Engineering Acidic Deoxyribozyme with Strongly Cooperative H⁺ Binding for Subcellular Imaging

Engineering deoxyribozymes under extreme conditions will expand their repertoires for unique applications. Although all known deoxyribozyme-based biosensors were derived to function under or near physiological conditions, no prior study has been conducted to purposely build them to absolutely function under atypical conditions. Here, we reported the design and characterization of an acidic RNA-cleaving deoxyribozyme, termed ET46, that displays strongly cooperative H⁺ binding for performing catalysis at acidic pH, yielding a Hill coefficient of 5.4. This feature of cooperativity significantly improves the responsiveness of ET46 to small pH variations within the pH range of 4.5-5.5. We further exploited the pH-sensitive properties of ET46 to track pH changes on the uptake and localization in lysosomes. Finally, we developed a lysosomal ATP-activated fluorescence-signaling aptazyme biosensor (named FabATP) using acidic ET46 and a known ATP-binding DNA aptamer. To demonstrate its ability to function in lysosomes, we applied FabATP in live subcellular fluorescence imaging of lysosomal ATP fluctuations upon stimulations.