Using nano-luciferase binary (NanoBiT) technology to assess the interaction between viral spike protein and angiotensin-converting enzyme II by aptamers

Authors: LIN, MENG-WEI - National Yang-Ming University; LIN, CHENG-HAN - National Yang-Ming University; CHIANG, HUA-HSIN - National Yang-Ming University; Quintela, Irwin; Wu, Vivian; LIN, CHIH-SHENG - National Yang-Ming University

Submitted to: BioTech
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/13/2025
Publication Date: 3/15/2025
Citation: Lin, M., Lin, C., Chiang, H., Quintela, I.A., Wu, V.C., Lin, C. 2025. Using nano-luciferase binary (NanoBiT) technology to assess the interaction between viral spike protein and angiotensin-converting enzyme II by aptamers. BioTech. 14(1). https://doi.org/10.3390/biotech14010020.
DOI: https://doi.org/10.3390/biotech14010020

Interpretive Summary: The Coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has driven significant advancements in diagnostic and therapeutic strategies, particularly for rapid and precise detection of viral components and interactions. Understanding protein-protein interaction (PPI), especially those involving viral proteins and their host counterparts, is essential for developing effective countermeasures. Aptamers or single-stranded DNA (ssDNA) or RNA molecule have been selected for their ability to bind specific targets with high affinity, are valuable tools for detecting and modulating PPI. Numerous studies have identified aptamer sequences specific to SARS-CoV-2 through in silico analysis, green fluorescent protein (GFP), and luciferase-based selection methods. However, there remains a need for more sensitive and accurate biosensors to validate these aptamers for SARS-CoV-2 applications comprehensively. The current study systematically elucidated the difference among NanoBiT, GFP, and luciferase-based pseudoviral systems through a direct comparative analysis to assess the interaction between viral spike protein and angiotensin converting enzyme II by aptamer. The NanoBiT technology emerged as the superior platform, demonstrating significantly higher luminescence intensity and markedly shorter detection times as compared to GFP and luciferase systems. This enhanced performance is attributed to the robust bioluminescent signal generated by the NanoBiT system, which facilitates high sensitivity and rapid assay readouts. Furthermore, integrating NanoBiT with aptamer-based approaches synergistically combines precise detection of PPI with the capability to identify and develop targeted inhibitors for virus infection. This dual functionality not only improves the accuracy and efficiency of molecular interaction studies but also provides a versatile platform for advancing SARS-CoV-2 diagnostics and therapeutics interventions. By addressing the limitations of existing methodologies, NanoBiT offers a transformative tool that enhances our ability to manage current viral challenges and fortify preparedness against future pandemics.

Technical Abstract: Nano-luciferase binary technology (NanoBiT)-based pseudoviral sensors are innovative tools for monitoring viral infection dynamics. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells via its trimeric surface spike protein, which binds to the human angiotensin-converting enzyme II (hACE2) receptor. This interaction is crucial for viral entry and serves as a key target for therapeutic interventions against Coronavirus disease 2019 (COVID-19). Aptamers, short single-stranded DNA (ssDNA) or RNA molecules, serve as highly specific and high-affinity bio-recognition elements for detecting infective pathogens. Despite their potential, optimizing viral infection assays using traditional protein-protein interaction (PPI) methods often face challenges in optimizing viral infection assays. In this study, aptamers were selected and evaluated for their ability to interact with viral proteins, enabling dynamic visualization of in-fection progression. The NanoBiT-based pseudoviral sensor demonstrated a rapid increase in luminescence within 3 h, offering a real-time measure of viral infection. A comparison of detection technologies, including green fluorescent protein (GFP), luciferase and NanoBiT technologies for detecting the PPI between the pseudoviral spike protein and hACE2, highlighted NanoBiT’s supe-rior sensitivity and performance, particularly in aptamer selection. This bioluminescent system provides a robust, sensitive, and early-stage quantitative approach for studying viral infection dynamics.