Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University , Chongqing 400715, P. R. China.
Anal Chem. 2017 Sep 19;89(18):9934-9940. doi: 10.1021/acs.analchem.7b02247. Epub 2017 Aug 25.
The detection of specific intracellular microRNAs (miRNAs) in living cells can potentially provide insight into the causal mechanism of cancer metastasis and invasion. However, because of the characteristic nature of miRNAs in terms of small sizes, low abundance, and similarity among family members, it is a great challenge to monitor miRNAs in living cells, especially those with much lower expression levels. In this work, we describe the establishment of a DNA-fueled and catalytic molecule machinery in cell signal amplification approach for monitoring trace and under-expressed miRNAs in living cells. The presence of the target miRNA releases the hairpin sequences from the dsDNA (containing the fluorescence resonance energy transfer (FRET) pair-labeled and unfolded hairpin sequences)-conjugated gold nanoparticles (dsDNA-AuNPs), and the DNA fuel strands assist the recycling of the target miRNA sequences via two cascaded strand displacement reactions, leading to the operation of the molecular machine in a catalytic fashion and the release of many hairpin sequences. As a result, the liberated hairpin sequences restore the folded hairpin structures and bring the FRET pair into close proximity to generate significantly amplified signals for detecting trace miRNA targets. Besides, the dsDNA-AuNP nanoprobes have good nuclease stability and show low cytotoxicity to cells, and the application of such a molecular system for monitoring trace and under-expressed miRNAs in living cells has also been demonstrated. With the advantages of in cell signal amplification and reduced background noise, the developed method thus offers new opportunities for detecting various trace intracellular miRNA species.
在活细胞中检测特定的细胞内 microRNA（miRNA）可能为癌症转移和侵袭的因果机制提供深入的了解。然而，由于 miRNA 在大小、丰度和家族成员之间的相似性方面的特征性质，因此监测活细胞中的 miRNA，尤其是那些表达水平较低的 miRNA，是一个巨大的挑战。在这项工作中，我们描述了一种在细胞信号放大方法中建立 DNA 驱动和催化分子机器的方法，用于监测活细胞中痕量和低表达的 miRNA。目标 miRNA 的存在从 dsDNA（包含荧光共振能量转移（FRET）对标记和展开的发夹序列）-缀合的金纳米粒子（dsDNA-AuNPs）上释放发夹序列，并且 DNA 燃料链通过两个级联的链置换反应辅助目标 miRNA 序列的循环，导致分子机器以催化方式操作并释放许多发夹序列。结果，释放的发夹序列恢复折叠的发夹结构，并使 FRET 对靠近以产生用于检测痕量 miRNA 靶标的显著放大信号。此外，dsDNA-AuNP 纳米探针具有良好的核酸酶稳定性，对细胞的细胞毒性低，并且已经证明了这种分子系统在活细胞中监测痕量和低表达的 miRNA 的应用。该方法具有细胞内信号放大和降低背景噪声的优势，因此为检测各种痕量细胞内 miRNA 物种提供了新的机会。
