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P6-9 ᐅ

TOTAL INTERNAL REFLECTION ELLIPSOMETRY FOR THE INVESTIGATION OF SARS-CoV-2 NUCLEOCAPSID PROTEIN AND SPECIFIC ANTIBODY BINDING KINETICS

Silvija Juciute1, Ieva Plikusiene1, 2, Vincentas Maciulis2, Almira Ramanaviciene1, Zigmas Balevicius2, Ernesta Buzavaite-Verteliene2, Evaldas Ciplys1, Rimantas Slibinskas1, Martynas Simanavicius3, Aurelija Zvirbliene3, Arunas Ramanavicius1, 2

1 Institute of Chemistry, Faculty of Chemistry and Geosciences, Vilnius University, Naugarduko 24, Vilnius, Lithuania

2 Laboratory of Nanotechnology, State Research Institute Centre for Physical Sciences and Technology, Sauletekio ave. 3, Vilnius, Lithuania

3 Institute of Biotechnology, Life Sciences Center, Vilnius University, Sauletekio ave. 7, LT-10257 Vilnius, Lithuania

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a virus that causes COVID-19 disease. This illness started global pandemic in late 2019 which is still going. It is extremely important to investigate SARS-CoV-2 proteins structure and their binding mechanisms because this knowledge could help scientists to develop new virus detection and monitoring methods. SARS-CoV-2 virus contains 4 structural proteins: nucleocapsid (N), spike (S), envelope (E) and membrane (M) [1]. N-protein enters host's cell and releases RNA which starts its replication and virus spreads to other cells [2]. Nucleocapsid protein is highly immunogenic and causes strong immune response. Thus, we chose to study N-protein in this investigation.

Various methods can be applied to study kinetics of antigen and specific antibodies binding. In this case, optical methods get special attention. Many of them are nondestructive and label-free which are huge advantages when investigating protein-protein interactions. One of such methods is total internal reflection ellipsometry (TIRE). The real time measurements can be performed and information about protein binding mechanisms can be obtained using TIRE [3].

In this study TIRE was applied for the investigation of SARS-CoV-2 N-protein and polyclonal antibodies against this protein interaction kinetics. The obtained results showed that the two steps binding kinetic model is suitable to describe the immune complex formation. Association and dissociation rate constants, affinity and dissociation constants were evaluated from two-steps binding kinetic model. Calculated thermodynamic properties of such immune complex showed that it has very strict steric requirements. Estimated Gibbs free energy ($\Delta G_{Form}$) was –34 kJ/mol. These findings could be useful for the design of new analytical systems for the determination of specific antibodies formed in the organism after infection, for the development of new SARS-CoV-2 detection methods, and for production of medications that are blocking viral SARS-CoV-2 proteins.

Acknowledgments: This project has received funding from European Social Fund (project No 09.3.3-LMT-K-712-19-0106) under grant agreement with the Research Council of Lithuania (LMTLT).

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[2] K. Narayanan, C.-J. Chen, J. Maeda, S. Makino, Nucleocapsid-Independent Specific Viral RNA Packaging via Viral Envelope Protein and Viral RNA Signal, J. Virol. 77 (2003) 2922–2927.

[3] I. Plikusiene, Z. Balevicius, A. Ramanaviciene, J. Talbot, G. Mickiene, S. Balevicius, A. Stirke, A. Tereshchenko, L. Tamosaitis, G. Zvirblis, A. Ramanavicius, Evaluation of affinity sensor response kinetics towards dimeric ligands linked with spacers of different rigidity: Immobilized recombinant granulocyte colony-stimulating factor based synthetic receptor binding with genetically engineered dimeric analyte d, Biosens. Bioelectron. 156 (2020) 112112.