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 Tb(3+) luminescence spectroscopy. Na(+) displaces Tb(3+) from the DNAzyme, and thus quenches the emission from Tb(3+) . 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 Kd of ∼20 mm with Na(+) (other monovalent cations: 40-60 mm). The Kd 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 Kd 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.
Keywords: aptamers; luminescence; sensors; sodium; terbium.
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