Dissecting metal ion–dependent folding and catalysis of a single DNAzyme

Abstract

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...

In vitro selection of a sodium-specific DNAzyme and its application in intracellular sensing

“…Being able to obtain the NaA43 DNAzyme with such a high selectivity for Na + will not only provide a highly selective sensor for Na + , as demonstrated here, it will also give us an opportunity to elucidate the origin of such a high selectivity. Previous studies of a Pb 2+ -DNAzyme have shown the high selectivity is mainly due to the DNAzyme forming a specific binding pocket for the metal ion (77). It would be interesting to find out if the NaA43 DNAzyme uses the same mechanism for selectivity.…”

RNA dynamics: it is about time

“…Clearly, slow global conformational dynamics significantly impact the overall catalytic rate constant [23••-25]. Single molecule FRET also allowed for the dissection of the metal-ion dependent multi-step folding pathways of an in vitro selected Diels-Alderase ribozyme [26] and a DNAzyme [27]. Sometimes FRET can demonstrate the lack of significant conformational dynamics of a ribozyme, for example of the glmS ribozyme upon cofactor binding [18].…”

Tandem DNAzymes for mRNA cleavage: Choice of enzyme, metal ions and the antisense effect

“…19 At the same time, it is also studied as a model DNAzyme due to its recurrence in many different in vitro selections and high catalytic efficiency. [20][21][22][23][24][25] Examples of using the 8-17 DNAzyme for gene silencing are also known. 18,26 While many examples of DNAzyme-based intracellular mRNA cleavage are reported, 13 concerns have also been raised that the 10-23 DNAzyme is basically inactive with physiological concentrations of free Mg 2+ .…”

“…For example, when an rAC junction was used, barely any cleavage was observed even with 500 mM Mg 2+ ( Figure S1, ESI). Therefore, it is also true for the [10][11][12][13][14][15][16][17][18][19][20][21][22][23] DNAzyme that its cleavage activity for every junction is not the same.…”