Dual miRNAs Imaging Platform Based on HRCA-Cas12a by Replacing PAM with Bubble to Reduce False Positive

Abstract

Detection and imaging of dual miRNAs based on AND logic gates can improve the accuracy of the early diagnosis of disease. However, a single target may lead to false positive. Hence, this work rationally integrates hyperbranched rolling circle amplification (HRCA) with Cas12a by replacing the PAM sequence with a bubble to sensitively detect and image miRNA-10b and miRNA-21 based on the AND logic gate. When miRNA-10b and miRNA-21 are both present, the two padlocks are linked into circular DNA as a template for RCA. Long ssDNA products are generated under the catalysis of phi29 DNA polymerase, which are cis-cleaved by Cas12a and activated the trans-cleavage of Cas12a to generate fluorescent signals. Subsequently, the primer hybridizes with the products of cis-cleavage and is extended as the dsDNA substrate of Cas12a to produce more fluorescent signals. However, a single target produces significant fluorescent signals leading to false positive due to the presence of protospacer adjacent motif (PAM) on the padlock. After PAM is removed from the padlock, the primer and RCA products form bubbles to replace PAM, which activate Cas12a without affecting sensitivity and reduce false positive. The introduction of a primer enables the second utilization of phi29 and Cas12a, increasing the signal-to-noise ratio. HRCA and Cas12a exhibit optimal activity in the T4 ligase buffer, achieving one-pot detection of dual miRNAs. In addition, the HRCA-Cas12a method enables the intracellular visualization of dual miRNAs. It exhibits the ability to distinguish different types of cancer cells based on the expression level of miRNAs.