Cell Reports Physical Science

DNA-nano/microparticle motors are burnt-bridge Brownian ratchets (BBR) moving on an RNA-modified two-dimensional surface driven by Ribonuclease H (RNase H), and are one of the fastest artificial molecular motors. Interestingly, these motors show a maximum speed of [~]30 nm s-{superscript 1} irrespective of the particle size ranging from 100 to 5000 nm, whereas the run-length increases with the particle size. Here we performed geometry-based kinetic simulations of DNA-nano/microparticle motors with the sizes of 100, 500, 1000, and 5000 nm to identify the factors governing speed, run-length, and unidirectionality. The simulations reproduced the experiments quantitatively, and the speed remained constant while the run-length and the unidirectionality increased with the particle size. The constant speed was caused by a trade-off between the step size and the pause length, both of which increased with the particle size. In contrast, the run-length and the unidirectionality increased with the particle size because large particles had high multivalency which suppresses stochastic detachment of the motor, high RNA hydrolysis efficiency under the motor trajectory which realizes almost perfect BBR motion, and stepping direction highly biased to forward. For the smaller motors with 100, 500, and 1000 nm particles, the speed increased from 20 to 200 nm s-{superscript 1} by 10-fold increases in DNA/RNA hybridization, RNase H binding, and RNA hydrolysis rates (from 0.8 to 8.0, 7.2 to 72, and 3.0 to 30 s-{superscript 1}, respectively), even when considering the rotational diffusion of these particles. On the other hand, the speed for the largest motor with 5000 nm particle was limited to 100 nm s-{superscript 1}, because the time required for rolling motion ([~]0.3 s) became comparable to the pause length. Our results indicate that DNA-particle motors must possess a nanoscale body to achieve a speed exceeding 100 nm s-{superscript 1}. SignificanceAutonomous artificial molecular motors have a potential to power nano- and micron-scale actuators and devices, but their performances such as speed, run-length, and unidirectionality are inferior to natural motor proteins. Using geometry-based kinetic simulations, we quantitatively analyzed performance metrics of artificial DNA-nano/microparticle motors which autonomously move on RNA-modified two-dimensional surfaces by a burnt-bridge Brownian ratchet mechanism. Our study revealed the mechanism why their speed is almost independent of the particle size, while the run-length and unidirectionality increases with the particle size. We also identified how multivalent binding, mode of detachment, and rotational diffusion set fundamental limits of the speed, run-length, and unidirectionality. Our results provide a general design strategy for engineering high-performance artificial molecular motors.

Optogenetic neuropathway inhibition is a powerful approach for dissecting circuit functions. This strategy, however, frequently encounters practical challenges due to insufficient expression or performance of the optogenetic silencer on axonal projections/terminals. HcKCR1, a light-gated potassium-selective channel from Hyphochytrium catenoides, has shown great promise for optogenetic inhibition. Unfortunately, the application of HcKCR1 in neuropathway manipulations is hindered by its unsatisfactory gating properties and poor axonal trafficking. To overcome these hurdles, we first engineered a performance-improved HcKCR1 (piKCR) that allowed more reliable neuronal inhibition at low intensities of green or red light. We next engineered an axon-targeted piKCR (piKCR.AT) that demonstrated long-range axonal trafficking and optical presynaptic inhibition in the mouse hippocampus. When piKCR.AT was expressed in the cerebellar Purkinje Cells (PCs), optical manipulation of PC outputs to the deep cerebellar nuclei robustly disrupted mouse movement on the balance beam. With enhanced performance and axonal distribution, piKCR.AT may provide new opportunities for elucidating neuropathway functions in health and diseases.

The advent of CRISPR-Cas systems has revolutionized multiple fields, including basic science, biotechnology, and medicine. Central to this versatility is the use of programmable guide RNAs (gRNAs), which enable flexible and specific gene targeting. Building on this principle, various CRISPR-associated tools have been developed, including Cas9, base editors, prime editors, and gene-regulation platforms. However, because most CRISPR modalities share a common gRNA platform, the simultaneous use of multiple tools is constrained by interactions among different gRNAs, limiting orthogonal genome editing. This study presents a method for orthogonal multiplexed gene editing by packaging distinct CRISPR effectors and their corresponding gRNAs into separate virus-like particles (VLPs), thereby virtually eliminating the gRNA crosstalk. Furthermore, we demonstrate that this VLP-based strategy enables orthogonal, multiplexed gene editing in vivo in mouse eyes and ears. This platform expands the CRISPR toolkit by enabling simultaneous, non-interfering genetic manipulations in both cultured cells and living organisms.

Living organisms achieve adaptive actuation through the seamless integration of neural motor control circuitry and proprioceptive feedback. While biohybrid robotics aims to replicate these capabilities by merging engineered muscle with synthetic scaffolds, the field remains limited by interfaces that lack the efficiency and closed-loop regulation of natural neuromuscular systems. Here, we introduce a biohybrid muscle actuator system featuring a bioelectronic interface based on soft poly(3,4-ethylenedioxythiophene) (PEDOT) fibers for stimulation and sensing. These fibers conformally couple to muscle tissues, eliciting robust contractions at voltages as low as 1 V--requiring ultra-low power (0.376 {+/-} 0.034 mW) and preserving long-term tissue viability. By leveraging the independent addressability of these fibers, we demonstrate selective actuation of individual muscle units to achieve precise spatiotemporal control of a two-muscle-powered walking biohybrid robot, reaching a locomotion speed of 5.43 {+/-} 0.79 mm/min. When configured as strain sensors, the fibers exhibit a high gauge factor of 155.45 {+/-} 6.59 and resolve contractile displacements within tens of micrometers. We demonstrate that this sensing modality can be integrated into a closed-loop controller to autonomously modulate stimulation based on real-time feedback, significantly mitigating muscle fatigue (p = 0.038) during continuous operation. This work establishes a versatile platform for efficient actuation and intrinsic feedback sensing, providing a blueprint for efficient, autonomous, and adaptive biohybrid machines. SummarySoft conductive fibers enable a bioelectronic interface for low-power actuation and closed-loop control in biohybrid robots.

The precise integration of large DNA fragments into the human genome holds significant therapeutic potential. Here, we demonstrate that combining engineered piggyBac (PB) transposase with CRISPR/Cas9 enables targeted integration of PB transposons into specified genomic loci. Our engineered PB transposase (PBase) retains high excision activity while substantially reducing endogenous integration activity. In the developed Cas9-PBase fusion system, PBase excises the transposon to generate linear DNA fragments, while Cas9 introduces site-specific double-strand breaks (DSBs), facilitating insertion of the excised fragment at the target locus. The optimized tool achieves 6.1-7.3 kb transposon integration at multiple genomic sites with 10-15% efficiency, demonstrating 60-80% targeted integration specificity. As a proof of concept, we inserted a 7.1 kb transposon encoding three genes into the {beta}2M locus of human induced pluripotent stem cells (iPSCs), conferring protection against allogeneic natural killer (NK) cell-mediated cytotoxicity in derived iNK cells. These results establish Cas9-PBase as a precise and programmable platform for large DNA sequence insertion with potential clinical applications.

Ganoderma adspersum is a white-rot fungus (WRF) that produces amphipathic, surface-active proteins, known as hydrophobins. To explore sustainable routes for protein production and waste valorization, G. adspersum was cultivated under defined shaking conditions using different carbon and nitrogen sources, including apple skin food waste. From these cultures, we report for the first time the isolation and characterization of a novel class I hydrophobin, designated as Gad1. Atomic force microscopy revealed abundant rodlet-like nanostructures consistent with class I hydrophobin assemblies, and Raman spectroscopy confirmed {beta}-sheet enrichment typical of amyloid-like organization. MALDI-TOF mass spectrometry further identified Gad1 along with additional hydrophobin-like proteins in the foam. Hydrophobin-enriched foam extracts were used to form stable oil-in-water emulsions that could be converted into porous, freezedried composite aerogels. These findings expand the known diversity of hydrophobins in whiterot fungi and demonstrate that food-waste-derived substrates can support hydrophobin production and functional biomaterial formation.

Precise manipulation of gene expression is pivotal for gene function studies and the optimization of gene therapy. RNA-based gene switches are attractive tools due to their robust tunability by FDA-approved small molecules, the absence of exogenous immunogenic proteins, and the small size for gene delivery vectors such as adeno-associated virus (AAV). However, existing RNA switches only target a single step of gene expression such as transcription or RNA splicing, exhibiting intrinsic limitations in gene regulation. To overcome this issue, this study integrated the aptamer-based polyA regulator (pA), the drug-elicitable alternative splicing module (DreAM) and an engineered translation modulator with conditional upstream open reading frames (uORFs) to construct the DreAM-plus RNA switch. The pA-DreAM concatenation led to 1.5[~]5.0-fold and 1.2[~]4.4-fold increase of inducible fold changes than pA and DreAM, respectively. The uORF module further enhanced the switching performance by 1.4[~]6.3-fold. DreAM-plus-mediated transient transgene expression demonstrated a temporal resolution of about 24 hours and high tissue specificity to liver or heart. Critically, DreAM-plus achieved transient expression of an array of gene editors (SpCas9, SaCas9, Un1Cas12f1, OsCas12f1, AcCas12n, IsDra2 TnpB etc.) that significantly mitigated off-target effects by 1.4[~]2.8 folds in plasmids, lentivirus and AAV. In a new mouse model with lipid-nanoparticle-delivered pre-existing immunity, DreAM-plus attenuated AAV-delivered Cas-specific CD8 T cell immune toxicity in the liver and the heart. Therefore, multiple RNA switches could be synergistically integrated to build more sophisticated genetic cassettes for enhanced manipulation of gene expression.

The microbial extracellular matrix (ECM) is a complex network of self-secreted biopolymers uniting the cells in biofilms, providing them with structural integrity, and contributing to their elevated resistance to antibiotic treatments. Recently, there is a growing realization that a regulated, bidirectional cross-talk of bacteria and ECM confers biofilms with tissue-like traits, however, the mechanisms of spatio-temporal self-organisation of ECM and its regulation are still poorly understood. In the model organism for biofilm formation Bacillus subtilis, TasA is the major protein component of the extracellular matrix. We recently showed that TasA, isolated in the form of stable and structured globules, assembles into elongated and ordered fibers via a donor-strand complementation mechanism. In this study, we discovered that in the presence of zinc metal ions, TasA is able to form hydrogels with > 97% water content. Electron- and atomic force-microscopies as well as small angle X-ray scattering measurements show that cross-linking with zinc ions induces a transition in TasA morphology from one-dimensional fibers to two-dimensional sheets. Electron paramagnetic resonance measurements then show that such a significant morphological shift is associated with molecular changes in the coordination environment of zinc ions, which lead to structural changes at the protein level. When assembling into macroscopic networks, TasA-Zn metallogels exhibit viscoelastic properties and a fast recovery following an excessive strain. These metallogels represent a novel class of bacterially-derived ECMs that form easily at room temperature without covalent crosslinking, and may be used as a natural matrix-mimics in biofilm models for infection studies.

Scaffolded DNA origami has become a valuable nanoscale tool for applications in biomedical and physical sciences. Critical to leveraging the modular and programmable properties of DNA origami nanodevices is access to the scaffold strand, a long single-stranded DNA (ssDNA) of precise length and sequence, which is folded into a compact shape via piecewise base-pairing with many staple strands, short ssDNA oligonucleotides. Current methods to produce and manipulate long ssDNA scaffolds can be costly, time-consuming, and cumbersome. In contrast, methods to produce and manipulate the sequence of double-stranded DNA (dsDNA) are efficient and scalable. Here, we present a method for the rapid isolation of target ssDNA sequences from a variety of dsDNA sources using oligonucleotides as blocking strands that bind continuously to the undesired strand, thereby releasing the target scaffold strand. We report successful ssDNA isolation from linear and supercoiled dsDNAs of various sequences and lengths, ranging from 769 to 15,101 nucleotides. In addition to isolating ssDNA, we demonstrated this approach enables folding of DNA origami directly from dsDNA templates using both blocking and staple strands in a single-pot thermally controlled reaction. Furthermore, we explore multi-scaffold and gene-encoding DNA origami structures, expanding the framework for application-based designs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/709872v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1cc75dcorg.highwire.dtl.DTLVardef@4df8e2org.highwire.dtl.DTLVardef@10ed113org.highwire.dtl.DTLVardef@1c05bdd_HPS_FORMAT_FIGEXP M_FIG C_FIG

Efficient intracellular delivery of nucleic acids, proteins, and other biomolecules is critical to advancing therapeutic strategies and genome-editing technologies. Lipid nanoparticles (LNPs) have emerged as highly promising delivery vehicles owing to their self-assembly properties, biocompatibility, and capacity to encapsulate large molecular cargos. Their biological performance is determined largely by lipid composition, which influences particle stability, cellular uptake, membrane fusion, and intracellular trafficking. In this study, we designed and optimized LNP formulations inspired by the lipid architecture of enveloped viruses. Four distinct formulations were generated and systematically evaluated in mammalian cell culture, leading to the identification of two lead candidates with superior delivery characteristics. The biodistribution and translocation properties of these formulations were subsequently assessed using an in vitro brain endothelial barrier model to mimic brain environment. Furthermore, we demonstrated that the selected LNPs enable efficient and functional delivery of CRISPR-Cas ribonucleoprotein complexes to mammalian cells. Together, these findings underscore the potential of rationally engineered LNPs as versatile, safe, and effective non-viral delivery platforms for advanced genome-editing applications.

Mushrooms are increasingly recognized as delicious, nutritious, and sustainable foods, with an intrinsic umami flavor and fibrous microstructure that can approximate meat-like texture. Among them, lions mane mushroom has emerged as a promising candidate for whole-cut meat alternatives. Yet, its mechanical, rheological, and sensory properties remain largely unquantified. Here we show that a minimally processed lions mane mushroom steak exhibits distinctive mechanical, rheological, and sensory characteristics that position it favorably among existing meat alternatives. Despite its pronounced fibrous morphology, lions mane steak behaves predominantly as an isotropic material under both mechanical loading and rheological testing, with elastic stiffnesses of E = 33.2 kPa and E = 34.8 kPa in-plane and cross-plane. A fundamental challenge in alternative protein development is to understand how these measurable physical properties relate to human texture perception. In a complementary sensory survey, n = 21 participants ranked lions mane steak as more fatty, fibrous, moist, and meaty than eight animal- and plant-based comparison meats. Strikingly, our perceived sensory softness correlates inversely with our experimentally measured mechanical stiffness ({tau}=-0.60, p = 0.02) and rheological loss modulus ({tau}=-0.56, p = 0.03). Taken together, our results demonstrate that lions mane steak combines favorable mechanical performance with desirable sensory attributes and provide a mechanistic link between physics and taste. Our findings highlight lions mane mushroom as a compelling whole-cut alternative protein and underscore the value of integrated mechanical-sensory characterization for rational food design.

The enzymatic depolymerization of polyethylene terephthalate (PET) presents a sustainable route for plastic circularity, but its industrial viability is disadvantaged by the need for thermostable enzymes that remain active under mild, energy-efficient conditions. While the Polyester Hydrolase Leipzig 7 (PHL7) rapidly degrades amorphous PET near its melting point, its poor protein expression, inactivation issues at temperatures above 60{degrees}C and slow depolymerization activity below 60{degrees}C limit its practical application. Here, we employ inverse folding models ProteinMPNN and LigandMPNN, informed by structural and evolutionary information, to redesign the sequence of PHL7, aiming to improve protein expression, thermal stability and activity. From 36 designed variants, we identified two (termed D5 and D11) with significantly enhanced PET depolymerization rates at lower temperatures, where enzymatic performance is typically limited. Remarkably, design D5 at 50{degrees}C achieved the same product yield as PHL7 at 70{degrees}C in 24 h PET microparticle degradation assays, with a shifted product profile favoring mono-(2-hydroxyethyl) terephthalate (MHET) over terephthalic acid (TPA). Molecular dynamics simulations revealed that the active redesigns exhibit enhanced local flexibility in key active site regions at 50{degrees}C, providing a mechanistic understanding of their low-temperature catalysis. This work demonstrates that computational sequence redesign can optimize biocatalysts for lower production costs and milder operational conditions. Furthermore, the D5 variant enables a potential route to resynthesize virgin PET via MHET polycondensation, offering an efficient circular economy pathway.

Micronutrient deficiencies in soils are a critical challenge in agriculture, particularly in acidic soil environments where nutrient availability is strongly limited by fixation, leaching, and altered metal speciation. These constraints contribute to inefficient nutrient uptake and reduced crop yields. Conventional micronutrient supplementation methods are often inefficient, environmentally harmful, and unsustainable, underscoring the need for smarter delivery systems tailored to soil pH conditions. In this study, we developed biodegradable, pH-responsive microbeads from {kappa}-carrageenan ({kappa}-CG) and trans-ferulic acid (TFA) for targeted micronutrient release. The {kappa}-CG-TFA microbeads were synthesized via an eco-friendly process and optimized for size, morphology, stability, and nutrient retention. Characterization confirmed the successful incorporation of functional groups, while swelling, degradation, and release studies demonstrated efficient delivery of essential micronutrients (Mn2+, Zn2+, Cu2+, and Fe3+) under acidic conditions (pH 4.0), mimicking acidic soil environments. The inherent antioxidant activity of TFA conferred strong radical-scavenging capacity, further enhancing its functionality. Soil water and plant growth assays revealed that the microbeads improved micronutrient availability, significantly increased chlorophyll content and leaf area, promoted vigorous seedling growth, and caused no phytotoxic effects. Collectively, these findings establish {kappa}-CG-TFA microbeads as a promising, eco-friendly platform for sustainable micronutrient delivery and stress reduction, thereby improving crop productivity in agriculture.

Bacterial cellulose (BC) hydrogels produced by Gluconacetobacter species hold considerable promise for a wide range of applications owing to their exceptional mechanical properties, biocompatibility, and biodegradability. Achieving precise control over their structural and mechanical characteristics is crucial for the engineering of BC-based materials. In this study, we investigated the formation dynamics and structural features of BC hydrogels, emphasizing the complex interplay between cellulose nanofibril secretion and bacterial motility. Comprehensive tracking of bacterial movement during hydrogel formation has validated mechanisms underlying the development of branching and merging junctions, which are key elements that define the networks physical properties. Additionally, we observed the emergence of vortex-lattice and chiral-nematic structures during hydrogel development, depending on bacterial and cellulose densities. These insights contribute to a fundamental understanding of bottom-up 3D fabrication of BC hydrogels that harness the collective behavior of cellulose-producing bacteria.

Although biohybrid robots offer the potential for soft, adaptive actuation by harnessing living muscle, practical operation in cell culture environments is often limited by the requirement of immersed leads or cumbersome stimulation equipment. Here, we present a thin, miniaturized, wireless bioelectronic stimulator that can electrically drive biohybrid robots while maintaining stability in aqueous cell culture media. Built on a 50-{micro}m liquid crystal polymer (LCP) substrate, the device integrates a planar receiving coil, interconnects, a diode-based rectifier, and a tank capacitor. This enables the device to convert an approximately 4.9-MHz radio-frequency (RF) input into pulsed direct current (DC), which is delivered through integrated stimulation electrodes. The stimulator has a footprint of [~]23 mm2 and a total thickness and mass of [~]100 {micro}m and [~]7 mg, respectively. We integrated the stimulator with a nanopatterned carbon nanotube (CNT)/gelatin hydrogel fin seeded with human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to generate propulsion through fin flapping. By optimizing the thickness of the polydimethylsiloxane (PDMS) encapsulation layer, the density was tuned, and the robot remained freely floating and retained shape integrity during operation. This produced autonomous forward locomotion of [~]70 {micro}m/s. The stimulator generated distance-dependent output voltage pulses of [~]2-6 V and reliably synchronized fin flapping rates of up to 2 Hz without an observable loss of cell attachment or sarcomeric organization. Together, these results establish a compact, media-compatible, wireless, bioelectronic interface suitable for closed-system biohybrid robotics.

RNA structure plays a crucial role in diverse biological processes beyond the translation of genetic information. Therefore, the development of reliable methods for RNA structure prediction is essential for understanding RNA structure-related functions, however accurate and comprehensive RNA structure prediction remains challenging. Here, we focus on secondary structure prediction of transfer RNA (tRNA) using structure probing coupled with next-generation sequencing (tRNA Structure-seq). In silico prediction of Saccharomyces cerevisiae tRNA secondary structures achieves only 56.9% accuracy on average. Incorporation of dimethyl sulfate (DMS) probing data improve prediction accuracy to 87.4%, which is still not sufficient for practical tRNA structure prediction. To overcome this, we optimized the tRNA Structure-seq analysis pipeline by explicitly incorporating natural tRNA modifications detected in tRNA sequencing data and by refining pseudo-free energy parameters specifically optimized for tRNA structure prediction. Using this optimized pipeline, the average prediction accuracy is remarkably improved to 94%. Furthermore, analysis of multiple structural conformations predicted from DMS probing data indicates that S. cerevisiae tRNAs predominantly adopt the canonical cloverleaf secondary structure under in vivo conditions. Finally, we examined tRNA structures under mild stress conditions, including heat stress, osmotic stress, and antibiotic stress. These perturbations had minimal effects on in vivo tRNA secondary structure, demonstrating that S. cerevisiae tRNAs maintain structural stability under physiologically relevant stress conditions. In summary, our results establish an optimized tRNA Structure-seq analysis that enables highly accurate tRNA secondary structure prediction and reveals the intrinsic robustness of tRNA structures in living cells.

There has been a surge in antibiotic resistance in recent years, making traditional antibiotics less effective against key pathogens. RNA has recently emerged as a potential target for antibiotics due to its involvement in crucial microbial functions. It is possible to expand the range of therapeutic targets by using RNA-based therapies, but it remains necessary to improve the molecular-level understanding of interactions between RNA and known and potential binders. The SAM-I riboswitch, which controls the transcriptional termination of gene expression involved in sulfur metabolism in most bacteria, is an excellent ligand target. Thus, understanding its behavior with and without ligand complexes would be very helpful for drug design applications. In this manuscript, we studied the interactions between the SAM-I riboswitch and its natural ligand, SAM, which controls riboswitch function, and compared those interactions to its interactions with the very similar small molecular SAH, which does not control riboswitch function, and to its interactions with a potential binder JS4, identified via virtual screening. From our simulations, we gain a deeper understanding of small molecule interactions with the SAM-I riboswitch. The results reveal how differently the small molecules (SAM, SAH and JS4) bind to and potentially induce conformational changes in the riboswitch. Our findings offer valuable insight into the molecular mechanisms underlying riboswitch RNA-ligand interactions for the design of more effective RNA-targeting therapeutics.

The CRISPR-Cas9 system has revolutionized genome engineering, yet its full therapeutic potential remains constrained by challenges in precisely modulating its activity and specificity. Here we report a fully computational end-to-end pipeline for the de novo design of a single-domain VHH nanobody (NbSpCas9-v1) targeting a structurally conserved, non-catalytic epitope at the PAM-interacting (PI) and RuvC-III interface of Streptococcus pyogenes Cas9 (SpCas9; PDB: 4UN3). Nanobody sequences were generated using BoltzGen, a generative diffusion binder design framework, and co-folded with SpCas9 using Boltz-2 to evaluate structural confidence and binding affinity. The top-ranked model (SpCas9_4UN3_Bivalent_Hub_v1) achieved a complex pLDDT of 0.8406, an aggregate score of 0.8016, and an ipTM of >0.8, indicating high confidence in the nanobody-antigen interface. The designed 1,616-residue quaternary complex (SpCas9 + sgRNA + DNA + nanobody) was subjected to 10 ns of all-atom molecular dynamics (MD) simulation using the AMBER14SB force field within the GROMACS/OpenMM framework. The complex stabilized at RMSD [~]6 [A] with a radius of gyration of 39-44 [A], confirming thermodynamic stability under physiological conditions (310 K, 0.15 M NaCl). A conserved 96.3 [A] inter-molecular distance between the nanobody centroid and the HNH catalytic residue H840 establishes NbSpCas9-v1 as a distal, non-inhibitory binder -- ideally suited for a Bivalent Hub architecture recruiting secondary effectors to the Cas9 ribonucleoprotein (RNP). The nanobody-Cas9 interface is stabilized by 8 hydrogen bonds, 4 salt bridges, and [~]1,850 [A]2 of buried solvent-accessible surface area. These results provide a rigorous structural and dynamic foundation for experimental validation of VHH-based CRISPR-Cas9 enhancers and modulators. GRAPHICAL ABSTRACTThe computational workflow proceeds from SpCas9 crystal structure acquisition (PDB: 4UN3) through BoltzGen nanobody design, Boltz-2 structural co-folding, 10 ns explicit-solvent MD validation, and Bivalent Hub functional characterization. The PyMOL rendering below shows the full quaternary complex at atomistic resolution.

To achieve a therapeutic effect, nanoparticles delivering nucleic acids must facilitate endosomal escape (EE) of their cargo. Despite extensive research, the mechanisms that lead to an effective EE are not sufficiently understood. Herein, we utilized Molecular Dynamics (MD) simulations in All Atom (AA) and Coarse Grained (CG) resolutions to differentiate the interaction of four polymeric formulations (polyplexes) and one lipid nanoparticle (LNP) with endosomal membranes. On the one hand, the results emphasize the benefit of hydrophobic residues in the nanoparticles. On the other hand, the role of anionic lipids in the biological membranes is demonstrated. Furthermore, the identified interaction patterns were successfully correlated to the in vitro performance of the formulations. For the first time, different EE mechanisms of polyplex formulations are visualized in simulation and therefore distinguishable from one another. Hence, this work highlights the power of MD simulations for taking a big step towards better understanding EE efficiency. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/711661v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@abba74org.highwire.dtl.DTLVardef@5e2b8eorg.highwire.dtl.DTLVardef@7db144org.highwire.dtl.DTLVardef@1034e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Rotavirus is a major cause of severe diarrheal disease in children under the age of five, with reduced vaccine effectiveness in low-resource settings causing substantial morbidity and mortality. In the absence of approved antiviral therapeutics, treatment is largely supportive, urging the need for targeted and precision-based interventions. VP4 protein plays an essential role in viral attachment, entry, and infectivity, making it a suitable target for targeted therapy. In this context, RNA interference is a specific method for inhibiting viral gene expression with its efficacy depending on sequence conservation, target accessibility, and compatibility with the RISC-loading machinery. In the present study, an integrative in silico approach was employed to design and evaluate siRNAs targeting conserved regions of the VP4 gene across six geographically diverse countries. Candidate siRNAs were screened using established design rules and regression-based scoring with off-target filtering. Three optimized siRNAs were further assessed through structural modeling, molecular docking, and molecular dynamics simulations to examine interactions with human Dicer, TRBP, and Argonaute-2. Comparative dynamic analyses identified one siRNA with enhanced structural compatibility, reduced conformational fluctuations, and stable interactions with RISC-loading proteins. These findings provide a rational computational basis for VP4-targeted siRNA development, facilitating experimental validation.