Dual Catalytic Hairpin Assembly-Based Automatic Molecule Machine for Amplified Detection of Auxin Response Factor-Targeted MicroRNA-160
Lei Wang,
Xing Dai,
Yujian Feng,
Qiyang Zhao,
Lin Liu,
Chang Xue,
Langtao Xiao,
Ruozhong Wang
Affiliations
Lei Wang
Hunan Provincial Key Laboratory of Phytohormones and Growth Development, College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China
Xing Dai
Hunan Provincial Key Laboratory of Phytohormones and Growth Development, College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China
Yujian Feng
Hunan Provincial Key Laboratory of Phytohormones and Growth Development, College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China
Qiyang Zhao
Hunan Provincial Key Laboratory of Phytohormones and Growth Development, College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China
Lin Liu
Guangdong Provincial Key Laboratory of Plant Epigenetics, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, China
Chang Xue
Key Laboratory of Laboratory Medicine, Ministry of Education of China, Zhejiang Provincial Key Laboratory of Medicine Genetics, School of Laboratory Medicine and Life Sciences, Institute of Functional Nucleic Acids and Personalized Cancer Theranostics, Wenzhou Medical University, Wenzhou 325035, China
Langtao Xiao
Hunan Provincial Key Laboratory of Phytohormones and Growth Development, College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China
Ruozhong Wang
Hunan Provincial Key Laboratory of Phytohormones and Growth Development, College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China
MicroRNA160 plays a crucial role in plant development by negatively regulating the auxin response factors (ARFs). In this manuscript, we design an automatic molecule machine (AMM) based on the dual catalytic hairpin assembly (D-CHA) strategy for the signal amplification detection of miRNA160. The detection system contains four hairpin-shaped DNA probes (HP1, HP2, HP3, and HP4). For HP1, the loop is designed to be complementary to miRNA160. A fragment of DNA with the same sequences as miRNA160 is separated into two pieces that are connected at the 3′ end of HP2 and 5′ end of HP3, respectively. In the presence of the target, four HPs are successively dissolved by the first catalytic hairpin assembly (CHA1), forming a four-way DNA junction (F-DJ) that enables the rearrangement of separated DNA fragments at the end of HP2 and HP3 and serving as an integrated target analogue for initiating the second CHA reaction, generating an enhanced fluorescence signal. Assay experiments demonstrate that D-CHA has a better performance compared with traditional CHA, achieving the detection limit as low as 10 pM for miRNA160 as deduced from its corresponding DNA surrogates. Moreover, non-target miRNAs, as well as single-base mutation targets, can be detected. Overall, the D-CHA strategy provides a competitive method for plant miRNAs detection.
