Limits of economy and fidelity for programmable assembly of size-controlled triply periodic polyhedra.

We propose and investigate an extension of the Caspar-Klug symmetry principles for viral capsid assembly to the programmable assembly of size-controlled triply periodic polyhedra, discrete variants of the Primitive, Diamond, and Gyroid cubic minimal surfaces. Inspired by a recent class of programmable DNA origami colloids, we demonstrate that the economy of design in these crystalline assemblies-in terms of the growth of the number of distinct particle species required with the increased size-scale (e.g., periodicity)-is comparable to viral shells. We further test the role of geometric specificity in these assemblies via dynamical assembly simulations, which show that conditions for simultaneously efficient and high-fidelity assembly require an intermediate degree of flexibility of local angles and lengths in programmed assembly. Off-target misassembly occurs via incorporation of a variant of disclination defects, generalized to the case of hyperbolic crystals. The possibility of these topological defects is a direct consequence of the very same symmetry principles that underlie the economical design, exposing a basic tradeoff between design economy and fidelity of programmable, size controlled assembly.

Hierarchical assembly is more robust than egalitarian assembly in synthetic capsids.

Self-assembly of complex and functional materials remains a grand challenge in soft material science. Efficient assembly depends on a delicate balance between thermodynamic and kinetic effects, requiring fine-tuning affinities and concentrations of subunits. By contrast, we introduce an assembly paradigm that allows large error-tolerance in the subunit affinity and helps avoid kinetic traps. Our combined experimental and computational approach uses a model system of triangular subunits programmed to assemble into T = 3 icosahedral capsids comprising 60 units. The experimental platform uses DNA origami to create monodisperse colloids whose three-dimensional geometry is controlled to nanometer precision, with two distinct bonds whose affinities are controlled to kBT precision, quantified in situ by static light scattering. The computational model uses a coarse-grained representation of subunits, short-ranged potentials, and Langevin dynamics. Experimental observations and modeling reveal that when the bond affinities are unequal, two distinct hierarchical assembly pathways occur, in which the subunits first form dimers in one case and pentamers in another. These hierarchical pathways produce complete capsids faster and are more robust against affinity variation than egalitarian pathways, in which all binding sites have equal strengths. This finding suggests that hierarchical assembly may be a general engineering principle for optimizing self-assembly of complex target structures.

Photocleavable Ortho-Nitrobenzyl-Protected DNA Architectures and Their Applications.

This review article introduces mechanistic aspects and applications of photochemically deprotected ortho-nitrobenzyl (ONB)-functionalized nucleic acids and their impact on diverse research fields including DNA nanotechnology and materials chemistry, biological chemistry, and systems chemistry. Specific topics addressed include the synthesis of the ONB-modified nucleic acids, the mechanisms involved in the photochemical deprotection of the ONB units, and the photophysical and chemical means to tune the irradiation wavelength required for the photodeprotection process. Principles to activate ONB-caged nanostructures, ONB-protected DNAzymes and aptamer frameworks are introduced. Specifically, the use of ONB-protected nucleic acids for the phototriggered spatiotemporal amplified sensing and imaging of intracellular mRNAs at the single-cell level are addressed, and control over transcription machineries, protein translation and spatiotemporal silencing of gene expression by ONB-deprotected nucleic acids are demonstrated. In addition, photodeprotection of ONB-modified nucleic acids finds important applications in controlling material properties and functions. These are introduced by the phototriggered fusion of ONB nucleic acid functionalized liposomes as models for cell-cell fusion, the light-stimulated fusion of ONB nucleic acid functionalized drug-loaded liposomes with cells for therapeutic applications, and the photolithographic patterning of ONB nucleic acid-modified interfaces. Particularly, the photolithographic control of the stiffness of membrane-like interfaces for the guided patterned growth of cells is realized. Moreover, ONB-functionalized microcapsules act as light-responsive carriers for the controlled release of drugs, and ONB-modified DNA origami frameworks act as mechanical devices or stimuli-responsive containments for the operation of DNA machineries such as the CRISPR-Cas9 system. The future challenges and potential applications of photoprotected DNA structures are discussed.

Geometrically programmed self-limited assembly of tubules using DNA origami colloids.

Self-assembly is one of the most promising strategies for making functional materials at the nanoscale, yet new design principles for making self-limiting architectures, rather than spatially unlimited periodic lattice structures, are needed. To address this challenge, we explore the tradeoffs between addressable assembly and self-closing assembly of a specific class of self-limiting structures: cylindrical tubules. We make triangular subunits using DNA origami that have specific, valence-limited interactions and designed binding angles, and we study their assembly into tubules that have a self-limited width that is much larger than the size of an individual subunit. In the simplest case, the tubules are assembled from a single component by geometrically programming the dihedral angles between neighboring subunits. We show that the tubules can reach many micrometers in length and that their average width can be prescribed through the dihedral angles. We find that there is a distribution in the width and the chirality of the tubules, which we rationalize by developing a model that considers the finite bending rigidity of the assembled structure as well as the mechanism of self-closure. Finally, we demonstrate that the distributions of tubules can be further sculpted by increasing the number of subunit species, thereby increasing the assembly complexity, and demonstrate that using two subunit species successfully reduces the number of available end states by half. These results help to shed light on the roles of assembly complexity and geometry in self-limited assembly and could be extended to other self-limiting architectures, such as shells, toroids, or triply periodic frameworks.

Self-assembly coupled to liquid-liquid phase separation.

Liquid condensate droplets with distinct compositions of proteins and nucleic acids are widespread in biological cells. While it is known that such droplets, or compartments, can regulate irreversible protein aggregation, their effect on reversible self-assembly remains largely unexplored. In this article, we use kinetic theory and solution thermodynamics to investigate the effect of liquid-liquid phase separation on the reversible self-assembly of structures with well-defined sizes and architectures. We find that, when assembling subunits preferentially partition into liquid compartments, robustness against kinetic traps and maximum achievable assembly rates can be significantly increased. In particular, both the range of solution conditions leading to productive assembly and the corresponding assembly rates can increase by orders of magnitude. We analyze the rate equation predictions using simple scaling estimates to identify effects of liquid-liquid phase separation as a function of relevant control parameters. These results may elucidate self-assembly processes that underlie normal cellular functions or pathogenesis, and suggest strategies for designing efficient bottom-up assembly for nanomaterials applications.

A machine learning approach to robustly determine director fields and analyze defects in active nematics.

Active nematics are dense systems of rodlike particles that consume energy to drive motion at the level of the individual particles. They exist in natural systems like biological tissues and artificial materials such as suspensions of self-propelled colloidal particles or synthetic microswimmers. Active nematics have attracted significant attention in recent years due to their spectacular nonequilibrium collective spatiotemporal dynamics, which may enable applications in fields such as robotics, drug delivery, and materials science. The director field, which measures the direction and degree of alignment of the local nematic orientation, is a crucial characteristic of active nematics and is essential for studying topological defects. However, determining the director field is a significant challenge in many experimental systems. Although director fields can be derived from images of active nematics using traditional imaging processing methods, the accuracy of such methods is highly sensitive to the settings of the algorithms. These settings must be tuned from image to image due to experimental noise, intrinsic noise of the imaging technology, and perturbations caused by changes in experimental conditions. This sensitivity currently limits automatic analysis of active nematics. To address this, we developed a machine learning model for extracting reliable director fields from raw experimental images, which enables accurate analysis of topological defects. Application of the algorithm to experimental data demonstrates that the approach is robust and highly generalizable to experimental settings that are different from those in the training data. It could be a promising tool for investigating active nematics and may be generalized to other active matter systems.

Active liquid crystals powered by force-sensing DNA-motor clusters.

Cytoskeletal active nematics exhibit striking nonequilibrium dynamics that are powered by energy-consuming molecular motors. To gain insight into the structure and mechanics of these materials, we design programmable clusters in which kinesin motors are linked by a double-stranded DNA linker. The efficiency by which DNA-based clusters power active nematics depends on both the stepping dynamics of the kinesin motors and the chemical structure of the polymeric linker. Fluorescence anisotropy measurements reveal that the motor clusters, like filamentous microtubules, exhibit local nematic order. The properties of the DNA linker enable the design of force-sensing clusters. When the load across the linker exceeds a critical threshold, the clusters fall apart, ceasing to generate active stresses and slowing the system dynamics. Fluorescence readout reveals the fraction of bound clusters that generate interfilament sliding. In turn, this yields the average load experienced by the kinesin motors as they step along the microtubules. DNA-motor clusters provide a foundation for understanding the molecular mechanism by which nanoscale molecular motors collectively generate mesoscopic active stresses, which in turn power macroscale nonequilibrium dynamics of active nematics.

Engineering Metastability into a Virus-like Particle to Enable Triggered Dissociation.

For a virus-like particle (VLP) to serve as a delivery platform, the VLP must be able to release its cargo in response to a trigger. Here, we use a chemical biology approach to destabilize a self-assembling capsid for a subsequent triggered disassembly. We redesigned the dimeric hepatitis B virus (HBV) capsid protein (Cp) with two differentially addressable cysteines, C150 for reversibly crosslinking the capsid and C124 to react with a destabilizing moiety. The resulting construct, Cp150-V124C, assembles into icosahedral, 120-dimer VLPs that spontaneously crosslink via the C-terminal C150, leaving C124 buried at a dimer-dimer interface. The VLP is driven into a metastable state when C124 is reacted with the bulky fluorophore, maleimidyl BoDIPY-FL. The resulting VLP is stable until exposed to modest, physiologically relevant concentrations of reducing agent. We observe dissociation with FRET relaxation of polarization, size exclusion chromatography, and resistive-pulse sensing. Dissociation is slow, minutes to hours, with a characteristic lag phase. Mathematical modeling based on the presence of a nucleation step predicts disassembly dynamics that are consistent with experimental observations. VLPs transfected into hepatoma cells show similar dissociation behavior. These results suggest a generalizable strategy for designing a VLP that can release its contents in an environmentally responsive reaction.

From disks to channels: dynamics of active nematics confined to an annulus.

Confinement can be used to systematically tame turbulent dynamics occurring in active fluids. Although periodic channels are the simplest geometries to study confinement numerically, the corresponding experimental realizations require closed racetracks. Here, we computationally study 2D active nematics confined to such a geometry-an annulus. By systematically varying the annulus inner radius and channel width, we bridge the behaviors observed in the previously studied asymptotic limits of the annulus geometry: a disk and an infinite channel. We identify new steady-state behaviors, which reveal the influence of boundary curvature and its interplay with confinement. We also show that, below a threshold inner radius, the dynamics are insensitive to the presence of the inner hole. We explain this insensitivity through a simple scaling analysis. Our work sheds further light on design principles for using confinement to control the dynamics of active nematics.

Effect of ionic strength on the assembly of simian vacuolating virus capsid protein around poly(styrene sulfonate).

Virus-like particles (VLPs) are noninfectious nanocapsules that can be used for drug delivery or vaccine applications. VLPs can be assembled from virus capsid proteins around a condensing agent, such as RNA, DNA, or a charged polymer. Electrostatic interactions play an important role in the assembly reaction. VLPs assemble from many copies of capsid protein, with a combinatorial number of intermediates. Hence, the mechanism of the reaction is poorly understood. In this paper, we combined solution small-angle X-ray scattering (SAXS), cryo-transmission electron microscopy (TEM), and computational modeling to determine the effect of ionic strength on the assembly of Simian Vacuolating Virus 40 (SV40)-like particles. We mixed poly(styrene sulfonate) with SV40 capsid protein pentamers at different ionic strengths. We then characterized the assembly product by SAXS and cryo-TEM. To analyze the data, we performed Langevin dynamics simulations using a coarse-grained model that revealed incomplete, asymmetric VLP structures consistent with the experimental data. We found that close to physiological ionic strength, [Formula: see text] VLPs coexisted with VP1 pentamers. At lower or higher ionic strengths, incomplete particles coexisted with pentamers and [Formula: see text] particles. Including the simulated structures was essential to explain the SAXS data in a manner that is consistent with the cryo-TEM images.

Two-step crystallization and solid-solid transitions in binary colloidal mixtures.

Crystallization is fundamental to materials science and is central to a variety of applications, ranging from the fabrication of silicon wafers for microelectronics to the determination of protein structures. The basic picture is that a crystal nucleates from a homogeneous fluid by a spontaneous fluctuation that kicks the system over a single free-energy barrier. However, it is becoming apparent that nucleation is often more complicated than this simple picture and, instead, can proceed via multiple transformations of metastable structures along the pathway to the thermodynamic minimum. In this article, we observe, characterize, and model crystallization pathways using DNA-coated colloids. We use optical microscopy to investigate the crystallization of a binary colloidal mixture with single-particle resolution. We observe classical one-step pathways and nonclassical two-step pathways that proceed via a solid-solid transformation of a crystal intermediate. We also use enhanced sampling to compute the free-energy landscapes corresponding to our experiments and show that both one- and two-step pathways are driven by thermodynamics alone. Specifically, the two-step solid-solid transition is governed by a competition between two different crystal phases with free energies that depend on the crystal size. These results extend our understanding of available pathways to crystallization, by showing that size-dependent thermodynamic forces can produce pathways with multiple crystal phases that interconvert without free-energy barriers and could provide approaches to controlling the self-assembly of materials made from colloids.

Switchable and dynamic G-quadruplexes and their applications.

G-Quadruplexes attract growing interest as functional constituents in biology, chemistry, nanotechnology, and material science. In particular, the reversible dynamic reconfiguration of G-quadruplexes provides versatile means to switch DNA nanostructures, reversibly control catalytic functions of DNA assemblies, and switch material properties and functions. The present review article discusses the switchable dynamic reconfiguration of G-quadruplexes as central functional and structural motifs that enable diverse applications in DNA nanotechnology and material science. The dynamic reconfiguration of G-quadruplexes has a major impact on the development of DNA switches and DNA machines. The integration of G-quadruplexes with enzymes yields supramolecular assemblies exhibiting switchable catalytic functions guided by dynamic G-quadruplex topologies. In addition, G-quadruplexes act as important building blocks to operate constitutional dynamic networks and transient dissipative networks mimicking complex biological dynamic circuitries. Furthermore, the integration of G-quadruplexes with DNA nanostructures, such as origami tiles, introduces dynamic and mechanical features into these static frameworks. Beyond the dynamic operation of G-quadruplex structures in solution, the assembly of G-quadruplexes on bulk surfaces such as electrodes or nanoparticles provides versatile means to engineer diverse electrochemical and photoelectrochemical devices and to switch the dynamic aggregation/deaggregation of nanoparticles, leading to nanoparticle assemblies that reveal switchable optical properties. Finally, the functionalization of hydrogels, hydrogel microcapsules, or nanoparticle carriers, such as SiO2 nanoparticles or metal-organic framework nanoparticles, yields stimuli-responsive materials exhibiting shape-memory, self-healing, and controlled drug release properties. Indeed, G-quadruplex-modified nanomaterials find growing interest in the area of nanomedicine. Beyond the impressive G-quadruplex-based scientific advances achieved to date, exciting future developments are still anticipated. The review addresses these goals by identifying the potential opportunities and challenges ahead of the field in the coming years.

Photoresponsive DNA materials and their applications.

Photoresponsive nucleic acids attract growing interest as functional constituents in materials science. Integration of photoisomerizable units into DNA strands provides an ideal handle for the reversible reconfiguration of nucleic acid architectures by light irradiation, triggering changes in the chemical and structural properties of the nanostructures that can be exploited in the development of photoresponsive functional devices such as machines, origami structures and ion channels, as well as environmentally adaptable 'smart' materials including nanoparticle aggregates and hydrogels. Moreover, photoresponsive DNA components allow control over the composition of dynamic supramolecular ensembles that mimic native networks. Beyond this, the modification of nucleic acids with photosensitizer functionality enables these biopolymers to act as scaffolds for spatial organization of electron transfer reactions mimicking natural photosynthesis. This review provides a comprehensive overview of these exciting developments in the design of photoresponsive DNA materials, and showcases a range of applications in catalysis, sensing and drug delivery/release. The key challenges facing the development of the field in the coming years are addressed, and exciting emergent research directions are identified.

Enzyme-Loaded Hemin/G-Quadruplex-Modified ZIF-90 Metal-Organic Framework Nanoparticles: Bioreactor Nanozymes for the Cascaded Oxidation of N-hydroxy-l-arginine and Sensing Applications.

Biocatalytic cascades are challenging to operate in homogeneous solution, where diffusional mass transport hinders efficient communication between the reactive components. There is great interest in developing devices to perform such transformations in confined environments, which increase the efficiency of the cascaded process by generating high local concentrations of the reactive species. Herein, a bioreactor-nanozyme assembly is introduced for the cascaded aerobic oxidation of N-hydroxy-l-arginine (NOHA) to citrulline in the presence of glucose. The reaction mimics a key step in the nitric oxide synthase oxidation of l-arginine in nature. The system consists of glucose oxidase (GOx)-loaded hemin/G-quadruplex (hemin/G4)-modified ZIF-90 metal-organic framework nanoparticles. The aerobic oxidation of glucose by GOx yields H2 O2 that fuels the hemin/G4-catalyzed oxidation of NOHA into citrulline. The process driven by the bioreactor-nanozyme system is ≈sixfold enhanced compared to the homogeneous mixture of the biocatalysts, due to its operation in the confined environment of the nanoparticles. Extension to a three-step cascade is then demonstrated using a bioreactor composed of ß-galactosidase/GOx-loaded hemin/G4-modified ZIF-90 nanoparticles activating the cascaded oxidation of NOHA to citrulline, in the presence of lactose. Moreover, the bioreactor-nanozyme hybrid is applied as a functional optical sensor of glucose, using fluorescence or chemiluminescence as readout signals.

Programmable icosahedral shell system for virus trapping.

Broad-spectrum antiviral platforms that can decrease or inhibit viral infection would alleviate many threats to global public health. Nonetheless, effective technologies of this kind are still not available. Here, we describe a programmable icosahedral canvas for the self-assembly of icosahedral shells that have viral trapping and antiviral properties. Programmable triangular building blocks constructed from DNA assemble with high yield into various shell objects with user-defined geometries and apertures. We have created shells with molecular masses ranging from 43 to 925 MDa (8 to 180 subunits) and with internal cavity diameters of up to 280 nm. The shell interior can be functionalized with virus-specific moieties in a modular fashion. We demonstrate this virus-trapping concept by engulfing hepatitis B virus core particles and adeno-associated viruses. We demonstrate the inhibition of hepatitis B virus core interactions with surfaces in vitro and the neutralization of infectious adeno-associated viruses exposed to human cells.

Data-Driven Discovery of Active Nematic Hydrodynamics.

Active nematics can be modeled using phenomenological continuum theories that account for the dynamics of the nematic director and fluid velocity through partial differential equations (PDEs). While these models provide a statistical description of the experiments, the relevant terms in the PDEs and their parameters are usually identified indirectly. We adapt a recently developed method to automatically identify optimal continuum models for active nematics directly from spatiotemporal data, via sparse regression of the coarse-grained fields onto generic low order PDEs. After extensive benchmarking, we apply the method to experiments with microtubule-based active nematics, finding a surprisingly minimal description of the system. Our approach can be generalized to gain insights into active gels, microswimmers, and diverse other experimental active matter systems.

Bioinspired Artificial Photosynthetic Systems.

Mimicking photosynthesis using artificial systems, as a means for solar energy conversion and green fuel generation, is one of the holy grails of modern science. This perspective presents recent advances towards developing artificial photosynthetic systems. In one approach, native photosystems are interfaced with electrodes to yield photobioelectrochemical cells that transform light energy into electrical power. This is exemplified by interfacing photosystem I (PSI) and photosystem II (PSII) as an electrically contacted assembly mimicking the native Z-scheme, and by the assembly of an electrically wired PSI/glucose oxidase biocatalytic conjugate on an electrode support. Illumination of the functionalized electrodes led to light-induced generation of electrical power, or to the generation of photocurrents using glucose as the fuel. The second approach introduces supramolecular photosensitizer nucleic acid/electron acceptor complexes as functional modules for effective photoinduced electron transfer stimulating the subsequent biocatalyzed generation of NADPH or the Pt-nanoparticle-catalyzed evolution of molecular hydrogen. Application of the DNA machineries for scaling-up the photosystems is demonstrated. A third approach presents the integration of artificial photosynthetic modules into dynamic nucleic acid networks undergoing reversible reconfiguration or dissipative transient operation in the presence of auxiliary triggers. Control over photoinduced electron transfer reactions and photosynthetic transformations by means of the dynamic networks is demonstrated.

Polymorphic self-assembly of helical tubules is kinetically controlled.

In contrast to most self-assembling synthetic materials, which undergo unbounded growth, many biological self-assembly processes are self-limited. That is, the assembled structures have one or more finite dimensions that are much larger than the size scale of the individual monomers. In many such cases, the finite dimension is selected by a preferred curvature of the monomers, which leads to self-closure of the assembly. In this article, we study an example class of self-closing assemblies: cylindrical tubules that assemble from triangular monomers. By combining kinetic Monte Carlo simulations, free energy calculations, and simple theoretical models, we show that a range of programmable size scales can be targeted by controlling the intricate balance between the preferred curvature of the monomers and their interaction strengths. However, their assembly is kinetically controlled-the tubule morphology is essentially fixed shortly after closure, resulting in a distribution of tubule widths that is significantly broader than the equilibrium distribution. We develop a simple kinetic model based on this observation and the underlying free-energy landscape of assembling tubules that quantitatively describes the distributions. Our results are consistent with recent experimental observations of tubule assembly from triangular DNA origami monomers. The modeling framework elucidates design principles for assembling self-limited structures from synthetic components, such as artificial microtubules that have a desired width and chirality.

Structure-activity relationship studies on divalent naphthalene diimide G quadruplex ligands with anticancer and antiparasitic activity.

Naphthalene diimide (NDI) is a central scaffold that has been commonly used in the design of G-quadruplex (G4) ligands. Previous work revealed notable anticancer activity of a disubstituted N-methylpiperazine propyl NDI G4 ligand. Here, we explored structure-activity relationship studies around ligand bis-N,N-2,7-(3-(4-methylpiperazin-1-yl)propyl)-1,4,5,8-naphthalenetetracarboxylic diimide, maintaining the central NDI core whilst modifying the spacer and the nature of the cationic groups. We prepared new disubstituted NDI derivatives of the original compound and examined their in vitro antiproliferative and antiparasitic activity. Several N-methylpiperazine propyl NDIs showed sub-micromolar activity against Trypanosoma brucei and Leishmania major parasites with up to 30 fold selectivity versus MRC-5 cells. The best compound was a dimorpholino NDI with an IC50 of 0.17 µM against T.brucei and 40 fold selectivity versus MRC-5 cells. However, no clear correlation between G4 binding of the new NDI derivatives and antiproliferative or antiparasitic activity was observed, indicating that other mechanisms of action may be responsible for the observed biological activity.

Optimization of non-equilibrium self-assembly protocols using Markov state models.

The promise of self-assembly to enable the bottom-up formation of materials with prescribed architectures and functions has driven intensive efforts to uncover rational design principles for maximizing the yield of a target structure. Yet, despite many successful examples of self-assembly, ensuring kinetic accessibility of the target structure remains an unsolved problem in many systems. In particular, long-lived kinetic traps can result in assembly times that vastly exceed experimentally accessible timescales. One proposed solution is to design non-equilibrium assembly protocols in which system parameters change over time to avoid such kinetic traps. Here, we develop a framework to combine Markov state model (MSM) analysis with optimal control theory to compute a time-dependent protocol that maximizes the yield of the target structure at a finite time. We present an adjoint-based gradient descent method that, in conjunction with MSMs for a system as a function of its control parameters, enables efficiently optimizing the assembly protocol. We also describe an interpolation approach to significantly reduce the number of simulations required to construct the MSMs. We demonstrate our approach with two examples; a simple semi-analytic model for the folding of a polymer of colloidal particles, and a more complex model for capsid assembly. Our results show that optimizing time-dependent protocols can achieve significant improvements in the yields of selected structures, including equilibrium free energy minima, long-lived metastable structures, and transient states.