UBC Theses and Dissertations
Advanced technologies for improved discovery of DNA aptamers and characterization of biologic affinity reagents Ouellet, Eric
Biological reagents that recognize target molecules with high affinity and specificity are widely used as capture agents, diagnostic reagents, and therapeutics. Through their ability to adopt structures that confer binding affinity for a target, aptamers represent one major class of such reagents. However, their use is limited by the general inability of current selection methods to reliably discover high-quality aptamers. Inefficiencies in their selection are due in part to a lack of fundamental understanding of the mechanisms underpinning each step in the screening process. This thesis reports on a series of studies conducted to define the factors and mechanisms currently limiting aptamer selections. That knowledge is then used to create highly effective strategies and technologies for ameliorating each limitation affecting their selection. The resulting collection of improvements is integrated into a novel selection workflow termed “Hi-Fi SELEX”. Those improvements include i) application of a novel “competent library” that eliminates fixed-region interference effects during selection, ii) development of effective chemistries to optimally retain desirable library members, iii) invention of simple methods to accurately quantify retained library diversity and mean binding affinity after each selection round, and iv) development of emulsion PCR methods to eliminate generation of amplification artifacts and v) achieve stoichiometric recovery of the desired single-stranded aptamer library. The resulting discovery platform greatly improves the reliability and speed in which useful panels of lead aptamers against several clinically-relevant targets are discovered. Following initial selection of candidate aptamers based on binding affinity, further screening is typically required, in part to ensure target-specific binding – a performance need shared by antibodies selected against specific targets. However, moderate to high-throughput methods to efficiently screen panels of candidates for binding specificity are lacking. A new technology enabling label-free specificity screening of antibody or aptamer populations at suitable throughputs was therefore established at the proof-of-concept level. The novel microfluidic SPRi arrays described permit multiplexed detection of lead candidates by quantifying both equilibrium binding constants and binding kinetics for each interaction in an element-addressable fashion. The technology offers the ability to independently interrogate candidate affinity reagents and then recover those samples for downstream analysis.
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Attribution-NonCommercial-NoDerivs 2.5 Canada