplex-specific way. The overall fluorescence intensities of the resulting complexes were dependent on the formation of proper base-pairing interactions in the duplex regions, and on the exact identity of the fluorescent probe. Compared with samples lacking any "target" DNA, the fluorescence intensities of Zn-DIGP-containing samples were lower, and the fluorescence intensities of NMM-containing samples were higher on addition of the target DNA. The resulting biosensors based on Zn-DIGP are therefore termed "turn-off" whereas the biosensors containing NMM are defined as "turn-on". Both of these biosensors can detect target DNAs with a limit of detection in the nanomolar range, and can discriminate mismatched from perfectly matched target DNAs. In contrast with previous biosensors based on the peroxidase activity of heme-bound split G-quadruplex probes, the use of fluorescent dyes eliminates the need for unstable sensing components (H2O2, hemin, and ABTS). Our approach is direct, easy to conduct, and fully compatible with the detection of specific DNA sequences in biological fluids. Having two different types of probe was highly valuable in the context of applied studies, because Zn-DIGP was found to be compatible with samples containing both serum and urine whereas NMM was compatible with urine, but not with serum-containing samples.