A dual amplification strategy integrating entropy-driven circuit with Cas14a for sensitive detection of miRNA-10b

Cas14a may recognize and randomly cleave ssDNAs without protospacer adjacent motif (PAM) sequence restriction to benefit nucleic acid sensing. However, the sensitivity of individual Cas proteins is limited. Herein a protocol based on entropy-driven circuit (EDC) and Cas14a/sgRNA (EDC-Cas14a) was developed to achieve dual signal amplification of miRNA. The target can continuously catalyze the reaction of EDC to generate multiple ssDNA activators, which triggers the trans-cleavage of Cas14a to cleave reporters and achieves 2nd signal amplification. The background signal is effectively reduced by adjusting the number of bases in the EDC. Therefore, two parts of the protocol can coexist in a system without interfering with each other. EDC significantly improves the sensitivity and specificity of Cas14a/sgRNA. A 100-fold improvement on sensitivity was achieved with a limit of detection of 2.1 pM. The specificity of EDC-Cas14a was characterized by outstanding dis-tinguishing capability of miRNA-10b and its counterpart with one-base mismatch. EDC-Cas14a can be readily adjusted to detect various miRNAs by simply manipulating EDC modules. Eventually, the promising assay performances of EDC-Cas14a was demonstrated by the detection of miRNA-10b in serum and cell lysate to provide a general approach for sensitive assay of miRNA biomarker in molecular diagnosis.