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bioRxiv2025

Moremi Bio Agent: Using Neisseria meningitidis Reference Data For The Double Blinded Validation of A General Purpose Biology-Trained Reasoning Model for Pathogen and Antigen Discovery

Antibodies serve as vital diagnostic and therapeutic agents due to their exceptional specificity toward antigenic targets. Mapping antibody-antigen interactions is essential for understanding immune responses and developing vaccines or biologics. Traditional antigen identification relies on labor-intensive wet-lab techniques such as phage display, peptide microarrays, and ELISA, while computational methods employ sequence alignment, epitope mapping, and structure prediction. Despite progress, to our knowledge, no existing AI framework has demonstrated the ability to blindly inference--predicting an antibodys antigen target solely from its amino acid sequence without prior biological context. This research employed Moremi Bio Nano, a general agentic reasoning large language model (LLM), to infer the antigen and pathogen targets of an anonymized monoclonal antibody sequence from Imperial College London. The model received only the VH and VL chain sequences and autonomously hypothesized, ranked, and validated probable targets. Of ten independent inference tests, four were completed successfully, with three correctly identifying the experimentally validated antigen and pathogen; with SARS-CoV-2 Spike RBD, Neisseria meningitidis fHbp v1.1, and SARS-CoV Spike emerging as the top 3-ranked candidates across both reranking strategies. Validation of the models predictions with experimental wet-lab data confirmed its capacity for correct antigen inference, marking Moremi Bio Nano as a first-of-its-kind AI system demonstrating reasoning-driven antigen discover; complementing experimental immunology and advancing automated biological inference.

molecular biology↗

bioRxiv2026

DORA: a dose-response autoencoder for interpretable transcriptome-to-viability prediction

Predicting the effect of drugs on cell viability is a central challenge in drug discovery. Artificial intelligence holds the promise to considerably accelerate this process by leveraging rich cellular data such as transcriptomics. Current models focus on either transcriptomes or inhibitory concentrations, but they fall short in integrating these sources of information. Here, we propose DORA (Dose-Response Autoencoder), a deep learning model that predicts changes in transcriptomes and viability in a dose-dependent manner, knowing the unperturbed cell state. By enforcing a latent space consistent with cumulative dose effects, DORA matches other methods at predicting transcriptomes and substantially outperforms existing latent representations at viability prediction. The transcriptome-viability relationship provided by the model further allows the recovery of known biomarkers of cell viability while suggesting novel ones. Overall, DORA provides a unified framework delivering actionable biological insights for phenotypic drug screening and personalized medicine.

molecular biology↗

bioRxiv2026

Small RNA-guided transgene repression systems enable toxic gene cloning in bacteria

Multiple vectors and bacterial strains have been developed to enable cloning and amplification of DNA plasmids used in bioengineering applications when transgenic components are toxic to the host. These include plasmids that limit readthrough transcription into transgenic sequences and host strains carrying mutations to minimize recombination or plasmid copy number. However, these techniques are insufficient in cases where transgene expression elements are recognized by the bacterial transcriptional apparatus, or the translation products have functions in cellular metabolism. Here we demonstrate two platforms that mitigate bacterial expression of transgenes driven by the prokaryotic-like promoters of chloroplast transgenes destined for use in plant plastid genetic engineering applications. Both an engineered CRISPRi approach and utilization of the native E. coli Hfq repression system resulted in significant knockdown of plasmid-borne transgene expression, resulting in reproducibly successful cloning and plasmid amplification. The advancements reported here will facilitate synthetic biology studies generally, and enable complex transgenic studies in prokaryotic-like organelles.

molecular biology↗

bioRxiv2024

Biological aging of different blood cell types

A biological age (BA) indicator is intended to capture detrimental age-related changes occurring with passing time. To date, the best-known and used BA indicators include DNA-methylation-based epigenetic ages (epigenetic clocks) and telomere length. The most common biological sample material for epidemiological aging studies is composed of different cell types, whole blood. We aimed to compare differences in BAs between blood cell types and assessed BA indicators cell type-specific associations with donors calendar age. Analysis on DNA methylation-based BA indicators including telomere length, methylation level at cg16867657 (a CpG-site in ELOVL2) and the Hannum, Horvath, DNAmPhenoAge and DunedinPACE epigenetic clocks was performed in 428 biological samples from 12 blood cell types. BA values were different (p<0.05) in the majority of pairwise comparisons between the cell types. Most cell types also displayed differences as compared to whole blood (p<0.05). Some of the observed differences persisted across blood donors calendar ages from 20 to 80 years (50-years-difference in DNAmPhenoAge between naive CD4+ T cells and monocytes), while others did not (up to four-fold difference in DunedinPACE values between monocytes and B cells). All BA indicators, except DunedinPACE, had mostly a very strong correlation with donors calendar age within a cell type. Our findings demonstrate that DNA methylation-based indicators of biological age exhibit cell type-specific characteristics, underscoring the importance of accounting for cell composition in related studies. Our results have implications for understanding the molecular mechanisms underlying epigenetic clocks and and provide guidance for utilizing them as indicators for success of aging interventions.

molecular biology↗

bioRxiv2026

Inositol hexakisphosphate Functions as a Cofactor and Modulator of ADAR1 Activity

Adenosine-to-inosine (A-to-I) RNA editing by ADAR1 is a key post-transcriptional modification, and mutations in ADAR1 lead to Aicardi-Goutieres syndrome (AGS), an autoimmune disorder. Despite its biological and clinical relevance, the regulation of ADAR1 activity remains incompletely understood. Using a combination of biochemical approaches, inositol-pentakisphosphate 2-kinase (IPPK)-knockout cells, molecular dynamics simulations, and a cell-permeable inositol hexakisphosphate (IP6) prodrug (Pro-IP6), we demonstrate that IP6 depletion drastically reduces global RNA editing, while supplementation with Pro-IP6 restores and even enhances editing levels. Furthermore, we identify the C6-phosphate of IP6 as a critical determinant of ADAR1 catalytic efficiency, functioning within a hydrogen-bonding network that indirectly coordinates a Zn{superscript 2}-ion. Finally, we show that the AGS-associated ADAR1 mutation N907S impairs RNA editing activity, most likely by altering the hydrogen-bond interaction network linking IP6 to the ADAR1 catalytic center. Together, these findings identify IP6 as an essential cofactor and regulator of ADAR1 activity and highlight cofactor availability and interaction networks as strategies for therapeutically modulating RNA editing.

molecular biology↗

bioRxiv2026

Modular split-trigger based cooperative activation platform for Cas13-12 tandem biosensing system

Achieving precise control of CRISPR/Cas trans-cleavage depends on understanding how nucleic acid activators engage Cas effectors, yet the fundamental principles of split-trigger activation of Cas12a remain unclear. Here, we uncover the mechanistic determinants that enable fragmented nucleic acids to collectively initiate Cas12a activity. We show that split triggers bearing external extensions fully support the R-loop formation, whereas internal extensions which disrupt the spacer complementarity abolish Csa12a activation. We further demonstrate that covalent linkage of split-trigger fragments prevents R-loop propagation, revealing that Cas12as activation strictly requires two physically independent split fragments. Together, these findings establish a synergistic split-trigger activation mechanism in which cooperative hybridization of two individually fragments nucleates and extends the Cas12a R-loop with high efficiency. Conceptually, this mechanism enables a cascade architecture that transforms CRISPR diagnostics from a one-target one-Cas ribonucleoprotein (RNP) paradigm into a highly amplifying process in which a single target molecule activates numerous downstream Cas RNPs. Building on this principle, we show that the cleavage of a rationally designed linear DNA-RNA-DNA mediator by LbuCas13a generates optimally configured split triggers for Cas12a activation, thereby coupling RNA recognition to large-scale Cas12a activation without enzymatic preamplification. The resulting Split Trigger Activated Cas13-Cas12 Cascade System (STACS) achieves amplification-free detection down to 1 copy/{micro}L within 15 minutes and maintains robust performance in complex biological (serum, saliva) and environmental (mud) matrices. This work establishes a generalizable strategy for engineering programmable CRISPR cascades with high Cas RNP activation multiplicity for ultrasensitive molecular diagnostics. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/734747v2_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@104a609org.highwire.dtl.DTLVardef@30cebcorg.highwire.dtl.DTLVardef@54b15borg.highwire.dtl.DTLVardef@47f89e_HPS_FORMAT_FIGEXP M_FIG C_FIG Mechanism and detection workflow of the Split Trigger Activated Cas13-12 Cascade System (STACS).

Molecular Biology↗

bioRxiv2020

The 3'tsRNAs are aminoacylated: Implications for their biogenesis

Emerging evidence indicates that tRNA-derived small RNAs (tsRNAs) are involved in fine-tuning gene expression and become dysregulated in various cancers. We recently showed that the 22nt LeuCAG tsRNA from 3 end of tRNALeu is required for efficient translation of a ribosomal protein mRNA and ribosome biogenesis. Inactivation of this tsRNA induced apoptosis in rapidly dividing cells and suppressed the growth of a patient derived orthotopic hepatocellular carcinoma in mice. The mechanism involved in the generation of the 3-tsRNAs remains elusive and it is unclear if the 3-ends of 3-tsRNAs are aminoacylated. Here we report an enzymatic method utilizing exonuclease T to determine the 3charging status of tRNAs and tsRNAs. Our results showed that the LeuCAG 3-tsRNA is fully charged and originated solely from charged mature tRNA. When the leucyl-tRNA synthetase was knocked down, less tsRNA was generated while the mature tRNA was not reduced further supporting that tsRNA generation is regulated. The fact that the 3-tsRNA is aminoacylated has implications for their biogenesis and provides additional insights into their biological role in post-transcriptional gene regulation.

molecular biology↗

bioRxiv2022

Circuit-seq: Circular reconstruction of cut in vitro transposed plasmids using Nanopore sequencing

Recombinant DNA is a fundamental tool in biotechnology and medicine. Validation of the resulting plasmid sequence is a critical and time-consuming step, which has been dominated for the last 35 years by Sanger sequencing. As plasmid sequences grow more complex with new DNA synthesis and cloning techniques, we need new approaches that address the corresponding validation challenges at scale. Here we prototype a high-throughput plasmid sequencing approach using DNA transposition and Oxford Nanopore sequencing. Our method, Circuit-seq, creates robust, full-length, and accurate plasmid assemblies without prior knowledge of the underlying sequence for approximately $1.50 per plasmid. We demonstrate the power of Circuit-seq across a wide range of plasmid sizes and complexities, generating accurate and contiguous plasmid maps. We then leverage our long read-data to characterize epigenetic marks and estimate plasmid contamination levels. Circuit-seq scales to large numbers of samples at a lower cost than commercial Sanger sequencing, accelerating a key step in synthetic biology, with low startup costs make it practical for individual laboratories.

molecular biology↗

bioRxiv2022

A new insight into RecA filament regulation by RecX from the analysis of conformation-specific interactions

RecA protein mediates homologous recombination repair in bacteria through assembly of long helical filaments on single-stranded DNA (ssDNA) in an ATP dependent manner. RecX, an important negative regulator of RecA, is known to inhibit RecA activity by stimulating the disassembly of RecA nucleoprotein filaments. Here we use a single-molecule approach to address the regulation of (E. coli) RecA-ssDNA filaments by RecX (E. coli) within the framework of distinct conformational states of RecA-ssDNA filament. Our findings revealed that RecX effectively binds the inactive conformation of RecA-ssDNA filaments and slows down the transition to the active state. Results of this work provide new mechanistic insights into the RecX-RecA interactions and highlight the importance of conformational transitions of RecA filaments as an additional level of regulation of its biological activity.

molecular biology↗

bioRxiv2022

Structural basis for differential targeting properties of small RNAs in plants and animals

microRNAs (miRNAs) regulate gene expression in plants and animals. Animals use miRNAs to sculpt the transcriptome, with each miRNA modestly repressing hundreds of otherwise unrelated targets. By contrast, each plant miRNA potently silences a small number of physiologically related targets. Here, we show that this major functional distinction depends on a minor structural difference between plant and animal Argonaute (AGO) proteins. A 7-amino-acid loop in the PIWI domain of Arabidopsis Argonaute10 (AtAGO10) reduces the affinity of the miRNA seed region for target RNAs. Swapping the PIWI-loop from human Argonaute2 (HsAGO2) into AtAGO10 increases seed strength and target-binding promiscuity, resembling animal miRNA targeting. Conversely, swapping the plant PIWI-loop into HsAGO2 increases targeting stringency and elevates target cleavage rates. The loop-swapped HsAGO2-siRNA complex silences targets more potently, with reduced off-targeting, than wild-type HsAGO2 and small interfering RNA (siRNA) duplexes in mammalian cells. Thus, tiny structural differences can tune the targeting properties of AGO proteins for distinct biological roles and HsAGO2 can be engineered for potential applications in siRNA therapeutics.

molecular biology↗

bioRxiv2022

Pharmacological profiling of a Brugia malayi muscarinic acetylcholine receptor as a putative antiparasitic target

The diversification of anthelmintic targets and mechanisms of action will help ensure the sustainable control of nematode infections in response to the growing threat of drug resistance. G protein-coupled receptors (GPCRs) are established drug targets in human medicine but remain unexploited as anthelmintic substrates despite their important roles in nematode neuromuscular and physiological processes. Bottlenecks in exploring the druggability of parasitic nematode GPCRs include a limited helminth genetic toolkit and difficulties establishing functional heterologous expression. In an effort to address some of these challenges, we profile the function and pharmacology of muscarinic acetylcholine receptors in the human parasite Brugia malayi, an etiological agent of human lymphatic filariasis. While acetylcholine-gated ion channels are intensely studied as targets of existing anthelmintics, comparatively little is known about metabotropic receptor contributions to parasite cholinergic signaling. Using multivariate phenotypic assays in microfilariae and adults, we show that nicotinic and muscarinic compounds disparately affect parasite fitness traits. We identify a putative G protein-linked acetylcholine receptor (Bma-GAR-3) that is highly expressed across intra-mammalian life stages and adapt spatial RNA in situ hybridization to map receptor transcripts to critical parasite tissues. Tissue-specific expression of Bma-gar-3 in Caenorhabditis elegans (body wall muscle, sensory neurons, and pharynx) enabled receptor deorphanization and pharmacological profiling in a nematode physiological context. Lastly, we developed an image-based feeding assay as a reporter of pharyngeal activity to facilitate GPCR screening in parasitized strains. We expect that these receptor characterization approaches and improved knowledge of GARs as putative drug targets will further advance the study of GPCR biology across medically important nematodes.

molecular biology↗

bioRxiv2022

Pisces: A combo-wise contrastive learning approach to synergistic drug combination prediction

Drug combination therapy is promising for cancer treatment through simultaneously reducing resistance and improving efficacy. Machine learning approaches to drug combination response prediction can prioritize experiments and discover new combinations, but require lots of training data in order to fit the nonlinearity of synergistic effect. Here, we propose Pisces, a novel machine learning approach for drug combination synergy prediction. The key idea of Pisces is to augment the sparse drug combination dataset by creating multiple views for each drug combination based on its different modalities. We combined eight different modalities of a single drug to create 64 augmented views for a pair of drugs, effectively expanding the size of the original data 64 times. Pisces obtained state-of-the-art results on cell-line-based drug synergy prediction, xenograft-based drug synergy prediction, and drug-drug interaction prediction. By interpreting Piscess predictions using a genetic interaction network, we further identified a breast cancer drug-sensitive pathway from BRCA cell lines in GDSC. We validated this pathway on an independent TCGA-BRCA tumor dataset and found that patients with this pathway activated had substantially longer survival time. Collectively, Pisces effectively predicts drug synergy and drug-drug interactions through augmenting the original dataset 64 times, and can be broadly applied to various biological applications that involve a pair of drugs.

molecular biology↗

bioRxiv2024

A Comprehensive Method on Black-legged Tick Larvae and Nymph Feeding on Mice to Study Lyme Disease Transmission and Acquisition

Tick-borne diseases are a growing public health concern in the United States, with cases rising steadily each year. Lyme borreliosis, or Lyme disease, remains the most prevalent, affecting approximately 476,000 individuals annually. Human-driven changes in climate and ecosystems have expanded the habitat of pathogen-carrying ticks, facilitating the spread of these infections. Additionally, increased instances of tick-borne diseases transmission through human tissues have been reported. Despite ongoing efforts to manage these infections, their incidence continues to rise. To develop effective control measures against these diseases and prevent the transmission of tick-borne infections through human and animal tissues, it is very important to develop detection assays and understand the transmission mechanisms of tick-borne infections. In this study, we provide detailed descriptions and visual references for larval and nymphal tick feeding on mice, focusing on the transmission and acquisition of Borrelia burgdorferi (sensu stricto). These methodologies can be applied to study other tick-borne diseases, tick vectorial capacity, and tick biology, aiding in the development of detection strategies to combat these infections.

molecular biology↗

bioRxiv2025

CryoDataBot: a pipeline to curate cryoEM datasets for AI-driven structural biology

Cryogenic electron microscopy (cryoEM) has revolutionized structural biology by enabling atomic-resolution visualization of biomacromolecules. To automate atomic model building from cryoEM maps, artificial intelligence (AI) methods have emerged as powerful tools. Although high-quality, task-specific datasets play a critical role in AI-based modeling, assembling such resources often requires considerable effort and domain expertise. We present CryoDataBot, an automated pipeline that addresses this gap. It streamlines data retrieval, preprocessing, and labeling, with fine-grained quality control and flexible customization, enabling efficient generation of robust datasets. CryoDataBots effectiveness is demonstrated through improved training efficiency in U-Net models and rapid, effective retraining of CryoREAD, a widely used RNA modeling tool. By simplifying the workflow and offering customizable quality control, CryoDataBot enables researchers to easily tailor dataset construction to the specific objectives of their models, while ensuring high data quality and reducing manual workload. This flexibility supports a wide range of applications in AI-driven structural biology.

molecular biology↗

bioRxiv2018

Real-time structural dynamics of late steps in bacterial translation initiation visualized using time-resolved cryogenic electron microscopy

Bacterial translation initiation entails the tightly regulated joining of the 50S ribosomal subunit to an initiator transfer RNA (fMet-tRNAfMet)-containing 30S ribosomal initiation complex (IC) to form a 70S IC that subsequently matures into a 70S elongation-competent complex (70S EC). Rapid and accurate 70S IC formation is promoted by 30S IC-bound initiation factor (IF) 1 and the guanosine triphosphatase (GTPase) IF2, both of which must ultimately dissociate from the 70S IC before the resulting 70S EC can begin translation elongation1. Although comparison of 30S2-6 and 70S5,7-9 IC structures have revealed that the ribosome, IFs, and fMet-tRNAfMet can acquire different conformations in these complexes, the timing of conformational changes during 70S IC formation, structures of any intermediates formed during these rearrangements, and contributions that these dynamics might make to the mechanism and regulation of initiation remain unknown. Moreover, lack of an authentic 70S EC structure has precluded an understanding of ribosome, IF, and fMet-tRNAfMet rearrangements that occur upon maturation of a 70S IC into a 70S EC. Using time-resolved cryogenic electron microscopy (TR cryo-EM)10 we report the first, near-atomic-resolution view of how a time-ordered series of conformational changes drive and regulate subunit joining, IF dissociation, and fMet-tRNAfMet positioning during 70S EC formation. We have found that, within ~20-80 ms, rearrangements of the 30S subunit and IF2, uniquely captured in its GDP*Pi-bound state, stabilize fMet-tRNAfMet in its intermediate, 70S P/I, configuration7 and trigger dissociation of IF1 from the 70S IC. Within the next several hundreds of ms, dissociation of IF2 from the 70S IC is coupled to further remodeling of the ribosome that positions fMet-tRNAfMet into its final, P/P, configuration within the 70S EC. Our results demonstrate the power of TR cryo-EM to determine how a time-ordered series of conformational changes contribute to the mechanism and regulation of one of the most fundamental processes in biology.

molecular biology↗

bioRxiv2026

Dissection of Poly(A)-binding protein (PABPC) cellular function using degron-mediated depletion with replacement

Cytoplasmic poly(A)-binding proteins (PABPCs) are essential and highly abundant regulators of mRNA stability and translation, but their cellular functions have been difficult to dissect due to slow turnover and the lethality observed upon loss of both major paralogs, PABPC1 and PABPC4. To enable structure-function analysis of PABPC in human cells, we developed a protein-replacement platform that couples rapid auxin-inducible degradation of endogenous PABPC1 and PABPC4 with doxycycline-controlled expression of engineered PABPC variants. This approach enables acute removal of native PABPC and real-time assessment of how specific domains, paralogs, and sequence alterations support cellular fitness, shape transcriptome profiles, and regulate poly(A)-tail length. Using this system, we show that the RRM4 domain of PABPC1 is essential for growth, whereas post-translationally modified lysines within RRM4 are individually dispensable. Paralogs and variants with heterologous RRM4 domains vary in their ability to substitute for PABPC1, revealing functional divergence among PABPCs. Transcriptome profiling identifies variant-specific regulatory signatures, and dose-controlled rescue further delineates the relationship between PABPC variant abundance and global poly(A)-tail lengths in vivo. Together, this platform provides a generalizable strategy for dissecting PABPC biology in the cellular context using rationally designed variants.

molecular biology↗

bioRxiv2026

Tension TRAAKer: a chemigenetic fluorescent membrane tension reporter

Mechanical force transduction is essential to survival, underlying biological processes as fundamental as morphogenesis, somatosensation, audition, and interoception; and driving pathologies as diverse as hypertension and cancer metastasis. Exogenous forces are translated to intracellular signals through transient changes in membrane tension which are currently not possible to directly monitor in situ. To remedy this, we have designed and validated Tension TRAAKer, a chemigenetic fluorescent membrane tension reporter for the visualization of tension induction, propagation, and dissipation in living cells. Tension TRAAKer is derived from inserting a tension-sensitive nonconductive variant of the mechanosensitive potassium ion channel TRAAK into a self-labelling HaloTag. Increasing membrane tensions effect conformational changes in the TRAAK channel that are optically monitored by a HaloTag-conjugated fluorogenic (environment-sensitive) dye. EGFP incorporation C-terminal to the HaloTag enables unambiguous tension reporting in mobile membranes via dual-color ratiometric imaging that controls for variations in sensor density. Tension TRAAKer reports membrane tension changes rapidly, reversibly, and with spatiotemporal precision--its fluorescence scaling to both stimulus magnitude and area, with consistent effect sizes observed between diverse cell types. It better distinguishes among elevated membrane tensions than do available indirect chemical reporters, with the additional advantage of being readily genetically targetable. We thus expect Tension TRAAKer to be a powerful tool for the study of membrane tension across biological systems and disease states.

molecular biology↗

bioRxiv2026

ATAC-seq and MNase-seq Detect Distinct Modes of Chromatin Accessibility

Chromatin accessibility shapes the ability of transcription factors (TFs) and the transcriptional machinery to engage genomic DNA and therefore plays a central role in gene regulation. Two widely used approaches for profiling chromatin accessibility are micrococcal nuclease (MNase)-seq and assay for transposase-accessible chromatin (ATAC)-seq. ATAC-seq peaks are often thought to be equivalent to nucleosome-depleted regions (NDRs) that are defined by MNase-seq; however, these two measurements have not been systematically compared. Here, we perform a side-by-side comparison of ATAC-seq and MNase-seq in budding yeast and find a large discrepancy between ATAC-seq peaks and MNase-defined NDRs. We show that this discrepancy is not primarily due to the intrinsic differences between MNase and Tn5 enzymatic activity. Instead, ATAC-seq peaks and NDRs capture distinct chromatin states. Specifically, ATAC-seq peaks are enriched at dynamic nucleosomes associated with transcriptional co-regulators, including SAGA and SWI/SNF, whereas ATAC- NDRs mark more static nucleosome-free regions at promoters. Depletion of SWI/SNF reduces ATAC-seq signals without affecting most NDRs. Generation of NDRs and ATAC-seq peaks requires distinct TF properties, and native TFs differ in their ability to produce these two types of open chromatin. Finally, we find that the functional distinctions between ATAC-seq peaks and NDRs are widespread across eukaryotic species, including human cells. Together, our results provide new insights into the biological meaning of chromatin accessibility measured by these two assays.

molecular biology↗