Polydispersion control in enzymatic polymerization of oligonucleotides

• Autor(a) principal: Cordeiro, Ana Alexandra Barrinha
• Data de Publicação: 2011
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/7835
• topic: Biotecnologia; Polimerização; Cinética química; Dispersão

The main goal of this work is the development of a stochastic-based kinetic model to predict the polydispertion in enzymatic polymerization reactions. The fact that the polymerization occurs simultaneously in several enzymatic sites which possess different processivities results in a wide size distribution with different product sizes. To besto control the observed polydispersion, the following was considered: the enzyme selected to this study was Terminal Deoxynucleotidyl Transferase (TdT) as it does not require a template sequence to catalyze DNA synthesis; and 4-methylcoumarin derivatives were the selected molecules to act as caging agents for different nucleotides making them inaccessible to the enzyme. The nucleotide is then released by light due to the break of the phosphodiester bond between the two molecules when submitted to monochromatic radiation. The referred system would bring a unique possibility of creating oligonucleotides in solution with a desired sequence. Different reaction stoichiometries were tested by the incorporation of deoxynucelotides and ribonucleotides into an oligonucleotide initiator, where distinct incorporation patterns were identified. Irradiation studies and controls for the incorporation reaction of DEACM-ATP, the cage molecule for ATP, were successfully performed. A comparison was established between the mathematic model data obtained through simulation and the experimental data related to the incorporation experiments where the polydispersion effect was observed. From this study resulted the observation of distinct incorporation velocities for each nucleotide. Several hypothesis were put together to justify why the adenine profile was so different from other nucleotides. The enzymesubstrate complex stability was considered to be one of the hypotheses. This fact can be related to the biologic function of TdT due to the presence of a “lariat-like” loop that is supposed to be related to the correct positioning of the incoming nucleotides.