CRISPR/Cas13a catalyzed self-assembly of quantum dot-DNA hydrogel for microRNA assay

Taking the advantages of outstanding precision in target recognition and trans-cleavage ability, the RNA-guided CRISPR-Cas systems have shown great promise in cancer diagnostics. Here, we developed a novel detection system based on CRISPR-Cas-catalyzed formation of quantum dot-DNA (QD-DNA) hydrogel for sensitive miRNA assay. After target miRNA recognition and Cas-mediated cleavage of the uracil ribonucleotide-embedded hairpin DNA (rU-HD), the cleaved rU-HD released primer DNA, which triggered toehold-mediated strand displacement (TMSD) amplification to form trefoil DNA quenchers. The trefoil DNA quenchers hybridized with DNA functionalized quantum dots (DNA-QDs) to construct self-assembled QD-DNA hydrogel and the fluorescence of the QDs could be efficiently quenched by the modified black hole quencher at the 3′ terminal of the trefoil DNA quenchers. This assay, termed CRISPR-Cas-catalyzed TMSD based QD-DNA hydrogel (Cas-TMSD-QDH), was applied for the sensitively detection of miR-17 and the limit of detection was as low as 182aM. The Cas-TMSD-QDH assay showed an accurate detection of miR-17 levels in different cell lines and the detection accuracy is confirmed by qRT-PCR. Besides, the assay was able to assess miR-17 levels in serum sample, revealing great potential for miRNAs assay in clinical molecular diagnostics.