High-throughput discovery and optimization of high affinity and high specificity aptamers
Abstract/Contents
- Abstract
- Aptamers are synthetic affinity reagents useful for measuring and studying small molecules in biological systems. Unfortunately, the bottleneck in aptamer discovery platforms is the low throughput and high failure rate of canonical aptamer selection methods. To address these issues, I created a high-throughput screening platform to enable multiplexed, simultaneous discovery of specific aptamers against different targets. Canonically, aptamer selections must use techniques such as "counterselection" to ensure aptamer specificity. Unfortunately, using counterselection forces aptamer selections to be single-plex. I developed a platform to identify specific aptamers and forgo the need for techniques such as counterselection. Using a modified a benchtop DNA sequencer, I measured the affinity and specificity for ~10^6 aptamer clusters. I identified rare, specific aptamers that are able to distinguish between small-molecule metabolites differing by a single hydroxyl group. Further, the platform allows us to optimize and utilize existing aptamers previously limited to fixed environmental conditions. Many published aptamers have been discovered under non-physiological environments. However, changing from selection conditions can reduce aptamer affinity and prevents the use of many aptamers. I demonstrated that small mutations in aptamer sequence can stabilize aptamer affinity in physiological conditions. Understanding structure-mechanism relationships that enable aptamer specificity has often been limited to specialized groups. Thus, improving aptamers through rational design guided by structural information has been largely inaccessible. I developed a novel method to investigate aptamer specificity; the pipeline uses experimental data to guide a computational pipeline for reliable structure determination. My work seeks to address the need for high affinity and high specificity aptamers by enabling multiplexed aptamer discovery and broadening aptamer usability for previously discovered aptamers.
Description
| Type of resource | text |
|---|---|
| Form | electronic resource; remote; computer; online resource |
| Extent | 1 online resource. |
| Place | California |
| Place | [Stanford, California] |
| Publisher | [Stanford University] |
| Copyright date | 2023; ©2023 |
| Publication date | 2023; 2023 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Wan, Leighton Terrance |
|---|---|
| Degree supervisor | Soh, H. Tom |
| Thesis advisor | Soh, H. Tom |
| Thesis advisor | Duchi, John |
| Thesis advisor | Huang, Possu |
| Degree committee member | Duchi, John |
| Degree committee member | Huang, Possu |
| Associated with | Stanford University, School of Engineering |
| Associated with | Stanford University, Department of Bioengineering |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Leighton Terrance Wan. |
|---|---|
| Note | Submitted to the Department of Bioengineering. |
| Thesis | Thesis Ph.D. Stanford University 2023. |
| Location | https://purl.stanford.edu/tp231xg1210 |
Access conditions
- Copyright
- © 2023 by Leighton Terrance Wan
- License
- This work is licensed under a Creative Commons Attribution 3.0 Unported license (CC BY).
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