Luciferase Fragments Research Articles

A modular platform for bioluminescent RNA tracking

AbstractA complete understanding of RNA biology requires methods for tracking transcripts in vivo. Common strategies rely on fluorogenic probes that are limited in sensitivity, dynamic range, and depth of interrogation, owing to their need for excitation light and tissue autofluorescence. To overcome these challenges, we report a bioluminescent platform for serial imaging of RNAs. The RNA tags are engineered to recruit light-emitting luciferase fragments (termed RNA lanterns) upon transcription. Robust photon production is observed for RNA targets both in cells and in live animals. Importantly, only a single copy of the tag is necessary for sensitive detection, in sharp contrast to fluorescent platforms requiring multiple repeats. Overall, this work provides a foundational platform for visualizing RNA dynamics from the micro to the macro scale.

Subcellular imaging of MGAT1/MGAT2 homo- and heteromers in living cells using bioluminescence microscopy

Protein-protein interactions (PPIs) play fundamental roles in many biological processes including the functioning of glycosylation machineries present in the endoplasmic reticulum (ER) and Golgi apparatus of mammalian cells. For the last couple of years, we have been successfully employing the most advanced version of the split luciferase complementation assay, termed NanoBiT, to demonstrate PPIs between solute carrier 35 (SLC35) family members with nucleotide sugar transporting activity and functionally related glycosyltransferases. NanoBiT has several unmatched advantages as compared with other strategies for studying PPIs. Firstly, the tendency of the free luciferase fragments to spontaneously associate is strongly reduced. As a consequence, the fragments of the reconstituted luciferase may dissociate upon the disruption of the PPI of interest. Secondly, the recombinant fusion proteins are expressed at low (near-endogenous) levels. Both of these features significantly minimize the possibility of obtaining false positive results. In this study we pushed the boundaries of this already powerful technique even further by coupling it with bioluminescence imaging of PPIs. Specifically, we visualized homo- and heterologous complexes formed by MGAT1 and MGAT2 glycosylation enzymes tagged with NanoBiT fragments and demonstrated ER-to-Golgi transitions between enzyme homo- and heteromers.

Development of Luminescent Biosensors for Calprotectin.

Calprotectin, a metal ion-binding protein complex, plays a crucial role in the innate immune system and has gained prominence as a biomarker for various intestinal and systemic inflammatory and infectious diseases, including inflammatory bowel disease (IBD) and tuberculosis (TB). Current clinical testing methods rely on enzyme-linked immunosorbent assays (ELISAs), limiting accessibility and convenience. In this study, we introduce the Fab-Enabled Split-luciferase Calprotectin Assay (FESCA), a novel quantitative method for calprotectin measurement. FESCA utilizes two new fragment antigen binding proteins (Fabs), CP16 and CP17, that bind to different epitopes of the calprotectin complex. These Fabs are fused with split NanoLuc luciferase fragments, enabling the reconstitution of active luciferase upon binding to calprotectin either in solution or in varied immobilized assay formats. FESCA's output luminescence can be measured with standard laboratory equipment as well as consumer-grade cell phone cameras. FESCA can detect physiologically relevant calprotectin levels across various sample types, including serum, plasma, and whole blood. Notably, FESCA can detect abnormally elevated native calprotectin from TB patients. In summary, FESCA presents a convenient, low-cost, and quantitative method for assessing calprotectin levels in various biological samples, with the potential to improve the diagnosis and monitoring of inflammatory diseases, especially in at-home or point-of-care settings.

Nanobody engineering for SARS-CoV-2 neutralization and detection.

Nanobodies, small protein binders derived from the camelid antibody, are highly potent inhibitors of respiratory viruses that offer several advantages over conventional antibodies as candidates for specific therapies, including high stability and low production costs. In this work, we leverage the unique properties of nanobodies and apply them as building blocks for new therapeutic and diagnostic tools. We report ultra-potent SARS-CoV-2 inhibition by engineered nanobodies comprising multiple modules in structure-guided combinations and develop nanobodies that carry signal molecules, allowing rapid detection of the SARS-CoV-2 spike protein. Our results highlight the potential of engineered nanobodies in the development of effective countermeasures, both therapeutic and diagnostic, to manage outbreaks of emerging viruses.

CRISPR-Based Split Luciferase as a Biosensor for Unique DNA Sequences In Situ.

To date, CRISPR-based DNA targeting approaches have typically used fusion proteins between full fluorescent reporters and catalytically inactive Cas9 (dCas9) for imaging rather than detection of endogenous genomic DNA sequences. A promising alternative strategy for DNA targeting is the direct biosensing of user-defined sequences at single copy with single-cell resolution. Our recently described DNA biosensing approach using a dual fusion protein biosensor comprised of two independently optimized fragments of NanoLuc luciferase (NLuc) directionally fused to dCas9 paired with user-defined single-guide RNAs (sgRNAs) could allow users to sensitively detect unique copies of a target sequence in individual living cells using common laboratory equipment such as a microscope or a luminescence-equipped microplate reader. Here we describe a protocol for using such a DNA biosensor noninvasively in situ.

SAT271 Ligand Induced GR Heterodimerization With MR In Living Cells

Abstract Disclosure: T. Sueyoshi: None. L. Perera: None. J.A. Cidlowski: None. We have established methods for monitoring heterodimer formation between GR and MR in living cells upon the addition of ligands. Two protein fragments derived from NanoBit luciferase (Small Bit peptide composed of 11 amino acids and Large Bit peptide sized 17.6 kDa) can form functional enzyme when protein-protein interaction brings these fragments close proximity. Utilizing the enzyme fragments for fusion protein partners for the hormone receptors, we have constructed expression plasmids for every combination of receptor and enzyme fragments comprising N-terminal or C-terminal fusion proteins. GR and MR fusion proteins with the luciferase fragments were expressed in HEK293 cells and the enzyme activities were determined in time course before and after the addition of ligands (dexamethasone and aldosterone). We observed robust increases of NanoBit luciferase activity in GR and MR transfected cells less than 20 min after the addition of ligands suggesting the heterodimer was formed in the time period. Similar assays detected these receptors formed homodimer after the addition of ligand for each receptor suggesting homodimer and heterodimer co-exists when appropriate ligands are in this assay system. DNA binding null mutations of GR DNA binding domain effectively abolished GR homodimers but not GR - MR heterodimers suggesting mechanistical differences between the homodimer and heterodimer formation. Based on the results only C-terminal NanoBit fusion receptors showed positive heterodimerization-induced enzyme activity, we proposed ligand binding domain 3D structure heterodimer models and found amino acid residues in helix 9, helix 10, and near C-terminus are involved in the heterodimer interaction interface. Thus, this method will be a powerful tool for analyzing molecular mechanisms of GR - MR heterodimer formation in living cells. Presentation: Saturday, June 17, 2023

Intramolecular interaction of NEP regulated by CRM1 ensures the unidirectional transport of M1 for the nuclear export of influenza viral ribonucleoprotein

IntroductionThe influenza virus genome consists of single-stranded RNAs and forms viral ribonucleoprotein (RNP) complexes. After viral genome replication in the nucleus, the viral RNP interacts with viral protein M1. The M1-viral RNP complex is exported to the cytoplasm via the CRM1-dependent pathway using NS2/NEP as an export adaptor protein. NEP is a 14 kDa protein and diffusely localizes in the nucleus and cytoplasm. Upon binding to the NLS motif of M1, NEP inhibits the nuclear accumulation of M1 and promotes the nuclear export of M1-viral RNP complex. However, the detail mechanism by which NEP binds to M1 only in the nucleus remains unclear.MethodsTo visualize the interaction of NEP with M1 in the formation of vRNP export complexes, we performed in situ proximity ligation assays. The close proximity of N-terminal and C-terminal domains of NEP was tested by split Renilla luciferase complementation assays in which the N-terminal and C-terminal fragments of Renilla luciferase were fused to the N-terminus and C-terminus of NEP, respectively.Results and discussionWe found that the intramolecular interaction of NEP inhibits the interaction of NEP with M1. The intramolecular interaction of NEP was mediated through the interaction of the N-terminal NES motif with the M1-binding domain at the C-terminus. By adding leptomycin B, a potent inhibitor of CRM1, the interaction of NEP with M1 was impaired. These results suggest that CRM1 disrupts the intramolecular interaction of NEP by recognizing the NES motif at the N-terminus of NEP, thereby promoting the interaction of NEP with M1. We also found that NEP mutant deficient in the intramolecular interaction was co-localized with M1 at the plasma membrane and did not show nuclear localization with M1. Based on these results, we propose that the intramolecular interaction of NEP regulated by CRM1 ensures the unidirectional transport of M1.

Real-time monitoring of cell surface protein arrival with split luciferases.

Each cell in a multicellular organism permanently adjusts the concentration of its cell surface proteins. In particular, epithelial cells tightly control the number of carriers, transporters and cell adhesion proteins at their plasma membrane. However, sensitively measuring the cell surface concentration of a particular protein of interest in live cells and in real time represents a considerable challenge. Here, we introduce a novel approach based on split luciferases, which uses one luciferase fragment as a tag on the protein of interest and the second fragment as a supplement to the extracellular medium. Once the protein of interest arrives at the cell surface, the luciferase fragments complement and generate luminescence. We compared the performance of split Gaussia luciferase and split Nanoluciferase by using a system to synchronize biosynthetic trafficking with conditional aggregation domains. The best results were achieved with split Nanoluciferase, for which luminescence increased more than 6000-fold upon recombination. Furthermore, we showed that our approach can separately detect and quantify the arrival of membrane proteins at the apical and basolateral plasma membrane in single polarized epithelial cells by detecting the luminescence signals with a microscope, thus opening novel avenues for characterizing the variations in trafficking in individual epithelial cells.

Fast and bioluminescent detection of antibiotic contaminants by on-demand transcription of RNA scaffold arrays

Cell-free biosensors have inspired low-cost and field-applicable methods to detect antibiotic contaminants. However, the satisfactory sensitivity of current cell-free biosensors is mostly achieved by sacrificing the rapidity, which prolongs turnaround time by hours. Additionally, the software-based result interpretation provides an obstacle for delivering these biosensors to untrained individuals. Here, we present a bioluminescence-based cell-free biosensor, termed enhanced Bioluminescence sensing of Ligand-Unleashed RNA Expression (eBLUE). The eBLUE leveraged antibiotic-responsive transcription factors to regulate the transcription of RNA arrays that can serve as scaffolds for reassembling and activating multiple luciferase fragments. This process converted target recognition into an amplified bioluminescence response, enabling smartphone-based quantification of tetracycline and erythromycin directly in milk within 15 min. Moreover, the detection threshold of eBLUE can be easily tuned according to the maximum residue limits (MRLs) established by government agencies. Owing to this tunable nature, the eBLUE was further repurposed as an on-demand semi-quantification platform, allowing for fast (∼20 min) and software-free identification of safe and MRL-exceeding milk samples only by glancing over the smartphone photographs. Overall, the sensitivity, rapidity and user-friendliness of eBLUE demonstrate its potentials for practical applications, especially in resource-limited and household settings.

Abstract 3538: Endogenous HiBiT-tagging of PAX3-FOXO1 identifies potent suppressors of PAX3-FOXO1 protein levels by high-throughput screening

Abstract Background: Oncogenic fusion genes are attractive therapeutic targets due to their tumor-specific expression and driver roles in cancers. PAX3-FOXO1 (P3F) is the dominant oncogenic driver of fusion-positive rhabdomyosarcoma (FP-RMS) with no targeted therapy. We developed methods to directly measure endogenous P3F protein levels amenable to high-throughput drug screens to identify suppressors of P3F. Methods: HiBiT tag, an 11 amino acid peptide of the small fragment of NanoLuc luciferase, was inserted into the endogenous P3F using CRISPR-Cas9 in FP-RMS cell lines RH4 and SCMC. Western analysis was used for HiBiT tag validation and confirmation of P3F suppression. RNA-seq and ChIP-seq were used to assess transcriptomics and DNA binding of HiBiT-tagged P3F (P3F-HiBiT) respectively. High-throughput drug screen using Nano-Glo luciferase assay was performed using the Mechanism Interrogation PlatE (MIPE 5.0) drug library, which included 2,480 drugs with known mechanisms of action. CellTiter-Glo was used to monitor cell viability. We identified drugs that suppressed P3F by Nano-Glo without acute cytotoxicity by CellTiter-Glo at an early 24-hour timepoint. Mouse xenograft model of FP-RMS was used to investigate in vivo efficacy of top hits. Results: We validated HiBiT tagging of P3F and not the wild-type FOXO1 by Western analysis. We showed that the HiBiT tag did not change the function of P3F by transducing human fibroblasts with P3F-HiBiT versus unmodified P3F. Gene Set Enrichment Analysis (GSEA) of RNA-seq showed that P3F-HiBiT activated the same downstream target genes as unmodified P3F. ChIP-seq using HiBiT antibody in HiBiT-tagged FP-RMS cell lines RH4 and SCMC matched the genomic locations from ChIP-seq with P3F antibody in parental RH4 and SCMC. Using a cutoff of Area Under the Curve (AUC) of CellTiter-Glo - AUC of Nano-Glo > 90, in both RH4 and SCMC, identified 182 compounds. Filtering for drugs with ≥ 3 hits for the same target identified 14 drug classes that suppressed P3F protein level including HDAC inhibitors (3), mTOR inhibitors (4), CDK inhibitors (8), and BRD4 inhibitors (3). One top hit was the CDK inhibitor TG02 (Zotiraciclib), currently in human trials. TG02 suppressed P3F protein levels by Nano-Glo and Western analysis. We confirmed induction of apoptosis by PARP cleavage in a panel of FP-RMS cell lines. GSEA analysis of RNA-seq after treatment with TG02 showed marked suppression of P3F target gene sets. TG02 also significantly delayed tumor progression of established tumors in a mouse xenograft model of FP-RMS without weight loss. Conclusion and Future Directions:By HiBiT tagging the fusion oncogene P3F, we identified 182 compounds that suppress P3F levels of which TG02 was a top hit that also showed in vivo efficacy. Drug combination studies are currently underway to identify synergistic suppressors of P3F protein levels that can be translated into clinical trials. Citation Format: Yong Yean Kim, Robert G. Hawley, Mehal Churiwal, Teresa S. Hawley, Christine N. Evans, Raj Chari, David Milewski, Ranuka Sinniah, Young K. Song, Hsien-Chao Chou, Xinyu Wen, Ying Pang, Jing Wu, Craig J. Thomas, Jun S. Wei, Michele Ceribelli, Javed Khan. Endogenous HiBiT-tagging of PAX3-FOXO1 identifies potent suppressors of PAX3-FOXO1 protein levels by high-throughput screening. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3538.

Bioluminescent RIPoptosome Assay for FADD/RIPK1 Interaction Based on Split Luciferase Assay in a Human Neuroblastoma Cell Line SH-SY5Y

Different programed cell death (PCD) modalities involve protein-protein interactions in large complexes. Tumor necrosis factor α (TNFα) stimulated assembly of receptor-interacting protein kinase 1 (RIPK1)/Fas-associated death domain (FADD) interaction forms Ripoptosome complex that may cause either apoptosis or necroptosis. The present study addresses the interaction of RIPK1 and FADD in TNFα signaling by fusion of C-terminal (CLuc) and N-terminal (NLuc) luciferase fragments to RIPK1-CLuc (R1C) or FADD-NLuc (FN) in a caspase 8 negative neuroblastic SH-SY5Y cell line, respectively. In addition, based on our findings, an RIPK1 mutant (R1C K612R) had less interaction with FN, resulting in increasing cell viability. Moreover, presence of a caspase inhibitor (zVAD.fmk) increases luciferase activity compared to Smac mimetic BV6 (B), TNFα -induced (T) and non-induced cell. Furthermore, etoposide decreased luciferase activity, but dexamethasone was not effective in SH-SY5Y. This reporter assay might be used to evaluate basic aspects of this interaction as well as for screening of necroptosis and apoptosis targeting drugs with potential therapeutic application.

A Cellular Assay for Spike/ACE2 Fusion: Quantification of Fusion-Inhibitory Antibodies after COVID-19 and Vaccination.

Not all antibodies against SARS-CoV-2 inhibit viral entry, and hence, infection. Neutralizing antibodies are more likely to reflect real immunity; however, certain tests investigate protein/protein interaction rather than the fusion event. Viral and pseudoviral entry assays detect functionally active antibodies but are limited by biosafety and standardization issues. We have developed a Spike/ACE2-dependent fusion assay, based on a split luciferase. Hela cells stably transduced with Spike and a large fragment of luciferase were co-cultured with Hela cells transduced with ACE2 and the complementary small fragment of luciferase. Cell fusion occurred rapidly allowing the measurement of luminescence. Light emission was abolished in the absence of Spike and reduced in the presence of proteases. Sera from COVID-19-negative, non-vaccinated individuals or from patients at the moment of first symptoms did not lead to a significant reduction of fusion. Sera from COVID-19-positive patients as well as from vaccinated individuals reduced the fusion. This assay was more correlated to pseudotyped-based entry assay rather than serology or competitive ELISA. In conclusion, we report a new method measuring fusion-inhibitory antibodies in serum, combining the advantage of a complete Spike/ACE2 interaction active on entry with a high degree of standardization, easily allowing automation in a standard bio-safety environment.

Experimental and Analytical Framework for "Mix-and-Read" Assays Based on Split Luciferase.

The use of immunodetection assays including the widely used enzyme-linked immunosorbent assay (ELISA) in applications such as point-of-care detection is often limited by the need for protein immobilization and multiple binding and washing steps. Here, we describe an experimental and analytical framework for the development of simple and modular “mix-and-read” enzymatic complementation assays based on split luciferase that enable sensitive detection and quantification of analytes in solution. In this assay, two engineered protein binders targeting nonoverlapping epitopes on the target analyte were each fused to nonactive fragments of luciferase to create biosensor probes. Binding proteins to two model targets, lysozyme and Sso6904, were isolated from a combinatorial library of Sso7d mutants using yeast surface display. In the presence of the analyte, probes were brought into close proximity, reconstituting enzymatic activity of luciferase and enabling detection of low picomolar concentrations of the analyte by chemiluminescence. Subsequently, we constructed an equilibrium binding model that relates binding affinities of the binding proteins for the target, assay parameters such as the concentrations of probes used, and assay performance (limit of detection and concentration range over which the target can be quantified). Overall, our experimental and analytical framework provides the foundation for the development of split luciferase assays for detection and quantification of various targets.

Imaging Unique DNA Sequences in Individual Cells Using a CRISPR-Cas9-Based, Split Luciferase Biosensor.

An extensive arsenal of biosensing tools has been developed based on the clustered regularly interspaced short palindromic repeat (CRISPR) platform, including those that detect specific DNA sequences both in vitro and in live cells. To date, DNA imaging approaches have traditionally used full fluorescent reporter-based fusion probes. Such “always-on” probes differentiate poorly between bound and unbound probe and are unable to sensitively detect unique copies of a target sequence in individual cells. Herein we describe a DNA biosensor that provides a sensitive readout for such low-copy DNA sequences through proximity-mediated reassembly of two independently optimized fragments of NanoLuc luciferase (NLuc), a small, bright luminescent reporter. Applying this “turn-on” probe in live cells, we demonstrate an application not easily achieved by fluorescent reporter-based probes, detection of individual endogenous genomic loci using standard epifluorescence microscopy. This approach could enable detection of gene edits during ex vivo editing procedures and should be a useful platform for many other live cell DNA biosensing applications.

Castanospermine suppresses CD44 ectodomain cleavage as revealed by transmembrane bioluminescent sensors.

Cluster of differentiation 44 (CD44) is a single-pass transmembrane glycoprotein that is a widely distributed cell-surface adhesion molecule. CD44 undergoes ectodomain cleavage by membrane-associated metalloproteinases in breast cancer cells. Cleavage plays a critical role in cancer cell migration by mediating the interaction between CD44 and the extracellular matrix. To explore inhibitors of CD44 ectodomain cleavage, we developed two bioluminescent sensors for the detection of CD44 ectodomain cleavage. The sensors were designed as two-transmembrane proteins with split-luciferase fragments, one of which was cyclized by protein trans-splicing of a DnaE intein. These two sensors emit light by the cyclization or the spontaneous complementation of the luciferase fragments. The luminescence intensities decreased upon cleavage of the ectodomain in breast cancer cells. The sensors revealed that castanospermine, an α-glucosidase inhibitor, suppressed the ectodomain cleavage of endogenous CD44 in breast cancer cells. Castanospermine also inhibited breast cancer cell invasion. Thus, the sensors are beneficial tools for evaluating the effects of different inhibitors.

“Ready-to-use” immunosensor for the detection of small molecules with fast readout

Immunoassays are commonly used methods for detection of small molecules that typically require numerous steps of the labeling between immune-recognition reagents and tracers, immobilization and recurrent washing, making them time consuming and difficult to adapt into point of care formats. Here we describe a “ready-to-use” homogeneous competitive immunosensor with an assay time of 10 min that is based exclusively on recombinant reagents. The signal is produced when the split fragments of the nano luciferase (Nluc) are brought together by the interaction of a heavy chain only variable domain (VHH) with a peptidomimetic of the target small molecule. A VHH to 2,4-dichlorophenoxyacetic acid (2,4-D) was used to isolated the peptidomimetic (NGFFEPWQVVYV) from phage display libraries using six panning conditions. Then the peptidomimetic and VHH were fused with the larger (LgN) and smaller piece (SmN) of split fragments of Nluc, respectively. In order to optimize the signal and sensitivity of the immunosensor, we explored the effects of the spacer between the peptidomimetic and LgN, the copy number of peptidomimetics, and the spacer between SmN and VHH, generating 24 combinations that allowed to conclude on their respective roles. Eventually, the developed “ready-to-use” immunosensor performed excellent signal-to-noise ratio and sensitivity, and could be applied to the detection of 2,4-D in real samples. Meanwhile, the immunosensor totally realizes labeling-free, immobilization-free and washing-free, also can be produced in a highly cost effective way.

Quantitative Imaging of Retinoic Acid Activities in Living Mammalian Cells.

Retinoic acid (RA) is an intriguing metabolite that is necessary for embryonic development and differentiation in vertebrates. The present protocol demonstrates how to image RA activities indirectly in mammalian cells with ligand-activatable single-chain bioluminescence (BL) probes. We introduce 13 different molecular designs for characterizing an efficient single-chain probe that quantitatively visualizes RA activities with significant sensitivity. The key components included in the probes are (i) the N- and C-terminal fragments of artificial luciferase 16 (ALuc16), (ii) the ligand-binding domain of human retinoic acid receptor α (RAR LBD), and (iii) an LXXLL motif derived from common coactivators of nuclear receptors. The probe is highly selective and sensitive to all-trans-RA (at-RA) in animal cells. This protocol exemplifies quantitative imaging of the RA levels in serum and cerebrospinal fluid with a linear range in two orders. The present protocol is an important addition to conventional techniques on quantitative imaging of endogenous at-RA levels in live mammalian cells.

A bioluminescent and homogeneous SARS-CoV-2 spike RBD and hACE2 interaction assay for antiviral screening and monitoring patient neutralizing antibody levels

Here we describe a homogeneous bioluminescent immunoassay based on the interaction between Fc-tagged SARS-CoV-2 Spike RBD and human ACE2, and its detection by secondary antibodies labeled with NanoLuc luciferase fragments LgBit and SmBit. The assay utility for the discovery of novel inhibitors was demonstrated with a panel of anti-RBD antibodies, ACE2-derived miniproteins and soluble ACE2. Studying the effect of RBD mutations on ACE2 binding showed that the N501Y mutation increased RBD apparent affinity toward ACE2 tenfold that resulted in escaping inhibition by some anti-RBD antibodies. In contrast, while E484K mutation did not highly change the binding affinity, it still escaped antibody inhibition likely due to changes in the epitope recognized by the antibody. Also, neutralizing antibodies (NAbs) from COVID-19 positive samples from two distinct regions (USA and Brazil) were successfully detected and the results further suggest the persistence of NAbs for at least 6 months post symptom onset. Finally, sera from vaccinated individuals were tested for NAbs and showed varying neutralizing activity after first and second doses, suggesting the assay can be used to assess immunity of vaccinated populations. Our results demonstrate the broad utility and ease of use of this methodology both for drug discovery and clinical research applications.

Split-enzyme immunoassay to monitor EGFR-HER2 heterodimerization on cell surfaces

Over 30,000 protein-protein interactions with pathological implications have been identified; yet, discovering and investigating drugs that target these specific interactions is greatly limited by the inability to monitor native protein-protein interactions (PPIs) efficiently. The two most frequently used tools to monitor PPIs, resonance-energy transfer (RET) assays and protein complementation assays (PCA), face significant limitations. RET assays have a narrow working range of 10 to 50 Å, while PCA require permanent attachment of a reporter probe to a protein of interest by chemical conjugation or genetic engineering. We developed a non-invasive assay platform to measure PPIs without modifications to the proteins of interest and is functional at a greater working range than RET assays. We demonstrate our approach by monitoring the EGFR-HER2 heterodimerization on relevant cell surfaces, utilizing various EGFR- and HER2-specific binders (e.g., Fab, DARPin, and VHH) fused with small fragments of a tri-part split-luciferase derived from NanoLuc®. Following independent binding of the binder fusions to their respective targets, the dimerization of EGFR and HER2 induces complementation of the luciferase fragments into a functional native structure, producing glow-type luminescence. We have confirmed the functionality of the platform to monitor EGFR-HER2 dimerization induction and inhibition. Statement of SignificanceWe describe a platform technology for rapid monitoring of protein-protein interactions (PPIs). Our approach is uses a luciferase split into three parts – two short peptide “tags” and a large third fragment. Each of the short peptides can be fused to antibodies which bind to domains of a target antigens which orients the two tags and facilitates refolding of an active enzyme. To our knowledge this is the first example of a split-enzyme used to monitor PPIs without requiring any modification of the target proteins. We demonstrate our approach on the important PPI of HER2 and EGFR. Significantly, we quantify stimulation and inhibition of these partners, opening the possibility of using our approach to assess potential drugs without engineering cells.

A plug-and-play platform of ratiometric bioluminescent sensors for homogeneous immunoassays

Heterogeneous immunoassays such as ELISA have become indispensable in modern bioanalysis, yet translation into point-of-care assays is hindered by their dependence on external calibration and multiple washing and incubation steps. Here, we introduce RAPPID (Ratiometric Plug-and-Play Immunodiagnostics), a mix-and-measure homogeneous immunoassay platform that combines highly specific antibody-based detection with a ratiometric bioluminescent readout. The concept entails analyte-induced complementation of split NanoLuc luciferase fragments, photoconjugated to an antibody sandwich pair via protein G adapters. Introduction of a calibrator luciferase provides a robust ratiometric signal that allows direct in-sample calibration and quantitative measurements in complex media such as blood plasma. We developed RAPPID sensors that allow low-picomolar detection of several protein biomarkers, anti-drug antibodies, therapeutic antibodies, and both SARS-CoV-2 spike protein and anti-SARS-CoV-2 antibodies. With its easy-to-implement standardized workflow, RAPPID provides an attractive, fast, and low-cost alternative to traditional immunoassays, in an academic setting, in clinical laboratories, and for point-of-care applications.