Nucleic Acid-Based Nanodevices in Biological Imaging.

The nanoscale engineering of nucleic acids has led to exciting molecular technologies for high-end biological imaging. The predictable base pairing, high programmability, and superior new chemical and biological methods used to access nucleic acids with diverse lengths and in high purity, coupled with computational tools for their design, have allowed the creation of a stunning diversity of nucleic acid-based nanodevices. Given their biological origin, such synthetic devices have a tremendous capacity to interface with the biological world, and this capacity lies at the heart of several nucleic acid-based technologies that are finding applications in biological systems. We discuss these diverse applications and emphasize the advantage, in terms of physicochemical properties, that the nucleic acid scaffold brings to these contexts. As our ability to engineer this versatile scaffold increases, its applications in structural, cellular, and organismal biology are clearly poised to massively expand.

Engineering tertiary chirality in helical biopolymers.

Tertiary chirality describes the handedness of supramolecular assemblies and relies not only on the primary and secondary structures of the building blocks but also on topological driving forces that have been sparsely characterized. Helical biopolymers, especially DNA, have been extensively investigated as they possess intrinsic chirality that determines the optical, mechanical, and physical properties of the ensuing material. Here, we employ the DNA tensegrity triangle as a model system to locate the tipping points in chirality inversion at the tertiary level by X-ray diffraction. We engineer tensegrity triangle crystals with incremental rotational steps between immobile junctions from 3 to 28 base pairs (bp). We construct a mathematical model that accurately predicts and explains the molecular configurations in both this work and previous studies. Our design framework is extendable to other supramolecular assemblies of helical biopolymers and can be used in the design of chiral nanomaterials, optically active molecules, and mesoporous frameworks, all of which are of interest to physical, biological, and chemical nanoscience.

Hierarchy of topological transitions in a network liquid.

The representation of complex systems as networks has become a critical tool across many fields of science. In the context of physical networks, such as biological neural networks, vascular networks, or network liquids where the nodes and edges occupy volume in three-dimensional space, the question of how they become densely packed is of special importance. Here, we investigate a model network liquid, which is known to densify via two successive liquid-liquid phase transitions (LLPTs). We elucidate the importance of rings-cyclic paths formed by bonded particles in the networks-and their spatial disposition in understanding the structural changes that underpin the increase in density across the LLPTs. Our analyses demonstrate that the densification of these networks is primarily driven by the formation of linked rings, and the LLPTs correspond to a hierarchy of topological transitions where rings form the fundamental building blocks. We envisage entanglement to emerge as a general mechanism for densification, with wide implications for the embedding of physical networks, especially in confined spaces.

Programming crystallization kinetics of self-assembled DNA crystals with 5-methylcytosine modification.

Self-assembled DNA crystals offer a precise chemical platform at the ångström-scale for DNA nanotechnology, holding enormous potential in material separation, catalysis, and DNA data storage. However, accurately controlling the crystallization kinetics of such DNA crystals remains challenging. Herein, we found that atomic-level 5-methylcytosine (5mC) modification can regulate the crystallization kinetics of DNA crystal by tuning the hybridization rates of DNA motifs. We discovered that by manipulating the axial and combination of 5mC modification on the sticky ends of DNA tensegrity triangle motifs, we can obtain a series of DNA crystals with controllable morphological features. Through DNA-PAINT and FRET-labeled DNA strand displacement experiments, we elucidate that atomic-level 5mC modification enhances the affinity constant of DNA hybridization at both the single-molecule and macroscopic scales. This enhancement can be harnessed for kinetic-driven control of the preferential growth direction of DNA crystals. The 5mC modification strategy can overcome the limitations of DNA sequence design imposed by limited nucleobase numbers in various DNA hybridization reactions. This strategy provides a new avenue for the manipulation of DNA crystal structure, valuable for the advancement of DNA and biomacromolecular crystallography.

A DNA-based hydrogel for exosome separation and biomedical applications.

Exosomes (EXOs) have been proven as biomarkers for disease diagnosis and agents for therapeutics. Great challenge remains in the separation of EXOs with high-purity and low-damage from complex biological media, which is critical for the downstream applications. Herein, we report a DNA-based hydrogel to realize the specific and nondestructive separation of EXOs from complex biological media. The separated EXOs were directly utilized in the detection of human breast cancer in clinical samples, as well as applied in the therapeutics of myocardial infarction in rat models. The materials chemistry basis of this strategy involved the synthesis of ultralong DNA chains via an enzymatic amplification, and the formation of DNA hydrogels through complementary base-pairing. These ultralong DNA chains that contained polyvalent aptamers were able to recognize and bind with the receptors on EXOs, and the specific and efficient binding ensured the selective separation of EXOs from media into the further formed networked DNA hydrogel. Based on this DNA hydrogel, rationally designed optical modules were introduced for the detection of exosomal pathogenic microRNA, which achieved the classification of breast cancer patients versus healthy donors with 100% precision. Furthermore, the DNA hydrogel that contained mesenchymal stem cell-derived EXOs was proved with significant therapeutic efficacy in repairing infarcted myocardium of rat models. We envision that this DNA hydrogel-based bioseparation system is promising as a powerful biotechnology, which will promote the development of extracellular vesicles in nanobiomedicine.

Programming the morphology of DNA origami crystals by magnesium ion strength.

Harnessing the programmable nature of DNA origami for controlling structural features in crystalline materials affords opportunities to bring crystal engineering to a remarkable level. However, the challenge of crystallizing a single type of DNA origami unit into varied structural outcomes remains, given the requirement for specific DNA designs for each targeted structure. Here, we show that crystals with distinct equilibrium phases and shapes can be realized using a single DNA origami morphology with an allosteric factor to modulate the binding coordination. As a result, origami crystals undergo phase transitions from a simple cubic lattice to a simple hexagonal (SH) lattice and eventually to a face-centered cubic (FCC) lattice. After selectively removing internal nanoparticles from DNA origami building blocks, the body-centered tetragonal and chalcopyrite lattice are derived from the SH and FCC lattices, respectively, revealing another phase transition involving crystal system conversions. The rich phase space was realized through the de novo synthesis of crystals under varying solution environments, followed by the individual characterizations of the resulting products. Such phase transitions can lead to associated transitions in the shape of the resulting products. Hexagonal prism crystals, crystals characterized by triangular facets, and twinned crystals are observed to form from SH and FCC systems, which have not previously been experimentally realized by DNA origami crystallization. These findings open a promising pathway toward accessing a rich phase space with a single type of building block and wielding other instructions as tools to develop crystalline materials with tunable properties.

DNA hydrogels for bone regeneration.

DNA-based biomaterials have been proposed for tissue engineering approaches due to their predictable assembly into complex morphologies and ease of functionalization. For bone tissue regeneration, the ability to bind Ca2+ and promote hydroxyapatite (HAP) growth along the DNA backbone combined with their degradation and release of extracellular phosphate, a known promoter of osteogenic differentiation, make DNA-based biomaterials unlike other currently used materials. However, their use as biodegradable scaffolds for bone repair remains scarce. Here, we describe the design and synthesis of DNA hydrogels, gels composed of DNA that swell in water, their interactions in vitro with the osteogenic cell lines MC3T3-E1 and mouse calvarial osteoblast, and their promotion of new bone formation in rat calvarial wounds. We found that DNA hydrogels can be readily synthesized at room temperature, and they promote HAP growth in vitro, as characterized by Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, atomic force microscopy, and transmission electron microscopy. Osteogenic cells remain viable when seeded on DNA hydrogels in vitro, as characterized by fluorescence microscopy. In vivo, DNA hydrogels promote the formation of new bone in rat calvarial critical size defects, as characterized by micro-computed tomography and histology. This study uses DNA hydrogels as a potential therapeutic biomaterial for regenerating lost bone.

High spatial-resolved heat manipulating membrane heterogeneity alters cellular migration and signaling.

Plasma membrane heterogeneity is a key biophysical regulatory principle of membrane protein dynamics, which further influences downstream signal transduction. Although extensive biophysical and cell biology studies have proven membrane heterogeneity is essential to cell fate, the direct link between membrane heterogeneity regulation to cellular function remains unclear. Heterogeneous structures on plasma membranes, such as lipid rafts, are transiently assembled, thus hard to study via regular techniques. Indeed, it is nearly impossible to perturb membrane heterogeneity without changing plasma membrane compositions. In this study, we developed a high-spatial resolved DNA-origami-based nanoheater system with specific lipid heterogeneity targeting to manipulate the local lipid environmental temperature under near-infrared (NIR) laser illumination. Our results showed that the targeted heating of the local lipid environment influences the membrane thermodynamic properties, which further triggers an integrin-associated cell migration change. Therefore, the nanoheater system was further applied as an optimized therapeutic agent for wound healing. Our strategy provides a powerful tool to dynamically manipulate membrane heterogeneity and has the potential to explore cellular function through changes in plasma membrane biophysical properties.

Regulating phase behavior of nanoparticle assemblies through engineering of DNA-mediated isotropic interactions.

Self-assembly of isotropically interacting particles into desired crystal structures could allow for creating designed functional materials via simple synthetic means. However, the ability to use isotropic particles to assemble different crystal types remains challenging, especially for generating low-coordinated crystal structures. Here, we demonstrate that isotropic pairwise interparticle interactions can be rationally tuned through the design of DNA shells in a range that allows transition from common, high-coordinated FCC-CuAu and BCC-CsCl lattices, to more exotic symmetries for spherical particles such as the SC-NaCl lattice and to low-coordinated crystal structures (i.e., cubic diamond, open honeycomb). The combination of computational and experimental approaches reveals such a design strategy using DNA-functionalized nanoparticles and successfully demonstrates the realization of BCC-CsCl, SC-NaCl, and a weakly ordered cubic diamond phase. The study reveals the phase behavior of isotropic nanoparticles for DNA-shell tunable interaction, which, due to the ease of synthesis is promising for the practical realization of non-close-packed lattices.

Framework nucleic acid-based nanoparticles enhance temozolomide sensitivity in glioblastoma.

O6-methylguanine DNA methyltransferase (MGMT) is a crucial determinant of temozolomide (TMZ) sensitivity in patients with glioblastoma (GBM). The therapeutic potential of small interfering RNA (siRNA) targeting MGMT to enhance TMZ sensitivity has been hampered by serum nuclease degradation, off-target effects, poor accumulation at tumor sites, and low circulation in blood stream. In this study, we developed a framework nucleic acid-based nanoparticles (FNN), which is constructed from a six-helix DNA bundle, to encapsulate and protect siMGMT for improving TMZ sensitivity in GBM treatment. For better blood-brain barrier (BBB) penetration and GBM targeting, we conjugated Angiopep-2 (ANG) targeting modules to each end of the FNN. Nucleolin (NCL)-responsive locks were engineered along the sides of the six-helix DNA bundle, which safeguard siMGMT before tumor entry. Upon interaction with tumor-overexpressed NCL, these locks unlock, exposing siMGMT, this allows for effective suppression of MGMT, resulting in a significant improvement of TMZ therapeutic efficacy in GBM. This innovative strategy has the potential to transform the current treatment landscape for GBM.

Diverse Chiral Nanotubes Assembled from Identical DNA Strands.

DNA nanotubes with controllable geometries hold a wide range of interdisciplinary applications. When preparing DNA nanotubes of varying widths or distinct chirality, existing methods require repeatedly designing and synthesizing specific DNA sequences, which can be costly and laborious. Here, we proposed an intercalator-assisted DNA tile assembly method which enables the production of DNA nanotubes of diverse widths and chirality using identical DNA strands. Through adjusting the concentration of intercalators during assembly, the twisting direction and extent of DNA tiles could be modulated, leading to the formation of DNA nanotubes featuring controllable widths and chirality. Moreover, through introducing additional intercalators and secondary annealing, right-handed nanotubes could be reconfigured into distinct left-handed nanotubes. We expect that this method could be universally applied to modulating the self-assembly pathways of various DNA tiles and other chiral materials, advancing the landscape of DNA tile assembly.

DNA-Origami-Based Precise Molecule Assembly and Their Biological Applications.

Inspired by efficient natural biomolecule assembly with precise control on key parameters such as distance, number, orientation, and pattern, the constructions and applications of artificial precise molecule assembly are highly important in many research areas including chemistry, biology, and medicine. DNA origami, a sophisticated DNA nanotechnology with rational design, can offer a predictable, programmable, and addressable nanoscale scaffold for the precise assembly of various kinds of molecules. Herein, we summarize recent progress, particularly in the last three years, in DNA-origami-based precise molecule assembly and their emerging biological applications. We first introduce DNA origami and the progress on DNA-origami-based precise molecule assembly, including assembly of various kinds of molecules (e.g., nucleic acids, proteins, organic molecules, nanoparticles), and precise control of important parameters (e.g., distance, number, orientation, pattern). Their biological applications in sensing, imaging, therapy, bionics, biophysics, and chemical biology are then summarized, and current challenges and opportunities are finally discussed.

Modulating the DNA/Lipid Interface through Multivalent Hydrophobicity.

Lipids and nucleic acids are two of the most abundant components of our cells, and both molecules are widely used as engineering materials for nanoparticles. Here, we present a systematic study of how hydrophobic modifications can be employed to modulate the DNA/lipid interface. Using a series of DNA anchors with increasing hydrophobicity, we quantified the capacity to immobilize double-stranded (ds) DNA to lipid membranes in the liquid phase. Contrary to electrostatic effects, hydrophobic anchors are shown to be phase-independent if sufficiently hydrophobic. For weak anchors, the overall hydrophobicity can be enhanced following the concept of multivalency. Finally, we demonstrate that structural flexibility and anchor orientation overrule the effect of multivalency, emphasizing the need for careful scaffold design if strong interfaces are desired. Together, our findings guide the design of tailored DNA/membrane interfaces, laying the groundwork for advancements in biomaterials, drug delivery vehicles, and synthetic membrane mimics for biomedical research and nanomedicine.

Dynamic Assembly of DNA Nanostructures in Cancer Cells Enables the Coupling of Autophagy Activating and Real-Time Tracking.

Developing dynamic nanostructures for in situ regulation of biological processes inside living cells is of great importance in biomedical research. Herein we report the cascaded assembly of Y-shaped branched DNA nanostructure (YDN) during intracellular autophagy. YDN contains one arm with semi-i-motif sequence and Cy3-BHQ2, and another arm with an apurinic/apyrimidinic (AP) site and Cy5-BHQ3. Upon uptake by cancer cells, intermolecular i-motif structures are formed in response to lysosomal H+, causing the formation of YDN-dimer and the recovery of Cy3 fluorescence; when escapes occur from the lysosome to the cytoplasm, the YDN-dimer responds to the overexpressed APE1, leading to the assembly of YDN into the DNA network and the fluorescence recovery of Cy5. Simultaneously, the cascaded assembly activates autophagy, and thus the process of assembly of YDN and autophagy flux can be spatiotemporally coupled. This work illustrates the potential of DNA nanostructures for the in situ regulation of intracellular dynamic events with spatiotemporal control.

A Poly Aptamer Encoded DNA Nanocatcher Informs Efficient Virus Trapping.

Broad-spectrum antiviral platforms are always desired but still lack the ability to cope with the threats to global public health. Herein, we develop a poly aptamer encoded DNA nanocatcher platform that can trap entire virus particles to inhibit infection with a broad antiviral spectrum. Ultralong single-stranded DNA (ssDNA) containing repeated aptamers was synthesized as the scaffold of a nanocatcher via a biocatalytic process, wherein mineralization of magnesium pyrophosphate on the ssDNA could occur and consequently lead to the formation of nanocatcher with interfacial nanocaves decorated with virus-binding aptamers. Once the viruses were recognized by the apatmers, they would be captured and trapped in the nanocaves via multisite synergistic interactions. Meanwhile, the size of nanocatchers was optimized to prevent their cellular uptake, which further guaranteed inhibition of virus infection. By taking SARS-CoV-2 variants as a model target, we demonstrated the broad virus-trapping capability of a DNA nanocatcher in engulfing the variants and blocking the infection to host cells.

Welded Gold Nanoparticle Assemblies Defined Plasmonic Coupling.

Nanoparticle assemblies with interparticle ohmic contacts are crucial for nanodevice fabrication. Despite tremendous progress in DNA-programmable nanoparticle assemblies, seamlessly welding discrete components into welded continuous three-dimensional (3D) configurations remains challenging. Here, we introduce a single-stranded DNA-encoded strategy to customize welded metal nanostructures with tunable morphologies and plasmonic properties. We demonstrate the precise welding of gold nanoparticle assemblies into continuous metal nanostructures with interparticle ohmic contacts through chemical welding in solution. We find that the welded gold nanoparticle assemblies show a consistent morphology with welded efficiency over 90%, such as the rod-like, triangular, and tetrahedral metal nanostructures. Next, we show the versatility of this strategy by welding gold nanoparticle assemblies of varied sizes and shapes. Furthermore, the experiment and simulation show that the welded gold nanoparticle assemblies exhibit defined plasmonic coupling. This single-stranded DNA encoded welding system may provide a new route for accurately building functional plasmonic nanomaterials and devices.

Constructing Stiffness Tunable DNA Hydrogels Based on DNA Modules with Adjustable Rigidity.

DNA hydrogel represents a potent material for crafting biological scaffolds, but the toolbox to systematically regulate the mechanical property is still limited. Herein, we have provided a strategy to tune the stiffness of DNA hydrogel through manipulating the rigidity of DNA modules. By introducing building blocks with higher molecular rigidity and proper connecting fashion, DNA hydrogel stiffness could be systematically elevated. These hydrogels showed excellent dynamic properties and biocompatibility, thus exhibiting great potential in three-dimensional (3D) cell culture. This study has offered a systematic method to explore the structure-property relationship, which may contribute to the development of more intelligent and personalized biomedical platforms.

Construction of a Spatial-Confined Self-Stacking Catalytic Circuit for Rapid and Sensitive Imaging of Piwi-Interacting RNA in Living Cells.

Piwi-interacting RNAs (piRNAs) are small noncoding RNAs that repress transposable elements to maintain genome integrity. The canonical catalytic hairpin assembly (CHA) circuit relies on random collisions of free-diffused reactant probes, which substantially slow down reaction efficiency and kinetics. Herein, we demonstrate the construction of a spatial-confined self-stacking catalytic circuit for rapid and sensitive imaging of piRNA in living cells based on intramolecular and intermolecular hybridization-accelerated CHA. We rationally design a 3WJ probe that not only accelerates the reaction kinetics by increasing the local concentration of reactant probes but also eliminates background signal leakage caused by cross-entanglement of preassembled probes. This strategy achieves high sensitivity and good specificity with shortened assay time. It can quantify intracellular piRNA expression at a single-cell level, discriminate piRNA expression in tissues of breast cancer patients and healthy persons, and in situ image piRNA in living cells, offering a new approach for early diagnosis and postoperative monitoring.

Organization of an Artificial Multicellular System with a Tunable DNA Patch on a Membrane Surface.

Coordinating multiple artificial cellular compartments into a well-organized artificial multicellular system (AMS) is of great interest in bottom-up synthetic biology. However, developing a facile strategy for fabricating an AMS with a controlled arrangement remains a challenge. Herein, utilizing in situ DNA hybridization chain reaction on the membrane surface, we developed a DNA patch-based strategy to direct the interconnection of vesicles. By tuning the DNA patch that generates heterotrophic adhesion for the attachment of vesicles, we could produce an AMS with higher-order structures straightforwardly and effectively. Furthermore, a hybrid AMS comprising live cells and vesicles was fabricated, and we found the hybrid AMS with higher-order structures arouses efficient molecular transportation from vesicles to living cells. In brief, our work provides a versatile strategy for modulating the self-assembly of AMSs, which could expand our capability to engineer synthetic biological systems and benefit synthetic cell research in programmable manipulation of intercellular communications.

DNA Reaction Circuits to Establish Designated Biological Functions in Multicellular Community.

In multicellular organisms, individual cells are coordinated through complex communication networks to accomplish various physiological tasks. Aiming to establish new biological functions in the multicellular community, we used DNA as the building block to develop a cascade of nongenetic reaction circuits to establish a dynamic cell-cell communication network. Utilizing membrane-anchored amphiphilic DNA tetrahedra (TDN) as the nanoscaffold, reaction circuits were incorporated into three unrelated cells in order to uniquely regulate their sense-and-response behaviors. As a proof-of-concept, this step enabled these cells to simulate significant biological events involved in T cell-mediated anticancer immunity. Such events included cancer-associated antigen recognition and the presentation of antigen-presenting cells (APCs), APC-facilitated T cell activation and dissociation, and T cell-mediated cancer targeting and killing. By combining the excellent programmability and molecular recognition ability of DNA, our cell-surface reaction circuits hold promise for mimicking and manipulating many biological processes.