Metal-ion-dependent folding of a uranyl-specific DNAzyme: Insight into function from fluorescence resonance energy transfer studies

Fluorescence resonance energy transfer (FRET) has been used to study the global folding of an uranyl (UO₂²⁺)-specific 39E DNAzyme in the presence of Mg²⁺, Zn²⁺, Pb²⁺, or UO ₂²⁺. At pH 5.5 and physiological ionic strength (100 mM Na⁺), two of the three stems in this DNAzyme folded into a compact structure in the presence of Mg²⁺ or Zn²⁺. However, no folding occurred in the presence of Pb²⁺ or UO₂ ²⁺; this is analogous to the "lock-and-key" catalysis mode first observed in the Pb²⁺-specific 8-17 DNAzyme. However, Mg ²⁺ and Zn²⁺ exert different effects on the 8-17 and 39E DNAzymes. Whereas Mg²⁺ or Zn²⁺-dependent folding promoted 8-17 DNAzyme activity, the 39E DNAzyme folding induced by Mg²⁺ or Zn²⁺ inhibited UO₂²⁺-specific activity. Group IIA series of metal ions (Mg²⁺, Ca²⁺, Sr²⁺) also caused global folding of the 39E DNAzyme, for which the apparent binding affinity between these metal ions and the DNAzyme decreases as the ionic radius of the metal ions increases. Because the ionic radius of Sr²⁺ (1.12 Å) is comparable to that of Pb²⁺ (1.20 Å), but contrary to Pb²⁺, Sr²⁺ induces the DNAzyme to fold under identical conditions, ionic size alone cannot account for the unique folding behaviors induced by Pb²⁺ and UO₂²⁺. Under low ionic strength (30 mM Na⁺), all four metal ions (Mg²⁺, Zn ²⁺, Pb²⁺, and UO₂²⁺), caused 39E DNAzyme folding, suggesting that metal ions can neutralize the negative charge of DNA-backbone phosphates in addition to playing specific catalytic roles. Mg²⁺ at low (<2 mM) concentration promoted UO₂ ²⁺-specific activity, whereas Mg²⁺ at high (>2 mM) concentration inhibited the UO₂²⁺-specific activity. Therefore, the lock-and-key mode of DNAzymes depends on ionic strength, and the 39E DNAzyme is in the lock-and-key mode only at ionic strengths of 100 mM or greater.