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

Our view of the interaction between Na + and nucleic acids was changed by a few recently discovered Na + ‐specific RNA‐cleaving DNAzymes. In addition to nonspecific electrostatic interactions, highly specific recognition is also possible. Herein, two such DNAzymes, named EtNa and Ce13d, are compared to elucidate their mechanisms of Na + binding. Mutation studies indicate that they have different sequence requirements. Phosphorothioate (PS) substitution at the scissile phosphate drops the activity of EtNa 140‐fold, and it cannot be rescued by thiophilic Cd 2+ or Mn 2+ , whereas the activity of PS‐modified Ce13d can be rescued. Na + ‐dependent activity assays indicate that two Na + ions bind cooperatively in EtNa, and each Na + likely interacts with a nonbridging oxygen atom in the scissile phosphate, whereas Ce13d binds only one Na + ion in a well‐defined Na + aptamer, and this Na + ion does not directly interact with the scissile phosphate. Both DNAzymes display a normal pH–rate profile, with a single deprotonation reaction required for catalysis. For EtNa, Na + fails to protect the conserved nucleotides from dimethyl sulfate attack, and no specific Na + binding is detected by 2‐aminopurine fluorescence, both of which are different from those observed for Ce13d. This work suggests that EtNa binds Na + mainly through its scissile phosphate without significant involvement of the nucleotides in the enzyme strand, whereas Ce13d has a well‐defined aptamer for Na + binding. Therefore, DNA has at least two distinct ways to achieve highly selective Na + binding.