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A light-up “G-quadruplex nanostring” for label-free and selective detection of miRNA via duplex-specific nuclease mediated tandem rolling circle amplification
Nanomedicine: Nanotechnology, Biology and Medicine ( IF 4.2 ) Pub Date : 2020-11-21 , DOI: 10.1016/j.nano.2020.102339
Li-qi Liu, Fei Yin, Yu Lu, Xi-luan Yan, Ching-Chou Wu, Xia Li, Chenzhong Li

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.



中文翻译:

用于通过双链特异性核酸酶介导的串联滚环扩增无标记和选择性检测 miRNA 的发光“G-四链体纳米线”

MicroRNA(miRNA)由于其表达水平与恶性疾病的进展密切相关,已成为一种有前景的癌症诊断和治疗遗传标志物。在此,基于通过双链特异性核酸酶(DSN)介导的串联滚环扩增(RCA)点亮“G-四链体纳米线”的miRNA,提出了一种无标记和选择性荧光平台。首先,从上游 RCA 产生的长 DNA 被设计为具有 miR-21 和下游 RCA 引物的反义序列。在识别 miR-21 后,产生的 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-四链体纳米线”以进行无标记荧光反应。在我们的策略中,DSN 对完美匹配的 DNA/RNA 的强烈偏好确保了其出色的选择性。开发的方法产生了 LOD 为 1.019 fM 的宽线性响应。此外,还评估了细胞提取物中的 miR-21 水平,揭示了该工具在生物医学研究和临床诊断中的实用性。

更新日期:2020-12-05
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