PAIT effect: Padlock activator inhibits the trans-cleavage activity of CRISPR/Cas12a

Abstract

The CRISPR/Cas12a system is increasingly used in biosensor development. However, high background signal and low sensitivity for the non-nucleic acid targets detection is challenging. Here, a padlock activator which could inhibit the trans-cleavage activity of CRISPR/Cas12a system in the intact form by steric hindrance effect (PAIT effect) was designed for non-nucleic acid targets detection. The PAIT effect disappeared when padlock activator was separated into two split activators. To verify the feasibility of padlock activator, a Ca²⁺ sensor was developed based on PAIT effect with the assistance of DNAzyme, activity of which was Ca²⁺ dependent. In the presence of Ca²⁺, DNAzyme was activated to cleave its substrate, a padlock activator modified with adenine ribonucleotide, into split padlock activators which would trigger the trans-cleavage activity of Cas12a to generate fluorescence. There was a mathematical relationship between the fluorescence intensity and the logarithm of Ca²⁺ concentration ranging from 10 pM to 1 nM, with a limit of detection of 3.98 pM. The little interference of Mg²⁺, Mn²⁺, Cd²⁺, Cu²⁺, Na⁺, Al³⁺, K⁺, Fe²⁺, and Fe³⁺ indicated high selectivity. Recovery ranged from 93.32% to 103.28% with RSDs from 1.87% to 12.74% showed a good accuracy and reliability. Furthermore, the proposed sensor could be applied to detect Ca²⁺ in mineral water, milk powder and urine. The results were consistent with that of flame atomic absorption spectroscopy. Thus, PAIT effect is valuable for expanding the application boundary of CRISPR/Cas12a system.