| Summary: | DNAzymes are strands of catalytic DNA first discovered in 1994. These species are isolated through in vitro selection and are capable of catalyzing many different types of reactions. RNA-cleaving DNAzymes are one subset that have many biological implications; however, more work needs to be done to make them suitable for therapeutic in vivo applications. Modifying DNAzymes with protein functionalities represents a promising strategy to evolve efficient cleavage in vivo.
Chapter 2 described the syntheses of five modified 2'-deoxyuridine triphosphates and the enzymatic incorporation of these modified dUTPs. The modifications were introduced at the 5-position, and consist of a carboxylate group, indole group and napthyl group. The enzymatic incorporation of these modified nucleotide triphosphates evaluated their suitability for use in an in vitro selection. It was found that Vent (exo-) DNA polymerase was able to incorporate all the modified dUTPs successfully.
In Chapter 3 and Chapter 4, two all-RNA-cleaving DNAzyme selections were described. DNAzyme clone 25 was selected in Chapter 3 against an HIV RNA target, which had a self-cleavage rate constant of 3.3 min-¹. However, when the DNAzyme was tested for intermolecular cleavage activity, the result was unsatisfactory. It was found that the maximum rate constant had not been reached under 2 µM substrate, indicating a low substrate binding affinity. With this disappointing result, DNAzyme clone 25 was not considered for in vivo studies.
Conversely, DNAzyme clone 11 was selected in Chapter 4 and displayed a robust trans-cleavage activity and a high binding affinity towards a c-Myc oncogene target sequence. DNAzyme clone 11 was obtained from this process, which had a self-cleavage rate constant of 0.84 min-¹. The intermolecular cleavage study showed that it had a cleavage kmax of 4.3 min-¹ and Km of 297 nM. The DNAzyme was then shown to be highly sequence-specific. Solid-phase synthesis of the modified DNAzyme was attempted, and the crude oligonucleotide mixture obtained showed trans-cleavage activity. Lastly, Chapter 5 described several failed DNAzyme selections in which no promising active strands were obtained. === Science, Faculty of === Chemistry, Department of === Graduate
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