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Branched RCA Coupled with NESA-based Fluorescence Assay for Ultrasensitive Detection of miRNA


We report the development of a novel fluorescence biosensing strategy based on branched-rolling circle amplification (RCA) coupled with nicking endonuclease signal amplification (NESA) for ultrasensitive and highly specific detection of miRNA. This biosensor relies on target miRNA-triggered quadratic RCA and endonuclease-assisted cyclic nicking of molecule beacon (MB). The specific binding between target miRNA and hairpin probe 1 (HAP1) leads to the conformation transform of HAP1, releasing exposed DNA segment that can hybridize with the circular probe 1 and subsequently induce primary RCA reaction. The generated RCA product combines specifically to hairpin probe 2 (HAP2), resulting in the unfolded HAP2 and liberating a new exposed DNA segment that can initiate quadratic RCA. Then MB hybridizes with the tandem repeated DNA segments produced by quadratic RCA, forming the specific nicking site of the nicking endonuclease. Therefore, the nicking endonuclease cleaves MB into two pieces, liberating strong fluorescence signal. Meanwhile, the quadratic RCA products would initiate the next cycle of hybridization and cleavage, thereby acquiring significantly increased fluorescence signal. Using the branched-RCA coupled with NESA strategy, an ultrasensitive miRNA-155 assay method has been developed with a wide linear range from 5 aM to 500 fM and a low detection limit of 3.9 aM. The work is to our knowledge the first report that branched-RCA coupled with NESA has been used for fluorescence assay of miRNA. The developed biosensing strategy might hold a great potential for detecting miRNA and early diagnosis in miRNA-related diseases.

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Publication details

The article was received on 03 Feb 2017, accepted on 22 Apr 2017 and first published on 24 Apr 2017

Article type: Paper
DOI: 10.1039/C7NJ00404D
Citation: New J. Chem., 2017, Accepted Manuscript
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    Branched RCA Coupled with NESA-based Fluorescence Assay for Ultrasensitive Detection of miRNA

    C. Xu, X. Wang, C. Han, J. Wang, H. Li, Y. Wang, S. Liu and J. Huang, New J. Chem., 2017, Accepted Manuscript , DOI: 10.1039/C7NJ00404D

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