High-throughput screening methodologies for non-natural aptamers with high affinity and specificity
Keywords: Bioengineering.

High quality, specific affinity reagents are vital for major advances in basic science and medicine. Monoclonal antibodies are the gold standard for affinity reagents. However, there are many targets for which developing antibodies is difficult or impossible. Aptamers, single-stranded nucleic acids that fold into three-dimensional structures and bind specifically to a molecule, are a promising alternative to antibodies. A major limitation for aptamers made of natural DNA and RNA is that their "chemical repertoire" is limited. Furthermore, the process for generating aptamers remains laborious and resource intensive. If these two limitations are addressed, researchers will be able to rapidly generate custom aptamers for many analytes for which antibody generation is not possible. In this dissertation, I describe the development of high throughput methods for screening non-natural aptamers. My first project focused on expanding the chemical repertoire of aptamers via non-natural nucleic acids. I used copper free click particle display to select a novel boronic acid modified aptamer for epinephrine, a small molecule neurotransmitter. This work demonstrates that using tailored functional groups can help generate aptamers for difficult targets, such as small molecules. However, characterizing thoroughly and selecting a final non-natural aptamer is still a major hurdle. My major scientific contribution is the non-natural aptamer array (N2A2), a system that enables the screening of custom functional group modifications on millions of aptamers, thereby 1) increasing the chemical diversity of aptamers using base-modified nucleic acids and 2) accelerating non-natural aptamer discovery by taking advantage of next generation sequencing instrumentation. The N2A2 platform is built on the current Illumina Miseq, with minor hardware and software changes to allow for the introduction of additional reagents. N2A2 will enable researchers to identify the optimal combination of base modification and aptamer sequences for a desired function. To demonstrate the capabilities and power of N2A2, I screened for two critical aptamer characteristics, affinity and specificity. First, I screened aptamers for VEGF with different base modifications, tyrosine and tryptophan, which resulted in aptamers with four-fold higher affinity for VEGF compared to previously published natural DNA aptamers. Affinity enhancement is necessary for aptamers to have a lower limit of detection in biosensor applications. Next, I screened aptamers for specificity using phenylalanine-modified aptamers that bind insulin in the presence of serum. This function enhances the clinical applicability of aptamers, by allowing them to maintain performance in complex biological matrices such as blood or serum. N2A2 can screen 10,000,000 non-natural aptamers for affinity and specificity and return the best modified sequence to the researcher. This system accelerates aptamer discovery from several months to just under two days, which will assist in the development of aptamer biosensors or therapeutics for new targets. In conclusion, N2A2 has expanded our ability to tailor non-natural aptamers for desired performance at an unprecedented scale. Going forward, I see N2A2 as a foundational tool to elucidate the rules governing the sequence to function relationships.