Expanding quantitative tools for secondary structure analysis of DNA aptamer candidates selected via CompELS

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Sullivan, Richard Stanley
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Aptamers are single stranded RNA or DNA oligonucleotides with high affinity and specificity for a particular non-nucleotide target. Historically, aptamers are selected from combinatorial libraries of 10E12-10E15 sequences through an evolutionary in vitro selection and screening processes for targets ranging from ions and small molecules to proteins and even whole cells. Aptamers have persistently garnered interest as recognition-based capture agents, particularly for biologically relevant targets. Though sequencing tools have significantly expanded to yield large data sets among aptamers with an evolutionary selection pathway, analytical tools to assess self-hybridized segments within sequences are lacking, particularly for DNA aptamers. This thesis focuses on developing analytical tools to systematically identify, quantify, and classify secondary structure elements (SSE) and corresponding secondary structures families (SSF) in aptamer sequences for comparison to a large random sequence population. Key differences are found in the distribution, location, and base length of SSE such as single-stranded segments and hairpins among the aptamers compared to the population background of random sequences that may point to motifs to include in future screening libraries. Importantly, since aptamers in this work were identified through a nonevolutionary screening approach we call CompELS (Competition-Enhanced Ligand Selection), these tools for determining secondary structure patterns do not presume an evolutionary connection in the primary structure of the aptamer sequences and thus may broadly serve as a useful structural analysis tool for DNA sequences. Gold nanorods (AuNR) serve as the first target for CompELS-based aptamer screening with analytical tool development focusing on the 42 resulting aptamer sequences; however, this thesis also demonstrates the successful adaption of CompELS into a two-stage screening platform for either reduced or oxidized mitogen-activated protein kinase (MKP3red and MKP3ox).
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