UBC Theses and Dissertations
Generation of modified DNA aptamers toward a complex whole cell target and investigations into improving selection methodology with modified DNA Miller, Emily W.
Since the discovery was made that DNA is capable of functions beyond genetic storage and propagation, much work has been done to explore the non-canonical abilities of this molecule. Two functionalities of DNA are of particular interest. The first is the ability of DNA to act as a catalyst, and the second is the ability of the molecule to bind with high affinity to various biological and non-biological surfaces. Although numerous DNA catalysts and binders (termed “aptamers”) have been identified using the five nucleoside triphosphates found in nature, the functionality of the molecule can be expanded with the addition of protein-like functional groups to the base moiety of the nucleotide. Several modified nucleotides have been developed previously and used to discover novel catalysts and aptamers. However, the successful development of modified-DNA aptamers has thus far been limited, particularly with respect to biological targets that might be of use in a clinical or commercial context. Moreover, technical challenges in the field exist that have not yet been adequately addressed. Most significantly, the employment of the modified molecules in the discovery process can be problematic as they are not ideal substrates for many basic molecular biology procedures, notably polymerase chain reaction. New techniques to overcome these difficulties are needed and few have been developed. This thesis will focus on two aspects of modified DNA research. The first is the application of modified DNA in aptamer discovery. Starting from the hypothesis that additional functional groups will confer chemical advantages in traditionally challenging aptamer selections, the objective was to identify a modified DNA aptamer for whole bacterial cells in vitro. Several promising phenol-dUTP-modified aptamer sequences were identified using a modified SELEX procedure. The specificity and affinity of these sequences for the target bacterial strain were investigated. The second aim of this work is to address technical problems encountered when working with modified DNA in order to develop catalysts or aptamers. Specifically, a novel selection scheme was designed that eliminated the need to amplify modified DNA. Research was conducted to develop and validate this selection system as a viable route to discovery of DNA catalysts.
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Attribution-NonCommercial-NoDerivs 2.5 Canada