DNAzyme-Based Rolling-Circle Amplification DNA Machine for Ultrasensitive Analysis of MicroRNA in Drosophila Larva
DNAzyme-Based Rolling-Circle Amplification DNA Machine for Ultrasensitive Analysis of MicroRNA in Drosophila Larva
复制标题
基于 DNAzyme 的滚环扩增 DNA 机器，用于果蝇幼虫中 MicroRNA 的超灵敏分析
DOI：
10.1021/ac300616z
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发表时间：
2012-09-18
影响因子：
7.4
通讯作者：
Li, Di
中科院分区：
文献类型：
--
作者：
Wen, Yanqin;Xu, Yan;Li, Di
我们提出了一种高灵敏度的microRNA（miRNA）检测方法。该方法基于滚环扩增（RCA）DNA仪，其集成了RCA、切口酶信号放大和DNAzyme信号放大。DNA机器由靶miRNA与合理设计的挂锁DNA模板杂交触发，并由RCA激活。然后，所得的RCA产品自主地复制多个机械切割机循环，并产生累积量的产品。具体来说，本工作中的DNA产物被设计为辣根过氧化物酶（HRP）模拟DNA酶，它可以催化比色反应并产生有色产物。通过这些级联扩增，可以检测到低至2aM的microRNA（miRNA）。作为体内应用的一个例子，成功地分析了来自单个果蝇幼虫的miRNA。果蝇是一种模式生物，为研究基因功能提供了强有力的遗传工具。对果蝇miRNAs的研究使我们对它的生物起源和生物学功能有了更深入的了解。miRNA的分析通常需要从靶细胞中提取总RNA的预处理过程，然后对总RNA样品中的靶miRNA进行定量分析，然而，其具有费力的总RNA提取和耗时的过程以及差的检测限。与此同时，果蝇的微小尺寸使得很难准确测量其细胞miRNA水平的微小变化。所提出的方法检测超低浓度miRNA的能力使得能够分析单个果蝇幼虫中mir-1的表达。因此，我们期望该策略可以为单细胞或活体动物的原位miRNA分析开辟新的途径。
We present a highly sensitive colorimetric method for microRNA (miRNA) detection. This method is based on a rolling-circle amplification (RCA) DNA machine, which integrates RCA, nicking enzyme signal amplification and DNAzyme signal amplification. The DNA machine is triggered by the hybridization of target miRNA with a rational designed padlock DNA template and activated by RCA. The resulting RCA product then autonomously replicates a multiple machinery cutter cycle and generates accumulated amount of products. Specifically, the DNA product in the present work is designed as a horseradish peroxidase (HRP)-mimicking DNAzyme, which could that catalyze a colorimetric reaction and generate colored product. Through these cascade amplifications, microRNA (miRNA) as low as 2 aM could be detected. As an example of in vivo application, miRNA from single Drosophila larva was successfully analyzed. Drosophila is a model organism that provides a powerful genetic tool to study gene functions. Study of Drosophila miRNAs has brought us knowledge of its biogenesis and biological functions. The analysis of miRNA typically requires a pretreatment process of extracting total RNAs from target cells, followed by quantitative analysis of target miRNA in total RNA samples, which nevertheless suffers from laborious total RNA extraction and time-consuming processes and poor limit of detection. Meanwhile, the tiny size of Drosophila makes it difficult to accurately measure trivial changes of its cellular miRNA levels. The ability to detect ultralow concentration of miRNA of the proposed method enables the analysis the expression of mir-1 in single Drosophila larva. We thus expect that the strategy may open new avenues for in situ miRNA analysis in single cell or living animals.