Construction of molecular logic gates using heavy metal ions as inputs based on catalytic hairpin assembly and CRISPR-Cas12a

We successfully constructed several molecular logic gates using heavy metal ions as inputs based on catalytic hairpin assembly (CHA) and CRISPR-Cas12a. The corresponding DNAzymes were used to recognize heavy metal ions (Hg2+, Cd2+, Pb2+, and Mn2+). The specific cleavage between heavy metal ions and DNAzymes leads to the release of the trigger DNA, which can be used to activate CHA through logic computation. The CHA-generated DNA duplexes contain the protospacer adjacent motifs (PAM) sequence, which can be distinguished by CRISPR-Cas12a. The hybridization interactions between the duplexes and gRNA will activate the trans-cleavage capability of Cas12a, which can cleave the single-stranded DNA (ssDNA) reporter. The separation of the fluorescence group and quench group in ssDNA will generate a high fluorescence signal for readout. Using Hg2+ and Cd2+ as the two inputs, several basic logic gates were constructed, including OR, AND, and INHIBT. Using Hg2+, Cd2+, Pb2+, and Mn2+ as the four inputs, cascaded logic gates were further fabricated. With the advantages of scalability, versatility, and logic computing capability, our proposed molecular logic gates can provide an intelligent sensing system for heavy metal ions monitoring.