A Selective Na+ Aptamer Dissected by Sensitized Tb3+ Luminescence

A previous study of two RNA‐cleaving DNAzymes, NaA43 and Ce13d, revealed the possibility of a common Na⁺ aptamer motif. Because Na⁺ binding to DNA is a fundamental biochemical problem, the interaction between Ce13d and Na⁺ was studied in detail by using sensitized Tb³⁺ luminescence spectroscopy. Na⁺ displaces Tb³⁺ from the DNAzyme, and thus quenches the emission from Tb³⁺. The overall requirement for Na⁺ binding includes the hairpin and the highly conserved 16‐nucleotide loop in the enzyme strand, along with a few unpaired nucleotides in the substrate. Mutation studies indicate good correlation between Na⁺ binding and cleavage activity, thus suggesting a critical role of Na⁺ binding for the enzyme activity. Ce13d displayed a K_d of ～20 mm with Na⁺ (other monovalent cations: 40–60 mm ). The K_d values for other metal ions are mainly due to non‐specific competition. With a single nucleotide mutation, the specific Na⁺ binding was lost. Another mutant improved K_d to 8 mm with Na⁺. This study has demonstrated a Na⁺ aptamer with important biological implications and analytical applications. It has also defined the structural requirements for Na⁺ binding and produced an improved mutant.