A pursuit of self-cleaving RNase-A mimicking DNAzymes without an imidazole functionality and their further characterization

DNAzymes are catalytic DNA molecules that resemble ribozymes and, to a lesser extent, protein-based enzymes. Many of the known DNAzymes have been selected in the presence of metal ions (M²⁺) that are present at non-physiological levels. As a result, there has been much less interest in developing a candidate DNAzyme for therapeutic use. M²⁺-independent DNAzymes are intriguing as they can function even under the low magnesium (Mg²⁺) concentrations usually found in an intracellular environment. Previous works in our lab relied on the use of DNAzymes containing three or two modified nucleosides such as an amine (lysine side chain), a guanidine (arginine side chain) and an imidazole (histidine side chain) with an emphasis on the imidazole group to select metal-independent DNAzymes resembling the RNase A mechanism. Using the functional group modifications they have been able to select several fast cleaving DNAzymes. However, to date there has not been a systematic study on the necessity of all three modifications (in particular the imidazole) as the value of each nucleoside have been contextualized at the beginning of the selection. I hypothesized that two of the positively charged dNTP analogs, excluding the imidazole, i.e. 5-aminoallyl-2'-deoxycytidine triphosphate (dCªªTP) and 5-guanidinoallyl-2'-deoxyuridine triphosphate (dUᵍªTP), are sufficient to select in-vitro self-cleaving DNAzymes in the absence of Mg²⁺ ions in a pH-independent fashion. I was able to identify two DNAzyme candidates that performed the best (Dz11-23 and Dz11-33). The DNAzymes appeared to show a monophasic rate constant (~0.03 min⁻¹) on-par with previous selections without the imidazole; when the DNAzyme species were inquired using a biphasic kinetics model two phases were identified with one of the phases showing a rate constant that was similar to the fast phase in the imidazole containing DNAzymes (kcat= 0.7-0.9 min⁻¹). When studied over different pH values and Mg²⁺ concentration the DNAzymes also appeared to show a self-cleavage activity that was independent of pH and Mg²⁺ with an optimal temperature around 25 °C and sufficiently high cleavage observed even at 37 °C. The results in the thesis support my hypothesis that the imidazole moiety may be redundant in selecting fast cleaving metal-independent DNAzymes.Science, Faculty ofGraduat