Self-assembly of nanocrystal checkerboard patterns via non-specific interactions.

Checkerboard lattices-where the resulting structure is open, porous, and highly symmetric-are difficult to create by self-assembly. Synthetic systems that adopt such structures typically rely on shape complementarity and site-specific chemical interactions that are only available to biomolecular systems (e.g., protein, DNA). Here we show the assembly of checkerboard lattices from colloidal nanocrystals that harness the effects of multiple, coupled physical forces at disparate length scales (interfacial, interparticle, and intermolecular) and that do not rely on chemical binding. Colloidal Ag nanocubes were bi-functionalized with mixtures of hydrophilic and hydrophobic surface ligands and subsequently assembled at an air-water interface. Using feedback between molecular dynamics simulations and interfacial assembly experiments, we achieve a periodic checkerboard mesostructure that represents a tiny fraction of the phase space associated with the polymer-grafted nanocrystals used in these experiments. In a broader context, this work expands our knowledge of non-specific nanocrystal interactions and presents a computation-guided strategy for designing self-assembling materials.

Mechanism of DNA origami folding elucidated by mesoscopic simulations.

Many experimental and computational efforts have sought to understand DNA origami folding, but the time and length scales of this process pose significant challenges. Here, we present a mesoscopic model that uses a switchable force field to capture the behavior of single- and double-stranded DNA motifs and transitions between them, allowing us to simulate the folding of DNA origami up to several kilobases in size. Brownian dynamics simulations of small structures reveal a hierarchical folding process involving zipping into a partially folded precursor followed by crystallization into the final structure. We elucidate the effects of various design choices on folding order and kinetics. Larger structures are found to exhibit heterogeneous staple incorporation kinetics and frequent trapping in metastable states, as opposed to more accessible structures which exhibit first-order kinetics and virtually defect-free folding. This model opens an avenue to better understand and design DNA nanostructures for improved yield and folding performance.

A Programmable DNAzyme for the Sensitive Detection of Nucleic Acids.

Nucleic acids in biofluids are emerging biomarkers for the molecular diagnostics of diseases, but their clinical use has been hindered by the lack of sensitive detection assays. Herein, we report the development of a sensitive nucleic acid detection assay named SPOT (sensitive loop-initiated DNAzyme biosensor for nucleic acid detection) by rationally designing a catalytic DNAzyme of endonuclease capability into a unified one-stranded allosteric biosensor. SPOT is activated once a nucleic acid target of a specific sequence binds to its allosteric module to enable continuous cleavage of molecular reporters. SPOT provides a highly robust platform for sensitive, convenient and cost-effective detection of low-abundance nucleic acids. For clinical validation, we demonstrated that SPOT could detect serum miRNAs for the diagnostics of breast cancer, gastric cancer and prostate cancer. Furthermore, SPOT exhibits potent detection performance over SARS-CoV-2 RNA from clinical swabs with high sensitivity and specificity. Finally, SPOT is compatible with point-of-care testing modalities such as lateral flow assays. Hence, we envision that SPOT may serve as a robust assay for the sensitive detection of a variety of nucleic acid targets enabling molecular diagnostics in clinics.

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.

Prediction and Control in DNA Nanotechnology.

DNA nanotechnology is a rapidly developing field that uses DNA as a building material for nanoscale structures. Key to the field's development has been the ability to accurately describe the behavior of DNA nanostructures using simulations and other modeling techniques. In this Review, we present various aspects of prediction and control in DNA nanotechnology, including the various scales of molecular simulation, statistical mechanics, kinetic modeling, continuum mechanics, and other prediction methods. We also address the current uses of artificial intelligence and machine learning in DNA nanotechnology. We discuss how experiments and modeling are synergistically combined to provide control over device behavior, allowing scientists to design molecular structures and dynamic devices with confidence that they will function as intended. Finally, we identify processes and scenarios where DNA nanotechnology lacks sufficient prediction ability and suggest possible solutions to these weak areas.

Controlling Silicification on DNA Origami with Polynucleotide Brushes.

DNA origami has been used as biotemplates for growing a range of inorganic materials to create novel organic-inorganic hybrid nanomaterials. Recently, the solution-based silicification of DNA has been used to grow thin silica shells on DNA origami. However, the silicification reaction is sensitive to the reaction conditions and often results in uncontrolled DNA origami aggregation, especially when growth of thicker silica layers is desired. Here, we investigated how site-specifically placed polynucleotide brushes influence the silicification of DNA origami. Our experiments showed that long DNA brushes, in the form of single- or double-stranded DNA, significantly suppress the aggregation of DNA origami during the silicification process. Furthermore, we found that double-stranded DNA brushes selectively promote silica growth on DNA origami surfaces. These observations were supported and explained by coarse-grained molecular dynamics simulations. This work provides new insights into our understanding of the silicification process on DNA and provides a powerful toolset for the development of novel DNA-based organic-inorganic nanomaterials.

Interplay between stochastic enzyme activity and microtubule stability drives detyrosination enrichment on microtubule subsets.

Microtubules in cells consist of functionally diverse subpopulations carrying distinct post-translational modifications (PTMs). Akin to the histone code, the tubulin code regulates a myriad of microtubule functions, ranging from intracellular transport to chromosome segregation. However, how individual PTMs only occur on subsets of microtubules to contribute to microtubule specialization is not well understood. In particular, microtubule detyrosination, the removal of the C-terminal tyrosine on α-tubulin subunits, marks the stable population of microtubules and modifies how microtubules interact with other microtubule-associated proteins to regulate a wide range of cellular processes. Previously, we found that in certain cell types, only ∼30% of microtubules are highly enriched with the detyrosination mark and that detyrosination spans most of the length of a microtubule, often adjacent to a completely tyrosinated microtubule. How the activity of a cytosolic detyrosinase, vasohibin (VASH), leads to only a small subpopulation of highly detyrosinated microtubules is unclear. Here, using quantitative super-resolution microscopy, we visualized nascent microtubule detyrosination events in cells consisting of 1-3 detyrosinated α-tubulin subunits after nocodazole washout. Microtubule detyrosination accumulates slowly and in a dispersed pattern across the microtubule length. By visualizing single molecules of VASH in live cells, we found that VASH engages with microtubules stochastically on a short timescale, suggesting limited removal of tyrosine per interaction, consistent with the super-resolution results. Combining these quantitative imaging results with simulations incorporating parameters from our experiments, we provide evidence for a stochastic model for cells to establish a subset of detyrosinated microtubules via a detyrosination-stabilization feedback mechanism.

Spatiotemporal Control over Polynucleotide Brush Growth on DNA Origami Nanostructures.

DNA nanotechnology provides an approach to create precise, tunable, and biocompatible nanostructures for biomedical applications. However, the stability of these structures is severely compromised in biological milieu due to their fast degradation by nucleases. Recently, we showed how enzymatic polymerization could be harnessed to grow polynucleotide brushes of tunable length and location on the surface of DNA origami nanostructures, which greatly enhances their nuclease stability. Here, we report on strategies that allow for both spatial and temporal control over polymerization through activatable initiation, cleavage, and regeneration of polynucleotide brushes using restriction enzymes. The ability to site-specifically decorate DNA origami nanostructures with polynucleotide brushes in a spatiotemporally controlled way provides access to "smart" functionalized DNA architectures with potential applications in drug delivery and supramolecular assembly.

Versatile computer-aided design of free-form DNA nanostructures and assemblies.

Recent advances in structural DNA nanotechnology have been facilitated by design tools that continue to push the limits of structural complexity while simplifying an often-tedious design process. We recently introduced the software MagicDNA, which enables design of complex 3D DNA assemblies with many components; however, the design of structures with free-form features like vertices or curvature still required iterative design guided by simulation feedback and user intuition. Here, we present an updated design tool, MagicDNA 2.0, that automates the design of free-form 3D geometries, leveraging design models informed by coarse-grained molecular dynamics simulations. Our GUI-based, stepwise design approach integrates a high level of automation with versatile control over assembly and subcomponent design parameters. We experimentally validated this approach by fabricating a range of DNA origami assemblies with complex free-form geometries, including a 3D Nozzle, G-clef, and Hilbert and Trifolium curves, confirming excellent agreement between design input, simulation, and structure formation.

Transcending shift-invariance in the paraxial regime via end-to-end inverse design of freeform nanophotonics.

Traditional optical elements and conventional metasurfaces obey shift-invariance in the paraxial regime. For imaging systems obeying paraxial shift-invariance, a small shift in input angle causes a corresponding shift in the sensor image. Shift-invariance has deep implications for the design and functionality of optical devices, such as the necessity of free space between components (as in compound objectives made of several curved surfaces). We present a method for nanophotonic inverse design of compact imaging systems whose resolution is not constrained by paraxial shift-invariance. Our method is end-to-end, in that it integrates density-based full-Maxwell topology optimization with a fully iterative elastic-net reconstruction algorithm. By the design of nanophotonic structures that scatter light in a non-shift-invariant manner, our optimized nanophotonic imaging system overcomes the limitations of paraxial shift-invariance, achieving accurate, noise-robust image reconstruction beyond shift-invariant resolution.

Thermally reversible pattern formation in arrays of molecular rotors.

Control over the mesoscale to microscale patterning of materials is of great interest to the soft matter community. Inspired by DNA origami rotors, we introduce a 2D nearest-neighbor lattice of spinning rotors that exhibit discrete orientational states and interactions with their neighbors. Monte Carlo simulations of rotor lattices reveal that they exhibit a variety of interesting ordering behaviors and morphologies that can be modulated through rotor design parameters. The rotor arrays exhibit diverse patterns including closed loops, radiating loops, and bricklayer structures in their ordered states. They exhibit specific heat peaks at very low temperatures for small system sizes, and some systems exhibit multiple order-disorder transitions depending on inter-rotor interaction design. We devise an energy-based order parameter and show via umbrella sampling and histogram reweighting that this order parameter captures well the order-disorder transitions occurring in these systems. We fabricate real DNA origami rotors which themselves can order via programmable DNA base-pairing interactions and demonstrate both ordered and disordered phases, illustrating how rotor lattices may be realized experimentally and used for responsive organization. This work establishes the feasibility of realizing structural nanomaterials that exhibit locally mediated microscale patterns which could have applications in sensing and precision surface patterning.

Steric Communication between Dynamic Components on DNA Nanodevices.

Biomolecular nanotechnology has helped emulate basic robotic capabilities such as defined motion, sensing, and actuation in synthetic nanoscale systems. DNA origami is an attractive approach for nanorobotics, as it enables creation of devices with complex geometry, programmed motion, rapid actuation, force application, and various kinds of sensing modalities. Advanced robotic functions like feedback control, autonomy, or programmed routines also require the ability to transmit signals among subcomponents. Prior work in DNA nanotechnology has established approaches for signal transmission, for example through diffusing strands or structurally coupled motions. However, soluble communication is often slow and structural coupling of motions can limit the function of individual components, for example to respond to the environment. Here, we introduce an approach inspired by protein allostery to transmit signals between two distal dynamic components through steric interactions. These components undergo separate thermal fluctuations where certain conformations of one arm will sterically occlude conformations of the distal arm. We implement this approach in a DNA origami device consisting of two stiff arms each connected to a base platform via a flexible hinge joint. We demonstrate the ability for one arm to sterically regulate both the range of motion and the conformational state (latched or freely fluctuating) of the distal arm, results that are quantitatively captured by mesoscopic simulations using experimentally informed energy landscapes for hinge-angle fluctuations. We further demonstrate the ability to modulate signal transmission by mechanically tuning the range of thermal fluctuations and controlling the conformational states of the arms. Our results establish a communication mechanism well-suited to transmit signals between thermally fluctuating dynamic components and provide a path to transmitting signals where the input is a dynamic response to parameters like force or solution conditions.

Discovery of two-dimensional binary nanoparticle superlattices using global Monte Carlo optimization.

Binary nanoparticle (NP) superlattices exhibit distinct collective plasmonic, magnetic, optical, and electronic properties. Here, we computationally demonstrate how fluid-fluid interfaces could be used to self-assemble binary systems of NPs into 2D superlattices when the NP species exhibit different miscibility with the fluids forming the interface. We develop a basin-hopping Monte Carlo (BHMC) algorithm tailored for interface-trapped structures to rapidly determine the ground-state configuration of NPs, allowing us to explore the repertoire of binary NP architectures formed at the interface. By varying the NP size ratio, interparticle interaction strength, and difference in NP miscibility with the two fluids, we demonstrate the assembly of an array of exquisite 2D periodic architectures, including AB-, AB2-, and AB3-type monolayer superlattices as well as AB-, AB2-, A3B5-, and A4B6-type bilayer superlattices. Our results suggest that the interfacial assembly approach could be a versatile platform for fabricating 2D colloidal superlattices with tunable structure and properties.

A bistable and reconfigurable molecular system with encodable bonds.

Molecular systems with ability to controllably transform between different conformations play pivotal roles in regulating biochemical functions. Here, we report the design of a bistable DNA origami four-way junction (DOJ) molecular system that adopts two distinct stable conformations with controllable reconfigurability by using conformation-controlled base stacking. Exquisite control over DOJ's conformation and transformation is realized by programming the stacking bonds (quasi-blunt-ends) within the junction to induce prescribed coaxial stacking of neighboring junction arms. A specific DOJ conformation may be achieved by encoding the stacking bonds with binary stacking sequences based on thermodynamic calculations. Dynamic transformations of DOJ between various conformations are achieved by using specific environmental and molecular stimulations to reprogram the stacking codes. This work provides a useful platform for constructing self-assembled DNA nanostructures and nanomachines and insights for future design of artificial molecular systems with increasing complexity and reconfigurability.

Influence of Polymer Characteristics on the Self-Assembly of Polymer-Grafted Metal-Organic Framework Particles.

Polymer-grafted metal-organic frameworks (MOFs) can combine the properties of MOFs and polymers into a single, matrix-free composite material. Herein, we examine polymer-grafted MOF particles (using UiO-66 as a model system) to examine how the molecular weight, grafting density, and chemical functionality of the polymer graft affects the preparation of free-standing self-assembled MOF monolayers (SAMMs). The physical properties of the monolayers are influenced by the choice of polymer, and robust, flexible monolayers were achieved more readily with poly(methyl acrylate) when compared to poly(methyl methacrylate) or poly(benzyl methacrylate). Molecular dynamics simulations were carried out to provide insights into the orientation and ordering of MOFs in the monolayers with respect to MOF size, graft length, and hydrophobicity. The relationship between molecular weight and graft density of the polymer brush was investigated and related to polymer brush conformation, offering design rules for further optimizations to balance mechanical strength, MOF weight fraction, and processability for this class of hybrid materials.

Molecular dynamics of DNA translocation by FtsK.

The bacterial FtsK motor harvests energy from ATP to translocate double-stranded DNA during cell division. Here, we probe the molecular mechanisms underlying coordinated DNA translocation in FtsK by performing long timescale simulations of its hexameric assembly and individual subunits. From these simulations we predict signaling pathways that connect the ATPase active site to DNA-gripping residues, which allows the motor to coordinate its translocation activity with its ATPase activity. Additionally, we utilize well-tempered metadynamics simulations to compute free-energy landscapes that elucidate the extended-to-compact transition involved in force generation. We show that nucleotide binding promotes a compact conformation of a motor subunit, whereas the apo subunit is flexible. Together, our results support a mechanism whereby each ATP-bound subunit of the motor conforms to the helical pitch of DNA, and ATP hydrolysis/product release causes a subunit to lose grip of DNA. By ordinally engaging and disengaging with DNA, the FtsK motor unidirectionally translocates DNA.

Binding kinetics of harmonically confined random walkers.

Diffusion-mediated binding of molecules under the influence of discrete spatially confining potentials is a commonly encountered scenario in systems subjected to explicit fields or implicit fields arising from tethering restraints. Here, we derive analytical expressions for the mean binding time of two random walkers geometrically confined by means of two harmonic potentials in one- and two-dimensional systems, which show excellent agreement with Brownian dynamics simulations. As a demonstration of its utility, we use this theory to maximize the communication speed in existing DNA walkers, obtaining quantitative agreement with previously reported experimental findings. The analytical expressions derived in this paper are broadly applicable to diverse systems, providing ways to characterize communication processes and optimize the rate of signal propagation for sensing and computing applications at the nanoscale.

Redox-sensitive E2 Rad6 controls cellular response to oxidative stress via K63-linked ubiquitination of ribosomes.

Protein ubiquitination is an essential process that rapidly regulates protein synthesis, function, and fate in dynamic environments. Within its non-proteolytic functions, we showed that K63-linked polyubiquitinated conjugates heavily accumulate in yeast cells exposed to oxidative stress, stalling ribosomes at elongation. K63-ubiquitinated conjugates accumulate mostly because of redox inhibition of the deubiquitinating enzyme Ubp2; however, the role and regulation of ubiquitin-conjugating enzymes (E2) in this pathway remained unclear. Here, we show that the E2 Rad6 associates and modifies ribosomes during stress. We further demonstrate that Rad6 and its human homolog UBE2A are redox regulated by forming a reversible disulfide with the E1 ubiquitin-activating enzyme (Uba1). This redox regulation is part of a negative feedback regulation, which controls the levels of K63 ubiquitination under stress. Finally, we show that Rad6 activity is necessary to regulate translation, antioxidant defense, and adaptation to stress, thus providing an additional physiological role for this multifunctional enzyme.

Tunable Orientation and Assembly of Polymer-Grafted Nanocubes at Fluid-Fluid Interfaces.

Self-assembly of faceted nanoparticles is a promising route for fabricating nanomaterials; however, achieving low-dimensional assemblies of particles with tunable orientations is challenging. Here, we demonstrate that trapping surface-functionalized faceted nanoparticles at fluid-fluid interfaces is a viable approach for controlling particle orientation and facilitating their assembly into unique one- and two-dimensional superstructures. Using molecular dynamics simulations of polymer-grafted nanocubes in a polymer bilayer along with a particle-orientation classification method we developed, we show that the nanocubes can be induced into face-up, edge-up, or vertex-up orientations by tuning the graft density and differences in their miscibility with the two polymer layers. The orientational preference of the nanocubes is found to be governed by an interplay between the interfacial area occluded by the particle, the difference in interactions of the grafts with the two layers, and the stretching and intercalation of grafts at the interface. The resulting orientationally constrained nanocubes are then shown to assemble into a variety of unusual architectures, such as rectilinear strings, close-packed sheets, bilayer ribbons, and perforated sheets, which are difficult to obtain using other assembly methods. Our work thus demonstrates a versatile strategy for assembling freestanding arrays of faceted nanoparticles with possible applications in plasmonics, optics, catalysis, and membranes, where precise control over particle orientation and position is required.

Assembly mechanism of surface-functionalized nanocubes.

Faceted nanoparticles can be used as building blocks to assemble nanomaterials with exceptional optical and catalytic properties. Recent studies have shown that surface functionalization of such nanoparticles with organic molecules, polymer chains, or DNA can be used to control the separation distance and orientation of particles within their assemblies. In this study, we computationally investigate the mechanism of assembly of nanocubes grafted with short-chain molecules. Our approach involves computing the interaction free energy landscape of a pair of such nanocubes via Monte Carlo simulations and using the Dijkstra algorithm to determine the minimum free energy pathway connecting key states in the landscape. We find that the assembly pathway of nanocubes is very rugged involving multiple energy barriers and metastable states. Analysis of nanocube configurations along the pathway reveals that the assembly mechanism is dominated by sliding motion of nanocubes relative to each other punctuated by their local dissociation at grafting points involving lineal separation and rolling motions. The height of energy barriers between metastable states depends on factors such as the interaction strength and surface roughness of the nanocubes and the steric repulsion from the grafts. These results imply that the observed assembly configuration of nanocubes depends not only on their globally stable minimum free energy state but also on the assembly pathway leading to this state. The free energy landscapes and assembly pathways presented in this study along with the proposed guidelines for engineering such pathways should be useful to researchers aiming to achieve uniform nanostructures from self-assembly of faceted nanoparticles.