Absolute and Direct MicroRNA Quantification Using DNA-Gold Nanoparticle Probes
Absolute and Direct MicroRNA Quantification Using DNA-Gold Nanoparticle Probes
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DOI：
10.1021/ja412152x
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发表时间：
2014-02-12
影响因子：
15
通讯作者：
Fiammengo, Roberto
中科院分区：
文献类型：
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作者：
Degliangeli, Federica;Kshirsagar, Prakash;Fiammengo, Roberto
DNA-金纳米颗粒探针以简单的策略实现，用于直接microRNA（miRNA）定量。将荧光标记的DNA探针链固定在聚乙二醇化的金纳米颗粒（AuNP）上。在靶miRNA的存在下，DNA-RNA异源双链体形成并成为内切核酸酶DSN（miRNA特异性核酸酶）的底物。DNA链的酶促水解由于荧光团远离金表面的扩散而产生荧光信号。我们表明，我们的DNA-AuNP探针的分子设计，与固定在基于PEG的钝化层的顶部上的DNA链，结果在几乎不变的酶活性对固定的异源双链体相比，在溶液中的基板自由。以实时格式开发的该测定法允许对低至0.2 fmol的miR-203进行绝对定量。我们还展示了该测定法在从细胞培养物提取的总RNA样品中直接定量癌症相关miR-203和miR-21的应用。直接和绝对定量的可能性可能会显着推进microRNA作为生物标志物在临床实践中的使用。
DNA-gold nanoparticle probes are implemented in a simple strategy for direct microRNA (miRNA) quantification. Fluorescently labeled DNA-probe strands are immobilized on PEGylated gold nanoparticles (AuNPs). In the presence of target miRNA, DNA-RNA heteroduplexes are formed and become substrate for the endonuclease DSN (duplex-specific nuclease). Enzymatic hydrolysis of the DNA strands yields a fluorescence signal due to diffusion of the fluorophores away from the gold surface. We show that the molecular design of our DNA-AuNP probes, with the DNA strands immobilized on top of the PEG-based passivation layer, results in nearly unaltered enzymatic activity toward immobilized heteroduplexes compared to substrates free in solution. The assay, developed in a real-time format, allows absolute quantification of as little as 0.2 fmol of miR-203. We also show the application of the assay for direct quantification of cancer-related miR-203 and miR-21 in samples of extracted total RNA from cell cultures. The possibility of direct and absolute quantification may significantly advance the use of microRNAs as biomarkers in the clinical praxis.