A Highly Thermostable Heme–G‐Quadruplex DNAzyme: Structural Integrity and Catalytic Function Under Elevated‐Temperature Aqueous Conditions

Heme-G-quadruplex DNAzymes are promising biomolecular catalysts; however, their structural stability and catalytic function under elevated-temperature aqueous conditions remain poorly understood. We systematically demonstrate that rationally increasing the number of consecutively stacked G-quartets in the DNA scaffold provides a useful strategy for constructing a highly thermostable heme-G-quadruplex DNAzyme. Our results establish a clear relationship between G-quartet stacking and enhanced thermal robustness of heme-G-quadruplex DNAzymes. The engineered DNAzyme retained its peroxidase activity at temperatures up to 90 °C, far exceeding the thermal stability of conventional protein-based peroxidases, which typically denature under such conditions. These results demonstrate the potential of G-quadruplex engineering for designing robust biocatalysts and establish a general strategy for engineering thermostable DNAzymes.