Tetrahedral DNA Nanostructures Research Articles

Self-assembled tetrahedral DNA nanostructures-based ultrasensitive label-free detection of ampicillin.

Antibiotics are widely used for improving the living conditions of livestock. However, residual antibiotics present in animal products induce several human diseases. Therefore, a simple, rapid, and cost-effective system for detecting and monitoring the presence of antibiotics in foods is in great demand to alleviate safety concerns. In this study, a highly sensitive and selective aptameric electrochemical sensing platform was designed based on nanomaterial modification and DNA nanotechnology. Electrochemically reduced graphene oxide and gold nanoparticles were used to modify the working surface of a screen-printed electrode to enhance electrical conductivity and biocompatibility. The electrode surface was further modified with self-assembled tetrahedral DNA nanostructures (TDN) to improve the detection sensitivity. The TDN allowed controlling the nano-spacing of aptamers immobilized on the electrode surface and placing aptamers in a solution-phase-like detecting environment to improve the target-binding efficiency without signal amplification modules. Differential pulse voltammetry was employed to measure electrical signals in proportion to the amount of ampicillin, the target antibiotic, present in buffer and spiked milk samples. The designed aptasensor was able to detect and measure the target ampicillin in less than 30min over a wide concentration range of 10 pM to 1mM, with a limit of detection of 1 pM, which is 100 times better than when using the same sensing probe without TDN modification. The aptasensor was reusable by simply rinsing with deionized water, remained stable during 15-day storage, and yielded reproducible results.

Study on the self-assembly and signal amplification ability of nucleic acid nanostructure with the nanopipette

Nanopipette as a member of solid-state nanopores has been developed to detect a set of biological molecules. Although the size of the nanopipette can be adjusted artificially, the direct detection of small molecules is still challenging, which is limited by the precision of the nanopipette preparation. In this work, the translocation characteristics of DNA tetrahedrons with varying degrees of the assembly were studied to evaluate their self-assembly process and the advantage of tetrahedral DNA nanostructures as a signal amplification label for biomarkers detection based on current blockages. Self-assembly of different number of single-stranded DNA causes the DNA nanostructure in different shapes. Through the complete assembly of DNA tetrahedron, we demonstrate that the nanopore signal-to-noise ratio is significantly increased and the event signal becomes more stable and significant. Therefore, these attributes allow us to gain more insight into the assembly process of DNA nanostructures and we can use tetrahedral DNA nanostructure to develop an effective assay for identifying small molecules with the nanopipette.

A sensitive aflatoxin B1 electrochemical aptasensor based on ferrocene-functionalized hollow porous carbon spheres as signal amplifier

Due to the high toxicity and carcinogenicity of Aflatoxin B1 (AFB1), a sensitive, low-cost and simple method to accurately detect AFB1 in foodstuffs is highly desirable. Herein, a sensitive and selective AFB1 electrochemical aptasensor was developed by using ferrocene immobilized hollow porous carbon spheres (HPCS-Fc) as the electrochemical signal tag. Attributed to the large surface and high conductivity of HPCS, the prepared Fc-HPCS showed the high load amount of Fc and HPCS-Fc labelled GC electrode displayed large oxidation peak current. To further improve the biosensing ability, tetrahedral DNA nanostructures (TDNs) was attached on the biosensing interface to control the orientation of HPCS-Fc-cDNA. Based on the above advantages, the HPCS-Fc labelled electrochemical aptasensor exhibited high sensitivity towards AFB1 in a wide linear range from 1.0 × 10−2 pg mL−1 to 100.0 μg mL−1 and a low detection limit down to 3.3 × 10−5 ng mL−1. Furthermore, the developed aptasensor was applied to detect AFB1 in wheat powder samples with satisfactory recoveries of 91.7%∼107.7%, demonstrating its potential application in testing AFB1 in food samples.

Tetrahedral DNA Nanostructure with Multiple Target-Recognition Domains for Ultrasensitive Electrochemical Detection of Mucin 1.

In this work, a tetrahedral DNA nanostructure (TDN) designed with multiple biomolecular recognition domains (m-TDN) was assembled to construct an ultrasensitive electrochemical biosensor for the quantitative detection of tumor-associated mucin 1 (MUC-1) protein. This new nanostructure not only effectively increased the capture efficiency of target proteins compared to the traditional TDN with a single recognition domain but also enhanced the sensitivity of the constructed electrochemical biosensors. Once the target MUC-1 was captured by the protein aptamers, the ferrocene-marked DNA strands as electrochemical signal probes at the vertices of m-TDN would be released away from the electrode surface, causing significant reduction of the electrochemical signal, thereby enhancing significantly the detection sensitivity. As a result, this well-designed biosensor achieved ultrasensitive detection of the biomolecule at a linear range from 1 fg mL-1 to 1 ng mL-1, with the limit of detection down to 0.31 fg mL-1. This strategy provides a new approach to enhance the detection sensitivity for the diagnosis of diseases.

Gold nanorods/tetrahedral DNA composites for chemo-photothermal therapy.

Combination therapy is extensively developed for cancer treatment in recent years due to its high efficiency. Herein, we constructed a nanocomposite based on gold nanorods (GNRs) and drug-loaded tetrahedral DNA nanostructures (TDN) for chemo-photothermal combinational therapy. Anti-tumor drug doxorubicin (DOX) was loaded via the insertion within GC base pairs of TDN. The aptamer AS1411 was attached to the apex of TDN (ATDN) to target tumor cells. The DOX-loaded DNA tetrahedron (ATDN-DOX) was compressed by the GNRs coated with PEI (GNRs@ATDN-DOX) to realize the photothermal function and lysosome escape. GNRs under the illumination of 808 nm infrared laser showed high photothermal conversion and stability due to the protection of PEI layer. The drug-loading capacity of ATDN-DOX was as high as 314 DOX molecules in per ATDN. The positive charge of PEI in GNRs@ATDN-DOX nanocomposites was utilized to achieve excellent cell penetration and induce proton sponge effect for lysosomal escape. The nanocomposites presented HeLa and 4T1 cells targeting and resulted in efficient anticancer activity.

A tetrahedral DNA nanostructure functionalized paper-based platform for ultrasensitive colorimetric mercury detection

A novel tetrahedral DNA nanostructure (TDN) functionalized paper-based colorimetric sensor was developed for ultrasensitive analysis of mercury ion (Hg2+). On the paper-based platform, TDN was used as scaffold to anchor aptamer to improve the capture efficiency owing to the controllably specific orientation, low steric hindrance effect and well-defined spacing. In the absence of Hg2+, biotinylated aptamer would bind with streptavidin-labeled HRP. Whereas, in the presence of Hg2+, aptamer would preferentially bind to Hg2+ owing to the higher binding constant of T–Hg–T than that of T–A, which consequently leading to the biotinylated aptamer releasing from TDN. After introducing TMB-H2O2 solution, strong variation of color signal could be observed. Therefore, the quantitative analysis of Hg2+ was carried out by using smartphone to monitor the color intensity on paper chip. The proposed strategy exhibits good linearity over a wide range of 10–13–10–7 M with a low detection limit of 30 fM. And this method had been successfully applied in real water samples. Our strategy provided a versatile, low-cost and user-friendly paper-based monitoring platform for rapid and ultrasensitive analysis of heavy metal ions and thus had potential applications in food safety and public health.

High Mannose-Specific Aptamers for Broad-Spectrum Virus Inhibition and Cancer Targeting

High Mannose-Specific Aptamers for Broad-Spectrum Virus Inhibition and Cancer Targeting

Ionic Current Fluctuation and Orientation of Tetrahedral DNA Nanostructures in a Solid-State Nanopore.

Understanding the dynamic behavior of a nanostructure translocating through a nanopore is important for various applications. In this paper, the characteristics in ion current traces of tetrahedral DNA nanostructures (TDN) translocating through a solid-state nanopore are examined, by combined experimental and theoretical simulations. The results of finite element analysis reveal the correlation between orientation of TDN and the conductance blockade. The experimentally measured fluctuations in the conductance blockade, expressed as voltage-dependent histogram profiles, are consistent with the simulation, revealing the nature of a random distribution in orientation and weak influence of electrostatic and viscous torques. The step changes in orientation of a TDN during translocation are further explained by the collision with the nanopore, while the gradual changes in orientation illustrate the impact of a weak torque field in the nano-fluidic channel. The results demonstrate a general method and basic understanding in the dynamic behavior of nanostructures translocating through solid-state nanopores.

Repair of infected bone defect with Clindamycin-Tetrahedral DNA nanostructure Complex-loaded 3D bioprinted hybrid scaffold

Infection of bone defects is a common clinical problem that negatively impacts bone repair and may result in the development of antibiotic resistance due to the long-term use of antibiotics. To the best of our knowledge, integrating tetrahedral DNA nanostructure (TDN) drug delivery with 3D bioprinting has not been reported to treat infected bone defects. However, numerous studies have focused on effectively controlling infected bone defects and achieving functional reconstruction. In this study, TDN was proposed as a drug delivery vehicle to enhance cell penetration and the antibacterial properties of clindamycin (CLI), a common antibiotic used to treat osteomyelitis. A 3D hybrid scaffold loaded with TDN-CLI complexes was constructed using bioprinting technology. Its structure and biological properties were characterized, and the scaffold was further applied to treat methicillin-resistant Staphylococcus aureus (MRSA) infection in a rat model of bone defect. The results demonstrated that the TDN-CLI-loaded 3D bioprinted hybrid scaffold possessed excellent biocompatibility, outstanding osteogenic and antimicrobial activity, and significantly improved the repair of infected bone defects in the rat model. The TDN-CLI-loaded hybrid scaffold developed in the current study has broad application prospects for treating infected bone defects.

Ultrasensitive fluorescent detection of telomerase activity based on tetrahedral DNA nanostructures as carriers for DNA-templated silver nanoclusters

Precise evaluation of telomerase activity is essential for the clinical diagnosis of early tumors. Herein, we have ingeniously designed a tetrahedral DNA nanostructure, with hairpin-shaped DNA probes rich in cytosine bases at four vertices for telomerase detection. The DNA-templated silver nanoclusters can be formed after the addition of Ag. Then the introduction of telomerase adds the single-strand TTAGGG extension, which can "turn on" the fluorescence of silver nanoclusters quickly by the proximity of the resulting guanine-rich sequences to silver nanoclusters and realize accurate detection of telomerase activity. In this study, integration of high stability tetrahedral DNA nanostructure and fluorescence signal amplification of four DNA-templated silver nanoclusters offers the advantage of high sensitivity, with a low detection limit of 1 cell. More than that, this method is low-cost, facile, and feasible for practical clinical applications.

Loading of DOX into a tetrahedral DNA nanostructure: the corner does matter.

With the rapid development of nanotechnology, various DNA nanostructures have been synthesized and widely used in drug delivery. However, the underlying mechanisms of drug molecule loading into the DNA nanostructure are still elusive. In this work, we systematically investigate the interactions of a tetrahedral DNA nanostructure (TDN) with the anti-cancer drug doxorubicin (DOX) by combining molecular docking and all-atom molecular dynamics simulations. It is found that there are five possible binding modes in the single TDN–DOX interactions, namely the outside-corner mode, the inside-corner mode, the major-groove mode, the minor-groove mode, and the intercalation mode, where the van der Waals (VDW) interaction and the electrostatic (ELE) interaction dominate in the case of unionized DOX and ionized DOX, respectively. Moreover, with the increase of the DOX number, some of the interaction modes may disappear and the inside-corner mode is the most energy-favorable mode. The present study enhances the molecular understanding of the role of TDN as the drug carrier, which may provide a useful guideline for the future design of DNA nanostructures.

Epigallocatechin gallate-loaded tetrahedral DNA nanostructures as a novel inner ear drug delivery system.

The study of drug delivery systems to the inner ear is a crucial but challenging field. The sensory organ (in the inner ear) is protected by the petrous bone labyrinth and the membranous labyrinth, both of which need to be overcome during the drug delivery process. The requirements for such a delivery system include small size, appropriate flexibility and biodegradability. DNA nanostructures, biomaterials that can arrange multiple functional components with nanometer precision, exhibit characteristics that are compatible with the requirements for inner ear drug delivery. Herein, we report the development of a novel inner ear drug delivery system based on epigallocatechin gallate (EGCG)-loaded tetrahedral DNA nanostructures (TDNs, EGCG@TDNs). The TDNs self-assembled via base-pairing of four single-stranded DNA constructs and EGCG was loaded into the TDNs through non-covalent interactions. Cy5-labeled TDNs (Cy5-TDNs) were significantly internalized by the House Ear Institute-Organ of Corti 1 cell line, and this endocytosis was energy-, clathrin-, and micropinocytosis-dependent. Cy5-TDNs penetrated the round window membrane (RWM) rapidly in vivo. Local application of EGCG@TDNs onto the RWM of guinea pigs in a single dose continuously released EGCG over 4 hours. Drug concentrations in the perilymph were significantly elevated compared with the administration of free EGCG at the same dose. EGCG@TDNs were found to have favorable biocompatibility and strongly affected the RSL3-induced down-regulation of GPX4 and the generation of reactive oxygen species, on the basis of 2',7'-dichlorodihydrofluorescein diacetate staining. JC-1 staining suggested that EGCG@TDNs successfully reversed the decrease in mitochondrial membrane potential induced by RSL-3 in vitro and rescued cells from apoptosis, as demonstrated by the analysis of Annexin V-FITC/PI staining. Further functional studies showed that a locally administered single-dose of EGCG@TDNs effectively preserved spiral ganglion cells in C57/BL6 mice after noise-induced hearing loss. Hearing loss at 5.6 and 8 kHz frequencies was significantly attenuated when compared with the control EGCG formulation. Histological analyses indicated that the administration of TDNs and EGCG@TDNs did not induce local inflammatory responses. These favorable histological and functional effects resulting from the delivery of EGCG by TDNs through a local intratympanic injection suggest potential for therapeutic benefit in clinical applications.

A novel label-free electrochemiluminescence aptasensor using a tetrahedral DNA nanostructure as a scaffold for ultrasensitive detection of organophosphorus pesticides in a luminol-H2O2 system.

In this work, a new type of Au-tetrahedral DNA nanostructure (Au-TDN) was originally proposed and successfully applied in an electrochemiluminescence aptasensor to detect organophosphorus pesticides (Ops). The aptamers modified with -SH could be covalently bonded with gold nanoparticles (AuNPs) to form a tetrahedron structure, and there were independent probes at each vertex of the tetrahedron, which could increase the probability of specific binding with Ops. The originally designed structure could not only maintain a stable tetrahedral configuration, but also combined with the target to improve the sensitivity of the sensor. Meanwhile, silver nanoparticles (AgNPs) could catalyze the chemical reaction between luminol and H2O2 to generate a variety of intermediates called reactive oxygen species (ROS) for signal enhancement. Factors that had important influences on the aptasensor, such as the concentration of Au-TDN, the incubation time, and the pH value of the buffer, were optimized in this trial. According to the final results, the limit of detection (LOD) of 3 pg mL-1 (S/N = 3) for methyl parathion, the LOD of 0.3 pg mL-1 (S/N = 3) for parathion and the LOD of 0.03 pg mL-1 (S/N = 3) for phoxim were obtained, respectively. Moreover, the novel tetrahedral structure could be replaced by different types of aptamers to expand its application range and lay a foundation for the development of portable rapid detection devices for pesticide residues.

DNA self-assembled Au nanoparticle clusters on silver nanorod arrays for high-sensitive and multiplex detection of cancer-related biomarkers.

To sensitively detect multiple and cross-species disease-related targets from a single biological sample in a quick and reliable manner is of high importance in accurately diagnosing and monitoring diseases. Herein, a surface-enhanced Raman scattering (SERS) sensor based on a functionalized multiple-armed tetrahedral DNA nanostructure (FMTDN) immobilized silver nanorod (AgNR) array substrate and Au nanoparticle (AuNP) SERS tags is constructed to achieve both multiplex detection and enhanced sensitivity using a sandwich strategy. The sensor can achieve single, dual, and triple biomarker detections of three lung cancer-related nucleic acid and protein biomarkers, i.e., miRNA-21, miRNA-486 and carcinoembryonic antigen (CEA) in human serum. The enhanced SERS signals in multiplex detections are due to the DNA self-assembled AuNP clusters on the silver nanorod array during the assay, and the experimentally obtained relative enhancement factor ratios, 150 for AuNP dimers and 840 for AuNP trimers, qualitatively agree with the numerically calculated local electric field enhancements. The proposed FMTDN-functionalized AgNR SERS sensor is capable of multiplex and cross-species detection of nucleic acid and protein biomarkers with improved sensitivity, which has great potential for the screening and clinical diagnosis of cancer in the early stage.

Targeting drug delivery and efficient lysosomal escape for chemo-photodynamic cancer therapy by a peptide/DNA nanocomplex.

A peptide/DNA nanocomplex was developed for the targeted delivery of chemotherapeutics and photosensitizers to cancer cells for efficient combination therapy. The chemotherapeutic drug doxorubicin (DOX) and the photosensitizer 5,10,15,20-tetra-(1-methylpyridine-4-yl)-porphyrin (TMPyP4) were physically incorporated by an aptamer (AS1411)-modified tetrahedral DNA nanostructure, where the tetrahedral DNA and aptamer-induced G-quadruplex provide binding sites of DOX and TMPyP4. The co-loaded 3A-TDN/DT displayed a targeted uptake by HeLa cancer cells through the high affinity and specificity between AS1411 and nucleolin, a protein overexpressed on many types of cancer cells. A polycationic polymer, mPEG-PAsp(TECH), was synthesized to complex with the DNA nanostructure to efficiently escape from lysosomes via the proton sponge effect upon the enhanced internalization by tumor cells. Under the irradiation of 660 nm laser light, TMPyP4 induced an upregulation of intracellular reactive oxygen species, which combined with DOX to fulfill the efficient inhibition of HeLa cells. Our study demonstrated a biocompatible peptide/DNA composite nanoplatform for combinational cancer therapy via the targeted delivery of therapeutic agents and efficient lysosomal escape.

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.

Electrochemically driven assembly of framework nucleic acids

• The utilization of an electrochemical chip to synthesis DNA nanostructures was firstly developed. • Tetrahedron DNA nanostructures of different sizes (20 bp, 26 bp and 37 bp) and octahedral DNA nanostructure were successfully assembled within 6 min. • The prepared DNA nanostructures exhibit the same structural characteristics and electrochemical performance in comparison to the ones fabricated with thermal annealing method. • The relations between electric potential and pH value were built. Electrochemically controlled synthesis of small molecules, DNA and various nanostructures provided unparalleled advantages to remotely control chemical reactions with high spatial and temporal precision. Although electrochemistry has been reported to actuate the reversible movement of DNA switch or tune the formation of hydrogen bonds in double strand DNA, electrochemically driven formation of DNA nanostructures remains a challenge as the result of their three-dimensional shapes and complex DNA folding pathway. Herein, through integrating a microfabricated chip with three-electrode system, we reported an electrochemical method to control the formation of tetrahedral DNA nanostructures. Using this method, the relation between the potential and solution pH value, and the melting temperature of double strand DNA was built, which facilitates synthesis of the tetrahedron DNA nanostructures in 6 min. Structure characterization of the prepared DNA nanostructures with atomic force microscopy (AFM) proved the physical integrity of the prepared DNA structures. By modifying the prepared DNA structure on the gold electrodes, a DNA nanostructure-based electrochemical biosensor with high sensitivity toward target DNA was fabricated. Based on these advantages, this method holds promise to develop high-throughput electrochemical microdevices capable of in-situ production of DNA nanostructures and the consequent multichannel biosensing.

Tetrahedral DNA nanostructures synergize with MnO2 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 Mn2+ 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 Mn2+, we constructed a TDN–MnO2 complex and found that it displayed a much higher efficacy than TDN plus Mn2+ 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 MnO2. These findings provide new therapeutic opportunities for cancer therapy.

Tetrahedral DNA nanostructure based biosensor for high-performance detection of circulating tumor DNA using all-carbon nanotube transistor

Adopting carbon nanotube (CNT) transistors as biosensors has been developed as a promising method for cancer biomarker detection, which has shown superior sensitivity and selectivity. However, the detection of circulating tumor DNA (ctDNA) by the CNT transistor based biosensors is still a challenge and no work has been reported. Here, direct label-free DNA detection of AKT2 gene related to triple-negative breast cancer by all-CNT thin-film transistor (TFT) biosensors incorporated with tetrahedral DNA nanostructures (TDNs) is proposed and achieved for the first time. The adoption of TDNs enables improved biosensor response for at least 35% and even as high as 98% as compared with single-stranded DNA (ssDNA) probes owing to the enhanced DNA hybridization efficiency. Influence of the TDNs’ linker length on the biosensor performance is important and has been investigated. Concentration-dependent DNA detection is achieved by the all-CNT TFT biosensors with a broad linear detection range of six orders of magnitude and a theoretical limit of detection (LOD) of 2 fM. In addition, the all-CNT TFT biosensors exhibit favorable selectivity and repeatability. The platform of all-CNT TFT biosensors incorporated with TDNs has great potential for multiplexed detection of various cancer biomarkers, providing a simple yet high performance universal strategy for low-cost clinical applications.

Rapid SARS-CoV-2 Nucleic Acid Testing and Pooled Assay by Tetrahedral DNA Nanostructure Transistor.

Direct SARS-CoV-2 nucleic acid testing with fast speed and high frequency is crucial for controlling the COVID-19 pandemic. Here, direct testing of SARS-CoV-2 nucleic acid is realized by field-effect transistors (FETs) with an electro-enrichable liquid gate (LG) anchored by tetrahedral DNA nanostructures (TDNs). The applied gate bias electrostatically preconcentrates nucleic acids, while the liquid gate with TDNs provides efficient analyte recognition and signal transduction. The average diagnosis time is ∼80 s, and the limit of detection approaches 1-2 copies in 100 μL of clinical samples without nucleic acid extraction and amplification. As such, TDN-LG FETs solve the dilemma of COVID-19 testing on mass scale that diagnosis accuracy and speed undergo trade-off. In addition, TDN-LG FETs achieve unamplified 10-in-1 pooled nucleic acid testing for the first time, and the results are consistent with PCR. Thus, this technology promises on-site and wide population COVID-19 screening and ensures safe world-reopening.