UBC Theses and Dissertations
Quantifying and modeling the melting thermodynamics of chemically modified duplex DNA Fakhfakh, Kareem
Biological reagents that bind a target selectively and with high affinity are widely used as recognition molecules within diagnostic assays and as therapeutics, among other applications. By leveraging their Watson-Crick base pairing ability, short DNA oligonucelotides represent one class of such biological agents that is particularly well suited to analyzing specific elements of the human genome. Such analyses are routinely used by clinics to detect and manage disease, and those analyses are increasingly providing the richer data content and improved performance necessary for effective clinical decision-making by employing chemically modified nucleic acids. To date, the use of these unnatural nucleotides has largely been achieved empirically, but their growing use is motivating the development of new tools and guidelines that accelerate and improve their implementation in novel assays. This thesis describes how two experimental methods may be tailored to accurately measure the melting thermodynamics of short duplex DNA containing chemical modifications – specifically locked nucleic acids (LNAs) – and then reports on a study that used those methods to measure the thermal stabilities of a large panel of DNA duplexes containing LNA substitutions in one or both strands. Those data and insights gleaned from them are used to extend a molecular thermodynamic model, the “Single Base Thermodynamic” (SBT) model, to enable accurate predictions of the melting thermodynamics of short B-form DNA duplexes containing i) LNA:LNA base pair and/or ii) oppositely oriented LNA:DNA base pair structures. It is the only thermodynamic model with this ability, and its value is demonstrated through its use to guide the development of a entirely new type of quantitative real-time PCR based diagnostic assay – in this case directed against clinically relevant BRAFV600 mutations in cancer – that improves upon commercially available assays by bettering their throughput and limit of detection.
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