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Rapid and sensitive DNA detection methods that can be conducted at the point of need may aid in disease diagnosis and monitoring. However, translation of current assays has proven challenging, as they typically require specialized equipment or probe-specific modifications for every new target DNA. Here, we present Luminescent Multivalent Intercalating Dye (LUMID), off-the-shelf bioluminescent sensors consisting of intercalating dyes conjugated to a NanoLuc luciferase, which allow for nonspecific detection of double-stranded DNA through a blue-to-green color change. Through the incorporation of multiple, tandem-arranged dyes separated by positively charged linkers, DNA-binding affinities were improved by over 2 orders of magnitude, detecting nanomolar DNA concentrations with an 8-fold change in green/blue ratio. We show that LUMID is easily combined with loop-mediated isothermal amplification (LAMP), enabling sequence-specific detection of viral DNA with attomolar sensitivity and a smartphone-based readout. With LUMID, we have thus developed a tool for simple and sensitive DNA detection that is particularly attractive for point-of-need applications.

Fast and reliable virus diagnostics is key to prevent the spread of viruses in populations. A hallmark of viruses is the presence of multivalent surface proteins, a property that can be harnessed to control conformational switching in sensor proteins. Here, we introduce a new sensor platform (dark-LUX) for the detection of viral surface proteins consisting of a general bioluminescent framework that can be post-translationally functionalized with separately expressed binding domains. The platform relies on (1) plug-and-play bioconjugation of different binding proteins via SpyTag/SpyCatcher technology to create branched protein structures, (2) an optimized turn-on bioluminescent switch based on complementation of the split-luciferase NanoBiT upon target binding and (3) straightforward exploration of the protein linker space. The influenza A virus (IAV) surface proteins hemagglutinin (HA) and neuraminidase (NA) were used as relevant multivalent targets to establish proof of principle and optimize relevant parameters such as linker properties, choice of target binding domains and the optimal combination of the competing NanoBiT components SmBiT and DarkBiT. The sensor framework allows rapid conjugation and exchange of various binding domains including scFvs, nanobodies and de novo designed binders for a variety of targets, including the construction of a heterobivalent switch that targets the head and stem region of hemagglutinin. The modularity of the platform thus allows straightforward optimization of binding domains and scaffold properties for existing viral targets, and is well suited to quickly adapt bioluminescent sensor proteins to effectively detect newly evolving viral epitopes.

Molecular strategies that allow for reversible control of antibody activity have drawn considerable interest for both therapeutic and diagnostic applications. Protein M is a generic antibody-binding protein that binds to the Fv domain of IgGs and, in doing so, blocks antigen binding. However, the dissociation of protein M is essentially irreversible, which has precluded its use as an antibody affinity reagent and molecular mask to control antibody activity. Here, we show that introduction of 8 histidine residues on the Fv binding interface of protein M results in a variant that shows pH-switchable IgG binding. This protein M-8his variant provides an attractive and universal affinity resin for the purification of IgGs, antibody fragments (Fab and single-chain variable fragments (scFv)), and antibody conjugates. Moreover, protein M-8his enables the pH-dependent blocking of therapeutic antibodies, allowing the selective targeting of cells at pH 6.0.

Fast and reliable virus diagnostics is key to prevent the spread of viruses in populations. A hallmark of viruses is the presence of multivalent surface proteins, a property that can be harnessed to control conformational switching in sensor proteins. Here, we introduce a new sensor platform (dark-LUX) for the detection of viral surface proteins consisting of a general bioluminescent framework that can be post-translationally functionalized with separately expressed binding domains. The platform relies on (1) plug-and-play bioconjugation of different binding proteins via SpyTag/SpyCatcher technology to create branched protein structures, (2) an optimized turn-on bioluminescent switch based on complementation of the split-luciferase NanoBiT upon target binding and (3) straightforward exploration of the protein linker space. The influenza A virus (IAV) surface proteins hemagglutinin (HA) and neuraminidase (NA) were used as relevant multivalent targets to establish proof of principle and optimize relevant parameters such as linker properties, choice of target binding domains and the optimal combination of the competing NanoBiT components SmBiT and DarkBiT. The sensor framework allows rapid conjugation and exchange of various binding domains including scFvs, nanobodies and de novo designed binders for a variety of targets, including the construction of a heterobivalent switch that targets the head and stem region of hemagglutinin. The modularity of the platform thus allows straightforward optimization of binding domains and scaffold properties for existing viral targets, and is well suited to quickly adapt bioluminescent sensor proteins to effectively detect newly evolving viral epitopes.

The rational design and implementation of synthetic mammalian communication systems can unravel fundamental design principles of cell communication circuits and offer a framework for engineering of designer cell consortia with potential applications in cell therapeutics. Here, we develop the foundations of an orthogonal, and scalable mammalian synthetic communication platform that exploits the programmability of synthetic receptors and selective affinity and tunability of diffusing coiled-coil peptides. Leveraging the ability of coiled-coils to exclusively bind to a cognate receptor, we demonstrate orthogonal receptor activation and Boolean logic operations at the receptor level. We show intercellular communication based on synthetic receptors and secreted multidomain coiled-coils and demonstrate a three-cell population system that can perform AND gate logic. Finally, we show CC-GEMS receptor-dependent therapeutic protein expression. Our work provides a modular and scalable framework for the engineering of complex cell consortia, with the potential to expand the aptitude of cell therapeutics and diagnostics.

Membraneless organelles are important for spatial organization of proteins and regulation of intracellular processes. Proteins can be recruited to these condensates by specific protein–protein or protein–nucleic acid interactions, which are often regulated by post-translational modifications. However, the mechanisms behind these dynamic, affinity-based protein recruitment events are not well understood. Here, a coacervate system that incorporates the 14-3-3 scaffold protein to study enzymatically regulated recruitment of 14-3-3-binding proteins is presented, which mostly bind in a phosphorylation-dependent manner. Synthetic coacervates are efficiently loaded with 14-3-3, and phosphorylated binding partners, such as the c-Raf pS233/pS259 peptide (c-Raf), show 14-3-3-dependent sequestration with up to 161-fold increase in local concentration. The c-Raf domain is fused to green fluorescent protein (GFP-c-Raf) to demonstrate recruitment of proteins. In situ phosphorylation of GFP-c-Raf by a kinase leads to enzymatically regulated uptake. The introduction of a phosphatase into coacervates preloaded with the phosphorylated 14-3-3-GFP-c-Raf complex results in a significant cargo efflux mediated by dephosphorylation. Finally, the general applicability of this platform to study protein–protein interactions is demonstrated by the phosphorylation-dependent and 14-3-3-mediated active reconstitution of a split-luciferase inside artificial cells. This work presents an approach to study dynamically regulated protein recruitment in condensates, using native interaction domains.

Immunoparalysis is a compensatory and persistent anti-inflammatory response to trauma, sepsis or another serious insult, which increases the risk of opportunistic infections, morbidity and mortality. Here, we show that in cultured primary human monocytes, interleukin-4 (IL4) inhibits acute inflammation, while simultaneously inducing a long-lasting innate immune memory named trained immunity. To take advantage of this paradoxical IL4 feature in vivo, we developed a fusion protein of apolipoprotein A1 (apoA1) and IL4, which integrates into a lipid nanoparticle. In mice and non-human primates, an intravenously injected apoA1-IL4-embedding nanoparticle targets myeloid-cell-rich haematopoietic organs, in particular, the spleen and bone marrow. We subsequently demonstrate that IL4 nanotherapy resolved immunoparalysis in mice with lipopolysaccharide-induced hyperinflammation, as well as in ex vivo human sepsis models and in experimental endotoxemia. Our findings support the translational development of nanoparticle formulations of apoA1-IL4 for the treatment of patients with sepsis at risk of immunoparalysis-induced complications.

Cytokines have pivotal roles in immunity, making them attractive as therapeutics for a variety of immune-related disorders. However, the widespread clinical use of cytokines has been limited by their short blood half-lives and severe side effects caused by low specificity and unfavourable biodistribution. Innovations in bioengineering have aided in advancing our knowledge of cytokine biology and yielded new technologies for cytokine engineering. In this Review, we discuss how the development of bioanalytical methods, such as sequencing and high-resolution imaging combined with genetic techniques, have facilitated a better understanding of cytokine biology. We then present an overview of therapeutics arising from cytokine re-engineering, targeting and delivery, mRNA therapeutics and cell therapy. We also highlight the application of these strategies to adjust the immunological imbalance in different immune-mediated disorders, including cancer, infection and autoimmune diseases. Finally, we look ahead to the hurdles that must be overcome before cytokine therapeutics can live up to their full potential.

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.

Scaffold proteins operate as organizing hubs to enable high-fidelity signaling, fulfilling crucial roles in the regulation of cellular processes. Bottom-up construction of controllable scaffolding platforms is attractive for the implementation of regulatory processes in synthetic biology. Here, we present a modular and switchable synthetic scaffolding system, integrating scaffold-mediated signaling with switchable kinase/phosphatase input control. Phosphorylation-responsive inhibitory peptide motifs were fused to 14-3-3 proteins to generate dimeric protein scaffolds with appended regulatory peptide motifs. The availability of the scaffold for intermolecular partner protein binding could be lowered up to 35-fold upon phosphorylation of the autoinhibition motifs, as demonstrated using three different kinases. In addition, a hetero-bivalent autoinhibitory platform design allowed for dual-kinase input regulation of scaffold activity. Reversibility of the regulatory platform was illustrated through phosphatase-controlled abrogation of autoinhibition, resulting in full recovery of 14-3-3 scaffold activity.

Fluorescent Zn2+sensors play a pivotal role in zinc biology, but their application in complex media such as blood serum or plate reader-based cellular assays is hampered by autofluorescence and light scattering. Bioluminescent sensor proteins provide an attractive alternative to fluorescent sensors for these applications, but the only bioluminescent sensor protein developed so far, BLZinCh, has a limited sensor response and a suboptimal Zn2+affinity. In this work, we expanded the toolbox of bioluminescent Zn2+sensors by developing two new sensor families that show a large change in the emission ratio and cover a range of physiologically relevant Zn2+affinities. The LuZi platform relies on competitive complementation of split NanoLuc luciferase and displays a robust, 2-fold change in red-to-blue emission, allowing quantification of free Zn2+between 2 pM and 1 nM. The second platform was developed by replacing the long flexible GGS linker in the original BLZinCh sensor by rigid polyproline linkers, yielding a series of BLZinCh-Pro sensors with a 3-4-fold improved ratiometric response and physiologically relevant Zn2+affinities between 0.5 and 1 nM. Both the LuZi and BLZinCh-Pro sensors allowed the direct determination of low nM concentrations of free Zn2+in serum, providing an attractive alternative to more laborious and/or indirect approaches to measure serum zinc levels. Furthermore, the genetic encoding of the BLZinCh-Pro sensors allowed their use as intracellular sensors, where the sensor occupancy of 40-50% makes them ideally suited to monitor both increases and decreases in intracellular free Zn2+concentration in simple, plate reader-based measurements, without the need for fluorescence microscopy.

In therapies involving therapeutic antibodies, therapeutic drug monitoring (TDM) has the potential to substantially increase clinical efficacy and to enable more economical drug use, because the clearance from the bloodstream after administration is patient dependent. Since TDM must be performed continuously and over extended time periods, it should ideally be achieved through an economical and easy to perform point-of-care (POC) assay. Here, we introduce a new analytical method for POC TDM of the clinically important therapeutic antibody cetuximab, using microfluidic thread-based analytical devices (µTADs) as a platform for a bioluminescence resonance energy-transfer (BRET) switching sensor protein for simple quantitative antibody detection. The µTAD is pre-treated with NaCl for blood plasma separation and a BRET switching sensor protein and its substrate for cetuximab detection, integrating all elements required for a reagent-free, single-step assay. A change in bioluminescence emission color depending on cetuximab concentrations occurs after whole blood application. The device design enables the quantification of cetuximab within the therapeutically relevant blood concentration range in 2.5 min using only 2 µL of whole blood. The results obtained are independent of the blood volume. This µTAD can be stored for about one month at − 20 °C, a temperature compatible with commonly available freezers. The application of mathematical morphology recognition (MMR)-integrated image processing techniques for the automatic selection of regions of interest (ROIs) yields results with minimal error. The simplicity of use, rapidity, and cost-effectiveness of this µTAD are expected to offer the possibility of achieving TDM of therapeutic antibodies at POC for individual patients.

Scaffold proteins operate as organizing hubs to enable high-fidelity signaling, fulfilling crucial roles in the regulation of cellular processes. Bottom-up construction of controllable scaffolding platforms is attractive for the implementation of regulatory processes in synthetic biology. Here, we present a modular and switchable synthetic scaffolding system, integrating scaffold-mediated signaling with switchable kinase/phosphatase input control. Phosphorylation-responsive inhibitory peptide motifs were fused to 14-3-3 proteins to generate dimeric protein scaffolds with appended regulatory peptide motifs. The availability of the scaffold for intermolecular partner protein binding could be lowered up to 35-fold upon phosphorylation of the autoinhibition motifs, as demonstrated using three different kinases. In addition, a hetero-bivalent autoinhibitory platform design allowed for dual-kinase input regulation of scaffold activity. Reversibility of the regulatory platform was illustrated through phosphatase-controlled abrogation of autoinhibition, resulting in full recovery of 14-3-3 scaffold activity.

Bioluminescent sensor proteins have recently gained popularity in both basic research and point-of-care diagnostics. Sensor proteins based on intramolecular complementation of split NanoLuc are particularly attractive because their intrinsic modular design enables for systematic tuning of sensor properties. Here we show how the sensitivity of these sensors can be enhanced by the introduction of catalytically inactive variants of the small SmBiT subunit (DarkBiTs) as intramolecular inhibitors. Starting from previously developed bioluminescent antibody sensor proteins (LUMABS), we developed single component, biomolecular switches with a strongly reduced background signal for the detection of three clinically relevant antibodies, anti-HIV1-p17, cetuximab (CTX), and an RSV neutralizing antibody (101F). These new dark-LUMABS sensors showed 5-13-fold increases in sensitivity which translated into lower limits of detection. The use of DarkBiTs as competitive intramolecular inhibitor domains is not limited to the LUMABS sensor family and might be used to boost the performance of other bioluminescent sensor proteins based on split luciferase complementation.

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.

Fluorescent Zn2+sensors play a pivotal role in zinc biology, but their application in complex media such as blood serum or plate reader-based cellular assays is hampered by autofluorescence and light scattering. Bioluminescent sensor proteins provide an attractive alternative to fluorescent sensors for these applications, but the only bioluminescent sensor protein developed so far, BLZinCh, has a limited sensor response and a suboptimal Zn2+affinity. In this work, we expanded the toolbox of bioluminescent Zn2+sensors by developing two new sensor families that show a large change in the emission ratio and cover a range of physiologically relevant Zn2+affinities. The LuZi platform relies on competitive complementation of split NanoLuc luciferase and displays a robust, 2-fold change in red-to-blue emission, allowing quantification of free Zn2+between 2 pM and 1 nM. The second platform was developed by replacing the long flexible GGS linker in the original BLZinCh sensor by rigid polyproline linkers, yielding a series of BLZinCh-Pro sensors with a 3-4-fold improved ratiometric response and physiologically relevant Zn2+affinities between 0.5 and 1 nM. Both the LuZi and BLZinCh-Pro sensors allowed the direct determination of low nM concentrations of free Zn2+in serum, providing an attractive alternative to more laborious and/or indirect approaches to measure serum zinc levels. Furthermore, the genetic encoding of the BLZinCh-Pro sensors allowed their use as intracellular sensors, where the sensor occupancy of 40-50% makes them ideally suited to monitor both increases and decreases in intracellular free Zn2+concentration in simple, plate reader-based measurements, without the need for fluorescence microscopy.

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.

In therapies involving therapeutic antibodies, therapeutic drug monitoring (TDM) has the potential to substantially increase clinical efficacy and to enable more economical drug use, because the clearance from the bloodstream after administration is patient dependent. Since TDM must be performed continuously and over extended time periods, it should ideally be achieved through an economical and easy to perform point-of-care (POC) assay. Here, we introduce a new analytical method for POC TDM of the clinically important therapeutic antibody cetuximab, using microfluidic thread-based analytical devices (µTADs) as a platform for a bioluminescence resonance energy-transfer (BRET) switching sensor protein for simple quantitative antibody detection. The µTAD is pre-treated with NaCl for blood plasma separation and a BRET switching sensor protein and its substrate for cetuximab detection, integrating all elements required for a reagent-free, single-step assay. A change in bioluminescence emission color depending on cetuximab concentrations occurs after whole blood application. The device design enables the quantification of cetuximab within the therapeutically relevant blood concentration range in 2.5 min using only 2 µL of whole blood. The results obtained are independent of the blood volume. This µTAD can be stored for about one month at − 20 °C, a temperature compatible with commonly available freezers. The application of mathematical morphology recognition (MMR)-integrated image processing techniques for the automatic selection of regions of interest (ROIs) yields results with minimal error. The simplicity of use, rapidity, and cost-effectiveness of this µTAD are expected to offer the possibility of achieving TDM of therapeutic antibodies at POC for individual patients.

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.

Contains fulltext : 236879.pdf (Publisher’s version ) (Open Access)

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.

Contains fulltext : 236879.pdf (Publisher’s version ) (Open Access)

Contains fulltext : 236879.pdf (Publisher’s version ) (Open Access)

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.

Engineered living materials have the potential for wide-ranging applications such as biosensing and treatment of diseases. Programmable cells provide the functional basis for living materials; however, their release into the environment raises numerous biosafety concerns. Current designs that limit the release of genetically engineered cells typically involve the fabrication of multilayer hybrid materials with submicrometer porous matrices. Nevertheless the stringent physical barriers limit the diffusion of macromolecules and therefore the repertoire of molecules available for actuation in response to communication signals between cells and their environment. Here, we engineer a novel living material entitled "Platform for Adhesin-mediated Trapping of Cells in Hydrogels" (PATCH). This technology is based on engineered E. coli that displays an adhesion protein derived from an Antarctic bacterium with a high affinity for glucose. The adhesin stably anchors E. coli in dextran-based hydrogels with large pore diameters (10-100 μm) and reduces the leakage of bacteria into the environment by up to 100-fold. As an application of PATCH, we engineered E. coli to secrete the bacteriocin lysostaphin which specifically kills Staphyloccocus aureus with low probability of raising antibiotic resistance. We demonstrated that living materials containing this lysostaphin-secreting E. coli inhibit the growth of S. aureus, including the strain resistant to methicillin (MRSA). Our tunable platform allows stable integration of programmable cells in dextran-based hydrogels without compromising free diffusion of macromolecules and could have potential applications in biotechnology and biomedicine. ©

Antibodies are important biomarkers in clinical diagnostics in addition to being increasingly used for therapeutic purposes. Although numerous methods for their detection and quantification exist, they predominantly require benchtop instruments operated by specialists. To enable the detection of antibodies at point-of-care (POC), the development of simple and rapid assay methods independent of laboratory equipment is of high relevance. In this study, we demonstrate microfluidic thread-based analytical devices (μTADs) as a new platform for antibody detection by means of bioluminescence resonance energy-transfer (BRET) switching sensor proteins. The devices consist of vertically assembled layers including a blood separation membrane and a plastic film with a sewn-in cotton thread, onto which the BRET sensor proteins together with the substrate furimazine have been predeposited. In contrast to intensity-based signaling, the BRET mechanism enables time-independent, ratiometric readout of bioluminescence signals with a digital camera in a darkroom or a smartphone camera with a 3D-printed lens adapter. The device design allows spatially separated deposition of multiple bioluminescent proteins on a single sewn thread, enabling quantification of multiple antibodies in 5 μL of whole blood within 5 min. The bioluminescence response is independent of the applied sample volume within the range of 5-15 μL. Therefore, μTADs in combination with BRET-based sensor proteins represent user-friendly analytical tools for POC quantification of antibodies without any laboratory equipment in a finger prick (5 μL) of whole blood.

The ability to continuously measure concentrations of small molecules is important for biomedical, environmental and industrial monitoring. However, due to their low molecular mass it is difficult to quantify concentrations of such molecules, particularly at low concentrations. Here we describe a small-molecule sensor that is generalizable, sensitive, specific, reversible, and suited for continuous monitoring over long durations. The sensor consists of particles attached to a sensing surface via a double stranded DNA tether. The particles transiently bind to the sensing surface via single molecular affinity interactions and the transient binding is optically detected as digital binding events via the Brownian motion of the particles. The rate of binding events decreases with increasing analyte concentration, because analyte molecules inhibit binding of the tethered particle to the surface. The sensor enables continuous measurements of analyte concentrations due to the reversibility of the inter-molecular bonds and digital read-out of particle motion. We show results for the monitoring of short single-stranded DNA sequences and creatinine, a small-molecule biomarker (113 Da) for kidney function, demonstrating a temporal resolution of a few minutes. The precision of the sensor is determined by the statistics of the digital switching events, which means that the precision is tunable by the number of particles and the measurement time.

The ability to control antibody activity by pH has important applications in diagnostics, therapeutic antibody targeting, and antibody-guided imaging. Here, we report the rational design of bivalent peptide-DNA ligands that allow pH-dependent control of antibody activity. Our strategy uses a pH-responsive DNA triple helix to control switching from a tight-binding bivalent peptide-DNA lock into a weaker-binding monovalent ligand. Different designs are introduced that allow antibody activation at both basic and acidic pHs, either autonomously or in the presence of an additional oligonucleotide trigger. The pH of antibody activation could be precisely tuned by changing the DNA triple helix sequence. The peptide-DNA locks allowed pH-dependent antibody targeting of tumor cells both in bulk and for single cells confined in water-in-oil microdroplets. The latter approach enables high-throughput antibody-mediated detection of single tumor cells based on their distinctive metabolic activity.

The ability to control antibody activity by pH has important applications in diagnostics, therapeutic antibody targeting, and antibody-guided imaging. Here, we report the rational design of bivalent peptide-DNA ligands that allow pH-dependent control of antibody activity. Our strategy uses a pH-responsive DNA triple helix to control switching from a tight-binding bivalent peptide-DNA lock into a weaker-binding monovalent ligand. Different designs are introduced that allow antibody activation at both basic and acidic pHs, either autonomously or in the presence of an additional oligonucleotide trigger. The pH of antibody activation could be precisely tuned by changing the DNA triple helix sequence. The peptide-DNA locks allowed pH-dependent antibody targeting of tumor cells both in bulk and for single cells confined in water-in-oil microdroplets. The latter approach enables high-throughput antibody-mediated detection of single tumor cells based on their distinctive metabolic activity.

The ability to continuously measure concentrations of small molecules is important for biomedical, environmental and industrial monitoring. However, due to their low molecular mass it is difficult to quantify concentrations of such molecules, particularly at low concentrations. Here we describe a small-molecule sensor that is generalizable, sensitive, specific, reversible, and suited for continuous monitoring over long durations. The sensor consists of particles attached to a sensing surface via a double stranded DNA tether. The particles transiently bind to the sensing surface via single molecular affinity interactions and the transient binding is optically detected as digital binding events via the Brownian motion of the particles. The rate of binding events decreases with increasing analyte concentration, because analyte molecules inhibit binding of the tethered particle to the surface. The sensor enables continuous measurements of analyte concentrations due to the reversibility of the inter-molecular bonds and digital read-out of particle motion. We show results for the monitoring of short single-stranded DNA sequences and creatinine, a small-molecule biomarker (113 Da) for kidney function, demonstrating a temporal resolution of a few minutes. The precision of the sensor is determined by the statistics of the digital switching events, which means that the precision is tunable by the number of particles and the measurement time.

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.

Bioluminescent sensor proteins provide attractive tools for applications ranging from in vivo imaging to point-of-care testing. Here we introduce a new class of ratiometric bioluminescent sensor proteins that do not rely on direct modulation of BRET efficiency, but are based on competitive intramolecular complementation of split NanoLuc luciferase. Proof of concept for the feasibility of this sensor principle was provided by developing a blue-red light emitting sensor protein for the detection of anti-HIV1-p17 antibodies with a 500% change in emission ratio and a K d of 10 pM. The new sensor design also improved the dynamic response of a sensor for the therapeutic antibody cetuximab 4-fold, allowing the direct quantification of this anti-EGFR antibody in undiluted blood plasma. The modular sensor architecture allows easy and systematic tuning of a sensor's dynamic range and should be generally applicable to allow rational engineering of bioluminescent sensor proteins.

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.

Bioluminescent sensor proteins provide attractive tools for applications ranging from in vivo imaging to point-of-care testing. Here we introduce a new class of ratiometric bioluminescent sensor proteins that do not rely on direct modulation of BRET efficiency, but are based on competitive intramolecular complementation of split NanoLuc luciferase. Proof of concept for the feasibility of this sensor principle was provided by developing a blue-red light emitting sensor protein for the detection of anti-HIV1-p17 antibodies with a 500% change in emission ratio and a K d of 10 pM. The new sensor design also improved the dynamic response of a sensor for the therapeutic antibody cetuximab 4-fold, allowing the direct quantification of this anti-EGFR antibody in undiluted blood plasma. The modular sensor architecture allows easy and systematic tuning of a sensor's dynamic range and should be generally applicable to allow rational engineering of bioluminescent sensor proteins.

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.

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.

Protease signaling and scaffold-induced control of protein-protein interactions represent two important mechanisms for intracellular signaling. Here we report a generic and modular approach to control the activity of scaffolding proteins by protease activity, creating versatile molecular platforms to construct synthetic signaling networks. Using 14 3 3 proteins as a structurally well-characterized and important class of scaffold proteins, three different architectures were explored to achieve optimal protease-mediated control of scaffold activity, fusing either one or two monovalent inhibitory ExoS peptides or a single bivalent ExoS peptide to T14 3 3 using protease-cleavable linkers. Analysis of scaffolding activity before and after protease-induced cleavage revealed optimal control of 14-3-3 activity for the system that contained monovalent ExoS peptides fused to both the N-and C-terminus, each blocking a single T14 3 3 binding site. The protease-activatable 14 3 3 scaffolds were successfully applied to construct a three-step signaling cascade in which dimerization and activation of FGG-caspase-9 on an orthogonal supramolecular platform resulted in activation of a 14 3 3 scaffold, which in turn allowed 14-3-3-templated complementation of a split-luciferase. In addition, by combining 14 3 3-templated activation of caspase-9 with a caspase-9-activatable 14 3 3 scaffold the first example of a synthetic self-activating protease signaling network was created. Protease-activatable 14 3 3 proteins thus represent a modular platform whose properties can be rationally engineered to fit different applications, both to create artificial in vitro synthetic molecular networks and as a novel signaling hub to re-engineer intracellular signaling pathways.

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.

Protease signaling and scaffold-induced control of protein-protein interactions represent two important mechanisms for intracellular signaling. Here we report a generic and modular approach to control the activity of scaffolding proteins by protease activity, creating versatile molecular platforms to construct synthetic signaling networks. Using 14 3 3 proteins as a structurally well-characterized and important class of scaffold proteins, three different architectures were explored to achieve optimal protease-mediated control of scaffold activity, fusing either one or two monovalent inhibitory ExoS peptides or a single bivalent ExoS peptide to T14 3 3 using protease-cleavable linkers. Analysis of scaffolding activity before and after protease-induced cleavage revealed optimal control of 14-3-3 activity for the system that contained monovalent ExoS peptides fused to both the N-and C-terminus, each blocking a single T14 3 3 binding site. The protease-activatable 14 3 3 scaffolds were successfully applied to construct a three-step signaling cascade in which dimerization and activation of FGG-caspase-9 on an orthogonal supramolecular platform resulted in activation of a 14 3 3 scaffold, which in turn allowed 14-3-3-templated complementation of a split-luciferase. In addition, by combining 14 3 3-templated activation of caspase-9 with a caspase-9-activatable 14 3 3 scaffold the first example of a synthetic self-activating protease signaling network was created. Protease-activatable 14 3 3 proteins thus represent a modular platform whose properties can be rationally engineered to fit different applications, both to create artificial in vitro synthetic molecular networks and as a novel signaling hub to re-engineer intracellular signaling pathways.

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.

Inspired by signaling networks in living cells, DNA-based programming aims for the engineering of biochemical networks capable of advanced regulatory and computational functions under controlled cell-free conditions. While regulatory circuits in cells control downstream processes through hierarchical layers of signal processing, coupling of enzymatically driven DNA-based networks to downstream processes has rarely been reported. Here, we expand the scope of molecular programming by engineering hierarchical control of enzymatic actuators using feedback-controlled DNA-circuits capable of advanced regulatory dynamics. We developed a translator module that converts signaling molecules from the upstream network to unique DNA strands driving downstream actuators with minimal retroactivity and support these findings with a detailed computational analysis. We show our modular approach by coupling of a previously engineered switchable memories circuit to downstream actuators based on β-lactamase and luciferase. To the best of our knowledge, our work demonstrates one of the most advanced DNA-based circuits regarding complexity and versatility.