Tetrahedral DNA Nanostructures Research Articles

Aptamer-targeted DNA nanostructures with doxorubicin to treat protein tyrosine kinase 7-positive tumours.

ObjectivesAptamer sgc8c is a short DNA sequence that can target protein tyrosine kinase 7 (PTK7), which was overexpressed on many tumour cells. This study aimed to fabricate a novelty DNA nanostructure drug delivery system target on PTK7‐positive cells—CCRF‐CEM (human T‐cell ALL).MethodsAptamer‐modified tetrahedron DNA was synthesized through one‐step thermal annealing process. The sgc8c‐TDNs (s‐TDNs) loading DOX complexes were applied to investigate the effect to PTK7‐negative and ‐positive cells.ResultsWhen s‐TDN:DOX acted on PTK7‐positive and ‐negative cells respectively, the complexes exhibited specific toxic effect on PTK7‐positive cells but not on PTK7‐negative Ramos cells in vitro research.ConclusionsIn this work, we successfully constructed a PTK7‐targeting aptamer‐guided DNA tetrahedral nanostructure (s‐TDN) as a drug delivery system via a facile one‐pot synthesis method. The results showed that s‐TDN:DOX exhibited enhanced cytotoxicity against PTK7‐positive CCRF‐CEM cells, with a minor effect against PTK7‐negative Ramos cells. Hence, this functionalized TDNs drug delivery system displayed its potential application in targeting PTK7‐positive tumour T‐cell acute lymphoblastic leukaemia.

High performance electrochemical biosensor based on 3D nitrogen-doped reduced graphene oxide electrode and tetrahedral DNA nanostructure

In this study, an electrochemical biosensor was developed for highly sensitive and specific detection of target miRNA-155. The structure was formed by the hybridization of a tetrahedral DNA nanostructure-based biomolecular probe assembled on 3D nitrogen-doped reduced graphene oxide/ gold nanoparticles (3D N-doped rGO/AuNPs) electrode surface. Upon addition of target miRNA-155, the gold and silver nanorod/ thionine/ complementary DNA (AuAgNR/Thi/F) was hybridized with the target, and used for signal amplification, catalyzing the reduction of Thi as an electron mediator. Due to the signal amplification by the enhanced immobilization of DNA on the surface of 3D N-doped rGO/AuNPs electrode and AuAgNR/Thi, coupling the low background signal produced by blank solution, electrochemical performance of the device was optimized to be proportional to miRNA-155 concentration in the range of 1 × 10–11 to 1 × 10−4 M with a detection limit of 1 × 10–12 M. In addition, direct detection in serum is demonstrated with high specificity. Thus, this biosensor is potentially applicable for microRNA detection in medical research and early clinical diagnosis.

DNA Nanostructure-Programmed Like-Charge Attraction at the Cell-Membrane Interface.

Cell entry of anionic nano-objects has been observed in various types of viruses and self-assembled DNA nanostructures. Nevertheless, the physical mechanism underlying the internalization of these anionic particles across the negatively charged cell membrane remains poorly understood. Here, we report the use of virus-mimicking designer DNA nanostructures with near-atomic resolution to program “like-charge attraction” at the interface of cytoplasmic membranes. Single-particle tracking shows that cellular internalization of tetrahedral DNA nanostructures (TDNs) depends primarily on the lipid-raft-mediated pathway, where caveolin plays a key role in providing the short-range attraction at the membrane interface. Both simulation and experimental data establish that TDNs approach the membrane primarily with their corners to minimize electrostatic repulsion, and that they induce uneven charge redistribution in the membrane under the short-distance confinement by caveolin. We expect that the nanoscale like-charge attraction mechanism provides new clues for viral entry and general rules for rational design of anionic carriers for therapeutics.

Tetrahedral DNA Nanomaterial Regulates the Biological Behaviors of Adipose-Derived Stem Cells via DNA Methylation on Dlg3.

As a simple and classical three-dimensional shape, tetrahedral DNA nanostructures (TDNs) can provide robust properties for better stability and can serve as a versatile platform for biosensing and drug delivery. More in-depth, its safety should be assessed by sensitive detection methods. However, the effect of TDNs at the epigenetic level has not received much attention. Here, DNA methylation alteration in adipose-derived stem cells (ASCs) after exposure to TDNs was comprehensively evaluated. The results from reduced representation bisulfite sequencing, bisulfite-specific polymerase chain reaction, and further gene function analysis revealed that TDNs induced a few differentially methylated regions where negatively correlated gene expressions occur. Moreover, TDNs facilitated ASC proliferation and attenuated apoptosis via DNA hypermethylation of the Dlg3 gene promotor. This study may help pave the way for potential applications with the nanosafety of TDNs and offer deep insights into the proliferation promotion effect and antiapoptosis ability of TDNs.

Advances in biological applications of self-assembled DNA tetrahedral nanostructures

Abstract In recent years, there has been a rapid progress in the development of DNA nanomaterials for biological applications. Owing to their unsurpassed editability, various DNA nanomaterials with well-documented biocompatibility and relative stability have been developed. Among these tetrahedral DNA nanostructures (TDNs), as typical multi-arm DNA tiles for 3D polyhedral, is a major research focus owing to their structural and biological features. TDNs have yielded satisfactory results as biological regulators, biosensors, and targeted delivery vehicles. Extensive studies on TDNs have revealed that TDNs alone could regulate several cellular processes, such as migration, proliferation, differentiation, and autophagy. Further, their editability enables control of the spatial distribution of biosensors, thereby promoting accuracy and efficiency. Finally, various modification procedures allow attachment of nucleic acids, aptamers, peptides, antibodies, and various low-molecular-weight drugs to the TDNs, thereby yielding a novel targeted delivery system. This review summarizes recent research progress on TDNs in these aforementioned major areas. These discoveries might be of great therapeutic value in regenerative medicine, gene delivery, targeted chemotherapy, and other related fields. We also illustrate the emerging challenges in the clinical application of TDNs, as well as future development opportunities.

Ultrasensitive aptasensor with DNA tetrahedral nanostructure for Ochratoxin A detection based on hemin/G-quadruplex catalyzed polyaniline deposition

An ultrasensitive electrochemical aptasensor with DNA tetrahedral nanostructure (DTN) for Ochratoxin A (OTA) detection was developed based on hemin/G-quadruplex catalyzed polyaniline (PANI) deposition in the presence of H2O2. DTN, which modified with OTA aptamer on top vertex and thiol group on other three vertices, was constructed on the modified electrode surface via Au-S bond and prominent template for PANI deposition owing to their numerous negative charges. The prepared gold nanorods (AuNRs) was used as a carrier to immobilize DTN and promote electron transfer. In the presence of OTA, the aptamer specific for OTA was formed G-quadruplex structure. The hemin/G-quadruplex was formed when hemin was intercalated into G-quadruplex, which had high catalytic activity to catalyze PANI deposition in the presence of H2O2 and amplify DPV signal. The amount of generated PANI was dependent on the number of hemin/G-quadruplex induced by OTA. Therefore, DPV currents was related to the concentration of OTA. The fabricated aptasensor exhibited a good linear response toward OTA over a wide range of concentrations from 0.001 ng/mL to 0.5 ng/mL, and the limit of detection of the proposed aptasensor was 0.26 pg/mL.

Tetrahedral DNA nanostructures facilitate neural stem cell migration via activating RHOA/ROCK2 signalling pathway.

The main purpose of current study was to explore the effects of tetrahedral DNA nanostructures (TDNs) on neuroectodermal (NE-4C) stem cells migration and unveil the potential mechanisms. The successfully self-assembled TDNs were also determined by dynamic light scattering (DLS). A bidirectional wound-healing assay and transwell chamber assay were employed to test the migrating behaviour of NE-4C stem cells cultured under different conditions. Through an in vitro study, we found that stem cells could internalize TDNs quickly, and the cells' parallel and vertical migration was promoted effectively. Besides, the effects of TDNs were found being exerted by upregulating the gene and protein expression levels of RhoA, Rock2 and Vinculin, indicating that the RHOA/ROCK2 pathway was activated by the TDNs during the cell migration. In conclusion, TDNs could enter NSCs without the aid of other transfection reagents in large amounts, whereas only small amounts of ssDNA could enter the cells. TDNs taken up by NSCs activated the RHOA/ROCK2 signalling pathway, which had effects on the relevant genes and proteins expression, eventually promoting the migration of NE-4C stem cells. These findings suggested that TDNs have great potential in application for the repair and regeneration of neural tissue.

Inhibiting Methicillin-Resistant Staphylococcus aureus by Tetrahedral DNA Nanostructure-Enabled Antisense Peptide Nucleic Acid Delivery.

One of the biggest obstacles for the use of antisense oligonucleotides as antibacterial therapeutics is their limited uptake by bacterial cells without a suitable carrier, especially in multi-drug-resistant bacteria with a drug efflux mechanism. Existing vectors, such as cell-penetrating peptides, are inefficient and nontargeting, and accordingly are not ideal carriers. A noncytotoxic tetrahedral DNA nanostructure (TDN) with a controllable conformation has been developed as a delivery vehicle for antisense oligonucleotides. In this study, antisense peptide nucleic acids (asPNAs) targeting a specific gene ( ftsZ) were efficiently transported into methicillin-resistant Staphylococcus aureus cells by TDNs, and the expression of ftsZ was successfully inhibited in an asPNA-concentration-dependent manner. The delivery system specifically targeted the intended gene. This novel delivery system provides a better platform for future applications of antisense antibacterial therapeutics and provides a basis for the development of a new type of antibacterial drug for multi-drug-resistant bacterial infections.

Neuroprotective Effect of Tetrahedral DNA Nanostructures in a Cell Model of Alzheimer's Disease.

Accumulating evidence supports the abnormal deposition of amyloid β-peptide (Aβ) as the main cause of Alzheimer's disease (AD). Therefore, fighting against the formation, deposition, and toxicity of Aβ is a basic strategy for the treatment of AD. In the process of in vitro nerve cell culture, screening out drugs that can antagonize a series of toxic reactions caused by β-amyloid deposition has become an effective method for the follow-up treatment of AD. Our previous studies showed that tetrahedral DNA nanostructures (TDNs) had good biocompatibility and had some positive effects on the biological behavior of cells. In this study, the main aim of our work was to explore the effects and potential mechanism of TDNs in protecting neuronal PC12 cells from the toxicity of Aβ. Our study demonstrated that TDNs can protect and rescue PC12 cell death through Aβ25-35-induced PC12 cell apoptosis. Further studies showed that TDNs significantly improved the apoptosis by affecting the abnormal cell cycle, restoring abnormal nuclear morphology and caspase activity. Western blot analysis showed that TDNs could prevent the damage caused by Aβ deposition by activating the ERK1/2 pathway and thus be a potential therapeutic agent with a neuroprotective effect in Alzheimer's disease. Our finding provides a potential application of TDNs in the prevention and treatment of AD.

Aflatoxin B1 Electrochemical Aptasensor Based on Tetrahedral DNA Nanostructures Functionalized Three Dimensionally Ordered Macroporous MoS2-AuNPs Film.

In food safety evaluation, aflatoxin B1 (AFB1) is an important indicator. In this work, we developed an AFB1 electrochemical aptasensor based on a tetrahedral DNA nanostructures (TDNs) immobilized three dimensionally ordered macroporous MoS2-AuNPs hybrid (3DOM MoS2-AuNPs) recognition interface and horseradish peroxidase (HRP) functionalized magnetic signal amplifier. To greatly enhance the recognition efficiency, sensitivity, and stability of the aptasensor, the AFB1 aptamer-incorporated TDNs were ingeniously combined with the 3DOM MoS2-AuNPs film for the construction of the sensing interface. The aptamers would release from the electrode surface after they reacted with AFB1, and then the hybridization-free TDNs formed. Thus, the biocomposite of DNA helper strands (H1)/HRP functionalized AuNPs-SiO2@Fe3O4 nanospheres would combine with the hybridization-free TDNs due to the hybridization of H1 and TDNs. The more AFB1 existed in the solution, the more H1/HRP-AuNPs-SiO2@Fe3O4 could be combined onto the 3DOM MoS2-AuNPs surface. The current response coming from HRP-catalyzed reduction of H2O2 using thionine (Thi) as electrochemical probe was proportional with the AFB1 concentration. Upon optimal conditions, the aptasensor showed specificity for AFB1, achieving a good linear range of 0.1 fg/mL-0.1 μg/mL and the detection limit of 0.01 fg/mL. Furthermore, the developed aptasensor was also applied for detecting AFB1 content in rice and wheat powder samples, obtaining good results in conformity with those achieved from the high-performance liquid chromatography tandem mass spectrometry (HPLC-MS) method.

Systematic Study in Mammalian Cells Showing No Adverse Response to Tetrahedral DNA Nanostructure

The advent of DNA technology has demonstrated great potential in a wide range of applications, especially in the field of biology and biomedicine. However, current understanding of the toxicological effects and cellular responses of DNA nanostructures remains to be improved. Here, we chose tetrahedral DNA nanostructures (TDNs), a type of nanocarriers for delivering molecular drugs, as a model for systematic live-cell analysis of the biocompatibility of TDNs to normal bronchial epithelial cells, carcinoma cells, and macrophage. We found that the interaction behaviors of TDNs in different cell lines were very different, whereas after internalization, most of the TDNs in diverse cell lines were positioned to lysosomes. By a systematic assessment of cell responses after TDN exposure to various cells, we demonstrate that internalized TDNs have good innate biocompatibility. Interestingly, we found that TDN-bearing cells would not affect the cell cycle progression and accompany cell division and that TDNs were separated equally into two daughter cells. This study improves our understanding of the interaction of DNA nanostructures with living systems and their biocompatibility, which will be helpful for further designing DNA nanostructures for biomedical applications.

Synthesis of an Ethyleneimine/tetrahedral DNA Nanostructure Complex and its Potential Application as a Multi‐Functional Delivery Vehicle

Nowadays, DNA nanostructures are extensively researched for their biocompatibility, editable functionality, and structural stability. Tetrahedral DNA nanostructures (TDNs), widely known for their membrane permeability, are regarded as potential candidates for drug delivery. However, the stability and membrane permeability of TDNs call for further enhancement if in vivo usage is ascribed. To overcome the drawbacks of TDNs, ethylene imine (PEI, 25 kDa, branched)-a classic cationic polymer in the field of gene delivery-was applied. Via a facile one-pot synthesis method, a PEI/TDNs complex was formed. Subsequently, a DNase protection assay, a cytotoxicity assay, endocytosis-related experiments, and lysosome staining were performed to examine the potential of PEI/TDNs as a delivery vehicle. The combination of PEI and TDNs not only overcame the drawbacks of each substance but also retained their individual merits. Traditionally, drug-delivery vehicles that enable enhanced cell entry and lysosome escape are often compromised by their toxicity and poor multifunctionality. We believe this novel PEI/TDNs complex with enhanced systemic stability, biocompatibility, cell-entry ability, and lysosome-escape ability and unsurpassed editable functionality could be a powerful tool as a multi-functional delivery vehicle in targeted drug delivery, in vivo imaging, and other related fields.

Effect of tetrahedral DNA nanostructures on proliferation and osteo/odontogenic differentiation of dental pulp stem cells via activation of the notch signaling pathway

Dental pulp stem cells (DPSCs) derived from the human dental pulp tissue have multiple differentiation capabilities, such as osteo/odontogenic differentiation. Therefore, DPSCs are deemed as ideal stem cell sources for tissue regeneration. As new nanomaterials based on DNA, tetrahedral DNA nanostructures (TDNs) have tremendous potential for biomedical applications. Here, the authors aimed to explore the part played by TDNs in proliferation and osteo/odontogenic differentiation of DPSCs, and attempted to investigate if these cellular responses could be driven by activating the canonical Notch signaling pathway. Upon exposure to TDNs, proliferation and osteo/odontogenic differentiation of DPSCs were dramatically enhanced, accompanied by up regulation of Notch signaling. In general, our study suggested that TDNs can significantly promote proliferation and osteo/odontogenic differentiation of DPSCs, and this remarkable discovery can be applied in tissue engineering and regenerative medicine to develop a significant and novel method for bone and dental tissue regeneration.

Self-Assembled Tetrahedral DNA Nanostructures Promote Neural Stem Cell Proliferation and Neuronal Differentiation.

Stem cell-based therapy is considered a promising approach for the repair of nervous tissues. Neural stem cells (NSCs) cannot proliferate or differentiate efficiently; hence, different biomaterials have been explored to improve NSC proliferation and differentiation. However, these agents either had low bioavailability or poor biocompatibility. In this work, our group investigated the effects of tetrahedral DNA nanostructures (TDNs), a novel DNA biological material, on the self-renew and differentiation of neuroectodermal (NE-4C) stem cells. We observed that TDN treatment promoted self-renew of the stem cells via activating the Wnt/β -catenin pathway. In addition, our findings suggested that NE-4C stem cells' neuronal differentiation could be promoted effectively by TDNs via inhibiting the notch signaling pathway. In summary, this is the first report about the effects of TDNs on the proliferation and differentiation of NE-4C stem cells and the results demonstrate that TDNs have a great potential in nerve tissue regeneration.

Programmable Modulation of Copper Nanoclusters Electrochemiluminescence via DNA Nanocranes for Ultrasensitive Detection of microRNA

The DNA nanocrane with functionalized manipulator and fixed-size base offered a programmable approach to modulate the luminous efficiency of copper nanoclusters (Cu NCs) for achieving remarkable electrochemiluminescence (ECL) enhancement, further the Cu NCs as signal label was constructed in biosensor for ultrasensitive detection of microRNA-155. Herein, the DNA nanocrane was first constructed by combining binding-induced DNA assembly as manipulator and tetrahedral DNA nanostructure (TDN) as base, which harnessed a small quantity of specific target (microRNA (miRNA)-155) binding to trigger assembly of separate DNA components for producing numerous AT-rich double-stranded DNA (dsDNA) on the vertex of TDN. Upon the incubation of Cu2+ on the AT-rich dsDNA, each DNA-stabilized Cu NCs probe could be in situ electrochemically generated on an individual TDN owing to the A-Cu2+-T bond. Thus, the generation of Cu NCs was highly regulated with AT-rich dsDNA as the template, and its lateral distance was tuned by the TDN size, which were two key factors to influence the luminous efficiency of Cu NCs. By coordinate modulation, the detection limit of the ultrasensitive biosensor for miRNA-155 down to 36 aM and the programmable modulation strategy paved the way for comprehensive applications of DNA nanomachines and metal nanoclusters in biosensing and clinical diagnosis.

Anti-inflammatory and Antioxidative Effects of Tetrahedral DNA Nanostructures via the Modulation of Macrophage Responses.

Tetrahedral DNA nanostructures (TDNs) are a new type of nanomaterials that have recently attracted attention in the field of biomedicine. However, the practical application of nanomaterials is often limited owing to the host immune response. Here, the response of RAW264.7 macrophages to TDNs was comprehensively evaluated. The results showed that TDNs had no observable cytotoxicity and could induce polarization of RAW264.7 cells to the M1 type. TDNs attenuated the expression of NO IL-1β (interleukin-1β), IL-6 (interleukin-6), and TNF-α (tumor necrosis factor-α) in LPS-induced RAW264.7 cells by inhibiting MAPK phosphorylation. In addition, TDNs inhibited LPS-induced reactive oxygen species (ROS) production and cell apoptosis by up-regulating the mRNA expression of antioxidative enzyme heme oxygenase-1 (HO-1). The findings of this study demonstrated that TDNs have great potential as a novel theranostic agent because of their anti-inflammatory and antioxidant activities, high bioavailability, and ease of targeting.

Targeted Imaging of Brain Tumors with a Framework Nucleic Acid Probe.

Development of agents for delivering drugs and imaging probes across the blood-brain barrier (BBB) remains a major challenge. In this study, we designed a biocompatible framework nucleic acid (FNA)-based imaging probe for brain tumor-targeting. We employed a typical type of FNAs, tetrahedral DNA nanostructures (TDNs), as the building block, which were modified with angiopep-2 (ANG), a 19-mer peptide derived from human Kunitz domain of aprotinin. This probe exhibited high binding efficiency with low-density lipoprotein receptor-related protein-1 (LRP-1) of BBB and glioma. We found that ANG-functionalized TDNs (ANG-TDNs) stayed intact for at least 12 h in serum, and that ANG modification effectively enhanced cellular uptake of TDNs in brain capillary endothelial cells and Uppsala 87 malignant glioma (U87MG) cells. Remarkably, studies in both in vitro and in vivo models revealed that ANG-TDNs could cross the BBB. Especially, in vivo imaging showed strong fluorescent signals in U87MG human glioblastoma xenograft in nude mice. This study establishes that the FNA-based platform provides a new theranostic tool for the study and therapy of brain tumors.

High-Discrimination Factor Nanosensor Based on Tetrahedral DNA Nanostructures and Gold Nanoparticles for Detection of MiRNA-21 in Live Cells

While detection of microRNA with or without signal amplification is highly informative, nanosensors with high specificity for cell-specific RNA detection are rare.Methods: In this study, a tetrahedral DNA nanostructure (TDN) with a specific function was combined with gold nanoparticles (Au-NP) possessing fluorescence quenching effects and a large surface area to fabricate a fluorescence resonance energy transfer based nanosensor (Au-TDNN). The presence of miR-21 (target) can separate the fluorescent dye-labeled detection probe on Au-TDNNs from Au-NPs, which separates the donor and acceptor, thus inducing an intensive fluorescence signal. High specificity for discerning point mutation targets was achieved by rationally designing the nucleic acid strand displacement reaction to occur spontaneously with ΔG0 ≈ 0 based on thermodynamic parameters; under this condition, slight thermodynamic changes caused by base mismatch exert significant effects on hybridization yield.Results: Chemically synthesized DNA of three single-base-changed analogues of target, let-7d, and miR-200b were tested. A discrimination factor (DF) of 15.4 was produced by the expected detection probe on Au-NPs for proximal single-base mismatch. As the control group, the DF produced by an ordinary detection probe on Au-NPs only reached 2.4. The feasibility of the proposed strategy was also confirmed using hepatocyte cancer cells (HepG2).Conclusion: This improved nanosensor opens a new avenue for the specific and easy detection of microRNA in live cells.

Effects of tetrahedral DNA nanostructures on autophagy in chondrocytes.

Tetrahedral DNA nanostructures (TDNs) have gathered great attention and are being widely used in biomedicine. We demonstrated that autophagy increased after exposure to TDNs (250 nM) along with the up-regulation of several autophagy-related genes and proteins. TDNs enhanced cell autophagy through the PI3K/AKT/mTOR signaling pathway.

Overcoming drug-resistant lung cancer by paclitaxel loaded tetrahedral DNA nanostructures.

Paclitaxel (PTX) is an effective drug against diseases such as lung cancer, ovarian cancer, and breast cancer. However, multidrug resistance limits the clinical applications of this drug. Tetrahedral DNA nanostructures (TDNs) offer great promise as a drug delivery candidate. In our study, we prepared TDNs that were subsequently loaded with PTX (PTX/TDNs). The cytotoxicity of PTX/TDNs and PTX alone on non-small cell lung cancer (NSCLC) cells (A549) and the PTX-resistant cell line (A549/T) was determined using a cell count kit-8 (CCK-8) assay. PTX/TDNs exerted strong lethality on both cell lines. Moreover, drug resistance was overcome. Furthermore, the mechanisms used by PTX/TDNs to overcome drug resistance were studied. The expression of mdr 1 gene and P-glycoprotein (P-gp) in A549/T was found to be downregulated, thus indicating that TDNs serve as a P-gp inhibitor. We also showed that PTX/TDNs killed cancer cells via apoptosis. Thus, PTX/TDNs have great potential for use as a nanodelivery system for the treatment of PTX-resistant NSCLC.