Self-assembled DNA Nanostructures Research Articles

Stress in DNA Gridiron Facilitates the Formation of Two-Dimensional Crystalline Structures.

Programmable DNA nanotechnology has generated some of the most intricate self-assembled nanostructures and has been employed in a growing number of applications, including functional nanomaterials, nanofabrication, biophysics, photonics, molecular machines, and drug delivery. An important design rule for DNA nanostructures is to minimize the mechanical stress to reduce the potential energy in these nanostructures whenever it is possible. This work revisits the DNA gridiron design consisting of Holliday junctions and compares the self-assembly of the canonical DNA gridiron with a new design of DNA gridiron, which has a higher degree of mechanical stress because of the interweaving of DNA helices. While the interweaving DNA gridiron indeed exhibits lower yield, compared to its canonical counterpart of a similar size, we discover that the mechanical stress within the interweaving gridiron can promote the formation of the two-dimensional crystalline lattice instead of nanotubes. Furthermore, tuning the design of interweaving gridiron leads to the change of overall crystal size and regularity of geometry. Interweaving DNA double helices represents a new design strategy in the self-assembly of DNA nanostructures. Furthermore, the discovery of the new role of mechanical stress in the self-assembly of DNA nanostructures provides useful knowledge to DNA nanotechnology practitioners: a more balanced view regarding mechanical stress can be considered when designing future DNA nanostructures.

Recent Advances in Self-Assembled DNA Nanostructures for Bioimaging.

DNA nanotechnology has been proven to be a powerful platform to assist the development of imaging probes for biomedical research. The attractive features of DNA nanostructures, such as nanometer precision, controllable size, programmable functions, and biocompatibility, have enabled researchers to design and customize DNA nanoprobes for bioimaging applications. However, DNA probes with low molecular weights (e.g., 10-100 nt) generally suffer from low stability in physiological buffer environments. To improve the stability of DNA nanoprobes in such environments, DNA nanostructures can be designed with relatively larger sizes and defined shapes. In addition, the established modification methods for DNA nanostructures are also essential in enhancing their properties and performances in a physiological environment. In this review, we begin with a brief recap of the development of DNA nanostructures including DNA tiles, DNA origami, and multifunctional DNA nanostructures with modifications. Then we highlight the recent advances of DNA nanostructures for bioimaging, emphasizing the latest developments in probe modifications and DNA-PAINT imaging. Multiple imaging modules for intracellular biomolecular imaging and cell membrane biomarkers recognition are also summarized. In the end, we discuss the advantages and challenges of applying DNA nanostructures in bioimaging research and speculate on its future developments.

ATP-Triggered Drug Release of Self-Assembled 3D DNA Nanostructures for Fluorescence Imaging and Tumor Therapy.

Stimulus-responsive materials are ideal carriers for precisely controlled drug delivery due to their high selectivity. However, the complex physiological environment hinders its development in clinical medicine. Here, we aim to design a self-assembled three-dimensional (3D) DNA nanostructure drug delivery system with adenosine-5'-triphosphate (ATP)-triggered drug release for tumor fluorescence imaging analysis and targeted drug delivery. Dox@3D DNA nanostructures are self-assembled by a simple one-pot annealing reaction and embedded with drugs, which are structurally stable but can be induced using high concentrations of ATP in tumor cells to cleave and release drugs rapidly, facilitating the rapid accumulation of drugs in tumors and exerting therapeutic effects, thus effectively avoiding damage to normal tissues. This work demonstrates that 3D DNA nanostructures can be used as efficient drug nanocarriers with promising applications in tumor therapy.

An mTOR siRNA-Loaded Spermidine/DNA Tetrahedron Nanoplatform with a Synergistic Anti-Inflammatory Effect on Acute Lung Injury.

Acute lung injury (ALI) is characterized by severe inflammation and damage to the lung air-blood barrier, resulting in respiratory function damage and life-threatening outcomes. Macrophage polarization plays an essential role in the occurrence, development, and outcome of ALI. As drug carriers, self-assembled DNA nanostructures can potentially overcome the drawbacks and limitations of traditional anti-inflammatory agents owing to their nontoxicity, programmability, and excellent structural control at the nanoscale. A small interfering RNA (siRNA) and drug dual therapy nanoplatform are proposed and constructed here to combat ALI. The nanoplatform consists of a spermidine-assembled DNA tetrahedron and four mammalian target of rapamycin siRNAs. Spermidine serves as a mediator of drug delivery vehicle synthesis and a drug that alters macrophage polarization. Both spermidine and siRNA exert anti-inflammatory effects in vitro and in vivo by regulating the macrophage phenotype. More importantly, these factors exhibit a synergistic anti-inflammatory effect by promoting macrophage autophagy. For the first time, an anti-inflammatory dual therapy strategy that uses self-assembled DNA nanostructures as nontoxic, programmable delivery vehicles is proposed and demonstrated through this work. Future work on utilizing DNA nanostructures for the treatment of noncancerous diseases such as ALI is highly promising and desirable.

Ultrasensitive Electrochemical Detection of cancer-Related Point Mutations Based on Surface-Initiated Three-Dimensionally Self-Assembled DNA Nanostructures from Only Two Palindromic Probes.

Sensitive and selective detection of proto-oncogenes, especially recognition of point mutation, is of great importance in cancer diagnosis. Here, a ligation-mediated technique is demonstrated for the construction of an intertwined three-dimensional DNA nanosheet (3D SDN) on an electrode surface from only two palindromic hairpin probes (HP1 and HP2), creating a powerful electrochemical biosensor (E-biosensor) for the detection of the p53 gene. First, a capturing probe (CP) is immobilized on an electrode surface via Au-S chemistry, forming an electrochemical sensing interface. In the presence of the target p53 (T), the triggering probe is covalently linked to CP by a ligase. Moreover, target hybridization/ligation/dehybridization process is repeated, amplifying the target hybridization event and increasing the content of surface-confined triggering fragments. As a result, HP1 is opened and in turn interacts with HP2, forming intertwined 3D SDN where HP1 and HP2 are alternately arranged in parallel. Common hybridization and interaction between palindromic fragments are responsible for the assembly in the horizontal and vertical directions, respectively. An electrochemical indicator, methylene blue (MB), can be inserted into 3D SDN, generating a strong electrochemical signal. Utilizing the 3D SDN-based E-biosensor, the target DNA is detected down to 3 fM with a linear response range from 10 fM to 10 nM. Single point mutations are reliably identified even in fetal bovine serum and cellular homogenate. Because of the several advantages of simple design, good universality, inexpensive instrumentation, high assay specificity, and sensitivity, the 3D SDN-based E-biosensor is expected to provide a potential platform for screening point mutation required by early clinical diagnostics and medical research.

Self-assembled DNA nanotrains for targeted delivery of mithramycin dimers coordinated by different metal ions: Effect of binding affinity on drug loading, release and cytotoxicity

Self-assembled DNA nanostructures, as a novel type of nano-biomaterials, are widely used for smart and targeted drug delivery due to their precise structures and versatile functions. The binding affinity of drugs to DNA nanostructures plays an important role in drug loading, release and efficacy. The interaction of mithramycin (MTR) with AS1411-tethered DNA nanotrains (AS1411NTrs) in the presence of different divalent metal ions (Mg2+, Zn2+, and Mn2+) was studied by fluorescence spectroscopy, differential scanning calorimetry and viscosity measurement. The binding mode between the MTR dimers and AS1411NTrs was determined by viscosity experiment and melting temperature measurement. The thermodynamic parameters of the binding process (binding stoichiometry, binding constant, enthalpy change, entropy change and Gibbs free energy change) were determined by fluorescence spectroscopy and differential scanning calorimetry, and the effect of different metal ions on the binding was compared. The release of the MTR dimers loaded by AS1411NTrs in the absence and presence of DNase I was analyzed. The in vitro cytotoxicity of the MTR dimers to HepG2 cancer cells and L02 normal cells and their intracellular uptake efficiency were evaluated. The binding parameters were correlated with the loading, release and cytotoxicity of the MTR dimers. These studies showed that AS1411NTrs had high drug loading and sustained release effect on (MTR)2Mg2+, (MTR)2Zn2+ and (MTR)2Mn2+. This drug loading system improved the lethality to cancer cells and reduced the side effects on normal cells. This study may provide a theoretical basis for the clinical application of MTR loaded by DNA nanostructures.

Self-AssembledDNA Nanostructure as a Carrier for Targeted siRNA Delivery in Glioma Cells.

IntroductionRNA interference is a promising therapy in glioma treatment. However, the application of RNA interference has been limited in glioma therapy by RNA instability and the lack of tumor targeting. Here, we report a novel DNA tetrahedron, which can effectively deliver small interfering RNA to glioma cells and induce apoptosis.MethodssiRNA, a small interfering RNA that can suppress the expression of survivin in glioma, was loaded into the DNA tetrahedron (TDN). To enhance the ability of active targeting of this nanoparticle, we modified one side of the DNA nanostructure with aptamer as1411 (As-TDN-R), which can selectively recognize the nucleolin in the cytomembrane of tumor cells. The modified nanoparticles were characterized by agarose gel electrophoresis, dynamic light scattering, and transmission electron microscopy. The serum stability was evaluated by agarose gel electrophoresis. Nucleolin was detected by Western blot and immunofluorescence, and targeted cellular uptake was examined by flow cytometry. The TUNEL assay, flow cytometry, and Western Blot were used to detect apoptosis in U87 cells. The gene silencing of survivin was examined by qPCR, Western Blot, and immunofluorescence.ResultsAs-TDN-R alone showed better stability towards siRNA, indicating that TDN was a good siRNA protector. Compared with TDN alone, there was increased intercellular uptake of As-TDN-R by U87 cells, evidenced by overexpressed nucleolin in glioma cell lines. TUNEL assay, flow cytometry, and Western Blot revealed increased apoptosis in the As-TDN-R group. The downregulation of survivin protein and mRNA expression levels indicated that As-TDN-R effectively silenced the target gene.ConclusionThe novel nanoparticle can serve as a good carrier for targeting siRNA delivery in glioma. Further exploration of the DNA nanostructure can greatly promote the application of DNA-based drug systems in glioma.

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.

Rationally Programming Nanomaterials with DNA for Biomedical Applications.

DNA is not only a carrier of genetic information, but also a versatile structural tool for the engineering and self‐assembling of nanostructures. In this regard, the DNA template has dramatically enhanced the scalability, programmability, and functionality of the self‐assembled DNA nanostructures. These capabilities provide opportunities for a wide range of biomedical applications in biosensing, bioimaging, drug delivery, and disease therapy. In this review, the importance and advantages of DNA for programming and fabricating of DNA nanostructures are first highlighted. The recent progress in design and construction of DNA nanostructures are then summarized, including DNA conjugated nanoparticle systems, DNA‐based clusters and extended organizations, and DNA origami‐templated assemblies. An overview on biomedical applications of the self‐assembled DNA nanostructures is provided. Finally, the conclusion and perspectives on the self‐assembled DNA nanostructures are presented.

Biophysical interaction between self-assembled branched DNA nanostructures with bovine serum albumin and bovine liver catalase

Branched DNA (bDNA) nanostructures have emerged as self-assembled biomaterials and are being considered for biomedical applications. Herein, we report the biophysical interaction between self-assembled bDNA nanostructure with circulating protein bovine serum albumin (BSA) and cellular enzyme bovine liver catalase (BLC). The binding between bDNA and BSA or BLC was confirmed through the decrease in fluorescence spectra. The Stern-Volmer data supports for non-covalent bonding with ~1 binding site in case of BSA and BLC thus advocating a static binding. Furthermore, FTIR and ITC study confirmed the binding of bDNAs with proteins through hydrogen bonding and van der Waals interaction. The negative free energy observed in ITC represent spontaneous reaction for BLC-bDNA interaction. The biophysical interaction between bDNA nanostructures and proteins was also supported by DLS and zeta potential measurement. With an increase in bDNA concentrations up to 100 nM, no significant change in absorbance and CD spectra was observed for both BLC and BSA which suggests structural stability and unaffected secondary conformation of proteins in presence of bDNA. Furthermore, the catalytic activity of BLC was unaltered in presence of bDNAscr even with increasing the incubation period from 1 h to 24 h. Interestingly, the time-dependent decrease in activity of BLC was protected by bDNAmix. The thermal melting study suggests a higher Tm value for proteins in presence of bDNAmix which demonstrates that interaction with bDNAmix increases the thermal stability of proteins. Collectively these data suggest that self-assembled DNA nanostructure may bind to BSA for facilitating circulation in plasma or binding to intracellular proteins like BLC for stabilization, however the secondary conformation of protein or catalytic activity of enzyme is unaltered in presence of bDNA nanostructure. Thus, the newly established genomic sequence-driven self-assembled DNA nanostructure can be explored for in vitro or in vivo experimental work in recent future.

DNA nanostructures as templates for biomineralization.

Nature uses extracellular matrix scaffolds to organize biominerals into hierarchical structures over various length scales. This has inspired the design of biomimetic mineralization scaffolds, with DNA nanostructures being among the most promising. DNA nanotechnology makes use of molecular recognition to controllably give 1D, 2D and 3D nanostructures. Thecontrol we have over these structures makes them attractive templates for the synthesis of mineralized tissues, such as bones and teeth. In this Review, we first summarize recent work on the crystallization processes and structural features of biominerals on the nanoscale. We then describe self-assembled DNA nanostructures and come to the intersection of these two themes: recent applications of DNA templates in nanoscale biomineralization, a crucial process to regenerate mineralized tissues.

A self-assembled DNA nanostructure as a FRET nanoflare for intracellular ATP imaging.

Due to the incorporation of gold nanoparticles (AuNPs), previously reported AuNP-based FRET nanoflares still have some problems, such as non-negligible cytotoxicity and a time-consuming preparation procedure. In this communication, a novel AuNP-free FRET nanoflare for intracellular ATP imaging is developed based on a DNA nanostructure, which is self-assembled through cyclic U-type hybridization only involving a certain number of DNA strands.

Rationally Designed DNA Nanostructures for Drug Delivery.

DNA is an excellent biological material that has received growing attention in the field of nanotechnology due to its unique capability for precisely engineering materials via sequence specific interactions. Self-assembled DNA nanostructures of prescribed physicochemical properties have demonstrated potent drug delivery efficiency in vitro and in vivo. By using various conjugation techniques, DNA nanostructures may be precisely integrated with a large diversity of functional moieties, such as targeting ligands, proteins, and inorganic nanoparticles, to enrich their functionalities and to enhance their performance. In this review, we start with introducing strategies on constructing DNA nanostructures. We then summarize the biological barriers ahead of drug delivery using DNA nanostructures, followed by introducing existing rational solutions to overcome these biological barriers. Lastly, we discuss challenges and opportunities for DNA nanostructures toward real applications in clinical settings.

In situ Surface Charge Density Visualization of Self-assembled DNA Nanostructures after Ion Exchange.

The charge density of DNA is a key parameter in strand hybridization and for the interactions occurring between DNA and molecules in biological systems. Due to the intricate structure of DNA, visualization of the surface charge density of DNA nanostructures under physiological conditions was not previously possible. Here, we perform a simultaneous analysis of the topography and surface charge density of DNA nanostructures using atomic force microscopy and scanning ion conductance microscopy. The effect of in situ ion exchange using various alkali metal ions is tested with respect to the adsorption of DNA origami onto mica, and a quantitative study of surface charge density reveals ion exchange phenomena in mica as a key parameter in DNA adsorption. This is important for structure‐function studies of DNA nanostructures. The research provides an efficient approach to study surface charge density of DNA origami nanostructures and other biological molecules at a single molecule level.

RNA Origami Nanostructures for Potent and Safe Anticancer Immunotherapy.

Rapid developments in nucleic acid nanotechnology have enabled the rational design and construction of self-assembling DNA and RNA nanostructures that are highly programmable. We recently developed a replicable single-stranded RNA origami (RNA-OG) technology that allows a long RNA molecule to be programmed to self-assemble into nanostructures of various shapes. Here, we show that such RNA-OG is highly stable in serum/plasma, and we thus exploited its immunostimulatory potential. We demonstrated that the RNA-OG stimulates a potent innate response primarily through a Toll-like receptor 3 (TLR3) pathway. In a murine peritoneal metastatic colon cancer model, intraperitoneally injected RNA-OG induced significant tumor retardation or regression by activating NK- and CD8-dependent antitumor immunity and antagonizing the peritoneal immunosuppressive environment. Unlike polyinosinic/polycytidylic acid (PolyIC), a well-known double-stranded RNA analogue, the RNA-OG treatment did not cause a high level of type-I interferons in the blood nor apparent toxicity upon its systemic administration in the animals. This work establishes the function of RNA-OG as a potent line of TLR3 agonists that are safe and effective for cancer immunotherapy.

DNA-Scaffolded Proximity Assembly and Confinement of Multienzyme Reactions.

Cellular functions rely on a series of organized and regulated multienzyme cascade reactions. The catalytic efficiencies of these cascades depend on the precise spatial organization of the constituent enzymes, which is optimized to facilitate substrate transport and regulate activities. Mimicry of this organization in a non-living, artificial system would be very useful in a broad range of applications—with impacts on both the scientific community and society at large. Self-assembled DNA nanostructures are promising applications to organize biomolecular components into prescribed, multidimensional patterns. In this review, we focus on recent progress in the field of DNA-scaffolded assembly and confinement of multienzyme reactions. DNA self-assembly is exploited to build spatially organized multienzyme cascades with control over their relative distance, substrate diffusion paths, compartmentalization and activity actuation. The combination of addressable DNA assembly and multienzyme cascades can deliver breakthroughs toward the engineering of novel synthetic and biomimetic reactors.

DNA framework-engineered electrochemical biosensors.

Self-assembled DNA nanostructures have shown remarkable potential in the engineering of biosensing interfaces, which can improve the performance of various biosensors. In particular, by exploiting the structural rigidity and programmability of the framework nucleic acids with high precision, molecular recognition on the electrochemical biosensing interface has been significantly enhanced, leading to the development of highly sensitive and specific biosensors for nucleic acids, small molecules, proteins, and cells. In this review, we summarize recent advances in DNA framework-engineered biosensing interfaces and the application of corresponding electrochemical biosensors.

High payload and targeted release of anthracyclines by aptamer-tethered DNA nanotrains — Thermodynamic and release kinetic study

As one of the most promising drug delivery carriers, self-assembled DNA nanostructures are characterized of well-defined sizes, excellent biocompatibility, high drug loading and ability to control drug release. Studying the interactions between anticancer drugs and DNA nanostructures can help to associate microstructure-drug loading-release rate-therapeutic effect. Herein AS1411 aptamer-tethered DNA nanotrains (AS1411NTrs) were constructed and used as anthracyclines carrier with high payload for targeted delivery. The bindings of doxorubicin (DOX), epirubicin (EPI), and daunorubicin (DAU) to AS1411NTrs were investigated by isothermal titration calorimetry and fluorescence spectroscopy, and thermodynamic parameters were obtained. The high drug payload capacity of AS1411NTrs was verified by the large number of binding sites (~20). The binding mode was determined by differential scanning calorimetry and potassium iodide (KI) quenching experiments. The release experiment data showed that DNase I facilitated drug release and the release followed the first-order kinetic model. MTT cell viability assay demonstrated that the drug-loaded AS1411NTrs had significantly higher cytotoxicity against target HeLa cells than normal human liver L02 cells. These findings revealed that AS1411NTrs had high payload and targeted release capacity for DOX, EPI, and DAU. This result can provide a theoretical basis for constructing reasonable DNA nanostructures based on drug carriers.

Characterization of 3D DNA Assemblies Using Cryogenic Electron Microscopy

DNA nanotechnology utilizes DNA double strands as building units for self-assembly of DNA nanostructures. The specific base-pairing interaction between DNA molecules is the basis of these assemblies. After decades of development, this technology has been able to construct complex and programmable structures. With the increase in delicate nature and complexity of the synthesized nanostructures, a characterization technology that can observe these structures in three dimensions has become necessary, and developing such a technology is considerably challenging. DNA assemblies have been studied using different characterization methods including atomic force microscopy (AFM), scanning electron microscopy(SEM), and transmission electron microscopy(TEM). However, the three-dimensional(3D) DNA assemblies always collapse locally due to the dehydration during the drying process. Cryogenic electron microscopy(cryo-EM) can overcome the challenge by maintaining three-dimensional morphologies of the cryogenic samples and reconstruct the 3D models from cryogenic samples accordingly by collecting thousands of two-dimensional(2D) projection images, which can restore their original morphologies in solution. Here, we have reviewed several typical cases of 3D DNA-assemblies and highlighted the applications of cryo-EM in characterization of these assemblies. By comparing with some other characterization methods, we have shown how cryo-EM promoted the development of structural characterization in the field of DNA nanotechnology.

Self-Assembled DNA Nanostructures-Based Nanocarriers Enabled Functional Nucleic Acids Delivery.

Nucleic acids are recognized as the blueprints of life and the repositories of genetic information, which play essential roles in the "central dogma of molecular biology". As classical and specific nucleic acids, apart from carrying genetic information, functional nucleic acids (FNAs) perform many intriguing functions such as targeted recognition, therapy and enzymatic catalysis. FNAs with excellent biocompatibility provide great promise for future applications in bioanalysis, material science, and nanotechnology. Nevertheless, effective delivery of FNAs to the targeted location still suffers from one or more challenges, especially in the field of biomedicine. Encouragingly, DNA nanotechnology shows great potential for applications in bioimaging, biosensing, and molecules delivery. Importantly, it provides an unprecedented opportunity to design and synthesize a series of self-assembled DNA nanostructures with well-defined size, shape, surface chemistry, and function. Through Watson-Crick base-pairing rules, FNAs, including aptamers, DNAzymes, small interfere RNA (siRNA), antisense oligonucleotides (ASOs), and unmethylated cytosine-phosphate-guanine dinucleotide oligonucleotides (CpG ODNS), are successfully decorated with self-assembled DNA nanostructures for delivery. In this progress report, we focus on self-assembled DNA nanostructures-based nanocarriers to deliver FNAs for applications in cellular analysis and cancer theranostics. First, we briefly summarize the superior properties of the prospective nanocarriers and introduce the self-assembled DNA nanostructures. Then we mainly highlight the most recent achievements of self-assembled DNA nanostructures as comers for the delivery of FNAs. Finally, this review points to some of the current challenges in the applications of self-assembled DNA nanostructures as FNAs nanocarriers and provides an insight into the future perspectives of self-assembled DNA nanostructures-based nanocarriers for FNAs delivery.