Improving the Sensitivity of a Mn(II)-Specific DNAzyme for Cellular Imaging Sensor through Sequence Mutations

Area of Science:

• Biochemistry and Molecular Biology
• Chemical Biology
• Bioanalytical Chemistry

Background:

• Manganese (II) (Mn²⁺) detection in living cells is crucial for understanding its roles in cellular functions and disease.
• Existing Mn²⁺-specific DNAzyme sensors require improved activity and clearer mechanistic understanding for effective biological applications.

Purpose of the Study:

• To identify critical nucleotides within the catalytic domain of the 11-5 DNAzyme for improved performance.
• To develop a more sensitive and selective fluorescence sensor for intracellular Mn²⁺ detection.

Main Methods:

• Site-specific mutations were introduced into the catalytic domain of the 11-5 DNAzyme.
• The activity, selectivity, and fluorescence response of the mutated DNAzymes were evaluated.
• Intracellular Mn²⁺ sensing was performed in tumor cells using the developed DNAzyme variants.

Main Results:

• A variant DNAzyme, Mn5V, was identified with a twofold increase in activity compared to the original 11-5 DNAzyme.
• Mn5V demonstrated maintained high selectivity for Mn²⁺ over other metal ions.
• Mn5V exhibited enhanced fluorescence signals in tumor cells upon Mn²⁺ addition compared to the original DNAzyme.

Conclusions:

• This study provides insights into the functional mechanism of the 11-5 DNAzyme through site-specific mutagenesis.
• The developed Mn5V DNAzyme serves as a more sensitive and selective probe for investigating Mn²⁺ in biological systems.
• Enhanced Mn²⁺ sensing capabilities facilitate deeper understanding of cellular processes and disease implications.