Global Folding of a Na⁺ -Specific DNAzyme Studied by FRET

Recently, a few RNA-cleaving DNAzymes have been isolated with excellent specificity for Na⁺ , and some of them contain a Na⁺ -binding aptamer. This metal recognition mechanism is different from that of most previously reported DNAzymes. When using 2-aminopurine (2AP) as a probe, interesting local folding induced by Na⁺ was recently observed. In this work, FRET was used to probe the global folding of the Ce13d DNAzyme; one of the Na⁺ -specific DNAzymes. FRET pairs were at different locations, which yielded a total of five constructs to probe the three-way junction structure with a large loop. With endlabeled DNAzymes, the global structure appears to be quite rigid with little folding upon adding up to 200 mm monovalent metal ions, although some minor differences were observed between Li⁺ , Na⁺ , and K⁺ . This lack of significant conformational change is also consistent with circular dichroism spectroscopy data. The loop was then labeled with an internal tetramethylrhodamine fluorophore at the G14 position, and its cleavage activity was partially retained. A clear Na⁺ -dependent folding was observed with spectral crossover. From a biosensing standpoint, global folding based sensors are unlikely to work due to the overall rigid structure of the DNAzyme. Therefore, the best way to use this DNAzyme to discriminate Na⁺ from K⁺ is based on cleavage activity, followed by probing local folding, whereas global folding is the least effective for metal discrimination.