Hairpin Switches-Based Isothermal Transcription Amplification for Simple, Sensitivity Detection of MicroRNA.

ABSTRACT

The ability to simply, selectively, and sensitively detect low numbers of miRNAs in clinical samples is highly valuable but remains a challenge. In this work, we present a novel miRNA detection system by using the elaborately designed hairpin switch, where the T7 primer, template, target recognize sequence, and light-up RNA aptamer template are edited and embedded in one single-stranded DNA hairpin structure. In the beginning, the hairpin switch maintained the hairpin structure 1, in which the ds promoter of T7 polymerase was disrupted, thus the transcription reaction of T7 polymerase was inhibited. After binding to the target, the hairpin switch 1 was unfolded and turned to the hairpin structure 2. This switch initiates the in vitro T7 transcription reaction, producing plenty of RNA transcripts containing RNA aptamers. Consequently, transcribed tremendous RNA aptamers lighted up the fluorophore for quantitative analysis. Compared with the existing T7 polymerase-based amplification system, this strategy exhibits several advantages, including simplicity, convenience, and high selectivity and sensitivity. The experimental results demonstrated that we could achieve the quantification of miRNA in buffer and complex biological samples.