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Comparative analysis of aptamers binding to SARS-CoV-2 N protein using capillary electrophoresis and bio-layer interferometry

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Abstract

Aptamers are increasingly employed in SARS-CoV-2 theragnostics in recent years. Characterization of aptamers, testing affinity and kinetic parameters (e.g., equilibrium dissociation constant (KD), kon, and koff), can be done by several methods and influenced by many factors. This study aims to characterize the binding of aptamers to SARS-CoV-2 nucleocapsid (N) protein using capillary electrophoresis (CE) and bio-layer interferometry (BLI). These two analytical methods differ by how the aptamer binds to its target protein once the aptamer, as a capture ligand, is partitioned in solution (CE) or immobilized on the biosensor (BLI). With CE, the KD values of the N-binding aptamers (tNSP1, tNSP2, and tNSP3) were determined to be 18 ± 4 nM, 45 ± 11 nM, and 32 ± 7 nM, respectively, while the KD measurements by BLI yielded 4.8 ± 0.6, 4.5 ± 0.5, and 2.9 ± 0.3 nM, respectively. CE results showed a higher KD across all aptamers tested. The differences in the steric hindrance and confirmational structures of the aptamers immobilized on the BLI biosensors versus those suspended in the CE sample solution affect the molecular interactions between aptamers and the target proteins. Moreover, the buffer composition including pH and ionic strength can influence the stability of aptamer structures, or aptamer-protein complexes. All these variables affect the binding and calculated KD. In this sense, a KD value alone is not sufficient to make comparisons between aptamers; instead, the entire experimental setup should also be considered. This is particularly important when implementing aptamers in different bioanalytical systems.

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Funding

This work was supported by the Canadian Institutes of Health Research grant OV1-170353.

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Authors and Affiliations

  1. Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, ON, K1N 6N5, Canada

    Gurcharan K. Uppal, Suttinee Poolsup, Emil Zaripov, Yuxuan Gu & Maxim V. Berezovski

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  1. Gurcharan K. Uppal
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  2. Suttinee Poolsup
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  3. Emil Zaripov
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  5. Maxim V. Berezovski
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Contributions

Gurcharan Uppal and Suttinee Poolsup: data collection, data analysis; writing, reviewing, and editing the original draft. Emil Zaripov and Yuxuan Gu: developing CE instrument. Maxim Berezovski: project conceptualization; reviewing and editing; supervision; funding acquisition.

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Correspondence to Maxim V. Berezovski.

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Gurcharan K. Uppal and Suttinee Poolsup contributed equally to this work.

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Uppal, G.K., Poolsup, S., Zaripov, E. et al. Comparative analysis of aptamers binding to SARS-CoV-2 N protein using capillary electrophoresis and bio-layer interferometry. Anal Bioanal Chem 416, 1697–1705 (2024). https://doi.org/10.1007/s00216-024-05174-3

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