An Exceptionally Selective DNA Cooperatively Binding Two Ca²⁺ Ions.

Area of Science:

• Biochemistry
• Molecular Biology
• Biosensor Technology

Background:

• Calcium ions (Ca²⁺) are crucial in biological and environmental systems, necessitating effective detection methods.
• While Ca²⁺-binding proteins are common, nucleic acid-based Ca²⁺ binders are rare.
• DNA-based biosensors offer stability and programmability for metal ion detection.

Purpose of the Study:

• To develop and characterize a novel RNA-cleaving DNAzyme, EtNa, with high selectivity for Ca²⁺.
• To investigate the cooperative binding mechanism of Ca²⁺ by the DNAzyme.
• To engineer the DNAzyme for practical Ca²⁺ sensing applications.

Main Methods:

• Comparative analysis of four Ca²⁺-dependent DNAzymes.
• Investigation of Ca²⁺ binding using phosphorothioate (PS) modification.
• Determination of pH-rate profiles to elucidate reaction mechanisms.
• Engineering of the EtNa DNAzyme for Ca²⁺ sensing and validation in tap water using ICP-MS.

Main Results:

• The EtNa DNAzyme exhibits superior Ca²⁺ selectivity, showing 90 times higher activity with Ca²⁺ than with Mg²⁺.
• EtNa cooperatively binds two Ca²⁺ ions, with equal contribution from non-bridging oxygen atoms at the scissile phosphate.
• Engineered EtNa achieved a Ca²⁺ detection limit of 17 μm with excellent selectivity, validated in tap water samples.
• The study provides fundamental insights into Ca²⁺ binding by nucleic acids.

Conclusions:

• The EtNa DNAzyme represents a significant advancement in selective Ca²⁺ detection using nucleic acid-based biosensors.
• Multiple cooperative metal binding sites enhance the metal selectivity of nucleic acid sensors.
• This work contributes to the understanding of Ca²⁺-nucleic acid interactions and the development of novel biosensing platforms.