Research Center for Bioengineering and Sensing Technology, Beijing Key Lab for Bioengineering and Sensing Technology, School of Chemistry and bioengineering, University of Science & Technology Beijing, Beijing 100083, P.R. China.
School of Petrochemical Engineering, School of Food Science and Technology, Changzhou University, Changzhou 213164, P.R. China.
Theranostics. 2018 Apr 3;8(10):2646-2656. doi: 10.7150/thno.24480. eCollection 2018.
As important modulators of gene expression, microRNAs (miRNAs) have been identified as promising biomarkers with powerful predictive value in diagnosis and prognosis for several diseases, especially for cancers. Here we report a facile, multiple and sensitive miRNA detection method that uses conventional gel electrophoresis and catalytic hairpin assembly (CHA) system without any complex nanomaterials or enzymatic amplification. In this study, three pairs of hairpin probes are rationally designed with thermodynamically and kinetically preferable feasibility for the CHA process. In the present of target miRNA, the stem of the corresponding hairpin detection probe (HDP) will be unfolded and expose the concealed domain. The corresponding hairpin assistant probe (HAP) then replaces the hybridized target miRNA to form specific HDP/HAP complexes and releases miRNA based on thermodynamically driven entropy gain process, and the released miRNA triggers the next recycle to produce tremendous corresponding HDP/HAP complexes. The results showed that the CHA gel assay can detect miRNA at fM levels and shows good capability of discriminating miRNA family members and base-mismatched miRNAs. It is able to analyze miRNAs extracted from cell lysates, which are consistent with the results of conventional polymerase chain reaction (PCR) method. Depending on the length of the designed hairpin probes, the CHA gel assay consisting of different hairpin probes effectively discriminated and simultaneously detected multiple miRNAs in homogenous solution and miRNAs extracted from cell lysates. The work highlights the practical use of a conventional gel electrophoresis for sensitive interesting nucleic acid sequences detection.
作为基因表达的重要调节剂，microRNAs（miRNAs）已被确定为具有强大预测价值的有前途的生物标志物，可用于多种疾病的诊断和预后，尤其是癌症。在这里，我们报告了一种简单、多重且敏感的 miRNA 检测方法，该方法使用常规凝胶电泳和催化发夹组装（CHA）系统，而无需任何复杂的纳米材料或酶放大。在本研究中，我们合理设计了三对发夹探针，以具有 CHA 过程热力学和动力学上优选的可行性。在靶 miRNA 的存在下，相应的发夹检测探针（HDP）的茎将展开并暴露隐藏的结构域。相应的发夹辅助探针（HAP）然后取代杂交的靶 miRNA，形成特异性的 HDP/HAP 复合物，并基于热力学驱动的熵增益过程释放 miRNA，释放的 miRNA 触发下一次循环以产生大量相应的 HDP/HAP 复合物。结果表明，CHA 凝胶分析可在 fM 水平检测 miRNA，并显示出良好的区分 miRNA 家族成员和碱基错配 miRNA 的能力。它能够分析从细胞裂解物中提取的 miRNA，这与常规聚合酶链反应（PCR）方法的结果一致。根据设计的发夹探针的长度，由不同发夹探针组成的 CHA 凝胶分析能够有效区分和同时检测同质溶液中和细胞裂解物中提取的多个 miRNA。这项工作突出了常规凝胶电泳在敏感核酸序列检测中的实际应用。
