Two Completely Different Mechanisms for Highly Specific Na⁺ Recognition by DNAzymes

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Recent discoveries of sodium ion (Na+)-specific RNA-cleaving DNAzymes have revealed mechanisms beyond simple electrostatic interactions.
• These findings challenge previous understandings of Na+ and nucleic acid interactions, highlighting the potential for highly specific recognition.

Purpose of the Study:

• To compare the Na+-binding mechanisms of two distinct DNAzymes, EtNa and Ce13d.
• To elucidate the specific molecular interactions and sequence requirements governing Na+ recognition in these DNAzymes.

Main Methods:

• Comparative analysis of EtNa and Ce13d using mutation studies and phosphorothioate (PS) substitution at the scissile phosphate.
• Na+-dependent activity assays and pH-rate profiles to investigate catalytic mechanisms.
• Dimethyl sulfate protection assays and 2-aminopurine (2-AP) fluorescence to probe nucleotide involvement and specific Na+ binding.

Main Results:

• Mutation studies revealed different sequence requirements for EtNa and Ce13d.
• EtNa activity was highly sensitive to PS substitution, unlike Ce13d, indicating different interactions with the scissile phosphate.
• EtNa binds two Na+ ions cooperatively at the scissile phosphate, whereas Ce13d binds one Na+ ion in a separate aptameric structure.
• EtNa showed no specific Na+ binding protection of nucleotides, contrasting with Ce13d's behavior.

Conclusions:

• DNAzymes exhibit at least two distinct mechanisms for highly selective Na+ binding.
• EtNa primarily utilizes its scissile phosphate for Na+ binding, with minimal nucleotide involvement.
• Ce13d employs a well-defined aptamer for specific Na+ binding, independent of the scissile phosphate.