Biology subjects

Castro, C.

Publications and source records attributed to Castro, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Organoid-to-organoid transplantation and pro-neurogenic treatment promote graft integration and neuronal repair in a human ischemic model

Neuronal loss resulting from brain injury and neurodegenerative diseases currently lacks effective regenerative therapies. While neurogenic compounds and cell transplantation hold promise, clinical translation is hindered by poor graft integration and limited cell survival. Here, we developed a novel organoid-to-organoid transplantation model using human pluripotent stem cell-derived cortical organoids (hCOs) to evaluate a dual cell transplantation and pro-neurogenic strategy for brain repair. We demonstrate that pro-neurogenic treatment with ER272 stimulates early-stage hCO growth and enhances neurogenesis through a PKC- and Notch-dependent pathway. Mechanistically, ER272 upregulates genes associated with extracellular matrix, cell adhesion and growth factor signaling. Notably, combining early-stage hCO transplantation with ER272 treatment significantly enhanced graft integration and improved neuronal repair in an ischemic-like model of late-stage host hCOs. This novel human organoid-to-organoid platform offers a robust translational tool to test regenerative therapies and highlight the potential of combining cell transplantation with pro-neurogenic stimulation for the treatment of neurological conditions associated with neuronal loss.

neuroscience↗

bioRxiv2026

Hepatic HIF2α modulates extra-hepatic disease-associated phenotypes during metabolic dysfunction-associated steatotic liver disease

Metabolic dysfunction-associated steatotic liver disease (MASLD) afflicts more than one-third of adults globally, contributing significantly to an increased cardiovascular disease risk. Further, patients with severe liver disease experience muscle weakness (sarcopenic obesity) and fatigue. Hypoxia-inducible factor 2 (HIF2) accumulates in the livers of MASLD patients and has been implicated in disease progression. Here we sought to understand the role of hepatic HIF2 in mediating hepatic and extra-hepatic features of MASLD. Using a well-validated obese mouse model of MASLD, we investigated the impact of hepatocyte-specific HIF2 deletion (hHIF2-/-) on hepatic, cardiac and skeletal muscle metabolism, and cardiac function. Over 28 weeks, mice were exposed to a high-fat, high-fructose, high-cholesterol (GAN) diet, which induced obesity alongside hepatic steatosis, fibrosis and inflammation. In contrast to observations in lean mouse models of liver disease, hHIF2-/- did not protect against MASLD, despite greater hepatic NADH-supported mitochondrial respiration and higher intracellular sphingomyelin levels. Instead, in the hearts of GAN-fed mice, hHIF2-/- caused diacylglycerol accumulation independent of diet, accumulation of long-chain acyl-carnitines and exacerbation of ceramide accumulation. Langendorff-perfused hearts from hHIF2-/- mice showed systolic and diastolic dysfunction, including 24% lower left ventricular developed pressure and 34% lower maximal rate of relaxation (dP/dtmin). However, isolated hearts from hHIF2-/- mice were protected against MASLD-associated sympathetic dominance, determined using autonomic receptor agonist stimulation. Both GAN-feeding and hHIF2-/- were associated with lower lean mass (14% and 5.4% lower than respective controls), whilst hHIF2-/- enhanced OXPHOS-associated protein levels in gastrocnemius muscle. Overall, hHIF2-/- resulted in detrimental extra-hepatic effects, including myocardial lipid accumulation, impaired cardiac function, and loss of whole-body lean mass, with no apparent protection against MASLD disease progression.

physiology↗

bioRxiv2026

Estrogen receptor-positive cell line xenograft models recapitulate metastatic dissemination and endocrine response of invasive lobular breast carcinoma

HighlightsO_LIER+, CDH1-deficient ILC xenografts recapitulate single-file histology in vivo C_LIO_LISpontaneous metastases faithfully mirror unique ILC clinical dissemination patterns C_LIO_LIER+ leptomeningeal metastases confirmed histologically in ILC xenograft models C_LIO_LIFulvestrant inhibits ILC tumor growth and metastatic burden, improving survival C_LIO_LIER signaling is enriched in ILC brain metastases, with upregulation of RET C_LI Invasive lobular breast carcinoma (ILC), the most common special histological subtype of breast cancer, is characterized by nearly universal expression of estrogen receptor alpha (ER) and unique sites of metastases, neither of which is fully recapitulated by genetically engineered mouse models. Using reporter-labeled ILC mouse xenografts, herein we used mammary fat pad, tail vein and intracardiac orthotopic growth to analyze spontaneous and experimental metastasis and gene expression. We observed ER-positive primary tumors with single-file histology and collagen deposition, and spontaneous metastasis from the mammary fat pad to bones, ovaries, and brain including the leptomeninges, thereby closely mirroring the growth and metastatic spread of human ILC. Brain metastases showed strong ER staining, confirmed by sequencing analyses which identified estrogen signaling as top activated pathway, and the lesions exhibited robust response to endocrine therapy. In summary, we report endocrine responsive mammary fat pad, tail vein and intracardiac xenografts that faithfully demonstrate unique ILC features and can serve as invaluable pre-clinical translational platforms for validating candidate ILC genetic drivers and testing novel therapeutics.

cancer biology↗

bioRxiv2026

Tex11 Mutant Mouse Models of Human Azoospermia

Non-obstructive azoospermia (NOA) is the absence of sperm in the ejaculate due to spermatogenic failure. Fifty percent of NOA cases are unexplained but may arise from unidentified genetic mutations. Variants in TEX11 have been identified in men with NOA; and Tex11 knockout in mice causes NOA. Here we attempt to validate three TEX11 variants discovered in NOA patients by knocking them into the orthologous region of the mouse genome using CRISPR/Cas9 gene editing. Compared to wild type (144.2 {+/-} 9.87 mg; 1.7 {+/-} 0.5 million sperm/cauda epididymis 4.8 {+/-} 1.3 pups/breeding), Tex11D mice (frameshift mutation) had reduced testis weight (28.33 {+/-} 1.16 mg); no sperm in the epididymis; and were infertile with a maturation arrest testicular phenotype. We did not observe any spermatogenesis or fertility defects Tex11A mice (missense mutation). Tex11L mice had reduced testis weight (87.5 {+/-} 14.79 mg) and epididymal sperm counts (0.33{+/-}0.13 million/cauda epididymis) but an incompletely penetrant infertility phenotype (5.4 {+/-} 1.13 pups/breeding) with one third of mice being infertile. Infertile Tex11L mice also had a distinct epididymal phenotype with reduced sperm density in the caput and no sperm in the cauda, which was filled with amorphous material.

genetics↗

bioRxiv2025

Sex-Specific neurogenic and cognitive responses in a murine model of accelerated aging

Aging is associated with cognitive deterioration accompanied by a reduction in hippocampal neurogenesis. Murine models have been widely used to study aging and age-related cognitive decline, as they recapitulate many of the key features of the degenerative process. Among these, the SAMP8 strain represents a well-established model of accelerated aging, characterized by early-onset and progressive cognitive impairment, Alzheimers disease-like neuropathology, and an initial increase in hippocampal neurogenesis that ultimately depletes the neural stem cell pool. Notably, most studies using murine models of aging or neurodegeneration have focused on males or mixed-sex cohorts, leaving sex-specific differences in neurogenesis and cognitive decline largely unexplored. Recent evidence indicates that diterpenoid treatment ameliorates cognitive decline and enhances neurogenesis in 6-month-old male SAMP8 mice. However, whether females exhibit similar responses remains unknown. In this study, we characterized sex differences in hippocampal neurogenesis in 6-month-old SAMP8 mice and examined potential sex-dependent effects of diterpenoid therapy. Our findings reveal marked sex differences in neurogenic capacity and treatment responsiveness. While diterpenoid treatment enhanced hippocampal neurogenesis and cognitive performance in males, these effects were largely absent in females. Overall, female SAMP8 mice exhibited reduced baseline neurogenesis and a diminished response to therapy, underscoring the importance of considering biological sex in the design of therapeutic strategies for age-related cognitive decline.

pharmacology and toxicology↗

bioRxiv2025

Tunable DNA Origami Nanosensors for Detection of Multiscale Spatial Ion Concentration Gradients

Ion gradients play a vital role in cellular signaling, mechanobiology, and organ-level homeostasis. Despite their importance, accurately mapping these spatial gradients at biologically relevant length scales remains a challenge due to the limited tunability and spatial resolution of conventional fluorescent sensors. Here, we present a DNA origami-based sensor (NanoDyn) with tunable sensitivity that enables the detection of Na+ ion gradients across micron to millimeter scales. The sensor design leverages programmable DNA base-pairing interactions to control both the detection range and sensitivity of the sensor. Using fluorescence spectroscopy, we show that NanoDyn can exhibit programmable sensing ranges spanning [~]100 - 1675 mM Na+. To validate the ability to quantify ion gradients and investigate its spatial resolution, we use a custom microfluidic gradient generator, showing that NanoDyn can resolve changes in ion gradients across multiple scales and over distances as little as [~]6 {micro}m, which here, is limited by the resolution of the microfluidic device. In highlighting the potential of DNA nanodevices as multiscale tunable ion gradient sensors, together with their biocompatibility, high temporal resolution, and potential for multiplexed functionalization, this work expands on the role that DNA nanodevices can play in spatial sensing to study ion-mediated processes in microenvironments. Overall, this work advances DNA nanotechnology as a versatile foundation for biosensing with capabilities to probe ion-mediated signaling in health and disease.

bioengineering↗

bioRxiv2025

Free energy spectroscopy reveals the mechanistic landscape of chromatin compaction

Eukaryotic genomic DNA is repeatedly wrapped into nucleosome spools: the basic building block of chromatin. This organization regulates the physical accessibility of the genome to gene transcription, replication, and repair regulatory factors. Chromatin compaction is controlled by multivalent weak interactions, resulting in a complicated conformational landscape that remains challenging to characterize. This work reports a method for characterizing chromatin compaction, Free Energy Spectroscopy (FES), which is based on DNA nanotechnology and transmission electron microscopy. This method experimentally determines the chromatin compaction free energy landscape in terms of end-to-end distance and nucleosome stacking interactions. By deconvolving the free energy landscapes of partially and fully compact tetranucleosomes, FES revealed three separate mechanisms by which linker histones reshape the compaction energetics to condense chromatin. This study establishes FES as a method with the potential to help answer a broad range of mechanistic questions about genome and epigenome function.

biophysics↗

bioRxiv2025

Bi-allelic mutations in KCTD11 cause a new form of autosomal recessive intermediate Charcot-Marie-Tooth disease

Charcot-Marie-Tooth disease (CMT) is the most common inherited neuromuscular disorder, characterized by progressive, length-dependent degeneration of peripheral nerves, resulting in distal muscle atrophy and weakness, foot and hand deformities, and sensory deficits. The disease is clinically and genetically heterogeneous, with over 125 disease-causing genes identified to date. Here, genetic studies in ten patients from 5 unrelated families of diverse ethnic background, led to the identification of KCTD11 as a novel CMT gene, responsible for a new autosomal recessive intermediate CMT subtype, RI-CMTE. The variants identified are loss of function. KCTD11 encodes KCTD11/REN, a protein of yet unknown function in the Peripheral Nervous System, known to regulate HDAC1, {beta}-catenin, and mTORC1, key regulators of myelination and neuronal differentiation in the PNS. To explore KCTD11s role in the PNS, we used a constitutive Kctd11-/- mouse model and the derived in vitro myelin model of sensory neuron and Schwann cell co-culture (DRGN/SC), to mimic the loss-of-function induced by patient mutations. We first demonstrate that the loss of KCTD11 is due to enhanced degradation of the mutated protein via autophagy. Both in vitro and in vivo, we demonstrate abnormal myelination in vivo and altered myelination dynamics in vitro. These defects were associated with dysregulation of the expression of key transcription factors in Schwann cells, such as Egr2 and Sox10, along with other myelin-related genes, as revealed by mRNA-sequencing data. Regarding pathophysiological mechanisms, we identified dysregulation of HDAC1 expression, as well as alterations in the Wnt/{beta}-catenin, Sonic Hedgehog and Hippo/YAP signaling pathways. The deregulation of these pathways seem to converge to altered autophagy and altered balance between proliferation, differentiation and apoptosis, at least in Schwann cells. These mechanisms remain to be explored in axons from PNS neurons. Altogether, our results identify KCTD11 as a novel gene defective in autosomal recessive intermediate RI-CMTE and highlight the key role of KCTD11 in maintaining myelin homeostasis through regulation of HDAC1 and phosphorylated {beta}-catenin levels, thereby preventing late-onset myelin abnormalities and degradation.

genetics↗

bioRxiv2025

Red-wine gene networks associated with exceptional longevity in humans

Moderate consumption of red wine has been associated with healthy aging and longevity, defined as one drink per day for women and two drinks per day for men (approximately 142 ml or 5 oz per drink). Previous research has revealed the health benefits of red wine, particularly in relation to cardiovascular disease. However, the influence of genetic factors on these benefits remains to be elucidated. In this study, we explored genes linked to red wine and created a curated gene set that intersects with those related to centenarians, who are markers of exceptional longevity. Utilizing over 190 databases, we identified and validated a curated list of genes, and conducted gene set enrichment analysis as well as enrichment analysis of annotations and diseases. Our findings highlighted 43 genes connected to centenarians, suggesting that these genes play a crucial role in stress response and apoptosis, which are essential for cell survival and renewal. Additionally, we noted that these genes were enriched in pathways associated with smooth muscle cell proliferation, neuroinflammation, nucleotide excision repair, and lipoprotein metabolism (false discovery rate, FDR < 3 x 10-07). Gene set enrichment analysis indicated significant tissue expression in the gastrointestinal, cardiovascular, and respiratory systems. Furthermore, the disease-gene enrichment analysis pointed to associations with the diseases related to the tissues, including cardiovascular disease (heart disease and stroke), type 2 diabetes, gastrointestinal diseases and metabolic diseases, immune diseases, and cancer (FDR < 9.37 x 10-6); notably, cardiovascular diseases, diabetes, and cancer are leading causes of death, suggesting that the genes may be protective against those diseases. Although further research is necessary to uncover additional genes, this study provides the first genetic overview of the health benefits of red wine, emphasizing its potential in supporting healthy aging and longevity.

genomics↗

bioRxiv2024

Cyclooxygenase-2 disruption predisposes to heart failure with preserved ejection fraction

Heart failure (HF) is one of the most strongly associated adverse cardiovascular events linked to the use of cyclooxygenase (COX)-2 selective and non-selective nonsteroidal anti-inflammatory drug (NSAID). Nevertheless, it remains uncertain whether NSAID exposure is more likely to lead to heart failure with reduced ejection fraction (HFrEF) or preserved ejection fraction (HFpEF). In adult mice, postnatal genetic deletion or pharmacological inhibition of COX-2 did not affect cardiac function. In contrast, aged female inducible COX-2 (iCOX-2) knockout (KO) mice displayed diastolic dysfunction, cardiac hypertrophy, pulmonary congestion, and elevated levels of plasma N-terminal pro B-type natriuretic peptide (BNP) when compared to age- and sex- matched controls, while their ejection fraction (EF) remained preserved ([≥] 50%). No such phenotype was observed in aged male iCox-2 KO mice. Aged female iCox-2 KO mice showed a shift from prostanoid to leukotriene biosynthesis, along with changes in the expression of mitochondrial genes and calcium-handling proteins in the myocardium. The ratio of phospholamban to SERCA2a was increased, indicating an inhibitory effect on SERCA2a activity, which may contribute to impaired myocardial relaxation. In larval zebrafish, COX-2 inhibition by celecoxib caused a modest yet significant reduction in heart rate and diastolic function, while EF was preserved. Additionally, celecoxib increased BNP expression and ventricular calcium transient amplitude. Diabetic patients in the Harvard-Partners electronic medical record exposed to NSAIDs selective for COX-2 inhibition were more strongly associated with an increased risk of HFpEF compared to HFrEF. Collectively, these findings indicate that COX-2 deletion or inhibition does not impair systolic cardiac function but instead leads to an HFpEF phenotype in mice, zebrafish, and humans. An imbalance in calcium handling may mediate the impairment of myocardial relaxation following COX-2 suppression. SummaryGenetic deletion or pharmacological inhibition of COX-2 results in heart failure with preserved ejection fraction across zebrafish, mice, and humans.

pharmacology and toxicology↗

bioRxiv2024

Control of alveolar bone development, homeostasis, and socket healing by salt inducible kinases

Alveolar bone supports and anchors teeth. The parathyroid hormone-related protein (PTHrP) pathway plays a key role in alveolar bone biology. Salt inducible kinases (SIKs) are important downstream regulators of PTH/PTHrP signaling in the appendicular skeleton where SIK inhibition increases bone formation and trabecular bone mass. However, the function of these kinases in alveolar bone remains unknown. Here, we report a critical role for SIK2/SIK3 in alveolar bone development, homeostasis, and socket healing after tooth extraction. Inducible SIK2/SIK3 deletion led to dramatic alveolar bone defects without changes in tooth eruption. Ablating these kinases impairs alveolar bone formation due to disrupted osteoblast maturation, a finding associated with ectopic periostin expression by fibrous cells in regions of absent alveolar bone at steady state and following molar extraction. Distinct phenotypic consequences of SIK2/SIK3 deletion in appendicular versus craniofacial bones prompted us to identify a specific transcriptomic signature in alveolar versus long bone osteoblasts. Thus, SIK2/SIK3 deletion illuminates a key role for these kinases in alveolar bone biology and highlights the emerging concept that different osteoblast subsets utilize unique genetic programs. Summary statementSIK2/SIK3 deletion in alveolar bone reduces bone formation and mass by impairing osteoblast maturation, unlike in long bones, where it increases bone formation and mass.

developmental biology↗

bioRxiv2024

Altered NRG1/ErbB4 signaling and cholesterol metabolism dysregulation are key pathomechanisms in VRK1-related motor neuropathies and motor neuron diseases

Hereditary Motor and Sensory Neuropathy (HMSN), or Charcot-Marie-Tooth disease (CMT), are the most common group of Inherited peripheral neuropathies (IPN), characterized by a strong clinical and genetic heterogeneity. Among them, distal Hereditary Motor Neuropathy (dHMN), also known as neuronopathy, is a subgroup, where only motor nerves are affected. This subgroup is also genetically heterogeneous, with 25 genes described to date, of which VRK1, that we have recently described as responsible for dHMN, associated to upper motor neuron signs. There are now more than thirty mutations in VRK1, which cause a range of neurological diseases affecting motor neurons (mainly lower, but also upper) or their axons in the peripheral nervous system, that we design as VRK1-related motor neuron diseases. In two previous studies, we have demonstrated that dHMN due to VRK1 mutations lead to reduced levels of VRK1 in the nucleus, and that this depletion alters the dynamics of coilin, a phosphorylation target of VRK1. hiPSC-derived Motor Neurons (hiPSC-MN) from these patients, display Cajal Bodies (CBs) disassembly and defects in neurite outgrowth and branching, altered Action Potential (AP) waveform and decreased Axonal Initial Segment (AIS) length. In this study, we have further studied the link between the loss of VRK1 function and the defects observed in hiPSC-MNs, by realizing bulk mRNA-Seq sequencing in this in vitro model of the disease. Our results evidenced altered NRG1/ERBB4 signaling, leading to cholesterol metabolism dysregulation and deregulation of genes encoding the glutamate receptors AMPAR and NMDAR, which role in the Axonal Initial Segment and abnormal AP initiation in hiPSC-MNs remains to be investigated.

genetics↗

bioRxiv2024

Cardiomyocyte PGC-1α enables physiological adaptations to endurance exercise through suppression of GDF15 and cardiac atrophy

Exercise training induces physiological cardiac hypertrophy, enhanced mitochondrial biogenesis and myocardial contractility. In skeletal muscle, the transcriptional coactivator PGC-1 is a key orchestrator of these responses. The heart expresses abundant and exercise-responsive PGC-1, but it is unclear whether cardiomyocyte PGC-1 is necessary for cardiac adaptation to endurance training. Here we demonstrate that cardiomyocyte PGC-1 is required for physiological cardiac hypertrophy during exercise training in mice. In the absence of cardiomyocyte PGC-1, voluntary wheel running does not improve exercise capacity and instead confers immune-fibrotic-atrophic heart failure after just 6 weeks of training. We identify cardiomyocyte PGC-1 as a negative regulator of stress-responsive senescence gene expression. The most enriched of these is the myomitokine GDF15. GDF15 is secreted locally but not systemically in PGC-1-deficient mouse hearts and reduces cardiomyocyte size. Cardiomyocyte-specific reduction of GDF15 expression preserves exercise tolerance and cardiac contractility in PGC-1-deficient mice during endurance training. Finally, we show that cardiomyocyte PPARGC1A expression correlates with cardiomyocyte number and negatively with GDF15 expression in human cardiomyopathies through single nucleus RNA sequencing. Our data implicate cardiomyocyte PGC-1 as a vital safeguard against stress-induced atrophy and local GDF15-induced dysfunction during exercise.

physiology↗

bioRxiv2024

Piggybacking functionalized DNA nanostructures into live cell nuclei

DNA origami (DO) are promising tools for in vitro or in vivo applications including drug delivery; biosensing, detecting biomolecules; and probing chromatin sub-structures. Targeting these nanodevices to mammalian cell nuclei could provide impactful approaches for probing visualizing and controlling important biological processes in live cells. Here we present an approach to deliver DO strucures into live cell nuclei. We show that labelled DOs do not undergo detectable structural degradation in cell culture media or human cell extracts for 24 hr. To deliver DO platforms into the nuclei of human U2OS cells, we conjugated 30 nm long DO nanorods with an antibody raised against the largest subunit of RNA Polymerase II (Pol II), a key enzyme involved in gene transcription. We find that DOs remain structurally intact in cells for 24hr, including within the nucleus. Using fluorescence microscopy we demonstrate that the electroporated anti-Pol II antibody conjugated DOs are efficiently piggybacked into nuclei and exihibit sub-diffusive motion inside the nucleus. Our results reveal that functionalizing DOs with an antibody raised against a nuclear factor is a highly effective method for the delivery of nanodevices into live cell nuclei.

bioengineering↗

bioRxiv2023

Construction and reconfiguration of dynamic DNA origami assemblies with coiled-coil patches and patterns

DNA origami nanodevices achieve programmable structure and tunable mechanical and dynamic properties by leveraging the sequence specific interactions of nucleic acids. Previous advances have also established DNA origami as a useful building block to make well-defined micron-scale structures through hierarchical self-assembly, but these efforts have largely leveraged the structural features of DNA origami. The tunable dynamic and mechanical properties also provide an opportunity to make assemblies with adaptive structure and properties. Here we report the integration of DNA origami hinge nanodevices and coiled-coil peptides into hybrid reconfigurable assemblies. With the same dynamic device and peptide interaction, we make multiple higher order assemblies by organizing clusters of peptides (i.e. patches) or arranging single peptides (i.e. patterns) on the surfaces of DNA origami to control the relative orientation of devices. We use coiled-coil interactions to construct circular and linear assemblies whose structure and mechanical properties can be modulated with DNA-based actuation. Actuation of linear assemblies leads to micron scale motions and [~]2.5-10-fold increase in bending stiffness. Our results provide a foundation for stimulus responsive hybrid assemblies that can adapt their structure and properties in response to nucleic acid, peptide, protein, or other triggers.

bioengineering↗

bioRxiv2023

Cooperative control of a DNA origami force sensor

Most biomolecular systems are dependent on a complex interplay of forces. Modern force spectroscopy techniques provide means of interrogating these forces. These techniques, however, are not optimized for studies in constrained or crowded environments as they typically require micron-scale beads in the case of magnetic or optical tweezers, or direct attachment to a cantilever in the case of atomic force microscopy. We implement a nanoscale force-sensing device using a DNA origami which is highly customizable in geometry, functionalization, and mechanical properties. The device, referred to as the NanoDyn, functions as a binary (open or closed) force sensor that undergoes a structural transition under an external force. The transition force is tuned with minor alterations of 1 to 3 DNA oligonucleotides and spans tens of picoNewtons (pN). This actuation of the NanoDyn is reversible and the design parameters strongly influence the efficiency of resetting the initial state, with higher stability devices ({gtrsim}10 pN) resetting more reliably during repeated force-loading cycles. Finally, we show that the opening force can be adjusted in real time by the addition of a single DNA oligonucleotide. These results establish the NanoDyn as a versatile force sensor and provide fundamental insights into how design parameters modulate mechanical and dynamic properties.

bioengineering↗

bioRxiv2023

Mechanism of DNA origami folding elucidated by mesoscopic simulations

DNA nanotechnology leverages the canonical base-pairing rules and geometry of DNA to create highly precise nanoscale structures with many potential applications. While the design and fabrication of DNA nanostructures is well-established, the self-assembly process that produces these structures is still poorly understood, especially for DNA origami that involve the assembly of hundreds of strands. Many experimental and computational efforts have sought to better understand DNA origami folding, but the small length and time scales of individual binding events and the long timescale over which folding occurs have posed significant challenges. Here, we present a new mesoscopic model that uses a switchable force field to capture the mechanical behavior of single- and double-stranded DNA motifs and transition between them at a coarseness level of up to 8 nucleotides per particle, allowing access to the long assembly timescales of DNA origami up to several kilobases in size. Brownian dynamics simulations of 4-helix bundle (4HB) structures using this model reveal a hierarchical folding process involving the zipping of structural domains into a partially folded precursor structure followed by gradual crystallization into the final structure. We elucidate the role of hybridization strength, scaffold routing, and staple design in the folding order and kinetics. Simulation of larger 32HB structures reveals heterogeneous staple incorporation kinetics and frequent trapping in metastable states, as opposed to smaller, more accessible structures like the 4HB, which exhibit first-order kinetics and virtually defect-free folding. The development of this model opens an avenue to better understand and design DNA nanostructures for improved yield and folding performance.

biophysics↗

bioRxiv2023

Accelerating the Characterization of Dynamic DNA Origami Devices with Deep Neural Networks

Mechanical characterization of dynamic DNA nanodevices is essential to facilitate their use in applications like molecular diagnostics, force sensing, and nanorobotics that rely on device reconfiguration and interactions with other materials. A common approach to evaluate the mechanical properties of dynamic DNA nanodevices is by quantifying conformational distributions, where the magnitude of fluctuations correlates to the stiffness. This is generally carried out through manual measurement from experimental images, which is a tedious process and a critical bottleneck in the characterization pipeline. While many tools to support analysis of static of molecular structures, there is a need for tools to facilitate the rapid characterization of dynamic DNA devices that undergo large conformational fluctuations. Here, we develop a data processing pipeline based on Deep Neural Networks (DNNs) to address this problem. The YOLOv5 and Resnet50 network architecture were used for the two key subtasks: particle detection and pose (i.e. conformation) estimation. We demonstrate effective network performance (F1 score of 0.85 in particle detection) and good agreement with experimental distributions with limited user input and small training sets. We also demonstrate this pipeline can be applied to multiple nanodevices, providing a robust approach for the rapid characterization of dynamic DNA devices.

bioengineering↗