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.

A Simple and Robust Exponential Amplification Reaction (EXPAR)-Based Hairpin Template (exp-Hairpin) for Highly Specific, Sensitive, and Universal MicroRNA Detection

“…In the let-7 family, the interference ratio of let-7i relative to let-7a was only 0.9%, while the interference rate of let7b/7c/7f/7g ranged from 11.3% to 14.9% (inset of Figure B), demonstrating a high specificity for miRNA let-7a detection in the homology family sequence. The reason for the high specificity is that the stable secondary structure of the exp-Hairpin template greatly resists the interaction with mismatched miRNA, thereby inhibiting nonspecific amplification reaction . Further, we compared the EXPAR performance of the exp-Hairpin template with that of a previously reported template.…”

“…A stem-loop hairpin structure is a single-stranded oligonucleotide that folds back on itself to form a paired double helix, which has an excellent selectivity to distinguish highly similar miRNA sequences with a single-base difference. , Hairpin structures have also been designed to improve the EXPAR amplification efficiency, specificity ,− , and versatility . A structure-switchable symmetric toehold dumbbell-template was designed for the primer hybrid on either side of this template, which enables the theoretical amplification efficiency up to 2 n .…”

In Vivo Imaging MicroRNA with Bright Fluorescent RNA Aptamer Through Target-Mediated Entropy-Driven Toehold Exchange

“…Universality and photostability are crucial in imaging intracellular targets in living systems. miRNA-21 (miR-21) is a cancer-related biomarker, and Hela cells with high content of miR-21 are selected as representative cell lines for further experiment. , After verification of the EDTE–DFHO amplified miR-125b imaging in living cells, we further explored its utilization and photostability in imaging miR-21. By designing the specific binding region with target in strand of S1, S2, and S3 (as shown in Table S2), this EDTE–DFHO strategy is utilized for recording imaging miR-21 in Hela cells.…”

Label-Free Direct Identification of MicroRNAs Based on a Narrow Constant-Inner-Diameter Emitter Mass Spectrometry Analysis

“…Studying and detecting miRNAs are vital for understanding their biological function, disease diagnosis, and drug development . In addition to conventional techniques such as real-time polymerase chain reaction, , microarray, , and RNA sequencing, several novel detection technologies have been reported in recent years, including isothermal amplification, , rolling ring amplification, electrochemistry, , mass spectrometry − (MS), and pico-high performance liquid chromatography . Most of these methods are based on signal and sequence amplification; only a few methods can achieve nonamplifying and nonenlarging detection of miRNA.…”