Xue Chang, Luo Mengxue, Wang Lei, Li Congcong, Hu Shuyao, Yu Xin, Yuan Pei, Wu Zai-Sheng
College of Chemical Engineering, Cancer Metastasis Alert and Prevention Center, Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, 350108, China.
Hunan Provincial Key Laboratory of Phytohormones and Growth Development, College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China.
Anal Chem. 2021 Jul 20;93(28):9869-9877. doi: 10.1021/acs.analchem.1c01856. Epub 2021 Jul 7.
DNAzymes with enzymatic activity identified from random DNA pools by selection have recently attracted considerable attention. In this work, a DNAzyme-based autonomous-motion (AM) molecular machine is demonstrated for sensitive simultaneous imaging of different intracellular microRNAs (miRNAs). The AM molecular machine consists of two basic elements, one of which is a target-analogue-embedded double-stem hairpin substrate (TDHS) and the other is a locking-strand-silenced DNAzyme (LSDz). LSDz can be activated by target miRNA and catalytically cleave TDHS, generating Clv-TDHS and releasing free target analogue capable of triggering the next round of cleavage reaction. As such, the molecular machine can exert sustainable autonomous operation, producing an enhanced signal. Because the active target analogue comes from the machine itself and offers cyclical stimulation in a feedback manner, this target-induced autonomous cleavage circuit is termed a self-feedback circuit (SFC). The SFC-based molecular machine can be used to quantify miRNA-21 down to 10 pM without interference from nontarget miRNAs, indicating a substantial improvement in assay performance compared with its counterpart system without an SFC effect. Moreover, due to the enzyme-free process, the AM molecular machine is suitable for miRNA imaging in living cells, and the quantitative results are consistent with the gold standard PCR assay. More interestingly, the AM molecular machine can be used for the simultaneous fluorescence imaging of several intracellular miRNAs, enabling the accurate discrimination of cancerous cells (e.g., HeLa and MCF-7) from healthy cells. The SFC-based autonomous-motion machine is expected to be a promising tool for the research of molecular biology and early diagnosis of human diseases.
通过筛选从随机DNA文库中鉴定出的具有酶活性的DNAzyme最近引起了广泛关注。在这项工作中，展示了一种基于DNAzyme的自主运动（AM）分子机器，用于对不同细胞内微小RNA（miRNA）进行灵敏的同时成像。该AM分子机器由两个基本元件组成，其中一个是嵌入靶标类似物的双链发夹底物（TDHS），另一个是锁定链沉默的DNAzyme（LSDz）。LSDz可被靶标miRNA激活，并催化切割TDHS，产生Clv-TDHS并释放能够触发下一轮切割反应的游离靶标类似物。因此，该分子机器可以实现可持续的自主运行，产生增强的信号。由于活性靶标类似物来自机器本身并以反馈方式提供循环刺激，这种靶标诱导的自主切割回路被称为自反馈回路（SFC）。基于SFC的分子机器可用于将miRNA-21定量至10 pM，而不受非靶标miRNA的干扰，这表明与没有SFC效应的对应系统相比，检测性能有了显著提高。此外，由于无酶过程，AM分子机器适用于活细胞中的miRNA成像，定量结果与金标准PCR检测一致。更有趣的是，AM分子机器可用于同时对几种细胞内miRNA进行荧光成像，从而能够准确地区分癌细胞（如HeLa和MCF-7）与健康细胞。基于SFC的自主运动机器有望成为分子生物学研究和人类疾病早期诊断的有前途的工具。
