DNAzymes are catalytically active, single-stranded DNA that coordinate with metal ions to catalyze a number of different reactions in the cell, including mRNA cleavage. This activity makes them an attractive, potential therapeutic for diseases caused by the over expression of specific proteins. However, for DNAzymes to be viable in the cell environment, they must be resistant to chemical insult. This project investigates the consequences of the spontaneous deamination of cytosine on the activity and selectivity of Dz10-23 by replacing such residues in the catalytic core with uracil and observing changes in catalytic activity of mRNA cleavage. Our study demonstrates that, in the presence of a DNA/RNA chimeric substrate with a single RNA base incorporated into the dinucleotide cleavage junction, the Dz10-23 uracil mutants have a reduced activity when compared to the wild type and require the addition of incredibly high concentrations of Mg<sup>2+</sup> (500mM) and Mn<sup>2+</sup> (10mM). We also show that, when the amount of RNA bases in the substrate is increased, the reactions are extremely accelerated and are efficient at much lower, physiologically relevant metal ion concentrations. Alteration of the substrate RNA content also causes mutations at specific positions to become more or less sensitive to deamination damage. To fully understand the potential impact of DNA damage on mRNA cleavage, we report an exploration of how deamination impacts substrate sequence selectivity. These results indicate that substrate composition plays as equally vital of a role in cleavage efficiency as the DNAzyme core sequence. By comparing our results to the recently determined structure of Dz10-23, we provide new insight into the residues integral for the cleavage reaction and their possible mechanistic roles. The results of this project clearly indicate that the effects of spontaneous deamination of cytosine, and likely other forms of DNA damage, must be considered when designing therapeutic DNAzymes.

Investigating the effects of uracil damage on the therapeutic potential of DNAzymes

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