Dual mature microRNA-responsive logic biosensing platform based on CRISPR/Cas12a and DNA nanocage.

Mature microRNAs play crucial roles in tumorigenesis and progression. However, their potential as cancer biomarkers is limited by the sequence interference of precursor microRNAs and the occurrence of false positive signals mediated by single microRNAs. Herein, we reported a dual mature microRNA-responsive second-order (YES-AND) logic biosensing platform for accurate cancer diagnosis. Specifically, DNA nanocages were conceived as the first stage of "YES" gates, capable of signal transduction through strand displacement reactions, and realizing size-selective discrimination of mature microRNAs and pre-microRNAs. Subsequently, CRISPR/Cas12a system served as the second stage of "AND" gate, wherein dual activators cooperatively triggered trans-cleavage. As a proof-of-concept, this second-order logic biosensing platform was successfully applied to detect non-small cell lung cancer-related mature microRNA in clinical serum, and showed remarkable sensitivity (Lod = 100 pM) and trueness (recovery ≥90 %). Our study represents a significant step forward in the development of intelligent biosensors capable of performing complex computations within pathological networks, and opens up broader possibilities for applications in biological science study and clinic disease diagnosis.