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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 271 records · Page 15Linked to original sources
bioRxiv2025

A High-Dimensional Interfacial Wave Assay for Early Biophysical Profiling of Therapeutic Antibodies

Therapeutic antibody performance depends not only on sequence and structure, but also on how the molecule responds to physico-chemical perturbations encountered in its environment. Whereas sequence and static structure can be inferred under controlled conditions, behaviour is a conditional, multidimensional response to environment, most directly characterised by applying defined perturbations and quantifying the resulting dynamics. Conventional early-stage developability methods capture isolated dimensions of this response under near-equilibrium conditions, deferring integrated behavioural assessment to late-stage characterisation, when the cost of correction is highest. We introduce Variations in Interfacial Behaviour under Excitation (VIBE), an interfacial wave method implemented via Liquid State Intelligence (LSI), a sensing architecture that transduces molecular perturbations at the air-liquid interface into high-dimensional wave patterns. A colloidal liquid substrate operated near a thermodynamic transition couples small molecular perturbations to large dynamical responses, integrating structural flexibility, charge distribution, and surface hydrophobicity into a single behavioural readout from microgram-scale samples. Applied to antibodies previously characterised by industrial benchmarks, the primary behavioural descriptor, VIBE1, functioned as a high-precision triage tool, flagging candidates carrying multiple biophysical liabilities. In a clinical-stage cohort, the proportion of high-VIBE1 antibodies declined progressively from early trials through approval, and high-VIBE1 candidates showed an elevated clinical failure rate. Concordance analysis against estab-lished methods confirmed that VIBE1 captures a composite signal spanning hydrophobicity, polyreactivity, self-interaction, and thermal stability rather than recapitulating any single conventional readout. These findings establish interfacial wave sensing as a low-material modality for early-stage developability assessment, repositioning molecular behaviour from late-stage validation to discovery-phase characterisation.

biophysics↗
bioRxiv2025

Cell Morphology and Biophysical Mechanisms-Informed Traction Force Microscopy Using Machine Learning

AO_SCPLOWBSTRACTC_SCPLOWQuantitative evaluation of cell motility is essential for understanding biological functions. Traction force microscopy (TFM) is a method for quantifying cellular traction forces. By performing inverse mathematical analysis of substrate deformations induced by cellular forces, it is possible to estimate the underlying traction forces. Among various approaches for detecting substrate deformations, the most widely used is to track the displacement of fluorescent beads randomly embedded in the substrate. However, it is well known that the accuracy of force estimation deteriorates when the observation density is low. Furthermore, standardized datasets have not been established, as validation settings--such as random force distributions on the substrate, cell size, and bead density--vary across studies. To enable quantitative evaluation, we constructed a dataset based on the biophysical mechanism by which cellular traction forces are transmitted to the substrate through stress fibers, clutch proteins, and focal adhesions. In addition, we proposed a novel machine learning model that incorporates cell shape information obtained from observations, and we designed a loss function that accounts for both force magnitude and direction. As a result, our method enables highly accurate force estimation even under sparse observation conditions.

biophysics↗
bioRxiv2025

Two peas in a pod: retroviral RNA dimers organize Gag-RNA nanoclusters with novel biophysical properties

The continued effective control of retroviral infection will no doubt require the development of new clinical interventions targeting underexploited areas of retroviral biology such as genome selection and virion assembly. In our previous work, we demonstrated that both the Gag-psi ({Psi}) interaction and genomic RNA (gRNA) dimerization each uniquely contribute to the formation, morphology, and stability of RSV Gag-viral RNA (vRNA) biomolecular condensates (BMCs). The present work builds upon those observations, utilizing atomic force microscopy (AFM) and fluorescence correlation spectroscopy (FCS) to elucidate the nanoscale morphology, resistance to mechanical deformation, and constituent diffusivity of RSV Gag-vRNA BMCs. These approaches revealed a novel role for gRNA dimerization in nanoscale condensate architecture and mechanical stability that aids in our understanding of why gRNA dimerization is so critical for efficient packaging of the retroviral genome. Further biophysical characterization of RSV Gag-gRNA BMCs therefore possesses great potential to reveal novel avenues for therapeutic intervention.

biophysics↗
bioRxiv2025

Biophysical and Colloidal Properties Govern Anti-Drug Antibody Generation: A Case Study Using the Tumoricidal mAb 4713

Anti-drug antibodies (ADA) are a well-recognized clinical problem, but their sporadic occurrence has limited detailed biophysical characterization of causative factors. We investigated how different stress conditions influence aggregation of a murine monoclonal antibody, mAb 4713, and the resulting potential to induce ADA in mice. Dynamic light scattering (DLS) revealed that stirring, freeze-thaw, and heat stress each generated subvisible aggregates (30-700 nm), whereas non-stressed mAb 4713 remained predominantly monomeric. Stirring- and freeze-thaw-induced aggregates retained native-like secondary and tertiary structures, thermal and biochemical stability, and anti-tumor cytotoxicity, as shown by circular dichroism, tryptophan fluorescence, thermal shift assay, limited proteolysis, and cell death assay. In contrast, heat-induced aggregates displayed complete structural denaturation and loss of cytotoxic activity. Immunization of Jcl: ICR mice demonstrated that heat-induced aggregates were only mildly immunogenic, while freeze-thawed and non-stressed mAb 4713 elicited no detectable immune response, and ADA generated under these conditions did not affect anti-tumor activity. Strikingly, stirred aggregates triggered a strong anti-mAb antibody response, as measured by ELISA, and the resulting antisera completely abolished mAb 4713 cytotoxicity against Raji cells, a Burkitt lymphoma cell line, indicating induction of neutralizing ADA. These results show that aggregate immunogenicity depends not simply on aggregate presence but critically on their structural and colloidal properties and formation pathways, highlighting the need to assess both structural and colloidal properties of aggregates in biopharmaceutical development to mitigate ADA risks.

biophysics↗
bioRxiv2025

Classifying Biophysical Subpopulations of Insulin Secretory Granules using Quantitative Whole Cell Structure Analysis

Pancreatic {beta}-cells contain insulin secretory granules (ISGs), organelles where proinsulin is converted into insulin. As ISGs mature, they undergo extensive biophysical remodeling, producing a spectrum of subpopulations with heterogeneous molecular and spatial characteristics. However, systematic methods to define ISG subpopulations remain underdeveloped. To address this gap in knowledge, we employed soft x-ray tomography (SXT), which can quantitatively measure the biochemical density of ISGs within whole {beta}-cells. Using unsupervised clustering, we classified subpopulations based on molecular density, size, and spatial positioning. Across different insulin secretory stimuli, we observed shifts towards mature and releasable subtypes, demonstrating that exogenous signals can dynamically remodel ISG subpopulation distributions. We extended this methodology to primary {beta}-cells characterized using volume electron microscopy (vEM). Integrating subpopulations from SXT and vEM uncovered insights inaccessible by a single method in isolation. This strategy establishes a framework for defining therapeutic approaches aimed at enriching physiologically beneficial ISG subpopulations.

biophysics↗
bioRxiv2025

Phase diagrams for biophysical fitness landscape design

Populations evolve toward the top of fitness landscapes in the way that physical particles experience forces toward the bottom of a potential energy landscape. We recently introduced fitness landscape design (FLD) as a means of customizing the shape of a proteins biophysical fitness landscape by using antibody sequences and concentrations as tunable control parameters. Until now, previous evidence for the feasibility of FLD has been numerical and simulated. Here, we derive an explicit analytical theory for the FLD phase diagram, providing tight bounds on the designability frontiers describing the extent to which fitnesses of different protein sequences can be tuned independently of each other. We then leverage a recently published experimental dataset of binding affinities between over 62,000 antibody variants and each of three glycoprotein antigens to construct experimental FLD phase diagrams, which show close agreement with theory. These results support the feasibility of engineering quantitatively programmable fitness landscapes for laboratory protein evolution experiments.

biophysics↗
bioRxiv2025

Computational biophysical characterization of a superradiant virus-like particle in its ground state

Superradiance (SR) arises when fluorophores radiate collectively rather than independently, producing ultrabright emission bursts. Recent experiments show that dye-labeled virus-like particles (VLPs) can exhibit SR at room temperature, where the VLP scaffold organizes dyes into a macrodipole of coherent emitters under pulsed excitation. To elucidate the biophysical basis of this phenomenon, microsecond-scale all-atom molecular dynamics (MD) simulations were performed on intact Brome mosaic virus (BMV) VLPs, comparing the native scaffold to a capsid decorated with Oregon Green 488 (OG). Results show that OG conjugation at K105 induces only subtle structural changes: a slight expansion of VLP volume and marginally higher inter-subunit flexibility, while overall shell morphology and pore architecture remain intact. However, dianionic OG alters VLP electrostatics, increasing water exchange across the shell and reducing chloride permeability. Dyes bound in pentamers are more closely packed and conformationally restricted than those in hexamers, and their transition dipoles more frequently adopt parallel orientations predicted to favor excitonic coupling and cooperative emission. By examining the role of scaffold architecture in dye interactions and dynamics, this work offers a mechanistic framework and design principles for engineering SR-VLPs as next-generation fluorescent probes and photonic materials.

biophysics↗
bioRxiv2025

Massively parallel evolution reveals a biophysical scaling law between cell size and internal cell density

Using a massively parallel evolution platform, we selected Saccharomyces cerevisiae for increased cell size to test how cellular architecture adapts to biophysical constraints. As cells evolved larger size, they became less spherical and showed reduced carrying capacity without changes in maximum growth rate. Optical diffraction tomography revealed that individual cells with greater volume consistently exhibited lower internal density, a relationship that persisted across replicate populations and evolved isolates. The largest cells often contained enlarged vacuoles, suggesting that vacuole expansion may sometimes contribute to reduced density. Extending this analysis beyond yeast, 68 species spanning major phylogenetic clades also demonstrate a negative scaling between cell density and cell size. Together, these results suggest that decreased internal density is a conserved consequence of increasing cell size, revealing a fundamental cellular trade-off between volume expansion and material concentration.

biophysics↗
bioRxiv2025

Biophysical Basis of the in vivo ERG of the Mouse: Rod, RPE and Müller glial Cell Contributions To Dark and to Light-driven Changes in Sub-Retinal Space o

The subretinal space (SRS) is a contiguous extracellular volume bounded by the external limiting membrane (ELM) and the retinal pigment epithelium (RPE). Approximately 90% of mouse rod photoreceptor dark current circulates in the SRS, and rod photoresponses materially alter SRS concentrations of permeant ions, especially K+. Following intravitreal injection of drugs that block glutamatergic synapses, and drugs that inhibit Kir4.1 and Kir7.1 channels expressed in Muller and RPE cells respectively, we measured electroretinograms (ERGs) in response to light flashes that completely suppress rod dark current, and compared ERG c-waves with predictions of a biophysical model of extracellular current sources generated by rod, Muller and RPE cells. SRS K+ fluxes in the dark steady-state were dominated by rods - NKX (-6.6 mM s-1), Kv2.1 channels (+5.4 mM s-1), NCKX (0.3 mM s-1) and HCN1 channel fluxes (1.0 mM s-1) - with much smaller contributions from Kir4.1 and Kir7.1 channels and trans-ELM and trans-RPE fluxes combining to zero net flux at resting SRS Ko of 5 mM (re 3.5 mM [K+] in blood plasma). In response to rod-saturating stimulation, Ko was predicted to decline to [~]3.5 mM with a half-time of [~] 0.5 s, driven mainly by a hyperpolarization-dependent decline in Kv2.1 current. The kinetics and amplitude of the c-wave are explained by the decline in SRS Ko, which negatively shifts the Nernst potential of apical RPE Kir7.1 channels, increasing outward current that sinks through the paracellular resistance to basal Bestrophin and CFTR chloride channels causing a trans-epithelial potential of [~] 2 mV.

biophysics↗
bioRxiv2025

Joint Biophysical Modeling of Paired Single-Cell RNA and Protein Measurements

Surface protein measurements can supplement gene expression information from single-cell RNA sequencing to provide a more complete assessment of cell identity and function. Recently developed multiomic assays facilitate such measurements, and can, in principle, be utilized to understand the dynamics of transcription and translation. We develop a framework for biophysical modeling of transcription jointly with translation from single-cell data, along with a suitable technical noise model for sequencing data. We demonstrate its efficacy using simulations, and illustrate how it can be useful in practice with 10x multiomic data. Our proof-of-principle highlights the potential for jointly modeling transcription and translation as data quality and measurement accuracy improves.

biophysics↗
bioRxiv2025

An Integrated Biophysical Fragment Screening Approach Identifies Novel Binders of the CD28 Immune Receptor

CD28 is an essential costimulatory receptor required for full T cell activation, and its dysregulation contributes to multiple immune-mediated pathologies. Despite its central immunological role, CD28 remains largely unexplored as a target for small-molecule modulation, primarily due to the shallow and large interface of its ligand-binding site. Here, we applied a fragment-based high-throughput screening (HTS) strategy to identify low molecular weight chemotypes capable of engaging with human CD28. A 3,200-member library composed of structurally diverse fragments, enriched for scaffolds designed to target protein-protein interaction (PPI) interfaces, was screened in single-dose format using temperature-related intensity change (TRIC) technology, yielding 36 primary hits (1.13% hit rate). Follow-up surface plasmon resonance (SPR) validation confirmed two fragments as direct CD28 binders. Molecular docking analysis revealed a plausible binding orientation for PPIF3 within the CD28 extracellular domain, suggesting potential interaction hotspots that may be exploited during future optimization. Together, these findings provide the first demonstration that fragment-based screening can successfully identify chemotypes capable of engaging with the CD28 PPI interface. This work establishes a scalable, biophysics-driven workflow for CD28 ligand discovery and lays the foundation for subsequent hit-to-lead development of small molecule CD28 modulators.

biophysics↗
bioRxiv2019

Biophysical characterization of DNA origami nanostructures reveals inaccessibility to intercalation binding sites

Intercalation of drug molecules into synthetic DNA nanostructures formed through self-assembled origami has been postulated as a valuable future method for targeted drug delivery. This is due to the excellent biocompatibility of synthetic DNA nanostructures, and high potential for flexible programmability including facile drug release into or near to target cells. Such favourable properties may enable high initial loading and efficient release for a predictable number of drug molecules per nanostructure carrier, important for efficient delivery of safe and effective drug doses to minimise non-specific release away from target cells. However, basic questions remain as to how intercalation-mediated loading depends on the DNA carrier structure. Here we use the interaction of dyes YOYO-1 and acridine orange with a tightly-packed 2D DNA origami tile as a simple model system to investigate intercalation-mediated loading. We employed multiple biophysical techniques including single-molecule fluorescence microscopy, atomic force microscopy, gel electrophoresis and controllable damage using low temperature plasma on synthetic DNA origami samples. Our results indicate that not all potential DNA binding sites are accessible for dye intercalation, which has implications for future DNA nanostructures designed for targeted drug delivery.

biophysics↗
bioRxiv2026

A biophysical framework for accurately identifying antigen single-amino acid escape variants and corresponding variant-specific compensatory TCR sequences

The impact of single amino acid substitution on T-cell receptor (TCR) recognition is central to understanding the molecular determinants of TCR specificity and degeneracy during viral mutational escape, cancer recognition, and autoimmunity. In this study, we developed a biophysics-informed computational approach integrating experimental alanine-scan mutagenesis data from the autoimmune-associated ALWGPDPAAA peptide bound to HLA-A*02:01 together with coarse-grained structural modeling. Our approach reconstructs the energetics and structural determinants underpinning the observed loss of recognition by the diabetogenic 1E6 TCR upon single-point mutations, specifically at the critical Pro5 and Asp6 residues. Leveraging the computational models ability to incorporate multiple structural templates into binding predictions, this approach quantitatively reproduces experimentally measured affinity disruptions. Additionally, we apply our approach to identify potential compensatory interactions capable of restoring binding affinity through alternative residue interactions. This integrative computational framework contributes a strategy for inferring TCR-peptide binding energetics at the single amino acid level, guiding the rational design of peptide-based immunotherapeutics, and predicting the functional impacts of clinically relevant peptide variants.

biophysics↗
bioRxiv2026

Quantitative biophysical analysis of human septin hexamer and octamer self-assembly on model membranes

1Septins are GTP-binding cytoskeletal proteins that shape and compartmentalize the plasma membrane. Their complex interactome has made it difficult to understand the molecular factors that govern their assembly. Moreover, it is unclear whether human septin hexamers and octamers form distinct higher-order assemblies, especially at the plasma membrane. Here, we address this question by using label-free methods to probe binding and self-assembly of recombinant human septins on supported lipid bilayers. Quartz crystal microbalance with dissipation (QCM-D) monitoring revealed that septin-membrane binding is diffusion-limited and concentration-dependent. Hexamers and octamers showed distinct viscoelastic properties, suggestive of structural differences. Imaging by atomic force microscopy (AFM) revealed that septin hexamers formed aligned nematic filamentous networks, whereas septin octamers formed aligned curved structures including spirals. QCM-D and AFM measurements both showed that septins form double-layered filament networks. However, upon C-terminal truncation of the SEPT6 and SEPT7 subunits, hexamers no longer bound the membrane while octamers formed a single-layered network of filament spirals. Our findings reveal that human septin hexamers and octamers interact differently with membranes, providing a baseline to understand their functions in the cell. 2 Significance statementO_LISeptins are cytoskeletal proteins that control cell membrane shape and stiffness. It is poorly understood how septin oligomers, the basic building blocks of septin filaments, bind and assemble on membranes. C_LIO_LIWe used label-free biophysical assays to quantitatively compare the binding kinetics and self-assembly behavior of recombinant human septin hexamers and octamers on supported lipid bilayer membranes. C_LIO_LIOur findings reveal that human septin hexamers and octamers both form organized filamentous networks on membranes, but with different structural properties that may potentially translate into different biological functions. C_LI

biophysics↗
bioRxiv2026

Inference of enhancer-specific transcription factor interactions from gene expression data using a biophysical model

Transcription factors (TFs) play a central role in gene expression and regulation. In recent years, numerous experimental techniques have generated large-scale datasets, alongside computational methods aimed at inferring the role of TF-TF interactions in gene regulation. However, these approaches typically yield global interaction patterns across datasets, which may not accurately reflect local regulatory interactions at specific enhancers. Here, we model transcription using an Ising-type biophysical framework and introduce approximations based on its mean-field representation to infer TF-TF interactions at the level of individual enhancers from expression data, such as STARR-seq or fluorescent protein measurements. We validate our approach using simulated data and evaluate the effect of the strengths of TF-TF and TF-DNA interactions on inference accuracy. We then apply the model to experimental fluorescence data of gap genes for the eve stripe-2 (eve2) enhancer in the fruit fly embryo. The model successfully infers the established roles of the gap genes and predicts the possibility of cooperative and antagonistic interactions among them, which can be experimentally investigated.

biophysics↗
bioRxiv2026

Dynamic organizational strategies of multidomain glycosyltransferases revealed by high-speed AFM and solution biophysics

Glycosyltransferases often contain multiple structural modules that contribute to substrate recognition, catalytic coordination, and higher-order molecular organization. However, how multidomain glycosyltransferases dynamically organize their catalytic domains in solution remains poorly understood. Here, we investigated the assembly states and conformational dynamics of POMGNT2, LARGE1, K4CP, and L137 using high-speed atomic force microscopy (HS-AFM) integrated with complementary solution biophysical analyses. POMGNT2 formed a stable dimeric architecture with limited large-scale conformational fluctuation, consistent with its role in site-selective substrate recognition. In contrast, LARGE1 and K4CP exhibited concentration-dependent and heterogeneous assembly behavior. K4CP displayed pronounced open-closed interdomain motion and substrate-dependent conformational compaction, indicating dynamic catalytic-domain reorganization during glycan elongation. By comparison, the mimivirus glycosyltransferase candidate L137 predominantly behaved as a monomeric species under the tested conditions. These findings demonstrate that multidomain glycosyltransferases employ diverse dynamic organizational strategies ranging from rigid recognition architectures to highly flexible and reversible catalytic assemblies. Our results further suggest that glycosyltransferase function is governed not only by catalytic-domain structure, but also by dynamic conformational coordination adapted to distinct catalytic demands.

biophysics↗
bioRxiv2026

Biophysical characterization of novel biomarkers and bioaffinity reagents (NanoMIPs): on the road for a low-cost diagnostic test for intestinal schistosomiasis

Schistosoma mansoni is a vector-borne intestinal parasite, endemic in [~]70 tropical countries. Most of the World Health Organization interventions are based on mass drug administration (MDA) and sanitation. Since the parasite does not induce permanent immunity, reinfection rate is high, inducing scheduling of several MDA campaigns. To follow up after treatment and avoid blind MDA, providing a new rapid and cheap diagnostic test is needed, since the most used method is fifty years-old, lacks sensitivity and efficiency. Venom Allergen-Like proteins (SmVALs) have been previously identified as secreted/excreted proteins and potential biomarkers. Here we present the biophysical characterization of SmVAL11 and SmVAL13, their recognition by both specific antibodies and molecularly imprinted polymers (nanoMIPs), an easy-to-standardize alternative. The specific antibodies showed no cross-reactivity, while initial characterization of the nanoMIPs indicates a promising level of selectivity, establishing them as a reliable, cost-effective alternative. Therefore, our results are promising for the future development of a new cheap diagnostic test of intestinal schistosomiasis.

biophysics↗