Date of Award
Doctor of Philosophy
Plant Pathology and Microbiology
Genetics and Genomics
Steven A. Whitham
Salicylic acid (SA) is a phenolic phytohormone regulating immune responses against pathogens. SA and its derivatives can be found in diverse food products, medicines, cosmetics and preservatives. While salicylates have potential disease-preventative activity, they can also cause health problems to people who are hypersensitive. The current SA detection methods are costly, labor-intensive and require bulky instruments. In my study, a structure-switching aptamer-based nanopore thin film sensor was developed for cost-effective, rapid, sensitive and simple detection of SA. To investigate the mechanism of how immune signal initiates the translational reprogramming I discovered that SA induces stress granules (SGs) formation in vivo. And with the help of the sensor we developed, I found that SA directly binds an SGs marker protein with high affinity and facilitates its phase separation in vitro.
In Chapter 2, I developed an SA sensor based on nanopore thin film sensor and an SA aptamer I screened. SA is a challenging target for aptamer selection using conventional systemic evolution of ligands by exponential enrichment (SELEX) due to its small size and scarcity of reactive groups for immobilization. By immobilizing the SELEX library instead of SA and screening the library using a structure-switching SELEX approach, a high affinity SA aptamer was identified. The dissociation constant of this aptamer to SA was found to be 34.57 nM which suggests a very high affinity. The binding kinetics assay illustrated that it took about 5 minutes for the reaction to achieve equilibrium when 0.2 μM SA was used. To test its binding specificity, several SA structural analogues were also detected by this sensor and we found it can distinguish SA from these structural analogues. Circular dichroism (CD) spectra confirmed the structural change of SA aptamer induced by SA and its binding specificity. The inter-day variation test demonstrated that this sensor has good reproducibility in addition to its high sensitivity and specificity. This platform was employed to measure SA concentrations in plant extracts and it has a similar performance to high performance liquid chromatography. In summary, the integration of SA aptamer and nanopore thin film sensor provides a promising solution for low-cost, rapid, sensitive on-site detection of SA.
In Chapter 3, I explored SA’s role in SGs formation. The quantification of newly synthesized proteins showed that SA represses global translation. This SA-induced translation inhibition was then found to trigger SGs formation. SA was surprisingly detected in the proteins isolated from SGs. And in vitro saturation binding assay using nanoFPI sensor proved that SA binds an SGs marker protein with high affinity. Through turbidity assay, we observed this protein underwent spontaneous phase separation and SA can further promote its phase separation. This reveals a novel SGs formation mechanism and provides certain clues for how immune signal impacts translational reprogramming.
In Chapter 4, I summarized the conclusions and discussed future directions. Taken together, I developed an SA sensor and found that SA triggers SGs formation through directly binding a critical SGs component and inducing its phase separation.
Chen, Changtian, "Development of an aptamer-based nanosensor for SA detection and exploration of the role of SA in stress granules formation" (2019). Graduate Theses and Dissertations. 17424.
Available for download on Friday, July 02, 2021