Integrated Bioluminescent Immunoassays for High-Throughput Sampling and Continuous Monitoring of Cytokines.

Immunoassays show great potential for the detection of low levels of cytokines, due to their high sensitivity and excellent specificity. There is a particular demand for biosensors that enable both high-throughput screening and continuous monitoring of clinically relevant cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα). To this end, we here introduce a novel bioluminescent immunoassay based on the ratiometric plug-and-play immunodiagnostics (RAPPID) platform, with an improved intrinsic signal-to-background and an >80-fold increase in the luminescent signal. The new dRAPPID assay, comprising a dimeric protein G adapter connected via a semiflexible linker, was applied to detect the secretion of IL-6 by breast carcinoma cells upon TNFα stimulation and the production of low concentrations of IL-6 (∼18 pM) in an endotoxin-stimulated human 3D muscle tissue model. Moreover, we integrated the dRAPPID assay in a newly developed microfluidic device for the simultaneous and continuous monitoring of changes in IL-6 and TNFα in the low-nanomolar range. The luminescence-based read-out and the homogeneous nature of the dRAPPID platform allowed for detection with a simple measurement setup, consisting of a digital camera and a light-sealed box. This permits the usage of the continuous dRAPPID monitoring chip at the point of need, without the requirement for complex or expensive detection techniques.

Bottom-up de novo design of functional proteins with complex structural features.

De novo protein design has enabled the creation of new protein structures. However, the design of functional proteins has proved challenging, in part due to the difficulty of transplanting structurally complex functional sites to available protein structures. Here, we used a bottom-up approach to build de novo proteins tailored to accommodate structurally complex functional motifs. We applied the bottom-up strategy to successfully design five folds for four distinct binding motifs, including a bifunctionalized protein with two motifs. Crystal structures confirmed the atomic-level accuracy of the computational designs. These de novo proteins were functional as components of biosensors to monitor antibody responses and as orthogonal ligands to modulate synthetic signaling receptors in engineered mammalian cells. Our work demonstrates the potential of bottom-up approaches to accommodate complex structural motifs, which will be essential to endow de novo proteins with elaborate biochemical functions, such as molecular recognition or catalysis.

Design and rationale of the REStoring mood after early life trauma with psychotherapy (RESET-psychotherapy) study: a multicenter randomized controlled trial on the efficacy of adjunctive trauma-focused therapy (TFT) versus treatment as usual (TAU) for adult patients with major depressive disorder (MDD) and childhood trauma.

BACKGROUND: Major depressive disorder (MDD) is a common, recurrent mental disorder and a leading cause of disability worldwide. A large part of adult MDD patients report a history of childhood trauma (CT). Patients with MDD and CT are assumed to represent a clinically and neurobiologically distinct MDD subtype with an earlier onset, unfavorable disease course, stress systems' dysregulations and brain alterations. Currently, there is no evidence-based treatment strategy for MDD that specifically targets CT. Given the central role of trauma in MDD patients with CT, trauma-focused therapy (TFT), adjunctive to treatment as usual (TAU), may be efficacious to alleviate depressive symptoms in this patient population. METHODS: The RESET-psychotherapy study is a 12-week, single-blind, randomized controlled trial testing the efficacy of TFT in 158 adults with moderate to severe MDD, as a 'stand-alone' depression diagnosis or superimposed on a persistent depressive disorder (PDD), and CT. TFT (6-10 sessions of Eye Movement Desensitization and Reprocessing and/or imagery rescripting) + TAU is compared to TAU only. Assessments, including a wide range of psychological/psychiatric and biological characteristics, take place before randomization (T0), during treatment (T1), at post-treatment (T2) and at 6-month follow-up (T3). Pre-post treatment stress-related biomarkers in hair (cortisol) and blood (epigenetics and inflammation) will be assessed to better understand working mechanisms of TFT. A subgroup of 60 participants will undergo structural and functional Magnetic Resonance Imaging (MRI) assessments to determine pre-post treatment brain activity. The primary outcome is self-reported depression symptom severity at post-treatment, measured with the 30-item Inventory of Depressive Symptomatology - Self Report (IDS-SR). DISCUSSION: If adjunctive TFT efficaciously alleviates depressive symptoms in MDD patients with CT, this novel treatment strategy could pave the way for a more personalized and targeted MDD treatment. TRIAL REGISTRATION: ClinicalTrials.gov, registered at 08-12-2021, number of identification: NCT05149352.

Bioluminescent RAPPID Sensors for the Single-Step Detection of Soluble Axl and Multiplex Analysis of Cell Surface Cancer Biomarkers.

Early diagnosis of cancer is essential for the efficacy of treatment. Our group recently developed RAPPID, a bioluminescent immunoassay platform capable of measuring a wide panel of biomarkers directly in solution. Here, we developed and systematically screened different RAPPID sensors for sensitive detection of the soluble fraction of Axl (sAxl), a cell surface receptor that is overexpressed in several types of cancer. The best-performing RAPPID sensor, with a limit of detection of 8 pM and a >9-fold maximal change in emission ratio, was applied to measure Axl in three different contexts: clinically relevant sAxl levels (∼0.5 and ∼1 nM) in diluted blood plasma, proteolytically cleaved Axl in the cell culture medium of A431 and HeLa cancer cells, and Axl on the membrane of A431 cells. We further extended the sensor toolbox by developing dual-color RAPPID for simultaneous detection of Axl and EGFR on A431 and HeLa cells, as well as an AND-gate RAPPID that measures the concurrent presence of these two cell surface receptors on the same cell. These new RAPPID sensors provide attractive alternatives for more laborious protein detection and quantification methods such as FACS and immunostainings, due to their simple practical implantation and low intrinsic background signal.

iFLinkC-X: A Scalable Framework to Assemble Bespoke Genetically Encoded Co-polymeric Linkers of Variable Lengths and Amino Acid Composition.

Linker engineering is rapidly gaining prominence as protein engineers and synthetic biologists construct increasingly sophisticated protein assemblies capable of executing complex molecular functions in the context of biosensing, biocatalysis, or biotherapeutics. Depending on the application, the structural and functional requirements imposed on the underlying linkers can differ vastly. At the same time, there is a distinct lack of methods to effectively code linkers at the level of DNA and tailor them to the functional requirements of different fusion proteins. Addressing these limitations, a scalable framework is presented to compose co-polymeric linkers of variable lengths and amino acid composition based on a limited number of linker fragments stored in sequence-verified entry plasmids. The assembly process is exemplified for Pro-rich linkers in the context of a Zn2+-responsive dual-readout BRET/FRET sensor while examining how linker composition impacts key functional properties such as ligand affinity, dynamic range, and their ability to separate structurally distinct domains.

Pathway-Dependent Co-Assembly of Elastin-Like Polypeptides.

In natural systems, temperature-induced assembly of biomolecules can lead to the formation of distinct assembly states, created out of the same set of starting compounds, based on the heating trajectory followed. Until now it has been difficult to achieve similar behavior in synthetic polymer mixtures. Here, a novel pathway-dependent assembly based on stimulus-responsive polymers is shown. When a mixture of mono- and diblock copolymers, based on elastin-like polypeptides, is heated with a critical heating rate co-assembled particles are created that are monodisperse, stable, and have tunable hydrodynamic radii between 20 and 120 nm. Below this critical heating rate, the constituents separately form polymer assemblies. This process is kinetically driven and reversible in thermodynamically closed systems. Using the co-assembly pathway, fluorescent proteins and bioluminescent enzymes are encapsulated with high efficiency. Encapsulated cargo shows unperturbed function even after delivery into cells. The pathway-dependent co-assembly of elastin-like polypeptides is not only of fundamental interest from a materials science perspective, allowing the formation of multiple distinct assemblies from the same starting compounds, which can be interconverted by going back to the molecularly dissolved states. It also enables a versatile way for constructing highly effective vehicles for the cellular delivery of biomolecular cargo.

Engineering with NanoLuc: a playground for the development of bioluminescent protein switches and sensors.

The small engineered luciferase NanoLuc has rapidly become a powerful tool in the fields of biochemistry, chemical biology, and cell biology due to its exceptional brightness and stability. The continuously expanding NanoLuc toolbox has been employed in applications ranging from biosensors to molecular and cellular imaging, and currently includes split complementation variants, engineering techniques for spectral tuning, and bioluminescence resonance energy transfer-based concepts. In this review, we provide an overview of state-of-the-art NanoLuc-based sensors and switches with a focus on the underlying protein engineering approaches. We discuss the advantages and disadvantages of various strategies with respect to sensor sensitivity, modularity, and dynamic range of the sensor and provide a perspective on future strategies and applications.

Bioluminescent Antibodies through Photoconjugation of Protein G-Luciferase Fusion Proteins.

Bioluminescent antibodies represent attractive detection agents in both bioanalytical assays and imaging. Currently, their preparation relies on genetic fusion of luciferases to antibodies or nonspecific chemical conjugation strategies. Here, we report a generic method to generate well-defined covalent antibody-luciferase conjugates starting from commercially available monoclonal antibodies. Our approach uses fusion proteins consisting of the bright blue light-emitting luciferase NanoLuc (NL) and an Fc-binding protein domain (Gx) that can be photo-cross-linked to the antibody using UV light illumination. Green and red color variants were constructed by tight fusion of the NanoLuc with a green fluorescent acceptor domain and introduction of Cy3, respectively. To increase the already bright NanoLuc emission, tandem fusions were successfully developed in which the Gx domain is fused to two or three copies of the NanoLuc domain. The Gx-NL fusion proteins can be efficiently photo-cross-linked to all human immunoglobulin G (IgG) isotypes and most mammalian IgG's using 365 nm light, yielding antibodies with either one or two luciferase domains. The bioluminescent antibodies were successfully used in cell immunostaining and bioanalytical assays such as enzyme-linked immunosorbent assay (ELISA) and Western blotting.

A Self-Reported Version of the Measurements in the Addictions for Triage and Evaluation-Q: Concurrent Validity with the MATE 2.1.

INTRODUCTION: Substance abuse treatment centers require reliable and valid instruments to monitor treatment progress, to evaluate treatment effectiveness, and to initiate clinical trials. Currently the Measurements in the Addictions for Triage and Evaluation (MATE) 2.1, an instrument that serves these purposes, is considered quite lengthy and intensive, especially in the case of allocation to milder treatment intensity. Therefore, a self-reported version of the MATE-Q was designed for patients with mild to moderate substance-abuse and co-occurring problems. The aim of the present study was to assess concurrent validity with the interviewer version of the MATE (version 2.1). MATERIALS AND METHODS: Data were collected at 2 locations of a Dutch substance abuse treatment center, one location in a large city and one in a suburban area. A correlational design was employed, where each included participant completed a MATE-Q and a MATE 2.1 within 3 days or less (administered at intake, before treatment initiation). A total of 98 treatment-seeking patients were included (51.0% alcohol as a primary problem, 19.4% cannabis, 14.3% gambling and 6.1% cocaine). Measurements included the MATE-Q and the MATE 2.1. Intraclass correlation coefficients (ICCs) for single measures were calculated, deploying the 2-way mixed procedure with absolute agreement. Descriptives of scores comprise means and Cronbach's alpha for internal consistency. RESULTS: For the majority (15 out of 24) of the scores ICCs were equal or above 0.7. For 93 patients (95%), the primary problem substance or problem behavior was reported correspondingly. Nine MATE-Q mean scores differed significantly from their MATE 2.1 counterparts. DISCUSSION/CONCLUSION: For the majority of scores, the MATE-Q has acceptable concurrent validity for the assessment of patients with mild to moderate substance abuse and co-occurring problems.

Optical Control of Antibody Activity by Using Photocleavable Bivalent Peptide-DNA Locks.

Antibody-based molecular recognition plays a central role in today's life sciences, ranging from immunoassays to molecular imaging and antibody-based therapeutics. Control over antibody activity by using external triggers such as light could further increase the specificity of antibody-based targeting. Here we present bivalent peptide-DNA ligands containing photocleavable linkers as a noncovalent approach by which to allow photoactivation of antibody activity. Light-triggered cleavage of the 3-amino-3-(2-nitrophenyl)propionic acid peptide linker converted the high-affinity bivalent peptide-DNA lock into weakly binding monovalent ligands, effectively restoring antibody targeting of cell-surface receptors. In this work, a proof of principle was provided with an anti-hemagglutinin antibody, but the molecular design of the lock is generic and applicable to any monoclonal antibody for which an epitope or mimotope of sufficient affinity is available.

DNA-Functionalized Supramolecular Polymers: Dynamic Multicomponent Assemblies with Emergent Properties.

Recent years have witnessed an increasing interest in hybrid molecular systems in which the programmability of DNA hybridization is used to introduce enhanced molecular control in synthetic systems. The first examples of DNA-functionalized supramolecular polymers have been reported only recently, but have already revealed structural and functional properties that are not easily obtained in either synthetic supramolecular polymers or DNA-only based systems. In this Topical Review, we provide an overview of the various forms of additional control offered by DNA hybridization for different types of supramolecular polymers and discuss how orthogonal supramolecular interactions in these hybrid systems can give rise to emergent structural and functional properties.

Affinity Maturation of a Cyclic Peptide Handle for Therapeutic Antibodies Using Deep Mutational Scanning.

Meditopes are cyclic peptides that bind in a specific pocket in the antigen-binding fragment of a therapeutic antibody such as cetuximab. Provided their moderate affinity can be enhanced, meditope peptides could be used as specific non-covalent and paratope-independent handles in targeted drug delivery, molecular imaging, and therapeutic drug monitoring. Here we show that the affinity of a recently reported meditope for cetuximab can be substantially enhanced using a combination of yeast display and deep mutational scanning. Deep sequencing was used to construct a fitness landscape of this protein-peptide interaction, and four mutations were identified that together improved the affinity for cetuximab 10-fold to 15 nm Importantly, the increased affinity translated into enhanced cetuximab-mediated recruitment to EGF receptor-overexpressing cancer cells. Although in silico Rosetta simulations correctly identified positions that were tolerant to mutation, modeling did not accurately predict the affinity-enhancing mutations. The experimental approach reported here should be generally applicable and could be used to develop meditope peptides with low nanomolar affinity for other therapeutic antibodies.

Accelerating DNA-Based Computing on a Supramolecular Polymer.

Dynamic DNA-based circuits represent versatile systems to perform complex computing operations at the molecular level. However, the majority of DNA circuits relies on freely diffusing reactants, which slows down their rate of operation substantially. Here we introduce the use of DNA-functionalized benzene-1,3,5-tricarboxamide (BTA) supramolecular polymers as dynamic scaffolds to template DNA-based molecular computing. By selectively recruiting DNA circuit components to a supramolecular BTA polymer functionalized with 10-nucleotide handle strands, the kinetics of strand displacement and strand exchange reactions were accelerated 100-fold. In addition, strand exchange reactions were also favored thermodynamically by bivalent interactions between the reaction product and the supramolecular polymer. The noncovalent assembly of the supramolecular polymers enabled straightforward optimization of the polymer composition to best suit various applications. The ability of supramolecular BTA polymers to increase the efficiency of DNA-based computing was demonstrated for three well-known and practically important DNA-computing operations: multi-input AND gates, Catalytic Hairpin Assembly and Hybridization Chain Reactions. This work thus establishes supramolecular BTA polymers as an efficient platform for DNA-based molecular operations, paving the way for the construction of autonomous bionanomolecular systems that confine and combine molecular sensing, computation, and actuation.

Dual-Color Bioluminescent Sensor Proteins for Therapeutic Drug Monitoring of Antitumor Antibodies.

Monitoring the levels of therapeutic antibodies in individual patients would allow patient-specific dose optimization, with the potential for major therapeutic and financial benefits. Our group recently developed a new platform of bioluminescent sensor proteins (LUMABS; LUMinescent AntiBody Sensor) that allow antibody detection directly in blood plasma. In this study, we targeted four clinically important therapeutic antibodies, the Her2-receptor targeting trastuzumab, the anti-CD20 antibodies rituximab and obinutuzumab, and the EGFR-blocking cetuximab. A strong correlation was found between the affinity of the antibody binding peptide and sensor performance. LUMABS sensors with physiologically relevant affinities and decent sensor responses were obtained for trastuzumab and cetuximab using mimotope and meditope peptides, respectively, with affinities in the 10-7 M range. The lower affinity of the CD20-derived cyclic peptide employed in the anti-CD20 LUMABS sensor ( Kd = 10-5 M), translated in a LUMABS sensor with a strongly attenuated sensor response. The trastuzumab and cetuximab sensors were further characterized with respect to binding kinetics and their performance in undiluted blood plasma. For both antibodies, LUMABS-based detection directly in plasma compared well to the analytical performance of commercial ELISA kits. Besides identifying important design parameters for the development of new LUMABS sensors, this work demonstrates the potential of the LUMABS platform for point-of-care detection of therapeutic antibodies.

Paper-Based Antibody Detection Devices Using Bioluminescent BRET-Switching Sensor Proteins.

This work reports on fully integrated "sample-in-signal-out" microfluidic paper-based analytical devices (µPADs) relying on bioluminescence resonance energy transfer (BRET) switches for analyte recognition and colorimetric signal generation. The devices use BRET-based antibody sensing proteins integrated into vertically assembled layers of functionalized paper, and their design enables sample volume-independent and fully reagent-free operation, including on-device blood plasma separation. User operation is limited to the application of a single drop (20-30 µL) of sample (serum, whole blood) and the acquisition of a photograph 20 min after sample introduction, with no requirement for precise pipetting, liquid handling, or analytical equipment except for a camera. Simultaneous detection of three different antibodies (anti-HIV1, anti-HA, and anti-DEN1) in whole blood was achieved. Given its simplicity, this type of device is ideally suited for user-friendly point-of-care testing in low-resource environments.

Tuning the Flexibility of Glycine-Serine Linkers To Allow Rational Design of Multidomain Proteins.

Flexible polypeptide linkers composed of glycine and serine are important components of engineered multidomain proteins. We have previously shown that the conformational properties of Gly-Gly-Ser repeat linkers can be quantitatively understood by comparing experimentally determined Förster resonance energy transfer (FRET) efficiencies of ECFP-linker-EYFP proteins to theoretical FRET efficiencies calculated using wormlike chain and Gaussian chain models. Here we extend this analysis to include linkers with different glycine contents. We determined the FRET efficiencies of ECFP-linker-EYFP proteins with linkers ranging in length from 25 to 73 amino acids and with glycine contents of 33.3% (GSSGSS), 16.7% (GSSSSSS), and 0% (SSSSSSS). The FRET efficiency decreased with an increasing linker length and was overall lower for linkers with less glycine. Modeling the linkers using the WLC model revealed that the experimentally observed FRET efficiencies were consistent with persistence lengths of 4.5, 4.8, and 6.2 Å for the GSSGSS, GSSSSS, and SSSSSS linkers, respectively. The observed increase in linker stiffness with reduced glycine content is much less pronounced than that predicted by a classical model developed by Flory and co-workers. We discuss possible reasons for this discrepancy as well as implications for using the stiffer linkers to control the effective concentrations of connected domains in engineered multidomain proteins.

Small-Molecule-Induced and Cooperative Enzyme Assembly on a 14-3-3 Scaffold.

Scaffold proteins regulate cell signalling by promoting the proximity of putative interaction partners. Although they are frequently applied in cellular settings, fundamental understanding of them in terms of, amongst other factors, quantitative parameters has been lagging behind. Here we present a scaffold protein platform that is based on the native 14-3-3 dimeric protein and is controllable through the action of a small-molecule compound, thus permitting study in an in vitro setting and mathematical description. Robust small-molecule regulation of caspase-9 activity through induced dimerisation on the 14-3-3 scaffold was demonstrated. The individual parameters of this system were precisely determined and used to develop a mathematical model of the scaffolding concept. This model was used to elucidate the strong cooperativity of the enzyme activation mediated by the 14-3-3 scaffold. This work provides an entry point for the long-needed quantitative insights into scaffold protein functioning and paves the way for the optimal use of reengineered 14-3-3 proteins as chemically inducible scaffolds in synthetic systems.

Detection of Antibodies in Blood Plasma Using Bioluminescent Sensor Proteins and a Smartphone.

Antibody detection is of fundamental importance in many diagnostic and bioanalytical assays, yet current detection techniques tend to be laborious and/or expensive. We present a new sensor platform (LUMABS) based on bioluminescence resonance energy transfer (BRET) that allows detection of antibodies directly in solution using a smartphone as the sole piece of equipment. LUMABS are single-protein sensors that consist of the blue-light emitting luciferase NanoLuc connected via a semiflexible linker to the green fluorescent acceptor protein mNeonGreen, which are kept close together using helper domains. Binding of an antibody to epitope sequences flanking the linker disrupts the interaction between the helper domains, resulting in a large decrease in BRET efficiency. The resulting change in color of the emitted light from green-blue to blue can be detected directly in blood plasma, even at picomolar concentrations of antibody. Moreover, the modular architecture of LUMABS allows changing of target specificity by simple exchange of epitope sequences, as demonstrated here for antibodies against HIV1-p17, hemagglutinin (HA), and dengue virus type I. The combination of sensitive ratiometric bioluminescent detection and the intrinsic modularity of the LUMABS design provides an attractive generic platform for point-of-care antibody detection that avoids the complex liquid handling steps associated with conventional immunoassays.

Supramolecular Control over Split-Luciferase Complementation.

Supramolecular split-enzyme complementation restores enzymatic activity and allows for on-off switching. Split-luciferase fragment pairs were provided with an N-terminal FGG sequence and screened for complementation through host-guest binding to cucurbit[8]uril (Q8). Split-luciferase heterocomplex formation was induced in a Q8 concentration dependent manner, resulting in a 20-fold upregulation of luciferase activity. Supramolecular split-luciferase complementation was fully reversible, as revealed by using two types of Q8 inhibitors. Competition studies with the weak-binding FGG peptide revealed a 300-fold enhanced stability for the formation of the ternary heterocomplex compared to binding of two of the same fragments to Q8. Stochiometric binding by the potent inhibitor memantine could be used for repeated cycling of luciferase activation and deactivation in conjunction with Q8, providing a versatile module for in vitro supramolecular signaling networks.

Efficient Synthesis of Peptide and Protein Functionalized Pyrrole-Imidazole Polyamides Using Native Chemical Ligation.

The advancement of DNA-based bionanotechnology requires efficient strategies to functionalize DNA nanostructures in a specific manner with other biomolecules, most importantly peptides and proteins. Common DNA-functionalization methods rely on laborious and covalent conjugation between DNA and proteins or peptides. Pyrrole-imidazole (Py-Im) polyamides, based on natural minor groove DNA-binding small molecules, can bind to DNA in a sequence specific fashion. In this study, we explore the use of Py-Im polyamides for addressing proteins and peptides to DNA in a sequence specific and non-covalent manner. A generic synthetic approach based on native chemical ligation was established that allows efficient conjugation of both peptides and recombinant proteins to Py-Im polyamides. The effect of Py-Im polyamide conjugation on DNA binding was investigated by Surface Plasmon Resonance (SPR). Although the synthesis of different protein-Py-Im-polyamide conjugates was successful, attenuation of DNA affinity was observed, in particular for the protein-Py-Im-polyamide conjugates. The practical use of protein-Py-Im-polyamide conjugates for addressing DNA structures in an orthogonal but non-covalent manner, therefore, remains to be established.