COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID 19 is detected by RT-PCR tests and serological tests. RT-PCR tests detect viral RNA and require trained individuals to run the test as well as a lengthy analysis time. Serological tests detect antibodies produced in response to viral infection. Rapid antigen detection (RAD) tests, such as the at-home COVID test kits, are quick and easy to run. RAD tests detect viral antigen in the test sample binding to the antibody-coated testing device. However, production of antibodies is a long and costly process. Aptamers can replace antibodies with advantages including low-cost, stability, tunable selectivity, and ability to be chemically modified. Aptamers are short single-stranded oligonucleotides selected for specific targets using Systematic Evolution of Ligands by Exponential Enrichment (SELEX).
This project aims to characterize the binding of aptamers to SARS-CoV-2 nucleocapsid (N) protein using capillary electrophoresis (CE) and compare with bio-layer interferometry (BLI). DNA aptamers were selected via SELEX and screened using BLI in which protein was immobilized on the BLI sensor tip and dipped into aptamer solution. Three aptamers specific to N protein were selected for further binding affinity (Kd) determination.
In CE, the aptamer and protein are free in solution to bind and unbind, providing an alternative approach in characterizing the binding. A greater Kd was observed with CE compared to BLI. Using CE, the apparent Kd of the 3 aptamers was determined to be 18 ± 4 nM, 45 ± 11 nM, and 32 ± 7 nM, respectively. When tested with BLI, the apparent Kd were 4.83 ± 0.63, 4.51 ± 0.87, and 2.91 ± 0.59 nM, respectively. This discrepancy in affinity can be due to steric differences between immobilized (BLI) and in solution (CE) binding, buffer composition and stability of aptamer structures, or buffer pH and difference in electrostatic interactions. All three of these variables will impact binding and the calculated Kd. This work offers insight into aptamer affinity when used in a different system from which they were selected. This work would lead to a better understanding when employing aptamers to different assays and assay mediums.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/45252 |
| Date | 11 August 2023 |
| Creators | Uppal, Gurcharan |
| Contributors | Berezovski, Maxim |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Attribution 4.0 International, http://creativecommons.org/licenses/by/4.0/ |
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