Development of Novel ssDNA Aptamers for Detection of Receptor-Binding Domain of SARS-COV-2.

Publication date: Jun 17, 2025

The highly virulent and transmissible SARS-CoV-2 causes COVID-19 and poses a global public health threat. Herein cloned, expressed, and the molecular weight of the receptor-binding domain (RBD) of the SARS-CoV-2 gene encoding protein was confirmed by SDS-PAGE electrophoresis and Western blot analysis. The pivotal aim is to develop single-stranded DNA (ssDNA) aptamers for the rapid detection of SARS-COV-2 infections in humans. In this investigation, a library of nine novel ssDNA aptamers was developed by several rounds of systematic evolution of ligands by an exponential enrichment approach and assessed by an enzyme-linked aptamer assay for binding affinity against RBD antigen (Ag). An in vitro assay resulted in a varied colorimetric signal that depends on the nature of aptamer. Quantitative determination of AptRBD3, AptRBD6, and AptRBD8 aptamers exhibited excellent binding affinity against Ag in the range of 5-10 ng/mL. The putative AptRBD3, AptRBD6, and AptRBD8 aptamers were converted into peptide sequences and docked against RBD, exhibiting good binding energy of -6. 8, -6. 3, and -7. 1 kcal/mol respectively, which were recorded. Furthermore, docking studies of ssDNA aptamers were performed using HDOCK web server to ascertain the binding mechanism and docking score perceived as -389. 74, -404. 28, and -390. 37. Despite this, we engineered a high-affinity AptRBD3. 3 aptamer that formed a single and bulged loop, which improved binding affinity, resulted in a docking score of -361. 56, and exhibited sensitivity at 4 ng of Ag of SARS-CoV-2. Moreover, computational modeling of AptRBD3. 3 revealed an intriguing significant binding affinity with the RBD mutant SARS-CoV-2 S-UK variant (PDB ID: 7EDG) with a docking score of -350. 21. In conclusion, the AptRBD3. 3 aptamer can be used for the development of lateral flow device and electrochemical sensors for rapid, low-cost, and accurate detection of COVID-19 infection in humans for point of care diagnostics.

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Semantics

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