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, Tb-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 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.