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.
(© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)