Biology subjects

Merkx, M.

Publications and source records attributed to Merkx, M..

11 recordsLinked to original sources

bioRxiv2025

De novo design of protein competitors for small molecule immunosensing

Competition-based immunoassays are a common strategy for detecting small-molecule biomarkers. However, these assays rely on the availability of a custom competitor molecule that can effectively be displaced upon analyte binding, often requiring time-consuming synthesis and conjugation steps. De novo designed protein binders present a compelling alternative, as their binding properties can be tuned and they allow for straightforward genetic-incorporation into existing immunoassays. Here, we leverage the BindCraft pipeline to design competitive binders by targeting antigen-binding sites, followed by in silico filtering to select for steric clashes with the small-molecule analyte. As a proof of concept, we designed digoxin competitors and experimentally screened the binders using a simple bioluminescent assay, identifying 7/10 successful binders directly in bacterial lysate. These binders exhibited low to moderate binding affinities (Kd = 42 nM - 1.1 {micro}M) and were displaced by digoxin. Two de novo binders were encoded into a previously established competition-based immunosensor, enabling sensitive digoxin detection (Kd = 109 nM). These results demonstrate that deep learning-based models can rapidly yield effective competitor binders, enabling straightforward adaptation and optimization of sensing platforms for small-molecule targets.

synthetic biology↗

bioRxiv2025

Homogeneous antibody-DNA-conjugates using unmodified oligonucleotides and photo-crosslinkable protein G-HUH endonuclease fusion proteins

Antibody-DNA conjugates are increasingly used in analytical biochemistry and nanotechnology. However, current methods for their preparation often lack specificity, resulting in heterogeneous products. Here, we present a novel strategy to covalently label the Fc domain of antibodies with single-strand DNA in a site-specific and stoichiometrically controlled manner. This method employs unmodified oligonucleotides and a fusion protein consisting of a protein G dimer linked to an HUH endonuclease. We first evaluated the sequence specificity and optimal conditions for DNA attachment to three HUH endonuclease variants, DCV, PCV2, and WDV. Our results show increased sequence specificity in the presence of Mg2+ compared to the more commonly used Mn2+ cofactor ion. Although formation of the phospho-tyrosine bond is found to be reversible, no significant hydrolysis of the protein-DNA conjugates is observed for up to 8 days at room temperature. The DCV domain allowed essentially complete formation of DNA-fusion protein conjugates at a 1:1 protein-DNA ratio, eliminating the need for removal of excess oligos. Subsequent photo-crosslinking yielded antibody-DNA conjugates that were successfully used in a proximity extension assay (PEA) to detect TNF and IL-6. Our method provides an efficient, low cost and generally applicable method to prepare homogeneous antibody-DNA conjugates with 1:1 stoichiometry.

bioengineering↗

bioRxiv2025

"Visualize, describe, compare" - nanoinformatics approaches for material-omics

Bioinformatics and cheminformatics are established disciplines, but nanoinformatics, the development of computational tools for understanding and designing nanomaterials, is still in its infancy. In light of the new data-driven approaches for nanomaterials discovery, there is a growing need for in silico tools tailored to analyze nanomaterials datasets. This is particularly crucial for soft materials, where a crystalline structure cannot be obtained and therefore the characterization datasets are less structured, and there are no standard methods for data mining. Here we present a computational package capable of visualizing, describing, and comparing nanoparticle datasets obtained with super-resolution microscopy at the single-particle and single-molecule level. Our method allows us to: i) visualize multiparametric nanoparticle datasets to grasp material properties and heterogeneity; ii) have a quantitative evaluation of a material through a series of molecular descriptors, and iii) compare different materials quantitatively and globally, going beyond comparison of a single property. We applied this method to a library of targeted nanoparticles revealing particle heterogeneity, similarities, and correlations between the synthesis and the physicochemical properties of the different nanomaterials. Finally, we show the potential of this approach to reveal batch-to-batch variations in time and between users hidden in standard analysis.

bioinformatics↗

bioRxiv2024

Direct and ultrasensitive bioluminescent detection of intact respiratory viruses

Respiratory viruses such as SARS-CoV-2, influenza, and respiratory syncytial virus (RSV) represent pressing health risks. Rapid diagnostic tests for these viruses detect single antigens or nucleic acids, which do not necessarily correlate with the amount of intact virus. Instead, specific detection of intact respiratory virus particles may better assess the contagiousness of a patient. Here, we report GLOVID, a modular biosensor platform to detect intact virions against a background of free viral proteins in solution. Our approach harnesses the multivalent display of distinct proteins on the surface of a viral particle to template the reconstitution of a split luciferase, allowing specific, single-step detection of intact influenza A and RSV virions corresponding to 0.1 - 0.3 fM of genomic units. The protein ligation system used to assemble GLOVID sensors is compatible with a broad range of binding domains, including nanobodies, scFv fragments and cyclic peptides, which allows straightforward adjustment of the sensor platform to target different viruses.

bioengineering↗

bioRxiv2023

Bioluminescent detection of viral surface proteins using branched multivalent protein switches

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.

biochemistry↗

bioRxiv2023

Critical thermal maxima and oxygen uptake in Elysia viridis (Montagu, 1804), a sea slug capable of photosynthesis

Photosynthetic animals produce oxygen internally, providing an ideal lens for studying how oxygen dynamics influence thermal sensitivity. The sea slug, Elysia viridis, can retain functional chloroplasts from its food alga Bryopsis plumosa for months, but retention is limited when fed Chaetomorpha sp., limiting potential oxygenic benefits. We fed slugs each alga and exposed them to 17{degrees}C (their current yearly maximum temperature) and 22{degrees}C (the increase predicted for 2100), to examine plasticity in thermal tolerance and changes in oxygen uptake when fed and starving. We also examined slugs under increased illumination to examine a potential tradeoff between increased oxygen production, and a faster rate of chloroplast degradation. Following exposure to these conditions, we performed ramping trials, subjecting them to acute thermal stress to determine their thermal tolerance. We also measured oxygen uptake before and after ramping. We observed increases in thermal tolerance for specimens exposed to 22{degrees}C, indicating they acclimated to temperatures higher than they naturally experience. Fed slugs exhibited higher rates of oxygen consumption before exposure to acute thermal stress, and suppressed their oxygen uptake more after it, than starved slugs. Under higher light, slugs exhibited improved thermal tolerance, possibly because increased oxygen production alleviated host oxygen limitation. Accordingly, this advantage disappeared later in the starvation period when photosynthesis ceased due to chloroplast digestion. In conclusion, E. viridis can suppress metabolism to cope with heat waves, however, starvation influences a slugs thermal tolerance and oxygen uptake, so continuous access to algal food for chloroplast retention is critical when facing thermal stress. Summary StatementOxygen has been implicated in determining an ectotherms thermal sensitivity. Examining photosynthetic (and therefore oxygen-producing) sea slugs under various conditions helps elucidate how oxygen and other factors impact thermal tolerance.

physiology↗

bioRxiv2023

Engineering a Scalable and Orthogonal Platform for Synthetic Communication in Mammalian Cells

The rational design and implementation of synthetic, orthogonal mammalian communication systems has the potential to unravel fundamental design principles of mammalian cell communication circuits and offer a framework for engineering of designer cell consortia with potential applications in cell therapeutics and artificial tissue engineering. We lay here the foundations for the engineering of an orthogonal, and scalable mammalian synthetic intercellular communication platform that exploits the programmability of synthetic receptors and selective affinity and tunability of diffusing coiled-coil (CC) peptide heterodimers. Leveraging the ability of CCs to exclusively bind to a selected cognate receptor, we demonstrate orthogonal receptor activation, as well as Boolean logic computations. Next, we reveal synthetic intercellular communication based on synthetic receptors and secreted multidomain CC ligands and demonstrate a minimal, three-cell population system that can perform distributed AND gate logic. 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.

synthetic biology↗

bioRxiv2022

Fast bioluminescent nucleic acid detection using one-pot isothermal amplification and dCas9-based split luciferase complementation

Nucleic acid detection methods based on isothermal amplification techniques show great potential for point-of-care diagnostic applications. However, most current methods rely on fluorescent or lateral flow assay readout, requiring external excitation or post-amplification reaction transfer. Here, we developed a bioluminescent nucleic acid sensor (LUNAS) platform in which target dsDNA is sequence-specifically detected by a pair of dCas9-based probes mediating split NanoLuc luciferase complementation. Whereas LUNAS itself features a detection limit of [~]1 pM for dsDNA targets, the LUNAS platform is easily integrated with recombinase polymerase amplification (RPA), providing attomolar sensitivity in a single-pot assay. We designed a one-pot RT-RPA-LUNAS assay for detecting SARS-CoV-2 RNA without the need for RNA isolation and demonstrated the diagnostic performance for COVID-19 patient nasopharyngeal swab samples using a digital camera to record the ratiometric signal. Detection of SARS-CoV-2 from samples with viral RNA loads of [~]200 cp/L was achieved within [~]20 minutes, showing that RPA-LUNAS is attractive for point-of-care diagnostic applications.

bioengineering↗

bioRxiv2020

RAPPID: a platform of ratiometric bioluminescent sensors for homogeneous immunoassays

Heterogeneous immunoassays such as ELISA have become indispensable in modern bioanalysis, yet translation into easy-to-use 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 that can be detected using a basic digital camera. The concept entails analyte-induced complementation of split NanoLuc luciferase fragments, photoconjugated to an antibody sandwich pair via protein G adapters. We also introduce the use of a calibrator luciferase that provides a robust ratiometric signal, allowing 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. RAPPID combines ratiometric bioluminescent detection with antibody-based target recognition into an easy-to-implement standardized workflow, and therefore represents an attractive, fast, and low-cost alternative to traditional immunoassays, both in an academic setting and in clinical laboratories for point-of-care applications.

bioengineering↗

bioRxiv2020

A bottom-up approach for the de novo design of functional proteins

De novo protein design has enabled the creation of novel protein structures. To design novel functional proteins, state-of-the-art approaches use natural proteins or first design protein scaffolds that subsequently serve as templates for the transplantation of functional motifs. In these approaches, the templates are function-agnostic and motifs have been limited to those with regular secondary structure. Here, we present a bottom-up approach to build de novo proteins tailored to structurally complex functional motifs. We applied a bottom-up strategy to design scaffolds for four different binding motifs, including one bi-functionalized protein with two motifs. The de novo proteins were functional as biosensors to quantify epitope-specific antibody responses and as orthogonal ligands to activate a signaling pathway in engineered mammalian cells. Altogether, we present a versatile strategy for the bottom-up design of functional proteins, applicable to a wide range of functional protein design challenges.

bioengineering↗

bioRxiv2019

Engineered Living Materials based on Adhesin-mediated Trapping of Programmable Cells

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 multi-layer hybrid materials with sub-micron 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 first-of-its-kind 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 high affinity for glucose. The adhesin stably anchors E. coli in dextran-based hydrogels with large pore diameters (10-100 m) 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 lysostaphin via the Type 1 Secretion System and demonstrated that living materials containing this 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.

synthetic biology↗