Using Nano-Luciferase Binary (NanoBiT) Technology to Assess the Interaction Between Viral Spike Protein and Angiotensin-Converting Enzyme II by Aptamers

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, are highly specific, high-affinity biorecognition 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, we selected and evaluated aptamers for their ability to interact with viral proteins, enabling the dynamic visualization of infection 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 PPI between the pseudoviral spike protein and hACE2, highlighted NanoBiT's superior sensitivity and performance, particularly in aptamer selection. This bioluminescent system provides a robust, sensitive, and early-stage quantitative approach to studying viral infection dynamics.

Keywords: angiotensin-converting enzyme type II (ACE2); aptamer; nano-luciferase binary technology (NanoBiT); severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); spike protein.

Figure 1

NanoLuc^® Binary Technology (NanoBiT) is a structural complementation reporter system composed of a large BiT (LgBiT) subunit and a small complimentary peptide (SmBiT). For the study of protein–protein interaction (PPI), the complimentary peptide is small BiT (SmBiT; 11 amino acid peptides, a.a.), which has been optimized to have low affinity for LgBiT (17.6 kDa). The LgBiT and SmBiT subunits are expressed as fusions to target proteins of interest and are expressed in cells. When the two proteins interact, the subunits come together to form an active enzyme and generate a bright luminescent signal in the presence of substrate.

Figure 2

Establishment of the NanoBiT-based pseudoviral system and Western blot analysis of HEK293T/LgBiT-hACE2 cells and SmBiT-BA.2 pseudovirus. (A) Plasmids of Lg-hACE2 and BA.2-SmBiT. (B) Protein expression levels of LgBiT-hACE2, ACE2, and β-actin in HEK293T-hACE2 and HEK293T/LgBiT-hACE2 cells. (C) Viral spike protein in the BA.2 and SmBiT-BA.2 pseudoviruses. β-actin and Coomassie blue staining were used as protein loading controls.

Figure 3

Detections of the luminescent intensity by NanoBiT-based and luciferase-based pseudovirus infection. (A) The bioluminescence imaging of HEK293T/LgBiT-hACE2 cells infected with SmBiT-BA.2 pseudovirus by 3 h, 24 h, and 48 h were shown. (B) The bioluminescence imaging of HEK293T-hACE2 cells infected with BA.2 pseudovirus (Luc) by 3 h, 24 h and 48 h were shown. The NanoBiT- and luciferase-derived luminescence intensity in HEK293T/LgBiT-hACE2 cells was quantified [total flux photons per second and per area (p/sec/cm²) intensity]. Data from all experimental values were shown as the mean ± SD for each group (n = 3). * p < 0.05 and *** p < 0.001 compared with Control group (blank).

Figure 4

The HEK293T-hACE2 cells were infected by GFP-based SARS-CoV-2 pseudovirus. (A) The represented GFP images of HEK293T-hACE2 cells infected with BA.2 pseudovirus (GFP) by 3 h, 24 h, and 48 h were shown. (B) The GFP intensity in HEK293T-hACE2 cells was quantified. Data from all experimental values were shown as the mean ± SD for each group (n = 3). *** p < 0.001 compared with the Control group.

Figure 5

Prediction of the possible secondary structure of the selected aptamers and their free energy. (A) 76-mer aptamer; (B) 67-mer aptamer; (C) 46-mer aptamer. The 76-mer, 67-mer, and 46-mer aptamer presented 2, 3, and 2 typical stem-loop structures and had the lowest ΔG of −4.62, −5.21, and −4.70 kcal/mol, respectively.

Figure 6

Comprehensive analysis of aptamer performance in NanoBiT-based SARS-CoV-2 pseudoviral detection. (A) Inhibition efficiency of the aptamers assessed using the NanoBiT-based pseudoviral detection system. Data from all experimental values were shown as the mean ± SD for each group (n = 3). *** p < 0.001 compared with the Control group. (B) Tertiary structure of the SARS-CoV-2 BA.2 variant spike protein in complex with the 46-mer aptamer. The three putative binding sites of the 46-mer aptamer in spike protein are highlighted in yellow (Model 1), green (Model 2), and red (Model 3).

Figure 7

Confocal microscopy images of pseudovirus-infected HEK293T-hACE2 cells transfected with Cy5-46-mer aptamer. Green fluorescence signal was generated by GFP in cells. Red fluorescence signal was generated by Cy5-labeled aptamer. The nuclei were stained blue with Hoechst.