Discovery of a Dual-Target DNAzyme for Clostridioides difficile and Listeria monocytogenes Diagnostics

Rapid and reliable detection of Clostridioides difficile is essential for clinical diagnosis and infection control. Building on our group’s prior development of RNA-cleaving fluorogenic DNAzymes (RFDs) for bacterial recognition, we report RFD-CD3, a fluorogenic DNAzyme with the highest catalytic efficiency reported to date for C. difficile-responsive DNAzymes, achieving a detection limit of 10³ CFU/mL and a catalytic rate constant (kobs) of 0.16 min⁻¹ for its engineered trans-acting variant (RFD-CD3-TM2). This trans-acting form also retains high activity and provides modularity for biosensor design, further extending the versatility of this platform. Unexpectedly, RFD-CD3 also responds to Listeria monocytogenes, making it the first DNAzyme known to be selectively activated by two phylogenetically distinct bacterial species. As no RFD has previously been reported for L. monocytogenes, this discovery opens new opportunities for DNAzyme-based biosensing of a clinically and food-relevant pathogen. The distinct clinical contexts of the two targets—C. difficile in stool and L. monocytogenes in blood or food—highlight the potential of RFD-CD3 as a versatile molecular tool suited to multiple diagnostic applications. RFD-CD3 also remains catalytically active at alkaline pH, enhancing its stability in nuclease-rich biological samples. Together, these features position RFD-CD3 as a promising dual-target platform for high-performance diagnostics in both clinical and food safety applications.