Designer Nanomaterials through Programmable Assembly.

Nanoparticles have long been recognized for their unique properties, leading to exciting potential applications across optics, electronics, magnetism, and catalysis. These specific functions often require a designed organization of particles, which includes the type of order as well as placement and relative orientation of particles of the same or different kinds. DNA nanotechnology offers the ability to introduce highly addressable bonds, tailor particle interactions, and control the geometry of bindings motifs. Here, we discuss how developments in structural DNA nanotechnology have enabled greater control over 1D, 2D, and 3D particle organizations through programmable assembly. This Review focuses on how the use of DNA binding between nanocomponents and DNA structural motifs has progressively allowed the rational formation of prescribed particle organizations. We offer insight into how DNA-based motifs and elements can be further developed to control particle organizations and how particles and DNA can be integrated into nanoscale building blocks, so-called "material voxels", to realize designer nanomaterials with desired functions.

Ordered three-dimensional nanomaterials using DNA-prescribed and valence-controlled material voxels.

The ability to organize nanoscale objects into well-defined three-dimensional (3D) arrays can translate advances in nanoscale synthesis into targeted material fabrication. Despite successes in nanoparticle assembly, most extant methods are system specific and not fully compatible with biomolecules. Here, we report a platform for creating distinct 3D ordered arrays from different nanomaterials using DNA-prescribed and valence-controlled material voxels. These material voxels consist of 3D DNA frames that integrate nano-objects within their scaffold, thus enabling the object's valence and coordination to be determined by the frame's vertices, which can bind to each other through hybridization. Such DNA material voxels define the lattice symmetry through the spatially prescribed valence decoupling the 3D assembly process from the nature of the nanocomponents, such as their intrinsic properties and shapes. We show this by assembling metallic and semiconductor nanoparticles and also protein superlattices. We support the technological potential of such an assembly approach by fabricating light-emitting 3D arrays with diffraction-limited spectral purity and 3D enzymatic arrays with increased activity.

Stability of the pH-Dependent Parallel-Stranded d(CGA) Motif.

Noncanonical DNA structures that retain programmability and structural predictability are increasingly being used in DNA nanotechnology applications, in which they offer versatility beyond traditional Watson-Crick interactions. The d(CGA) triplet repeat motif is structurally dynamic and can transition between parallel-stranded homo-base paired duplex and antiparallel unimolecular hairpin in a pH-dependent manner. Here, we evaluate the thermodynamic stability and nuclease sensitivity of oligonucleotides composed of the d(CGA) motif and several structurally related sequence variants. These results show that the structural transition resulting from decreasing the pH is accompanied by both a significant energetic stabilization and decreased nuclease sensitivity as unimolecular hairpin structures are converted to parallel-stranded homo-base paired duplexes. Furthermore, the stability of the parallel-stranded duplex form can be altered by changing the 5'-nucleobase of the d(CGA) triplet and the frequency and position of the altered triplets within long stretches of d(CGA) triplets. This work offers insight into the stability and versatility of the d(CGA) triplet repeat motif and provides constraints for using this pH-adaptive structural motif for creating DNA-based nanomaterials.

Practitioner review: Co-design of digital mental health technologies with children and young people.

BACKGROUND: There is increasing interest in digital technologies to help improve children and young people's mental health, and the evidence for the effectiveness for these approaches is rising. However, there is concern regarding levels of user engagement, uptake and adherence. Key guidance regarding digital health interventions stress the importance of early user input in the development, evaluation and implementation of technologies to help ensure they are engaging, feasible, acceptable and potentially effective. Co-design is a process of active involvement of stakeholders, requiring a change from the traditional approaches to intervention development. However, there is a lack of literature to inform the co-design of digital technologies to help child and adolescent mental health. METHODS: We reviewed the literature and practice in the co-design of digital mental health technologies with children and young people. We searched Medline, PsycInfo and Web of Science databases, guidelines, reviews and reference lists, contacted key authors for relevant studies, and extracted key themes on aspects of co-design relevant to practice. We supplemented this with case studies and methods reported by researchers working in the field. RESULTS: We identified 25 original articles and 30 digital mental health technologies that were designed/developed with children and young people. The themes identified were as follows: principles of co-design (including potential stakeholders and stages of involvement), methods of involving and engaging the range of users, co-designing the prototype and the challenges of co-design. CONCLUSIONS: Co-design involves all relevant stakeholders throughout the life and research cycle of the programme. This review helps to inform practitioners and researchers interested in the development of digital health technologies for children and young people. Future work in this field will need to consider the changing face of technology, methods of engaging with the diversity in the user group, and the evaluation of the co-design process and its impact on the technology.

Stimuli-Responsive DNA-Based Hydrogels: From Basic Principles to Applications.

The base sequence of nucleic acids encodes structural and functional information into the DNA biopolymer. External stimuli such as metal ions, pH, light, or added nucleic acid fuel strands provide triggers to reversibly switch nucleic acid structures such as metal-ion-bridged duplexes, i-motifs, triplex nucleic acids, G-quadruplexes, or programmed double-stranded hybrids of oligonucleotides (DNA). The signal-triggered oligonucleotide structures have been broadly applied to develop switchable DNA nanostructures and DNA machines, and these stimuli-responsive assemblies provide functional scaffolds for the rapidly developing area of DNA nanotechnology. Stimuli-responsive hydrogels undergoing signal-triggered hydrogel-to-solution transitions or signal-controlled stiffness changes attract substantial interest as functional matrices for controlled drug delivery, materials exhibiting switchable mechanical properties, acting as valves or actuators, and "smart" materials for sensing and information processing. The integration of stimuli-responsive oligonucleotides with hydrogel-forming polymers provides versatile means to exploit the functional information encoded in the nucleic acid sequences to yield stimuli-responsive hydrogels exhibiting switchable physical, structural, and chemical properties. Stimuli-responsive DNA-based nucleic acid structures are integrated in acrylamide polymer chains and reversible, switchable hydrogel-to-solution transitions of the systems are demonstrated by applying external triggers, such as metal ions, pH-responsive strands, G-quadruplex, and appropriate counter triggers that bridge and dissociate the polymer chains. By combining stimuli-responsive nucleic acid bridges with thermosensitive poly(N-isopropylacrylamide) (pNIPAM) chains, systems undergoing reversible solution ↔ hydrogel ↔ solid transitions are demonstrated. Specifically, by bridging acrylamide polymer chains by two nucleic acid functionalities, where one type of bridging unit provides a stimuli-responsive element and the second unit acts as internal "bridging memory", shape-memory hydrogels undergoing reversible and switchable transitions between shaped hydrogels and shapeless quasi-liquid states are demonstrated. By using stimuli-responsive hydrogel cross-linking units that can assemble the bridging units by two different input signals, the orthogonally-triggered functions of the shape-memory were shown. Furthermore, a versatile approach to assemble stimuli-responsive DNA-based acrylamide hydrogel films on surfaces is presented. The method involves the activation of the hybridization chain-reaction (HCR) by a surface-confined promoter strand, in the presence of acrylamide chains modified with two DNA hairpin structures and appropriate stimuli-responsive tethers. The resulting hydrogel-modified surfaces revealed switchable stiffness properties and signal-triggered catalytic functions. By applying the method to assemble the hydrogel microparticles, substrate-loaded, stimuli-responsive microcapsules are prepared. The signal-triggered DNA-based hydrogel microcapsules are applied as drug carriers for controlled release. The different potential applications and future perspectives of stimuli responsive hydrogels are discussed. Specifically, the use of these smart materials and assemblies as carriers for controlled drug release and as shape-memory matrices for information storage and inscription and the use of surface-confined stimuli-responsive hydrogels, exhibiting switchable stiffness properties, for catalysis and controlled growth of cells are discussed.

Controlling the Catalytic Functions of DNAzymes within Constitutional Dynamic Networks of DNA Nanostructures.

Mimicking complex cellular dynamic chemical networks being up-regulated or down-regulated by external triggers is one of the challenges in systems chemistry. Constitutional dynamic networks (CDNs), composed of exchangeable components that respond to environmental triggers by self-adaption, provide general means to mimic biosystems. We use the structural and functional information encoded in nucleic acid nanostructures to construct effector (input)-triggered constitutional dynamic networks that reveal adaptable catalytic properties. Specifically, CDNs composed of four exchangeable constituents, AA', BA', AB', and BB', are constructed. In the presence of an effector (input) that controls the stability of one of the constituents, the input-guided up-regulation or down-regulation of the CDN's constituents proceeds. As effectors we apply the fuel-strand stabilization of one of the CDN constituents by the formation of the T-A·T triplex structure, or by the K+-ion-induced stabilization of one of the CDN constituents, via the formation of a K+-ion-stabilized G-quadruplex. Energetic stabilization of one of the CDN constituents leads to a new dynamically adapted network composed of up-regulated and down-regulated constituents. By applying counter triggers to the effector units, e.g., an antifuel strand or 18-crown-6-ether, reconfiguration to the original CDNs is demonstrated. The performance of the CDNs is followed by the catalytic activities of the constituents and by complementary quantitative gel electrophoresis experiments. The orthogonal triggered and switchable operation of the CDNs is highlighted.

DNA Scaffolds for the Dictated Assembly of Left-/Right-Handed Plasmonic Au NP Helices with Programmed Chiro-Optical Properties.

Within the broad interest of assembling chiral left- and right-handed helices of plasmonic nanoparticles (NPs), we introduce the DNA-guided organization of left- or right-handed plasmonic Au NPs on DNA scaffolds. The method involves the self-assembly of stacked 12 DNA quasi-rings interlinked by 30 staple-strands. By the functionalization of one group of staple units with programmed tether-nucleic acid strands and additional staple elements with long nucleic acid chains, acting as promoter strands, the promoter-guided assembly of barrels modified with 12 left- or right-handed tethers is achieved. The subsequent hybridization of Au NPs functionalized with single nucleic acid tethers yields left- or right-handed structures of plasmonic NPs. The plasmonic NP structures reveal CD spectra at the plasmon absorbance, and the NPs are imaged by HR-TEM. Using geometrical considerations corresponding to the left- and right-handed helices of the Au NPs, the experimental CD spectra of the plasmonic Au NPs are modeled by theoretical calculations.

Reversible Modulation of DNA-Based Hydrogel Shapes by Internal Stress Interactions.

We present the assembly of asymmetric two-layer hybrid DNA-based hydrogels revealing stimuli-triggered reversibly modulated shape transitions. Asymmetric, linear hydrogels that include layer-selective switchable stimuli-responsive elements that control the hydrogel stiffness are designed. Trigger-induced stress in one of the layers results in the bending of the linear hybrid structure, thereby minimizing the elastic free energy of the systems. The removal of the stress by a counter-trigger restores the original linear bilayer hydrogel. The stiffness of the DNA hydrogel layers is controlled by thermal, pH (i-motif), K+ ion/crown ether (G-quadruplexes), chemical (pH-doped polyaniline), or biocatalytic (glucose oxidase/urease) triggers. A theoretical model relating the experimental bending radius of curvatures of the hydrogels with the Young's moduli and geometrical parameters of the hydrogels is provided. Promising applications of shape-regulated stimuli-responsive asymmetric hydrogels include their use as valves, actuators, sensors, and drug delivery devices.

Layered Metal Nanoparticle Structures on Electrodes for Sensing, Switchable Controlled Uptake/Release, and Photo-electrochemical Applications.

Layered metal nanoparticle (NP) assemblies provide highly porous and conductive composites of unique electrical and optical (plasmonic) properties. Two methods to construct layered metal NP matrices are described, and these include the layer-by-layer deposition of NPs, or the electropolymerization of monolayer-functionalized NPs, specifically thioaniline-modified metal NPs. The layered NP composites are used as sensing matrices through the use of electrochemistry or surface plasmon resonance (SPR) as transduction signals. The crosslinking of the metal NP composites with molecular receptors, or the imprinting of molecular recognition sites into the electropolymerized NP matrices lead to selective and chiroselective sensing interfaces. Furthermore, the electrosynthesis of redox-active, imprinted, bis-aniline bridged Au NP composites yields electrochemically triggered "sponges" for the switchable uptake and release of electron-acceptor substrates, and results in conductive surfaces of electrochemically controlled wettability. Also, photosensitizer-relay-crosslinked Au NP composites, or electrochemically polymerized layered semiconductor quantum dot/metal NP matrices on electrodes, are demonstrated as functional nanostructures for photoelectrochemical applications.

Orthogonal Dual-Triggered Shape-Memory DNA-Based Hydrogels.

DNA-based shape-memory hydrogels revealing switchable shape recovery in the presence of two orthogonal triggers are described. In one system, a shaped DNA/acrylamide hydrogel is stabilized by duplex nucleic acids and pH-responsive cytosine-rich, i-motif, bridges. Separation of the i-motif bridges at pH 7.4 transforms the hydrogel into a quasi-liquid, shapeless state, that includes the duplex bridges as permanent shape-memory elements. Subjecting the quasi-liquid state to pH 5.0 or Ag(+) ions recovers the hydrogel shape, due to the stabilization of the hydrogel by i-motif or C-Ag(+) -C bridged i-motif. The cysteamine-induced transformation of the duplex/C-Ag(+) -C bridged i-motif hydrogel into a quasi-liquid shapeless state results in the recovery of the shaped hydrogel in the presence of H(+) or Ag(+) ions as triggers. In a second system, a shaped DNA/acrylamide hydrogel is generated by DNA duplexes and bridging Pb(2+) or Sr(2+) ions-stabilized G-quadruplex subunits. Subjecting the shaped hydrogel to the DOTA or KP ligands eliminates the Pb(2+) or Sr(2+) ions from the respective hydrogels, leading to shapeless, memory-containing, quasi-liquid states that restore the original shapes with Pb(2+) or Sr(2+) ions.

DNA Microgels as a Platform for Cell-Free Protein Expression and Display.

Protein expression and selection is an essential process in the modification of biological products. Expressed proteins are selected based on desired traits (phenotypes) from diverse gene libraries (genotypes), whose size may be limited due to the difficulties inherent in diverse cell preparation. In addition, not all genes can be expressed in cells, and linking genotype with phenotype further presents a great challenge in protein engineering. We present a DNA gel-based platform that demonstrates the versatility of two DNA microgel formats to address fundamental challenges of protein engineering, including high protein yield, isolation of gene sets, and protein display. We utilize microgels to show successful protein production and capture of a model protein, green fluorescent protein (GFP), which is further used to demonstrate a successful gene enrichment through fluorescence-activated cell sorting (FACS) of a mixed population of microgels containing the GFP gene. Through psoralen cross-linking of the hydrogels, we have synthesized DNA microgels capable of surviving denaturing conditions while still possessing the ability to produce protein. Lastly, we demonstrate a method of producing extremely high local gene concentrations of up to 32 000 gene repeats in hydrogels 1 to 2 µm in diameter. These DNA gels can serve as a novel cell-free platform for integrated protein expression and display, which can be applied toward more powerful, scalable protein engineering and cell-free synthetic biology with no physiological boundaries and limitations.

Integration of Switchable DNA-Based Hydrogels with Surfaces by the Hybridization Chain Reaction.

A novel method to assemble acrylamide/acrydite DNA copolymer hydrogels on surfaces, specifically gold-coated surfaces, is introduced. The method involves the synthesis of two different copolymer chains consisting of hairpin A, HA, modified acrylamide copolymer and hairpin B, HB, acrylamide copolymer. In the presence of a nucleic acid promoter monolayer associated with the surface, the hybridization chain reaction between the two hairpin-modified polymer chains is initiated, giving rise to the cross-opening of hairpins HA and HB and the formation of a cross-linked hydrogel on the surface. By the cofunctionalization of the HA- and HB-modified polymer chains with G-rich DNA tethers that include the G-quadruplex subunits, hydrogels of switchable stiffness are generated. In the presence of K(+)-ions, the hydrogel associated with the surface is cooperatively cross-linked by duplex units of HA and HB, and K(+)-ion-stabilized G-quadruplex units, giving rise to a stiff hydrogel. The 18-crown-6-ether-stimulated elimination of the K(+)-ions dissociates the bridging G-quadruplex units, resulting in a hydrogel of reduced stiffness. The duplex/G-quadruplex cooperatively stabilized hydrogel associated with the surface reveals switchable electrocatalytic properties. The incorporation of hemin into the G-quadruplex units electrocatalyzes the reduction of H2O2. The 18-crown-6-ether stimulated dissociation of the hemin/G-quadruplex bridging units leads to a catalytically inactive hydrogel.

A Shape-Memory DNA-Based Hydrogel Exhibiting Two Internal Memories.

The synthesis of a shape-memory acrylamide-DNA hydrogel that includes two internal memories is introduced. The hydrogel is stabilized, at pH 7.0, by two different pH-responsive oligonucleotide crosslinking units. At pH 10.0, one of the T-A⋅T triplex DNA bridging units is dissociated, resulting in the dissociation of the hydrogel into a shapeless quasi-liquid state that includes the other oligonucleotide bridges as internal memory. Similarly, at pH 5.0, the second type of bridges is separated, through the formation of C-G⋅C(+) triplex units to yield the shapeless quasi-liquid state that includes the other oligonucleotide bridges as internal memory. By reversible pH triggering of the hydrogel between the values 10.0⇔7.0⇔5.0, the two internal memories cycle the material across shaped hydrogel and shapeless quasi-liquid states. The two memories enable the pH-dictated formation of two different hydrogel structures.

Rationally designed coiled-coil DNA looping peptides control DNA topology.

Artificial DNA looping peptides were engineered to study the roles of protein and DNA flexibility in controlling the geometry and stability of protein-mediated DNA loops. These LZD (leucine zipper dual-binding) peptides were derived by fusing a second, C-terminal, DNA-binding region onto the GCN4 bZip peptide. Two variants with different coiled-coil lengths were designed to control the relative orientations of DNA bound at each end. Electrophoretic mobility shift assays verified formation of a sandwich complex containing two DNAs and one peptide. Ring closure experiments demonstrated that looping requires a DNA-binding site separation of 310 bp, much longer than the length needed for natural loops. Systematic variation of binding site separation over a series of 10 constructs that cyclize to form 862-bp minicircles yielded positive and negative topoisomers because of two possible writhed geometries. Periodic variation in topoisomer abundance could be modeled using canonical DNA persistence length and torsional modulus values. The results confirm that the LZD peptides are stiffer than natural DNA looping proteins, and they suggest that formation of short DNA loops requires protein flexibility, not unusual DNA bendability. Small, stable, tunable looping peptides may be useful as synthetic transcriptional regulators or components of protein-DNA nanostructures.

DNA, flexibly flexible.

Investigators have constructed dsDNA molecules with several different base modifications and have characterized their bending and twisting flexibilities using atomic force microscopy, DNA ring closure, and single-molecule force spectroscopy with optical tweezers. The three methods provide persistence length measurements that agree semiquantitatively, and they show that the persistence length is surprisingly similar for all of the modified DNAs. The circular dichroism spectra of modified DNAs differ substantially. Simple explanations based on base stacking strength, polymer charge, or groove occupancy by functional groups cannot explain the results, which will guide further high-resolution theory and experiments.

Tethered particle motion reveals that LacI·DNA loops coexist with a competitor-resistant but apparently unlooped conformation.

The lac repressor protein (LacI) efficiently represses transcription of the lac operon in Escherichia coli by binding to two distant operator sites on the bacterial DNA and causing the intervening DNA to form a loop. We employed single-molecule tethered particle motion to observe LacI-mediated loop formation and breakdown in DNA constructs that incorporate optimized operator binding sites and intrinsic curvature favorable to loop formation. Previous bulk competition assays indirectly measured the loop lifetimes in these optimized DNA constructs as being on the order of days; however, we measured these same lifetimes to be on the order of minutes for both looped and unlooped states. In a range of single-molecule DNA competition experiments, we found that the resistance of the LacI-DNA complex to competitive binding is a function of both the operator strength and the interoperator sequence. To explain these findings, we present what we believe to be a new kinetic model of loop formation and DNA competition. In this proposed new model, we hypothesize a new unlooped state in which the unbound DNA-binding domain of the LacI protein interacts nonspecifically with nonoperator DNA adjacent to the operator site at which the second LacI DNA-binding domain is bound.

FRET studies of a landscape of Lac repressor-mediated DNA loops.

DNA looping mediated by the Lac repressor is an archetypal test case for modeling protein and DNA flexibility. Understanding looping is fundamental to quantitative descriptions of gene expression. Systematic analysis of LacI•DNA looping was carried out using a landscape of DNA constructs with lac operators bracketing an A-tract bend, produced by varying helical phasings between operators and the bend. Fluorophores positioned on either side of both operators allowed direct Förster resonance energy transfer (FRET) detection of parallel (P1) and antiparallel (A1, A2) DNA looping topologies anchored by V-shaped LacI. Combining fluorophore position variant landscapes allows calculation of the P1, A1 and A2 populations from FRET efficiencies and also reveals extended low-FRET loops proposed to form via LacI opening. The addition of isopropyl-ß-D-thio-galactoside (IPTG) destabilizes but does not eliminate the loops, and IPTG does not redistribute loops among high-FRET topologies. In some cases, subsequent addition of excess LacI does not reduce FRET further, suggesting that IPTG stabilizes extended or other low-FRET loops. The data align well with rod mechanics models for the energetics of DNA looping topologies. At the peaks of the predicted energy landscape for V-shaped loops, the proposed extended loops are more stable and are observed instead, showing that future models must consider protein flexibility.

Crystallization of DNA-capped gold nanoparticles in high-concentration, divalent salt environments.

The multiparametric nature of nanoparticle self-assembly makes it challenging to circumvent the instabilities that lead to aggregation and achieve crystallization under extreme conditions. By using non-base-pairing DNA as a model ligand instead of the typical base-pairing design for programmability, long-range 2D DNA-gold nanoparticle crystals can be obtained at extremely high salt concentrations and in a divalent salt environment. The interparticle spacings in these 2D nanoparticle crystals can be engineered and further tuned based on an empirical model incorporating the parameters of ligand length and ionic strength.

Direct observation of dynamic mechanical regulation of DNA condensation by environmental stimuli.

Gene delivery is a promising way to treat hereditary diseases and cancer; however, there is little understanding of DNA:carrier complex mechanical properties, which may be critical for the protection and release of nucleic acids. We applied optical tweezers to directly measure single-molecule mechanical properties of DNA condensed using 19-mer poly-L-lysine (PLL) or branched histidine-lysine (HK) peptides. Force-extension profiles indicate that both carriers condense DNA actively, showing force plateaus during stretching and relaxation cycles. As the environment such as carrier concentration, pH, and the presence of zinc ions changes, DNA:HK complexes showed dynamically regulated mechanical properties at multiple force levels. The fundamental knowledge from this study can be applied to design a mechanically tailored complex which may enhance transfection efficiency by controlling the stability of the complex temporally and spatially.