Splitting a DNAzyme enables a Na

Recently, a few Na+-specific RNA-cleaving DNAzymes have been reported, and a Na+ aptamer was identified from the NaA43 and Ce13d DNAzymes. These DNAzymes and the embedded aptamer have been used for Na+ detection. In this work, we studied the Na+-dependent folding of the Ce13d DNAzyme using fluorescence resonance energy transfer (FRET). When a FRET donor and an acceptor were respectively labeled at the ends of the DNAzyme, Na+ failed to induce an obvious end-to-end distance change, suggesting a rigid global structure. To relax this rigidity, the Ce13d DNAzyme was systematically split at various sites on both the enzyme and the substrate strands. The Na+ binding activity of the split structures was characterized by 2-aminopurine fluorescence, enzymatic activity, Tb3+-sensitized luminescence, and DMS footprinting. Among the various constructs, the only one that retained Na+ binding was the split at the cleavage site, and this construct was further labeled with two dyes near the split site. This FRET result showed Na+-dependent folding with a Kd of 26 mM Na+. This study provides important structural information related to Na+ binding to this new aptamer, which might also be useful for future work in biosensor design.