Accurate Detection of Target MicroRNA in Mixed Species of High Sequence Homology Using Target-Protection Rolling Circle Amplification

Abstract

The close relationships of miRNAs with human diseases highlight the urgent needs for miRNA detection. However, the accurate detection of a target miRNA in mixed miRNAs of high sequence homology presents a great challenge. Herein, a novel method called target-protection rolling circle amplification (TP-RCA) is proposed for this purpose. The protective probe is designed so that it can form a fully complementary duplex with the target miRNA and can also mismatch duplexes with other nontarget miRNAs. These duplexes are treated with a single strand-specific nuclease. Consequently, only the target miRNA in a perfect-match duplex can resist the cleavage of nuclease, whereas the nontarget miRNAs in mismatched duplexes will be digested completely. The protected target miRNA can be detected using RCA reactions. MicroRNA let-7 family members (let-7a-let-7f) and nuclease CEL I were used as proof-of-concept models to evaluate the feasibility of the TP-RCA method under different experimental conditions. The experimental results show that the TP-RCA method can unambiguously detect the target let-7 species in mixtures of let-7 family members even though they may differ by only a single nucleotide. This TP-RCA method significantly improves the detection specificity of miRNAs.

Figure 1

Scheme showing the principle of the TP-RCA method for high-specificity detection of miRNAs.

Figure 2

Electrophoresis analyses of the sss-nuclease CEL I cleavage of perfect-match and mismatch RNA/DNA heteroduplexes at different temperatures.

Figure 3

(a) Fluorescence curves of the RCA reactions initiated by single-strand let-7a and let-7a/pro-7a duplexes at different concentrations. (b) Result of the linear analysis between the fluorescence intensity and the single-strand let-7a concentration. (c) Result of the linear analysis between the fluorescence intensity and the let-7a/pro-7a duplex concentration.

Figure 4

Detection specificity of miRNA let-7a using the proposed TP-RCA method. (a) Fluorescence curves of the TP-RCA reaction for the protective probe pro-7a to detect let-7 family members. (b) Histogram showing the detection specificity in TP-RCA of Figure 4a. (c) Fluorescence curves of TP-RCA detection of let-7a in sample mixtures mix-a1–mix-a6. (d) Histogram showing the linear relationship between the fluorescence intensity and the let-7a quantity in sample mixtures in Figure 4c. Detection specificities of other let-7 family members using the TP-RCA method are given in Figures S8–S10.