PAM-free hairpin target binding activates t rans -cleavage activity of Cas12a

CRISPR-Cas12a has been demonstrated to be activated for its trans-cleavage activity by single- and double-stranded DNA containing a protospacer adjacent motif (PAM), but other types of activators have remained undiscovered. In this work, we found that a hairpin-structured substrate can activate the trans-cleavage activity of Cas12a without a PAM, and the parameters of the hairpin loop obviously affect the activity. Cas12a exhibits sequence preference for proximal loops, preferring to recognize polyadenine hairpin loop activators. Molecular docking and dynamic calculations provide a theoretical basis for the activation of Cas12a by hairpin activators. Leveraging the efficient activation capability of the hairpin activator, we constructed an allosteric detection platform for non-nucleic acid targets, capable of sensitively and specifically detecting hypochlorous acid and calcium ions. This novel activator of Cas12a holds enormous potential for the development of multi-functional biological platforms.