Protein metalloenzymes use various modes for functions where metal-dependent global conformational change is required in some cases, but not in others. In contrast, most ribozymes appear to require a global folding that almost always precedes enzyme reactions. Herein we studied metal-dependent folding and cleavage activity of the 8-17 DNAzyme using single molecule fluorescence resonance energy transfer (FRET). Addition of Zn 2+ and Mg 2+ resulted in a folding step followed by cleavage reaction, suggesting that the DNAzyme may require metaldependent global folding for activation. In the presence of Pb 2+ , however, cleavage reaction occurred without a precedent folding step, suggesting that the DNAzyme may be prearranged to accept Pb 2+ for the activity. This feature may contribute to the remarkably fast Pb 2+ -dependent reaction of the DNAzyme. These results suggest that DNAzymes can use all modes of activation that metalloproteins use.Metal ion-dependent folding can play a critical role in metalloenzyme function. Understanding the relationship between folding and reaction is important in obtaining deeper insight into the enzyme mechanism. For protein metalloenzymes, an active-site metal-dependent global folding precedes enzymatic reaction in some cases while such a folding is not required in others 1 . These different modes of activation may fulfill different functions. For example, a reaction preceded by a folding step may be responsible for an allosteric effect in many enzyme functions, or such a folding step may contribute to the overall reaction. Competing Financial Interests StatementThe authors declare there are no competing financial interests. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptIn the early 1980s, RNA molecules that can catalyze enzymatic reactions were discovered and named ribozymes 2,3 . This discovery was then followed by demonstrations in the 1990s that DNA can also act as enzymes, termed deoxyribozymes or DNAzymes 4-7 . With only four nucleotides as building blocks versus twenty in proteins, nucleic acid enzymes may need to recruit cofactors to perform some functions. Metal ions are a natural choice and indeed most nucleic acid enzymes require metal ions for function under physiological conditions and therefore, are metalloenzymes. Even though DNAzymes constitute the newest metalloenzyme family, they have already been used in a number of applications such as therapeutic agents 8 , biosensors [9][10][11][12] , and nanomaterials assembly 13 , often because DNAzymes are more stable against hydrolysis and more cost-effective to produce than proteins or ribozymes. A primary example is the 8-17 DNAzyme which cleaves a DNA substrate containing one RNA base at the cleavage site (Fig. 1a) Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript DNAzyme constructsThe original 8-17 DNAzyme was labeled with a fluorophore (Alexa fluoro 488) and two quenchers (Dabcyl) for bulk cleavage activity assays (Fig. 1a). To carry out the smFRET...

The 8-17 DNAzyme is a DNA metalloenzyme catalyzing RNA transesterification in the presence of divalent metal ions, with activity following the order Pb2+ >> Zn2+ >>Mg2+. Since the DNAzyme has been used as a metal ion sensor, its metal-induced global folding was studied by fluorescence resonance energy transfer (FRET) by labeling the three stems of the DNAzyme with the Cy3/Cy5 FRET pair two stems at a time in order to gain deeper insight into the role of different metal ions in its structure and function. FRET results indicated that, in the presence of Zn2+ and Mg2+, the DNAzyme folds into a compact structure, stem III approaching a configuration defined by stems I and II without changing the angle between stems I and II. Correlations between metal-induced folding and activity were also studied. For Zn2+ and Mg2+, the metal ion with higher affinity for the DNAzyme in global folding (Kd(Zn) = 52.6 microM and Kd(Mg) = 1.36 mM) also displays higher affinity in activity (Kd(Zn) = 1.15 mM and Kd(Mg) = 53 mM) under the same conditions. Global folding was saturated at much lower concentrations of Zn2+ and Mg2+ than the cleavage activities, indicating the global folding of the DNAzyme occurs before the cleavage activity for those metal ions. Surprisingly, no Pb2+-dependent global folding was observed. These results suggest that for Pb2+ global folding of the DNAzyme may not be a necessary step in its function, which may contribute to the DNAzyme having the highest activity in the presence of Pb2+.

The effect of monovalent ions on both the reactivity and global folding of the 8–17 DNAzyme is investigated and the results are compared with the hammerhead ribozyme, which has similar size and secondary structure. In contrast to the hammerhead ribozyme, the 8–17 DNAzyme activity is not detectable in the presence of 4 M K+, Rb+, and Cs+ and the complex, [Co(NH3)6]3+. Only Li+, NH4+ and to a lesser extent Na+ showed detectable activity. The observed rate constants (kobs ~10−3 min−1 for Li+ and NH4+) are ~1000-fold lower than that in the presence of 10 mM Mg2+, and ~200,000-fold slower than the estimated rate in the presence of 100 µM Pb2+. Since the hammerhead ribozyme displays monovalent ion-dependent activity that is often within ~10-fold of divalent metal ion-dependent activity, these results suggest that the 8–17 DNAzyme, obtained by in vitro selections has evolved to have a more stringent divalent metal ion requirement for high activity as compared to the naturally occurring ribozymes, making the 8–17 DNAzyme an excellent choice as a Pb2+ sensor with high selectivity. In contrast to the activity data, folding was observed in the presence of all the monovalent ions investigated, although those monovalent ions that do not support DNAzyme activity have weaker binding affinity (Kd ~0.35 M for Rb+ and Cs+), while those that confer DNAzyme activity possess stronger affinity (Kd ~0.22 M for Li+, Na+ and NH4 +). In addition, a correlation between metal ion charge density, binding affinity and enzyme activity was found among mono- and divalent metal ions except Pb2+; higher charge density resulted in stronger affinity and higher activity, suggesting that the observed folding and activity is at least partially due to electrostatic interactions between ions and the DNAzyme. Finally, circular dichroism (CD) study has revealed Z-DNA formation with the monovalent metal ions, Zn2+ and Mg2+; the Kd values obtained using CD were in the same range as those obtained from folding studies using FRET. However, Z-DNA formation was not observed with Pb2+. These results indicate that Pb2+-dependent function follows a different mechanism from the monovalent metal ions and other divalent metal ions; in the presence of latter metal ions, metal-ion dependent folding and structural changes, including formation of Z-DNA, play an important role in the catalytic function of the 8–17 DNAzyme.

Metal-dependent cleavage activities of the 8-17 DNAzyme were found to be inhibited by Tb(III) ions, and the apparent inhibition constant in the presence of 100 microM of Zn(II) was measured to be 3.3+/-0.3 microM. The apparent inhibition constants increased linearly with increasing Zn(II) concentration, and the inhibition effect could be fully rescued with addition of active metal ions, indicating that Tb(III) is a competitive inhibitor and that the effect is completely reversible. The sensitized Tb(III) luminescence at 543 nm was dramatically enhanced when Tb(III) was added to the DNAzyme-substrate complex. With an inactive DNAzyme in which the GT wobble pair was replaced with a GC Watson-Crick base pair, the luminescence enhancement was slightly decreased. In addition, when the DNAzyme strand was replaced with a complete complementary strand to the substrate, no significant luminescence enhancement was observed. These observations suggest that Tb(III) may bind to an unpaired region of the DNAzyme, with the GT wobble pair playing a role. Luminescence lifetime measurements in D(2)O and H(2)O suggested that Tb(III) bound to DNAzyme is coordinated by 6.7+/-0.2 water molecules and two or three functional groups from the DNAzyme. Divalent metal ions competed for the Tb(III) binding site(s) in the order Co(II)>Zn(II)>Mn(II)>Pb(II)>Ca(II) approximately Mg(II). This order closely follows the order of DNAzyme activity, with the exception of Pb(II). These results indicate that Pb(II), the most active metal ion, competes for Tb(III) binding differently from other metal ions such as Zn(II), suggesting that Pb(II) may bind to a different site from that for the other metal ions including Zn(II) and Tb(III).