Net-Shaped DNA Nanostructure-Based Lateral Flow Assays for Rapid and Sensitive SARS-CoV-2 Detection.

Lateral flow assay (LFA)-based rapid antigen tests are experiencing extensive global uptake as an expeditious and highly effective modality for the screening of viral infections during the COVID-19 pandemic. While these devices have played a significant role in alleviating the burden on the public healthcare system, their specificity and sensitivity fall short compared with molecular tests. In this study, we endeavor to address both limitations through the utilization of DNA nanotechnology in LFA format, wherein we substitute the target-specific antibody with designer DNA nanostructure-based molecular probes for recognizing the SARS-CoV-2 virus via multivalent, pattern-matching interactions. We meticulously designed a Net-shaped DNA nanostructure and strategically arranged trimeric clusters of aptamers that specifically recognize the spike proteins of SARS-CoV-2. This approach has proven instrumental in bolstering virus-binding affinity on the LFAs. Our findings indicate high LFA sensitivity, enabling the detection of viral loads ranging from 103 to 108 viral copies/mL. This notable sensitivity is maintained across various SARS-CoV-2 viral strains, obviating the need for intricate sample preparation protocols. The significance of this heightened sensitivity lies in the crucial role played by the designer DNA nanostructure, which facilitates the detection of extremely low levels of viral loads. This not only enhances the overall reliability of self-testing but also reduces the likelihood of false-negative results, especially in cases of low viral load within patient samples.

Surface-assisted self-assembly of 2D, DNA binary crystals.

Surface-assisted, tile-based DNA self-assembly is a powerful method to construct large, two-dimensional (2D) nanoarrays. To further increase the structural complexity, one idea is to incorporate different types of tiles into one assembly system. However, different tiles have different adsorption strengths to the solid surface. The differential adsorptions make it difficult to control the effective molar ratio between different DNA tile concentrations on the solid surface, leading to assembly failure. Herein, we propose a solution to this problem by engineering the tiles with comparable molecular weights while maintaining their architectures. As a demonstration, we have applied this strategy to successfully assemble binary DNA 2D arrays out of very different tiles. We expect that this strategy would facilitate assembly of other complicated nanostructures as well.

Mechanical deformation behaviors and structural properties of ligated DNA crystals.

DNA self-assembly has emerged as a powerful strategy for constructing complex nanostructures. While the mechanics of individual DNA strands have been studied extensively, the deformation behaviors and structural properties of self-assembled architectures are not well understood. This is partly due to the small dimensions and limited experimental methods available. DNA crystals are macroscopic crystalline structures assembled from nanoscale motifs via sticky-end association. The large DNA constructs may thus be an ideal platform to study structural mechanics. Here, we investigate the fundamental mechanical properties and behaviors of ligated DNA crystals made of tensegrity triangular motifs. We perform coarse-grained molecular dynamics simulations and confirm the results with nanoindentation experiments using atomic force microscopy. We observe various deformation modes, including untension, linear elasticity, duplex dissociation, and single-stranded component stretch. We find that the mechanical properties of a DNA architecture are correlated with those of its components. However, the structure shows complex behaviors which may not be predicted by components alone and the architectural design must be considered.

Programming DNA Self-Assembly by Geometry†.

This manuscript introduces geometry as a means to program the tile-based DNA self-assembly in two and three dimensions. This strategy complements the sequence-focused programmable assembly. DNA crystal assembly critically relies on intermotif, sticky-end cohesion, which requires complementarity not only in sequence but also in geometry. For DNA motifs to assemble into crystals, they must be associated with each other in the proper geometry and orientation to ensure that geometric hindrance does not prevent sticky ends from associating. For DNA motifs with exactly the same pair of sticky-end sequences, by adjusting the length (thus, helical twisting phase) of the motif branches, it is possible to program the assembly of these distinct motifs to either mix with one another, to self-sort and consequently separate from one another, or to be alternatingly arranged. We demonstrate the ability to program homogeneous crystals, DNA "alloy" crystals, and definable grain boundaries through self-assembly. We believe that the integration of this strategy and conventional sequence-focused assembly strategy could further expand the programming versatility of DNA self-assembly.

Powering ≈50 µm Motion by a Molecular Event in DNA Crystals.

A major challenge in material design is to couple nanoscale molecular and supramolecular events into desired chemical, physical, and mechanical properties at the macroscopic scale. Here, a novel self-assembled DNA crystal actuator is reported, which has reversible, directional expansion and contraction for over 50 µm in response to versatile stimuli, including temperature, ionic strength, pH, and redox potential. The macroscopic actuation is powered by cooperative dissociation or cohesion of thousands of DNA sticky ends at the designed crystal contacts. The increase in crystal porosity and cavity in the expanded state dramatically enhances the crystal capability to accommodate/encapsulate nanoparticles/proteins, while the contraction enables a "sponge squeezing" motion for releasing nanoparticles. This crystal actuator is envisioned to be useful for a wide range of applications, including powering self-propelled robotics, sensing subtle environmental changes, constructing functional hybrid materials, and working in drug controlled-release systems.

Structure-Guided Designing Pre-Organization in Bivalent Aptamers.

Multivalent interaction is often used in molecular design and leads to engineered multivalent ligands with increased binding avidities toward target molecules. The resulting binding avidity relies critically on the rigid scaffold that joins multiple ligands as the scaffold controls the relative spatial positions and orientations toward target molecules. Currently, no general design rules exist to construct a simple and rigid DNA scaffold for properly joining multiple ligands. Herein, we report a crystal structure-guided strategy for the rational design of a rigid bivalent aptamer with precise control over spatial separation and orientation. Such a pre-organization allows the two aptamer moieties simultaneously to bind to the target protein at their native conformations. The bivalent aptamer binding has been extensively characterized, and an enhanced binding has been clearly observed. This strategy, we believe, could potentially be generally applicable to design multivalent aptamers.

Kinetic DNA Self-Assembly: Simultaneously Co-folding Complementary DNA Strands into Identical Nanostructures.

DNA origami is a powerful method for constructing DNA nanostructures. It requires long single-stranded DNAs. The preparation of such long DNA strands is often quite tedious and has a limited production yield. In contrast, duplex DNAs can be easily prepared via enzymatic reactions in large quantities. Thus, we ask a question: can we design DNA nanostructures in such a way that the two complementary strands can simultaneously fold into the designed structures in the same solution instead of hybridizing with each other to form a DNA duplex? By engineering DNA interaction kinetics, herein we are able to provide multiple examples to concretely demonstrate a positive answer to this question. The resulting DNA nanostructures have been thoroughly characterized by electrophoresis and atomic force microscopy imaging. The reported strategy is compatible with the DNA cloning method and thus would provide a convenient method for the large-scale production of the designed DNA nanostructures.

5'-Phosphorylation Strengthens Sticky-End Cohesions.

Sticky-end cohesion plays a critical role in molecular biology and nucleic acid nanotechnology. Although free energy calculations and molecular mechanics can predict these interactions, chemical modification would compromise such predictions. Herein, we have used rationally designed 3D DNA crystals as a tool to experimentally investigate the modulation of 5'-phosphorylation on sticky-end cohesions. We have found that 5'-phosphorylation strengthens the sticky-end cohesion: in a DNA crystal self-assembled exclusively via sticky-end cohesions, 5'-phosphorylation not only promotes the crystallization process, in general, but also accelerates the crystal growth along designed directions. Such a finding allows the fine-tuning of DNA crystallization kinetics and the control of DNA crystal morphology. It also suggests a potential difference in self-assembly kinetics between natural DNA (with 5'-phosphorylation) and synthetic DNA (without 5'-phosphorylation).

Engineering the Nanoscaled Morphologies of Linear DNA Homopolymers.

Supramolecular polymers have unique characteristics such as self-healing and easy processing. However, the scope of their structures is limited to mostly either flexible, random coils or rigid, straight chains. By broadening this scope, novel properties, functions, and applications can be explored. Here, DNA is used as a model system to engineer innovative, nanoscaled morphologies of supramolecular polymers. Each polymer chain consists of multiple copies of the same short (38-46 nucleotides long) DNA strand. The component DNA strands first dimerize into homo-dimers, which then further assemble into long polymer chains. By subtly tuning the design, a range of polymer morphologies are obtained; including straight chains, spirals, and closed rings with finite sizes. Such structures are confirmed by AFM imaging and predicted by molecular coarse simulation.

Assembly of a DNA Origami Chinese Knot by Only 15% of the Staple Strands.

As a giant leap in DNA self-assembly, DNA origami has exhibited an unprecedented ability to construct nanostructures with arbitrary shapes and sizes. In typical DNA origami, hundreds of short DNA staple strands fold a long, single-stranded (ss) DNA scaffold cooperatively into designed nanostructures. However, large numbers of DNA strands are expensive and would hinder applications such as pharmaceutical investigations because of the complicated components. Therefore, one challenge is how to reduce the number of staple strands needed to construct DNA origami. For a DNA origami structure, the scale-free folding pattern of the scaffold strand is determined by staple strands at the branching vertexes. Simple duplex regions help to define the size-related features of the origami geometry. In this study, we hypothesized that a scaffold strand can be correctly folded into a designed topology by using only staple strands involved in branching vertexes. After assembly, any remaining, flexible, single-stranded regions of the scaffold could be converted into rigid duplexes by DNA polymerase to achieve the designed geometric structures. To demonstrate the concept, we used only 18 staple strands (covering 15 % of the scaffold strand) to assemble a porous DNA nanostructure, which was visualized by atomic force microscopy (AFM). This study helps understanding of the role of cooperativity in origami folding, and provides a cost-effective approach for small-scale prototyping DNA origami.

Making Engineered 3D DNA Crystals Robust.

Engineered 3D DNA crystals are promising scaffolds for bottom-up construction of three-dimensional, macroscopic devices from the molecular level. Nevertheless, this has been hindered by the highly constrained conditions for DNA crystals to be stable. Here we report a method to prepare robust 3D DNA crystals by postassembly ligation to remove this constraint. Specifically, sticky ends at crystal contacts were enzymatically ligated, and the covalent bonds significantly enhanced crystal stability, e.g., being stable at 65 °C. This method also enabled the fabrication of DNA crystals with complex architectures including crystal shell, core-shell, and matryoshka dolls. Furthermore, we have demonstrated the applications of the robust DNA crystals in biocatalysis and protein entrapment. Our study removes one key obstacle for the applications of DNA crystals and offers many new opportunities in DNA nanotechnology.

Patterning Nanoparticles with DNA Molds.

We report a nanopatterning strategy in which self-assembled DNA nanostructures serve as structural templates. In previous work, ordering of NPs primarily relied on specific recognition, e.g., DNA-DNA hybridization. Only a few cases have been reported on nonspecific adsorption. Unfortunately, these studies were limited by the integrity and homogeneity of templates and the variety of patterned nanoparticles (NPs). Herein, we have developed a general method to pattern various NPs. The NPs adsorb onto substrate via NP-substrate direct interactions and the substrates are patterned into large arrays (>4 × 4 µm) of tiny, accessible cavities by self-assembled DNA arrays. As a demonstration, DNA templates include tetragonal and hexagonal arrays and the NPs include individual DNA nanomotifs, gold nanoparticles (AuNPs), and proteins. All nanostructures have been confirmed by atomic force microscopy and corresponding fast Fourier transform (FFT) analysis.

In vivo production of RNA nanostructures via programmed folding of single-stranded RNAs.

Programmed self-assembly of nucleic acids is a powerful approach for nano-constructions. The assembled nanostructures have been explored for various applications. However, nucleic acid assembly often requires chemical or in vitro enzymatical synthesis of DNA or RNA, which is not a cost-effective production method on a large scale. In addition, the difficulty of cellular delivery limits the in vivo applications. Herein we report a strategy that mimics protein production. Gene-encoded DNA duplexes are transcribed into single-stranded RNAs, which self-fold into well-defined RNA nanostructures in the same way as polypeptide chains fold into proteins. The resulting nanostructure contains only one component RNA molecule. This approach allows both in vitro and in vivo production of RNA nanostructures. In vivo synthesized RNA strands can fold into designed nanostructures inside cells. This work not only suggests a way to synthesize RNA nanostructures on a large scale and at a low cost but also facilitates the in vivo applications.

Insight into the effects of modifying π-bridges on the performance of dye-sensitized solar cells containing triphenylamine dyes.

Herein we prepare four novel D-π-A dyes based on triphenylamine (ZHG1, ZHG2, ZHG3 and ZHG4) by modifying the π-bridges. Compared with ZHG1, the power conversion efficiency (PCE) of ZHG2 is improved to 6.1% after the introduction of ethynyl. But further extension of the conjugation of the π-bridges by introducing the chromophore 4,8-bis(n-octyloxy)-benzo[1,2-b:4,5-b']dithiophene (BDT) into ZHG3 conversely decreases the PCE to 4.6%. Improving the coplanarity by replacing cyclobenzene with thiophene in ZHG4 after introducing BDT further decreases the PCE of ZHG4 to 4.3%. Theoretical calculations indicate that the LUMOs of ZHG3 and ZHG4 were mainly delocalized over benzothiadiazole which is far from the anchoring groups. Cyclic voltammetry experiments indicate that the LUMO energy levels of ZHG3 and ZHG4 are lower than those of ZHG1 and ZHG2. Both of these results affect the ability to inject electrons into the TiO2 conduction band. X-ray photoelectron spectroscopy (XPS) analysis shows that the mean thickness of the dye coverage for ZHG1, ZHG2, ZHG3 and ZHG4 is 16 Å, 18 Å, 27 Å and 24 Å, respectively. So the tilt angle of the dye backbone anchored on the TiO2 film is in the order of ZHG1 > ZHG2 > ZHG4 > ZHG3, which is consistent with the dye coverage of the outermost TiO2 surfaces. This result indicates that the intermolecular π-π aggregation in ZHG3 and ZHG4 with overlong π-bridges is more serious compared with that in ZHG1 and ZHG2. Perhaps the above two factors are the reason that the PCEs of ZHG3 and ZHG4 are lower than those of ZHG1 and ZHG2. So it is very important to find a balance point between the electron injection ability, intermolecular π-π aggregation and the expansion of the light absorption range.

Macrocyclic Se4N2[7,7]ferrocenophane and Se2N[10]ferrocenophane containing benzyl unit: synthesis, complexation, crystal structures, electrochemical and optical properties.

Two novel macrocyclic polyselena[n]ferrocenophanes containing a pendent benzyl unit, 20-membered Se4N2[7,7]ferrocenophane (L1) and 10-membered Se2N[10]ferrocenophane (L2), were designed and synthesized. The reaction of L1 with two molar amounts of metal salts (M = Cu(+), Cu(2+), Pd(2+) and Hg(2+)) led to six dimetallic complexes 1-6. A crystallographic study revealed that each metal center in 1-5 was tetracoordinated to two selenium atoms from different ferrocene units, one aliphatic nitrogen atom and one co-ligand. The structures of the complexes contain a two-fold axis perpendicular to the molecular plane with two pendant benzyl moieties in an anti-conformation. Macrocycle L1 gives significant electrochemical, linear and third-order nonlinear optical responses to Cu(2+) and Hg(2+). The DFT/B3LYP calculations of 4 demonstrated a small HOMO-LUMO energy gap and delocalization of the π-electron cloud in the frontier molecular orbitals, which led to the enhancement of molecular NLO properties after complexation. The results show that the oxidation state of the ferrocene unit is accompanied by significant differences in the corresponding absorption spectra and third-order NLO properties.

Assembly of various degrees of interpenetration of Co-MOFs based on mononuclear or dinuclear cluster units: magnetic properties and gas adsorption.

Three new Co-based MOFs with a nanosized tetradentate pyridine ligand, N,N,N',N'-tetrakis(4-(4-pyridine)-phenyl) biphenyl-4,4'-diamine (TPPBDA) and carboxylate co-ligands, [Co(TPPBDA)(NO3)2]n·2H2O (1), [Co2(TPPBDA)(bpdc)2 (H2O)]n·2DMA (2) and [Co(TPPBDA)0.5(hfipbb)(H2O)]n·3.5H2O (3) (H2bpdc = biphenyldicarboxylic acid, H2hfipbb = 4,4'-(hexafluoroisopropylidene)bis-(benzoic acid), DMA = N,N-dimethylacetamide) have been synthesized under hydrothermal conditions. For complex 1, a large cavity causes a 4-fold interpenetration of the network, which can be classified as a type IIIa mode of interpenetration. Complex 2 reveals a non-interpenetrating three-dimensional (3D) framework based on the [Co2(µ2-H2O)(CO2)2] unit. Complex 3 is also a 2-fold interpenetrating 3D net based on the [Co2(CO2)2] cluster. These mononuclear or dinuclear cluster units are interconnected by TPPBDA and carboxylate co-ligands, resulting in interesting structural diversities and various degrees of interpenetration.

One non-interpenetrated chiral porous multifunctional metal-organic framework and its applications for sensing small solvent molecules and adsorption.

The herein obtained multifunctional compound is a promising fluorescent material that can give tunable fluorescence emissions by changing the solvent molecules. The fluorescence sensing behaviors are different for non-protonic and protonic solvents. To date, such a large response range of emission positions for fluorescent MOFs has not been reported before.