Biology subjects

Wanunu, M.

Publications and source records attributed to Wanunu, M..

17 recordsLinked to original sources

bioRxiv2026

NanoCortex: A Unified Agentic System for Nanopore Sequencing Analysis

Nanopore sequencing has enabled various layers of information about DNA and RNA sequence isoforms and chemical modifications. Yet, the archipelago of disjoint nanopore analysis tools makes navigating among these a significant challenge for the nanopore user. We present NanoCortex, a unified autonomous agentic framework designed to bridge this shortcoming by providing end-to-end data processing which ranges from raw signal basecalling to biological interpretation. Built upon Gemini API services that incur usage-based API costs and orchestrated through the Gemini Agent Development Kit (ADK), the system utilizes a multi-agent architecture to autonomously perform task parsing, code generation, iterative code-level self-correction of code, and scientific interpretation. Following code generation, the code can be used offline. Benchmarking reveals that NanoCortex achieves significantly higher usability across complex analytical tasks compared to general-purpose large language models. The framework seamlessly integrates experimental data with meta-analysis of publicly available, biological databases to facilitate the extraction of biologically meaningful insights from sequencing data without cumbersome computational steps.

bioinformatics↗

bioRxiv2025

Rare bioactive tau oligomers from Alzheimer brain support both templated misfolding and fibril formation

In Alzheimers disease, both classical neurofibrillary tangles, and diffusible, aqueous soluble (High Molecular Weight, or HMW) species are able to support templated misfolding. How these tau proteoforms relate is uncertain. Using sequential size exclusion and anion exchange chromatography, we fractionated the HMW tau population and found both seed competent, and seed not competent proteoforms. Super resolution, atomic force, and immunogold electron microscopy confirmed that the size and conformation of both bioactive and non-bioactive tau proteoforms are similar, with dimers, trimers, and tetramers predominating. The presence of surface phosphorylation correlates with seeding capacity. Bioactive tau at fMol concentrations can induce seeding in a reporter cell. The soluble bioactive species support aggregation of a truncated repeat domain tau construct into thioflavin T positive fibrils and retain seeding activity over serial amplification in vitro and in cellulo, whereas non-bioactive oligomeric species do not. Together, these findings indicate that oligomeric assembly is required but not sufficient for seeding; instead, specific biochemical attributes of a rare oligomeric tau subset confer self-propagating, prion-like templated misfolding.

neuroscience↗

bioRxiv2025

Pore-Based RNA Evaluation for Control of Integrity, Sequence, and Errors - Quality Control (PRECISE-QC)

RNA is at the forefront of therapeutics and gene editing technologies. Yet, RNA synthesis remains expensive and low-yield. Consequently, most oligo manufacturers abstain from synthesizing RNA oligos longer than 60-mers. Solid-phase synthesis is the current standard production method but is often fraught with low coupling yields for canonical nucleotides and even poorer coupling for modifications. This results in high levels of byproducts such as truncations and RNA infidelity. Existing analytical methods can only provide quality control metrics such as RNA length distribution or limited composition information for short oligos. Here, we developed a standard quality control metric using Oxford Nanopore direct RNA sequencing to obtain direct insight into RNA length distribution, sequence, and presence of RNA modification sites. Our pipeline identifies error-prone regions and truncation sites that occur during synthesis. Furthermore, problematic steps in the synthesis are identified and repaired. We show that our platform can produce and assess CRISPR guide RNAs with high-fidelity and higher cleavage activity, and further, that modifications can be reliably detected. We envision that our tool will serve as an integral method for quality control pipelines that assess the integrity and accuracy of synthetic RNAs and guide the improved synthesis and yield of synthesized RNAs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/677417v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1163a7forg.highwire.dtl.DTLVardef@5a709corg.highwire.dtl.DTLVardef@19737e6org.highwire.dtl.DTLVardef@d71a60_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

Direct RNA sequencing of primary human T cells reveals the impact of immortalization on mRNA pseudouridine modifications.

Immortalized cell lines are commonly used as proxies for primary cells in human biology research. For example, Jurkat leukemic T cells fundamentally contributed to uncovering T cell signaling, activation, and immune responses. However, the immortalization process can alter key cellular properties, and researchers widely believe this process could significantly change RNA modification machinery and modification sites. In this study, we focus on pseudouridine ({Psi}), one of the most abundant mRNA modifications, and compare {Psi} profiles in mRNA from primary and immortalized T cells using direct RNA sequencing (DRS). Surprisingly, 87% of {Psi}-sites were shared between the two cell types, primarily in transcripts encoding proteins involved in essential cellular processes, including RNA-modification regulation. Furthermore, the analysis of the 13% of sites unique to each cell type reveals that Jurkat cells contained transcripts linked to immune activation and oncogenesis, while primary T cells contained transcripts associated with calcium signaling and intracellular trafficking. We provide a list of these genes, which should be considered when using immortalized cells to study RNA modifications in immunology contexts. Most differences were driven by whether the mRNA was present or absent in the immortalized or primary cell type. Interestingly, RNA-modification enzyme expression levels were highly conserved in both cell types. This suggests that site-specific differences in {Psi} levels arise from regulatory processes acting in trans rather than differences in modification enzyme levels. Significance StatementIt is widely believed that RNA modification machinery and modification sites could be significantly altered in immortalized cells, yet this has never been tested. Focusing on pseudouridine ({Psi}), we map {Psi} in the transcriptomes of primary and immortalized human T cells. Surprisingly, most sites are conserved in the primary and immortalized T cells, with several important examples of cases with cell type specificity and should be considered on a case-by-case basis. Furthermore, we evaluated RNA-modification machinery levels in primary and immortalized T cells, finding high conservation across the cell lines. Our findings demonstrate that RNA modifications are largely conserved between primary and immortalized cells, and the edge cases can be considered individually.

genomics↗

bioRxiv2024

mRNA psi profiling using nanopore DRS reveals cell-type-specific pseudouridylation

Pseudouridine (psi) is one of the most abundant human mRNA modifications yet its functional impact on translation has remained unclear. Using direct RNA nanopore sequencing coupled with our Mod-p ID analytical framework, we mapped psi at single-nucleotide resolution across six immortalized human cell lines derived from diverse tissue types. Psi sites identified by nanopore sequencing were cross-validated using Illumina-based methods, confirming both positional accuracy and reproducibility. Unlike prior short-read approaches, nanopore sequencing provided the unique ability to quantify relative occupancy at each site and to detect multiple modifications on the same RNA molecule, revealing combinatorial modification patterns that cannot be captured otherwise. Integrating these psi maps with matched proteomic and ribosome profiling datasets, we find that psi modulates translation through two mechanistic modes: (i) single high-occupancy psi sites enhance translational efficiency and protein output, whereas (ii) clustered psi modifications promote ribosome pausing, decoupling translation efficiency from protein yield. This integrative, multi-omics framework provides a quantitative model of how psi stoichiometry and distribution along transcripts shape ribosome dynamics and proteome composition across human cell types.

genomics↗

bioRxiv2024

Probing enzyme-dependent pseudouridylation using direct RNA sequencing to assess neuronal epitranscriptome plasticity

Chemical modifications in mRNAs, such as pseudouridine (psi), can control gene expression. Yet, we know little about how they are regulated, especially in neurons. We applied nanopore direct RNA sequencing to investigate psi dynamics in SH-SY5Y cells in response to two perturbations that model a natural and unnatural cellular state: retinoic-acid-mediated differentiation (healthy) and exposure to the neurotoxicant, lead (unhealthy). We discovered that the expression of some psi writers change significantly in response to physiological conditions. We also found that globally, lead-treated cells have more psi sites but lower relative occupancy than untreated cells and differentiated cells. Interestingly, examples of highly plastic sites were accompanied by constant expression for psi writers, suggesting trans-regulation. Many positions were static throughout all three cellular states, suggestive of a "housekeeping" function. This study enables investigations into mechanisms that control psi modifications in neurons and its possible protective effects in response to cellular stress.

systems biology↗

bioRxiv2023

Electro-Osmotic Flow Generation via a Sticky Ion Action

Selective transport of ions through nanometer-sized pores is fundamental to cell biology and central to many technological processes such as water desalination and electrical energy storage. Conventional methods for generating ion selectivity include placement of fixed electrical charges at the inner surface of a nanopore through either point mutations in a protein pore or chemical treatment of a solid-state nanopore surface, with each nanopore type requiring a custom approach. Here, we describe a general method for transforming a nanoscale pore into a highly selective, anion-conducting channel capable of generating a giant electro-osmotic effect. Our molecular dynamics simulations and reverse potential measurements show that exposure of a biological nanopore to high concentrations of guanidinium chloride renders the nanopore surface positively charged due to transient binding of guanidinium cations to the protein surface. A comparison of four biological nanopores reveals the relationship between ion selectivity, nanopore shape, composition of the nanopore surface, and electro-osmotic flow. Remarkably, guanidinium ions are also found to produce anion selectivity and a giant electro-osmotic flow in solid-state nanopores via the same mechanism. Our sticky-ion approach to generate electro-osmotic flow can have numerous applications in controlling molecular transport at the nanoscale and for detection, identification, and sequencing of individual proteins.

biophysics↗

bioRxiv2023

Post-Transcriptional Methylation of Mitochondrial-tRNA Differentially Contributes to Mitochondrial Pathology

Human mitochondrial tRNAs (mt-tRNAs), critical for mitochondrial biogenesis, are frequently associated with pathogenic mutations. These mt-tRNAs have unusual sequence motifs and require post-transcriptional modifications to stabilize their fragile structures. However, whether a modification that stabilizes a wild-type (WT) mt-tRNA structure would also stabilize its pathogenic variants is unknown. Here we show that the N1-methylation of guanosine at position 9 (m1G9) of mt-Leu(UAA), while stabilizing the WT tRNA, has an opposite and destabilizing effect on variants associated with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). This differential effect is further demonstrated by the observation that demethylation of m1G9, while damaging to the WT tRNA, is beneficial to the major pathogenic variant, improving its structure and activity. These results have new therapeutic implications, suggesting that the N1-methylation of mt-tRNAs at position 9 is a determinant of pathogenicity and that controlling the methylation level is an important modulator of mt-tRNA-associated diseases.

cell biology↗

bioRxiv2022

Synthesis of Long RNA with a Site-Specific Modification by Enzymatic Splint Ligation

Synthesis of RNA molecules that contain an internal site-specific modification is important for RNA research and therapeutics. While solid-state synthesis is attainable for such RNA in the range of 100 nucleotides (nts), it is currently impossible with kilobase (kb)-long RNA. Instead, long RNA with an internal modification is usually assembled in an enzymatic 3-part splint ligation to join a short RNA oligonucleotide, containing the site-specific modification, with both a left-arm and a right-arm long RNA that are synthesized by in vitro transcription. However, long RNAs have structural heterogeneity and those synthesized by in vitro transcription have 3-end sequence heterogeneity, which together substantially reduce the yield of 3-part splint ligation. Here we describe a method of 3-part splint ligation with an enhanced efficiency utilizing a ribozyme cleavage reaction to address the 3-end sequence heterogeneity and involving DNA disruptors proximal to the ligation sites to address the structural heterogeneity. The yields of the synthesized kb-long RNA are sufficiently high to afford purification to homogeneity for practical RNA research. We also verify the sequence accuracy at each ligation junction by nanopore sequencing.

biochemistry↗

bioRxiv2022

High-Voltage Biomolecular Sensing Using a Bacteriophage Portal Protein Covalently Immobilized Within a Solid-State Nanopore

The application of nanopores as label-free, single-molecule biosensors for electrical or optical probing of structural features in biomolecules has been widely explored. While biological nanopores (membrane proteins and bacteriophage portal proteins) and solid-state nanopores (thin films and two-dimensional materials) have been extensively employed, the third class of nanopores known as hybrid nanopores, where an artificial membrane substitutes the organic support membrane of proteins, has been only sparsely studied, due to challenges in implementation. G20c portal protein contains a natural DNA pore that is used by viruses for filling their capsid with viral genomic DNA. We have previously developed a lipid-free hybrid nanopore by "corking" the G20c portal protein into a SiNx nanopore. Herein, we demonstrate that through chemical functionalization of the synthetic nanopore, covalent linkage between the solid-state pore and the G20c portal protein considerably improves the hybrid pore stability, lifetime, and voltage resilience. Moreover, we demonstrate electric-field-driven and motor protein-mediated transport of DNA molecules through this hybrid pore. Our integrated protein/solid-state hybrid nanopore can serve as a robust and durable framework for sensing and sequencing at high voltages, potentially providing higher resolution, higher signal-to-noise ratio, and higher throughput compared to the more conventional membrane-embedded protein platforms.

bioengineering↗

bioRxiv2022

Messenger-RNA Modification Standards and Machine Learning Models Facilitate Absolute Site-Specific Pseudouridine Quantification

Enzyme-mediated chemical modifications to mRNA are important for fine-tuning gene expression, but they are challenging to quantify due to low copy number and limited tools for accurate detection. Existing studies have typically focused on the identification and impact of adenine modifications on mRNA (m6A and inosine) due to the availability of analytical methods. The pseudouridine ({Psi}) mRNA modification is also highly abundant but difficult to detect and quantify because there is no available antibody, it is mass silent, and maintains canonical basepairing with adenine. Nanopores may be used to directly identify {Psi} sites in RNAs using a systematically miscalled base, however, this approach is not quantitative and highly sequence dependent. In this work, we apply supervised machine learning models that are trained on sequence-specific, synthetic controls to endogenous transcriptome data and achieve the first quantitative {Psi} occupancy measurement in human mRNAs. Our supervised machine learning models reveal that for every site studied, different signal parameters are required to maximize {Psi} classification accuracy. We show that applying our model is critical for quantification, especially in low-abundance mRNAs. Our engine can be used to profile {Psi}-occupancy across cell types and cell states, thus providing critical insights about physiological relevance of {Psi} modification to mRNAs.

genomics↗

bioRxiv2021

Detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct, long-read sequencing

We developed and applied a semi-quantitative method for high-confidence identification of pseudouridylated sites on mammalian mRNAs via direct long-read nanopore sequencing. A comparative analysis of a modification-free transcriptome reveals that the depth of coverage and specific k-mer sequences are critical parameters for accurate basecalling. By adjusting these parameters for high-confidence U-to-C basecalling errors, we identified many known sites of pseudouridylation and uncovered new uridine-modified sites, many of which fall in k-mers that are known targets of pseudouridine synthases. Identified sites were validated using 1,000-mer synthetic RNA controls bearing a single pseudouridine in the center position which demonstrate systematical under-calling using our approach. We identify mRNAs with up to 7 unique modification sites. Our pipeline allows direct detection of low-, medium-, and high-occupancy pseudouridine modifications on native RNA molecules from nanopore sequencing data as well as multiple modifications on the same strand.

bioengineering↗

bioRxiv2021

Unidirectional Single-File Transport of Full-Length Proteins Through a Nanopore

Nanopore sensors could revolutionize single-molecule proteomics by providing a means for identification of known proteins through fingerprinting or by de novo sequencing. However, the complex chemical and physical properties of proteins present multiple challenges to the conventional nanopore sensing method, predominantly, the single-file threading of a protein chain into a nanopore and its transport through it. Herein we describe a general approach for realizing unidirectional transport of full-length proteins through nanopores. We show that the combination of a chemically resistant biological nanopore platform and a high concentration guanidinium chloride buffer enables protein unfolding and unidirectional transport through a pore, propelled by an electroosmotic effect that largely owes to the guanidinium chloride presence. The uniform and slow (~10 {micro}s/amino acid) single-file transport, when combined with supervised machine learning of the electrical current signatures obtained, allows us to use to discern the protein threading orientation and identity. In conjunction with a method for tail-modification of native proteins and higher-resolution nanopores, our approach could offer a path towards direct single-molecule protein fingerprinting without the requirement of a motor enzyme.

biophysics↗

bioRxiv2021

Control of subunit stoichiometry in single-chain MspA nanopores

Transmembrane protein channels enable fast and highly sensitive electrical detection of single molecules. Nanopore sequencing of DNA was achieved using an engineered Mycobacterium smegmatis porin A (MspA) in combination with a motor enzyme. Due to its favorable channel geometry, the octameric MspA pore exhibits the highest current level as compared to other pore proteins. To date, MspA is the only protein nanopore with a published record of DNA sequencing. While widely used in commercial devices, nanopore sequencing of DNA suffers from significant base-calling errors due to stochastic events of the complex DNA-motor-pore combination and the contribution of up to five nucleotides to the signal at each position. Asymmetric mutations within subunits of the channel protein offer an enormous potential to improve nucleotide resolution and sequencing accuracy. However, random subunit assembly does not allow control of the channel composition of MspA and other oligomeric protein pores. In this study, we showed that it is feasible to convert octameric MspA into a single-chain pore by connecting eight subunits using peptide linkers. We constructed single-chain MspA trimers, pentamers, hexamers and heptamers to demonstrate that it is feasible to alter the subunit stoichiometry and the MspA pore diameter. All single-chain MspA proteins formed functional channels in lipid bilayer experiments. Importantly, we demonstrated that single-chain MspA discriminated all four nucleotides identical to MspA produced from monomers. Thus, single-chain MspA constitutes a new milestone in its development and adaptation as a biosensor for DNA sequencing and many other applications. STATEMENT OF SIGNFICANCENanopore sequencing of DNA is a fast and cheap technology that uniquely delivers multi-kilobase reads. It is currently used world-wide in many applications such as genome sequencing, epigenetics, and surveillance of viral and bacterial pathogens and has started to revolutionize human lives in medicine, agriculture and environmental studies. However, the high base-calling error rates prevent nanopore DNA sequencing from reaching its full potential. In this study, we converted octameric MspA into a single-chain pore enabling asymmetric mutations to fine-tune the pore geometry and chemistry and address the shortcomings of nanopores. Thus, single-chain MspA constitutes a new milestone in its development and adaptation as a biosensor for DNA sequencing and many other applications.

biophysics↗

bioRxiv2021

CMOS electrochemical imaging arrays for thedetection and classification of microorganisms

Microorganisms account for most of the biodiversity on earth. Yet while there are increasingly powerful tools for studying microbial genetic diversity, there are fewer tools for studying microorganisms in their natural environments. In this paper, we present recent advances in CMOS electrochemical imaging arrays for detecting and classifying microorganisms. These microscale sensing platforms can provide non-optical measurements of cell geometries, behaviors, and metabolic markers. We review integrated electronic sensors appropriate for monitoring microbial growth, and present measurements of single-celled algae using a CMOS sensor array with thousands of active pixels. Integrated electrochemical imaging can contribute to improved medical diagnostics and environmental monitoring, as well as discoveries of new microbial populations.

bioengineering↗

bioRxiv2021

Electrical Unfolding of Cytochrome c During Translocation Through a Nanopore Constriction

Many small proteins move across cellular compartments through narrow pores. In order to thread a protein through a constriction, free energy must be overcome to either deform or completely unfold the protein. In principle, the diameter of the pore, along with the effective driving force for unfolding the protein, as well as its barrier to translocation, should be critical factors that govern whether the process proceeds via squeezing, unfolding/threading, or both. To probe this for a well-established protein system, we studied the electric-field-driven translocation behavior of cytochrome c (cyt c) through ultrathin silicon nitride (SiNx) solid-state nanopores of diameters ranging from 1.5 to 5.5 nm. For a 2.5 nm diameter pore we find that, in a threshold electric field regime of [~]30-100 MV/m, cyt c is able to squeeze through the pore. As electric fields inside the pore are increased, the unfolded state of cyt c is thermodynamically stabilized, facilitating its translocation. In contrast, for 1.5 nm and 2.0 nm diameter pores, translocation occurs only by threading of the fully unfolded protein after it transitions through a higher energy unfolding intermediate state at the mouth of the pore. The relative energies between the metastable, intermediate, and unfolded protein states are extracted using a simple thermodynamic model that is dictated by the relatively slow ([~]ms) protein translocation times for passing through the nanopore. These experiments map the various modes of protein translocation through a constriction, which opens new avenues for exploring protein folding structures, internal contacts, and electric field-induced deformability. Significance StatementCan localized electric fields drive the complete unfolding of a protein molecule? Protein unfolding prior to its translocation through a nanopore constriction is an important step in protein transport across biological membranes and also an important step in nanopore-based protein sequencing. We studied here the electric-field-driven translocation behavior of a model protein (cyt c) through nanopores of diameters ranging from 1.5 to 5.5 nm. These single molecule measurements show that electric fields at the nanopore constriction can select both partially and fully unfolded protein conformations. Zero-field free energy gaps between these conformations, found using a simple thermodynamic model, are in remarkable agreement with previously reported studies of cyt c unfolding energetics.

biophysics↗

bioRxiv2020

Stable Polymer Bilayers for Protein Channel Recordings at High Guanidinium Chloride Concentrations

Use of chaotropic reagents is common in biophysical characterization of biomolecules. When the study involves transmembrane protein channels, the stability of the protein channel and supporting bilayer membrane must be considered. In this letter we show that planar bilayers composed of poly(1,2-butadiene)-b-poly(ethylene oxide) diblock copolymer are stable and leak-free at high guanidinium chloride concentrations, in contrast to diphytanoyl phosphatidylcholine bilayers which exhibit deleterious leakage under similar conditions. Further, insertion and functional analysis of channels such as -hemolysin and MspA are straightforward in these polymer membranes. Finally, we demonstrate that -hemolysin channels maintain their structural integrity at 2M guanidinium chloride concentrations using blunt DNA hairpins as molecular reporters.

biophysics↗