Understand the mechanism of SARS-CoV-2 entry by single-molecule approaches

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and its transmission across the world was the cause of the COVID-19 pandemic. The CoV entry is mainly initiated by the binding of furin cleavage activated spike (S) protein with ACE2 receptor protein on the surface of host cell. Next, S2' cleavage by transmembrane protease serine 2 (TMPRSS2) on the cell surface induce the conformational change of S2 subunit to activate the membrane fusion and entry of virus genetic material. Therefore, S protein has been the major target to design the vaccines and therapeutics. While a handful of investigations have been reported about the structure of S protein:ACE2 complex, critical questions about the detachment of S1/S2 subunits, structural changes in S2 and their dynamics to activate the membrane fusion remain. These answers will help to improve the current understanding of the mechanism of virus entry and should be highly significant for the development of effective preventions and therapeutics. I propose to combine novel DNA nanoswitch calipers (DNC), single-molecule fluorescence integrated optical tweezers, and high-throughput magnetic tweezers to study the dynamics of the molecular events associated with the membrane fusion process of viral entry and analyze the heterogeneity of neutralizing antibodies (nAbs). Using DNC that I developed, we showed the measurement of multiple distances within a target biomolecule at angstrom level precision. Next, we showed the high-throughput measurements in magnetic tweezers to analyze the heterogeneous mixture of peptides. Therefore, DNCs are useful to study multicomponent protein-protein interactions and simultaneously monitor the structural changes associated with the process. By executing these projects, I will have the following three important answers. First, I will measure binding dynamics of full-length S protein and monomer/dimer ACE2 receptor proteins to understand the entire energy landscape governing the interaction of these proteins. Second, I will measure the detachment kinetics of S1/S2 subunits, induced by TMPRSS2 and determine the change in conformation of S2. These reactions are critical to understand the membrane fusion process. Third, I will utilize the similar approach to measure the binding dynamics of nAbs with S protein. By high-throughput measurements in magnetic tweezers, I will analyze the efficacy and heterogeneity of nAbs to understand the immune response of the patients and vaccinated individuals, and also validate DNC assay with a cell-based pseudovirus neutralization assay. Hence, the proposed work will provide detailed insight into understanding the mechanism of SARS- CoV-2 entry and rapid analysis of immune response of the patients. My quantitative approaches and advanced single-molecule approaches will bring insights, into the field of virology and immunology. This study will also give me firsthand experience in biochemistry, molecular biology, virology, and immunology, enabling me to better apply my quantitative and physical training to more topics in biological research in the future.