UBC Theses and Dissertations
Increasing the chemical functionality of DNA enzymes Lam, Curtis Hon Ming
Deoxyribonucleic acid (DNA) has long been known as the storage of genetic material. DNA is chemically stable and predominantly found as a double helix of antiparallel complementary strands. Due to the development of in vitro selection techniques, artificial single-stranded DNA molecules have been discovered that can catalyze a range of reactions including the sequence-specific cleavage of ribonucleic acids (RNA). Such DNA enzymes (DNAzymes) are being studied for the in vivo cleavage of mRNA. Compared to protein enzymes, DNAzymes have far fewer functional groups to employ for catalysis. In order to increase the chemical functionality of DNAzymes, eight modified nucleoside triphosphates were synthesized: three dUTP’s modified at the 5-position with either a phenol, phenylboronic acid or guanidinium and five dATP’s modified at the 8-position with imidazoles that are attached with linkers of various composition, length and flexibility. After the synthesis of the modified nucleotides, the incorporation of seven of them into oligonucleotides by DNA polymerases was studied. Under primer extension conditions, the guanidinium- and phenol-modified dUTP’s were found to be good substrates for several DNA polymerases including Pfu (exo–) and Vent (exo–). Under PCR conditions, both of the modified nucleotides gave rise to properly sized products as well. The modified dATP’s, however, were found to be very poor substrates. Only two of them were incorporated by Sequenase V2.0. After establishing conditions under which the modified nucleotides could be incorporated, the phosphoramidite of the guanidinium-modified nucleotide was used for the solid phase synthesis of oligonucleotides, and the phenol-modified nucleotide was used in an in vitro selection. Oligonucleotides containing the guanidinium groups were found to exhibit enhanced duplex stability. The guanidinium-modified nucleoside phosphoramidite was also used to synthesize variants of the divalent metal cation-dependent DNAzyme, 10-23. Variants containing guanidinium groups in the substrate binding regions were found to display reduced rates of RNA cleavage. Using the phenol-modified dUTP, an in vitro selection process gave rise to DNAzyme STA17, isolated from the eleventh generation, that could catalyze self-cleavage in the presence of divalent metal cations such as Mg⁺², Zn⁺² and Mn⁺². This DNAzyme was found to be inhibited by Hg⁺².
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