Biology subjects

Zhang, L.

Publications and source records attributed to Zhang, L..

At least 811 records · Page 45Linked to original sources

bioRxiv2023

A Computational Approach for the Identification of Novel L1 Transcriptional Regulators

Long interspersed element 1 (L1) are a family of autonomous, actively mobile transposons that occupy [~]17% of the human genome. A number of pleiotropic effects induced by L1 (promoting genome instability, inflammation, or cellular senescence) have been observed, and L1s contributions to aging and aging diseases is an area of active research. However, because of the cell type-specific nature of transposon control, the catalogue of L1 regulators remains incomplete. Here, we employ an eQTL approach leveraging transcriptomic and genomic data from the GEUVADIS and 1000Genomes projects to computationally identify new candidate regulators of L1 RNA levels in lymphoblastoid cell lines. To cement the role of candidate genes in L1 regulation, we experimentally modulate the levels of top candidates in vitro, including IL16, STARD5, HSDB17B12, and RNF5, and assess changes in TE family expression by Gene Set Enrichment Analysis (GSEA). Remarkably, we observe subtle but widespread upregulation of TE family expression following IL16 and STARD5 overexpression. Moreover, a short-term 24-hour exposure to recombinant human IL16 was sufficient to transiently induce subtle, but widespread, upregulation of L1 subfamilies. Finally, we find that many L1 expression-associated genetic variants are co-associated with aging traits across genome-wide association study databases. Our results expand the catalogue of genes implicated in L1 RNA control and further suggest that L1-derived RNA contributes to aging processes. Given the ever-increasing availability of paired genomic and transcriptomic data, we anticipate this new approach to be a starting point for more comprehensive computational scans for transposon transcriptional regulators.

bioinformatics↗

bioRxiv2023

Herpes zoster mRNA vaccine induces superior vaccine immunity over licensed vaccine in mice and rhesus macaques

Herpes zoster remains an important global health issue and mainly occurs in aged and immunocompromised individuals with an early exposure history to Varicella Zoster Virus (VZV). Although the licensed vaccine Shingrix has a remarkably high efficacy, undesired reactogenicity and increasing global demand causing vaccine shortage urged the development of improved or novel VZV vaccines. In this study, we developed a novel VZV mRNA vaccine candidate (named as ZOSAL) containing sequence-optimized mRNAs encoding full-length glycoprotein E encapsulated in an ionizable lipid nanoparticle. In mice and rhesus macaques, ZOSAL demonstrated superior immunogenicity and safety in multiple aspects over Shingrix, especially in the induction of strong T cell immunity. Transcriptomic analysis revealed that both ZOSAL and Shingrix could robustly activate innate immune compartments, especially Type-I IFN signaling and antigen processing/presentation. Multivariate correlation analysis further identified several early factors of innate compartments that can predict the magnitude of T cell responses, which further increased our understanding of the mode of action of two different VZV vaccine modalities. Collectively, our data demonstrated the superiority of VZV mRNA vaccine over licensed subunit vaccine. The mRNA platform therefore holds prospects for further investigations in next-generation VZV vaccine development.

immunology↗

bioRxiv2023

CDK19 and CDK8 Mediator kinases drive androgen-independent in vivo growth of castration-resistant prostate cancer

Castration-resistant prostate cancer (CRPC) remains incurable due to its high plasticity. We found that Mediator kinases CDK8 and CDK19, pleiotropic regulators of transcriptional reprogramming, are differentially affected by androgen, which downregulates CDK8 and upregulates CDK19. Accordingly, expression of CDK8 decreases while CDK19 increases during prostate carcinogenesis, but both CDK19 and CDK8 are upregulated in metastatic CRPC. Genetic inactivation of CDK8 and CDK19 suppresses CRPC tumor growth in castrated male mice and renders CRPC responsive to androgen deprivation. Restoration of active CDK19 or CDK8 kinases reverses this phenotype, indicating that CRPC becomes dependent on Mediator kinase activity for in vivo growth under the conditions of androgen deprivation. Selective CDK8/19 inhibitors suppress androgen-independent growth of cell line-based and patient-derived CRPC xenografts, whereas prolonged inhibitor treatment induces tumor regression and even leads to cures. Mediator kinase activity was found to affect tumor and stromal gene expression preferentially in castrated mice, orchestrating castration-induced transcriptional reprogramming. These results warrant the exploration of Mediator kinase inhibitors for CRPC therapy.

cancer biology↗

bioRxiv2023

Nutrient levels control root growth responses to high ambient temperature in plants

Global warming will lead to significantly increased temperatures on earth. Plants respond to high ambient temperature with altered developmental and growth programs, termed thermomorphogenesis. Here we show that thermomorphogenesis is conserved in Arabidopsis, soybean, and rice and that it is linked to a decrease in the levels of the two macronutrients nitrogen and phosphorus. We also find that low external levels of these nutrients abolish root growth responses to high ambient temperature. We show that in Arabidopsis, this is due to the function of the transcription factor ELONGATED HYPOCOTYL 5 (HY5) and its transcriptional regulation of the transceptor NITRATE TRANSPORTER 1.1 (NRT1.1). Soybean and Rice homologs of these genes are expressed consistently with a conserved role in regulating temperature responses in a nitrogen and phosphorus level dependent manner. Overall, our data show that root thermomorphogenesis is a conserved feature in species of the two major groups of angiosperms, monocots and dicots, that it leads to a reduction of nutrient levels in the plant, and that it is dependent on sufficient environmental nutrient supply, a regulatory process mediated by the HY5-NRT1.1 module.

plant biology↗

bioRxiv2023

libpspm: A feature-rich numerical package for solving physiologically structured population models

For a vast majority of organisms, life-history processes depend on their physiological state, such as body size, as well as on their environment. Size-structured population models, or more generally, physiologically structured population models (PSPMs), have emerged as powerful tools for modelling the population dynamics of organisms, as they account for the dependences of growth, mortality, and fecundity rates on an organisms physiological state and capture feedbacks between a populations structure and its environment, including all types of density regulation. However, despite their widespread appeal across biological disciplines, few numerical packages exist for solving PSPMs in an accessible and computationally efficient way. The main reason for this is that PSPMs typically involve solving partial differential equations (PDEs), and no single numerical method works universally best, or even at all, for all PDEs. Here, we present libpspm, a general-purpose numerical library for solving user-defined PSPMs. libpspm provides eight different methods for solving the PDEs underlying PSPMs, including four semi-implicit solvers that can be used for solving stiff problems. Users can choose the desired method without changing the code specifying the PSPM. libpspm allows for predicting the dynamics of multiple physiologically structured or unstructured species, each of which can have its own distinct set of physiological states and demographic functions. By separating model definition from model solution, libpspm can make PSPM-based modelling accessible to non-specialists and thus promote the widespread adoption of PSPMs.

ecology↗

bioRxiv2023

Optogenetic strategies for optimizing the performance of biosensors of membrane phospholipids in live cells

High-performance biosensors play a crucial role in elucidating the intricate spatiotemporal regulatory roles and dynamics of membrane lipids. However, enhancing their sensitivity and substrate-detecting capabilities remains a significant challenge. Here, we presented optogenetic-based strategies to optimize phospholipid biosensors. These strategies involved pre-sequestering unbound probes in the cell nucleus to minimize background signals in the cytoplasm. These stored probes could be released from the nucleus in response to blue light according to experimental requirements. Furthermore, we employed optically-controlled phase separation to generate punctate probes that amplified signals and facilitated the visualization of phospholipids in cells. These improved phospholipid biosensors hold great potential for enhancing our understanding of the spatiotemporal dynamics and regulatory roles of membrane lipids in live cells and this methodological insights might be valuable for developing other high-performance biosensors.

cell biology↗

bioRxiv2023

AnglesRefine: refinement of 3D protein structures using Transformer based on torsion angles

MotivationThe goal of protein structure refinement is to enhance the precision of predicted protein models, particularly at the residue level of the local structure. Existing refinement approaches primarily rely on physics, whereas molecular simulation methods are resource-intensive and time-consuming. In this study, we employ deep learning methods to extract structural constraints from protein structure residues to assist in protein structure refinement. We introduce a novel method, AnglesRefine, which focuses on a proteins secondary structure and employs a transformer model to refine various protein structure angles (psi, phi, omega, CA_C_N_angle, C_N_CA_angle, N_CA_C_angle), ultimately generating a superior protein model based on the refined angles. ResultsWe evaluate our approach against other cutting-edge protein structure refinement methods using the CASP11-14 and CASP15 datasets. Experimental outcomes indicate that our method generally surpasses other techniques on the CASP11-14 test dataset, while performing comparably or marginally better on the CASP15 test dataset. Our method consistently demonstrates the least likelihood of model quality degradation, e.g., the degradation percentage of our method is less than 10%, while other methods are about 50%. Furthermore, as our approach eliminates the need for conformational search and sampling, it significantly reduces computational time compared to existing protein structure refinement methods. Availabilityhttps://github.com/Cao-Labs/AnglesRefine.git Contactcaora@plu.edu CCS CONCEPTS*Computing methodologies [->] Machine learning. ACM Reference FormatLei Zhang, Junyong Zhu, Sheng Wang, Jie Hou, Dong Si, and Renzhi Cao. 2023. AnglesRefine: refinement of 3D protein structures using Transformer based on torsion angles. In Proceedings of 22nd International Workshop on Data Mining in Bioinformatics (BIOKDD 2023) (BIOKDD 2023). ACM, New York, NY, USA, 10 pages. https://doi.org/XXXXXXX.XXXXXXX

bioinformatics↗

bioRxiv2023

Crystal structure and biochemical activity of the macro domain from rubella virus p150

Rubella virus remains a global health threat. Rubella infections during pregnancy can cause serious congenital pathology and no antiviral treatments are available. Rubella virus encodes a nonstructural polyprotein with RNA polymerase, methyltransferase, and papain-like cysteine protease activities, along with a putative macro domain of unknown function. Macro domains bind ADP-ribose adducts, a post-translational modification that plays a key role in host-virus conflicts. Some macro domains can also remove the mono-ADP-ribose adduct or degrade poly-ADP-ribose chains. Here, we report high-resolution crystal structures of the macro domain from rubella virus nonstructural protein p150, with and without ADP-ribose bound. The overall fold is most similar to macroD-type macro domains from various nonviral species. The specific composition and structure of the residues that poised for catalysis or coordinate ADP-ribose in the rubella virus macro domain are most similar to those of macro domains from alphaviruses. Isothermal calorimetry and enzymatic assays show that the rubella virus macro domain binds ADP-ribose in solution and has mono-ADP-ribosylhydrolase (de-MARylation) activity. IMPORTANCEOur work demonstrates that, like alpha- and coronaviruses, rubiviruses encode a mono-ADP-ribosylhydrolase with a structurally conserved macro domain fold to counteract MARylation by PARPs in the host innate immune response. Our structural data will guide future efforts to develop novel antiviral therapeutics against rubella or infections with related viruses.

biochemistry↗

bioRxiv2023

Discovery of fibrotic intervertebral disc in mice

Intervertebral discs are crucial to spine flexibility and stability during locomotion, but the mechanism of their mechanoadaptation to stress remains inadequately elucidated. We discovered spontaneous collagenesis in discs at the tail base in mice occurring from 14 days post-natal development. These discs termed as type C, differ from typical discs by exhibiting intensive collagen II and reduced aggrecan deposition contributing to the stiffening of the extracellular matrix (ECM), without typical degenerative markers. The reinforced mechanical properties enable type C discs to withstand high mechanical stress during flexion. Further analysis revealed that type C discs experience a turnover in cell composition, with the emergence of Procr+ progenitor cells and depletion of notochord cells. We demonstrated the essential role of mechanical stress in type C disc formation, suggesting a mechanoadaptive process rather than a pathological condition. Mechanistically, we identified TRPV4, as pivotal factors in collagen II synthesis in type C discs, highlighting the role of mechanotransduction in this adaptation. Our findings introduce a novel mechanoadaptive process of intervertebral disc by orchestrating collagenesis, advancing understanding of diverse disc functions in spine development and homeostasis, thereby providing insights for leveraging this mechanism to address spinal disorders.

pathology↗

bioRxiv2023

Extracting herbicide and antibiotic natural products from a plant-microbe interaction system

Plants and their associated microbes live in complicated, changeable, and unpredictable environments. They usually interact with each other in many ways by proceeding in multi-dimensional, multi-scale and multi-level coupling manners, leading to challenges of the co-existence of randomness and determinism, or continuity and discreteness. Gaining a deeper understanding of these diverse interaction mechanisms can facilitate the development of new data mining theories and methods for complex systems, new coupled modelling for the system with different spatiotemporal scales and functional properties, or even universal theory of information and information interactions. In this study, we use a "close-loop" model to present a plant-microbe interaction system and describe the probable functions from the microbial natural products. Specifically, we report a rhizosphere species, Streptomyces ginsengnesis G7, which produces polyketide lydicamycins and other active metabolites. Interestingly, these distinct molecules have the potential to function both as antibiotics and herbicides for crop protection. Detailed laboratory experiments combined with comprehensive bioinformatics analysis allow us to rationalise a model for this specific plant-microbe interaction process. Our work reveals the benefits of exploring otherwise neglectable resources for the identification of novel functional molecules and provides a good reference to better understand the system biology in the complex ecosystems.

cell biology↗

bioRxiv2023

BL-Hi-C reveals the 3D genome structure of Brassica crops with high efficiency and sensitivity

High-throughput chromatin conformation capture (Hi-C) technologies can be used to investigate the three-dimensional genomic structure of plants. However, complex protocol and high background noise in Hi-C hinder its practical application in plant 3D genomics. Here, we took the approach of modified Bridge Linker Hi-C technology (BL-Hi-C) to explore plant 3D landscape. We modified the BL-Hi-C method by simplifing nuclei extraction step. By using Brassica rapa and Brassica oleracea, BL-Hi-C showed higher signal value and lower background noise than Hi-C. The high sensitivity of BL-Hi-C was further demonstrated by its capacity to identify gene loops involving BrFLC1, BrFLC2 and BrFLC3 which were undetectable in Hi-C. BL-Hi-C also showed promising performance with input as low as 100 mg leaf tissue. By analyzing of the generated data from BL-Hi-C, we found that the simulated 3D genome structure of B. rapa leaf cells was Bouquet configuration. Our results showed that the modified BL-Hi-C is a powerful tool for the investigation of plants genomic organization, gene regulation, and genome assembly. IN A NUTSHELL Background3D genome structure play a critical role in regulating spatiotemporal gene expression. However, there is a lack of simple, efficient and sensitive Hi-C technique in plants. QuestionHow to study plant 3D genomics more simple and efficientHow to detect plant chromatin loops more sensitive? FindingsWe showed that BL-Hi-C is more simple, efficient and sensitive than coventional Hi-C by using Brassica rapa and Brassica oleracea. Furthermore, BL-Hi-C demonstrated its high sensitivity by detecting gene loops involving BrFLC1, BrFLC2, and BrFLC3 which could not be detected by Hi-C. In addition, BL-Hi-C demonstrated promising performance with inputs as low as 100 mg leaf tissue. By analyzing BL-Hi-C data, we found that the simulated 3D genome structure of B. rapa leaf cells was Bouquet configuration. Next stepsHow chromatin loops are formed and regulated gene expression are key questions to be answered in plants. Our dataset of BL-Hi-C will enable future investigations to improve our understanding of chromatin loops.

genomics↗

bioRxiv2023

Structure, Dynamics and Free Energy Studies on the Effect of Spot Mutations on SARS-CoV-2 Spike Protein Binding with ACE2 Receptor

The ongoing COVID-19 pandemic continues to infect people worldwide, and the virus continues to evolve in significant ways which can pose challenges to the efficiency of available vaccines and therapeutic drugs and cause future pandemic. Therefore, it is important to investigate the binding and interaction of ACE2 with different RBD variants. A comparative study using all-atom MD simulations was conducted on ACE2 binding with 8 different RBD variants, including N501Y, E484K, P479S, T478I, S477N, N439K, K417N and N501Y-E484K-K417N on RBD. Based on the RMSD, RMSF, and DSSP results, the overall binding of RBD variants with ACE2 is stable, and the secondary structures of RBD and ACE2 are consistent after the spot mutation. Besides that, a similar buried surface area, a consistent binding interface and a similar amount of hydrogen bonds formed between RBD with ACE2 although the exact residue pairs on the binding interface were modified. The change of binding free energy from spot mutation was predicted using the free energy perturbation (FEP) method. It is found that N501Y, N439K, and K417N can strengthen the binding of RBD with ACE2, while E484K and P479S weaken the binding, and S477N and T478I have negligible effect on the binding. Spot mutations modified the dynamic correlation of residues in RBD based on the dihedral angle covariance matrix calculation. Doing dynamic network analysis, a common intrinsic network community extending from the tail of RBD to central, then to the binding interface region was found, which could communicate the dynamics in the binding interface region to the tail thus to the other sections of S protein. The result can supply unique methodology and molecular insight on studying the molecular structure and dynamics of possible future pandemics and design novel drugs.

biophysics↗

bioRxiv2023

The super-pangenome of Populus unveil genomic facets for adaptation and diversification in widespread forest trees

Understanding the underlying mechanisms between genome evolution and phenotypic and adaptive innovations is a key goal of evolutionary studies. Poplars are the worlds most widely distributed and cultivated trees, with extensive phenotypic diversity and environmental adaptability. Here we report a genus-level super-pangenome of 19 Populus genomes. After integrating pan-genomes with transcriptomes, methylomes and chromatin accessibility mapping, we reveal that the evolutionary fate of pan-genes and duplicated genes are largely associated with local genomic landscapes of regulatory and epigenetic architectures. Further comparative genomic analyses enabled to identify 142,202 structural variations (SVs) across species, which overlap with substantial genes and play key roles in both phenotypic and adaptive divergence. We experimentally validated a [~]180 bp presence/absence variant located in the promoter of the CUC2 gene, which contributed critically to leaf serration divergence between species. Together, this first super-pangenome resource in forest trees will not only accelerate molecular functional studies and genetic breeding of this globally important tree genus, but also lays a foundation for our understanding of tree biology.

evolutionary biology↗

bioRxiv2023

Regulation of BCR-mediated Ca2+ mobilization by MIZ1-TIMBIM4 safeguards IgG1+ GC B cell positive selection

The transition from IgM to affinity-matured IgG antibodies is vital for effective humoral immunity. This is facilitated by germinal centers (GCs) through affinity maturation and preferential accumulation of IgG+ B cells over IgM+ B cells. However, it is not known whether the positive selection of the different immunoglobulin isotypes within GCs varies in its dependency on specific transcriptional mechanisms. Here, we identified IgG1+ GC B cell transcription factor dependency using CRISPR-Cas9 and conditional mouse genetics. We found that MIZ1 was specifically required for IgG1+ GC B cell survival during positive selection, whereas IgM+ GC B cells were largely independent. Mechanistically, MIZ1 induced TMBIM4, an ancestral anti-apoptotic protein that regulated inositol trisphosphate receptor mediated Ca2+ mobilization downstream of IgG1. The MIZ1-TMBIM4 axis prevented mitochondrial dysfunction-induced IgG1+ GC cell death caused by excessive Ca2+ accumulation. This study uncovers a unique immunoglobulin isotype-specific dependency, on a hitherto unidentified mechanism in GC positive selection.

immunology↗

bioRxiv2023

Persistent effects of intramammary ceftiofur treatment on the gut microbiome and antibiotic resistance in dairy cattle

Intramammary (IMM) ceftiofur treatment is commonly used in dairy farms to prevent mastitis, though its impact on the cattle gut microbiome and selection of antibiotic-resistant bacteria has not been elucidated. Herein, we enrolled 40 healthy dairy cows after lactation: 20 were treated with IMM ceftiofur (Spectramast(R)DC) and a non-antibiotic internal teat sealant (bismuth subnitrate) and 20 (controls) received only bismuth subnitrate. Fecal samples were collected before (day -1) and after treatment (weeks 1, 2, 3, 5, 7, and 9) for bacterial quantification and metagenomic next-generation sequencing. Overall, 90% and 24% of the 278 samples had Gram-negative bacteria with resistance to ampicillin and ceftiofur, respectively. Most of the cows treated with ceftiofur did not have an increase in the number of resistant bacteria; however, a subset (25%) shed higher levels of ceftiofur-resistant bacteria for up to 2 weeks post-treatment. At week 5, the antibiotic-treated cows had lower microbiome abundance and richness, whereas a greater abundance of genes encoding extended-spectrum {beta}-lactamases (ESBLs), CfxA, ACI-1, and CMY, was observed at weeks 1, 5 and 9. Moreover, the contig and network analyses detected associations between {beta}-lactam resistance genes and phages, mobile genetic elements, and specific genera. Commensal bacterial populations belonging to Bacteroidetes most often possessed ESBL genes followed by members of Enterobacteriaceae. This study highlights variable, persistent effects of IMM ceftiofur treatment on the gut microbiome and resistome in dairy cattle. Antibiotic-treated cattle had an increased abundance of specific taxa and genes encoding ESBL production that persisted for 9 weeks, while fecal shedding of ESBL-producing Enterobacteriaceae varied across animals. Together, these findings highlight the need for additional studies that identify factors linked to shedding levels and the dissemination and persistence of resistance determinants on dairy farms in different geographic locations.

microbiology↗

bioRxiv2023

Ebola Virus VP35 Interacts Non-Covalently with Ubiquitin Chains to Promote Viral Replication Creating New Therapeutic Opportunities

Ebolavirus (EBOV) belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans. EBOV replication requires the activity of the viral polymerase complex, which includes the co-factor and Interferon antagonist VP35. We previously showed that the covalent ubiquitination of VP35 promotes virus replication by regulating interactions with the polymerase complex. In addition, VP35 can also interact non-covalently with ubiquitin (Ub); however, the function of this interaction is unknown. Here, we report that VP35 interacts with free (unanchored) K63-linked polyUb chains. Ectopic expression of Isopeptidase T (USP5), which is known to degrade unanchored polyUb chains, reduced VP35 association with Ub and correlated with diminished polymerase activity in a minigenome assay. Using computational methods, we modeled the VP35-Ub non-covalent interacting complex, identified the VP35-Ub interacting surface and tested mutations to validate the interface. Docking simulations identified chemical compounds that can block VP35-Ub interactions leading to reduced viral polymerase activity that correlated with reduced replication of infectious EBOV. In conclusion, we identified a novel role of unanchored polyUb in regulating Ebola virus polymerase function and discovered compounds that have promising anti-Ebola virus activity. Significance StatementEbola virus infection can result in high mortality rates with extreme risk of person-to-person transmission. Sporadic outbreaks in Africa have resulted in thousands of fatal cases, highlighting that there is still insufficient knowledge to develop effective antiviral therapies. Like other viruses, Ebola utilizes the host machinery to replicate. Understanding how viral and host proteins interact can help identifying targets for the rational design of antiviral drugs. Here, we identified interactions between the cellular ubiquitin machinery and the Ebola virus polymerase cofactor protein VP35, which are important for efficient virus replication. We developed an approach to identify and block these virus-host interactions using small chemical compounds, which provides a useful tool to study functional molecular mechanisms and at the same time a potential approach to antiviral therapies.

immunology↗

bioRxiv2023

MKP1 promotes nonalcoholic steatohepatitis by suppressing AMPK activity through LKB1 nuclear retention

Nonalcoholic steatohepatitis (NASH) is triggered by hepatocyte death through activation of caspase 6, as a result of decreased adenosine monophosphate (AMP)-activated protein kinase-alpha (AMPK) activity. Increased hepatocellular death promotes inflammation which drives hepatic fibrosis. We show that the nuclear-localized mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP1) is upregulated in NASH patients and in NASH diet fed mice. The focus of this work was to investigate whether and how MKP1 is involved in the development of NASH. Under NASH conditions increased oxidative stress, induces MKP1 expression leading to nuclear p38 MAPK dephosphorylation and decreased liver kinase B1 (LKB1) phosphorylation at a site required to promote LKB1 nuclear exit. Hepatic deletion of MKP1 in NASH diet fed mice released nuclear LKB1 into the cytoplasm to activate AMPK and prevent hepatocellular death, inflammation and NASH. Hence, nuclear-localized MKP1-p38 MAPK-LKB1 signaling is required to suppress AMPK which triggers hepatocyte death and the development of NASH.

physiology↗

bioRxiv2023

Improving the Circularization Efficiency, Stability and Translatability of Circular RNA by circDesign

Synthetic circular RNA (circRNA) has emerged as a promising platform for vaccine and therapeutic development, featuring its uniqueness in a closed-loop structure, cap-independent translation mechanism, and prolonged expression. However, the rational design of a circRNA sequence to jointly improve its stability and protein coding potential remains challenging. In this study, we present circDesign, an efficient algorithm to achieve the optimal design of circRNA by ensuring optimized folding of each segment, which leads to enhanced circularization efficiency, stability, and translatability. Using rabies virus glycoprotein (RABV-G) as the model antigen, we demonstrated that circDesign-generated circRNAs exhibited higher stability and protein translation efficiency in vitro and in vivo compared to other codon adaptation index (CAI)-optimized sequences, thus leading to enhanced in vivo immunogenicity. Ribosome and polysome profiling further revealed that an intact internal ribosome entry site (IRES) structure is critical for efficient translation. By intentionally disrupting the IRES motifs, we observed that the resulting circRNA sequences had lower translatability compared to circDesign-generated sequences. Taken together, our circular RNA design algorithm provides a general strategy to leverage the capability of circRNA as next-generation vaccines or therapeutics.

bioengineering↗