Identification of SARS-CoV-2-against aptamer with high neutralization activity by blocking the RBD domain of spike protein 1

Abstract

Dear Editor, The ongoing outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARSCoV-2) poses a great threat to the public health of people and the normal economic and social development around the world. As of January 8, 2021, more than 88 million people were infected with SARS-CoV-2, resulting in more than 1.9 million death. Early detection and treatment of SARS-CoV-2 are of prime significance for effective control of COVID-19. Currently, nucleic acid amplification (e.g., RT-PCR) and detection of IgG, IgM, or viral antigen are employed in clinical diagnosis, and there is only one drug, remdesivir, was approved for COVID-19 treatment. Although various virus-based and host-based therapeutics, such as remdesivir, lopinavir/ritonavir, umifenovir, ribavirin, chloroquine, hydroxychloroquine, interferon, Tocilizumab, convalescent plasma, neutralizing antibody, and traditional Chinese medicine, have been tested to treat COVID-19, developing novel therapeutic are urgently required. Nucleic acid modalities including aptamer oligonucleotide constitute a promising next-generation solution for disease diagnosis and treatment. Developing an aptamerbased theranostic regimen represents a potentially effective way to control COVID-19. We herein report that six novel DNA aptamers, screened via capillary electrophoresis (CE)-based systematic evolution of ligands by exponential enrichment (SELEX) method, can specifically recognize SARS-CoV-2 in human serum, potently inhibit SARS-CoV-2 infection by blocking the receptor-binding domain (RBD) domain of spike (S) protein subunit 1. The spike protein composed of S1 and S2 subunits, mediating the recognition and infection of SARS-CoV-2. Particularly, the S1 subunit-containing RBD is an important site for host neutralizing antibodies and a promising target for vaccine design. It can interact with the angiotensin-converting enzyme (ACE2) to infect human respiratory epithelial cells. Hence, we screened S1 protein against DNA aptamers, aiming to achieve specific detection and/or effective inhibition of SARS-CoV-2 infection. SELEX of oligonucleotide aptamers of active virulent viruses has extremely high safety requirements for separation equipment and environment. Therefore, in order to increase the feasibility of screening strategies, recombinant S1 protein was thrown to capture the entire SARS-CoV-2. In order to ensure the evolution efficiency of the affinity and specificity of aptamer candidates, we adopted a combined SELEX strategy of the positive, negative, and complex background screening process, and continuously reduced the concentration of S1 to increase the screening pressure to obtain high-performance aptamers (Supplementary Fig. S1). In the final round of screening, the evolved sub-library bound to S1 protein to form a stable complex in human serum. After high-throughput sequencing, the six most enriched aptamer candidates (nCoV-S1-Apts) were selected for performance verification (Supplementary Table S1). The interaction analysis results of capillary zone electrophoresis (CZE) showed that due to the different binding ratios, nCoV-S1Apts formed stable polymorphic complexes with S1 (Fig. 1a), and had nanomolar affinity (KD= 0.118 ± 0.033–85.610 ± 14.219 nM) (Fig. 1b). The interfering substances existing in vivo environments including human serum albumin (HSA), IgG Fc, normal human serum (NHS), and IgG Anti-S1 were introduced to evaluate the specificity of nCoV-S1-Apts. The results showed that the peak area of nCoV-S1-Apt1 did not change significantly during the interaction with the above interfering components (Fig. 1c and Supplementary Fig. S2). The results indicated that nCoV-S1-Apts exhibited stronger specificity and better anti-plasma protein interference properties, which is beneficial to reduce potential off-target effects and cytotoxicity (Supplementary Fig. S3). The affinity and specificity of nCoV-S1-Apt1 were verified by surface plasmon resonance (SPR) (Fig. 1d and Supplementary Fig. S4). In order to determine the possible binding site(s) of nCoV-S1Apt1, we docked nCoV-S1-Apt1 to S1. Visualization of docking possess was performed using PyMOL. The results showed that nCoV-S1-Apt1 residues from 45 to 71 bind to the two main active sites designated for ACE2 binding (Supplementary Fig. S5). Different types of interactions were observed in the S1-aptamer complex, e.g., 32 polar contacts, 20 VdW, 15 H-bonds, 26 hydrophobic, 4 ionic, and 18 aromatic, yielding −12.17 kcal/mol binding free energy with KD value of 0.1 nM. This data suggested that nCoV-S1-Apt1 may be used as a potent inhibitor of the RBD domain of S1. In addition, the binding affinities between nCoV-S1-Apt1 and RBD were determined via CE (Fig. 1e). Data showed that nCoV-S1Apt1 combined with RBD to form stable complexes with a KD of 1.56 ± 0.22 μM. Therefore, we sought to determine the binding kinetics of nCoV-S1-Apts and S1. The KD of nCoV-S1-Apts to S1 is 1.5 × 10 times lower than that of RBD (Supplementary Fig. S6), which indicates that the binding time of nCoV-S1-Apts to S1 is significantly longer than that of RBD. S1 provides a more stable binding site for the aptamer than RBD, thereby greatly extending the binding time and exhibiting the kinetic advantage of drugreceptor interaction, and further helping to significantly improve the interaction potency. To evaluate the recognition and detection potential of nCoV-S1Apt1, we developed an AuNPs colorimetric assay, which could quickly and specifically detect S1 protein in human serum with a detection limit of ~3.125 nM (Fig. 1f, g and Supplementary Fig. S7). Meanwhile, FAM-labeled nCoV-S1-Apt1 was employed as a fluorescent probe to capture S1 protein in human serum (Fig. 1h). The CE laser-induced fluorescence (LIF) analysis results showed that nCoV-S1-Apt1 formed a significant complex with