Biology subjects

Shi, J.

Publications and source records attributed to Shi, J..

At least 55 records · Page 3Linked to original sources

bioRxiv2026

Dynamic Consistency Reveals Predictable Genes in Cross-Cell Type Temporal scRNA-Seq Data

Understanding how gene expression evolves over time after trauma is central to modeling immune responses, yet single-cell temporal data remain sparse and heterogeneous across cell types. Using a temporal trauma scRNA-seq dataset, we formulate the task of predicting next-time gene expression from earlier observations under a cross-cell-type generalization setting. We introduce the Dynamic Consistency Index (DCI), which quantifies how consistently a genes temporal trajectory aligns across cell types, serving as a measure of biological regularity and predictability. High-DCI genes exhibit reproducible temporal dynamics and are markedly easier to model. By integrating DCI-based gene selection with a recurrent neural architecture trained under a Gaussian negative log-likelihood objective, we achieve superior accuracy and well-calibrated uncertainty compared to deterministic baselines. Overall, DCI reliably identifies dynamically consistent genes, and uncertainty-aware recurrent modeling provides a robust framework for capturing cross-cell-type gene-expression evolution.

bioinformatics↗

bioRxiv2026

Liver-specific loss of the transcriptional coregulator ARGLU1 protects against diet-induced obesity in mice through decreased lipid absorption

New medications that decrease food intake show promise in addressing the global obesity epidemic; however, additional therapies are needed for patients who are intolerant or unresponsive to these drugs. ARGLU1 is a transcriptional coregulator of several nuclear receptors. Herein, we detail the discovery that mice lacking ARGLU1 in hepatocytes (LKO) are resistant to diet-induced obesity, with no difference in food intake, locomotion, or energy expenditure compared to wildtype mice. Interestingly, LKO mice exhibited decreased CYP8B1, corresponding to fewer 12-hydroxylated bile acids, increasing the relative hydrophobicity of bile which decreases lipid emulsification. Notably, LKO mice have lower plasma and liver cholesterol levels, attributed to impaired lipid absorption. Furthermore, LKO mice showed preferential use of fatty acids as their fuel source. Herein, we establish a mechanistic basis for resistance of ARGLU1 LKO mice to diet-induced obesity, identifying a potential new molecular target for obesity.

physiology↗

bioRxiv2026

Principles of in situ protein sequencing: expansion microscopy-adapted Edman degradation and amino acid recognition

The ability to map protein identity, with resolution sufficient to infer interactions, would support analysis of how proteins work together, or malfunction, in biological processes and diseases. Although several emerging technologies aim towards single-molecule protein sequencing, they require proteins to be removed from the nanoscale spatial context of cells and tissues. Expansion microscopy (ExM) has facilitated a diversity of chemical analyses by isotropically separating molecules throughout a specimen after permeation via a charged hydrogel, followed by gel swelling. Here, we adapt key protein sequencing steps - Edman degradation and amino acid recognition - to the ExM gel context. Using testbed peptides in ExM gels, we show that N-terminal amino acids can be recognized over multiple cycles of in-gel Edman degradation. This principle-oriented study demonstrates sequencing chemistry on defined synthetic constructs, rather than endogenous proteins in biological samples. These results establish principles of in situ protein sequencing and provide a framework for future in situ protein sequencing developments, including the development of higher specificity and affinity amino acid binders.

biochemistry↗

bioRxiv2026

Precision CRISPR annotation of the functional enhancer landscape in primary human T cells

Precise modulation of T cell function through engineering the non-coding genome holds great promise for advancing next-generation immunotherapies. However, robust high-throughput approaches to annotate functional cis-regulatory elements (CRE) in human T cells remain limited. Here, we developed a simple and highly efficient CRISPR interference (CRISPRi) perturbation platform to systematically annotate CREs in human primary T cells. Using this platform, we identified novel CREs controlling PDCD1, HAVCR2, and TBX21 expression. Combinatorial CRE perturbations revealed synergistic CRE pairs that fine-tune PDCD1 and HAVCR2 expression, while Cas9-indel-based mutagenesis pinpointed the critical nucleotides within each enhancer that are essential for their activity. Functional experiments demonstrated that CRE-edited HAVCR2 outperformed conventional total gene knockout in enhancing CAR T cell anti-tumor efficacy. Moreover, CRE editing of PDCD1 and HAVCR2 repressed PD-1 and TIM-3 expression in human tumor-infiltrating lymphocyte CD8 T cells, highlighting regulatory role of these CREs in disease relevant exhausted T cells. Together, this approach offers a compact CRISPRi platform that enables high-throughput dissection of functionally relevant non-coding genomic regions in T cells, providing insights for mechanistic studies and precision genome engineering of advanced cellular therapies.

immunology↗

bioRxiv2026

SS18-SSX co-opts P300 to sustain oncogenic transcription independent of SWI/SNF activity

Synovial sarcoma is driven by the SS18-SSX fusion oncoprotein, which has been assumed to promote tumorigenesis through its incorporation into the SWI/SNF chromatin remodeling complexes. Accordingly, therapeutic efforts have focused on targeting SS18-SSX containing SWI/SNF assemblies, yet these approaches have produced limited clinical benefit. Here, we demonstrate that SS18-SSX sustains oncogenic transcription independent of SWI/SNF activity. Despite efficient degradation and dismantling of SWI/SNF complexes, fusion occupancy at target loci and associated gene expression programs remain largely intact. Instead, we identify the acetyltransferase P300 as an essential co-factor supporting SS18-SSX chromatin binding and transcriptional activation. Targeting P300 displaces the fusion from chromatin, suppresses its transcriptional output, compromising synovial sarcoma viability. Notably, dual PROTAC mediated degradation of P300 and SWI/SNF produces strong synergistic effects, broadly disrupting SS18-SSX localization and function. These findings redefine the mechanistic basis of synovial sarcoma and reveal a mechanistically anchored therapeutic strategy for targeting its core oncogenic driver.

cancer biology↗

bioRxiv2026

ANKS1B in the Nucleus Accumbens controls escalated cocaine self-administration via regulating CBP-FoxO3 complex

The transition from controlled to escalated drug intake is a core feature of cocaine use disorder (CUD), yet the molecular mechanisms underlying this behavioral escalation remain poorly defined. Our prior genome-wide association study (GWAS) identified ANKS1B as a significant shared genetic risk factor for heroin, methamphetamine, and alcohol dependence, suggesting a broad role in addiction vulnerability. However, the specific function of ANKS1B in cocaine addiction and its associated neural mechanisms were unknown. Here, we found that ANKS1B expression level in the Nucleus Accumbens (NAc) was downregulated after extended cocaine use, and manipulating ANKS1B could selectively influence the escalation of cocaine intake and the subsequent cocaine-seeking behavior in the long-access cocaine self-administration rat model. Molecular experiments reveal that ANKS1B interacts with the histone acetyltransferase CBP to control H3K27 acetylation and extended cocaine intake, via epigenetically repressing the transcription factor FoxO3. Overall, these findings suggest that ANKS1B is a crucial factor influencing the escalation of cocaine use. The ANKS1B-CBP-FoxO3 signaling pathway presents a promising target for potential therapeutic interventions for controlling extended cocaine use.

neuroscience↗

bioRxiv2026

A Hybrid Gag Lattice as a Structural Intermediate in HIV-1 Maturation

HIV-1 maturation converts the spherical immature Gag lattice into the conical mature capsid required for infectivity, yet the structural route linking these two lattice endpoints remains unclear. Using single-particle cryo-electron microscopy and cryo-electron tomography on reconstituted assemblies and virus-like particles (VLPs), we identify a distinct hybrid lattice in which the capsid protein (CA) N-terminal domain adopts a mature-like conformation, whereas the CA C-terminal domain-SP1 layer remains immature. This architecture is observed in native, enveloped VLPs produced in human cells, demonstrating formation under physiologically relevant conditions. The hybrid lattice engages three myo-inositol hexakisphosphate (IP6) molecules per CA-SP1 hexamer, underscoring IP6-dependent stabilization; accordingly, excess IP6 enriches the hybrid population. Tomographic mapping shows that hybrid regions coexist with the immature lattice within the same particle and are enriched near lattice discontinuities, consistent with edge-localized remodeling that can accommodate conformational rearrangements and partial disassembly and reassembly. Disrupting a hybrid-specific inter-hexamer contact preserves immature lattice assembly and particle release but abrogates infectivity, compromises core integrity, and prevents mature lattice formation, implicating the hybrid architecture as an on-pathway intermediate. Molecular dynamics simulations further support coordinated rearrangements that bias the system away from the immature configuration toward hybrid and then mature organizations. Together, these results support a maturation model in which localized displacive remodeling and partial disassembly/reassembly act in concert, with IP6 tuning the balance among lattice states. This study provides new insight into HIV-1 maturation and identifies the hybrid lattice as a potential therapeutic target.

biochemistry↗

bioRxiv2026

Functional characterization of a multi-cancer risk locus on chromosome band 2q33.1 near CASP8

Genome-wide association studies (GWAS) of melanoma have identified numerous susceptibility loci. However, causal genes and variants underlying risk have yet to be established for most. It is becoming apparent that many functional variants underlying complex traits act via cis-regulation that may be context-specific, dependent on availability of specific transcription factors/complexes in specific cell types and cell-states. To characterize a risk locus on chromosome band 2q33.1 associated with melanoma, breast cancer, and keratinocyte cancers, we integrated fine-mapping, cell-type specific expression quantitative trait locus (eQTL) analysis, a massively parallel reporter assay, individual luciferase assays, and SNP-based proteomics. Integrated analysis implicates the presence of multiple functional variants lying primarily within a promoter for CASP8. A haplotype containing rs3769823 appeared have the largest effect on expression. Strikingly, both tumor/normal context and this risk-associated haplotype play critical roles in mediating allelic cis-regulatory activity. Quantitative mass spectrometry for rs3769823 identified both E4F1, a transcriptional repressor, and IRF2, a transcriptional activator, as binding preferentially to risk-associated rs3969823-A. The binding of these transcription factors was validated via EMSA, supershift, and chromatin immunoprecipitation (ChIP) assays. The relative levels of E4F1 and IRF2 differ by cell-type and play a role in mediating transcriptional activity in a cell-type specific manner. Our results indicate that the top credible causal set variant rs3769823 likely influences expression of CASP8 and FLACC1 in a cell-type specific manner and may be a relevant functional variant for multiple cancers associated with this locus.

genetics↗

bioRxiv2025

Transcriptomic Profiling Reveals Key Molecular Events and Pathways Driving Acquired Chemotherapy Resistance in Relapsed Acute Myeloid Leukemia

BackgroundAcute myeloid leukemia (AML) is a highly heterogeneous hematologic malignancy. Although induction therapy induces remission in many patients, relapse and acquired chemoresistance remain the major causes of treatment failure. Defining the molecular mechanisms underlying relapse is essential for improving therapeutic strategies. MethodsBone marrow samples from nine AML patients were analyzed, including five newly diagnosed cases and four relapsed cases after chemotherapy. Transcriptome sequencing and integrative bioinformatics analyses were performed, including differential expression analysis, GO/KEGG enrichment, GSEA, and protein-protein interaction (PPI) network analysis, to delineate relapse-associated molecular alterations. ResultsPrincipal component analysis demonstrated clear transcriptional segregation between primary and relapsed AML, indicating extensive molecular reprogramming during relapse. A total of 2,025 differentially expressed genes were identified, enriched in pathways related to epithelial-mesenchymal transition-like programs, leukemia stem cell maintenance, apoptosis evasion, and bone marrow microenvironment remodeling. Marked upregulation of FOXC1, HOXA11/HOXA11-AS, and AXL suggests key roles in sustaining stemness and promoting drug resistance. GO/KEGG analysis revealed coordinated activation of small GTPase, Rho/Ras signaling, ion transport, and epigenetic regulatory pathways, reflecting multilayered adaptive responses. GSEA indicated metabolic-epigenetic reprogramming in relapsed AML, while primary AML showed enrichment of energy metabolism and chromatin assembly pathways. PPI network analysis highlighted a central inflammation-metabolism axis involving TP53, IL6, CXCL8, and CCL2, associated with apoptosis resistance and metabolic adaptation. ConclusionsRelapsed AML is characterized by transcriptional reprogramming, metabolic remodeling, and microenvironment-driven adaptive resistance. Targeting small GTPase signaling, AXL, or IL6-related inflammatory pathways, alone or combined with epigenetic modulators, may offer promising therapeutic strategies to overcome chemoresistance in AML.

cell biology↗

bioRxiv2025

Identifying High-Dimensional Genomic Factors Modulating Biological Networks Across Multi-Omics Data

Biological traits such as genes, metabolites, and microbial taxa interact within complex networks, yet how genomic factors shape these interactions remains poorly understood. Here, we introduce GFBioNet, a computationally efficient method for identifying factors that modulate direct associations between biological traits within network models. Our two-stage strategy first estimates a baseline network using Gaussian graphical models and then tests whether genomic factors modulate specific network edges (trait-trait relationships), enabling scalable analysis of high-dimensional multi-omics data while explicitly controlling the false discovery rate (FDR). Simulations demonstrate reliable FDR control and high statistical power across a broad range of settings. Applied to multiple datasets, GFBioNet reveals host genetic variants influencing oral microbiome relationships, gut microbial taxa modulating metabolite networks in colorectal cancer, and somatic mutations and copy-number alterations reshaping gene expression networks in lung adenocarcinoma. By expanding network analysis to evaluate modifiers of trait-trait relationships, GFBioNet offers a versatile tool for uncovering the genomic architecture of biological networks across multi-omics studies.

genomics↗

bioRxiv2025

Functional characterization of the 9q34.13 locus identifies RAPGEF1 as modulating risk for melanoma and nevi via RAS activation

Genome-wide association studies identified a melanoma- and nevus count-associated locus on chromosome band 9q34.13. Fine-mapping and melanocyte expression data collectively suggest two potential causal genes with opposite association with risk: higher levels of Rap guanine nucleotide exchange factor 1 (RAPGEF1) and lower levels of uridine-cytidine kinase 1 (UCK1). Colocalization analyses and conditional TWAS suggest multiple causal cis-regulatory sequence variants in partial linkage disequilibrium (LD) to each other. Melanocyte capture-HiC and CRISPR-inhibition demonstrated regulatory interactions between fine-mapped variants and the RAPGEF1 and UCK1 promoters. Focusing on RAPGEF1, we demonstrate RAPGEF1 expression promotes melanocyte growth and drives malignant transformation of human immortalized melanocytes. Following treatment with human EGF, RAPGEF1 overexpression activated both RAP1 and RAS. Further, we show RAPGEF1 expression is significantly enriched in melanomas lacking strongly activating RAS-MAPK mutations, suggesting that RAPGEF1 may promote oncogenic RAS-MAPK signaling in melanomas. Furthermore, in these tumors, we provide preliminary evidence to support the prognostic relevance of RAPGEF1 expression in patients lacking RAS or BRAF mutations. Together with other recent studies, these data suggest that germline variation influencing RAS activation may play a key role in nevus development and melanoma risk.

genetics↗

bioRxiv2025

Longitudinal Analysis of Matched Patient Biospecimens Reveals Neural Reprogramming of Cancer-Associated Fibroblasts Following Chemotherapy in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC), the most common subtype of pancreatic cancer, is a deadly disease with a complex tumor microenvironment (TME). How chemotherapy alters the TME, and whether these changes drive chemoresistance, is poorly understood. We examined matched pre- and post-treatment tissue specimens and found near-universal enrichment of axonal guidance genes in cancer-associated fibroblasts (CAFs) after treatment. These CAFs were enriched near sites of perineural invasion, coinciding with regions of increased tumor cell proliferation, and were enriched in tumor areas distant from nerves after chemotherapy. Metastatic recurrence lesions had the highest prevalence of these CAFs versus primary tumors and untreated metastasis. These CAFs showed elevated non-canonical Wnt mediators and axonal guidance genes, which complemented matching cognate binding partners in tumor epithelial cells, suggesting a role in tumor-stroma crosstalk. Our findings implicate fibroblast-derived axonal-guidance genes in promoting PDAC invasion and point to a promising target for this disease. Statement of SignificanceTherapeutic resistance remains a major challenge in pancreatic cancer. Our findings shed light on the changes in the complex tumor microenvironment in response to chemotherapy and identify fibroblasts high in axonal-guidance genes that may drive tumor progression and chemoresistance, thus uncovering a potential avenue for targeting pancreatic cancer.

cancer biology↗

bioRxiv2025

Comparative evaluation of reference genomes and cell-type annotation frameworks for single-nucleus transcriptomic analysis in apple

Single-nucleus RNA sequencing (snRNA-seq) provides a powerful tool to profile cell-type-specific transcriptional programs in woody fruit trees. Yet its application in Malus domestica has been limited by heterogeneous reference genomes and the lack of validated marker genes. Here, we first established a comprehensive set of marker genes corresponding to 29 plant cell types and identified their orthologs in apple. Using these resources, we systematically evaluated six publicly available apple genome assemblies as references for snRNA-seq analysis. Our results demonstrate that reference genome selection has a major impact on mapping performance and downstream cell type annotation, with the GDDH13 assembly consistently producing the highest transcript counts per nucleus and the greatest number of identifiable transcriptional clusters (15 clusters representing 11 distinct cell types across 35,557 high-quality nuclei). To achieve robust cell type annotation, we integrated classical marker gene-based annotation with two cross-species computational frameworks -- Orthologous Marker Gene Groups (OMGs) and XSpeciesSpanner. These three approaches enabled confident identification of 11 major cell types in apple seedlings and provided cross-validation for ambiguous or previously unannotated clusters. Pseudotime analysis reconstructed the developmental trajectories of both procambial and stomatal lineages, revealing their progressive transcriptional transitions and enabling the delineation of key molecular programs underlying vascular differentiation and early guard-cell formation. Together, this work provides a reference-quality single-nucleus transcriptomic atlas for apple and underlined the critical impact of genome choice and multi-method cell-type annotation on single-cell studies in non-model crops.

bioinformatics↗

bioRxiv2025

HalluDesign: Protein Optimization and de novo Design via Iterative Structure Hallucination and Sequence design

Deep learning has revolutionized biomolecular modeling, enabling the prediction of diverse structures with atomic accuracy. However, leveraging the atomic-level precision of the structure prediction model for de novo design remains challenging. Here, we present HalluDesign, a general all-atom framework for protein optimization and de novo design, which iteratively update protein structure and sequence. HalluDesign harnesses the inherent hallucination capabilities of AlphaFold3-style structure prediction models and enables fine-tune free, forward-pass only sequence-structure co-optimization. Structure conditioning at different noise level in the structure prediction stage allows precise control over the sampling space, facilitating tasks from local and global protein optimization to de novo design. We demonstrate the versatility of this framework by optimizing suboptimal structures, rescuing previously unsuccessful designs, designing new biomolecular interactions and generating new protein structures from scratch. Experimental characterization of a diverse set of proteins--including protein binders for small molecules, a metal ion and proteins; antibody design of phosphorylation-specific peptide; and monomeric proteins--revealed high design success rates and excellent structural accuracy. Together, our comprehensive computational and experimental results highlight the broad utility of this framework. We anticipate that HalluDesign will further unlock the modeling and design potential of AlphaFold3-like models, enabling the robust creation of complex biomolecules for a wide range of biotechnological applications.

bioinformatics↗

bioRxiv2025

A multi-tissue atlas of genetic regulatory effects in sheep

Characterizing the impact of genomic variants on genome function and ultimately complex traits in livestock is essential for the development of sustainable precision agriculture and comparative genomics. Here, as part of the Farm animal Genotype-Tissue Expression (FarmGTEx) project, we present the pilot phase of the SheepGTEx resource through analyzing 6,761 RNA-sequencing samples of 51 primary tissues in a multi-breed population of sheep. We identify millions of regulatory variants associated with seven types of molecular phenotypes, and fine-map 322,468 primary and 114,017 non-primary effects, revealing a high degree of regulatory allelic heterogeneity. We systematically characterize the pleiotropic effects of these variants on molecular phenotypes, assess their context-specific regulatory patterns across tissues, breeds, sexes, and developmental stages, as well as explore their evolutionary constraints across mammals. Finally, we demonstrate the substantial potential of the SheepGTEx resource (https://sheepgtex.farmgtex.org), by providing examples of regulatory mechanisms underpinning 34 complex traits, population divergence between European and Asian breeds, and adaptive evolution in sheep over the past ten millennia.

genomics↗

bioRxiv2025

Molecular mechanism of the key dormancy regulator DosR-dependent transcription activation in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is the etiological agent responsible for the worldwide disease Tuberculosis. DosR is recognized as the key dormancy regulator of Mtb due to its global transcriptional regulation in intracellular adaptation and long-term persistence prevailing in latent infection. However, the molecular mechanism regarding DosR-dependent transcription activation remains inadequately defined. Here, we successfully determined the cryo-EM structure of an intact DosR-dependent transcriptional activation complex (DosR-TAC), comprising Mtb RNA polymerase (RNAP), DosR, and the hypoxic promoter DNA. In DosR-TAC, two DosR monomers symmetrically dimerize through their N-terminal receiver domains (DosR_RECs) and the distinct 10 helices of the C-terminal DNA-binding domains (DosR_DBDs). Unlike its inhibitory configuration, the DosR dimer undergoes significant conformational changes within the original linker, isomerizing DosR_REC into a canonical ({beta})5 fold. This extended linker affords the DosR_DBDs and DosRI_REC to contact promoter DNA, while concurrently interacting with the conserved domains of RNAP ({sigma}AR4, CTD, and NTD). Additionally, RT-qPCR and growth curve assays of the wild-type and DosR mutant strains, also unravel the physiological importance of this divergent linker. These findings, together with prior hypotheses regarding DosR, support an allosteric activation-recruitment model for DosR. Altogether, these results highlight the molecular mechanism of DosR-dependent transcription activation critical for dormancy survival of Mtb, and offer potential targets for developing anti-dormancy strategies against persistent tuberculosis.

microbiology↗

bioRxiv2025

Development of a High-Throughput Screening Method for Anti-NNV Drugs

Nervous necrosis virus (NNV) is an infectious pathogen, characterized by rapid infection and high mortality, commonly encountered in the aquaculture industry. Therefore, there is an urgent need to develop effective antiviral drugs against NNV, which is crucial for targeted treatment strategies, pathogen control, and loss reduction. Screening of effective antiviral active compounds, namely drug precursors, is key to developing highly efficient drugs against NNV. Reverse transcription-quantitative real-time PCR (RT-qPCR) is widely applied to screen and evaluate various substances to prevent and control virus infection. However, RT-qPCR is a cumbersome procedure and not suitable for high-throughput rapid screening. The aptamer TNA1c, which specifically binds to NNV-infected cells, was used to construct a target-driven activatable aptamer probe (TAA). Then, the TAA probe was applied to establish a high-throughput screening (TAA-HTS) method for efficient evaluation of substances against NNV infection. TAA-HTS technology achieved rapid, sensitive, and specific screening of bioactive substances with significant anti-NNV effects. As compared to commonly used analytical methods, such as RT-qPCR, TAA-HTS has the advantages of easy operation and high sensitivity and specificity. The findings of this study provide data support and a theoretical basis for the development of effective antivirus preparations. IMPORTANCEIn this study, a target-driven activatable aptamer probe was employed to establish a high-throughput screening method for anti-NNV drugs. This method enables rapid, sensitive, and specific screening of anti-NNV compounds while reducing background interference. Although RT-qPCR is currently the most widely used technique for antiviral drug screening and validation, the screening approach developed in this study significantly shortens screening time while achieving results consistent with those of RT-qPCR. This demonstrates that the screening method presented here has the potential to become a universal strategy for future drug screening against viral diseases. Moreover, this study not only provides a novel technical solution for NNV control but also demonstrates the potential of aptamer-based probes in high-throughput antiviral screening, serving as a model for combating viral pathogens in aquaculture and other fields.

pathology↗

bioRxiv2025

De novo design of phospho-tyrosine peptide binders

Phosphorylation on tyrosine is a key step in many signaling pathways. Despite recent progress in de novo design of protein binders, there are no current methods for designing binders that recognize phosphorylated proteins and peptides; this is a challenging problem as phosphate groups are highly charged, and phosphorylation often occurs within unstructured regions. Here we introduce RoseTTAFold Diffusion 2 for Molecular Interfaces (RFD2-MI), a deep generative framework for the design of binders for protein, ligand, and covalently modified protein targets. We demonstrate the power and versatility of this method by designing binders for four critical phosphotyrosine sites on three clinically relevant targets: Cluster of Differentiation 3 (CD3{varepsilon}), Epidermal Growth Factor Receptor (EGFR), Insulin Receptor (INSR) and Signal Transducer and Activator of Transcription 5 (STAT5). Experimental characterization shows that the designs bind their phosphotyrosine containing targets with affinities comparable to native binding sites and have negligible binding to non-phosphorylated targets or phosphopeptides with different sequences. X-ray crystal structures of generated binders to CD3{varepsilon} and EGFR are very close to the design models, demonstrating the accuracy of the design approach. A designed binder to an EGFR intracellular region phosphorylated upon EGF activation co-localizes with the receptor following EGF stimulation in single-particle tracking (SPT) experiments, demonstrating pY specific recognition in living cells. RFD2-MI provides a generalizable all-atom diffusion framework for probing and modulating phosphorylation-dependent signaling, and more generally, for developing research tools and targeted therapeutics against post-translationally modified proteins.

bioengineering↗