Theranostics 2022; 12(12):5522-5536. doi:10.7150/thno.74428 This issue

Research Paper

DNA aptamers inhibit SARS-CoV-2 spike-protein binding to hACE2 by an RBD- independent or dependent approach

Achut Prasad Silwal, Siddhartha Kalpa Samadhi Thennakoon, Satya Prakash Arya, Rick Mason Postema, Raunak Jahan, Chien Minh Tran Phuoc, Xiaohong Tan

Department of Chemistry and Center for Photochemical Sciences, Bowling Green State University, Bowling Green, Ohio 43403, United States.

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Silwal AP, Thennakoon SKS, Arya SP, Postema RM, Jahan R, Phuoc CMT, Tan X. DNA aptamers inhibit SARS-CoV-2 spike-protein binding to hACE2 by an RBD- independent or dependent approach. Theranostics 2022; 12(12):5522-5536. doi:10.7150/thno.74428. Available from

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Graphic abstract

Objective: Nobody knows when the COVID-19 pandemic will end or when and where the next coronavirus will outbreak. Therefore, it is still necessary to develop SARS-CoV-2 inhibitors for different variants or even the new coronavirus. Since SARS-CoV-2 uses its surface spike-protein to recognize hACE2, mediating its entry into cells, ligands that can specifically recognize the spike-protein have the potential to prevent infection.

Methods: We have recently discovered DNA aptamers against the S2-domain of the WT spike-protein by exploiting the selection process called SELEX. After optimization, among all candidates, the aptamer S2A2C1 has the shortest sequence and the best binding affinity toward the S2-protein. More importantly, the S2A2C1 aptamer does not bind to the RBD of the spike-protein, but it efficiently blocks the spike-protein/hACE2 interaction, suggesting an RBD-independent inhibition approach. To further improve its performance, we conjugated the S2A2C1 aptamer with a reported anti-RBD aptamer, S1B6C3, using various linkers and constructed hetero-bivalent fusion aptamers. Binding affinities of mono and fusion aptamers against the spike-proteins were measured. The inhibition efficacies of mono and fusion aptamers to prevent the hACE2/spike-protein interaction were determined using ELISA.

Results: Anti-spike-protein aptamers, including S2A2C1 and S1B6C3-A5-S2A2C1, maintained high binding affinity toward the WT, Delta, and Omicron spike-proteins and high inhibition efficacies to prevent them from binding to hACE2, rendering them well-suited as diagnostic and therapeutic molecular tools to target SARS-CoV-2 and its variants.

Conclusions: Overall, we discovered the anti-S2 aptamer, S2A2C1, which inhibits the hACE2/spike-protein interaction via an RBD-independent approach. The anti-S2 and anti-RBD aptamers were conjugated to obtain the fusion aptamer, S1B6C3-A5-S2A2C1, which recognizes the spike-protein by an RBD-dependent approach. Our strategies, which discovered aptamer inhibitors targeting the highly conserved S2-protein, as well as the design of fusion aptamers, can be used to target new coronaviruses as they emerge.

Keywords: SARS-CoV-2, DNA aptamer, S2-protein, fusion aptamer, spike-protein