Abstract
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.
Highlights
MicroRNA is a type of non-coding single-stranded RNA widely found in eukaryotic cells, with a length of about 20–24 nucleotides [1,2]
MiRNA160 plays an important role in the auxin (IAA) response in plants by targeting auxin response factor (ARF) genes, including ARF10, ARF16, and ARF17 genes [7,8,9]
We present an automatic molecule machine (AMM) based on the dual catalytic hairpin assembly (D-CHA) process for the signal amplification detection of ARF-targeted miRNA160
Summary
MicroRNA (miRNA) is a type of non-coding single-stranded RNA widely found in eukaryotic cells, with a length of about 20–24 nucleotides [1,2]. RT-qRCR assay is accomplished with high speed and high sensitivity, the thermal-recycling controlling system and expensive instruments increase the detection cost [25,26,27] Due to these limitations above, the development of new sensing probes is one of the current areas driving the demand for convenient, simple, sensitive, and specific detection of miRNAs. Besides genetic molecules in living systems, DNA is known as a versatile tool in the fields of biochemical analysis and nanomaterials [28,29,30,31]. The usage of protein enzymes inevitably increases the detection cost To overcome those difficulties, various signal amplification strategies without protein enzymes were developed, such as hybrid chain reaction (HCR) and catalytic hairpin assembly (CHA) [39,40,41,42]. The comparative experiments have demonstrated that the D-CHA strategy displays a better assay performance compared with traditional CHA, possessing several advantages, such as simple operation procedure, high sensitivity, and specificity
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