DNAzyme-Based Rolling-Circle Amplification DNA Machine for Ultrasensitive Analysis of MicroRNA in <i>Drosophila</i> Larva

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 <i>Drosophila</i> larva was successfully analyzed. <i>Drosophila</i> is a model organism that provides a powerful genetic tool to study gene functions. Study of <i>Drosophila</i> 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 <i>Drosophila</i> 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 <i>Drosophila</i> larva. We thus expect that the strategy may open new avenues for in situ miRNA analysis in single cell or living animals