Biology subjects

Suresh, S.

Publications and source records attributed to Suresh, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Repetitive suprathreshold paired-pulse transcranial magnetic stimulation alters corticospinal excitability

Background: Suprathreshold paired-pulse transcranial magnetic stimulation (TMS) at interstimulus intervals (ISIs) of 20-30 ms evokes cumulative online increases in corticospinal excitability. Whether these changes persist beyond stimulation remains unclear. This study investigated whether repetitive paired-pulse TMS targeting periods of net excitation or inhibition induces offline changes in corticospinal excitability. Methods: Nineteen healthy participants completed three sessions, separated by at least one week, involving 40 pairs of repetitive paired-pulse TMS delivered to the left primary motor cortex at an ISI of 20 ms (rPPS20) or 100 ms (rPPS100), or a single-pulse control condition (rSPS). Offline changes in corticospinal excitability were assessed using motor evoked potential (MEP) amplitudes recorded before and after stimulation. Results: During stimulation, MEP amplitudes decreased across trials in the rPPS100 condition, although online changes did not differ significantly between conditions. Following stimulation, MEP amplitudes were significantly reduced for up to 40 minutes after rPPS100 (p = 0.017) but were not changed following rPPS20 or rSPS (all p > 0.05). Conclusion: Contrary to our hypothesis, repetitive paired-pulse TMS targeting periods of net excitation did not induce offline changes in corticospinal excitability. In contrast, stimulation targeting long-interval intracortical inhibition (LICI) produced sustained reductions in corticospinal excitability, suggesting that repetitive LICI-inducing stimulation can induce lasting plasticity within the corticospinal system.

neuroscience↗

bioRxiv2026

ANCHOR : Atlas of Neurochemical Characterization of the Human Brainstem with 3D Reconstruction

The human brainstem is a complex division of the brain comprised of more than 200 nuclei and fiber tracts. The brainstem is essential for the functioning of the entire body. We introduce here the most detailed human brainstem Atlas across the human lifespan: fetus, child, adult. ANCHOR, the Atlas of Neurochemical Characterization of the Human Brainstem, is an online platform that includes more than 800 serial histological sections, stained for Nissl and seven immunochemical (IHC) markers, from the human brainstem of three ages: 25 fetal gestational weeks (GW), 9 years old, and 54 years old. This makes ANCHOR the most comprehensive human brainstem Atlas to date. In these three brainstems, we identified and manually annotated over 200 structures. We further characterized these structures with the seven IHC markers. We specifically describe the catecholaminergic groups in the human brainstem across all three age groups. In addition, we identified the protoplasmic commissural dendrites of the hypoglossal nucleus and we describe the pretectal nuclei in the Nissl-stained fetal 25 GW brainstem. ANCHOR includes an online viewer that integrates multimodal data, from magnetic resonance imaging and block face imaging to Nissl- and IHC-stained serial sections and 3D reconstruction of the entire brainstem. For the 9-year-old specimen, the online viewer allows simultaneous navigation of annotated sections with corresponding IHC, for viewing the specific region-wise cellular features accessible at https://anchor.humanbrain.in/.

neuroscience↗

bioRxiv2026

Early-Life Exposure to Warming Enhances Sea Urchin Heatwave Tolerance but Fails under Extreme Stress

The exposure to environmental stressors early in life can shape organisms to express more tolerant phenotypes to the same conditions during adulthood, a process called developmental plasticity. However, this acquired acclimation ability might depend on the intensity of the stimulus perceived later in life. Here, we took advantage of a purple sea urchin Paracentrotus lividus population developed at abnormally high sea temperature in the proximity of a power plant to test the limits of their developmentally acquired plasticity to increased water temperature. We simulated two marine heatwaves, a category I (moderate) and IV (extreme), and exposed the power plant population as well as a naive population developed in natural sea conditions. We measured their respiration rate and molecular responses to these two heatwaves. Regardless of the population of origin, sea urchins exposed to heatwaves showed higher oxygen consumption, indicating an increase in metabolic rates. At the molecular level, the biggest difference between the two populations was found following the moderate heatwave. Compared to the developmentally acclimated sea urchins, the naive population expressed genes coding for proteins with stress response, chromatin remodeling and RNA splicing functions, while suppressing immune response, revealing that developmental exposures can aid in priming the responses of adults to moderate temperature increases. However, a stronger heatwave leveled the differences between the two populations, with sea urchins from both locations expressing genes involved in proteostasis and detoxification. Nevertheless, regardless of the simulated marine heatwave intensity, sea urchins from the naive population always showed enrichment of the spliceosome pathway compared to power plant urchins, which activated immune response genes instead, reflecting fundamentally different thermal stress-coping strategies shaped by their developmental environments. Overall, our results demonstrate the critical yet context-dependent role of developmental plasticity in shaping the resilience of marine ectotherms to climate change.

evolutionary biology↗

bioRxiv2026

ARMH3 acts as a central scaffold at the Golgi/TGN through interactions with Arl5, GBF1, and PI4KB

The armadillo repeat protein ARMH3 regulates the activity and localisation of the Golgi resident lipid kinase phosphatidylinositol 4 kinase III{beta} (PI4KB) and the Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) that activates Arf1. ARMH3 localises to the trans Golgi network (TGN) via the GTPase Arl5. We used hydrogen deuterium exchange mass spectrometry (HDX-MS) and AI-enabled modeling to define the interfaces of ARMH3 with its binding partners Arl5, PI4KB, and GBF1. The ARMH3-Arl5 interface was determined to consist of the N and C termini of ARMH3, with Arl5 binding causing conformational changes in ARMH3 located at a shared PI4KB/GBF1 interface. Both PI4KB and GBF1 form mutually exclusive complexes with ARMH3, with GBF1 binding to ARMH3 through a disordered loop we have named the ARMH3 binding region (ABR). The ARMH3 interfaces in PI4KB and GBF1 contain phosphosites, with the phosphomimetic mutation of GBF1 blocking complex formation. These findings provide new insights into the role of ARMH3 as a master coordinator of GTPase and phosphoinositide signaling at the Golgi/TGN.

biochemistry↗

bioRxiv2026

Interspecific adaptations in root system architecture define host tolerance of Arabidopsis to biotic stresses by root feeding nematodes

Belowground, plants are exposed to a wide range of biotic stresses that vary in severity and nature, including tissue damage, disruption of vascular connectivity, and depletion of assimilates. How plants adapt their root systems to cope with different types of belowground biotic stresses is not well known. In this paper we compare above- and belowground plant adaptations to three nematode species with distinct tissue migration and feeding behaviours to study mechanisms underlying tolerance to different types of biotic stresses. We monitored both green canopy growth and changes in root system architecture of Arabidopsis inoculated with Pratylenchus penetrans, Heterodera schachtii, and Meloidogyne incognita. This revealed three distinct phases in aboveground plant responses: (i) initial growth inhibition associated with host invasion and tissue damage, (ii) persistent growth reduction associated with nematode sedentarism, and (iii) late growth stimulus in more advanced stages of infection. Specific adaptations in the root systems further revealed fundamentally different stress coping strategies. Tissue damage and intermittent feeding by P. penetrans in the root cortex did not induce significant changes in root system architecture. Tissue damage to the root cortex and prolonged feeding on host vascular cells by H. schachtii induced secondary root formation compensating for primary root growth inhibition. Prolonged feeding on host vascular cell by M. incognita alone did not induce secondary root formation, but was accompanied by typical local tissue swelling instead. Our data suggest that local secondary root formation and tissue swelling are two distinct compensatory mechanisms underlying tolerance to sedentarism by root-feeding nematodes. HighlightHow plants utilize root system plasticity to cope with different types of biotic stresses by root feeding nematodes remains largely unknown. Here, we report on specific adaptive growth responses in Arabidopsis roots to three nematode species, Pratylenchus penetrans, Heterodera schachtii, and Meloidogyne incognita, with fundamentally different strategies for host invasion, subsequent migration through host tissue, and feeding on host cells.

plant biology↗

bioRxiv2026

Phosphorylation of the C-terminus of PI4KA inhibits lipid kinase activity

Phosphatidylinositol 4-kinase alpha (PI4KA) is a lipid kinase that generates phosphatidylinositol 4-phosphate (PI4P) from phosphatidylinositol (PI) at the plasma membrane (PM). PI4P generated by PI4KA is essential for both plasma membrane identity and for PIP2 and PIP3 signalling driven by the PLC and PI3K family of enzymes. While the structure of PI4KA is known, the regulatory mechanisms that control its activity are undefined. Here, we discovered PI4KA can be inhibited through phosphorylation of Y2090 in the kinase domain, which can be phosphorylated by multiple tyrosine kinases. Y2090 phosphorylation causes local conformational changes in the k12 C-terminal helix. PI4KA activity is predominantly controlled through membrane recruitment by EFR3, with Y2090 phosphorylation inhibiting EFR3 bound PI4KA. Phosphorylation of the k12 C-terminal helix is found in PI3Ks and PI4Ks, suggesting an evolutionarily conserved regulatory mechanism for diverse phosphoinositide kinases. Overall, our work reveals novel molecular insight into inhibitory post-translational regulation of PI4KA.

biochemistry↗

bioRxiv2025

Shifts in attentional demand are accompanied by sex-specific patterns of insula-frontal cortical interactions in mice

The insula is a brain region that is correlated with the cognitive process of attention in humans. Aberrant insula function is also implicated in attentional impairments in several psychiatric disorders, such as attention deficit hyperactivity disorder (ADHD), major depressive disorder (MDD), and schizophrenia. Although the insulas role in attention has been well-studied in humans, we do not yet have an understanding of how the insula communicates with other brain areas to promote attentional processing. Here, we show that neurons in the mouse insula send direct axonal projections to the frontal cortex, and that these two brain areas functionally interact in different ways between male and female mice performing a touchscreen-based attention task. Specifically, we recorded and analyzed local field potentials (LFPs) from the insula and frontal cortex while mice performed variants of a rodent analogue of the continuous performance test of attention, or rCPT. In male mice, we found that activity in the frontal cortex preceded insular activity when the task was well-learned, but that insula-frontal cortex synchrony decreased when the need to inhibit responding was high. In female mice, synchrony between the insula and frontal cortex was largely bidirectional, but was high during all session types. We further demonstrate that patterns of theta-gamma coupling between the frontal cortex and insula distinguish between correct choices and errors during the rCPT, and that these patterns diverge between sexes. Our results suggest that functional differences in insula-frontal cortical circuitry at least partially underlie sex differences in attention-guided behavior in mice. HighlightsO_LIWe measured local field potentials (LFPs) in the mouse insula and frontal cortex during a translationally-relevant touchscreen-based task of attention C_LIO_LIManipulation of stimulus frequency during the task increased the demand on vigilance and response inhibition C_LIO_LIIn male mice, frontal cortex-driven synchrony in slow frequency bands reflected task rules, while theta-gamma coupling reflected errors when demand on response inhibition was increased C_LIO_LIIn female mice, bidirectionally-driven synchrony in slow frequency bands also reflected task rules, while theta-gamma coupling reflected errors when demand on vigilance was increased C_LI

neuroscience↗

bioRxiv2025

Cumulative timing-dependent changes in corticospinal excitability during suprathreshold paired-pulse transcranial magnetic stimulation

Transcranial magnetic stimulation (TMS) is widely used to assess inhibitory and facilitatory circuits within the primary motor cortex. However, accumulating evidence suggests that even brief TMS paradigms may induce unintended changes in corticospinal excitability. Here, we examined whether suprathreshold paired-pulse TMS delivered at inter-stimulus intervals (ISIs) associated with intracortical facilitation or long-interval cortical inhibition (LICI) elicits cumulative changes in motor-evoked potential (MEP) amplitude. In experiment 1, we reanalysed data from 17 participants who received 20 suprathreshold paired-pulses at eight ISIs (10-200 ms) and 40 unconditioned single pulses. Stimulation was pseudo-randomised and distributed evenly across four blocks. A linear mixed-effects model assessed trial-wise changes in MEP amplitude across ISIs. We replicated the design in an independent sample (n=10, experiment 2). A significant trial-by-ISI interaction was observed in both cohorts. Specifically, MEP amplitudes increased across trials for ISIs of 20 and 30 ms (p<0.05), but remained stable at LICI-related ISIs (100-150 ms). A similar increase was also seen with single-pulse TMS. These findings demonstrate that suprathreshold paired-pulse TMS at short ISIs can cumulatively enhance corticospinal excitability during stimulation. Furthermore, the results suggest potential for using these protocols not just for probing cortical circuits, but also as interventions to modulate motor system excitability.

neuroscience↗

bioRxiv2025

An intron-based timer for circadian rhythms

Abstract: Circadian clocks regulate daily rhythms in all eukaryotes through [~]24-hour transcriptional-translational feedback loops driven by clock proteins. However, the molecular mechanisms that set the 24-hour period remain poorly defined. Here, using single-molecule imaging and nascent RNA sequencing, we uncover an unexpected RNA-based molecular timer: a single intron in the Drosophila timeless (tim) gene that regulates circadian period length by controlling mRNA localization. Strikingly, we find that [~]50% of tim mRNAs are localized to the nucleus due to inefficient post-transcriptional splicing of a single intron (which we named intron P), in contrast to other core clock transcripts that localize predominantly to the cytoplasm. CRISPR-mediated removal of intron P abolishes nuclear retention of tim transcripts, leading to accelerated TIM protein accumulation and a shortened [~]22-hour period with reduced rhythmic robustness. Remarkably, insertion of intron P alone into heterologous reporters is sufficient to promote nuclear retention in both Drosophila and human cells, acting as a conserved checkpoint that withholds transcripts in the nucleus until splicing is complete. Finally, we identify three RNA-binding proteins, two repressors (Hrb27C and Squid) and one activator (Qkr58E-2, a Sam68 homolog), that modulate intron P splicing in a rheostat-like manner. Together, these findings establish tim intron P as the first intron-based molecular timer in circadian clocks and reveal splicing kinetics as a critical regulatory layer in temporal gene expression programs, with broad implications for other processes such as development and immunity.

molecular biology↗

bioRxiv2025

Establishing a continuum of cell types in the visual cortex

The mammalian cerebral cortex is composed of neurons whose properties vary in a continuous fashion rather than falling into discrete cell types. In the mouse visual cortex, excitatory neurons in layer 2 and 3 (L2/3) form such a continuum along cortical depth, patterned by the graded expression of hundreds of genes. Here we sought to understand how this continuum develops and contributes to cortical wiring. Using single-nucleus multiomics (RNA- and ATAC-Seq) and spatial transcriptomics, we show that the L2/3 continuum is established in two phases. During the first postnatal week, a genetically hardwired program establishes a primitive continuum of cell identities spanning the depth of L2/3. The second program, promoted by visual experience, is later superimposed upon the preexisting continuum. This second phase is driven by activity-regulated transcription factors that drive the L2/3 depth-dependent expression of genes linked to synaptic function and plasticity. We show that neurons at different positions along the L2/3 continuum project preferentially to distinct higher visual areas and that visual deprivation disrupts targeting to some higher visual areas while sparing others. Thus, cortical continua emerge through a stepwise process in which genetic programs and sensory experience specify neuronal identity and sculpt intracortical wiring specificity.

neuroscience↗

bioRxiv2025

Internal and external contexts drive distinct dynamics in shared action-encoding striatal neurons

Animals can initiate movements either in response to external cues or from internal drive, yet how the brain flexibly supports both remains unclear. Disorders such as Parkinsons disease disrupt these modes differently, suggesting distinct underlying mechanisms. Such differences could arise from specialized circuits dedicated to each action mode or from shared neuronal populations that shift their dynamics across contexts. To distinguish between these possibilities, we performed two-photon calcium imaging in the dorsolateral striatum as mice executed the same lever press either spontaneously or in response to a cue, enabling direct comparison of internally and externally triggered actions. Unsupervised clustering revealed subpopulations of neurons modulated during cue, movement, or post-action periods. Critically, the same neurons were tuned to movement across initiation contexts. However, the dynamics of this shared population evolved within distinct "context" and "action" subspaces, even before movement initiation. Both dopamine D1- and D2-receptor-expressing spiny projection neurons contributed to both subspaces, with D1-SPNs more active at the time of the sensory stimulus. These results show that context shapes neural dynamics within a shared movement-encoding population, revealing a context-generalizable striatal code that supports flexible movement initiation across internal and external drives.

neuroscience↗

bioRxiv2025

Molecular drivers of mutualistic association between anemone and anemonefish

The anemone-anemonefish mutualism is one of the most iconic in the marine environment. While the evolution of this mutualistic relationship has contributed to the ecological success of both partners, the underlying molecular processes that establish and maintain it remain poorly understood, particularly how anemonefish tolerate anemone venom. Here we explored the transcriptional dynamics in the anemonefish Amphiprion clarkii and its host anemone Entacmaea quadricolor 48 hours after association to identify the molecular underpinnings of their mutualistic relationship. Upon acclimation with an anemone, anemonefish showed differential regulation of sensory perception and memory genes in key brain regions, indicating activation of neural pathways that may facilitate host recognition and mutualism establishment. In the fishs skin, altered expression of genes involved in neurotransmitter release, cytoskeleton organization, and venom receptor proteins points to mechanisms of resistance to anemone venom. This resistance is particularly remarkable since anemone hosting fish exhibited increased expression of genes encoding mechanoreceptors, venom proteins, and ion channels involved in nematocyst discharge, indicating the anemone does indeed mount an active response to their mutualistic partner. Overall, our results highlight the complex interplay of molecular events in both species that play a pivotal role in establishing this mutualistic relationship.

genomics↗

bioRxiv2025

ICEPIC: A Toolkit to Discover Ice Binding Proteins from Sequence

Ice binding proteins, such as antifreeze proteins (AFPs) and ice nucleation proteins (INPs), are critical for survival in subzero environments and have wide-ranging applications in biotechnology, agriculture, and materials science. Current discovery methods for these proteins are constrained by low throughput and limited datasets that are not conducive for engineering. Here, we present a high-throughput, sequence-based model that leverages contextual embeddings from protein language models to predict ice binding potential, as well as the expression and activity potential of candidate proteins. Using a curated data corpus of over 18,000 ice binding proteins -- far larger than previous datasets -- we fine-tuned a ProtBERT-based model, achieving 99% accuracy for prediction of ice binding potential. Sensitivity analyses through targeted mutagenesis (alanine and threonine substitutions) confirmed the models biological significance, revealing functionally important residues and sequence patterns. Additionally, we developed an expression prediction model that achieved an R2 score of 0.64 and low false-negative rates in identifying highly expressible candidates in Pichia pastoris. An additional regression model trained to predict ice activity as measured by thermal hysteresis achieved an R2 score of at least 0.79 with a clear difference in prediction between ice binding and non-ice binding proteins. Our toolkit advances the predictive accuracy, interpretability, and scalability of ice binding protein discovery, offering a powerful tool for protein engineering in cold-environment applications. Significance StatementIce binding proteins enable organisms to survive freezing temperatures and are essential for applications in cryopreservation, agriculture, and materials science. However, discovering and engineering these proteins has been limited by small datasets and inadequate predictive tools. We developed a machine learning model trained on over 18,000 ice binding protein sequences to predict not only ice binding potential but also protein activity and expression in engineered hosts. This approach integrates advanced protein language models with biological context, enabling faster, more reliable discovery of ice binding proteins. Our platform advances rational protein design for real-world applications in climate resilience, biotechnology, and beyond.

synthetic biology↗

bioRxiv2025

Promoter evolution in HIV-1C establishes latent reservoirs highly resistant to reversal

Latent viral reservoirs remain a major barrier to curing HIV-1, with the long-terminal repeat (LTR) and Tat playing crucial roles in regulating viral transcription. Subtype-specific transcription factor binding site (TFBS) variations within the LTR significantly influence latency and reservoir stability. In earlier work, we identified HIV-1C LTR variants with duplicated TFBS motifs, including NF-{kappa}B, AP1, RBEIII, and TCF-1/LEF-1. Using five cell models, including Jurkat and primary CD4 T cells, we compared canonical R-LTR and variant R2-LTR strains. Across sub-genomic reporters, single-round infections, and full-length viral vectors, we found that the balance between RBEIII and NF-{kappa}B motifs governs stability of latency. The two-viruses-one-cell system that normalized confounding environmental factors further revealed that latency is primarily controlled by intrinsic transcriptional circuits rather than external stimuli. In longitudinal studies of HIV-1 individuals from acute and chronic infection phases, we observed dominant R strains during early infection and the spontaneous emergence of R2 strains in nearly half of chronic-phase subjects, a process accelerated by ART. Upon CD4 T cell activation, R strains preferentially rebounded, while R2 strains showed strong resistance to reversal, even in subjects harbouring a co-infection. Together, these findings establish the clinical significance of LTR variation in latency regulation and identify the R2 phenotype as a critical determinant of reservoir persistence. These results underscore the importance of addressing reservoir heterogeneity in cure strategies, particularly in HIV-1C-prevalent regions.

microbiology↗

bioRxiv2025

Development of an inhibitory TTC7B selective nanobody that blocks EFR3 recruitment of PI4KA

Phosphatidylinositol 4 kinase III (PI4KIII/PI4KA) is an essential lipid kinase that plays a critical role in regulating plasma membrane identity. PI4KA is primarily recruited to the plasma membrane through the targeted recruitment by the proteins, EFR3A and EFR3B, which bind to the PI4KA accessory proteins TTC7 (TTC7A/B) and FAM126 (FAM126A/B). Here we characterised how both EFR3 isoforms interact with all possible TTC7-FAM126 combinations and developed a nanobody that specifically blocked EFR3-mediated PI4KA recruitment in TTC7B containing complexes. Most EFR3-TTC7-FAM126 combinations show similar binding affinities, with the exception of EFR3A-TTC7B-FAM126A, which binds with a [~]10-fold higher affinity. Moreover, we showed that EFR3B phosphorylation markedly decreased binding to TTC7-FAM126. Using a yeast display approach, we isolated a TTC7B selective nanobody that blocked EFR3 binding. Cryo-electron microscopy and hydrogen deuterium exchange mass spectrometry showed an extended interface with both PI4KA and TTC7B that sterically blocks EFR3 binding. The nanobody caused decreased membrane recruitment both on lipid bilayers and in cells, with decreased PM production of PI4P. Collectively, these findings provide new insights into PI4KA regulation and provide a tool for manipulating PI4KA complexes, that may be valuable for therapeutic targeting.

biochemistry↗

bioRxiv2025

CARM1-mediated methylation controls interactions of ALIX with key partners important for cytokinesis

CARM1 is an arginine methyltransferase with a well-established role in regulating gene expression, but its cytoplasmic functions remain largely uncharacterized. Here, we identified ALIX, a protein acting with the ESCRT-III machinery in numerous membrane remodeling events, as a main cytoplasmic partner and a relevant substrate of CARM1. We demonstrate that CARM1 methylates arginine residues within the proline-rich motif of ALIX. At the molecular level, ALIX methylation impairs SH3-dependent association with CD2AP, CIN85, and endophilin-A2 that are required for cytokinesis. Using a mutant of ALIX that is unable to bind these proteins, we further show that these interactions are essential for ALIX functions during cytokinesis. Altogether, this work highlights the significance of arginine methylation as a regulatory post-translational modification important for the final step of cell division, by modulating interactions between proline-rich domains and their SH3-containing partners.

cell biology↗

bioRxiv2025

Selective inhibition of MR1-restricted T cell activation by a novel MR1-targeting nanobody

MR1 is a non-polymorphic, ubiquitously expressed, MHC class I-like antigen-presenting molecule that presents small-molecule metabolites to T cells. Studies have shown that MR1 plays a role in microbial infection, inflammation, and tumor immunity. The antigens it presents include metabolites of microbial and self-origin as well as small-molecule drugs and form stable complexes with MR1 that are displayed on the cell surface to activate T cells. However, unlike classical MHC I and II molecules, the fundamental biology of MR1 remains poorly understood, particularly the mechanisms governing antigen loading and intracellular trafficking. This knowledge gap is largely due to the lack of molecular tools available to precisely manipulate MR1 function. In this study, we describe a high-affinity (1.6 nM KD) anti-MR1 nanobody, MR1Nb1. We characterize the binding of this nanobody including affinity by ELISA and kinetics by BLI. Crucially, we map the binding epitope of MR1Nb1 on MR1 by HDX-MS, providing key insights into the mechanism through which it blocks MR1T cell activation. In functional assays MR1Nb1 effectively and specifically blocks MAIT cell activation by cells infected with M. tuberculosis or treated with M. smegmatis supernatant. This nanobody represents a unique and versatile tool for the field, as it can be produced inexpensively and expressed intracellularly within antigen-presenting cells. Hence, our study provides a powerful new molecular probe for dissecting the mechanistic underpinnings of MR1 biology and uncover its broader roles in immunity.

immunology↗

bioRxiv2025

Nanopore Assay for Fingerprinting DNA Binding and Quantifying Real-Time Cleavage by Catalytically Active Cas9 Enzyme

Nanopore sensing, a high-resolution DNA sequencing technology, is fast expanding into novel and exciting direction of probing specific DNA-enzyme interactions. Although proven excellent for detection of structural features of bare DNA, quantitative measurements on enzyme-DNA complexes and its real-time activity are lagging and only starting to emerge for long DNA templates. Signal-to-noise requirement and high translocation speeds make it difficult to detect protein bound on biologically relevant plasmid length DNA. To this end we report accurate position detection of a catalytically active Cas9 bound to its single or multiple target sites on the DNA. Protein position is fingerprinted using event charge deficit (ECD) based analysis of the high signal-to-noise electrical signals as the complex translocates through a glass nanopore. Using a time dependent assay, we quantify kinetics of the released products upon enzymatic cleavage of the target DNA by the wild-type Cas9 nuclease. Our approach enables the nanopore based single molecule sensing of DNA-protein complexes, for real-time monitoring of biochemical reactions. This may help understand protein binding & localization as well as improve Cas9 based targeting in genome engineering applications. TOC Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/656938v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@9ea40org.highwire.dtl.DTLVardef@f214e6org.highwire.dtl.DTLVardef@13a10bborg.highwire.dtl.DTLVardef@12067ab_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗