Department of Chemistry, Liaocheng University, Liaocheng, China.
Department of Bio-industrial Mechatronics Engineering, National Chung Hsing University, Taiwan.
Nanomedicine. 2021 Feb;32:102339. doi: 10.1016/j.nano.2020.102339. Epub 2020 Nov 21.
MicroRNA (miRNA) has emerged as a promising genetic marker for cancer diagnosis and therapy because its expression level is closely related to the progression of malignant diseases. Herein, a label-free and selective fluorescence platform was proposed for miRNA based on light-up "G-quadruplex nanostring" via duplex-specific nuclease (DSN) mediated tandem rolling circle amplification (RCA). First, a long DNA generated from upstream RCA was designed with the antisense sequences for miR-21 and downstream RCA primer. Upon recognizing miR-21, the resulting DNA-RNA permitted DSN digestion and triggered downstream two-way RCA, and generation of abundant "G-quadruplex nanostring" binding with ZnPPIX for label-free fluorescent responses. In our strategy, the strong preference of DSN for perfectly matched DNA/RNA ensures its excellent selectivity. The developed method generated wide linear response with LOD of 1.019 fM. Additionally, the miR-21 levels in cell extracts have been evaluated, revealing the utility of this tool for biomedical research and clinical diagnosis.
微 RNA（miRNA）已成为癌症诊断和治疗有前途的遗传标志物，因为其表达水平与恶性疾病的进展密切相关。在此，我们提出了一种基于无标记和选择性荧光平台的 miRNA 检测方法，该方法通过双链特异性核酸酶（DSN）介导的串联滚环扩增（RCA）利用了点亮的“G-四链体纳米串”。首先，设计了一条长 DNA，其上游 RCA 产生的具有 miR-21 的反义序列和下游 RCA 引物。当识别出 miR-21 时，所得的 DNA-RNA 允许 DSN 消化并触发下游双向 RCA，并产生大量与 ZnPPIX 结合的“G-四链体纳米串”用于无标记荧光响应。在我们的策略中，DSN 对完全匹配的 DNA/RNA 的强烈偏好确保了其出色的选择性。该方法具有较宽的线性响应范围，LOD 为 1.019 fM。此外，还评估了细胞提取物中的 miR-21 水平，显示了该工具在生物医学研究和临床诊断中的应用。
