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

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 Ca sensor was developed based on PAIT effect with the assistance of DNAzyme, activity of which was Ca dependent. In the presence of Ca, 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 Ca concentration ranging from 10 pM to 1 nM, with a limit of detection of 3.98 pM. The little interference of Mg, Mn, Cd, Cu, Na, Al, K, Fe, and Fe 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 Ca 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.