SARS-CoV-2 S1 NanoBiT: A nanoluciferase complementation-based biosensor to rapidly probe SARS-CoV-2 receptor recognition

Abstract

As the COVID-19 pandemic continues, there is an imminent need for rapid diagnostic tools and effective antivirals targeting SARS-CoV-2. We have developed a novel bioluminescence-based biosensor to probe a key host-virus interaction during viral entry: the binding of SARS-CoV-2 viral spike (S) protein to its receptor, angiotensin-converting enzyme 2 (ACE2). Derived from Nanoluciferase binary technology (NanoBiT), the biosensor is composed of Nanoluciferase split into two complementary subunits, Large BiT and Small BiT, fused to the Spike S1 domain of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. The ACE2-S1 interaction results in reassembly of functional Nanoluciferase, which catalyzes a bioluminescent reaction that can be assayed in a highly sensitive and specific manner. We demonstrate the biosensor’s large dynamic range, enhanced thermostability and pH tolerance. In addition, we show the biosensor’s versatility towards the high-throughput screening of drugs which disrupt the ACE2-S1 interaction, as well as its ability to act as a surrogate virus neutralization assay. Results obtained with our biosensor correlate well with those obtained with a Spike-pseudotyped lentivirus assay. This rapid in vitro tool does not require infectious virus and should enable the timely development of antiviral modalities targeting SARS-CoV-2 entry.

Luciferase-Based Biosensors in the Era of the COVID-19 Pandemic

“…Rather than examining RBD–ACE2 binding, this biosensor specifically interrogates the spike S1 domain and ACE2 ectodomain interaction. 124 We characterized the stability, dynamic range, and sensitivity of this biosensor and further showed that it could be used to identify inhibitors of the protein–protein interaction as recombinant RBD and ACE2 could be used as competitive inhibitors to block the interaction and decrease signal intensity. In developing a second biosensor for detecting SARS-CoV-2–host cell binding, we foresee a potential application for the RBD-ACE2 biosensor and S1 domain–ACE2 biosensors to be used in parallel as controls for each other and to investigate specific aspects of this protein–protein interaction.…”

“…Right: Outline of the NanoBiT split luciferase assay for Spike S1 and ACE2 binding upon addition of substrate furimazine. Adapted with permission from ref ( 124 ). Copyright 2021 Elsevier B.V.…”

Detection of antibodies neutralizing historical and emerging SARS-CoV-2 strains using a thermodynamically coupled de novo biosensor system

“…To achieve this goal, we focused on antibodies that interfere with RBD:ACE-2 interactions as a proxy for antibody neutralization 12,13 ( Fig 1a ). We adapted a designed coronavirus spike RBD biosensor 14 consisting of a switchable lucCageRBD protein containing a “cage” domain that in the closed state of the sensor binds a “latch” domain containing the picomolar affinity RBD binding LCB1 protein, and a lucKey protein that binds to the open state of the sensor, reconstituting luciferase activity 14 .…”

“…Unlike previously described competition assays which directly assess the extent of ACE-2:RBD complex formation (by luciferase reconstitution 12 or capturing of enzyme conjugated to one component 13 ) ( Fig 1c ), in this thermodynamic coupling scheme, the RBD is unmodified and free in solution. This simplifies testing reactivity against RBD variants of concern, since no labeling is required.…”

“…Lateral flow antigen tests 9 have been introduced but they are used primarily as binary qualitative tests and report only binding between antibody-antigen rather than neutralization 10 . Recently developed cell-free tools can measure antibody titer, but cannot necessarily evaluate neutralization, and none of the currently available tools have estimated neutralization activity against the emerging set of SARS-CoV-2 VoCs [11][12][13] . We aimed to develop a sensor technology that can quantitatively measure nAb responses against different strains of SARS-CoV-2, be adapted for all-in-solution, multi-well format, and provide rapid results in 30 minutes (faster than established ELISA assays measuring SARS-CoV-2 antibody titer (~6 hours) or cell-based neutralization assays(~several days)).…”