Informatically-informed biochemical characterization of aptamer-protein complexes
Abstract
Nucleic acid aptamers are single-stranded oligonucleotides that fold into unique three-dimensional shapes that typically bind to targets of interest with high specificity and high affinity. However, a significant limiting step after performing an aptamer selection is the characterization of the resulting aptamers to identify those with the desired binding characteristics. By combining aptamer selections with high-throughput sequencing and bioinformatic analyses, we prioritized aptamers based on their enrichment profiles over the course of the selection for further characterization. The work described here uses biochemical techniques and bioinformatics to study aptamer-protein target interactions. We first describe the initial RNA aptamer selection against the assembled HIV-1 capsid lattice and the characterization of aptamer CA15-2. A capsid differentiation selection was then performed starting with the lattice round 15 library to identify subsets of aptamers capable of binding the assembled capsid lattice only or the capsid lattice and hexamer, and we utilized comparative sequence analyses and biochemical approaches to begin elucidating the sequence and structural requirements for aptamer binding to the capsid lattice. Finally, this work describes an aptamer reselection strategy to evaluate the effects of different 2' modifications on a pre-enriched aptamer library using an aptamer library with affinity for HIV-1 reverse transcriptase (RT). While we identified RT aptamers capable of tolerating the three 2' modifications used, we also observed that the presence of 2'-fluoro-modified pyrimidines caused all RNA sequences, including non-binding sequences, to bind and inhibit RT. The results described in this dissertation provide new strategies and insights for aptamer selections, bioinformatic analyses of aptamer populations from high-throughput sequencing, and biochemical characterization of aptamer-target interactions.
Degree
Ph. D.