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 UO22+. 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 UO22+; this is analogous to the lock-and-key catalysis mode first observed in the Pb2+-specific 817 DNAzyme. However, Mg2+ and Zn2+ exert different effects on the 817 and 39E DNAzymes. Whereas Mg2+ or Zn2+-dependent folding promoted 817 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 angstrom) is comparable to that of Pb2+ (1.20 angstrom), 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+, Zn2+, 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 UO22+-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.