Biology subjects

Amit, R.

Publications and source records attributed to Amit, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Revealing pan-cancer clonal niches of T cells from single-cell RNA sequencing using contrastive learning

T-cell clonal organization is central to anti-tumor immunity, yet its functional characterization typically requires paired single-cell RNA and TCR sequencing (scRNA/TCR-seq). Existing multimodal methods integrate transcriptome and TCR information but are limited when TCR data are unavailable. Here we present CloneTrast, a supervised contrastive-learning framework that uses paired scRNA/TCR-seq during training to learn clonal-functional states from gene expression, while requiring only scRNA-seq at inference. Using paired scRNA/TCR-seq profiles from 2.6 million human T cells across 16 cancer types, CloneTrast identified distinct T-cell clonal niches not explained solely by TCR sequence similarity or conventional cell-state annotations. Among these, an NK-like T-cell niche enriched for tumor-blood shared TCRs was consistently associated with poor immunotherapy response at the pan-cancer level. Analysis of antigen-specific T-cell clones revealed that tumor-resident and blood-borne antigen-specific T cells were preferentially associated with distinct clonal niches. Overall, CloneTrast enables scalable and interpretable inference of clonal organization from single-cell transcriptomes.

bioinformatics↗

bioRxiv2026

Expanding The Algal Hydrogen Toolbox: A Non-GMO Platform Reveals Multiple Physiological Routes To Sustained Hydrogen Production Across Microalgae

Sustainable hydrogen production from microalgae remains limited by intrinsic physiological constraints and the need to preserve biomass value for food and feed applications. Transgenic approaches to overcome these limitations were proven successful, yet result in genetically modified (GMO) strains that face major regulatory and deployment barriers. Here, we present a non-GMO experimental platform that enables systematic isolation of hydrogen-producing phenotypes through high-throughput UV mutagenesis pipline coupled with targeted physiological screening. Applying this approach across phylogenetically distinct algal species, including the industrial strain Chlorella vulgaris and the extremophile Chlorella ohadii, we achieve high discovery efficiency, recovering 0.4-0.6% validated hydrogen-producing mutants and achieving 6.7-25% validation rates among screen-positive candidates, indicating strong enrichment at the primary screening stage. We show that sustained hydrogen production represents a physiologically accessible state emerging across diverse genetic backgrounds. This state is consistently associated with reorganization of photosynthetic electron partitioning, yet arises through multiple distinct configurations that differentially balance hydrogen production, oxygen metabolism, and carbon fixation. This framework provides a scalable route to identify hydrogen-producing strains in industrially relevant algae without introducing foreign DNA and expands the accessible design space for photobiological hydrogen production.

plant biology↗

bioRxiv2026

High avidity phase-separated RNA-protein sialogranules sense lectins and inhibit influenza infection

We previously developed synthetic RNA-protein (sRNP) granules as stable, programmable, genetically encoded nanoparticles. Here, we adapt this platform into high-avidity sialogranules that inhibit influenza virus entry. Natural or synthetic 2,6-sialylated proteins and peptides were fused to RNA-binding domains and assembled with synthetic long non-coding RNA to form phase-separated glyco-nanoparticles. Using the sialic-acid-binding lectin Sambucus nigra agglutinin (SNA), we identified a selective transition from sialogranules to tri-component multiphasic biocondensates that occurs only for sialylated constructs. The structural features of this transition quantitatively correlate with predicted sialylation density, enabling extraction of an effective avidity constant. Enzymatic desialylation with neuraminidase or Endo H abolished SNA binding and restored native granule morphology. Guided by this assay, LAMP1-based sialogranules were selected and inhibited influenza entry by [~]50% in cell culture. These results establish sRNP sialogranules as programmable glyco-nanoparticles integrating glycan sensing with antiviral decoy activity.

synthetic biology↗

bioRxiv2026

Synthetic RNA-protein decoy granules to prevent SARS-CoV-2 infection

Synthetic receptor decoys offer a promising strategy to block viral entry but are often limited by instability and rapid clearance. Here we introduce AntiCoV decoy particles: phase-separated synthetic RNA-protein nanoparticles that display multivalent human ACE2 and act as robust decoy receptors for SARS-CoV-2. The granules remain structurally stable for at least two weeks at room temperature. Using confocal imaging and FRET, we show that the SARS-CoV-2 receptor-binding domain is absorbed into the granule matrix with nanometer-scale proximity to ACE2. In viral entry assays with Delta and Omicron (BA.1) variants, AntiCoV decoy particles achieve complete inhibition of infection at low micromolar concentrations and outperform soluble ACE2, which also exhibits infection enhancement at low doses. A minimal kinetic model suggests a multistep spike-priming mechanism that reconciles this biphasic response. AntiCoV decoy particles therefore provide a stable, modular, and pan-variant antiviral platform with potential for low-cost, room-temperature-stable prophylaxis.

synthetic biology↗

bioRxiv2025

scXpand: Pan-cancer detection of T-cell clonal expansion from single-cell RNA sequencing without paired single-cell TCR sequencing

Advances in single-cell sequencing have enabled detailed characterization of T-cell clonal dynamics in cancer. However, analyses aiming to link transcriptional landscape to T-cell clonality remain limited by confounding factors unequally controlled in different studies. To address this challenge, we developed scXpand, the first machine learning framework for pan-cancer detection of T-cell clonal expansion directly from single-cell RNA sequencing (scRNA-seq), without paired T-cell receptor (TCR) sequencing. Trained and tested using our in-house-constructed human pan-cancer database of paired scRNA/TCR-seq profiles from 2.6 million T cells, scXpand demonstrates robust and accurate detection of clonal expansion across tissues and T-cell subtypes. Applied to datasets lacking TCR sequencing, scXpand predictions correspond with known characteristics of the tumor microenvironment. Overall, scXpand is the first framework to detect T-cell clonal expansion across cancers directly from scRNA-seq, enabling broad use on datasets lacking scTCR-seq, while supporting scalable, memory-efficient processing, including pre-trained models with user-friendly documentation for flexible applications.

bioinformatics↗

bioRxiv2024

Formation of polyphasic RNP granules by intrinsically disordered Qβ coat proteins and hairpin-containing RNA

RNA-protein (RNP) granules are fundamental components in mammalian cells where they perform multiple crucial functions. Many RNP granules form via phase separation driven by protein-protein, protein-RNA, and RNA-RNA interactions. Notably, associated proteins frequently contain intrinsically disordered regions (IDRs) which can associate with multiple partners. Previously we have shown that synthetic RNA molecules containing multiple hairpin coat-protein binding sites can phase separate, forming granules capable of selectively incorporating proteins inside. Here, we expand this platform by introducing a phage coat protein with a known IDR which facilitates protein-protein interactions. We show that the coat protein phase-separates on its own in vivo, and that introduction of hairpin-containing RNA molecules can lead to dissolvement of the protein granules. We further demonstrate via multiple assays that RNA valency, determined by the number of hairpins present on the RNA, leads to distinctly different phase behaviors, effectively forming a polyphasic programmable RNP granule. Moreover, by incorporating the gene for a blue fluorescent protein into the RNA, we demonstrate a phase-dependent boost to protein titer. These insights not only shed light on the behavior of natural granules, but also hold profound implications for the biotechnology field, offering a blueprint for engineering cellular compartments with tailored functionalities.

biophysics↗

bioRxiv2024

Phase Separation-based Antiviral Decoy Particles as Basis for Programmable Broad-spectrum Therapeutics

To gain access to cells, viruses employ host proteins as receptors. In soluble form, these receptors are used as decoys to inhibit infection. We fused candidate soluble receptors to an RNA-binding protein, and using synthetic long non-coding RNA (slncRNA) cassettes that can undergo phase-separation we scaffolded the receptor fusions to generate antiviral decoy particles. Using confocal microscopy, we screened antiviral protein candidates by observing changes in phase-separation morphology when incubated with viral-mimicking components. We demonstrated that ACE2 decoy particles bind strongly to the coronavirus RBD, facilitating FRET, while sufficiently sialylated decoy particles form agglutinated structures with RNA peripheries in the presence of a sialolectin. Infection assays show ACE2 decoy particles fully inhibit the Delta and Omicron BA.1 coronavirus variants, and LAMP1 and GYPA decoy particles significantly reduce influenza infection in-cellulo. This work establishes a foundation for broad-spectrum antiviral decoy particles, composed of multiple receptors targeting various viruses.

synthetic biology↗

bioRxiv2024

GelMetrics: An algorithm for analyzing the dynamics of gel-like phase separated condensates

Cellular compartments and organelles are essential for the spatial organization of biological matter. Recently, membraneless organelles like paraspeckles, stress granules, and Cajal bodies have garnered significant scientific interest due to their lack of membrane boundaries and crucial cellular functions. These organelles self-assemble through phase separation, a process in which a homogeneous solution separates into distinct phases. The phases most commonly encountered in cells are liquids and gels. Although various microscopy techniques exist to study phase-separated compartments, they are often inadequate for investigating the dynamics of gel-like condensates, where molecular motion occurs over tens of minutes rather than seconds. Here, we introduce the GelMetrics algorithm to quantitatively measure the dynamics of gel-like phase-separated structures by tracking their fluorescence signals over extended durations. First, we show using simulations that this method can identify molecular motion amidst measurement noise and estimate biophysical parameters such as the number of molecules and the average fluorescence of a single molecule exiting a condensate, providing insights into the dynamics of phase-separated organelles. Second, we validate our approach on data from synthetic RNA-protein (sRNP) granules in E. coli and in vitro, establishing its applicability both in vitro and in vivo. Finally, we demonstrate the biological meaning of our algorithms output by quantitatively determining how the presence of an intrinsically disordered region causes sRNP granules to exhibit a more brittle phase-behavior. Thus, not only does the proposed method fill a gap in analysis methods for gel-like condensates, but it also elucidates biophysical mechanisms, improving our understanding of both synthetic and natural systems.

bioinformatics↗

bioRxiv2022

Wikipedia as a tool for contemporary history of science: A case study on CRISPR

Rapid developments and methodological divides hinder the study of how scientific knowledge accumulates, consolidates and transfers to the public sphere. Our work proposes using Wikipedia, the online encyclopedia, as a historiographical source for contemporary science. We chose the high-profile field of gene editing as our test case, performing a historical analysis of the English-language Wikipedia articles on CRISPR. Using a mixed method approach, we qualitatively and quantitatively analyzed its text, sections and references, alongside 50 affiliated articles. These, we found, documented CRISPRs maturation from a fundamental scientific discovery to a biotechnological revolution with vast social and cultural implications. We developed automated tools to support such research generically and demonstrated its applicability on two other scientific fields we have previously studied - COVID-19 and Circadian clocks. This method makes use of Wikipedia as a digital and free archive, documenting the incremental growth of knowledge and the manner scientific research accumulates and translates into public discourse. Using Wikipedia in this manner compliments and overcomes some issues with contemporary histories and can also augment existing bibliometric research.

scientific communication and education↗

bioRxiv2021

A cell-free assay for rapid screening of inhibitors of hACE2-receptor - SARS-CoV-2-Spike binding

We present a cell-free assay for rapid screening of candidate inhibitors of protein binding, focusing on inhibition of the interaction between the SARS-CoV-2 Spike receptor binding domain (RBD) and human angiotensin-converting enzyme 2 (hACE2). The assay has two components: fluorescent polystyrene particles covalently coated with RBD, termed virion-particles (v-particles), and fluorescently-labeled hACE2 (hACE2F) that binds the v-particles. When incubated with an inhibitor, v-particle - hACE2F binding is diminished, resulting in a reduction in the fluorescent signal of bound hACE2F relative to the non-inhibitor control, which can be measured via flow cytometry or fluorescence microscopy. We determine the amount of RBD needed for v-particle preparation, v-particle incubation time with hACE2F, hACE2F detection limit, and specificity of v-particle binding to hACE2F. We measure the dose response of the v-particles to a known inhibitor. Finally, we demonstrate that RNA-hACE2F granules trap v-particles effectively, providing a basis for potential RNA-hACE2F therapeutics.

synthetic biology↗

bioRxiv2020

Oculomotor inhibition precedes temporally expected auditory targets

Eye movements are inhibited prior to the onset of temporally-predictable visual targets. This oculomotor inhibition effect could be considered a marker for the formation of temporal expectations and the allocation of temporal attention in the visual domain. Here we show that eye movements are also inhibited before predictable auditory targets. In two experiments, we manipulate the period between a cue and an auditory target to be either predictable or unpredictable. The findings show that although there is no perceptual gain from avoiding gaze-shifts in this procedure, saccades and blinks are inhibited prior to predictable relative to unpredictable auditory targets. These findings show that oculomotor inhibition occurs prior auditory targets. This link between auditory expectation and oculomotor behavior, in combination with the results of our parallel study in the tactile domain, reveals a multimodal perception action coupling, which has a central role in temporal expectations.

neuroscience↗

bioRxiv2019

Identifying triplex binding rules in vitro leads to creation of a new synthetic regulatory tool in vivo

Nature provides a rich toolbox of dynamic nucleic acid structures that are widespread in cells and affect multiple biological processes1. Recently, non-canonical structures gained renewed scientific and biotechnological interest2,3. One particularly intriguing form of such structures are triplexes4 in which a single-stranded nucleic acid molecule interacts via Hoogsteen bonds with a DNA/RNA double helix5. Despite extensive research in vitro6-9, the underlying rules for triplex formation remain debated and evidence for triplexes in vivo is circumstantial10-12. Here, we demonstrate the development of a deep-sequencing platform termed Triplex-Seq to systematically refine the DNA triplex code and identify high affinity triplex forming oligo (TFO) variants. We identified a preference for short G-rich motifs using an oligo-library with a mix of all four bases. These high-information content motifs formed specific high-affinity triplexes in a pH-independent manner and stability was increased with G-rich double-stranded molecules. We then conjugated one high-affinity and one low-affinity variant to a VP48 peptide and studied these synthetic biomolecules in mammalian cells. Using these peptide-oligo constructs (POCs), we demonstrated possible triplex-induced down-regulation activity in 544 differentially expressed genes. Our results show that deep-sequencing platforms can substantially expand our understanding of triplex binding rules, which in turn has led to the development of a functional non-genetically encoded regulatory tool for in vivo applications.

synthetic biology↗

bioRxiv2019

Overcoming the design, build, test (DBT) bottleneck for synthesis of nonrepetitive protein-RNA binding cassettes for RNA applications

The design-build-test (DBT) cycle in synthetic biology is considered to be a major bottleneck for progress in the field. The emergence of high-throughput experimental techniques, such as oligo libraries (OLs), combined with machine learning (ML) algorithms, provide the ingredients for a potential "big-data" solution that can generate a sufficient predictive capability to overcome the DBT bottleneck. In this work, we apply the OL-ML approach to the design of RNA cassettes used in gene editing and RNA tracking systems. RNA cassettes are typically made of repetitive hairpins, therefore hindering their retention, synthesis, and functionality. Here, we carried out a high-throughput OL-based experiment to generate thousands of new binding sites for the phage coat proteins of bacteriophages MS2 (MCP), PP7 (PCP), and Q{beta} (QCP). We then applied a neural network to vastly expand this space of binding sites to millions of additional predicted sites, which allowed us to identify the structural and sequence features that are critical for the binding of each RBP. To verify our approach, we designed new non-repetitive binding site cassettes and tested their functionality in U2OS mammalian cells. We found that all our cassettes exhibited multiple trackable puncta. Additionally, we designed and verified two additional cassettes, the first containing sites that can bind both PCP and QCP, and the second with sites that can bind either MCP or QCP, allowing for an additional orthogonal channel. Consequently, we provide the scientific community with a novel resource for rapidly creating functional non-repetitive binding site cassettes using one or more of three phage coat proteins with a variety of binding affinities for any application spanning bacteria to mammalian cells.

synthetic biology↗

bioRxiv2019

Live dynamical tracking of slncRNA speckles in single E. coli cells reveals bursts of fluorescence degradation

Liquid-solid transition, also known as gelation, is a specific form of phase separation in which molecules cross-link to form a highly interconnected compartment with solid - like dynamical properties. Here, we utilize RNA hairpin coat-protein binding sites to form synthetic RNA based gel-like granules via liquid-solid phase transition. We show both in-vitro and in-vivo that hairpin containing synthetic long non-coding RNA (slncRNA) molecules granulate into bright localized puncta. We further demonstrate that upon introduction of the coat-proteins, less-condensed gel-like granules form with the RNA creating an outer shell with the proteins mostly present inside the granule. Moreover, by tracking puncta fluorescence signals over time, we detected addition or shedding events of slncRNA-CP nucleoprotein complexes. Consequently, our granules constitute a genetically encoded storage compartment for protein and RNA with a programmable controlled release profile that is determined by the number of hairpins encoded into the RNA. Our findings have important implications for the potential regulatory role of naturally occurring granules and for the broader biotechnology field.

biophysics↗

bioRxiv2018

Improved DNA based storage capacity and fidelity using composite DNA letters

DNA, with its remarkable density and long-term stability, is an appealing potential next generation data storage medium, most notably for long-term archiving. Megabyte scale DNA based storage was first reported in 2012. The Shannon information capacity of DNA was recently demonstrated, using fountain codes, to be [~]1.57 bit per synthesized position. However, synthesis and sequencing technologies process multiple nominally identical molecules in parallel, leading to significant information redundancies. We introduce composite DNA alphabets, using mixed DNA base types, to leverage this redundancy, enabling higher density. We develop encoding and decoding for composite DNA based storage, including error correction. Using current DNA synthesis technologies, we code 6.4 Megabyte data into composite DNA, achieving [~]25% increase in capacity as compared to literature. We further demonstrate, on smaller scales, how flexible synthesis leads to 2.7 fold increased capacity per synthesized position. Composite DNA can thus reduce costs for DNA based storage and can also serve in other applications.

synthetic biology↗

bioRxiv2017

Synthetic protein-sensing riboswitches

We study translational regulation by a 5 UTR sequence encoding the binding site of an RNA-binding protein (RBP) in bacteria, using a reporter assay and Selective 2-hydroxyl acylation analysed by primer extension sequencing (SHAPE-Seq). We tested constructs containing a single hairpin, based on the binding sites of the coat RBPs of bacteriophages GA, MS2, PP7, and Q{beta}, positioned in the 5 UTR of a reporter gene. With specifically-bound RBP present, either weak repression or up-regulation is observed, depending on the binding site and its flanking sequence. SHAPE-Seq data for a representative construct exhibiting up-regulation, indicates a partially-folded hairpin and non-reactive upstream and downstream flanking region, which we attribute to intermediate structures that apparently blocks translation. RBP binding stabilizes the fully-folded hairpin state and thus facilitates translation, suggesting that the up-regulating constructs are RBP-sensing riboswitches. This finding is further supported by lengthening the binding-site stem, which in turn destabilizes the translationally-inactive state, and abolishes the up-regulating behavior. Finally, we found that the combination of two binding sites, positioned in the 5 UTR and gene-header of the same transcript, can yield a cooperative regulatory response. Together, we show that the interaction of an RBP with its RNA target facilitates structural changes in the RNA, which is reflected by a controllable range of binding affinities and dose response behaviors. Thus, demonstrating that RNA-RBP interactions can provide a platform for constructing gene regulatory networks that are based on translational, rather than transcriptional, regulation.

synthetic biology↗

bioRxiv2017

An in vivo binding assay for RNA-binding proteins based on repression of a reporter gene

We employ a reporter assay and Selective 2'-hydroxyl acylation analysed by primer extension sequencing (SHAPE-seq) to study translational regulation by RNA-binding proteins, in bacteria. We designed 82 constructs, each with a single hairpin based on the binding sites of the RNA-binding coat proteins of phages MS2, PP7, GA, and Q{beta}, at various positions within the N-terminus of a reporter gene. In the absence of RNA-binding proteins, the translation level depends on hairpin location, and exhibits a three-nucleotide periodicity. For hairpin positions within the initiation region, we observe strong translational repression in the presence of its cognate RNA-binding protein. In vivo SHAPE-seq results for a representative construct indicate that the repression phenomenon correlates with a wide-swath of protection, including the hairpin and extending past the ribosome binding site. Consequently, our data suggest that the protection provided by the RBP-hairpin complex inhibits ribosomal initiation. Finally, utilizing the repression phenomenon for quantifying protein-RNA binding affinity in vivo, we both observe partially contrasting results to previous in vitro and in situ studies, and additionally, show that this method can be used in a high-throughput assay for a quantitative study of protein-RNA binding in vivo.

synthetic biology↗

bioRxiv2017

Demonstration of de novo chemotaxis in E. coli using a real-time, quantitative, and digital-like approach

Chemotaxis is the movement of an organism in response to an external chemical stimulus. This system enables bacteria to sense their immediate environment and adapt to changes in its chemical composition. Bacterial chemotaxis is mediated by chemoreceptors, membrane proteins that bind an effector and transduce the signal to the downstream proteins. From a synthetic biology perspective, the natural chemotactic repertoire is of little use since bacterial chemoreceptors have evolved to sense specific ligands that either benefit or harm the cell. Here we demonstrate that using a combined computational design approach together with a quantitative, real-time, and digital detection approach, we can rapidly design, manufacture, and characterize a synthetic chemoreceptor in E. coli for histamine (a ligand for which there are no known chemoreceptors). First, we employed a computational protocol that uses the Rosetta bioinformatics software together with high threshold filters to design mutational variants to the native Tar ligand binding domain that target histamine. Second, we tested different ligand-chemoreceptors pairs with a novel chemotaxis assay, based on optical reflectance interferometry of porous silicon (PSi) optical transducers, enabling label-free quantification of chemotaxis by monitoring real-time changes in the optical readout (expressed as the effective optical thickness, EOT). We found that different ligands can be characterized by an individual set of fingerprints in our assay. Namely, a binary, digital-like response in EOT change (i.e. positive or negative) that differentiates between attractants and repellants, the amplitude of change of EOT response, and the rate by which steady state in EOT change is reached. Using this assay, we were able to positively identify and characterize a single mutational chemoreceptor variant for histamine that mediated chemotaxis comparably to the natural Tar-aspartate system. Our results demonstrate the possibility of not only expanding the natural chemotaxis repertoire, but also provide a new quantitative assay by which to characterize the efficacy of the chemotactic response.

synthetic biology↗