A novel CRISPR/Cas14a1-Exo III aptasensor for melamine detection coupled with systematically studied binding mechanism of truncated aptamer

In this work, the aptamer of melamine was truncated and mutated. Based on isothermal titration calorimetry, circular dichroism and molecular dynamics, the thermodynamic information, structural changes and binding pocket of the truncated aptamer SJs32 with 32 bases to melamine were systematically studied. Meanwhile, a novel aptasensor coupled with CRISPR/Cas14a1 and Exo III was developed for melamine detection. SJs32 was first hybridized with the complementary DNA (cDNA) to form SJs32-cDNA. In the presence of melamine, SJs32 recognized melamine to form SJs32-melamine and performed structural transformation, which resulted in the releasing of cDNA into the solution. Exo III digested the SJs32-melamine and the released melamine led to the dissociation of more cDNA from SJs32-cDNA complex. Finally, the combination of cDNA and sgRNA activated the trans-cleavage activity of Cas14a1. According to the change of fluorescence intensity, the sensitive and rapid detection of melamine was realized with the linear detection range of 4 nmol/L-1 µmol/L. The developed strategy broadened the application of melamine aptamer in food safety and provided a new insight for the development of CRISPR/Cas14a1 aptasensor.