Splitting a DNAzyme enables a Na⁺-dependent FRET signal from the embedded aptamer

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 K_d 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.