Target-mediated assembly formation of multi-arm DNAzyme nanostructures for sensitive and accurate discrimination of single-nucleotide polymorphism in K-ras gene
Target-mediated assembly formation of multi-arm DNAzyme nanostructures for sensitive and accurate discrimination of single-nucleotide polymorphism in K-ras gene
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DOI：
10.1016/j.snb.2021.130535
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发表时间：
2021-11
影响因子：
8.4
通讯作者：
Jialun He;Xiaolong Li;Lei Liao;Wenjiao Zhou;Bingying Jiang
中科院分区：
文献类型：
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作者：
Jialun He;Xiaolong Li;Lei Liao;Wenjiao Zhou;Bingying Jiang
单核苷酸多态(SNP)是一种典型的遗传变异方式，常被用作指示人类各种疾病的遗传标记。在此，我们报道了一种非酶和无标记的检测方法，用于准确和超灵敏地区分K-ras基因中的SNP。靶突变体K-ras基因(MtDNA)的存在导致暴露于末端的“三向连接”(3WJ)结构的形成，从而引发末端链置换反应(TSDR)形成多臂DNAzyme纳米结构。这些DNAzyme进一步循环切割发夹DNA底物，释放许多富含G的片段，与有机染料硫代黄素T(THT)相互作用，产生显着增强的荧光，用于mtDNA的敏感识别。然而，野生型K-ras基因(WtDNA)的存在触发了3WJ结构上的碱基配对框架移动，导致脚趾部位关闭，从而既没有启动TSDR，也没有启动DNAzyme辅助的切割反应，没有观察到明显的荧光信号。重要的是，该方法具有很高的灵敏度，检测下限为3.63fM，对wtDNA和mtDNA浓度比高达5000：1的混合物中的mtDNA具有很高的选择性。此外，该方法还可以用于分析稀释的血清样品中的靶mtDNA，这表明它在未来的临床诊断中具有潜在的应用前景。
Single-nucleotide polymorphism (SNP) is a typical way of genetic variation and commonly used as genetic marker to indicate various types of human diseases. We herein report a non-enzymatic and label-free sensing method for accurate and ultrasensitive differentiation of SNP in K-ras gene. The presence of target mutant K-ras gene (MtDNA) leads to the formation of “three-way junctions” (3WJ) structure with an exposure toehold site, which initiates the toehold strand displacement reaction (TSDR) to form multi-arm DNAzyme nanostructures. These DNAzymes further cyclically cleave the hairpin DNA substrates and release many G-rich fragments to interact with organic dye thioflavin T (ThT) and yield significantly enhanced fluorescence for sensitive discrimination of MtDNA. However, the presence of wild K-ras gene (WtDNA) trigger a base pairing frame shift on the 3WJ structure and result in the closure of the toehold site, thus neither TSDR nor DNAzyme-assistant cleavage reaction is initiated, and no obvious fluorescent signal is observed. Importantly, this strategy shows high sensitivity with a detection limit of 3.63 fM and exhibits high selectivity to discriminate MtDNA from a mixture with a concentration ratio of WtDNA to MtDNA as high as 5000:1. Moreover, this approach can be used to analyze target MtDNA in diluted serum samples, which demonstrates its potential application for future clinical diagnostics.