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Results for “molecular biology”

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bioRxiv2020

Direct RNA targeted transcriptomic profiling in tissue using Hybridization-based RNA In Situ Sequencing (HybRISS)

Highly multiplexed spatial mapping of multiple transcripts within tissues allows for investigation of the transcriptomic and cellular diversity of mammalian organs previously unseen. Here we explore the possibilities of a direct RNA (dRNA) detection approach incorporating the use of padlock probes and rolling circle amplification in combination with hybridization-based in situ sequencing (HybISS) chemistry. We benchmark a dRNA targeting kit that circumvents the standard reverse transcription limiting, cDNA-based in situ sequencing (ISS). We found a five-fold increase in transcript detection efficiency when compared to cDNA-based ISS and also validated its multiplexing capability by targeting a curated panel of 50 genes from previous publications on mouse brain sections, leading to additional data interpretation such as de novo cell typing. With this increased efficiency, we maintain specificity, multiplexing capabilities and ease of implementation. Overall, the dRNA chemistry shows significant improvements in target detection efficiency, closing the gap between the gold standard of fluorescent in situ hybridization (FISH) based technologies and opens up possibilities to explore new biological questions previously not possible with cDNA-based ISS, nor with FISH.

molecular biology↗

bioRxiv2021

Subnuclear localisation is associated with gene activation not repression or parental origin at the imprinted Dlk1-Dio3 locus

At interphase, de-condensed chromosomes have a non-random three-dimensional architecture within the nucleus, however, little is known about the extent to which nuclear organisation might influence expression or vice versa. Here, using imprinting as a model, we use 3D RNA- and DNA-fluorescence-in-situ-hybridisation in normal and mutant mouse embryonic stem cells to assess the relationship between imprinting control, gene expression and allelic distance from the nuclear periphery. We compared the two parentally inherited imprinted domains at the Dlk1-Dio3 domain and find a small but reproducible trend for the maternally inherited domain to be further away from the periphery if the maternally expressed gene Gtl2/Meg3 is active compared to when it is silenced. Using Zfp57KO ES cells, which harbour a paternal to maternal epigenotype switch, we observe active alleles significantly further away from the nuclear periphery with the distance from the periphery being proportional to the number of alleles active within the cell. This distribution of alleles suggests an activating effect of the nuclear interior rather than a repressive association with the nuclear periphery. Although we see a trend for the paternally inherited copy of the locus to be closer to the nuclear periphery, this appears to be linked to stochastic gene expression differences rather than parental origin. Our results suggest that transcriptional activity, rather than transcriptional repression or parental origin, defines sub-nuclear localisation at an endogenous imprinted domain. Author summaryGenomic imprinting is an epigenetically regulated process that results in the preferential expression of a subset of developmentally regulated genes from maternally or paternally inherited chromosomes. We have used imprinted genes as a model system to investigate the relationship between the localisation of genes within the cell nucleus and their active expression while at the same time distinguishing gene repression by genomic imprinting, and gene repression by other mechanisms that act on the active allele. We find that there is a significant correlation between transcription and distance to the edge of the nucleus for the Gtl2/Meg3 gene in the imprinted Dlk1-Dio3 region. However, this correlation has a very small effect size and the nuclear envelope, which is commonly thought to act as a repressive environment for gene expression, does not appear to play a major role. We show that position effects, which have been shown for artificially lamina-targeted genes, also exist for endogenous loci and consider the possible biological relevance of the observed small effect.

molecular biology↗

bioRxiv2021

The developmentally-timed decay of an essential microRNA family is seed sequence-dependent

MicroRNA (miRNA) abundance is tightly controlled by regulation of biogenesis and decay. Here we show that the mir-35 miRNA family undergoes selective decay at the transition from embryonic to larval development in C. elegans. The seed sequence of the miRNA is necessary and largely sufficient for this regulation. Sequences outside the seed (3 end) regulate mir-35 abundance in the embryo but are not necessary for sharp decay at the transition to larval development. Enzymatic modifications of the miRNA 3 end are neither prevalent nor correlated with changes in decay, suggesting that miRNA 3 end display is not a core feature of this mechanism and further supporting a seed-driven decay model. Our findings demonstrate that seed sequence-specific decay can selectively and coherently regulate all redundant members of a miRNA seed family, a class of mechanism that has great biological and therapeutic potential for dynamic regulation of a miRNA familys target repertoire.

molecular biology↗

bioRxiv2023

Open-source, high-throughput targeted in-situ transcriptomics for developmental biologists

Multiplexed spatial profiling of mRNAs has recently gained traction as a tool to explore the cellular diversity and the architecture of tissues. We propose a sensitive, open-source, simple and flexible method for the generation of in-situ expression maps of hundreds of genes. We exploit direct ligation of padlock probes on mRNAs, coupled with rolling circle amplification and hybridization-based in situ combinatorial barcoding, to achieve high detection efficiency, high throughput and large multiplexing. We validate the method across a number of species, and show its use in combination with orthogonal methods such as antibody staining, highlighting its potential value for developmental biology studies. Finally, we provide an end-to-end computational workflow that covers the steps of probe design, image processing, data extraction, cell segmentation, clustering and annotation of cell types. By enabling easier access to highthroughput spatially resolved transcriptomics, we hope to encourage a diversity of applications and the exploration of a wide range of biological questions.

molecular biology↗

bioRxiv2024

cryoTIGER: Deep-Learning Based Tilt Interpolation Generator for Enhanced Reconstruction in Cryo Electron Tomography

Cryo-electron tomography enables the visualization of macromolecular complexes within native cellular environments, but is limited by incomplete angular sampling and the maximal electron dose that biological specimen can be exposed to. Here, we developed cryoTIGER (Tilt Interpolation Generator for Enhanced Reconstruction), a computational workflow leveraging a deep learning-based frame interpolation to generate intermediate tilt images. By interpolating between tilt series projections, cryoTIGER improves angular sampling, leading to enhanced 3D reconstructions, more refined particle localization, and improved segmentation of cellular structures. We evaluated our interpolation workflow on diverse datasets and compared its performance against non-interpolated data. Our results demonstrate that deep learning-based interpolation improves image quality and structural recovery. The presented cryoTIGER framework offers a computational alternative to denser sampling during tilt series acquisition, paving the way for enhanced cryo-ET workflows and advancing structural biology research.

molecular biology↗

bioRxiv2025

Deciphering ESR1-driven transcription in human endometrial stromal cells via transcriptome, cistrome, and integration with chromatin landscape

ObjectiveTo investigate ESR1 and estrogen-driven transcription in human endometrial stromal cells. DesignTelomerase-immortalized human endometrial stromal cells were engineered to activate ESR1 using the CRISPR activation system and treated with vehicle or E2. Primary endometrial stromal cells were treated with vehicle or decidualization cocktail. SubjectsBiopsies from two healthy, reproductive-aged volunteers with regular menstrual cycles and no history of gynecological malignancies. Main Outcome MeasuresBulk RNA-sequencing in ESR1-activated and E2-treated cells was compared to identify ligand-independent and -dependent ESR1 actions. Cut&Run was performed in ESR1-activated cells treated with E2 or vehicle to assess the ESR1 cistrome. H3K27ac HiChIP was conducted in primary endometrial stromal cells treated with vehicle or decidualization cocktail to identify hormonally induced chromatin interaction changes. ResultsAmong six tested guide RNAs, the ESR1-3 gRNA induced robust ESR1 activation, which restored E2 responsiveness in THESC. Bulk RNA-seq revealed ESR1-mediated E2-dependent and E2-independent transcriptional programs, regulating pathways involved in inflammation, proliferation, extracellular matrix organization, and cancer. Notably, 72% of differentially expressed genes (DEGs) overlapped with genes active in human endometrial tissue during the proliferative E2 dominant phase, supporting their physiological relevance. Cut&Run-seq identified genome-wide ESR1 binding sites, with most located at distal regulatory elements. To associate distal ESR1 binding sites with genes, we integrated H3K27ac HiChIP chromatin loops in hESC to identify distal ESR1 binding sites that loop to gene promoters. We identified genes regulated by ESR1/E2 through long-range chromatin looping that are involved in stromal cell decidualization, including FOXO1 and IL6R. Additionally, we identified genes implicated in endometrial cancer, including ERRFI1, NRIP1, and EPAS1, suggesting a role for stromal ESR1 driven endometrial pathologies. Functional assays confirmed that ESR1 promotes cell viability and, in the presence of E2, enhances migration. ConclusionsESR1 activation through CRISPR restores E2 responsiveness in endometrial stromal cells. Changes to chromatin architecture support gene expression changes that drive decidualization. Integration of ESR1/E2 transcriptome and cistrome with HiChIP data identifies its role in regulating inflammation, proliferation, and decidualization, as well as its implications in endometrial cancer. This model serves as a powerful tool to study estrogen signaling in endometrial stromal biology and related pathologies.

molecular biology↗

bioRxiv2025

eRNAkit: Expanding the Functional Atlas of human Enhancer RNAs Beyond the Nucleus

Enhancer RNAs (eRNAs) are a class of non-coding RNAs transcribed from active enhancers that regulate various aspects of transcription. Although traditionally viewed as nuclear-localised and unstable transcripts, large transcriptomic studies reveal they vary widely in localisation and biochemical properties, including detectable accumulation in cytoplasmic compartments. These observations suggest potential non-nuclear functions for eRNAs, yet existing databases remain focused on nuclear, cis-regulatory roles, limiting systematic exploration of their broader regulatory repertoire. Here we present eRNAkit, a comprehensive and accessible resource designed to address this gap by integrating subcellular localisation, RNA-RNA interaction and expression data for annotated eRNAs. Leveraging fractionation-based RNA-Seq datasets, eRNAkit profiles eRNA distribution across nuclear and cytoplasmic compartments. It incorporates gene expression data spanning major human organs and primary cell types, enabling tissue-specific analysis of eRNA function. Crucially, eRNAkit includes experimentally derived RNA-RNA interaction data from RIC-Seq, PARIS, and KARR-Seq, supporting exploration of trans-acting and cytoplasmic roles for eRNAs grounded in physical interaction evidence. eRNAkit expands current eRNA resources beyond the enhancer-promoter paradigm, offering a robust platform for dissecting non-canonical functions of eRNAs and advancing our understanding of their full regulatory potential in human biology. The eRNAkit resource is available to download at https://github.com/AneneLab/eRNAkit.

molecular biology↗

bioRxiv2019

Dengue and Zika virus 5′-UTRs harbor IRES functions

Members of Flavivirus, a genus of Flaviviridae, encompass numerous enveloped plus strand RNA viruses, of which globally dengue virus (DENV) is the leading cause of serious arthropod-borne disease. The genomes of DENV, just as those of yellow fever virus (YFV), West Nile fever virus (WNV), or Zika virus (ZIKV), control their translation by a 5-terminal capping group. Three other genera of Flaviviridae are remarkable because their viruses use internal ribosomal entry sites (IRESs) to control translation and they are not arthropod transmitted. In 2006 E. Harris group published work suggesting that DENV RNA does not stringently need a cap for translation. They proposed that instead DENV translation is controlled by an interplay between 5 and 3 termini. Here we present evidence that the DENV or ZIKV 5-untranslated regions (5-UTRs) alone have IRES competence. This conclusion is based, first, on the observation that uncapped mono-cistronic mRNAs 5 terminated with the DENV or ZIKV 5-UTRs can efficiently direct translation of a reporter gene in BHK and C6/36 cells; second, that either 5-UTR placed between two reporter genes can efficiently induce expression of the downstream gene in BHK but not in C6/36 cells. These experiments followed observations that uncapped DENV/ZIKV genomic transcripts, 5 terminated with pppAN... or GpppAN..., can initiate infections of mammalian (BHK) or mosquito (C6/36) cells. IRES competence of the 5-UTRs of DENV/ZIKV raises many open questions regarding the biology and control, as well as the evolution, of insect-borne flaviviruses. ImportanceMembers of the genus Flavivirus of Flaviviridae are important human pathogens of great concern because they cause serious diseases, sometimes death, in human populations living in tropical, subtropical (dengue, DENV; Zika, ZIKV; yellow fever virus), or moderate climates (West Nile virus). Flaviviruses are known to control their translation by a cap-dependent mechanism. We have observed, however, that the uncapped genomes of DENV or ZIKV can initiate infection of mammalian and insect cells. We provide evidence that the short 5 untranslated region (5-UTR) of DENV or ZIKV genomes can fulfill the function of an internal ribosomal entry site (IRES). This strategy frees these organisms from the cap-dependent mechanism of gene expression at an as yet unknown stage of proliferation. The data raise new questions about the biology and evolution of flaviviruses, possibly leading to new controls of flavivirus disease.

molecular biology↗

bioRxiv2026

Signal, noise, and sampling: How pool size and replication shape metabolomic inference

Metabolomics provides a direct readout of physiological state and is increasingly used in evolutionary and systems biology. In small organisms such as Drosophila melanogaster, metabolomic analyses typically require pooling individuals to obtain sufficient material, yet pool sizes vary widely across studies with little justification. How pooling and biological replication influence metabolome characterization and the detection of biological signal remains poorly understood. Here, we evaluate the effects of pool size and biological replication on metabolomic profiles and signal detection using two complementary experimental designs. In the first, we assess how pooling (5, 50, or 100 individuals) influences metabolomic structure and reproducibility in inbred and outbred populations. In the second, we test how pool size interacts with systematic variation in replicate number to affect detection of diet-associated metabolite changes under a high-sugar perturbation. Pool size strongly influenced metabolomic profiles, with samples pooled at five individuals consistently differing from larger pools, while profiles from 50 and 100 individuals were more similar. Larger pools improved reproducibility in a dataset-dependent manner. In the dietary experiment, smaller pool sizes substantially reduced sensitivity, leading to loss of true diet-associated metabolites without increasing false discoveries. Replicate downsampling further revealed that both pool size and biological replication jointly determine signal retention, with smaller pools accelerating the loss of detectable metabolites under reduced replication. Across all analyses, the ability to detect metabolite signals was strongly dependent on effect size and variability. Metabolites with larger and more stable effect estimates were consistently retained, whereas those with smaller or more variable effects were rapidly lost under reduced sampling. Linear mixed-effects modeling confirmed that detection probability is governed by a balance between biological signal strength and measurement variability, with pool size and replication jointly modulating this relationship. More broadly, our results demonstrate that metabolomic inference is governed by the interplay of signal, noise, and sampling design, with pool size and replication jointly shaping the detectability, stability, and interpretation of biological signals.

molecular biology↗

bioRxiv2026

Development of an Ethylenediaminetetraacetic Acid-Enhanced Deep Proteomic Profiling Method for Dried Blood Spots and Its Application in Mouse Disease Models

Dried blood spots (DBS) are well-established microsamples used in clinical testing and newborn screening. However, their use in deep proteomics is hindered by highly abundant blood proteins and inefficient protein recovery from filter paper matrices. The non-targeted analysis of non-specifically DBS-absorbed proteins (NANDA) workflow partially overcomes the impact of abundant blood proteins and has enabled the identification of over 5,000 proteins from DBS samples. Nonetheless, residual abundant proteins, including hemoglobin and fibrinogen, constrain deep proteomic analysis. Therefore, this study aimed to evaluate the effects of the metal chelator ethylenediaminetetraacetic acid (EDTA) on the depth of DBS proteomic analysis. An optimized EDTA-enhanced NANDA protocol that incorporated a 100 mM EDTA wash step was compatible with standard DBS collection procedures and required no modification of current clinical workflows, markedly enhancing the depletion of abundant proteins and facilitating its potential use in clinical and translational settings. When combined with Orbitrap Astral data-independent acquisition mass spectrometry, this approach enabled the single-shot identification of more than 7,000 proteins from DBS samples; to the best of our knowledge, this represents the deepest proteome coverage reported to date, and the workflow further supported high-throughput and highly reproducible analyses. Additionally, its application to mouse disease models revealed disease-specific systemic immune signatures from minimal blood volumes. Collectively, these results establish EDTA-enhanced NANDA as a practical and scalable workflow that overcomes longstanding limitations of DBS proteomics, thereby enabling deep, high-throughput, minimally invasive proteomic profiling across diverse biological and experimental contexts.

molecular biology↗

bioRxiv2024

Markers, Mechanisms and Metrics of Biological Aging: A Scoping Review

Biological aging is a rapidly growing area of research, which entails characterizing the rate of aging independent of an individuals chronological age. In this review, we analyze the results of biological aging research in 435 papers published in a twelve year window, revealing changing patterns of molecular markers of biological aging use over time, and the development of novel metrics of biological aging. We further identify consistent and discordant research findings, as well as areas of potential future research focusing on questions of measurement with methylation or biomarker-based assessment and other variables relevant to the study of biological age.

molecular biology↗

bioRxiv2025

MFPLI: A Computational Framework for Assessing Biological Authenticity of Protein-Ligand Interactions Using Molecular Fingerprints and Structural Features

Traditional computational drug discovery approaches struggle to accurately evaluate the biological authenticity of protein-ligand binding conformations due to inherent limitations in empirical scoring functions and force field approximations. This study proposes MFPLI - a deep learning framework integrating multimodal physicochemical features to systematically assess the biological authenticity alignment between molecular docking poses and true co-crystal structures. By establishing a continuous surface characterization system for protein-ligand interfaces, we concurrently incorporate geometric curvature features (radius, shape index) and chemical interaction fields (electrostatic potential, hydrogen-bond networks, hydrophobicity gradients). A contrastive learning architecture based on Siamese equivariant graph neural networks was developed to enable discriminative analysis between co-crystal conformations and parameter-perturbed pseudo-conformations generated through inverse docking. The five-channel fusion model demonstrates robust performance on the time-split PoseBuster validation set (AUC=0.91), with predicted Euclidean distance deviation ({Delta}E) effectively distinguishing native co-crystal conformations from aberrant docking poses in 80% of samples. Notably, 71% of {Delta}E-negative samples concentrate within the [-0.3, 0] interval, reflecting physical consistency between model predictions and conformational transition processes. This framework establishes a novel paradigm for biological authenticity assessment in virtual screening for computer-aided drug discovery through synergistic modeling of surface topology and interaction chemistry.

molecular biology↗

bioRxiv2025

SimMapNet: A Bayesian Framework for Gene Regulatory Network Inference Using Gene Ontology Similarities as External Hint

MotivationGene regulatory network (GRN) reconstruction is a fundamental challenge in computational biology, and is crucial for understanding gene interactions. In this study, we aim to incorporate Gene Ontology (GO) similarities into the construction of GRNs. Our key assumption is that genes with higher similarity in Molecular Function, Biological Process, or Cellular Component categories are more likely to be functionally related and, therefore, more likely to be connected in the network. We introduce SimMapNet, a Bayesian framework that estimates the precision matrix, which serves as the adjacency matrix in a Gaussian graphical model (GGM) for GRN inference. SimMapNet enhances network inference by integrating GO similarities, which inform the hyperparameters of the prior distribution through a kernel function, incorporating biological prior knowledge in a principled manner. ResultsWe evaluate SimMapNet on three datasets: two datasets from the SOS DNA-repair response pathway in Escherichia coli and one dataset from Drosophila melanogaster. The results demonstrate the algorithms superior performance compared to state-of-the-art methods such as GLASSO, GENIE3, and KBOOST in terms of F1-score. SimMapNet has low time complexity, making it suitable for constructing large networks. Our simulation results confirm that SimMapNet is particularly well-suited for scenarios with limited sample sizes, where traditional methods often struggle. Availability and implementationThe datasets and R package of SimMapNet are available in the github repository, https://github.com/maryam-shahdoust/SimMapNet.

bioinformatics↗

bioRxiv2019

Manumycin Polyketides Act as Molecular Glues Between UBR7 and P53 to Impair Breast Cancer Pathogenicity

Molecular glues are an intriguing therapeutic modality that harness small-molecules to induce interactions between proteins that typically do not interact, thus enabling the creation of novel protein functions not naturally encoded in biology. While molecular glues such as thalidomide and rapamycin have catalyzed drug discovery efforts, such molecules are rare and have often been discovered fortuitously, thus limiting their potential as a general strategy for therapeutic intervention of disease. Historically, natural products have proven to be important sources of molecular glues and we postulated that natural products bearing multiple electrophilic sites may be an unexplored source of such molecules, potentially through multi-covalent attachment. Using activity-based protein profiling (ABPP)-based chemoproteomic platforms, we show that members of the manumycin family of polyketides, which bear multiple potentially reactive sites, target C374 of the putative E3 ligase UBR7 in breast cancer cells to impair breast cancer pathogenicity through engaging in molecular glue interactions with the neo-substrate tumor-suppressor TP53, leading to the activation of p53 transcriptional activity and cell death. Our results reveal a previously undiscovered anti-cancer mechanism of this natural product family and highlight the potential for combining chemoproteomics and multi-covalent natural products for the discovery and characterization of new molecular glues.

biochemistry↗

bioRxiv2017

C-BERST: Defining subnuclear proteomic landscapes at genomic elements with dCas9-APEX2

Mapping proteomic composition at distinct genomic loci and subnuclear landmarks in living cells has been a long-standing challenge. Here we report that dCas9-APEX2 Biotinylation at genomic Elements by Restricted Spatial Tagging (C-BERST) allows the rapid, unbiased mapping of proteomes near defined genomic loci, as demonstrated for telomeres and centromeres. By combining the spatially restricted enzymatic tagging enabled by APEX2 with programmable DNA targeting by dCas9, C-BERST has successfully identified nearly 50% of known telomere-associated factors and many known centromere-associated factors. We also identified and validated SLX4IP and RPA3 as telomeric factors, confirming C-BERSTs utility as a discovery platform. C-BERST enables the rapid, high-throughput identification of proteins associated with specific sequences, facilitating annotation of these factors and their roles in nuclear and chromosome biology.

molecular biology↗

bioRxiv2021

Systematic in vivo quantification of microRNA affinities

The majority of mammalian genes are under regulation by microRNAs, yet predicting the extent of miRNA-mediated repression has remained elusive. Here we systematically quantified the biological impact of miRNAs conserved in vertebrates using stable mouse embryonic stem cell lines expressing sensitive fluorescent reporters. Differentiation of these 163 lines to the three germ layers revealed that the majority of conserved miRNAs have detectable changes in activity. We determined in vivo target affinity KD of 115 miRNAs by integrating activity measurements, CRISPR/Cas miRNA knockouts and miRNA sequencing. Target affinities of individual miRNAs spanned several orders of magnitude, with highly expressed miRNAs having overall higher KD. Scaling miRNA expression levels by their respective KD recapitulated the relative number of Argonaute-bound targets for individual miRNA families. Our results provide a rationale to determine the set of miRNAs with a biological activity in a given cell type, KD values setting expression thresholds for target repression.

molecular biology↗

bioRxiv2021

Protein Charge Neutralization is the Proximate Driver Dynamically Tuning a Nanoscale Bragg Reflector

Reflectin is a cationic, block copolymeric protein that mediates the dynamic fine-tuning of color and brightness of light reflected from nanostructured Bragg reflectors in iridocyte skin cells of squids. In vivo, neuronally activated phosphorylation of reflectin triggers its assembly, driving osmotic dehydration of the membrane-bounded Bragg lamellae containing the protein to simultaneously shrink the lamellar thickness and spacing while increasing its refractive index contrast, thus tuning the wavelength and increasing the brightness of reflectance. In vitro, we show that reduction in repulsive net charge of the purified, recombinant reflectin - either (for the first time) by generalized anionic screening with salt, or by pH titration - drives a finely tuned, precisely calibrated increase in size of the resulting multimeric assemblies. The calculated effects of phosphorylation in vivo are consistent with these effects observed in vitro. X-ray scattering analyses confirm the sphericity, size and low polydispersity of the assemblies. Precise proportionality between assembly size and charge-neutralization is enabled by the demonstrated rapid dynamic arrest of multimer growth. The resulting stability of reflectin assemblies with time ensures reciprocally precise control of the particle number concentration, thereby encoding a precise calibration between the extent of neuronal signaling, osmotic pressure, and the resulting optical changes. The results presented here strongly suggest that it is charge neutralization, rather than any change in aromatic content, that is the proximate driver of assembly, fine-tuning a colligative property-based nanostructured biological machine. A physical mechanism is proposed.

molecular biology↗

bioRxiv2023

The "replacing surgery" of cpDNA: de novo chemical synthesis and in vivo functional testing of Chlamydomonas chloroplast genome

We have successfully designed and synthesized the 221,372-bp cpDNA SynCpV1.0 with the native cpDNA of Chlamydomonas reinhardtii as the template. Homoplasmic SynCpv1.0-harboring algal strains were obtained by biolistic transformation and selected with an ascending gradient of antibiotic pressure. Meanwhile, we were pleasantly surprised to find that SynCpV1.0 was able to re-introduce and replicate normally after the total DNA of transplastomic algal strains were transformed to Escherichia coli, it indicated that SynCpV1.0 was able to shuttle between C. reinhardtii and E. coli. Finally, we analyzed the photosynthetic properties of SynCpV1.0-harboring transplastomic strains, the results showed that they exhibited the same photosynthetic efficiency as the wild strain of C. reinhardtii CC125, and could rescue the photosynthetic defect in mutant strain of C. reinhardtii CC5168. Herein, we have performed the "replacing surgery" of cpDNA and established an ideal platform to complete multiple cycles of "Design-Build-Test" for optimizing the cpDNA of photosynthetic organisms. HighlightO_LIAn artificial cpDNA SynCpV1.0 is constructed by de novo chemical synthesis. C_LIO_LIThe "replacing surgery" of cpDNA was performed in the chloroplast of C. reinhardtii C_LIO_LIIt is found that artificial cpDNA was able to shuttle between Chlamydomonas chloroplast and E. coli. C_LIO_LIEstablish an ideal platform to complete multiple cycles of "Design-Build-Test" for optimizing the cpDNA. C_LI One-Sentence SummaryThe chloroplast genome can be replaced by a complete synthesized genome and performs the designed biological function in C. reinhardtii.

molecular biology↗