Rational evolution of Cd2+-specific DNAzymes with phosphorothioate modified cleavage junction and Cd2+ sensing

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• In vitro selection is effective for isolating metal-specific DNAzymes.
• Targeting thiophilic metals like cadmium (Cd2+) is challenging due to DNA's limited functional groups.
• Modified DNA bases can enhance functionality, but simpler modifications are desirable.

Purpose of the Study:

• To report the first in vitro selection of a DNAzyme targeting cadmium (Cd2+).
• To investigate the utility of a single phosphorothioate modification for enhancing metal affinity.
• To develop a highly specific and efficient DNAzyme for Cd2+ detection.

Main Methods:

• Employed in vitro selection strategies, including blocking DNA and negative selections, to isolate Cd2+-specific DNA sequences.
• Introduced a single phosphorothioate modification to enhance affinity for thiophilic metals.
• Characterized the catalytic activity, substrate specificity, and metal selectivity of the isolated DNAzyme.

Main Results:

• Successfully isolated a 12-nucleotide DNAzyme with high specificity for Cd2+.
• The DNAzyme exhibits a cleavage rate of 0.12 min⁻¹ with 10 μM Cd2+ at pH 6.0.
• Demonstrated over 100,000-fold selectivity for Cd2+ against Zn2+ and preferential cleavage of the Rp substrate isomer.

Conclusions:

• A single phosphorothioate modification effectively boosts DNAzyme affinity for Cd2+ without complicating selection.
• The developed DNAzyme is highly specific and efficient for Cd2+ detection, with demonstrated application.
• This approach expands the scope of DNA/metal interactions by introducing minimal modifications.