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

Abstract

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.

1

Receptor-binding (RBD) domain of SARS-COV-2 protein purification and confirmation. A: illustrates SDS-PAGE analysis of RBD proteins of SARS-CoV-2, Lanes EL1, EL2, EL3, and EL4 are eluates 1, 2, 3, and 4 of RBD proteins. Lane M: Protein molecular weight marker. B: illustrates SDS-PAGE of Western blot analysis of RBD proteins in different elutions. EL1 and EL2 were detected in the presence of anti-His-tag antibodies. Lane 1: elution 1 (EL1), Lane 2: elution 1 (EL2), Lane M: protein ladder.

2

PCR amplification of the SELEX product resolved onto 2% agarose gel electrophoresis. Lane 1: Ladder 100 bp, Lane 2: Both forward and reverse primers, and lane 3: Eluted DNA pool has shown a specific amplicon at ∼80 bp.

3

Conversion of ssDNA from dsDNA using lambda exonuclease treatment: Lane M: DNA ladder; Lane 1: dsDNA; and lane 2: ssDNA.

4

Qualitative assessment of aptamers binding against RBD antigen using an enzyme-linked aptamer assay. The background control (BC) shows the absorbance containing only the RBD antigen.

5

Quantitative assessment of novel ssDNA aptamers for detection of RBD antigen using an enzyme-linked aptamer assay.

6

Quantitative assessment of promising aptamers for detection of RBD Antigen using 200 ng of aptamers.

7

Validation of cross reactivity of promising ssDNA aptamers against various nonspecific antigens (E. coli protein/lysate, OVA, BSA, and IgG) using enzyme-linked aptamer assay.

8

Visualization of docked structure of ssDNA aptamers against RBD Domain (PDB ID-6VSB) of SARS-CoV-2 with AptRBD3, AptRBD6, AptRBD8, and AptRBD3.3 as in A, B, C, and D, respectively.

9

Qualitative determination of AptRBD3.3 aptamer for detection of RBD antigen. Well 1 represents background control (only aptamer without RBD), well 2 indicates negative control aptamer (only RBD without aptamer), and wells 3–12 correspond to 1–10 ng of RBD antigen of SARS-CoV2.

10

Illustrates the docked structure of the SARS-CoV-2 S-UK variant (PDB ID: 7EDG) with AptRBD 3.3.