MicroRNAs (miRNAs) family members are usually different from each other in one-base variation. The high sequence homology poses a challenge for miRNA analysis with single-base selectivity. On the basis of G-quadruplex molecular beacons (G4MB) and duplex-specific nuclease (DSN), we developed a simple and highly selective amplification biosensor for miRNA detection. G4MB with a G4 motif stem is used as recognition probe. In the present of target miRNAs, G4MB hybridizes with target miRNA perfectly and forms a G4MB-miRNA duplex. Then, DSN subsequently cleaves the G4MB of the G4MB-miRNA duplex to recycle the target miRNA, which leads to fluorescence signal amplification. In the absence of target miRNAs, DSN can not digest the stem of G4MB because of the protection of G4 motif, which eliminates the false positive signal, and produces low fluorescence background. Importantly, the powerful discriminating abilities of both G4MB and DSN make the novel sensor suitable for miRNAs detection with high single-base selectivity. Comparing with traditional linear ssDNA probe-DSN-based method, the signal response of similar miRNA sequences with one-base difference has been reduced from 24% to 6% by using this G4MB-DSN-based method. Moreover, this simple sensor also exhibits a good applicability in cancer cell samples and a multiplex capability in one sample with different miRNA targets, making it a promising strategy for clinical diagnostics.
Keywords: Duplex-specific nuclease; G-quadruplex molecular beacon; MiRNA; Single-base selectivity.
Copyright © 2016. Published by Elsevier B.V.