In situ imaging and interfering Dicer-mediated cleavage process via a versatile molecular beacon probe
2019
Abstract A novel versatile locked nucleic acid modified molecular beacon probe (LNA-MB) was developed for imaging intracellular precursor miRNAs (pre-miRNAs) and disturbing Dicer-mediated cleavage process. The target recognition reaction between the smart probe and pre-miRNA can not only induce the conformational changes of probe and block the Dicer cleavage site, but also inhibit the cleavage process, and then achieve down-regulation of miRNA expression. Simultaneously, the target recognition reaction broke the fluorescence resonance energy transfer (FRET) between fluorophore donor FAM and acceptor TAMRA, which were labelled on the LNA-MB probe, further induced the relevant change of fluorescence signal, and then achieved imaging analysis of pre-miRNA and inhibition events in situ. Both in vitro and in single living cell studies showed that the versatile probes exhibited a remarkable performance in targeting with pre-miRNA-21, and nearly 65% downregulation of mature miRNA-21 was achieved with 100 nM probes. All investigations demonstrate that the proposed strategy represents a promising alternative for regulating and inhibiting endogenous disease-associated RNAs, then further for achieving therapeutic outcomes in personalized treatments.
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