Molecular Binder Research Articles

New methods of modification of α-cyclodextrin.

While being some of the oldest supramolecular hosts, cyclodextrins remain very popular as molecular binders in materials, devices, artificial enzymes and more. The popularity is undoubtedly connected to the ready availability, carbohydrate biomass origin, biodegradability and water solubility of the cyclodextrins. Many of these applications require synthetic modification of the cyclodextrin - at the simplest the attachment of a linker - but also often attachment of several functional groups, lids, bridges etc. Here we review state of the art methods of modifying α-cyclodextrin, which include direct modications of unprotected α-cyclodextrin and protection/deprotection method to partially modified cyclodextrins.

Unraveling the Properties of Phage Display Fab Libraries and Their Use in the Selection of Gliadin-Specific Probes by Applying High-Throughput Nanopore Sequencing.

Directed evolution is a pivotal strategy for new antibody discovery, which allowed the generation of high-affinity Fabs against gliadin from two antibody libraries in our previous studies. One of the libraries was exclusively derived from celiac patients' mRNA (immune library) while the other was obtained through a protein engineering approach (semi-immune library). Recent advances in high-throughput DNA sequencing techniques are revolutionizing research across genomics, epigenomics, and transcriptomics. In the present work, an Oxford Nanopore in-lab sequencing device was used to comprehensively characterize the composition of the constructed libraries, both at the beginning and throughout the phage-mediated selection processes against gliadin. A customized analysis pipeline was used to select high-quality reads, annotate chain distribution, perform sequence analysis, and conduct statistical comparisons between the different selection rounds. Some immunological attributes of the most representative phage variants after the selection process were also determined. Sequencing results revealed the successful transfer of the celiac immune response features to the immune library and the antibodies derived from it, suggesting the crucial role of these features in guiding the selection of high-affinity recombinant Fabs against gliadin. In summary, high-throughput DNA sequencing has improved our understanding of the selection processes aimed at generating molecular binders against gliadin.

A Multi-Faceted Binding Assessment of Aptamers Targeting the SARS-CoV-2 Spike Protein.

The COVID-19 pandemic has underscored the critical need for the advancement of diagnostic and therapeutic platforms. These platforms rely on the rapid development of molecular binders that should facilitate surveillance and swift intervention against viral infections. In this study, we have evaluated by three independent research groups the binding characteristics of various published RNA and DNA aptamers targeting the spike protein of the SARS-CoV-2 virus. For this comparative analysis, we have employed different techniques such as biolayer interferometry (BLI), enzyme-linked oligonucleotide assay (ELONA), and flow cytometry. Our data show discrepancies in the reported specificity and affinity among several of the published aptamers and underline the importance of standardized methods, the impact of biophysical techniques, and the controls used for aptamer characterization. We expect our results to contribute to the selection and application of suitable aptamers for the detection of SARS-CoV-2.

Proton Conductivity in a Metal-Organic Cube-Based Framework and Derived Hydrogel with Tubular Morphology.

The hydrogels, formed by self-assembly of predesigned, discrete metal-organic cubes (MOCs), have emerged as a new type of functional soft material whose diverse properties are yet to be explored. Here, we explore the proton conductivity of a MOC-based supramolecular porous framework {(Me2NH2)12[Ga8(ImDC)12]·DMF·29H2O} (1) (ImDC = 4,5-imidazole dicarboxylate) and derived hydrogel (MOC-G1). The intrinsic charge-assisted H-bonded (between anionic MOC {[Ga8(ImDC)12]12-} and dimethylammonium cations) framework 1 exhibits an ambient condition proton conductivity value of 2.3 × 10-5 S cm-1 (@40% RH) which increases with increasing temperature (8.2 × 10-4 S cm-1 at 120 °C and 40% RH) and follows the Grotthuss type of mechanism of proton conduction. Self-assembly of the MOCs in the presence of ammonium cations, as molecular binders, resulted in a hydrogel (MOC-G1) that shows directional H-bonded 1D nanotubular morphology. While guest water molecules are immensely important in deciding the proton conductivity of both 1 and MOC-G1, the presence of additional proton carriers, such as DMA and ammonium cations, resulted in at least 1 order increment in the proton conductivity of the latter (1.8 × 10-2 S cm-1) than the former (1.4 × 10-3 S cm-1) under 25 °C and 98% RH condition. The values of proton conductivity of 1 and MOC-G1 are comparable with those of the best proton conduction reports in the literature. This work may pave the way for the development of proton conductors with unique architecture and conductivity requisite for the state-of-the-art technologies by selecting appropriate MOC and molecular binders.

Advancing Mycotoxin Detection in Food and Feed: Novel Insights from Surface-Enhanced Raman Spectroscopy (SERS).

The implementation of low-cost and rapid technologies for the on-site detection of mycotoxin-contaminated crops is a promising solution to address the growing concerns of the agri-food industry. Recently, there have been significant developments in surface-enhanced Raman spectroscopy (SERS) for the direct detection of mycotoxins in food and feed. This review provides an overview of the most recent advancements in the utilization of SERS through the successful fabrication of novel nanostructured materials. Various bottom-up and top-down approaches have demonstrated their potential in improving sensitivity, while many applications exploit the immobilization of recognition elements and molecular imprinted polymers (MIPs) to enhance specificity and reproducibility in complex matrices. Therefore, the design and fabrication of nanomaterials is of utmost importance and are presented herein. This paper uncovers that limited studies establish detection limitsor conduct validation using naturally contaminated samples. One decade on, SERS is still lacking significant progress and there is a disconnect between the technology, the European regulatory limits, and the intended end-user. Ongoing challenges and potential solutions are discussed including nanofabrication, molecular binders, and data analytics. Recommendations to assay design, portability, and substrate stability are made to help improve the potential and feasibility of SERS for future on-site agri-food applications.

A comparative study of protein structure prediction tools for challenging targets: Snake venom toxins

Protein structure determination is a critical aspect of biological research, enabling us to understand protein function and potential applications. Recent advances in deep learning and artificial intelligence have led to the development of several protein structure prediction tools, such as AlphaFold2 and ColabFold. However, their performance has primarily been evaluated on well-characterised proteins and their ability to predict sturtctures of proteins lacking experimental structures, such as many snake venom toxins, has been less scrutinised. In this study, we evaluated three modelling tools on their prediction of over 1000 snake venom toxin structures for which no experimental structures exist. Our findings show that AlphaFold2 (AF2) performed the best across all assessed parameters. We also observed that ColabFold (CF) only scored slightly worse than AF2, while being computationally less intensive. All tools struggled with regions of intrinsic disorder, such as loops and propeptide regions, and performed well in predicting the structure of functional domains. Overall, our study highlights the importance of exercising caution when working with proteins with no experimental structures available, particularly those that are large and contain flexible regions. Nonetheless, leveraging computational structure prediction tools can provide valuable insights into the modelling of protein interactions with different targets and reveal potential binding sites, active sites, and conformational changes, as well as into the design of potential molecular binders for reagent, diagnostic, or therapeutic purposes.

Computational workflow for discovering small molecular binders for shallow binding sites by integrating molecular dynamics simulation, pharmacophore modeling, and machine learning: STAT3 as case study.

STAT3 belongs to a family of seven transcription factors. It plays an important role in activating the transcription of various genes involved in a variety of cellular processes. High levels of STAT3 are detected in several types of cancer. Hence, STAT3 inhibition is considered a promising therapeutic anti-cancer strategy. However, since STAT3 inhibitors bind to the shallow SH2 domain of the protein, it is expected that hydration water molecules play significant role in ligand-binding complicating the discovery of potent binders. To remedy this issue, we herein propose to extract pharmacophores from molecular dynamics (MD) frames of a potent co-crystallized ligand complexed within STAT3 SH2 domain. Subsequently, we employ genetic function algorithm coupled with machine learning (GFA-ML) to explore the optimal combination of MD-derived pharmacophores that can account for the variations in bioactivity among a list of inhibitors. To enhance the dataset, the training and testing lists were augmented nearly a 100-fold by considering multiple conformers of the ligands. A single significant pharmacophore emerged after 188ns of MD simulation to represent STAT3-ligand binding. Screening the National Cancer Institute (NCI) database with this model identified one low micromolar inhibitor most likely binds to the SH2 domain of STAT3 and inhibits this pathway.

Orientation-Controllable Enzyme Cascade on Electrode for Bioelectrocatalytic Chain Reaction.

The accomplishment of concurrent interenzyme chain reaction and direct electric communication in a multienzyme-electrode is challenging since the required condition of multienzymatic binding conformation is quite complex. In this study, an enzyme cascade-induced bioelectrocatalytic system has been constructed using solid binding peptide (SBP) as a molecular binder that coimmobilizes the invertase (INV) and flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase gamma-alpha complex (GDHγα) cascade system on a single electrode surface. The SBP-fused enzyme cascade was strategically designed to induce diverse relative orientations of coupling enzymes while enabling efficient direct electron transfer (DET) at the FAD cofactor of GDHγα and the electrode interface. The interenzyme relative orientation was found to determine the intermediate delivery route and affect overall chain reaction efficiency. Moreover, interfacial DET between the fusion GDHγα and the electrode was altered by the binding conformation of the coimmobilized enzyme and fusion INVs. Collectively, this work emphasizes the importance of interenzyme orientation when incorporating enzymatic cascade in an electrocatalytic system and demonstrates the efficacy of SBP fusion technology as a generic tool for developing cascade-induced direct bioelectrocatalytic systems. The proposed approach is applicable to enzyme cascade-based bioelectronics such as biofuel cells, biosensors, and bioeletrosynthetic systems utilizing or producing complex biomolecules.

Integrated Covalent Drug Design Workflow Using Site Identification by Ligand Competitive Saturation.

Covalent drug design is an important component in drug discovery. Traditional drugs interact with their target in a reversible equilibrium, while irreversible covalent drugs increase the drug-target interaction duration by forming a covalent bond with targeted residues and thus may offer a more effective therapeutic approach. To facilitate the design of this class of ligands, computational methods can be used to help identify reactive nucleophilic residues, frequently cysteines, on a target protein for covalent binding, to test various warhead groups for their potential reactivities, and to predict noncovalent contributions to binding that can facilitate drug-target interactions that are important for binding specificity. To further aid covalent drug design, we extended a functional group mapping approach based on explicit solvent all-atom molecular simulations (SILCS: site identification by ligand competitive saturation) that intrinsically considers protein flexibility, functional group, and protein desolvation along with functional group-protein interactions. Through docking of a library of representative warhead fragments using SILCS-Monte Carlo (SILCS-MC), reactive cysteines can be correctly identified for proteins being tested. Furthermore, a machine learning model was trained to quantify the effectiveness of various warhead groups for proteins using metrics from SILCS-MC as well as experimental model compound warhead reactivity data. The ability to rank covalent molecular binders with similar warheads using SILCS ligand grid free energy (LGFE) ranking was also tested for several proteins. Based on these tools, an integrated SILCS-based workflow was developed, named SILCS-Covalent, which can both qualitatively and quantitatively inform covalent drug discovery.

Suppression of TDP-43 aggregation by artificial peptide binder targeting to its low complexity domain

TAR DNA-binding protein 43 (TDP-43), aggregation prone protein, is a potential target of drug discovery for amyotrophic lateral sclerosis. The molecular binders, targeting the disordered low complexity domain (LCD) relevant to the aggregation, may suppress the aggregation. Recently, Kamagata et al. developed a rational design of peptide binders targeting intrinsically disordered proteins based on contact energies between residue pairs. In this study, we designed 18 producible peptide binder candidates to TDP-43 LCD by using this method. Fluorescence anisotropy titration and surface plasmon resonance assays demonstrated that one of the designed peptides bound to TDP-43 LCD at 30 μM. Thioflavin-T fluorescence and sedimentation assays showed that the peptide binder suppressed the aggregation of TDP-43. In summary, this study highlights the potential applicability of peptide binder design for aggregation prone proteins.

Development of Fluorescent Turn-On Probes for CAG-RNA Repeats.

Fluorescent sensing of nucleic acids is a highly sensitive and efficient bioanalytical method for their study in cellular processes, detection and diagnosis in related diseases. However, the design of small molecule fluorescent probes for the selective binding and detection of RNA of a specific sequence is very challenging because of their diverse, dynamic, and flexible structures. By modifying a bis(amidinium)-based small molecular binder that is known to selectively target RNA with CAG repeats using an environment-sensitive fluorophore, a turn-on fluorescent probe featuring aggregation-induced emission (AIE) is successfully developed in this proof-of-concept study. The probe (DB-TPE) exhibits a strong, 19-fold fluorescence enhancement upon binding to a short CAG RNA, and the binding and fluorescence response was found to be specific to the overall RNA secondary structure with A·A mismatches. These promising analytical performances suggest that the probe could be applied in pathological studies, disease progression monitoring, as well as diagnosis of related neurodegenerative diseases due to expanded CAG RNA repeats.

Robust Bioinspired MXene-Hemicellulose Composite Films with Excellent Electrical Conductivity for Multifunctional Electrode Applications.

MXene-based structural materials with high mechanical robustness and excellent electrical conductivity are highly desirable for multifunctional applications. The incorporation of macromolecular polymers has been verified to be beneficial to alleviate the mechanical brittleness of pristine MXene films. However, the intercalation of a large amount of insulating macromolecules inevitably compromises their electrical conductivity. Inspired by wood, short-chained hemicellulose (xylo-oligosaccharide) acts as a molecular binder to bind adjacent MXene nanosheets together; this work shows that this can significantly enhance the mechanical properties without introducing a large number of insulating phases. As a result, MXene-hemicellulose films can integrate a high electrical conductivity (64,300 S m-1) and a high mechanical strength (125 MPa) simultaneously, making them capable of being high-performance electrode materials for supercapacitors and humidity sensors. This work proposes an alternative method to manufacture robust MXene-based structural materials for multifunctional applications.

Illuminating the G-Quadruplex: A Review on Fluorescent Probes for Detecting Polymorphic G-Quartet DNA Structures

Abstract:Modulating crucial biological processes such as gene regulation, aging, and relationship to globally important human health issues such as cancer has significantly brought considerable attention to G-quadruplex over the past few decades. As the impact of Gquadruplex emerges on so many biological roles, cancer prognosis and pathogenesis have not been fully understood, and selective small molecular binders with suitable chemical, photophysical and biological properties are potentially applicable biophysical tools for tracking Gquadruplex functions. The chemical properties include suitable water solubility, liphophilicity, etc., and the photophysical properties include excitation, emission, stoke-shift, lifetime, quantum yield, and measurable, selective changes of former photophysical parameters within the ideal spectral window upon interaction with the target. The biological properties include; toxicity, cellular infiltration, and selective binding with G-quadruplex over non-specific targets (e.g., duplex DNA, RNA, non-specific biomolecules etc.) in the complex cellular matrix. The development of G-quadruplex-selective probes, therefore, continues to be an important but challenging task for molecular therapeutic, diagnostic, imaging, and sensing applications. In this review, we have classified and summarized several classes of probes; carbocyanine, porphyrins, ethidium, carbazoles, acridines, tripodal or tetrapodal probes, pyrimidine carboxamides, tianguleniums, anthraquinones, polyaromatic hydrocarbons, BODIPY dyes, berberines, acetones and their derivatives for the variation of selectivity, photophysical, and biological properties with respect to the structural modifications, which ultimately provide helpful guidance for designing novel probes with optimal characteristics.

Machine-designed biotherapeutics: opportunities, feasibility and advantages of deep learning in computational antibody discovery.

Antibodies are versatile molecular binders with an established and growing role as therapeutics. Computational approaches to developing and designing these molecules are being increasingly used to complement traditional lab-based processes. Nowadays, in silico methods fill multiple elements of the discovery stage, such as characterizing antibody–antigen interactions and identifying developability liabilities. Recently, computational methods tackling such problems have begun to follow machine learning paradigms, in many cases deep learning specifically. This paradigm shift offers improvements in established areas such as structure or binding prediction and opens up new possibilities such as language-based modeling of antibody repertoires or machine-learning-based generation of novel sequences. In this review, we critically examine the recent developments in (deep) machine learning approaches to therapeutic antibody design with implications for fully computational antibody design.

Supramolecular bridging strategy enables high performance and stable organic–inorganic halide perovskite solar cells

The manipulation of grain boundaries in polycrystalline perovskites is an indispensable consideration for the photovoltaic performance and environmental stability of solar cells, because perovskite films obtained by solution process will inevitably introduce many defects in the grain boundaries. Although additives based on small molecules have proven to be effective defect passivators, their high volatility and diffusibility cannot satisfy the stability requirements of perovskite films. As an upgraded approach, herein, we introduced a supramolecular bridging strategy with 2,5-diaminobenzotrifluorid (DTF) molecular binder as the repeating unit to form a supramolecular-perovskite composite. The supramolecular binder plays a role of bridging the perovskite crystal grains, passivating the traps states of the perovskite films and producing excellent environmental stability, which is superior to the conventional additive engineering. As a result, the power conversion efficiency of the MAPbI3 solar cell has been significantly increased from 18.8% to 21.0 %. The perovskite solar cells with S-DTF maintaining 93% of their original efficiency for over 30 days (∼70% humidity) in air ambient without encapsulation, exhibiting an excellent long-term stability. This will inspire a wider range of ideas beyond the regular additive engineering for improving the performance of polycrystalline perovskite-based photoelectronic devices.

Direct electrical contact of NAD+/NADH-dependent dehydrogenase on electrode surface enabled by non-native solid-binding peptide as a molecular binder

NAD+/NADH-dependent redox enzymes constitute most known oxidoreductases, but their inherent complexity due to their reliance on the diffusional nature of soluble cofactors limits their application in bioelectrocatalytic systems. Herein, a gold-binding peptide (gbp) is genetically introduced at the NAD+/NADH-dependent formate dehydrogenase (FDH) to add a non-native gold-binding activity to directly wiring the enzyme to the electrode surface. Our gold-binding kinetics studies on the native and synthetic FDHs revealed that the gold-binding properties of the fused enzymes are highly dependent on the fusion site and that the tertiary structure of the fusion enzyme controls the efficiency of gold-binding domain display fusion. As such, the highest gold-binding activity was observed with the fusion of gbp at the C-terminus (FDHgbpC), whereas binding at both termini FDHgbpNC and FDHgbpN appeared to be less active. Moreover, the presence of gbp increased the stability of an integrated enzyme electrode in the bioelectrocatalytic reactions occurring at the enzyme-electrode interface. Direct electrochemical NADH oxidation produced by the enzymatic reaction in the presence of formate and NAD+ was observed at a low overpotential range of −0.45 to −0.15 V vs Ag/Ag+ for all types of enzyme-electrodes, which indicates direct-electrical contact between the cofactor binding site and the electrode surface. The enhanced electron transfer kinetics could be explained by the shorter distance between the cofactor-binding site and electrode surface. Furthermore, the addition of NADH and CO2 increased the reductive catalytic current, which suggested the enzymatic CO2 reduction to formate using a hydride of NADH and a subsequent reduction of the generated NAD+. We demonstrated that biotechnology can directly contact enzymes onto the electrode surface, which has proven to be a reliable method for bio-electronic applications, especially involving NAD+/NADH-dependent enzymes.

Isoporous membrane with glucose mediated toughness and protein sieving prepared from novel block copolymers containing boronic acid moieties

Bio-responsiveness is widely found in organisms. Incorporation of this function to membranes is an effective way to endow membranes with new functionalities. In this work, phenylboronic acid groups were used as the glucose binding moieties to construct a novel block copolymer polystyrene-block-poly(4-vinylphenylboronic acid) (PS-b-P4VBA), which was observed to assemble easily into periodic nanostructures in annealed thin films due to strong immiscibility between PS and P4VBA segments. Isoporous membranes were then fabricated from this newly synthesized copolymer following the procedure of self-assembly combined with non-solvent induced phase separation (SNIPS). The prepared isoporous membrane was determined to show a notable glucose dependent permeability that was ascribed to the switch of P4VBA conformation when it was bonded with glucose molecules. This interpretation was demonstrated by the glucose tailored surface wettability and protein sieving. Moreover, the glucose was found to serve as a molecular binder for the P4VBA, and toughness of dried isoporous membranes increased by ∼4 times. The reason was ascribed to the formation of new linkage between glucose and P4VBA that was verified by X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy analysis. Our results prove that the binding between glucose and boronic acid can be used to regulate both the separation performance and mechanical strength for isoporous block copolymer membranes. As far as we know, this performance has been rarely achieved for such membranes before.

Design of hydrangea-type Co/Mo bimetal MOFs and MOF-derived Co/Mo2C embedded carbon composites for highly efficient oxygen evolution reaction

Transition metal organic framework (MOF)-derived catalysts are considered as promising electrocatalysts for the oxygen evolution reactions (OER) owing to their large surface area and abundant catalytic sites. However, to overcome the remaining limitations of their catalytic performance, it is imperative to develop catalysts with more efficient structure and sufficient intrinsic activity. Herein, we report a creative strategy for preparing efficient OER catalysts based on the Co/Mo MOFs with a two-dimensional (2D) hydrangea-type structure by employing molecular binders. In addition, these MOFs were successfully converted into hydrangea-type 2D Co/Mo2C heterostructure nanoparticles embedded in a N-doped carbon composite (H-2D Co/Mo2C@NC). Remarkably, H-2D Co/Mo2C@NC exhibited excellent OER performance with a low overpotential of 256 mV@η10 and a low Tafel slope of 48 mVdec−1. This can be attributed to the large accessible surface area of the nanoparticles owing to their hydrangea-type structure with open active sites and the modulation of the electronic structure of Co by Mo2C. This strategy will provide useful insights into the design of MOF-derived bimetal catalyst and heterojunction catalysts.

Peptide sequence-driven direct electron transfer properties and binding behaviors of gold-binding peptide-fused glucose dehydrogenase on electrode

SummaryOriented enzyme immobilization on electrodes is crucial for interfacial electrical coupling of direct electron transfer (DET)-based enzyme-electrode systems. As inorganic-binding peptides are introduced as molecular binders and enzyme-orienting agents, inorganic-binding peptide-fused enzymes should be designed and constructed to achieve efficient DET. In this study, it is aimed to compare the effects of various gold-binding peptides (GBPs) fused to enzymes on electrocatalytic activity, bioactivity, and material-binding behaviors. Here, GBPs with identical gold-binding properties but different amino acid sequences were fused to the FAD-dependent glucose dehydrogenase gamma-alpha complex (GDHγα) to generate four GDHγα variants. The structural, biochemical, mechanical, and bioelectrochemical properties of these GDHγα variants immobilized on electrode were determined by their fused GBPs. Our results confirmed that the GBP type is vital in the design, construction, and optimization of GBP-fused enzyme-modified electrodes for facile interfacial DET and practical DET-based enzyme-electrode systems.

Effect of the Binder on Properties of Molten Core and Co-Debinding Behavior in Silica-Based Ceramics

The effects of molecular weight of the binder on the properties of molten cores were studied The results showed that the filling parameter had an inverse correlation to molecular weight and was consistent with the Market-Houwink Equation. The surface roughness of the cavity surface deteriorated from 2.468 μm to 11.998 μm as the molecular weight of the binder climbed from 2000 to 6000 due to the presence of residual carbon, which was confirmed by SEM and EDS results. At the debinding stage, the pores that occurred in the ceramic core, were insufficient for the liquid PEG binder to flow out, some of the liquid binder moved to the fringe side. More pores formed as the temperature increased and the rest of the binder flowed out through this channel to flow to the filler. Some of the higher molecular weight PEG binder remained as it could not flow away completely and became residual carbon in the cavity surface.