Biology subjects

Zhang, H.

Publications and source records attributed to Zhang, H..

At least 523 records · Page 29Linked to original sources

bioRxiv2024

Upstream open reading frames buffer translational variability during Drosophila evolution and development

Protein abundance tends to be more evolutionarily conserved than mRNA levels both within and between species, yet the mechanisms underlying this phenomenon remain largely unknown. Upstream open reading frames (uORFs) are widespread cis-regulatory elements in eukaryotic genomes that regulate translation, but it remains unclear whether and how uORFs contribute to stabilizing protein levels. In this study, we performed ribosome translation simulations on mRNA to quantitatively assess the extent to which uORF translation influences the translational variability of downstream coding sequences (CDSs) across varying contexts. Our simulations revealed that uORF translation dampens CDS translational variability, with buffering capacity increasing in proportion to uORF efficiency, length, and number. We then compared the translatomes at different developmental stages of two Drosophila species, demonstrating that uORFs buffer mRNA translation fluctuations during both evolution and development. Experimentally, deleting a uORF in the bicoid (bcd) gene--a prominent example of translational buffering--resulted in extensive changes in gene expression and phenotypes in Drosophila melanogaster. Additionally, we observed uORF-mediated buffering between primates and within human populations. Together, our results reveal a novel regulatory mechanism by which uORFs stabilize gene translation during development and across evolutionary time.

evolutionary biology↗

bioRxiv2024

Curcumin attenuates microglia-mediated chronic neuropathic pain through CDK5 /p35 signaling pathway

Curcumin is a phenolic compound derived from turmeric, one of the main ingredients of curry powder, which is widely used for its antioxidant, anti-inflammatory and immunomodulatory effects. Curcumin has been reported to help relieve pain, such as neuropathic pain caused by injury or disease, but the specific mechanism of its antinociceptive effect on pathological pain is unclear. Cyclin-dependent kinase 5 (Cdk5) is a key control point for the release of neurotransmitters from presynaptic vesicles. Cdk5 and its activator p35 couple to regulate key signaling cascades, thereby participating in the pain process. In this study, we established a NP model by chronic constriction injury (CCI) of the bilateral sciatic nerve in rats and evaluated behavioral hyperalgesia using mechanical and hot and cold tests. Protein expression and distribution were evaluated using western blotting and immunofluorescence. The results showed that Iba-1 and Cdk5/p35 were co-localized in the dorsal horn and dorsal root ganglia, respectively. After CCI, the expression of Cdk5 and p35 was upregulated in the dorsal horn and dorsal root ganglia, while intraperitoneal injection of curcumin significantly reversed the activation of Cdk5/p35 protein and alleviated the hyperalgesia in rats. In addition, the injection of curcumin reduced the co-localization expression of Iba-1 and Cdk5/p35, indicating that curcumin inhibited the activation of Cdk5/p35 protein in the dorsal horn and dorsal root ganglia, thereby affecting the activation of microglia, thereby having a destructive effect on the neuronal cell plasticity and synaptic structure remodeling in the development of NP. Our study provides new evidence that Cdk5/p35 in the dorsal horn and dorsal root ganglia is related to the occurrence of NP, introduces microglia as the basis for the long-term maintenance of NP, and provides insights into the molecular mechanisms involved in the analgesic effect of curcumin. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/623498v1_ufig1.gif" ALT="Figure 1"> View larger version (80K): org.highwire.dtl.DTLVardef@125fc20org.highwire.dtl.DTLVardef@cba7d3org.highwire.dtl.DTLVardef@772291org.highwire.dtl.DTLVardef@17e87e3_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Structural basis of augment taurine uptake by taurine transporter alleviating cellular senescence

Taurine, a sulfur-containing amino acid, is critical for diverse physiological processes including liver function, cardiovascular health, and neurological development. Its deficiency has been linked to liver damage, cardiomyopathy, retinal degeneration, and accelerated aging. Despite its importance in metabolic regulation and disease prevention, the structural basis for taurines cellular uptake and its potential therapeutic applications have remained elusive. Here, we present high-resolution cryo-electron microscopy structures of the human taurine transporter (TauT) in multiple conformational states, revealing key insights into the taurine transport mechanism. We identify five distinct taurine-binding sites, elucidating a continuous pathway for taurine translocation across the cell membrane. Notably, we discover a novel gating mechanism involving a conserved salt bridge, which when disrupted, significantly enhances taurine uptake. Leveraging these structural insights, we demonstrate that augmented taurine uptake via TauT alleviates stress-induced senescence in biliary epithelial cells, potentially through the upregulation of the stress-responsive gene ATF3. Our findings not only advance the understanding of neurotransmitter transport but also unveil TauT as a promising therapeutic target for age-related liver diseases and other senescence-associated disorders. This work bridges structural biology with physiological function, offering new avenues for developing interventions to combat cellular aging and taurine-deficiency related diseases.

biochemistry↗

bioRxiv2024

ScisTree2: An Improved Method for Large-scale Inference of Cell Lineage Trees and Genotype Calling from Noisy Single Cell Data

In a multicellular organism, cell lineages share a common evolutionary history. Knowing this history can facilitate the study of development, aging, and cancer. Cell lineage trees represent the evolutionary history of cells sampled from an organism. Recent developments in single-cell sequencing have greatly facilitated the inference of cell lineage trees. However, single-cell data are sparse and noisy, and the size of single-cell data is increasing rapidly. Accurate inference of cell lineage tree from large single-cell data is computationally challenging. In this paper, we present ScisTree2, a fast and accurate cell lineage tree inference and genotype calling approach based on the infinite-sites model. ScisTree2 relies on an efficient local search approach to find optimal trees. ScisTree2 also calls single-cell genotypes based on the inferred cell lineage tree. Experiments on simulated and real biological data show that ScisTree2 achieves better overall accuracy while being significantly more efficient than existing methods. To the best of our knowledge, ScisTree2 is the first model-based cell lineage tree inference and genotype calling approach that is capable of handling datasets from tens of thousands of cells or more.

bioinformatics↗

bioRxiv2024

Assessing Conformation Validity and Rationality of Deep Learning-Generated 3D Molecules

Recent advancements in artificial intelligence (AI) have revolutionized the field of 3D molecule generation. However, the lack of effective evaluation methods for 3D conformations limits further improvements. Current techniques, in order to achieve the necessary speed for evaluating large numbers of AI-generated molecules, often rely on empirical geometric metrics that do not adequately capture various conformational anomalies, or on molecular mechanics (MM) energy metrics that exhibit low accuracy and lack atomic or torsional details. To address this gap, we propose a two-stage approach that achieves both high speed and quantum mechanical (QM) level accuracy. The first stage, termed the validity test, employs an AI-derived force field to identify atoms with elevated energy resulting from implausible neighboring environments. The second stage, known as the rationality test, utilizes a deep learning network trained on data with density functional theory (DFT) accuracy to detect rotatable bonds with high torsional energies. To demonstrate the functionality of our evaluation system, we applied our approach to five recently reported 3D molecule generation AI models across 102 targets in Directory of Useful Decoys-Enhanced (DUD-E) dataset. To facilitate accessibility for the academic community, our method is available as an open-source package.

bioinformatics↗

bioRxiv2024

Aurora kinase A promotes trained immunity via regulation of endogenous S-adenosylmethionine metabolism

Innate immune cells can acquire a memory phenotype, termed trained immunity, but the mechanism underlying the regulation of trained immunity remains largely elusive. Here, we demonstrate that inhibition of Aurora kinase A (AurA) dampens trained immunity induced by {beta}-glucan. ATAC-seq and RNA-seq analysis reveals that AurA inhibition restricts chromatin accessibility of genes associated with inflammatory pathways such as JAK-STAT, TNF and NF-{kappa}B pathways. Specifically, AurA inhibition promotes nuclear localization of FOXO3 and the expression of glycine N-methyltransferase (GNMT), a key enzyme responsible for S-adenosylmethionine (SAM) consumption. Metabolomic analysis confirms a reduction in SAM level upon AurA inhibition. As a result of SAM deficiency, trained mouse macrophages exhibit decreased H3K4me3 and H3K36me3 enrichment on gene regions of Il6 and Tnf. Additionally, the tumor inhibition effect of {beta}-glucan is notably abolished by AurA inhibition. Together, our findings identify an essential role of AurA in regulating trained immunity via a methylation-dependent manner by maintaining endogenous SAM level through mTOR-FOXO3-GNMT axis.

immunology↗

bioRxiv2024

A unified central thalamus mechanism underlying diverse recoveries in disorders of consciousness

Disorders of consciousness (DoC) encompass a range of states characterized by prolonged altered awareness due to heterogeneous brain damage and are associated with highly diverse prognoses. However, the neural mechanisms underlying such diverse recoveries in DoC remain unclear. To address this issue, we analysed neuronal spiking activities recorded from the central thalamus (CT), a key hub in arousal regulation, in a cohort of 23 DoC patients receiving deep brain stimulation treatment. Using machine learning techniques, we identified a core set of electrophysiological features of the CT, particularly the theta rhythm, that could account for individual recovery outcomes across highly varied etiologies (trauma, brainstem hemorrhage, and anoxia), clinical baselines and patient ages. These features also correctly identified one subgroup of patients who exhibited poor initial clinical manifestations but recovered unexpectedly. Simulating a conductance-based model further revealed the neurodynamics of the theta rhythm in the CT during different stages of consciousness recovery. Taken together, these findings uncover a previously unknown, unified CT mechanism that governs the recoveries in DoC.

neuroscience↗

bioRxiv2024

In vivo engineered quadrivalent CAR-macrophages break tumor barrier,remodel TME and overcome tumor heterogeneity through antigen spreading

CAR-macrophages have shown promising prospect in treating solid tumors. Ex vivo engineered CAR-macrophages face the challenge of low transfection efficiency, long preparation cycle, and limited cell number. Here, we designed two novel CAR molecules, GPC3-CAR-Super IL-2 and FAP-CAR-{bigtriangleup}TGF{beta}RII from the perspective of targeting tumor cells and improving tumor microenvironment, and generated quadrivalent CAR-macrophages in vivo for treating solid tumors by the LNP-mRNA system. We found that in vivo engineered quadrivalent CAR-macrophages can strongly activate tumor immunity and achieve complete tumor regression without significant side effects. Mechanically, in vivo engineered quadrivalent CAR-macrophages broke down physical barriers around the tumor constructed by CAFs and significantly promoted infiltration and expansion of CD8+ T cells. Moreover, the transiently formed CAR-macrophages in vivo are sufficient to form long-lasting T cell memory which can effectively prevent tumor recurrence. Most importantly, in vivo engineered CAR-macrophages also stimulated T cell memory against antigen-negative tumor cells through antigen spreading, which might effectively prevent the immune escape of heterogeneous tumor cells. Overall, we developed a platform of in vivo CAR-macrophages with dual roles as a tumor-killing effector cell and a recurrence-preventing vaccine.

bioengineering↗

bioRxiv2024

SARS-CoV-2 JN.1 reveals attenuated pathogenicity and airborne transmission

JN.1 is a subvariant of SARS-CoV-2 Omicron BA.2.86 lineage that was predominant worldwide in early 2024, of which the in vivo characteristics are largely unknown. Our results demonstrated that the replication of JN.1 was more efficient than that of the parental BA.2 in Vero cells, which demonstrated low dependence on TMPRSS2. Compared to Omicron variants BA.2 and XBB EG.5.1, JN.1 replicated less efficiently in hACE2 mouse lungs of which the intranasal infection was not lethal to hACE2 mice and led to weaker immune dysregulation. On a more sensitive, aged hACE2 hamster model, JN.1 led to a lower mortality rate and no weight loss, corresponding well with the low preference in lower airways. Lower amounts of viruses in nasal washes and exhaled aerosols were detected in JN.1 infected wildtype hamsters than EG.5.1, and consistently, JN.1 also exhibited largely reduced airborne transmission. Moreover, the poor transmission was also clearly demonstrated even by using hamsters expressing hACE2 receptors in the whole airway. Thus both pathogenicity and airborne transmission of JN.1 were demonstrated to be largely attenuated. ImportanceCurrently, SARS-CoV-2 JN.1 and its subvariants have fully replaced the previous dominant XBB lineage around the world. Although the strong immune evasion of JN.1 has been distinctly revealed, its in vivo pathogenicity and airborne transmission remained unclear. By using multiple Omicron-sensitive rodent models, our findings demonstrated that the pathogenicity of JN.1 was largely attenuated. The weak airborne transmission of JN.1 in wildtype and hACE2 hamsters was consistent with the reported relative lower transmissibility in human, and the using of airway-expressing hACE2 hamsters ulteriorly eliminates the potential bias in viral transmission studies induced by receptor divergence between animal models and human. These findings uncover the in vivo virological characteristics of SARS-CoV-2 novel lineage, providing insights for communicable disease control.

microbiology↗

bioRxiv2024

The Mla pathway promotes Vibrio cholerae re-expansion from stationary phase

Bacteria have evolved diverse strategies to ensure survival under nutrient-limited conditions, where rapid energy generation is not achievable. Here, we performed a transposon insertion site sequencing loss-of-function screen to identify Vibrio cholerae genes that promote the pathogens fitness in stationary phase. We discovered that the Mla (maintenance of lipid asymmetry) pathway, which is crucial for transferring phospholipids from the outer to the inner membrane, is critical for stationary phase fitness. Competition experiments with barcoded and fluorophore labeled wild-type and mlaE mutant V. cholerae revealed that the Mla pathway promotes re-expansion from 48h stationary phase cultures. The mutants defect in transitioning out of stationary phase into active growth (culturability) was also observed in monocultures at 48h. However, by 96h the culturability of the mutant and wild-type strains were equivalent. By monitoring the abundances of genomically barcoded libraries of wild-type and {Delta}mlaE strains, we observed that a few barcodes dominated the mutant culture at 96h, suggesting that the similarity of the population sizes at this time was caused by expansion of a subpopulation containing a mutation that suppressed the mlaE mutants defect. Whole genome sequencing revealed that mlaE suppressors inactivated flagellar biosynthesis. Additional mechanistic studies support the idea that the Mla pathway is critical for the maintenance of V. choleraes culturability as it promotes energy homeostasis, likely due to its role in regulating outer membrane vesicle shedding. Together our findings provide insights into the cellular processes that control re-expansion from stationary phase and demonstrate a previously undiscovered role for the Mla pathway. ImportanceBacteria regularly encounter conditions with nutrient scarcity, where cell growth and division are minimal. Knowledge of the pathways that enable re-growth following nutrient restriction are limited. Here, using the cholera pathogen, we uncovered a role for the Mla pathway, a system that enables phospholipid re-cycling, in promoting Vibrio cholerae re-expansion from stationary phase cultures. Cells labeled with DNA barcodes or fluorophores were useful to demonstrate that though the abundances of wild-type and Mla mutant cells were similar in stationary phase cultures, they had marked differences in their capacities to regrow on plates. Of note, Mla mutant cells lose cell envelope components including high energy phospholipids due to OMV shedding. Our findings suggest that the defects in cellular energy homeostasis which emerge in the absence of the Mla pathway underlie its importance in maintaining V. cholerae culturability.

microbiology↗

bioRxiv2024

Decoding Liver Cancer Prognosis: From Multi-omics Subtypes, Prognostic Models to Single Cell Validation

PurposeHepatocellular carcinoma (HCC) is a highly aggressive tumor characterized by significant heterogeneity and invasiveness, leading to a lack of precise individualized treatment strategies and poor patient outcomes. This necessitates the urgent development of accurate patient stratification methods and targeted therapies based on distinct tumor characteristics. Experimental DesignBy integrating gene expression data from The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC), and Gene Expression Omnibus (GEO), we identified subtypes through a multi-omics consensus clustering approach amalgamated from 10 clustering techniques. Subsequently, we developed a prognostic model, employing machine learning algorithms, based on subtype classification features. Finally, by analyzing single cell sequencing data, we investigated the mechanisms driving prognostic variations among distinct subtypes. ResultsFirst, we developed a novel consensus clustering method that categorizes liver cancer patients into two subtypes, CS1 and CS2. Second, we constructed a prognostic prediction model, which demonstrated superior predictive accuracy compared to several models published in the past five years. Finally, we observed differences between CS1 and CS2 in various metabolic pathways, biological processes, and signaling pathways, such as fatty acid metabolism, hypoxia levels, PI3K-AKT and MIF signaling pathway.

bioinformatics↗

bioRxiv2024

Land plant-specific H3K27 methyltransferases ATXR5 and ATXR6 control plant development and stress responses

The colonization of land by plants relies on numerous evolutionary innovations crucial for terrestrial adaptation. These include advances in sexual reproduction and the ability to properly respond to various environmental stresses, which involve precise control of their regulatory genes. A notable genetic innovation in land plants is the emergence of histone lysine methyltransferases ATXR5/6, which specifically catalyse the repressive histone H3 lysine 27 monomethylation (H3K27me1). However, the significance of the evolution of these enzymes in land plants remains unclear. In this study, we investigate the importance of ATXR5/6 by generating strong atxr5;atxr6 double mutants in Arabidopsis. Our results show that ATXR5/6 are essential for plant reproductive development and play a critical role in supporting normal plant growth by repressing the transcription of stress responsive genes. In addition, ATXR5/6 are necessary for maintaining H3K27 trimethylation (H3K27me3), likely by providing H3K27me1 as a substrate for further methylation. We also demonstrate that the function of ATXR5/6 in regulating reproductive development and responsive genes is conserved in the monocot rice. We propose that land plants may have evolved ATXR5/6 to assist in the transcriptional regulation necessary for terrestrial adaptation.

plant biology↗

bioRxiv2024

HumanTestisDB: A Comprehensive Atlas of Testicular Transcriptomics and Cellular Interactions

Advances in single-cell technology have enabled the detailed mapping of testicular cell transcriptomes, which is essential for understanding spermatogenesis. However, the fragmented nature of age-specific data from various literature sources has hindered comprehensive analysis. To overcome this, the Human Testis Database (HumanTestisDB) was developed, consolidating multiple human testicular sequencing datasets to address this limitation. Through extensive investigation, 38 unique cell types were identified, providing a detailed perspective on cellular variety. Furthermore, the database systematically categorizes samples into eight developmental stages, offering a structured framework to comprehend the temporal dynamics of testicular development. Each stage features comprehensive maps of cell-cell interactions, elucidating the complex communication network inside the testicular microenvironment at particular developmental stages. Moreover, by facilitating comparisons of interactions among various cell types at different stages, the database permits examining alterations that transpire during critical transitions in spermatogenesis. HumanTestisDB, available at https://shalab.njmu.edu.cn/humantestisdb, offers vital insights into testicular transcriptomics and interactions, serving as an essential resource for advancing research in reproductive biology.

cell biology↗

bioRxiv2024

Improved split prime editors enable efficient in vivo genome editing

Prime editor (PE) is a precise genome-editing tool that enables all 12 possible base-to-base conversions, as well as insertions and deletions, that does not require DSBs or donor DNA. The efficient delivery of prime editors in vivo is critical for realizing the full potential of prime editing in disease modeling and therapeutic correction. Although PE has been divided into two halves and delivered using dual adeno-associated viruses (AAVs), editing efficiency at different gene loci varies among split sites, and efficient split sites within Cas9 nickase are limited. In this study, by screening multiple split sites, we demonstrated that 1115 (Asn) is an efficient split site when delivering PE by dual-AAV. Besides, we utilized a feature reported by others recently that RNase could be detached from the Cas9n and designed split sites in the first half of Cas9n. We found that split-PE-367 enabled high prime editing efficiency with Rma intein. To test the editing efficiency in vivo, dual-AAV split-ePE3-367 was packaged in AAV9 and delivered by tail vein injection in mice, achieving 24.4% precise genome editing 4 weeks after injection. Our findings establish an alternative split-PE architecture that could achieve robust gene editing efficiency, facilitating the potential utility both in model organisms and as a therapeutic modality.

genomics↗

bioRxiv2024

Intelligence and Cognitive Flexibility Selectively Modulate Working Memory Functioning

Working memory is a key component of intelligence, which can be underpinned by task connectome, while cognitive flexibility plays a role in adaptive and executive function that supports working memory processes. However, it remains unknown how intelligence and cognitive flexibility modulate working memory functioning, in terms of behavioural performance and task-related brain hierarchy. Current study investigated the working memory task-related behaviour and brain connectomic gradient in healthy adults together with their intelligence and cognitive flexibility measures. Results showed cognitive flexibility and intelligence were not significantly correlated with each other but with working memory function. Principal component and partial least square analysis revealed that nearly half of variance was explained by their overlap component, which linked with task gradient values of frontoparietal and ventral attention network representing executive, control and cognitive functioning. Individuals with higher intelligence and cognitive flexibility showed less distance between frontoparietal/ventral attention network and unimodal regions in gradient axis during working memory performance. Further analyses demonstrated that task gradient of frontoparietal and ventral attention network was significantly associated with working memory function, and selectively mediated by cognitive flexibility and intelligence, which may suggest partly convergent neural mechanisms for higher order functions that are involved in working memory functioning. In conclusion, current study provides new insight into the relationship among task connectomic gradient, working memory, intelligence, and cognitive flexibility, which helps understand the multi-component model of working memory and underlying task gradient principle.

neuroscience↗

bioRxiv2024

SloR-SRE binding to the S. mutans mntH promoter is cooperative.

Streptococcus mutans is a commensal member of the plaque microbiome. It is especially prevalent when dietary sugars are available for S. mutans fermentation, generating acid byproducts that lower plaque pH and foster tooth decay. S. mutans can survive in the transient conditions of the mouth, in part because it can regulate the uptake of manganese and iron during periods of feast when metal ions are available, and famine when they are limiting. S. mutans depends on a 25kDa metalloregulatory protein, called SloR, to modulate uptake of these cations across the bacterial cell surface. When bound to manganese, SloR, binds to palindromic recognition elements in the promoter of the sloABC genes that encode the major manganese transporter in S. mutans. Reports in the literature describ MntH, an ancillary manganese transporter in S. mutans, that is also subject to SloR control. In the present study, we performed expression profiling experiments that reveal coordinate regulation of the sloABC and mntH genes at the level of transcription. In addition, we describe a role for the mntH gene product that is redundant with that of the sloABC-encoded metal ion uptake machinery. The results of DNA binding studies support direct SloR binding to the mntH promoter region which, like that at the sloABC promoter, harbors three palindromic recognition elements to which SloR binds cooperatively to repress downstream transcription. These findings expand our understanding of the SloR metalloregulome and elucidate SloR-DNA binding that is essential for S. mutans metal ion homeostasis and fitness in the oral cavity.

microbiology↗

bioRxiv2024

In vivo genome editing of human hematopoietic stem cells for treatment of blood disorders by mRNA delivery

Ex vivo autologous hematopoietic stem cells (HSCs) gene therapy provides promising new treatments for hematological disorders. However, current methods involve complex processes and chemotherapeutic conditioning, leading to limited accessibility for treatment and significant side effects. Here, we developed an antibody-free targeted lipid nanoparticles (LNPs) for mRNA delivery to HSCs in vivo, enabling efficient base editing of the HBG target in human HSCs to reactivate fetal hemoglobin in derived erythroid cells. Delivery of ABE8e/sgRNA mRNA with optimized structure LNPs achieves efficient in vivo base editing of HBG in transfusion-dependent {beta}-thalassemia (TDT) patients derived HSCs engrafted in immunodeficient NCG-X mice, showing restored globin chain balance in erythroid cells. Our research indicated that utilizing LNPs for delivery of genome editor achieves efficient editing of endogenous genes of human HSCs. Notably, this non-viral delivery system eliminates the need for harvesting or mobilizing HSCs, providing a potent and one-time treatment potential for blood disorders like sickle cell disease (SCD) and TDT. HighlightsO_LIAntibody-free targeted LNPs system achieves efficient mRNA delivery and base editing in human bone marrow cells including HSCs via one-time intravenous injection. C_LIO_LIDelivery of ABE8e/sgRNA mRNA with optimized LNPs achieves efficient in vivo base editing of HBG in TDT patients derived HSCs engrafted in immunodeficient NCG-X mice, showing restored globin chain balance in erythroid cells. C_LIO_LILNPs efficiently deliver gene editing system to the BM for in vivo editing of human HSCs, providing preclinical evidence for the next generation non-viral gene therapy for blood disorders. C_LI

bioengineering↗

bioRxiv2024

Revolutionizing large-scale DNA synthesis with microchip-based massive in parallel synthesis system

DNA synthesis serves as the fundamental enabling technology of engineering biology, aiming to provide DNA molecules of designed composition, length, and complexity at scale and low cost. Current high-throughput DNA synthesis technologies rely on intricate chip manufacturing and microfluidic systems to provide large-scale synthetic oligonucleotides, at the expense of low concentration and limited compatibility in the processing of longer DNA constructs assembly. Here, we report a microchip-based massive in parallel synthesis (mMPS), pioneering an "identification-sorting-synthesis-recycling" iteration mechanism to microchips for high throughput DNA synthesis. In comparison to microarray-based methods, we demonstrate that our method can increase the DNA product concentration by 4 magnitudes (to picomole-scale per sequence) and greatly simplifies the downstream processes for large-scale gene synthesis construction. By the construction of 1.97 million-diversity variant libraries that cover 1,254 human protein domains, we demonstrated the uniformity of the constructed variant libraries using mMPS-derived oligos is greatly improved, with amino acid distribution highly consistent as designed. In addition, by synthesizing 285 1kb-to-3kb genes with varying degrees of sequence complexity from previously reported strains A501 and 3DAC, potential ancestor of early archaea and bacteria, our result shows that the overall gene assembly success rate using mMPS-derived oligos is increased by 10-fold in comparison to other methods. Our mMPS technology holds the potential to close the gap between the quality and cost of writing DNA in increasing demand across many sectors of research and industrial activities.

synthetic biology↗