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

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Deoxyribozymes are DNA-based enzymes with catalytic activity.
• Existing deoxyribozyme biosensors are designed for physiological conditions.
• Engineering deoxyribozymes for extreme conditions expands their application scope.

Purpose of the Study:

• To design and characterize an RNA-cleaving deoxyribozyme functional at acidic pH.
• To develop a lysosomal biosensor for tracking adenosine triphosphate (ATP) fluctuations.
• To investigate deoxyribozyme cooperativity for pH sensing.

Main Methods:

• Design and characterization of an acidic RNA-cleaving deoxyribozyme (ET46).
• Assessment of cooperative H+ binding and pH responsiveness (Hill coefficient of 5.4).
• Development of a lysosomal ATP-activated aptazyme biosensor (FabATP) using ET46.

Main Results:

• ET46 exhibits strong cooperative H+ binding, enabling catalysis at acidic pH (pH 4.5-5.5).
• The biosensor FabATP successfully tracked pH changes related to lysosomal uptake.
• FabATP demonstrated live subcellular fluorescence imaging of lysosomal ATP fluctuations.

Conclusions:

• Engineered acidic deoxyribozymes can function under extreme cellular conditions.
• The developed biosensor provides a tool for real-time monitoring of lysosomal ATP.
• This work expands the utility of deoxyribozymes in cellular imaging and diagnostics.