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bioRxiv2022

The biophysical nature and not only the size of protein aggregates determines the strength of the immune response against dengue ED3

Here we used domain 3 of dengue virus serotype 3 envelope protein (D3ED3), a natively folded globular low-immunogenicity protein, to ask whether the biophysical nature of amorphous aggregates can affect immunogenicity. We prepared amorphous oligomers in five distinct ways. One oligomer type was produced using our SCP tag (Solubility Controlling Peptide) made of 5 Isoleucines (C5I). The others were prepared by miss-shuffling the SS bonds (Ms), heating (Ht), stirring (St), and freeze-thaw (FT). Dynamic light scattering showed that all five formulations contained oligomers of approximately identical sizes with hydrodynamic radii (Rh) between 30 and 55 nm. Circular dichroism (cd) indicated that the secondary structure content of oligomers formed by stirring and freeze-thaw was essentially identical to that of the native monomeric D3ED3. The secondary structure content of the Ms showed moderate changes, whereas the C5I and heat-induced (Ht) oligomers exhibited a significant change. Immunization in JcL:ICR mice showed that both C5I and Ms significantly increased the anti-D3ED3 IgG titer. Ht, St, and FT were barely immunogenic, similar to the monomeric D3ED3. Cell surface CD marker analysis by flow cytometry confirmed that immunization with Ms generated a strong central and effector T-cell memory. This result adds a new dimension to earlier studies where the strength of the immune response was associated solely with the presence and sizes of the oligomers. It also suggests that controlled oligomerization can provide a new, adjuvant-free method for increasing a proteins immunogenicity, yielding a potentially powerful platform for protein-based vaccines. SignificanceProtein aggregation is suspected to increase the immunogenicity of proteins. Here we show that the strength of the immune response depends not merely on the size of the oligomers/aggregates but also on their biophysical properties. Dengue virus 3 envelop protein domain 3 (D3ED3) was oligomerized/aggregated in five different ways. All five formulations contained oligomers with hydrodynamic radii between 30 and 55 nm. Two formulations, where D3 ED3 was natively folded, were not or poorly immunogenic. On the other hand, two others, where D3ED 3 was in a molten globule-like state, were strongly immunogenic. This result adds a new dimension to earlier studies where the strength of the immune response was associated solely with the presence and sizes of the oligomers.

biophysics↗

bioRxiv2023

A programmable and site-specific anchoring system for biophysical analysis of DNA:protein interactions on large topologically closed DNA molecules

Single-molecule and bulk biophysical approaches to study protein-DNA interactions on surface-immobilised nucleic acid templates typically rely on modifying the ends of linear DNA molecules to enable surface-DNA attachments. Unless both strands are constrained, this results in topologically-free DNA molecules and the inability to observe supercoiling-dependent biological processes, or requires additional means to micro-manipulate the free DNA end to impose rotational constraints or induce supercoiling. We developed a method using RecA protein to induce the formation of a circularised compliment-stabilised D-loop. The resulting joint molecule is topologically closed, surface anchorable and stable under microfluidic flow. Importantly, the method obviates the need for subsequent manipulation of surface tethered DNA; tethered molecules remain supercoiled and retain accessibility to DNA binding proteins. This approach adds to the toolkit for those studying processes on DNA that require supercoiled DNA templates or topologically constrained systems. WHY IT MATTERSSupercoiling plays an important role in regulating genetic processes such as DNA replication and transcription. We have developed a facile method to immobilise large (supra diffraction-limited) supercoiled DNA substrates without the need for complex trapping modalities to allow biophysical interrogation of the surface-tethered DNA molecules. This will expand the toolkit for experimentalists interested in studying protein-DNA interactions at the single-molecule or ensemble level requiring the use of topologically closed or supercoiling-dependent systems such as origin-dependent bacterial DNA replication.

biophysics↗

bioRxiv2023

Biophysical evolution of the receptor binding domains of SARS-CoVs

With hundreds of coronaviruses (CoVs) identified in bats that are capable of infecting humans, it is important to understand how CoVs that affected the human population have evolved. Seven known coronaviruses have infected humans, of which three CoVs caused severe disease with high mortality rates: SARS-CoV emerged in 2002, MERS-CoV in 2012, and SARS-CoV-2 in 2019. Both SARS-CoV and SARS-CoV-2 belong to the same family, follow the same receptor pathway, and use their receptor binding domain (RBD) of spike protein to bind to the ACE2 receptor on the human epithelial cell surface. The sequence of the two RBDs is divergent, especially in the receptor binding motif (RBM) that directly interacts with ACE2. We probed the biophysical differences between the two RBDs in terms of their structure, stability, aggregation, and function. Since RBD is being explored as an antigen in protein subunit vaccines against CoVs, determining these biophysical properties will also aid in developing stable protein subunit vaccines. Our results show that despite RBDs having a similar three-dimensional structure, they differ in their thermodynamic stability. RBD of SARS-CoV-2 is significantly less stable than that of SARS-CoV. Correspondingly, SARS-CoV-2 RBD shows a higher aggregation propensity. Regarding binding to ACE2, less stable SARS-CoV-2 RBD binds with a higher affinity than more stable SARS-CoV RBD. In addition, SARS-CoV-2 RBD is more homogenous in terms of its binding stoichiometry towards ACE2, compared to SARS-CoV RBD. These results indicate that SARS-CoV-2 RBD differs from SARS-CoV RBD in terms of its stability, aggregation, and function, possibly originating from the diverse RBMs. Higher aggregation propensity and decreased stability of SARS-CoV-2 RBD warrants further optimization of protein subunit vaccines that use RBD as an antigen either by inserting stabilizing mutations or formulation screening. Statement of SignificanceThis study holds significant relevance in the context of the COVID-19 pandemic and the broader understanding of coronaviruses. A comparison of the receptor binding domains (RBDs) of SARS-CoV and SARS-CoV-2 reveals significant differences in their structure, stability, aggregation, and function. Despite divergent sequences, the RBDs share a similar fold and ACE2 receptor binding capability, likely through convergent evolution. These findings are crucial for understanding coronavirus evolution, interactions with human receptors, and the spillover of coronaviruses from animals to humans. The study also has implications for vaccine design strategies for SARS-CoVs, where the RBD is used as an antigen in protein subunit vaccines. By anticipating future outbreaks and enhancing our understanding of zoonotic spillover, this research contributes to safeguarding human health.

biophysics↗

bioRxiv2024

Biophysical limits of ultrafast cellular motility

Many single-celled organisms and specialized cell types can surprisingly achieve speed and acceleration significantly faster than multicellular counterparts. These remarkable cellular machines must integrate energy storage and amplification in actuation, latches for triggered release, and energy dissipation without failure -- all implemented in macro-molecular assemblies inside a single cell. However, a universal biophysical framework that can comparatively evaluate extreme cellular motility remains lacking. Scaling laws have long been recognized as powerful tools for revealing universal principles in physical systems. We map the atlas of ultrafast motility for single cells across the tree of life. We then introduce a new quantitative framework that can be used to evaluate and compare extreme acceleration, speed, area strain rate, volume expansion strain rate, and density changes in single cells. Recognizing that many single cells operate in low-Reynolds number environments, we introduce a new dimensionless number, the "cellular acceleration number," based on energy dissipation at this scale. Using this new framework, we discover a scaling law between the cellular acceleration number and the transient Reynolds number, valid across six orders of magnitude in a range of single-cell organisms. We further generalize these ideas by placing various trigger, actuation, and dissipation mechanisms within the same framework and estimating the fundamental limits of speed and acceleration at the cellular scale. We conclude with a detailed summary of the range of functions implemented via ultrafast cellular phenomena, laying down a quantitative foundation for extreme biophysics at the cellular scale.

biophysics↗

bioRxiv2024

A minimalist model lipid system mimicking the biophysical properties of Escherichia coli membrane

Biological membrane are highly complex systems that are of fundamental importance to the development and survival of organisms. Native membranes typically comprise different types of lipids, biomolecules and proteins assembled around a lipid bilayer structure. This complexity can render investigations challenging, with many studies relying on model membranes such as artificial vesicles and supported lipid bilayers (SLBs). The purpose of a model system is to capture the desired dominant features of the native context while remaining uniquely defined and simpler. Here, we search for a minimal lipid-only model system of the Escherichia coli inner membrane. We aim to retain the main lipidomic components in their native ratio while mimicking the membrane thermal and mechanical properties. We design a collection of candidate model systems reflecting the main aspects of the known native lipidomic composition and narrow down our selection based on the systems phase transition temperature. We further test our candidate model systems by independently measuring their elastic properties. We identify 3 ternary model systems able to form stable bilayers that closely mimic E. colis inner membrane lipid composition and mechanical properties. These model systems are made of commercially available synthetic 16:0-18:1 phosphatidylethanolamine (POPE), 16:0-18:1 phosphatidylglycerol (POPG), and 16:0-18:1 Cardiolipin (CL). We anticipate our results to be of interest for future studies making use of E. coli models, for example investigating membrane proteins function or macromolecule-membrane interactions. Significance StatementEscherichia Coli membranes serve as model systems for numerous fundamental and technological applications in the field of membrane biophysics. Being a Gram-negative bacterium, E. Coli is characterized by a thin cell wall that separates two phospholipid membranes: the inner and outer membranes. These membranes are complex, comprising many different lipids, proteins and other biomolecules. Here we develop a minimalist system to mimic the biophysical properties and lipidic composition of E. Colis inner membrane. Using only commercially available lipids, we develop a model membrane that can be used for studies where simplicity is needed to aid interpretation of the results, for example to investigate protein-induced mechano-transduction across E. Coli membranes.

biophysics↗

bioRxiv2024

The biophysical mechanism of mitochondrial pearling

Mitochondrial networks exhibit remarkable dynamics that are driven in part by fission and fusion events. However, there are other reorganizations of the network that do not involve fission and fusion. One such exception is the elusive, "beads-on-a-string" morphological transition of mitochondria. During such transitions, the cylindrical tubes of the mitochondrial membrane transiently undergo shape changes to a string of "pearls" connected along thin tubes. These dynamics have been anecdotally observed in many contexts and given disparate explanations. Here we unify these observations by proposing a common underlying mechanism based on the biophysical properties of tubular fluid membranes for which it is known that, under particular regimes of tension and pressure, membranes reach an instability and undergo a shape transition to a string of connected pearls. First, we use high-speed light-sheet microscopy to show that transient, short-lived pearling events occur spontaneously in the mitochondrial network in every cell type we have examined, including primary fibroblasts, T-cells, neurons, and budding yeast. We present evidence that transient mitochondrial pearling occurs during important biological events, particularly during T cell activation, neuronal firing, and replicative senescence. Using our high-temporal resolution data, we identify two distinct categories of spontaneous pearling, i) internal pressure-driven pearling generated by ionic flux, and ii) external tension-driven pearling generated by the cytoskeleton. By applying live-cell STED and FIB-SEM imaging we document the structural reorganization of inner cristae membranes during mitochondrial pearling and the role of the MICOS complex in regulating the frequency of pearling events. We then establish numerous methods for inducing pearling, including the ability to induce these dynamics with single mitochondrion precision. These methods include ionophores, channel activators, osmotic shock, detergents, laser stimulation, membrane intercalating molecules, chemical fixation, and micro-needle force. These disparate inducers establish three main physical causes of pearling, i) ionic flux producing internal osmotic pressure, ii) membrane packing lowering bending elasticity, and iii) external mechanical force increasing membrane tension. Pearling dynamics thereby reveal a fundamental biophysical facet of mitochondrial biology. We suggest that pearling should take its place beside fission and fusion as a key process of mitochondrial dynamics, with implications for physiology, disease, and aging.

biophysics↗

bioRxiv2025

Biophysical modeling reveals the transcriptional regulatory mechanism of Spo0A, the master regulator in starving Bacillus subtilis

In starving Bacillus subtilis bacteria, the initiation of two survival programs - biofilm formation and sporulation - is both controlled by the same phosphorylated master regulator Spo0A[~]P. Its gene, spo0A, is transcribed from two promoters, Pv and Ps, that are respectively regulated by RNAP holoenzymes bearing {sigma}A and {sigma}H. Notably, transcription is directly autoregulated by Spo0A[~]P binding sites known as 0A1, 0A2, and 0A3 box, located in between the two promoters. It remains unclear whether, at the onset of starvation, these boxes activate or repress spo0A expression, and whether the Spo0A[~]P transcriptional feedback plays a role in the increase in spo0A expression. Based on the experimental data of the promoter activities under systematic perturbation of the promoter architecture, we developed a biophysical model of transcription regulation of spo0A by Spo0A[~]P binding to each of the 0A boxes. The model predicts that Spo0A[~]P binding to its boxes does not affect the RNA polymerase recruitment to the promoters but instead affects the transcriptional initiation rate. Moreover, the effects of Spo0A[~]P binding to 0A boxes are mainly repressive and saturated early at the onset of starvation. Therefore, the increase in spo0A expression is mainly driven by the increase in RNAP holoenzyme levels. Additionally, we reveal that Spo0A[~]P affinity to 0A boxes is strongest at 0A3 and weakest at 0A2 and that there are attractive forces between the occupied 0A boxes. Our findings, in addition to clarifying how the sporulation master regulator is controlled, offer a framework to predict regulatory outcomes of complex gene-regulatory mechanisms. ImportanceCell differentiation is often critical for survival. In bacteria, differentiation decisions are controlled by transcriptional master regulators under transcriptional feedback control. Therefore, understanding how master regulators are transcriptionally regulated is required to understand differentiation. However, in many cases, underlying regulation is complex with multiple transcription factor binding sites and multiple promoters, making it challenging to dissect the exact mechanisms. Here, we address this problem for the B. subtilis master regulator Spo0A. Using a biophysical model, we quantitatively characterize the effect of individual transcription factor binding sites on each spo0A promoter. Further, the model allows to identify the specific transcription step that is affected by transcription factor binding. Such model is promising for the quantitative study of a wide range of master regulators involved in transcriptional feedback.

biophysics↗

bioRxiv2025

Quantome: A Quantum Surrogate Model for Biophysical Landscapes

Accurately modelling the potential energy landscapes that govern molecular interactions is a central challenge in computational biophysics. While quantum computers promise to solve such problems with high fidelity, a key bottleneck is the encoding of complex spatial information into low-qubit Hamiltonians suitable for near-term devices. Here, we introduce a generalisable framework for creating quantum surrogate models of 2D biophysical landscapes. Our method translates a discrete, classically-derived potential energy grid into a continuous quantum Hamiltonian by fitting it to a high-degree polynomial, where the polynomials coefficients directly define the potential energy operator. We demonstrate this pipeline on a custom-designed landscape featuring two asymmetric potential wells. By systematically varying a kinetic hopping term in the Hamiltonian, our variational quantum eigensolver simulations, averaged over 100 runs, successfully reproduce the physical transition from a localised ground state to a delocalised state governed by tunnelling. The entire framework is made accessible through the Quantome web application, a pedagogical platform that allows users to both design custom landscapes and explore pre-calculated results on EF-hand protein binding sites, which serve as a simplified real-world test bed for this framework. The web app is freely available at: https://biosig.lab.uq.edu.au/quantumlabs/quantome.

biophysics↗

bioRxiv2025

A FINITE ELEMENT FRAMEWORK FOR BULK-SURFACE COUPLED PDES TO SOLVE MOVING BOUNDARY PROBLEMS IN BIOPHYSICS

We consider moving boundary problems for biophysics and introduce a new computational framework to handle the complexity of the bulk-surface PDEs. In our framework, interpretability is maintained by adapting the fast, generalizable and accurate structure preservation scheme in [29]. We show that mesh distortion is mitigated by adopting the pioneering work of [36], which is tied to an Arbitrary Lagrangian Eulerian (ALE) framework. We test our algorithms accuracy on moving surfaces with boundary for the following PDEs: advection-diffusion-reaction equations, phase-field models of Cahn-Hilliard type, and Helfrich energy gradient flows. We performed convergence studies for all the schemes introduced to demonstrate accuracy. We use a staggered approach to achieve coupling and further verify the convergence of this coupling using numerical experiments. Finally, we demonstrate broad applicability of our work by simulating state-of-the-art tests of biophysical models that involve membrane deformation.

biophysics↗

bioRxiv2018

Chromatin compaction states, nuclear shape fluctuations and auxeticity: A biophysical interpretation of the epigenetic landscape of stem cells

When embryonic stem cells differentiate, the mechanical properties of their nuclei evolve en route to their terminal state. Measurements of the deformability of cell nuclei in the transitional state that intervenes between the embryonic stem cell state and the differentiation primed state of mouse stem cells, indicate that such nuclei are auxetic i.e. have a negative Poissons ratio. We show, using a theoretical model, how this unusual mechanical behaviour results from the coupling between chromatin compaction states and nuclear shape. Our biophysical approach, which treats chromatin as an active polymer system whose mechanics is modulated by nucleosome binding and unbinding, reproduces experimental results while providing new predictions. We discuss ways of testing these predictions. Our model suggests a biophysical interpretation of the epigenetic landscape of stem cells.

biophysics↗

bioRxiv2019

Inferring biophysical models of evolution from genome-wide patterns of codon usage

Frequencies of synonymous codons are typically non-uniform, despite the fact that such codons correspond to the same amino acid in the genetic code. This phenomenon, known as codon bias, is broadly believed to be due to a combination of factors including genetic drift, mutational biases, and selection for speed and accuracy of codon translation; however, quantitative modeling of codon bias has been elusive. We have developed a biophysical population genetics model which explains genome-wide codon frequencies observed across 20 organisms. We assume that codons evolve independently of each other under the influence of mutation and selection forces, and that the codon population has reached evolutionary steady state. Our model implements codon-level treatment of mutations with transition/transversion biases, and includes two contributions to codon fitness which describe codon translation speed and accuracy. Furthermore, our model includes wobble pairing - the possibility of codon-anticodon base pairing mismatches at the 3 nucleotide position of the codon. We find that the observed patterns of genome-wide codon usage are consistent with a strong selective penalty for mistranslated amino acids. Thus codons undergo purifying selection and their relative frequencies are affected in part by mutational robustness. We find that the dependence of codon fitness on translation speed is weaker on average compared to the strength of selection against mistranslation. Although no constraints on codon-anticodon pairing are imposed a priori, a reasonable hierarchy of pairing rates, which conforms to the wobble hypothesis and is consistent with available structural evidence, emerges spontaneously as a model prediction. Finally, treating the translation process explicitly in the context of a finite ribosomal pool has allowed us to estimate mutation rates per nucleotide directly from the coding sequences. Reminiscent of Drakes observation that mutation rates are inversely correlated with the genome size, we predict that mutation rates are inversely proportional to the number of genes. Overall, our approach offers a unified biophysical and population genetics framework for studying codon bias across all domains of life.

biophysics↗

bioRxiv2019

Biophysical characterization reveals the similarities of liposomes produced using microfluidics and electroformation

Giant Unilamellar Vesicles (GUVs) are a versatile tool in many branches of science, including biophysics and synthetic biology. Octanol-Assisted Liposome Assembly (OLA), a recently developed microfluidic technique enables the production and testing of GUVs within a single device under highly controlled experimental conditions. It is therefore gaining significant interest as a platform for use in drug discovery, the production of artificial cells and more generally for controlled studies of the properties of lipid membranes. In this work, we expand the capabilities of the OLA technique by forming GUVs of tunable binary lipid mixtures of DOPC, DOPG and DOPE. Using fluorescence recovery after photobleaching we investigated the lateral diffusion coefficients of lipids in OLA liposomes and found the expected values in the range of 1 m2/s for the lipid systems tested. We studied the OLA derived GUVs under a range of conditions and compared the results with electroformed vesicles. Overall, we found the lateral diffusion coefficients of lipids in vesicles obtained with OLA to be quantitatively similar to those in vesicles obtained via traditional electroformation. Our results provide a quantitative biophysical validation of the quality of OLA derived GUVs, which will facilitate the wider use of this versatile platform.

biophysics↗

bioRxiv2026

A Biophysical Model of Human Colonic Motor Pattern Generation in Health and Disease

PurposeColonic motility disorders, including diarrhea-predominant irritable bowel syndrome and slow-transit constipation, impose a major clinical burden. Although high-resolution colonic manometry reveals characteristic spatiotemporal motor patterns, such as high-amplitude propagating contractions and cyclic motor pattern in healthy individuals, these patterns are often altered or absent in disease. Understanding how these patterns arise from underlying pacemaker, neural, and mechanical mechanisms is essential for improving treatment strategies. MethodsWe developed a biophysical whole-colon model that integrates an Interstitial Cells of Cajal-inspired oscillator network, enteric nervous system reflexes, a pressure-gated modulation element motivated by rectosigmoid brake behavior, and a nonlinear tube law describing colon wall mechanics. The model simulates spatiotemporal pressure patterns along the colon and allows systematic variation of physiological parameters associated with pacemaker activity, neural reflex control, and distal gating. ResultsA small set of parameters reproduces three illustrative motility patterns corresponding to healthy motility, diarrhea-predominant irritable bowel syndrome, and slow-transit constipation. The simulated pressure maps recapitulate key features observed in high-resolution manometry, including propagation direction, regional patterning of contractions, and case-specific changes in amplitude and coordination. Sensitivity analysis suggests that proximal excitation strength and waveform morphology strongly influence global motility metrics. ConclusionOur study presents a simple, biophysical framework for reproducing clinically observed colonic motor patterns and exploring their disruption in disease. More broadly, the model may help interpret clinical manometry in mechanistic terms and support hypothesis-driven in silico studies of colonic motility disorders.

biophysics↗

bioRxiv2026

Structural modelling and biophysical analyses reveal a dimeric coiled-coil architecture in the FAZ10 central region of Trypanosoma brucei

Trypanosoma brucei relies on the flagellum attachment zone (FAZ) to coordinate flagellum positioning, cell morphology, and cytokinesis. The giant FAZ10 protein, which contains both repetitive and structured regions, is essential for correct cleavage-furrow placement, yet its molecular organization remains unresolved due to its exceptional size. In this study, we define the architecture of the FAZ10 central region through structural modelling and biophysical validation. AlphaFold2 and molecular dynamics simulations defined a parallel coiled-coil dimer flanked by symmetric globular domains, with canonical hydrophobic core packing, complementary interhelical contacts, and local heptad discontinuities, including a stammer-stutter pair that modulates the superhelical geometry. Biophysical analyses show that this region forms a stable dimer in solution, mediated by the coiled-coil domain and consistent with a predominantly -helical structure. Together, these findings identify the FAZ10 central region as a semi-flexible dimeric scaffold that provides a structural framework for understanding FAZ supramolecular organization and the integration of large cytoskeletal assemblies in trypanosomatids.

biophysics↗

bioRxiv2026

Lipid-Driven Biophysical Selection of β-Furanoside-5'-Phosphate as the Sole Scaffold of Extant RNA

The emergence of RNAs primordial backbone presents a fundamental question in the study of lifes origins: why was {beta}-furanoside-5-phosphate selected over other isomeric alternatives as the predominant building block during the origin of life? In contrast to previous studies that have demonstrated selective synthetic routes to canonical RNA structures, we explore biophysical factors that selectively favor {beta}-furanoside and 5-nucleotide over their undesired isomers. Inspired by the chromatographic elution order of nucleoside and nucleotide isomers, we establish a new selection model to discriminate between them. {beta}-Furanoside is found to be more lipid-permeable than any other configurational isomer, making it the most abundant species after permeation. Outward permeation selectively screens intracellularly formed nucleotides, enriching 5-nucleotide as the primary RNA building block within a protocell. The unique properties of representative canonical nucleosides and nucleotides are rationalized based on both their structural and dynamic characteristics, as elucidated by DFT and MD calculations. A scenario in which {beta}-furanoside selectively accumulates in a lipid droplet is further investigated using a specifically designed micromixer. Together, these findings suggest the existence of a primitive selection mechanism driven by purely biophysical forces, which may have played a critical role in advancing key steps in the progression of primitive biomolecules.

biophysics↗

bioRxiv2026

A generalisable method for the purification and biophysical characterisation of bacterial membrane receptors

Membrane-embedded bacterial receptors are challenging to express and purify in soluble form, yet their isolated domains are essential tools for structural and ligand-discovery studies. Pseudomonas aeruginosa relies on the TonB-dependent heme receptor HasR for iron acquisition, a process central to its pathogenicity. Here, we report a robust strategy for the recombinant expression, purification, and biophysical characterisation of the two soluble HasR domains directly involved in heme uptake: the N-terminal plug and the Secretin/TonB short N-terminal domain. Each domain was expressed individually in E. coli and purified to homogeneity, adopting well-folded conformations as confirmed by circular dichroism, NMR spectroscopy, and mass spectrometry. We then engineered a fusion construct containing both domains and systematically evaluated multiple solubilisation tags. A GST-His dual-affinity strategy enabled efficient purification of the construct, whereas His-tag alone resulted in insoluble protein and HLT-tag fusions suffered from non-specific proteolysis. Biophysical analyses revealed that the Secretin/TonB short N-terminal domain remains stably folded within the fusion construct, while the N-terminal plug domain becomes partially disordered, a finding further supported by hydrogen/deuterium exchange mass spectrometry. Together, these results establish a generalizable workflow for producing soluble receptor domains from membrane proteins and provide validated HasR constructs suitable for downstream ligand-screening applications, including aptamer and nanobody discovery.

biophysics↗

bioRxiv2026

A modular DNA toolkit for single-molecule biophysics

Long, customizable DNA molecules are an essential tool for in vitro single-molecule biophysics. Most studies still rely on the {lambda}-phage genome, which has a fixed sequence, a non-uniform GC distribution, and limited options for modification. To overcome these limitations, we developed a modular DNA (ModDNA) toolkit that integrates in silico sequence design with cloning, to create long (up to 100 kb) DNA molecules with entirely user-defined sequences. We used a computational workflow with curated databases to design a motif-depleted sequence backbone, into which any desired sequence can be inserted at any position. Golden Gate assembly then builds these sequences into constructs, which we extend iteratively in defined increments up to 100 kb. We show precise placement of local features, e.g. a fluorophore, a bound protein, and a DNA-origami structure, and illustrate the construct's utility in single-molecule visualization assays, including DNA loop extrusion by human condensin I. ModDNA offers a scalable, sequence-programmable alternative to {lambda}-DNA and other restrictive substrates. More broadly, the motif-aware feature-free design connects single-molecule biophysics to emerging efforts in machine-guided regulatory-sequence design and synthetic-genome engineering, where controlled programmable sequence context is increasingly essential.

biophysics↗

bioRxiv2016

Biophysical Attributes of CpG Presentation Control TLR9 Signaling to Differentially Polarize Systemic Immune-Responses

It is currently unknown whether and how mammalian pathogen-recognition receptors (PRR) respond to biophysical patterns of pathogen-associated molecular danger-signals. Using synthetic pathogen-like particles (PLPs) that mimic physical properties of bacteria or large-viruses, we have discovered that the quality and quantity of Toll-like-receptor-9 (TLR9)-signaling by CpG in mouse dendritic cells (mDC) is uniquely dependent on biophysical attributes, specifically the surface-density of CpG and size of the presenting PLP. These physical patterns control DC-programming by regulating kinetics and magnitude of MyD88-IRAK4 signaling, NF{kappa}B-driven responses, and STAT3 phosphorylation, which in turn controls differential T cell responses and in vivo immune-polarization, especially T-helper 1 (Th1) versus T-helper 2 (Th2) antibody responses. Our findings suggest that innate immune cells can sense and respond not only to molecular, but also pathogen-associated physical patterns (PAPPs), broadening the tools for modulating immunity, helping to better understand innate response mechanisms to pathogens and develop new and improved vaccines.

Immunology↗