Biology subjects

Walter, N. G.

Publications and source records attributed to Walter, N. G..

At least 19 recordsLinked to original sources

bioRxiv2026

PINPOINT enables non-destructive nanoscopic quality control of individual lipid nanoparticles and their mRNA cargo

Quality control of lipid nanoparticles (LNPs) is central to development of mRNA therapeutics, yet ensemble measurements obscure critical heterogeneity in particle morphology and mRNA cargo loading. Here we introduce PINPOINT (Profiling Individual Nanoparticles with Precise Observation of INternalized Therapeutics), a single-particle nanoscopy platform for non-destructive, multiparametric profiling of preformulated LNP samples. Super-resolution imaging with Nile Red resolves size and morphology, while an optimized permeabilization strategy provides selective access to encapsulated mRNA without compromising LNP integrity. Single-particle fluorescence measurements, including RiboGreen and sequence-specific probes, enable quantification and validation of mRNA loading and identity. Comparative analyses of bare and encapsulated mRNA reveal kinetic and structural features, exposing heterogeneity masked by bulk assays. By integrating structural and molecular readouts at the single-particle level, PINPOINT establishes a quantitative framework for assessing of LNP composition and payload distribution, offering actionable insights for improving batch consistency and guiding the rational design of next-generation mRNA delivery systems.

biophysics↗

bioRxiv2026

FINDER converts zero-background kinetic fingerprinting into area-scalable attomolar biomarker detection

Background constrains analytical sensitivity: surveying larger sensor areas samples more analyte molecules but also accumulates false positives, limiting gains in detection performance. Here we introduce FINDER--Fluorogenic INstantaneous Digital Enumeration and Recognition--a single-molecule platform that combines kinetic fingerprinting with fluorogenic transient probes for rapid molecular classification under near-zero-background conditions. By suppressing both solution and surface-associated background at micromolar probe concentrations, FINDER classifies individual molecules within seconds-scale observation windows per field of view. This regime allows sensitivity to scale with surveyed sensor area, enabling amplification-free quantification of the miRNA cancer biomarker hsa-miR-16 with an 11 aM detection limit. FINDER further generalizes to HPV16 DNA biomarker detection, two-color RNA/DNA co-profiling, and rapid discrimination of clinically relevant EGFR single-nucleotide variants using multidimensional kinetic filtering. Rapid per-field classification permits tens of fields to be surveyed within minutes. By converting kinetic specificity into area-scalable sensitivity, FINDER enables semi-automated attomolar biomarker counting without amplification in practical workflows.

biophysics↗

bioRxiv2026

ASPL-driven subunit exchange remodels VCP/p97 hexamers and is impaired by a multisystem proteinopathy mutation

Valosin-containing protein (VCP/p97) is an essential homohexameric AAA+ ATPase that powers ubiquitin-dependent protein quality control by extraction and unfolding of clients for proteasomal degradation. Heterozygous, autosomal-dominant VCP missense mutants are associated with multisystem proteinopathy (MSP) through unclear molecular mechanisms. We developed a single-molecule pull-down assay to quantify VCP hexamer assembly and subunit exchange dynamics directly in human cell lysate. We show the common MSP-associated VCP variant R155H co-assembles with wild-type subunits to form heterohexamers. Wild-type VCP complexes readily undergo subunit exchange in cell lysates, but this exchange is markedly reduced for purified complexes in buffer. We identify the VCP-interactor ASPL as a selective mediator of monomer exchange that efficiently remodels wild-type, but only modestly exchanges R155H variants within multimers. Single-molecule kinetics analyses reveal [~]2-fold faster ASPL association with, and [~]4-fold slower dissociation from, wild-type VCP than R155H-VCP. We propose that ASPL-driven monomer exchange remodels VCP molecular machines to sustain proteostasis. The failure of ASPL-driven exchange of MSP variant monomers would be predicted to stabilize mutant VCP in assemblies, revealing a potentially targetable defect.

biophysics↗

bioRxiv2026

Npl4 decodes polyubiquitin length and gates D1-D2 coupling in human VCP/p97

VCP/p97 binds the Npl4-Ufd1 heterodimer adaptor to extract polyubiquitinated substrates for proteasomal degradation, but how it decodes K48-linked chain length and how D1-coupled events license downstream D2 power strokes remain unclear. Here we introduce smUbiRAD, or single-molecule ubiquitin recognition and dynamics, and identify a sharp chain-length threshold: Npl4 binds transiently to short chains but switches to long-lived, multivalent engagement on tetra- and penta-ubiquitin. Ufd1 and p97 further stabilize these complexes mainly by suppressing Npl4 dissociation without affecting initial encounter. In fully assembled p97-Ufd1-Npl4-substrate complexes, D1 ATP hydrolysis--rather than D2--drives rapid Npl4 exchange. These results support a model in which D1-powered conformational changes promote cofactor Npl4, but not Ufd1, turnover and gate iterative coupling to downstream D2-driven substrate processing. Finally, we show that multisystem proteinopathy variants R155H and A232E bias p97 toward a high-affinity resting state and accelerate Npl4 exchange, implicating hyperactive cofactor cycling as a disease-linked dysregulation.

biophysics↗

bioRxiv2026

Localized ribosome access and distal tuning via the Listeria prfA RNA thermometer

RNA thermometers (RNATs) are temperature-responsive structures in 5' untranslated regions (UTRs) of bacterial messenger RNA (mRNA) that control translation by modulating ribosome access. The Listeria monocytogenes prfA RNAT represses translation of PrfA (positive regulatory factor A), the master virulence regulator, at ambient temperature and activates it near the human host temperature ([~]37 {degrees}C) by modulating ribosome binding site (RBS) accessibility. However, the prfA RNAT shares no homology with known RNAT classes, and its unfolding mechanism remains unclear. Here, we used analytical ultracentrifugation and single-molecule kinetic analysis of RNA transient structure (SiM-KARTS) to map prfA RNAT unfolding. SiM-KARTS analysis demonstrates that thermal opening occurs predominantly at the RBS, while the upper helix of the RNAT hairpin remains largely folded at 37 {degrees}C. The RBS binding kinetics increases with temperature in parallel with translation output, establishing a quantitative link between structural unfolding and function. Mutations in the upper helix impair thermal regulation, indicating that this region tunes switching even as it stays structured at host temperature. Together, these data reveal a hierarchical unfolding pathway in which initial RBS opening triggers activation, whereas the upper helix remotely tunes temperature sensitivity. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/717274v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@a95daborg.highwire.dtl.DTLVardef@144bc42org.highwire.dtl.DTLVardef@1a39bf6org.highwire.dtl.DTLVardef@545e60_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2026

Directional co-transcriptional folding and pausing create kinetic checkpoints for riboswitch-controlled gene expression

Transcriptional riboswitches typically regulate gene expression by sensing small metabolites or ions that cannot be fluorophore-labeled without perturbing recognition, limiting direct observation of ligand binding during transcription. The GlyQS T-box riboswitch instead senses the uncharged 3' end of a macromolecular ligand, tRNAGly, enabling visualization of co-transcriptional ligand binding. Using single-molecule fluorescence microscopy, we show that tRNAGly dynamically samples discrete structural domains of the nascent riboswitch as transcription proceeds. Productive recognition depends on a hierarchical 5'-to-3' folding pathway shaped by transcription rate and pausing. Pausing enhances tRNA anchoring, stabilizes the riboswitch-tRNA complex, and selectively promotes RNA polymerase readthrough when an uncharged 3' end is presented. These results support a kinetic checkpoint model in which early recruitment increases local ligand concentration, while pause-mediated stabilization converts transient encounters into committed regulatory decisions. More broadly, our findings identify directional co-transcriptional folding and transcriptional pausing as principles synergistically enabling selective and robust riboswitch function.

biophysics↗

bioRxiv2026

pH-dependent allosteric remodeling of a bacterial riboswitch couples alkaline activation to metal sensing

The widespread yybP-ykoY riboswitches control bacterial manganese (Mn) homeostasis by activating exporter expression in response to intracellular Mn2+ levels. The E. coli alx riboswitch distinctively couples Mn2+ sensing to cytoplasmic alkalinity, but the mechanism is unknown. We show that pH tunes the alx aptamers conformational sampling to modulate Mn2+ sensitivity. Single-molecule FRET reveals that Mn2+ stabilizes a docked three-way-junction conformation, and alkaline pH shifts this equilibrium to sensitize metal-dependent folding. Molecular dynamics simulations identify a loop whose low-pH-induced base pairing perturbs the adjacent helix, predicted to allosterically disrupt the Mn2+-binding state. In vivo reporters indicate that both this loop and the Mn2+-binding core are required for optimal pH-dependent translational activation: replacing the core with the non-pH-responsive mntP sequence abolishes activation. These results define how RNA allosterically integrates orthogonal metal and proton cues to enable combinatorial environmental sensing during alkaline stress.

biochemistry↗

bioRxiv2025

Surface-Tethering Enhances Precision in Measuring Diffusion Within 3D Protein Condensates

Biomolecular condensates, or membraneless organelles, play pivotal roles in cellular organization by compartmentalizing biochemical reactions and regulating diverse processes such as RNA metabolism, signal transduction, and stress response. Super-resolved imaging and single-molecule tracking are essential for probing the internal dynamics of these condensates, yet intrinsic Brownian (thermal capillary wave) motion of the entire condensate in vitro could introduce artifacts into diffusion measurements, confounding the interpretation of molecular mobility. Here, we systematically assess and address this question using both experiments and simulations. We deploy three surface-tethering strategies--using biotinylated DNA, protein, or antibody tethers--to immobilize FUS protein condensates on passivated glass surfaces. We show that tethering effectively suppresses the global translational and rotational Brownian motion of the entire condensate, eliminating inherent measurement artifacts while preserving their spherical appearance and native liquid-like properties. Quantitative analysis reveals that untethered condensates systematically overestimate or underestimate molecular diffusion coefficients and step sizes, particularly for slowly diffusing structured mRNAs, while rapidly diffusing unstructured RNAs are unaffected due to temporal scale separation. Comparative evaluation of tethering strategies demonstrates tunable control over condensate stability and internal dynamics, with implications for optimizing experimental design. Finally, simulations spanning the full physiological parameter space enable us to provide practical guidelines for assessing whether, and to what extent, tethering is beneficial, based on condensate size and the diffusion properties of the biomolecule of interest. Our findings establish surface tethering as a necessary and robust approach for accurate quantification of intra-condensate molecular dynamics, providing a methodological framework for future studies of membraneless organelles. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/659185v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1f0ae11org.highwire.dtl.DTLVardef@341574org.highwire.dtl.DTLVardef@16c75daorg.highwire.dtl.DTLVardef@1ad5ed1_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

ER tethering and active transport govern condensate diffusion during hyperosmotic stress

BackgroundHyperosmotic shock and the resulting cell volume compression are commonly experienced by organs such as the kidneys, causing rapid formation of hyperosmotic phase separation (HOPS) condensates in the cytoplasm and nucleoplasm. Although the tight relationship between hyperosmotic shock and condensation has been characterized, the dynamics of biomolecular condensates in hyperosmotically compressed cells and their regulatory mechanisms remain largely unknown. ResultsWe used live-cell single-particle tracking (SPT) across different time scales to systematically characterize the dynamics of HOPS condensates formed by model protein mRNA decapping enzyme 1A (DCP1A). We found that HOPS condensates predominantly exhibited sub-diffusion rather than free diffusion, whereas some ([~]2%) exhibited short super-diffusion. Using tools measuring spatial accessibility inside cells and fluorescence labels for specific cellular organelles, we further revealed the origins of sub-diffusion and super-diffusion as endoplasmic reticulum (ER) attachment and coupling to microtubule-dependent active transport, respectively. Further, we reconstructed an accessibility map of the hyperosmotically compressed cell from trajectories of genetically encoded multimeric nanoparticles (GEMs), revealing that the cytoplasm of a compressed cell remains highly accessible without significant local corrals. ConclusionsIn contrast to prior portrayals of the cytosolic space as static and constrained, our data suggest that the cytosol of a hyperosmotically compressed cell remains dynamic and accessible. Meanwhile, hyperosmotic and potentially other condensates can be spatially organized through docking to membrane structures, with intermittent episodes of long-range transport. These insights broaden our understanding of the physical environment within cells under hyperosmotic shock and provide a model for spatiotemporal organization of condensates via docking or coupling to existing cellular structures and processes.

biophysics↗

bioRxiv2025

Co-transcriptional folding orchestrates sequential multi-effector sensing by a glycine tandem riboswitch

Riboswitches are non-coding RNA motifs that regulate gene expression in response to ligand binding. The glycine tandem riboswitch (GTR) is notable because it comprises two distinct glycine aptamers that interact extensively. These inter-aptamer contacts drive conformational changes in the downstream expression platform to control gene expression. Despite extensive studies, the role of glycine and RNA folding pathways in co-transcriptional regulation remains unclear. Here, we integrate single-molecule kinetic analysis, co-transcriptional RNA structure probing, and computational modeling to reveal that the GTR processes multiple molecular inputs sequentially, guided by polymerase pausing. Our findings elucidate its stepwise 5-to-3 folding pathway and demonstrate how sequential glycine binding to each aptamer, K+ binding to a kink-turn, non-native RNA folding intermediates, inter-aptamer docking that drives binding site pre-organization, and modulation by the transcription factor NusA collectively orchestrate co-transcriptional gene regulation. These results support a model in which glycine binding cooperativity arises through non-equilibrium mechanisms rather than a classical concerted model.

molecular biology↗

bioRxiv2025

A unifying model for microRNA-guided silencing of messenger RNAs

Silencing by the miRNA-guided RNA induced silencing complex (miRISC) is dependent on Ago2-chaperoned base pairing between the miRNA 5' seed (5'S) and a complementary sequence in the 3' untranslated region of an mRNA. Prevailing mechanistic understanding posits that initial 5'S pairing can further allow functional base pair expansion into the 3' non-seed (3'NS), while functionally distinct non-canonical pairing was reported between only the 3'NS and the mRNA coding sequence. We developed single-molecule kinetics through equilibrium Poisson sampling (SiMKEPS) to measure highly precise binding and dissociation rate constants of varying-length target sequences to 5'S and 3'NS in a paradigmatic miRISC isolated from human cells, revealing distinct stable states of miRISC with mutually exclusive 5'S and 3'NS pairing. Our data suggest conformational rearrangements of the Ago2-bound miRNA that regulate alternative 5'S-and 3'NS-driven target recognition. The resulting model reconciles previously disparate observations and deepens our acumen for successfully marshaling RNA silencing therapies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/643529v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1e92bb5org.highwire.dtl.DTLVardef@1348249org.highwire.dtl.DTLVardef@559fdborg.highwire.dtl.DTLVardef@1721b74_HPS_FORMAT_FIGEXP M_FIG C_FIG In brief: In this study, Chatterjee et al. developed a technique to characterize and identify multiple conformations of the RNA-induced silencing complex (RISC) present in human cells. Two distinct pathways of target recognition through different RISC conformations reconcile previously observed divergent findings on the microRNA (miRNA) binding mechanism to messenger RNA (mRNA). HighlightsO_LISiMKEPS is developed to precisely measure binding and dissociation of miRISC:mRNA C_LIO_LISingle human miRISC molecules exhibit mutually exclusive seed and non-seed pairing C_LIO_LISiMKEPS unveils conformational changes of single miRNAs bound to Ago2 C_LIO_LIResulting model reconciles previously divergent findings, supporting RNAi therapies C_LI

biophysics↗

bioRxiv2025

Chromato-kinetic fingerprinting enables multiomic digital counting of single disease biomarker molecules

Early and personalized intervention in complex diseases requires robust molecular diagnostics, yet the simultaneous detection of diverse biomarkers--microRNAs (miRNAs), mutant DNAs, and proteins--remains challenging due to low abundance and preprocessing incompatibilities. We present Biomarker Single-molecule Chromato-kinetic multi-Omics Profiling and Enumeration (Bio-SCOPE), a next-generation, triple-modality, multiplexed detection platform that integrates both chromatic and kinetic fingerprinting for molecular profiling through digital encoding. Bio-SCOPE achieves femtomolar sensitivity, single-base mismatch specificity, and minimal matrix interference, enabling precise, parallel quantification of up to six biomarkers in a single sample with single-molecule resolution. We demonstrate its versatility in accurately detecting low-abundance miRNA signatures from human tissues, identifying upregulated miRNAs in the plasma of prostate cancer patients, and measuring elevated interleukin-6 (IL-6) and hsa-miR-21 levels in cytokine release syndrome patients. By seamlessly integrating multiomic biomarker panels on a unified, high-precision platform, Bio-SCOPE provides a transformative tool for molecular diagnostics and precision medicine.

biophysics↗

bioRxiv2024

Mechanistic model for epigenetic maintenance by methyl-CpG-binding domain proteins

DNA methylation is a fundamental element of epigenetic regulation that is governed by the MBD protein superfamily, a group of "readers" that share a highly conserved methyl-CpG-binding domain (MBD) and mediate chromatin remodeler recruitment, transcription regulation, and coordination of DNA and histone modification. Previous work has characterized the binding affinity and sequence selectivity of MBD-containing proteins toward palindromes of 5-methylcytosine (5mC) containing 5mCpG dinucleotides, often referred to as single symmetrically methylated CpG sites. However, little is known about how MBD binding is influenced by the prototypical local clustering of methylated CpG sites and the presence of DNA structural motifs encountered, e.g., during DNA replication and transcription. Here, we use Single-Molecule Kinetics through Equilibrium Poisson Sampling (SiMKEPS) to measure precise binding and dissociation rate constants of the MBD of human protein MBD1 to DNAs with varying patterns of multiple methylated CpG sites and diverse structural motifs. MBD binding is promoted by two major properties of its DNA substrates: 1) tandem (consecutive) symmetrically methylated CpG sites in double-stranded DNA and secondary structures in single-stranded DNA; and 2) DNA forks. Based on our findings, we propose a mechanistic model for how MBD proteins contribute to epigenetic boundary maintenance between transcriptionally silenced and active genome regions.

biophysics↗

bioRxiv2024

Foundation model for efficient biological discovery in single-molecule data

Modern data-intensive techniques offer ever deeper insights into biology, but render the process of discovery increasingly complex. For example, exploiting the unique ability of single-molecule fluorescence microscopy (SMFM)1-5. to uncover rare but critical intermediates often demands manual inspection of time traces and iterative ad hoc approaches that are difficult to systematize. To facilitate systematic and efficient discovery from SMFM data, we introduce META-SiM, a transformer-based foundation model pre-trained on diverse SMFM analysis tasks. META-SiM achieves high performance--rivaling best-in-class algorithms--on a broad range of analysis tasks including trace selection, classification, segmentation, idealization, and stepwise photobleaching analysis. Additionally, the model produces high-dimensional embedding vectors that encapsulate detailed information about each trace, which the web-based META-SiM Projector (https://www.simol-projector.org) casts into lower-dimensional space for efficient whole-dataset visualization, labeling, comparison, and sharing. Combining this Projector with the objective metric of Local Shannon Entropy enables rapid identification of condition-specific behaviors, even if rare or subtle. As a result, by applying META-SiM to an existing single-molecule Forster resonance energy transfer (smFRET) dataset6, we discover a previously unobserved intermediate state in pre-mRNA splicing. META-SiM thus removes bottlenecks, improves objectivity, and both systematizes and accelerates biological discovery in complex single-molecule data.

biophysics↗

bioRxiv2024

Ultrasensitive amplification-free quantification of a methyl CpG-rich cancer biomarker by single-molecule kinetic fingerprinting

The most well-studied epigenetic marker in humans is the 5-methyl modification of cytosine in DNA, which has great potential as a disease biomarker in liquid biopsies of cell-free DNA. Currently, quantification of DNA methylation relies heavily on bisulfite conversion followed by PCR amplification and NGS or microarray analysis. PCR is subject to potential bias in differential amplification of bisulfite-converted methylated versus unmethylated sequences. Here, we combine bisulfite conversion with single-molecule kinetic fingerprinting to develop an amplification-free assay for DNA methylation at the branched-chain amino acid transaminase 1 (BCAT1) promoter. Our assay selectively responds to methylated sequences with a limit of detection below 1 fM and a specificity of 99.9999%. Evaluating complex genomic DNA matrices, we reliably distinguish 2-5% DNA methylation at the BCAT1 promoter in whole blood DNA from completely unmethylated whole-genome amplified DNA. Taken together, these results demonstrate the feasibility and sensitivity of our amplification-free, single-molecule quantification approach to improve the early detection of methylated cancer DNA biomarkers.

bioengineering↗

bioRxiv2024

Single molecule tracking reveals nanodomains in biomolecular condensates

Biomolecular condensates regulate cellular physiology by sequestering and processing RNAs and proteins, yet how these processes are locally tuned within condensates remains unclear. Moreover, in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), condensates undergo liquid-to-solid phase transitions, but capturing early intermediates in this process has been challenging. Here, we present a surface multi-tethering approach to achieve intra-condensate single-molecule tracking of fluorescently labeled RNA and protein molecules within liquid-like condensates. Using RNA-binding protein Fused in Sarcoma (FUS) as a model for condensates implicated in ALS, we discover that RNA and protein diffusion is confined within distinct nanometer-scale domains, or nanodomains, which exhibit unique connectivity and chemical environments. During condensate aging, these nanodomains reposition, facilitating FUS fibrilization at the condensate surface, a transition enhanced by FDA-approved ALS drugs. Our findings demonstrate that nanodomain formation governs condensate function by modulating biomolecule sequestration and percolation, offering insights into condensate aging and disease-related transitions.

biophysics↗

bioRxiv2024

Molecular basis of mRNA delivery to the bacterial ribosome

Protein synthesis begins with the formation of a ribosome-mRNA complex. In bacteria, the 30S ribosomal subunit is recruited to many mRNAs through base pairing with the Shine Dalgarno (SD) sequence and RNA binding by ribosomal protein bS1. Translation can initiate on nascent mRNAs and RNA polymerase (RNAP) can promote recruitment of the pioneering 30S subunit. Here we examined ribosome recruitment to nascent mRNAs using cryo-EM, single-molecule fluorescence co-localization, and in-cell crosslinking mass spectrometry. We show that bS1 delivers the mRNA to the ribosome for SD duplex formation and 30S subunit activation. Additionally, bS1 mediates the stimulation of translation initiation by RNAP. Together, our work provides a mechanistic framework for how the SD duplex, ribosomal proteins and RNAP cooperate in 30S recruitment to mRNAs and establish transcription-translation coupling.

biochemistry↗

bioRxiv2024

A nascent riboswitch helix orchestrates robust transcriptional regulation through signal integration

Widespread manganese-sensing transcriptional riboswitches effect the dependable gene regulation needed for bacterial manganese homeostasis in changing environments. Riboswitches - like most structured RNAs - are believed to fold co-transcriptionally, subject to both ligand binding and transcription events; yet how these processes are orchestrated for robust regulation is poorly understood. Through a combination of single molecule and bulk approaches, we discovered how a single Mn2+ ion and the transcribing RNA polymerase (RNAP), paused immediately downstream by a DNA template sequence, are coordinated by the bridging switch helix P1.1 in the paradigmatic Lactococcus lactis riboswitch. This coordination achieves a heretofore-overlooked semi-docked global conformation of the nascent RNA, P1.1 base pair stabilization, transcription factor NusA ejection, and RNAP pause extension, thereby enforcing transcription readthrough. Our work demonstrates how a central, adaptable RNA helix functions analogous to a molecular fulcrum of a first-class lever system to integrate disparate signals for finely balanced gene expression control.

molecular biology↗