Rapid and label-free fluorescence bioassay for microRNA based on exonuclease III-assisted cycle amplification

Abstract

The quantitative analysis of microRNA is extremely important in biological research and clinical diagnosis due to the relationship between microRNA and disease. In this study, we reported a new assay for the rapid and simple detection of microRNA based on G-quadruplex and exonuclease III (ExoIII) dual signal amplification. We specifically designed two hairpins with G-quadruplex sequence. In the absence of a target, the G-quadruplex sequences are enclosed in the hairpin and fluorescence signal shut down. However, when a target is added, the dual cycle is carried out because two hairpins are digested and X and Y sequences are released under the action of ExoIII. Then, these released sequences form the G-quadruplex sequence, and N-methylmorpholine (NMM) is embedded in the G-quadruplex to produce strong fluorescence. The linear range is from 2.5 × 10^-10 to 4 × 10^-9 mol L^-1 with a low detection limit of 6 pM. Compared to some of the previous strategies, this bioassay needs only a simple one-step reaction, and is easy for realizing the rapid detection of microRNAs. The time required for the entire analysis is only 1 hour. In addition, this bioassay has good specificity and can be applied to the actual samples.

Scheme 1. Schematic illustration of exonuclease III and G-quadruplex-based dual signal amplification strategy for miRNA detection.

Fig. 1. Fluorescence spectra for the feasibility of strategy. (1) HP1 + HP2 + Exo III + NMM; (2) HP1 + HP2 + Exo III + NMM + miRNA-122; the concentrations of HP1, HP2, Exo III, miRNA-122, NMM are 50 nM, 600 nM, 6 U, 15 nM, 1.5 μM.

Fig. 2. Agarose (4%) gel electrophoresis for feasibility of strategy. (1) HP2; (2) HP1; (3) HP1 + HP2 + Exo III, (4) HP1 + HP2 + Exo III + miRNA-122; (5) HP1 + HP2 + miRNA-122; (6) HP1 + Exo III + miRNA-122; (7) HP1 + miRNA-122; (8) Marker. The concentrations of HP1, HP2, Exo III, miRNA-122 are 2.0 μM, 2.0 μM, 10 U, 2.0 μM.

Fig. 3. The fluorescence changes for various concentration of miRNA-122 (A). The Inset in (B) displays the linear graph of miRNA-122 and fluorescence changes. The concentration of miRNA is 0, 0.08, 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 2.5, 3.5, 4.0 nM. The concentrations of HP1, HP2, Exo III, NMM are 50 nM, 600 nM, 6 U, 1.5 μM. The incubation time of Exo III is 40 min at 37 °C. Error bars were derived from three experiments.

Fig. 4. The Selectivity of the biosensor. The concentration of miRNA-122 and its analogue, HP1, HP2, Exo III, NMM are 15 nM, 750 nM, 50 nM, 600 nM, 6 U, 1.5 μM, respectively. The incubation time of Exo III is 40 min at 37 °C. Error bars were derived from three experiments.