RESUMEN
Self-assembled DNA crystals offer a precise chemical platform at the ångström-scale for DNA nanotechnology, holding enormous potential in material separation, catalysis, and DNA data storage. However, accurately controlling the crystallization kinetics of such DNA crystals remains challenging. Herein, we found that atomic-level 5-methylcytosine (5mC) modification can regulate the crystallization kinetics of DNA crystal by tuning the hybridization rates of DNA motifs. We discovered that by manipulating the axial and combination of 5mC modification on the sticky ends of DNA tensegrity triangle motifs, we can obtain a series of DNA crystals with controllable morphological features. Through DNA-PAINT and FRET-labeled DNA strand displacement experiments, we elucidate that atomic-level 5mC modification enhances the affinity constant of DNA hybridization at both the single-molecule and macroscopic scales. This enhancement can be harnessed for kinetic-driven control of the preferential growth direction of DNA crystals. The 5mC modification strategy can overcome the limitations of DNA sequence design imposed by limited nucleobase numbers in various DNA hybridization reactions. This strategy provides a new avenue for the manipulation of DNA crystal structure, valuable for the advancement of DNA and biomacromolecular crystallography.
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5-Metilcitosina , ADN , Cristalización , Catálisis , CristalografíaRESUMEN
Multienzyme assemblies mediated by multivalent interaction play a crucial role in cellular processes. However, the three-dimensional (3D) programming of an enzyme complex with defined enzyme activity in vitro remains unexplored, primarily owing to limitations in precisely controlling the spatial topological configuration. Herein, we introduce a nanoscale 3D enzyme assembly using a tetrahedral DNA framework (TDF), enabling the replication of spatial topological configuration and maintenance of an identical edge-to-edge distance akin to natural enzymes. Our results demonstrate that 3D nanoscale enzyme assemblies in both two-enzyme systems (glucose oxidase (GOx)/horseradish peroxidase (HRP)) and three-enzyme systems (amylglucosidase (AGO)/GOx/HRP) lead to enhanced cascade catalytic activity compared to the low-dimensional structure, resulting in â¼5.9- and â¼7.7-fold enhancements over homogeneous diffusional mixtures of free enzymes, respectively. Furthermore, we demonstrate the enzyme assemblies for the detection of the metabolism biomarkers creatinine and creatine, achieving a low limit of detection, high sensitivity, and broad detection range.
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Enzimas Inmovilizadas , Glucosa Oxidasa , Enzimas Inmovilizadas/química , Peroxidasa de Rábano Silvestre/química , Glucosa Oxidasa/química , ADN/químicaRESUMEN
DNA monolayers with inherent chirality play a pivotal role across various domains including biosensors, DNA chips, and bioelectronics. Nonetheless, conventional DNA chiral monolayers, typically constructed from single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), often lack structural orderliness and design flexibility at the interface. Structural DNA nanotechnology has emerged as a promising solution to tackle these challenges. In this study, we present a strategy for crafting highly adaptable twisted DNA origami-based chiral monolayers. These structures exhibit distinct interfacial assembly characteristics and effectively mitigate the structural disorder of dsDNA monolayers, which is constrained by a limited persistence length of â¼50 nm of dsDNA. We highlight the spin-filtering capabilities of seven representative DNA origami-based chiral monolayers, demonstrating a maximal one-order-of-magnitude increase in spin-filtering efficiency per unit area compared with conventional dsDNA chiral monolayers. Intriguingly, our findings reveal that the higher-order tertiary chiral structure of twisted DNA origami further enhances the spin-filtering efficiency. This work paves the way for the rational design of DNA chiral monolayers.
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ADN de Cadena Simple , ADN , ADN/química , Nanotecnología , Conformación de Ácido NucleicoRESUMEN
Generally, two-dimensional gold nanomaterials have unique properties and functions that offer exciting application prospects. However, the crystal phases of these materials tend to be limited to the thermodynamically stable crystal structure. Herein, we report a DNA framework-templated approach for the ambient aqueous synthesis of freestanding and microscale amorphous gold nanosheets with ultrathin sub-nanometer thickness. We observe that extended single-stranded DNA on DNA nanosheets can induce site-specific metallization and enable precise modification of the metalized nanostructures at predefined positions. More importantly, the as-prepared gold nanosheets can serve as an electrocatalyst for glucose oxidase-catalyzed aerobic oxidation, exhibiting enhanced electrocatalytic activity (~3-fold) relative to discrete gold nanoclusters owing to a larger electrochemical active area and wider band gap. The proposed DNA framework-templated metallization strategy is expected to be applicable in a broad range of fields, from catalysis to new energy materials.
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Oro , Nanoestructuras , Oro/química , Nanoestructuras/química , Oxidación-Reducción , ADN , AguaRESUMEN
Terahertz (THz) waves show nontrivial interactions with living systems, but the underlying molecular mechanisms have yet to be explored. Here, we employ DNA origami as a model system to study the interactions between THz waves and DNA structures. We find that a 3-min THz illumination (35.2 THz) can drive the unwinding of DNA duplexes at â¼10 °C below their melting point. Computational study reveals that the THz wave can resonate with the vibration of DNA bases, provoking the hydrogen bond breaking. The cooperation of thermal and nonthermal effects allows the unfolding of undesired secondary structures and the THz illumination can generate diverse DNA origami assemblies with the yield (>80%) â¼ 4-fold higher than that by the contact heating at similar temperatures. We also demonstrate the in situ assembly of DNA origami in cell lysate. This method enables remotely controllable assembly of intact biomacromolecules, providing new insight into the bioeffects of THz waves.
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ADN , Vibración , Enlace de HidrógenoRESUMEN
A myriad of DNA origami nanostructures have been demonstrated in various intriguing applications. In pursuit of facile yet high-yield synthesis, the mechanisms underlying DNA origami folding need to be resolved. Here, we visualize the folding processes of several multidomain DNA origami structures under ambient annealing conditions in solution using atomic force microscopy with submolecular resolution. We reveal the coexistence of diverse transitional structures that might result in the same prescribed products. Based on the experimental observations and the simulation of the energy landscapes, we propose the heterogeneity of the folding pathways of multidomain DNA origami structures. Our findings may contribute to understanding the high-yield folding mechanism of DNA origami.
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ADN , Nanoestructuras , ADN/química , Microscopía de Fuerza Atómica , Nanoestructuras/química , Nanotecnología , Conformación de Ácido NucleicoRESUMEN
DNA logic circuits are based on DNA molecular programming that implements specific algorithms using dynamic reaction networks. Particularly, DNA adder circuits are key building blocks for performing digital computation. Nevertheless, existing circuit architectures are limited by scalability for implementing multi-bit adder due to the number of required gates and strands. Here, we develop a compact-yet-efficient architecture using cooperative strand displacement reactions (cSDRs) to construct DNA full adder. By exploiting a parity-check algorithm, double-logic XOR-AND gates are constructed with a single set of double-stranded molecule. One-bit full adder is implemented with three gates containing 13 strands, with up to 90% reduction in strand complexity compared to conventional circuit designs. Using this architecture and a transmitter on magnetic beads, we demonstrate DNA implementation of 6-bit adder on a scale comparable to that of a classic electronic full adder chip, providing the potential for application-specific circuit customization for scalable digital computing with minimal reactions.
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Computadores Moleculares , ADN , Algoritmos , ADN/genética , Electrónica , LógicaRESUMEN
Nanozymes have emerged as a class of novel catalytic nanomaterials that show great potential to substitute natural enzymes in various applications. Nevertheless, spatial organization of multiple subunits in a nanozyme to rationally engineer its catalytic properties remains to be a grand challenge. Here, we report a DNA-based approach to encode the organization of gold nanoparticle clusters (GNCs) for the construction of programmable enzyme equivalents (PEEs). We find that single-stranded (ss-) DNA scaffolds can self-fold into nanostructures with prescribed poly-adenine (polyA) loops and double-stranded stems and that the polyA loops serve as specific sites for seed-free nucleation and growth of GNCs with well-defined particle numbers and interparticle spaces. A spectrum of GNCs, ranging from oligomers with discrete particle numbers (2-4) to polymer-like chains, are in situ synthesized in this manner. The polymeric GNCs with multiple spatially organized nanoparticles as subunits show programmable peroxidase-like catalytic activity that can be tuned by the scaffold size and the inter-polyA spacer length. This study thus opens new routes to the rational design of nanozymes for various biological and biomedical applications.
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Nanopartículas del Metal , Nanoestructuras , Catálisis , ADN de Cadena Simple , Oro/química , Nanopartículas del Metal/química , Nanoestructuras/químicaRESUMEN
MOTIVATION: Co-evolution analysis can be used to accurately predict residue-residue contacts from multiple sequence alignments. The introduction of machine-learning techniques has enabled substantial improvements in precision and a shift from predicting binary contacts to predict distances between pairs of residues. These developments have significantly improved the accuracy of de novo prediction of static protein structures. With AlphaFold2 lifting the accuracy of some predicted protein models close to experimental levels, structure prediction research will move on to other challenges. One of those areas is the prediction of more than one conformation of a protein. Here, we examine the potential of residue-residue distance predictions to be informative of protein flexibility rather than simply static structure. RESULTS: We used DMPfold to predict distance distributions for every residue pair in a set of proteins that showed both rigid and flexible behaviour. Residue pairs that were in contact in at least one reference structure were classified as rigid, flexible or neither. The predicted distance distribution of each residue pair was analysed for local maxima of probability indicating the most likely distance or distances between a pair of residues. We found that rigid residue pairs tended to have only a single local maximum in their predicted distance distributions while flexible residue pairs more often had multiple local maxima. These results suggest that the shape of predicted distance distributions contains information on the rigidity or flexibility of a protein and its constituent residues. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Aprendizaje Automático , Proteínas , Proteínas/química , Conformación Molecular , Alineación de Secuencia , Biología Computacional/métodosRESUMEN
Atomically precise gold nanoclusters (AuNCs) are an emerging class of quantum-sized nanomaterials. Intrinsic discrete electronic energy levels have endowed them with fascinating electronic and optical properties. They have been widely applied in the fields of optoelectronics, photovoltaics, catalysis, biochemical sensing, bio-imaging, and therapeutics. Nevertheless, most AuNCs are synthesized in organic solvents and do not disperse in aqueous solutions; this restricts their biological applications. In this review, we focus on the recent progress in the preparation of water-dispersible AuNCs and their biological applications. We first review different methods of synthesis, including direct synthesis from hydrophilic templates and indirect phase transfer of hydrophobic AuNCs. We then discuss their photophysical properties, such as emission enhancement and fluorescence lifetimes. Next, we summarize their latest applications in the fields of biosensing, biolabeling, and bioimaging. Finally, we outline the challenges and potential for the future development of these AuNCs.
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Nanopartículas del Metal , Nanoestructuras , Fluorescencia , Oro/química , Nanopartículas del Metal/química , AguaRESUMEN
The naïve antibody/B-cell receptor (BCR) repertoires of different individuals ought to exhibit significant functional commonality, given that most pathogens trigger an effective antibody response to immunodominant epitopes. Sequence-based repertoire analysis has so far offered little evidence for this phenomenon. For example, a recent study estimated the number of shared ('public') antibody clonotypes in circulating baseline repertoires to be around 0.02% across ten unrelated individuals. However, to engage the same epitope, antibodies only require a similar binding site structure and the presence of key paratope interactions, which can occur even when their sequences are dissimilar. Here, we search for evidence of geometric similarity/convergence across human antibody repertoires. We first structurally profile naïve ('baseline') antibody diversity using snapshots from 41 unrelated individuals, predicting all modellable distinct structures within each repertoire. This analysis uncovers a high (much greater than random) degree of structural commonality. For instance, around 3% of distinct structures are common to the ten most diverse individual samples ('Public Baseline' structures). Our approach is the first computational method to find levels of BCR commonality commensurate with epitope immunodominance and could therefore be harnessed to find more genetically distant antibodies with same-epitope complementarity. We then apply the same structural profiling approach to repertoire snapshots from three individuals before and after flu vaccination, detecting a convergent structural drift indicative of recognising similar epitopes ('Public Response' structures). We show that Antibody Model Libraries derived from Public Baseline and Public Response structures represent a powerful geometric basis set of low-immunogenicity candidates exploitable for general or target-focused therapeutic antibody screening.
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Anticuerpos , Diversidad de Anticuerpos , Linfocitos B , Bases de Datos Genéticas , Epítopos Inmunodominantes , Linfocitos B/química , Linfocitos B/inmunología , Linfocitos B/metabolismo , Biología Computacional , HumanosRESUMEN
The Therapeutic Structural Antibody Database (Thera-SAbDab; http://opig.stats.ox.ac.uk/webapps/therasabdab) tracks all antibody- and nanobody-related therapeutics recognized by the World Health Organisation (WHO), and identifies any corresponding structures in the Structural Antibody Database (SAbDab) with near-exact or exact variable domain sequence matches. Thera-SAbDab is synchronized with SAbDab to update weekly, reflecting new Protein Data Bank entries and the availability of new sequence data published by the WHO. Each therapeutic summary page lists structural coverage (with links to the appropriate SAbDab entries), alignments showing where any near-matches deviate in sequence, and accompanying metadata, such as intended target and investigated conditions. Thera-SAbDab can be queried by therapeutic name, by a combination of metadata, or by variable domain sequence - returning all therapeutics that are within a specified sequence identity over a specified region of the query. The sequences of all therapeutics listed in Thera-SAbDab (461 unique molecules, as of 5 August 2019) are downloadable as a single file with accompanying metadata.
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Anticuerpos/química , Anticuerpos/uso terapéutico , Anticuerpos/inmunología , Ensayos Clínicos como Asunto , Bases de Datos de Proteínas , Humanos , Internet , Metadatos , Alineación de Secuencia , Interfaz Usuario-ComputadorRESUMEN
Therapeutic mAbs must not only bind to their target but must also be free from "developability issues" such as poor stability or high levels of aggregation. While small-molecule drug discovery benefits from Lipinski's rule of five to guide the selection of molecules with appropriate biophysical properties, there is currently no in silico analog for antibody design. Here, we model the variable domain structures of a large set of post-phase-I clinical-stage antibody therapeutics (CSTs) and calculate in silico metrics to estimate their typical properties. In each case, we contextualize the CST distribution against a snapshot of the human antibody gene repertoire. We describe guideline values for five metrics thought to be implicated in poor developability: the total length of the complementarity-determining regions (CDRs), the extent and magnitude of surface hydrophobicity, positive charge and negative charge in the CDRs, and asymmetry in the net heavy- and light-chain surface charges. The guideline cutoffs for each property were derived from the values seen in CSTs, and a flagging system is proposed to identify nonconforming candidates. On two mAb drug discovery sets, we were able to selectively highlight sequences with developability issues. We make available the Therapeutic Antibody Profiler (TAP), a computational tool that builds downloadable homology models of variable domain sequences, tests them against our five developability guidelines, and reports potential sequence liabilities and canonical forms. TAP is freely available at opig.stats.ox.ac.uk/webapps/sabdab-sabpred/TAP.php.
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Regiones Determinantes de Complementariedad , Simulación por Computador , Modelos Moleculares , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Descubrimiento de Drogas , HumanosRESUMEN
In situ synthesis of DNA origami structures in living systems is highly desirable due to its potential in biological applications, which nevertheless is hampered by the requirement of thermal activation procedures. Here, we report a photothermal DNA origami assembly method in near-physiological environments. We find that the use of copper sulfide nanoparticles (CuS NPs) can mediate efficient near-infrared (NIR) photothermal conversion to remotely control the solution temperature. Under a 4 min NIR illumination and subsequent natural cooling, rapid and high-yield (>80%) assembly of various types of DNA origami nanostructures is achieved as revealed by atomic force microscopy and single-molecule fluorescence resonance energy transfer analysis. We further demonstrate the in situ assembly of DNA origami with high location precision in cell lysates and in cell culture environments.
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Nanopartículas , Fototerapia , Cobre , ADN , SulfurosRESUMEN
Repurposing the RNA-guided endonuclease Cas9 to develop artificial CRISPR molecular machines represents a new direction toward synthetic molecular information processing. The operation of CRISPR-Cas9-based machines, nevertheless, relies on the molecular recognition of freely diffused sgRNA/Cas9, making it practically challenging to perform spatially regulated localized searching or navigation. Here, we develop a DNA origami-based single-molecule CRISPR machine that can perform spatially resolved DNA cleavage via either free or localized searching modes. When triggered at a specific site on the DNA origami with nanoscale accuracy, the free searching mode leads to searching activity that gradually decays with the distance, whereas the localized mode generates spatially-confined searching activity. Our work expands the function of CRISPR molecular machines and lays foundations to develop integrated molecular circuits and high-throughput nucleic acid detection.
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Sistemas CRISPR-Cas , División del ADN , Sistemas CRISPR-Cas/genética , ADN/genética , Endonucleasas/metabolismo , Nanotecnología , ARN Pequeño no TraducidoRESUMEN
Multivalent interactions of biomolecules play pivotal roles in physiological and pathological settings. Whereas the directionality of the interactions is crucial, the state-of-the-art synthetic multivalent ligand-receptor systems generally lack programmable approaches for orthogonal directionality. Here, we report the design of programmable atom-like nanoparticles (aptPANs) to direct multivalent aptamer-receptor binding on the cell interface. The positions of the aptamer motifs can be prescribed on tetrahedral DNA frameworks to realize atom-like orthogonal valence and direction, enabling the construction of multivalent molecules with fixed aptamer copy numbers but different directionality. These directional-yet-flexible aptPAN molecules exhibit the adaptability to the receptor distribution on cell surfaces. We demonstrate the high-affinity tumor cell binding with a linear aptPAN oligomer (≈13-fold improved compared to free aptamers), which leads to ≈50 % suppression of cell growth.
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Aptámeros de Nucleótidos , Nanopartículas , Aptámeros de Nucleótidos/química , Membrana Celular/metabolismo , LigandosRESUMEN
Controlling the deposition and diffusion of adsorbed atoms (adatoms) on the surface of a solid material is vital for engineering the shape and function of nanocrystals. Here, we report the use of single-stranded DNA (oligo-adenine, oligo-A) to encode the wettability of gold seeds by homogeneous gold adatoms to synthesize highly tunable plasmonic nanostructures. We find that the oligo-A attachment transforms the nanocrystal growth mode from the classical Frank-van der Merwe to the Volmer-Weber island growth. Finely tuning the oligo-A density can continuously change the gold-gold contact angle (θ) from 35.1±3.6° to 125.3±8.0°. We further demonstrate the versatility of this strategy for engineering nanoparticles with different curvature and dimensions. With this unconventional growth mode, we synthesize a sub-nanometer plasmonic cavity with a geometrical singularity when θ>90°. Superfocusing of light in this nanocavity produces a near-infrared intraparticle plasmonic coupling, which paves the way to surface engineering of single-particle plasmonic devices.
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Nanopartículas del Metal , Nanopartículas , Nanoestructuras , Oro/química , Humectabilidad , ADN/química , Nanoestructuras/química , Nanopartículas/química , Nanopartículas del Metal/químicaRESUMEN
Fluorescence anisotropy (FA) holds great potential for multiplexed analysis and imaging of biomolecules since it can effectively discriminate fluorophores with overlapping emission spectra. Nevertheless, its susceptibility to environmental variation hampers its widespread applications in biology and biotechnology. In this study, we design FA DNA frameworks (FAFs) by scaffolding fluorophores in a fluorescent protein-like microenvironment. We find that the FA stability of the fluorophores is remarkably improved due to the sequestration effects of FAFs. The FA level of the fluorophores can be finely tuned when placed at different locations on an FAF, analogous to spectral shifts of protein-bound fluorophores. The high programmability of FAFs further enables the design of a spectrum of encoded FA barcodes for multiplexed sensing of nucleic acids and multiplexed labeling of live cells. This FAF system thus establishes a new paradigm for designing multiplexing FA probes for cellular imaging and other biological applications.
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ADN/análisis , Polarización de Fluorescencia , Colorantes Fluorescentes/química , Animales , Ratones , Imagen Óptica , Células Tumorales CultivadasRESUMEN
MOTIVATION: T-cell receptors (TCRs) are immune proteins that primarily target peptide antigens presented by the major histocompatibility complex. They tend to have lower specificity and affinity than their antibody counterparts, and their binding sites have been shown to adopt multiple conformations, which is potentially an important factor for their polyspecificity. None of the current TCR-modelling tools predict this variability which limits our ability to accurately predict TCR binding. RESULTS: We present TCRBuilder, a multi-state TCR structure prediction tool. Given a paired αßTCR sequence, TCRBuilder returns a model or an ensemble of models covering the potential conformations of the binding site. This enables the analysis of structurally driven polyspecificity in TCRs, which is not possible with existing tools. AVAILABILITY AND IMPLEMENTATION: http://opig.stats.ox.ac.uk/resources. CONTACT: deane@stats.ox.ac.uk. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Biología Computacional , Programas Informáticos , Algoritmos , Conformación Molecular , Receptores de Antígenos de Linfocitos TRESUMEN
The high sequence specificity and precise base complementary pairing principle of DNA provides a rich orthogonal molecular library for molecular programming, making it one of the most promising materials for developing bio-compatible intelligence. In recent years, DNA has been extensively studied and applied in the field of biological computing. Among them, the toehold-mediated strand displacement reaction (SDR) with properties including enzyme free, flexible design and precise control, have been extensively used to construct biological computing circuits. This review provides a systemic overview of SDR design principles and the applications. Strategies for designing DNA-only, enzymes-assisted, other molecules-involved and external stimuli-controlled SDRs are described. The recently realized computing functions and the application of DNA computing in other fields are introduced. Finally, the advantages and challenges of SDR-based computing are discussed.