title: DNAzyme-based rolling-circle amplification DNA machine for ultrasensitive analysis of microRNA in Drosophila larva.
abstract: 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.
authors.affiliation: Laboratory of Physical Biology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, China.
authors.forename: Yanqin
authors.initials: Y
authors.lastname: Wen
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
authors.forename: Xiuhai
authors.initials: X
authors.lastname: Mao
authors.forename: Yingliang
authors.initials: Y
authors.lastname: Wei
authors.forename: Haiyun
authors.initials: H
authors.lastname: Song
authors.forename: Nan
authors.initials: N
authors.lastname: Chen
authors.forename: Qing
authors.initials: Q
authors.lastname: Huang
authors.forename: Chunhai
authors.initials: C
authors.lastname: Fan
authors.forename: Di
authors.initials: D
authors.lastname: Li
mesh_terms: D000818:Animals; D052160:Benzothiazoles / Q000737:chemistry; D021881:DNA, Catalytic / Q000378:metabolism*; D004330:Drosophila / Q000254:growth & development / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D007814:Larva / Q000378:metabolism; D035683:MicroRNAs / Q000032:analysis*; D021141:Nucleic Acid Amplification Techniques; D013451:Sulfonic Acids / Q000737:chemistry
chemical_list: D052160:Benzothiazoles; D021881:DNA, Catalytic; C574927:MIRN1 microRNA, Drosophila; D035683:MicroRNAs; D013451:Sulfonic Acids; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D006861:Hydrogen Peroxide