Applying dynamic light scattering to investigate the self-assembly process of DNA nanostructures

Understanding the dynamic assembly process of DNA nanostructures is important for developing novel strategy to design and construct functional devices.In this work,temperature-controlled dynamic light scattering(DLS)strategy has been applied to study the global assembly process of DNA origami and DNA bricks.Through the temperature dependent size and intensity profiles,the self-assembly process of various DNA nanostructures with different morphologies have been well-studied and the temperature transition ranges could be observed.Taking advantage of the DLS information,rapid preparation of the DNA origami and the brick assembly has been realized through a constant temperature annealing.Our results demonstrate that the DLS-based strategy provides a convenient and robust tool to study the dynamic process of forming hieratical DNA structures,which will benefit understanding the mechanism of self-assembly of DNA nanostructures.

Octopus-like DNA nanostructure coupled with graphene oxide enhanced fluorescence anisotropy for hepatitis B virus DNA detection

Fluorescence Anisotropy(FA)is an effective biochemical detection method based on molecular rotations.Graphene oxide(GO)has been extensively used as an FA amplifier.However,the enhancement of FA by GO alone is limited and the strong scattering of GO will easily make the measurement of FA inaccurate.In order to address these problems,an octopus-like DNA nanostructure(ODN)was designed and coupled with GO to enhance the FA together in this work.By mimicking the multi-clawed structure of the octopus,the ODN can be adsorbed on GO tightly,which not only could improve the sensitivity because of the double FA enhancement abilities of GO and ODN,but also could improve the specificity due to the decrease of the nonspecific interaction in complex samples.Furthermore,ODN could maintain a certain distance between the fluorophore and GO to reduce the fluorescence quenching efficiency of GO,which could improve the accuracy.This method has been applied for the detection of hepatitis B virus DNA(HBV-DNA)in a range of 1-50 nmol/L and the limit of detection(LOD)was 330 pmol/L.In addition,the proposed method has been successfully utilized to detect HBV-DNA in human serum,indicating that this method has a great practical application prospect.

Tetrahedron DNA nanostructure/iron-based nanomaterials for combined tumor therapy

Triple-negative breast cancer,due to its aggressive nature and lack of targeted treatment,faces serious challenges in breast cancer treatment.Conventional therapies,such as chemotherapy,are encumbered by a range of limitations,and there is an urgent need for more effective treatment strategies.Ferroptosis,as an iron-dependent form of cell death,has exhibited promising potential in cancer treatment.Combining ferroptosis with other cancer therapies offers new avenues for treatment.Tetrahedral DNA nanostructure(TDN),a novel DNA-based three-dimensional(3D)nanomaterial,is promising drug delivery vehicle and can be utilized for functionalizing inorganic nanomaterials.In this work,we have demonstrated the preparation of Fe_(3)O_(4)-PEI@TDN-DOX nanocomposites and elucidated their antitumor mechanism.The TDN facilitated the enhanced cellular uptake of polyetherimide(PEI)-modified Fe_(3)O_(4),and the delivery of the chemotherapeutic drug doxorubicin(DOX)further augmented their anti-tumor effect.This novel strategy can destroy the tumor redox homeostasis and produce overwhelming lipid peroxides,consequently sensitizing the tumor to ferroptosis.The integration of ferroptosis with other cancer therapies opens up new possibilities for treatment.This research provides valuable mechanistic insights and practical strategies for leveraging nanotechnology to induce ferroptosis and amplify its impact on tumor cells.

DNA nanostructures prevent the formation of and convert toxic amyloid proteospecies into cytocompatible and biodegradable spherical complexes

The deposition of insoluble proteinaceous aggregates in the form of amyloidfibrils within the extracellular space of tissues is associated with numerous diseases.The development of molecular approaches to arrest amyloid formation and prevent cel-lular degeneration remains very challenging due to the complexity of the process of protein aggregation,which encompasses an infinite array of conformations and quaternary structures.Polyanionic biopolymers,such as glycosaminoglycans and RNAs,have been shown to modulate the self-assembly of amyloidogenic polypep-tides and to reduce the toxicity induced by the formation of oligomeric and/or pre-fibrillar proteospecies.This study evaluates the effects of double-stranded DNA(dsDNA)nanostructures(1D,2D,and 3D)on amyloid self-assembly,fibril dis-aggregation,and the cytotoxicity associated with amyloidogenesis.Using the islet amyloid polypeptide(IAPP)whose pancreatic accumulation is the hallmark of type 2 diabetes,it was observed that dsDNA nanostructures inhibit amyloid formation by inducing the formation of spherical complexes in which the peptide adopts a random coil conformation.Interestingly,the DNA nanostructures showed a per-sistent ability to disassemble enzymatically and thermodynamically stable amyloidfibrils into nanoscale DNA/IAPP entities that are fully compatible withβ-pancreatic cells and are biodegradable by proteolysis.Notably,dsDNA nanostructures avidly trapped highly toxic soluble oligomeric species in complete cell culture media and converted them into non-toxic binary complexes.Overall,these results expose the potent modulatory effects of dsDNA on amyloidogenic pathways,and these DNA nanoscaffolds could be used as a source of inspiration for the design of molecules tofight amyloid-related disorders.

Electrochemical single nucleotide polymorphisms genotyping on surface immobilized three-dimensional branched DNA nanostructure

An electrochemical assay for single nucleotide polymorphisms (SNPs) genotyping is reported. Although electrochemical method is sensitive for DNA detection on surfaces, the ability of surface assay to precisely recognize DNA hybridization event is sacrificed to some extent due to the crowded confined surfaces environments that disfavor DNA hybridization. In the present study, we employed branched tetrahedron structure probes (TSPs) to replace regular linear single stranded DNA capture probes that were immobilized on solid surfaces. This three-dimensional DNA nanostructure lowers the density of immobilized DNA probes on confined surfaces, providing a hybridization environment that is similar to homogenous solution. This TSP-based electrochemical assay reveals excellent performance for SNPs genotyping with concentration as low as 1 nM.

Self-Assembled DNA Nanostructures for Drug Delivery

Due to the uniform nanoscale sizes, well-defined shapes, precise spatial addressability and prominent biocom- patibility, self-assembled DNA nanostructures have been intensively studied for their biomedical applications. This review summarizes the recent development ofDNA nanotechnology in cancer therapy, and discusses the challenges and potential strategies to advance the methodologies of cancer treatments.

Tetrahedral DNA nanostructures synergize with MnO_(2) to enhance antitumor immunity via promoting STING activation and M1 polarization

Stimulator of interferon genes(STING) is a cytosolic DNA sensor which is regarded as a potential target for antitumor immunotherapy. However, clinical trials of STING agonists display limited anti-tumor effects and dose-dependent side-effects like inflammatory damage and cell toxicity. Here,we showed that tetrahedral DNA nanostructures(TDNs) actively enter macrophages to promote STING activation and M1 polarization in a size-dependent manner, and synergized with Mn^(2+) to enhance the expressions of IFN-β and iNOS, as well as the co-stimulatory molecules for antigen presentation. Moreover, to reduce the cytotoxicity of Mn^(2+),we constructed a TDN-MnO_(2) complex and found that it displayed a much higher efficacy than TDN plus Mn^(2+) to initiate macrophage activation and anti-tumor response both in vitro and in vivo. Together, our studies explored a novel immune activation effect of TDN in cancer therapy and its synergistic therapeutic outcomes with MnO_(2).These findings provide new therapeutic opportunities for cancer therapy.

Effect of tetrahedral DNA nanostructures on LPS-induced neuroinflammation in mice

Neuroinflammation plays a significant role in inducing depression-like behavior. Tetrahedral DNA nanostructures(TDNs) are molecules that exhibit anti-inflammatory properties and can effectively penetrate the blood-brain barrier. Thus, researchers have hypothesized that TDNs regulate the secretion of proinflammatory cytokines and consequently alleviate depression-like behavior. To test this hypothesis, we investigated the effect of TDNs on the depression-like behavior of C57 mice induced by lipopolysaccharide(LPS). We performed open-field, tail suspension, and sucrose preference tests on LPS-and LPS/TDNtreated mice. The results indicated that the injection of TDNs into LPS-treated mice resulted in increased velocity, center zone duration, frequency to the center zone, and sucrose preference, and decreased immobility time. Immunofluorescence results indicated that peripheral administration of LPS in the mice activated inflammation, which culminated in distinct depression-like behavior. However, TDNs effectively alleviated the inflammation and depression-like behavior through the reduction of the expression levels of proinflammatory cytokines, such as interleukin-1β and tumor necrosis factor-α in the brain. Additionally, TDNs normalized the expression level of microglia cell activation markers, such as ionized calcium binding adaptor molecule 1, in the hippocampus of mice. These results indicated that TDNs attenuated the LPS-induced secretion of inflammatory factors and consequently alleviated depression-like behavior.

Functionalizing DNA nanostructures with natural cationic amino acids

Complexing self-assembled DNA nanostructures with various functional guest species is the key to unlocking new and exciting biomedical applications.Cationic guest species not only induce magnesium-free DNA to self-assemble into defined structures but also endow the final complex nanomaterials with new properties.Herein,we propose a novel strategy that employs naturally occurring cationic amino acids to induce DNA self-assembly into defined nanostructures.Natural L-arginine and L-lysine can readily induce the assembly of tile-based DNA nanotubes and DNA origami sheets in a magnesium-free manner.The self-assembly processes are demonstrated to be pH-and concentration-dependent and are achieved at constant temperatures.Moreover,the assembled DNA/amino acid complex nanomaterials are stable at a physiological temperature of 37◦C.Substituting L-arginine with its D form enhances its serum stability.Further preliminary examination of this complex nanomaterial platform for biomedical applications indicates that DNA/amino acids exhibit distinct cellular uptake behaviors compared with their magnesium-assembled counterparts.The nanomaterial mainly clusters around the cell membrane and might be utilized to manipulate molecular events on the membrane.Our study suggests that the properties of DNA nanostructures can be tuned by complexing them with customized guest molecules for a designed application.The strategy proposed herein might be promising to advance the biomedical applications of DNA nanostructures.

Covalent stabilization of DNA nanostructures on cell membranes for efficient surface receptor-mediated labeling and function regulations

One major challenge of using DNA nanostructures for cellular and in vivo applications is their insufficiently structural integrity that stems from the non-covalent base pairing and stacking in complex cellular and physiological environment. The establishment of covalent bonds in DNA nanostructures can link individual strands more stably and therefore should improve the performance of DNA nanostructures in different scenarios where structural integrity is required. Here, we developed a convenient and effective method for constructing covalently stabilized DNA nanostructures by chemically inserting photo-crosslinker(^(CNV)K) in DNA sequences. These covalently linked DNA nanostructures were found to be more resistant to external interference, such as low cation concentrations and unspecific displacement on cell membranes. We also demonstrated that our strategy could improve the efficiency of cell surface receptor-mediated labeling and function regulations in living cells, which sheds light on broadening the biomedical applications of DNA nanostructures.

Applications of tetrahedral DNA nanostructures in wound repair and tissue regeneration

Tetrahedral DNA nanostructures(TDNs)are molecules with a pyramidal structure formed by folding four single strands of DNA based on the principle of base pairing.Although DNA has polyanionic properties,the special spatial structure of TDNs allows them to penetrate the cell membrane without the aid of transfection agents in a caveolin-dependent manner and enables them to participate in the regulation of cellular processes without obvious toxic side effects.Because of their stable spatial structure,TDNs resist the limitations imposed by nuclease activity and innate immune responses to DNA.In addition,TDNs have good editability and biocompatibility,giving them great advantages for biomedical applications.Previous studies have found that TDNs have a variety of biological properties,including promoting cell migration,proliferation and differentiation,as well as having anti-inflammatory,antioxidant,anti-infective and immune regulation capabilities.Moreover,we confirmed that TDNs can promote the regeneration and repair of skin,blood vessels,muscles and bone tissues.Based on these findings,we believe that TDNs have broad prospects for application in wound repair and regeneration.This article reviews recent progress in TDN research and its applications.

Lithographically directed assembly of one-dimensional DNA nanostructures via bivalent binding interactions

In order to exploit the outstanding physical properties of one-dimensional （1D） nanostructures such as carbon nanotubes and semiconducting nanowires and nanorods in future technological applications, it will be necessary to organize them on surfaces with precise control over both position and orientation. Here, we use a 1D rigid DNA motif as a model for studying directed assembly at the molecular scale to lithographically patterned nanodot anchors. By matching the inter-nanodot spacing to the length of the DNA nanostructure, we are able to achieve nearly 100% placement yield. By varying the length of single-stranded DNA linkers bound covalently to the nanodots, we are able to study the binding selectivity as a function of the strength of the binding interactions. We analyze the binding in terms of a thermodynamic model which provides insight into the bivalent nature of the binding, a scheme that has general applicability for the controlled assembly of a broad range of functional nanostructures.

DNA Nanostructure-Guided Plasmon Coupling Architectures

Plasmon coupling architectures with specific spatial and orientational arrangement configurations possess unique and tailored plasmonic properties and hold promise for advancements in nano-optics,nanoantennas,and biosensors.Numerous research has focused on the construction of plasmonic assemblies with predetermined configurations.DNA nanostructures with arbitrary geometry,high compatibility with metal nanoparticles,and spatial addressability meet the requirement for precise spatial and orientation arrangement.Currently,DNA nanostructures are widely exploited as structural materials to generate plasmonic structures with well-defined topologies.We review the evolution of DNA nanostructureguided plasmon coupling architectures,including the introduction of DNA nanostructures,DNA modification on the surface of plasmonic nanoparticles,and three strategies for constructing complex plasmonic nanostructures.Then we focus on the emerging applications of DNA nanostructure-guided architectures with engineered local electromagnetic enhancement for modulating plasmon coupling,amplifying emitter signals,and serving as biosensors.Finally,we will critically discuss the challenges and opportunities in this field.

Step-growth polymerization of traptavidin-DNA conjugates for plasmonic nanostructures

Here,we use two important biomaterials,protein and DNA,to construct self-assembled linear nanostructures through Watson-Crick base-paring of DNAs.We apply a simple magnetic separation method to purify traptavidin-DNA co njugates,and demonstrate synthesis of linear arrays of traptavidinDNA conjugates via the step-growth polymerization approach with pre-determined DNA sequences.Using the traptavidin-DNA array as a template,we assemble gold nanoparticles to form linear plasmonic nanostructures in a programmable manner.The traptavidin-DNA conjugates thus provide a convenient platform for one-dimensional assembly of biotinylated nanomaterials for many biomedical applications from drug delivery to bio-sensing.

DNA origami nanodevice with spatial regulation of CD95 signaling for rheumatoid arthritis treatment

Recently,a work jointly studied by Ling Li and coworkers1 was published in Nature Materials,describing a reconfigurable DNA origami nanodevice designed to regulate CD95 death-inducing signaling of immune cells.The researchers utilized the DNA origami nanodevice to establish selective local immune tolerance and demonstrated its ability to alleviate rheumatoid arthritis(RA)in the inflamed synovial tissue of mice without causing any obvious side effects(Fig.1).This approach presents a novel idea for the development of drug interventions involving ligandreceptor interactions.

Discrete DNA Three-dimensional Nanostructures： the Synthesis and Applications

Structural DNA nanotechnology, an emerging technique that utilizes the nucleic acid molecule as generic polymer to programmably assemble well-defined and nano-sized architectures, holds great promise for new material synthesis and constructing functional nanodevices for different purposes. In the past three decades, rapid development of this technique has enabled the syntheses of hundreds and thousands of DNA nanostructures with various morphologies at different scales and dimensions. Among them, discrete three-dimensional （3D） DNA nanostructures not only represent the most advances in new material design, but also can serve as an excellent platform for many important applications. With precise spatial addressability and capability of arbitrary control over size, shape, and function, these nanostructures have drawn particular interests to scientists in different research fields. In this review article, we will briefly summarize the development regarding the synthesis of discrete DNA 3D nanostructures with various size, shape, geometry, and topology, including our previous work and recent progress by other groups. In detail, three methods majorly used to synthesize the DNA 3D objects will be introduced accordingly. Additionally, the principle, design rule, as well as pros and cons of each method will be highlighted. As functions of these discrete 3D nanostructures have drawn great interests to researchers, we will further discuss their cutting-edge applications in different areas, ranging from novel material synthesis, new device fabrication, and biomedical applications, etc. Lastly, challenges and outlook of these promising nanostructures will be given based on our point of view.

Superresolution imaging of DNA tetrahedral nanostructures in cells by STED method with continuous wave lasers

DNA tetrahedral nanostructures are considered to be uew nanocarriers because they can be precisely controlled and hold excellent penetration ability to the cellular membrane. Although the DNA tetrahedral nanostructure is extensively studied in biology and medicine, its behavior in the cells with nanoscale resolution is not understood clearly. In this letter, we demonstrate superrcsolution fluorescence imaging of the distribution of DNA tetrahedral nanostructures in the cell with a simulated emission depletion （STED） microscope, which is built based on a conventional eonfocal microscope and can t）rovide a resolution of 70 nm.

Coating with flexible DNA network enhanced T-cell activation and tumor killing for adoptive cell therapy

Adoptive cell therapy(ACT)is an emerging powerful cancer immunotherapy,which includes a complex process of genetic modification,stimulation and expansion.During these in vitro or ex vivo manipulation,sensitive cells are inescapability subjected to harmful external stimuli.Although a variety of cytoprotection strategies have been developed,their application on ACT remains challenging.Herein,a DNA network is constructed on cell surface by rolling circle amplification(RCA),and T cell-targeted trivalent tetrahedral DNA nanostructure is used as a rigid scaffold to achieve high-efficient and selective coating for T cells.The cytoprotective DNA network on T-cell surface makes them aggregate over time to form cell clusters,which exhibit more resistance to external stimuli and enhanced activities in human peripheral blood mononuclear cells and liver cancer organoid killing model.Overall,this work provides a novel strategy for in vitro T cell-selective protection,which has a great potential for application in ACT.

Atomic force microscopy analysis of orientation and bending of oligodeoxynucleotides in polypod-like structured DNA

We previously demonstrated that polypod-like structured DNA, or polypodna, constructed with three or more oligodeoxynucleotides （ODNs）, is efficiently taken up by immune cells such as dendritic cells and macrophages, depending on its structural complexity. The ODNs comprising the polypodna should bend to form the polypod-like structure, and may do so by adopting either a bend- type conformation or a cross-type conformation. Here, we tried to elucidate the orientation and bending of ODNs in polypodnas using atomic force microscopy （AFM）. We designed two types of pentapodnas （i.e., a polypodna with five pods） using 60- to 88-base ODNs, which were then immobilized on DNA origami frames. AFM imaging showed that the ODNs in the pentapodna adopted bend-type conformations. Tetrapodna and hexapodna also adopted bend-type conformations when they were immobilized on frames under unconstrained conditions. These findings provide useful information toward the coherent design of, and the structure-activity relationships for, a variety of DNA nanostructures.

Functional DNA Structures and Their Biomedical Applications

Since the discovery of the double-helix structure in 1953,nucleic acids have been developed from natural genetic codes into functional building blocks in a wide range of biotechnology and materials sciences.Taking advantage of their design diversity and biocompatibility,functional nucleic acids facilitate the“bottom-up”fabrication of nanomaterials that are highly potential for molecular medicine to treat different diseases,such as cancers.The present perspective article introduces recent advances in the use of these unique properties of nucleic acid biopolymers for biomedical applications.Specifically,nanomaterial/nucleic acid hybrid structures for sensing,controlled drug release,programmable intracellular imaging,and apoptosis,as well as logic calculation,are discussed.Furthermore,the detailed operation for both extracellular and intracellular bioactivity regulation with these new design functional nucleic acid nanostructures are fully illustrated.