Preserving DNAzyme Stability and Activity in Stool Samples for Clinical Diagnostics

Stool is one of the most challenging biological matrices for functional nucleic acid–based diagnostics because of its high and variable nuclease activity. Here, we present a simple and robust strategy for stabilizing RNA‐cleaving fluorogenic DNAzymes (RFD) while preserving their catalytic function in stool. Using RFD‐CD3—a DNAzyme previously developed for Clostridioides difficile detection—as a model system, we evaluated two stabilization approaches. While nucleic acid competitors, including tRNA and salmon sperm DNA, provided only modest protection under alkaline conditions, heparin treatment combined with high‐pH buffer conditions significantly enhanced DNAzyme stability without compromising catalytic activity. This approach proved effective in both pooled and individual stool samples. The generality of the strategy was further demonstrated using RFD‐CD2, a sequence‐divergent C. difficile ‐responsive DNAzyme, as well as RFD‐EC1, which targets Escherichia coli K12. Across all tested DNAzymes, stabilization preserved both probe integrity and target‐responsive catalytic function. Together, these findings establish a practical and broadly applicable platform for deploying DNAzyme‐based diagnostics in complex clinical samples such as stool.