한빛사 논문
Youngwook Ham a,b, Nam-Chul Cho c, Daeyong Kim a,b, Jung-Hee Kim d, Min Ju Jo a,e, Min Seon Jeong a,b, Bo-Yeong Pak d, Sanghyeok Lee d, Mi-Kyung Lee f,g,h, Seung-Wook Chi f,h, Tae-Don Kim i,j, Nak Cheol Jeong d, Sungchan Cho a,b,*
a Nucleic Acid Therapeutics Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Chungbuk, 28116, Republic of Korea
b Department of Biomolecular Science, KRIBB School of Bioscience, Korea University of Science and Technology (KUST), Daejeon, 34113, Republic of Korea
c Korea Chemical Bank, Korea Research Institute of Chemical Technology (KRICT), Daejeon, 34114, Republic of Korea
d AM Science Corp., Gyeonggi, 12902, Republic of Korea
e College of Pharmacy, Chungbuk National University, Chungbuk, 28644, Republic of Korea
f Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea
g Critical Diseases Diagnostics Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea
h Department of Proteome Structural Biology, KRIBB School of Bioscience, Korea University of Science and Technology (KUST), Daejeon, 34113, Republic of Korea
i Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea
j Department of Functional Genomics, KRIBB School of Bioscience, Korea University of Science and Technology (KUST), Daejeon, 34113, Republic of Korea
*Corresponding author: Sungchan Cho
Abstract
The COVID-19 pandemic is an ongoing global public health threat. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and binding of the SARS-CoV-2 spike to its receptor, angiotensin-converting enzyme 2 (ACE2), on host cells is critical for viral infection. Here, we developed a luminescent biosensor that readily detects interactions of the spike receptor-binding domain (RBD) and ACE2 in cell culture medium (‘SpACE-CCM’), which was based on bimolecular complementation of the split nanoluciferase-fused spike RBD and ectodomain of ACE2 and further engineered to be efficiently secreted from cells by adding a heterologous secretory signal peptide (SSP). Screening of various SSPs identified ‘interferon-α+alanine-aspartate’ as the SSP that induced the highest activity. The SpACE-CCM biosensor was validated by observing a marked reduction of the activity caused by interaction-defective mutations or in the presence of neutralizing antibodies, recombinant decoy proteins, or peptides. Importantly, the SpACE-CCM biosensor responded well in assay-validating conditions compared with conventional cell lysate-based NanoLuc Binary Technology, indicating its advantage. We further demonstrated the biosensor's versatility by quantitatively detecting neutralizing activity in blood samples from COVID-19 patients and vaccinated individuals, discovering a small molecule interfering with the spike RBD-ACE2 interaction through high-throughput screening, and assessing the cross-reactivity of neutralizing antibodies against SARS-CoV-2 variants. Because the SpACE-CCM is a facile and rapid one-step reaction biosensor that aptly recapitulates the native spike-ACE2 interaction, it would be advantageous in many experimental and clinical applications associated with this interaction.
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