Improving DNAzyme catalysis through synthetically modified DNAzymes and probing DNA polymerase function to improve selection methodology

• Main Author: Hipolito, Christopher John
• Language: English
• Published: University of British Columbia 2011
• Online Access: http://hdl.handle.net/2429/30659

DNA enzymes (DNAzymes) are part of a growing field of nucleic acid-based catalysts. Unlike ribozymes, DNAzymes have no apparent precedence in nature to date and can only be discovered through in vitro selection. These selections can accommodate both natural and chemically-functionalized nucleotides. Functionalities provide DNAzymes with enhanced novel function or catalytic rates normally not attainable using natural nucleotides. The overall purpose of this thesis is to evaluate and improve the utility of modified nucleotides as components in a DNAzyme selection. This dissertation discusses an updated synthesis of the functionalized 8-(2-(4-imadazolyl)aminoethyl)-2'-deoxyadenosine triphosphate, dAimeTP, that was based on the synthesis used to produce the phosphoramidite analog. dAimeTP bears the imidazole which was crucial in the discovery of several divalent metal cation-independent DNAzymes. A DNAzyme selection using a derivative nucleoside triphosphate, 8-(4-imidazolyl)aminomethyl-2'-deoxyadenosine triphosphate, investigated shortening the linker of the appended imidazole group. The most efficient clone, Dz20-49, was determined to have a catalytic rate of 3.5 ± 0.4 • 10-³ min-¹. In the context of eight different DNAzyme selections, the replication of modified DNA is examined, and it was found that DNA templates modified with 8-(4-imidazolyl)aminomethyl-2'-deoxyadenosine are poorly amplified compared to an oligonucleotide template of the same sequence modified with 8-(2-(4-imidazolyl)aminoethyl)-2'-deoxyadenosine. Also examined was the use of 5-modified dUTP derivatives as substrates in PCR. The resulting doubly-modified dsDNA was used for restriction enzyme digestions and cloning. Restriction sites containing doubly-modified dsDNA were found to be resistant to restriction enzyme digestion. The doubly-modified amplicons were ligated into a vector and transfected into cells. Plasmids copied from the modified dsDNA were sequenced. Fidelity appeared to be maintained through PCR and cell-mediated replication. Due to the limitations of incorporation and read-through of modified nucleotides, steps were taken towards the directed evolution of Thermus aquaticus DNA polymerase I, commonly referred to as Taq, for improved incorporation and read-through of modified nucleotides. Short patch compartmentalized self-replication (spCSR) was chosen for the directed evolution. Three unnatural nucleoside triphosphates targeted for use in the polymerase evolution included 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)thymine 5'-triphosphate, 5-aminoallyl-2'-deoxycytidine triphosphate and 8-(4-imidazolyl)aminomethyl-2'-deoxyadenosine triphosphate.