RNA aptamer-based CRISPR-Cas12a system for enhanced small molecule detection and point-of-care testing.

The CRISPR-Cas12a system has emerged as a robust platform for small molecule detection. However, existing methodologies primarily emphasize DNA aptamer-based strategies. This study introduces an RNA aptamer-based CRISPR-Cas12a approach due to the fact that the majority of small molecules lack corresponding DNA aptamers. The approach employs theophylline RNA aptamer (TA) to regulate Cas12a activity through competitive inhibition of crRNA. The results demonstrate that this system effectively detects theophylline (TP) in various food, beverage, and human serum samples, exhibiting excellent selectivity and sensitivity. Additionally, a visual paper-based detection system showcases its applicability for real-time analysis in food matrices and human serum. The RNA aptamer-based CRISPR-Cas12a strategy holds significant potential for diverse biomedical applications, offering a versatile tool for future sensing applications through customized RNA aptamer designs for small molecules.