A CRISPR-Cas–driven positive feedback circuit with exponential dynamic enables ultrasensitive detection of genomic DNA.

CRISPR-based diagnostics (CRISPR-Dx) has shown great promise in molecular diagnostics, but its utility in the sensing of microRNA (miRNA) biomarkers is limited by sensitivity, cost and robustness. Here, we describe a CRISPR-Dx method for the sensitive and cost-effective detection of miRNAs by rationally integrating CRISPR-Cas12a with DNA circuits. In this work, a modular catalytic hairpin assembly (CHA) circuit is designed to convert and amplify each target into multiple programmable DNA duplexes, which serve as triggers to initiate the trans-cleavage activity of CRISPR-Cas12a for further signal amplification. Such rational integration provides a generic assay for the effectively amplified detection of miRNA biomarkers. By simply tuning the variable regions in the CHA modules, this assay achieves sub-femtomolar sensitivity for different miRNA biomarkers, which improves the detection limit of CRISPR-Dx in the analysis of miRNA by 3–4 orders of magnitude. With the usage of the proposed assay, the sensitive assessment of miR-21 levels in different cancer cell lines and clinical serum samples has been achieved, providing a generic method for the sensitive detection of miRNA biomarkers in molecular diagnosis.

G-quadruplex (G4) with stacked G-tetrads structure is able to bind hemin (iron (III)-protoporphyrin IX) to form a unique type of DNAzyme/RNAzyme with peroxidase-mimicking activity, which has been widely employed in multidisciplinary fields. However, its further applications are hampered by its relatively weak activity compared with protein enzymes. Herein, we report a unique intramolecular enhancement effect of the adjacent adenine (EnEAA) at 3′ end of G4 core sequences that significantly improves the activity of G4 DNAzymes. Through detailed investigations of the EnEAA, the added 3′ adenine was proved to accelerate the compound I formation in catalytic cycle and thus improve the G4 DNAzyme activity. EnEAA was found to be highly dependent on the unprotonated state of the N1 of adenine, substantiating that adenine might function as a general acid–base catalyst. Further adenine analogs analysis supported that both N1 and exocyclic 6-amino groups in adenine played key role in the catalysis. Moreover, we proved that EnEAA was generally applicable for various parallel G-quadruplex structures and even G4 RNAzyme. Our studies implied that adenine might act analogously as the distal histidine in protein peroxidases, which shed light on the fundamental understanding and rational design of G4 DNAzyme/RNAzyme catalysts with enhanced functions.