Development And Characterization Of High-Throughput Methods And Technologies For In Vitro Rna Aptamer Selections
Aptamers are an emerging class of molecules that are valued for their ability to bind with high affinity and specificity to a desired target. These DNA or RNA ligands can be synthesized easily in the lab making them much more stable, reproducible, and cost-effective than other affinity reagents such as antibodies. These molecules possess a diverse and versatile set of abilities upon binding, making them useful not only in biotechnology and diagnostics, but also in therapeutics. Aptamers are typically generated through an in vitro process called SELEX (Systematic Evolution of Ligands by EXponential Enrichment). SELEX is an iterative process whereby a library of random sequences is exposed to a target, and bound sequences are selected and amplified making a pool enriched for improved binding over the original library. This process can be repeated until the best binding sequence dominates the pool. In this way, aptamers can be generated to nearly any conceivable target or molecule, overcoming the immunological limitations in generating antibodies. By manipulating the conditions within this selection process, aptamers can also be generated to bind in non-physiological environments, giving them the ability to perform in diverse applications. To help accelerate the discovery of aptamers, we developed a modular microcolumn technology that decreases reagent consumption, while permitting multiplex SELEX, allowing for more sophisticated selection schemes. By characterizing aptamer enrichments for all the available parameters, we have significantly increased the selection efficiency, while illustrating the failures of simple binding theories to non-classical modes of selection. We have also demonstrated the power of high-throughput sequencing for early identification of aptamers before they fully converge. This was used to validate a new method which optimizes selection time over individual selection cycle efficiencies. Surprisingly, our results also show the new method significantly improves selection efficiency, bringing into question some common beliefs derived from SELEX theory. Finally, we scaled our microcolumn technology to an automatable 96-well microplate format, and demonstrated its utility by characterizing and validating specific, non-specific, and background binding behavior of several RNA aptamers. The results reveal binding behaviors that fundamentally limit the performance and sensitivity of aptamer selections.
Aptamer; high-throughput; SELEX
Craighead, Harold G
Lis, John T; Zipfel, Warren R.; Muller, David Anthony
Ph. D., Applied Physics
Doctor of Philosophy
dissertation or thesis