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Basak, S.

Publications and source records attributed to Basak, S..

At least 19 recordsLinked to original sources
bioRxiv2026

A generalizable interface-seeded framework for de novo design of functional oligomers

Protein oligomers are ubiquitous in biological systems and essential for function. However, the de novo design of oligomers that controllably assemble in response to exogenous stimuli remains challenging. Here, we present an AI-based generative approach that leverages an interface-seeded strategy for designing responsive homo-oligomers from isolated interaction modules. Experimentally validated designs are highly accurate and explore new-to-nature topologies. We show that designs effectively respond to their chemical triggers with conditional oligomerization or to phosphorylation-driven conformational changes with reversible oligomerization. We further functionalized our responsive assemblies to build ligand-dependent membrane binding systems and phosphorylation-controlled gene regulatory switches. Our framework enables the generalizable design of responsive protein complexes, opening novel possibilities for the engineering of biosynthetic systems with sophisticated regulatory mechanisms.

biochemistry↗
bioRxiv2026

Co-transcriptional Phase Separation of Nucleic Acids at Membrane Surfaces

Transcription is usually framed as information transfer, yet it also injects a new polymer into a crowded, confined environment. Here we demonstrate how spatial confinement to surfaces in a minimal membrane-bound transcription (MBT) system displays the physical consequences of RNA synthesis. Within a dense membrane-tethered DNA network, transcription drives co-transcriptional RNA phase separation: nascent RNA oligomerizes, gels and demixes from a surrounding fluid DNA phase, generating stable spatial patterns while mechanically remodeling the DNA layer. RNA gelation sequesters T7 RNA polymerase, whereas RNA-binding and translation-associated factors reverse gelation and restore fluidity. Thus, in the absence of downstream regulatory machinery, transcription under confinement is sufficient to trigger RNA condensation and nucleic-acid phase separation. The membrane as confining interface catalyzes the onset of DNA-RNA demixing and modulates the morphology of the resulting patterns. Since such large-scale spatial unmixing may be detrimental to cellular physiology, we suggest that one fundamental role of translation is to actively prevent condensation effects created by continuous RNA production.

biochemistry↗
bioRxiv2026

Nanobodies equipped with HaloTag variants enable rapid and straightforward one-step immunofluorescence lifetime multiplexing

Multiplexed fluorescence imaging is limited by spectral overlap, whereas fluorescence lifetime provides an orthogonal encoding dimension. We replace genetically fused HaloTags with recombinant nanobody-HaloTag constructs applied as immunofluorescence reagents and determine a lifetime palette. Integrating lifetime and spectral encoding enables up to eight targets in a single acquisition and supports multiplexed imaging in cells and tissue using a simple OneStep labeling strategy compatible with fluorescent proteins, STED nanoscopy, and any standard laboratory antibody.

cell biology↗
bioRxiv2026

High-Performance, Computer-Controlled Bipedal DNA Motor

Achieving precise, repeatable motion at the molecular scale remains a central challenge in the development of synthetic molecular machines. Here we report a high-fidelity, fast bipedal DNA walker that moves bidirectionally along a DNA origami track using a fuel-before-antifuel operational scheme that suppresses trap states typical of externally powered DNA motors. Automated, computer-controlled microfluidics enables programmable trajectory execution with >98% yield per step and sustained bidirectional walking over distances of up to 360 nm, as monitored by single-molecule FRET. Kinetic analysis reveals rapid leg lifting but slower leg placement due to inhibitory fuel-antifuel heterocomplex formation, identifying a mechanistic bottleneck that can be mitigated through optimization of fuel, antifuel and foothold design. The resulting motor operates with efficiencies of up to 4 orders of magnitude higher than previous externally controlled DNA walkers, establishing a robust framework for deterministic, programmable molecular transport.

biophysics↗
bioRxiv2025

Self-blinking dye restores efficient use of nanobodies in single-molecule localization microscopy.

Direct stochastic optical reconstruction microscopy (dSTORM) relies on controlled fluorophore blinking to achieve nanometer-scale resolution, yet the fields benchmark dye, Alexa Fluor 647, underperforms when conjugated to nanobodies, limiting the practical use of minimal-linkage labeling strategies. Here, we show that the self-blinking dye JF635b overcomes this limitation by maintaining robust, photostable blinking upon conjugation to nanobodies under buffer-independent conditions. This enables reliable single-molecule localization microscopy without the need for complex switching buffers. Using JF635b-labeled nanobodies, we demonstrate consistent performance across multiple imaging modalities, including wide-field dSTORM, fluorescence lifetime SMLM, and MINFLUX nanoscopy, achieving localization precisions from [~]15 nm down to the sub-nanometer regime. In addition, JF635b supports long-term sample preservation and efficient blinking even in pure water, enabling minimally perturbative imaging conditions. Together, these results establish self-blinking dSTORM as a robust and accessible platform for quantitative nanoscopy across experimental contexts.

biophysics↗
bioRxiv2025

Inhibition of systemic mammalian metabolism by carnitine mimics from the gut microbiota

BackgroundThe gut microbiota and microbiome-derived metabolites are implicated in various aspects of human health. Here we sought to determine the systemic effects, and mechanism of action, of microbiome-derived carnitine analogues in germ free and conventionally colonised mice. ResultsHere we report the systemic localization of the microbiome-derived carnitine analogues, 3-methyl-4-(trimethylammonio)butanoate (3M-4-TMAB) and 5-aminovalerate betaine (5-AVAB), post-administration to germ free mice, with systemic carnitine depletion and mitochondrial dysregulation, reflected in altered acylcarnitine profiles due to incomplete carnitine-mediated fatty acid oxidation. Studies on the inhibitory potency of 3M-4-TMAB at the enzymatic, cellular, and organism levels indicate that, in part, this is a result of inhibition of gammabutyrobetaine hydroxylase, which catalyses the final step in carnitine biosynthesis. Systemic administration of 13C- labelled 3M-4-TMAB to conventionally colonised animals to further investigate the physiological relevance of this inhibition, identified a significant reduction in systemic carnitine levels due to increased excretion in urine and faeces and disruption of carnitine-mediated metabolism across ten organs. ConclusionsThese results highlight the physiological relevance and significance of microbiome-derived metabolites in vivo. The depletion of carnitine and inhibition of its function have potentially long-term impacts on both mammalian energy generation and the protective, signalling and immune regulatory effects of this critical molecule.

microbiology↗
bioRxiv2025

A versatile microfluidics platform for enhanced multi-target super-resolution microscopy

DNA-based Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) is a powerful variant of single-molecule localization microscopy (SMLM) that overcomes the limitations of photobleaching, offers flexible fluorophore selection, and enables fine control of imaging parameters through tunable on- and off-binding kinetics. Its most distinctive feature is the capacity for multiplexing, which is achieved through a process known as Exchange-PAINT. This technique involves assigning orthogonal DNA strands to different targets within a sample and then sequentially adding and removing complementary imager strands that are specific to only one target at a time. However, manual Exchange-PAINT workflows are often inefficient, prone to drift and variability, and lack reproducibility. Here, we introduce a custom compressed-air-driven microfluidics system specifically designed for multiplexed SMLM. Featuring a stackable and modular design that is, in principle, not limited by the number of channels, the system ensures robust, reproducible, and material-efficient buffer exchange with minimal dead volume. It operates in both manual and automated modes and can be readily adapted to a wide range of commercial and custom microscopes, including wide-field, confocal, STED, and MINFLUX platforms. We demonstrate robust 5-plex Exchange-PAINT imaging in cancerous U2OS cells and, importantly, we establish multiplexed nanoscale imaging in fragile primary cardiomyocytes. These applications highlight the unique power of our platform to extend super-resolution multiplexing into physiologically relevant systems, thereby opening new avenues for biomedical research.

biophysics↗
bioRxiv2025

A dominant role of cell death in limiting Chandipura virus propagation at cell-saturating high multiplicity of infection

Viruses transit from a low to high multiplicity of infection (MOI) regime in infected tissues. Type-1 interferons (IFNs) enforce a cellular state refractory to virus multiplication, while the death of infected cells eliminates viral replicative niche. Here, we investigated how these two innate antiviral mechanisms cooperate at various MOIs upon cell infection by Chandipura virus (CHPV), a cytopathic RNA virus implicated in several outbreaks of acute encephalitis in India. We found that as expected, a gradual increase in the input MOI from 0.02 to 2 led to a proportionate surge in the viral yield. Surprisingly, a further rise to MOI 20 caused a reduction in the progeny titer. Our mathematical modeling together with ex vivo infection studies involving mutant cells suggested that cell death - more so than virus-induced type-1 IFNs - restricted CHPV propagation at cell-saturating high MOIs, leading to a net fall in the yield at MOI 20. We argue that the distinct involvement of innate immune pathways at varied MOIs imparts robust cellular defense against cytopathic viruses. Significance sentenceCell death - more so than type-1 interferons - limits Chandipura virus propagation at cell-saturating high multiplicity of infection. Highlights# Type-1 IFNs and cell death cooperate at varied MOI in limiting CHPV multiplication. # At sub-saturating low MOI, type-1 IFNs play a dominant role in controlling CHPV propagation. # At cell-saturating high MOI, cell death determines the progeny yield.

systems biology↗
bioRxiv2025

Growth of Paracoccus aminovorans is restricted by L-ascorbic acid (LA) and human gut isolate

Gut microbiota plays a crucial role in the outcome of cholera. In this context, Paracoccus aminovorans, a gut commensal, has been evidenced to positively influence the pathogenesis of cholera bacterium. In the present study, we examined the impact of various non-antibiotic strategies on the growth of Paracoccus aminovorans. We observed the growth of Paracoccus aminovarans was severely restricted in the presence of L-ascorbic acid as well as in co-culture growth assays with another gut commensal, Weissella confusa.

microbiology↗
bioRxiv2025

Modeling the Co-Activation of Inflammatory Genes1Mediated by NFκB and IRF-3 During Viral Infections

Innate immune responses of host cells are activated upon exposure to external stimuli such as viral infections. These activations trigger the signaling pathways involved in antiviral responses. Activated pathways play a crucial role in the regulation of inflammatory and antiviral responses involving transcription factors, cytokines, cell surface viral sensors, and the activation of target genes. Although the role of key transcription factors such as NF-{kappa}B and IRF-3 in the regulation of antiviral genes that encode type I interferons (IFN/{beta}) is identified and well explored. The precise mechanistic understanding that governs their complex context-dependent temporal activation remains elusive. Here, we have developed a mathematical model to demonstrate the co-regulation of the expression of the IFN{beta} gene by Nuclear Factor kappa B (NF{kappa}B) and IFN Regulatory Factor-3 (IRF-3) after their activation in host cells by the viral analogue Poly I:C. In our model, the binding activities of the transcription factors NF{kappa}B and IRF-3 at the IFN{beta} promoter are modeled using a probability-based framework. Our results demonstrate that the expression pattern of IFN{beta} postviral infections would be pertaining to the co-activation of that by NF{kappa}B and IRF-3. In addition, the immediate expression of the IFN{beta} gene and, thus, the initial host response would be mainly attributed to immediate activation of NF{kappa}B while IRF-3 would be responsible for delayed but persistent expression of the IFN{beta}. Our model provides an underlying molecular mechanism for a similar response observed for Newcastle disease virus and the Vesicular stomatitis virus infection. Furthermore, we suggest that the rapid as well as an elevated and persistent expression of IFN{beta}, would be observed later due to a temporal bias in binding of both transcription factors NF{kappa}B and IRF-3, highlighting the need for coregulation of IFN{beta} by two different signaling pathways in a sequential manner. Also, our model predicts that the binding affinity of IRF3 to the promoter region of IFN{beta} would be equal to or larger than that of NF{kappa}B for a strong and persistent antiviral responses.

systems biology↗
bioRxiv2025

A High-Performance and Recoverable DNA Origami Rotary Motor

Inspired by biological molecular machines, we developed a highly processive DNA origami rotary motor. The rotor consists of two disk-shaped DNA origamis that are connected by a single-stranded swivel that allows free rotation but prevents rotor dissociation in the event of operational error. The rotor is propelled by two bipedal walkers that stride on a circular DNA track propelled by a previously optimized DNA propulsion mechanism called fuel before antifuel. The DNA fuel and antifuel strands are delivered by a microfluidic device. The rotation is monitored by a single-molecule, light scattering, defocused imaging technique; light scattered from a gold nanorod attached to the rotor enables high angular and temporal resolution analyses of rotor orientation. Imaging movies show individual rotors performing 96 individual steps, corresponding to 8 full rotor revolutions, with direction determined by the fuel and antifuel sequence. In cases of operational errors, which result in free Brownian rotation, the rotors were able to recover and continue rotating as commanded. Our origami-based rotary motor design, microfluidics-based control system, and high-resolution monitoring of rotor orientation will facilitate the development of DNA-based machines driven by autonomous propulsion.

biophysics↗
bioRxiv2025

Prediction of Enzyme function using interpretable optimized Ensemble learning framework

Accurate prediction of enzyme function, particularly for newly discovered uncharacterized sequences, is immensely important for modern biology research. Recently machine learning (ML) based methods have shown promises. However, such tools often suffer from complexity in feature extraction, interpretability, and generalization ability. Here we present an interpretable ML method, SOLVE that addresses these issues by using only combination of tokenized subsequences from the proteins primary sequence for classification. Its optimized ensemble learning framework improves prediction accuracy, distinguishes enzymes from non-enzymes, and predicts enzyme commission (EC) numbers for mono- and multi-functional enzymes. Additionally, SOLVE provides interpretability through Shapley analyses, identifying functional motifs at catalytic and allosteric sites of enzymes. By focusing only on primary sequence data, SOLVE simplifies high-throughput enzyme function prediction for functionally uncharacterized sequences and outperforms existing tools. With high prediction accuracy and its identification ability of functional regions, SOLVE can become a promising tool in different fields of biology and therapeutic drug design.

biophysics↗
bioRxiv2024

The enzyme glutamate-cysteine ligase (GCL) is a target for ferroptosis induction in cancer

Despite glutathiones long-recognized role as a major cellular antioxidant and its central role in ferroptosis defense, inhibition of glutathione biosynthetic enzymes has received little attention as a target for the therapeutic induction of ferroptosis. Here, we report that small-molecule inhibition of glutamate-cysteine ligase (GCL), the rate-limiting enzyme of glutathione biosynthesis, selectively and potently kills cancer cells by ferroptosis. We further describe novel GCL inhibitors including KOJ-1 and KOJ-2, compounds with excellent cellular potency and pharmacological properties, representing valuable tools to study the biology of ferroptosis and glutathione.

cancer biology↗
bioRxiv2024

Three-dimensional multi-target super-resolution microscopy of cells using Metal-Induced Energy Transfer and DNA-PAINT

AbsrtactAchieving nanometer precision in 3D remains a major challenge in super-resolution microscopy. DNA-PAINT offers excellent lateral resolution and versatile multiplexing, but its axial localization precision is typically 3-5 times poorer, limiting quantitative 3D imaging. Here, we present MIET-PAINT, which combines DNA-PAINT with Metal-Induced Energy Transfer (MIET) to overcome this limitation. We implement MIET-PAINT on both wide-field fluorescence lifetime and confocal TCSPC platforms. Wide-field MIET-PAINT enables robust, multiplexed imaging of focal adhesion proteins and actin in fixed cells. To address the lateral resolution limits of lifetime cameras, we further developed confocal MIET-PAINT, which leverages optical background rejection and high-efficiency SPAD detection. This modality achieves [~]12 nm lateral precision and resolves the 3D architecture of microtubules, vimentin, and actin with high fidelity. MIET-PAINT thus unites nanometer-scale axial accuracy with the multiplexing versatility of DNA-PAINT, establishing a powerful tool for quantitative 3D cell biology.

biophysics↗
bioRxiv2024

Nanoscale architecture and dynamics of CaV1.3 channel clusters in cardiac myocytes revealed by single channel nanoscopy

The clustering of L-type calcium channels for functional regulation of intracellular calcium signaling remains poorly understood. Here we applied super-resolution imaging to study CaV1.3 channel clusters in human iPSC-derived atrial cardiomyocytes (hiPSC-aCM) to analyze subcellular localization, dimensions, architecture, and dynamics, which were largely unexplored previously. STimulated Emission Depletion (STED) imaging characterized the localization and structure of CaV1.3 channel clusters in living cardiomyocytes. DNA Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) achieved true molecular resolution, revealing an irregular channel distribution with substantial spacing. Single Particle Tracking (SPT) showed that channels co-diffuse in confined and stationary membrane nanodomains. The cytosolic C-terminal tail of CaV1.3 by itself was found sufficient for cluster formation. In conclusion, our LTCC clustering studies demonstrate that CaV1.3 channel clusters consist of mobile individual channels inside defined membrane nanodomains, in contrast to previous models of dense channel packing.

cell biology↗
bioRxiv2023

In-utero exposure to estrogen-mimicking bisphenols alters bone mineralization in the offspring

Exposure to plastic-derived estrogen-mimicking endocrine-disrupting bisphenols can have a long-lasting effect on bone health. However, gestational exposure to below tolerable daily intake (TDI) of bisphenol A (BPA) and its substitute, bisphenol S (BPS), on offsprings bone mineralization is unclear. This study examined the effects of in-utero bisphenol exposure on the growth and bone density of the offspring rats. Pregnant Wistar rats were exposed to BPA and BPS (0.0, 0.4 g/kg bw) via oral gavage from gestational day 4 to 21. The bone density, IGF-1, osteocalcin, and calcium levels were measured by DEXA, ELISA and AAS, respectively. The bisphenols action on canonical BMP signaling was examined in osteoblast SaOS-2 cells. Maternal exposure to bisphenols (BPA and BPS) increased the body weight, bone mineral content, and density in the offspring aged 30 and 90 days (p<0.05). Plasma IGF-1, calcium, osteocalcin, and alkaline phosphatase activities were altered in BPA-exposed offspring (p<0.05). The bisphenols exposure to SaOS-2 cells decreased its viability in a dose-dependent manner and promoted the cell cycle progression of the S/G2-M phase (p<0.05). The expression of BMP1, BMP4, and intracellular signalling mediators SMAD1, SMAD5, and RUNX2 mRNAs was altered upon bisphenol exposure in these cells (p<0.05). The bone mineralization index and expression of extracellular matrix proteins such as ALPL, COL1A1, DMP1, and FN1 were downregulated (p<0.05). Bisphenol co-incubation with noggin decreased TGF-{beta}1 expression, indicating its involvement in bone mineralization. Overall, exposure to bisphenols (BPA and BPS) during gestation altered growth and bone mineralization in the offspring by modulating canonical BMP/ TGF-{beta}1 signalling mediators. HighlightsO_LIGestational exposure to low doses of bisphenol increases whole-body BMC and BMD in the offspring. C_LIO_LIIn-utero BPA exposure increased plasma IGF-1 and gla-type osteocalcin, a marker of osteoblast activity in the offspring. C_LIO_LIBisphenol exposure modulates Smad-dependent BMP signaling in the SaOS-2 cells. C_LI

developmental biology↗
bioRxiv2023

In Silico Therapeutic Intervention on Cytokine Storm in COVID-19

The recent global COVID-19 outbreak, attributed by the World Health Organization to the rapid spread of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), underscores the need for an extensive exploration of virological intricacies, fundamental pathophysiology, and immune responses. This investigation is vital to unearth potential therapeutic avenues and preventive strategies. Our study delves into the intricate interaction between SARS-CoV-2 and the immune system, coupled with exploring therapeutic interventions to counteract dysfunctional immune responses like the cytokine storm (CS), a driver of disease progression. Understanding these immunological dimensions informs the design of precise multiepitopetargeted peptide vaccines using advanced immunoinformatics and equips us with tools to confront the cytokine storm. Employing a control theory-based approach, we scrutinize the perturbed behavior of key proteins associated with cytokine storm during COVID-19 infection. Our findings support ACE2 activation as a potential drug target for CS control and confirm AT1R inhibition as an alternative strategy. Leveraging deep learning, we identify potential drugs to individually target ACE2 and AT1R, with Lomefloxacin and Fostamatinib emerging as standout options due to their close interaction with ACE2. Their stability within the protein-drug complex suggests superior efficacy among many drugs from our deep-learning analysis. Moreover, there is a significant scope for optimization in fine-tuning protein-drug interactions. Strong binding alone may not be the sole determining factor for potential drugs; precise adjustments are essential. The application of advanced computational power offers novel solutions, circumventing time-consuming lab work. In scenarios necessitating both ACE2 and AT1R targeting, optimal drug combinations can be derived from our analysis of drug-drug interactions, as detailed in the manuscript.

bioinformatics↗