Tetrahedral DNA Nanostructures Research Articles

Tetrahedral DNA Nanoconjugates for Simultaneous Measurement of Telomerase Activity and miRNA.

In this study, a tetrahedral DNA nanostructure was first self-assembled; this was then conjugated with gold nanoparticles (AuNPs) and carbon nanodots (CDs). The fabricated nanocomposites allow simultaneous analysis of telomerase activity and miRNA with dual fluorescence channels. By further introducing an iRGD peptide sequence, the nanoconjuates can be conveniently transferred inside living cells for in situ imaging. The analytical performances and anti-jamming capabilities are excellent. Meanwhile, the materials are highly biocompatible for intracellular applications. Therefore, the proposed biosystem shows great promise as a powerful tool for quantitative analysis of the dual biomarkers. The strategy can also be further exploited as a versatile platform for in situ detection of many other targets for early disease diagnosis.

An electrochemical aptasensor based on tetrahedral DNA nanostructures as a signal probe carrier platform for sensitive detection of patulin

In this work, we proposed an electrochemical aptasensor for patulin (PAT) based on tetrahedral DNA nanostructures (TDNs) and thionine (Thi)-labeled Fe3O4 nanoparticles (Fe3O4NPs)/rGO signal amplification strategy. The rigid structure of TDNs could effectively improve the binding efficiency. Fe3O4NPs/rGO with excellent electrical conductivity and large specific surface area was used as a label material, which could load more Thi and accelerate electron transfer. Besides, the unique catalytic properties of Fe3O4NPs could achieve active signal amplification. Once PAT existed, PAT aptamer was released from the capture probe, thereby introducing Fe3O4NPs/rGO with Thi onto the electrode surface. Therefore, a noticeable increase in Thi current intensity was observed. Under the optimized conditions, the proposed aptasensor showed superior performance with a linear range from 5 × 10−8 to 5 × 10−1 μg mL−1 and a detection limit of 30.4 fg mL−1. The obtained sensor showed reliable specificity, stability and reproducibility, and was successfully applied to the determination of real samples.

Mono-dispersed nano-hydroxyapatite based MRI probe with tetrahedral DNA nanostructures modification for in vitro tumor cell imaging

Taking advantage of the superior biocompatibility, good stability in a wide pH and temperature range, as well as its strong affinity with DNA of hydroxyapatite (HAp), tetrahedral DNA nanostructures (TDNs) conjugated with AS1411 aptamer (anti-nucleolin overexpressed on tumor cell membranes) were employed as affinity ligands to construct a novel mono-dispersed HAp based probe with Gd3+ doping (Apt-TDNs-GdHAp) for MR imaging. The adsorption of TDNs on the nano-HAp surface facilely accomplished the construction of the Apt-TDNs-GdHAp probes. Meanwhile, the use of hydrophilic TDNs not only favored the phase-transfer from the oil phase to the aqueous phase, but also enhanced the mono-dispersion of this probe due to the well-ordered distribution of TDNs on the surface of nano-HAp. Moreover, Apt-TDNs-GdHAp probe with a better mono-dispersion and crystalinity achieved twice higher longitudinal relaxivity (r1 value) than that of GdHAp synthesized by microwave-assisted method (Microwave-GdHAp), exhibiting much more excellent T1-weighted imaging performance. With the introduction of TDNs, the stability and the tumor-targeting accessibility were also greatly improved, showing its great potential for further bio-applications.

Direct Analysis of Rare Circulating Tumor Cells in Whole Blood Based on Their Controlled Capture and Release on Electrode Surface.

The development of a simple, sensitive, and effective method for the analysis of circulating tumor cells (CTCs) is essential for cancer diagnosis and metastasis prediction. In this work, we have proposed an enzyme-free electrochemical method for specific capture, sensitive quantification, and efficient release of CTCs. To achieve this, the specific interaction between CTCs and the corresponding aptamer designed to be located in the identification probe (IP) will unfold the hairpin structure of IP. Consequently, IP will initiate a hybridization reaction to produce a duplex, which will further trigger the hybridization chain reaction (HCR) process to form a composite product of CTCs and double-stranded DNA polymers. Therefore, a significantly amplified signal readout can be obtained. Moreover, the composite product can be brought to the electrode surface by tetrahedral DNA nanostructures to achieve the purpose of capturing and quantifying CTCs. More significantly, these captured CTCs can be controlled released without compromising cell viability via a simple strand displacement reaction. Taking the breast cancer cell MCF-7 as a representative, the newly developed approach led to an ultralow detection limit of 3 cells mL-1, which is superior to several studies previously reported. The current method has also been demonstrated to analyze CTCs in human whole blood and hence revealed a great potential in the future.

Fe-MOFs as signal probes coupling with DNA tetrahedral nanostructures for construction of ratiometric electrochemical aptasensor

A ratiometric electrochemical aptasensor is proposed for the detection of thrombin. In the sensor, the iron metal-organic frameworks (Fe MOFs)-labeled aptamer as signal tags was used as signal probe (SP), and the electrolyte solution [Fe(CN)6]3-/4- was utilized as an inner reference probe (IR). In the presence of thrombin, the signal of Fe-MOFs can be detected. Meanwhile, the signal of [Fe(CN)6]3-/4-IR almost remains stable. Accordingly, thrombin concentration can be monitored with the ratio response of IFe-MOFs-SP/I[Fe(CN)6]3-/4--IR. The proposed ratiometric biosensor owns a strong ability to eliminate the disturbance that arises from different DNA loading densities, environmental impact and instrumental efficiency. DNA nanotetrahedron (NTH) with three-dimensional (3D) scaffold can effectively eliminate nonspecific adsorption of DNA and protein. The accessibility of target molecules and loading amounts of signal substances could be increased because of the enhanced mechanical rigidity of well-designed 3D NTH. Thus, detection reproducibility and sensitivity can be further improved. Moreover, the biosensor only requires conjugation with one electroactive substance. The modification procedure can be greatly simplified. The biosensor owns high sensitivity with the detection limit of 59.6 fM. We expect that it will emerge as a generalized ratiometric sensor that may be useful for detecting target analytes.

Extracellular vesicles engineered with valency-controlled DNA nanostructures deliver CRISPR/Cas9 system for gene therapy.

Extracellular vesicles (EVs) hold great promise for transporting CRISPR–Cas9 RNA-guided endonucleases (RNP) throughout the body. However, the cell-selective delivery of EVs is still a challenge. Here, we designed valency-controlled tetrahedral DNA nanostructures (TDNs) conjugated with DNA aptamer, and loaded the valency-controlled TDNs on EV surface via cholesterol anchoring for specific cell targeting. The targeting efficacy of different ratios of aptamer/cholesterol from 1:3 to 3:1 in TDNs on decorating EVs was investigated. TDNs with one aptamer and three cholesterol anchors (TDN1) efficiently facilitated the tumor-specific accumulation of the EVs in cultured HepG2 cells and human primary liver cancer-derived organoids, as well as xenograft tumor models. The intracellular delivery of RNP by TDN1-EVs successfully realized its subsequent genome editing, leading to the downregulation of GFP or WNT10B in specific cells. This system was ultimately applied to reduce the protein expression of WNT10B, which presented remarkable tumor growth inhibition in vitro, ex vivo and in vivo, and could be extended to other therapeutic targets. The present study provides a platform for the directional display of aptamer on surface labeling and the EVs-based Cas9 delivery, which provides a meaningful idea for future cell-selective gene editing.

Tetrahedral DNA nanostructures as drug delivery and bioimaging platforms in cancer therapy.

Structural DNA nanotechnology enables DNA to be used as nanomaterials for novel nanostructure construction with unprecedented functionalities. Artificial DNA nanostructures can be designed and generated with precisely controlled features, resulting in its utility in bionanotechnological and biomedical applications. A tetrahedral DNA nanostructure (TDN), the most popular DNA nanostructure, with high stability and simple synthesis procedure, is a promising candidate as nanocarriers in drug delivery and bioimaging platforms, particularly in precision medicine as well as diagnosis for cancer therapy. Recent evidence collectively indicated that TDN successfully enhanced cancer therapeutic efficiency both in vitro and in vivo. Here, we summarize the development of TDN and highlight various aspects of TDN applications in cancer therapy based on previous reports, including anticancer drug loading, photodynamic therapy, therapeutic oligonucleotides, bioimaging platforms, and other molecules and discuss a perspective in opportunities and challenges for future TDN‐based nanomedicine.

Design, fabrication and applications of tetrahedral DNA nanostructure-based multifunctional complexes in drug delivery and biomedical treatment.

Although organic nanomaterials and inorganic nanoparticles possess inherent flexibility, facilitating functional modification, increased intracellular uptake and controllable drug release, their underlying cytotoxicity and lack of specificity still cause safety concerns. Owing to their merits, which include natural biocompatibility, structural stability, unsurpassed programmability, ease of internalization and editable functionality, tetrahedral DNA nanostructures show promising potential as an alternative vehicle for drug delivery and biomedical treatment. Here, we describe the design, fabrication, purification, characterization and potential biomedical applications of a self-assembling tetrahedral DNA nanostructure (TDN)-based multifunctional delivery system. First, relying on Watson-Crick base pairing, four single DNA strands form a simple and typical pyramid structure via one hybridization step. Then, the protocol details four different modification approaches, including replacing a short sequence of a single DNA strand by an antisense peptide nucleic acid, appending an aptamer to the vertex, direct incubation with small-molecular-weight drugs such as paclitaxel and wogonin and coating with protective agents such as cationic polymers. These modified TDN-based complexes promote the intracellular uptake and biostability of the delivered molecules, and show promise in the fields of targeted therapy, antibacterial and anticancer treatment and tissue regeneration. The entire duration of assembly and characterization depends on the cargo type and modification method, which takes from 2 h to 3 d.

Nucleic acid based tetrahedral framework DNA nanostructures for fibrotic diseases therapy

As a type of novel biomaterial, DNA nanomaterial has been widely applied to biomedical area due to its self-assembly properties. Meanwhile, fibrotic diseases are common health problems, which can lead to multiple organs dysfunction and even death if without proper intervention and treatment. Therefore, we aim to develop the nucleic acids based tetrahedral framework DNA nanostructures (TFNAs) for the treatment of fibrotic diseases. Four single-strand DNA were self-assembled to form tetrahedral framework DNA nanostructures and the anti-fibrotic effects of TFNAs were investigated with an in vitro TGF-β1-induced fibrotic model. TFNAs were successfully assembled and showed obvious cellular uptake. After TFNAs treatment, ROS overproduction, fibrosis-related biomolecules expression including collagen I, fibronectin and α-SMA were obviously reduced. DNA-based TFNAs could be promising biomaterials for the treatment of fibrotic diseases.

Activity assay and intracellular imaging of APE1 assisted with tetrahedral DNA nanostructure modified-dnazyme and molecular beacon

Human apurinic/apyrimidinic endonuclease 1 (APE1) is an emerging target for sensitizing tumor cells to radiation and chemotherapy due to its critical function in DNA repair and reduction-oxidation regulation. Thus, accurate detection and intracellular imaging of APE1 hold great promise for drug screening, cancer diagnosis and prognosis evaluation. Here, a DNAzyme modified tetrahedral DNA nanostructure (DZ-TDN) and molecular beacon (MB) incorporated biosensing system was developed to reliably monitor intracellular APE1 activity. DZ-TDN was self-assembled from four single-stranded DNAs, one of which contained an extrude DNAzyme sequence on the 5′-end. At normal status, DNAzyme activity was plugged by performing a duplex with a blocker strand containing an AP site. The digestion of APE1 on this site can restore DNAzyme activity, which consequently trigger MB cleavage recycle to amplify detection signal. The results indicated that the proposed fluorescence method showed a linear response ranged for APE1 from 0.01 to 1 U mL−1 with a limit of detection (LOD) of 0.01 U mL−1 (S/N = 3). Moreover, the strategy was successfully used for effectors screening and intracellular imaging of APE1 with high reliability.

Treatment of Alzheimer's disease with framework nucleic acids.

ObjectivesTo provide a new research direction for nerve regeneration and strategy for Alzheimer's disease treatment, tetrahedral DNA nanostructures (TDNs)—novel tetrahedral framework nucleic acid molecule nanoparticles (tFNA) that can inhibit the apoptosis of nerve cells are employed in the experiment.Materials and methodsTo verify the successful preparation of TDNs, the morphology of TDNs was observed by atomic force microscopy (AFM) and transmission electron microscopy (TEM). The expression of apoptosis‐related genes and proteins was investigated by confocal microscope, flow cytometry, PCR and Western blot to detect the impact of TDNs on the Alzheimer's model. And finally, Morris water maze experiment was used to test behavioural changes and Nissl stain was detected to observe the morphology and quantity of neurons in the hippocampus. Immunofluorescence stain was used to observe the Aβ stain, and TUNEL dyeing was utilized to observe neuronal apoptosis.ResultsIn vitro and in vivo experiments confirm that TDNs, in a specific concentration range, have no toxic or side effects on nerve cells, can effectively inhibit apoptosis in an Alzheimer's disease cell model and effectively improve memory and learning ability in a rat model of Alzheimer's disease.ConclusionsThese findings suggest that TDNs may be a promising drug for the treatment of Alzheimer's disease.

An electrochemical aptasensor for ATP based on a configuration-switchable tetrahedral DNA nanostructure.

A novel electrochemical aptasensor for ATP was developed based on an aptamer-embedded configuration-switchable tetrahedral DNA nanostructure (TDN) and the formation of a G-quadruplex. This unique TDN was formed through the self-assembly of four specially designed single-stranded DNA (ssDNA) sequences (S1, S2, S3 and S4). The TDN was immobilized on the surface of a Au electrode through the thiol groups at the 5'-end of S1, S2 and S3. Five edges of the TDN were designed to form a double helix to preserve the structural robustness of the tetrahedron, while the ATP aptamer embedded sequence (S3) was designed to be located at the rest edge. The two terminals of S4 at the same edge were composed of two split G-quadruplex-forming sequences, which were non-complementary to the aptamer. This edge offered the configuration-switchable characteristic of the TDN. In the absence of ATP, the TDN remained in a relaxed state, and the G-quadruplex cannot form due to the large distance between the split G-quadruplex-forming sequences. However, in the presence of ATP, the aptamer combined with ATP and shortened the distance between the split sequences, resulting in the taut state of the TDN and the formation of a G-quadruplex at the edge. After the addition of hemin, the differential pulse voltammograms (DPVs) were used to quantify ATP. The sensor revealed a dynamic response range from 0.1 nM to 1 μM, with a detection limit of 50 pM. In addition, the specificity and practicability in real samples were also verified, indicating its potential applications.

A tetrahedral DNA nanostructure-decorated electrochemical platform for simple and ultrasensitive EGFR genotyping of plasma ctDNA.

Genotyping of the epidermal growth factor receptor (EGFR) mutation status is of great importance in the screening of appropriate patients with advanced non-small cell lung carcinoma (NSCLC) to receive superior tyrosine kinase inhibitor (TKIs) therapy. Yet conventional assays are generally costly with a relatively long turnaround time for obtaining results, which can lead to a bottleneck for immediately starting TKI therapy in late-staged patients. In this study, we propose an on-site electrochemical platform for sensitive simultaneous genotyping of the two major EGFR mutations (19del and L858R) through plasma ctDNA based on tetrahedral DNA nanostructure decorated screen-printed electrodes (SPE). Linear-after-the-exponential (LATE)-PCR combined with the amplification refractory mutation system (ARMS) was adopted to produce abundant biotin-labeled single-stranded DNA with high amplification efficiency and specificity. Disposable SPE decorated with self-assembled tetrahedral nanostructured DNA probes that showed ordered orientation and good target accessibility enabled the highly efficient hybridization of the specific amplicons through a sandwich-type and quantitatively translated the interfacial hybridization event into electrochemical signals via enzymatic amplification. Taking advantage of the ARMS-based LATE-PCR and the tetrahedral nanostructure-decorated SPE platform, we achieved the accurate detection of around 30 pg DNA of 19del or L858R, or as low as 0.1% of them in the presence of wild-type DNA. Moreover, the EGFR mutation profiles of 13 NSCLC patients we enlisted were accurately genotyped by our electrochemical platform, the results of which were in good agreement with those of commercial genetic detection methods.

A silver nanoparticle-assisted signal amplification electrochemiluminescence biosensor for highly sensitive detection of mucin 1.

A novel electrochemiluminescence (ECL) biosensor was developed in this study, which was based on the Ag-NP modified tetrahedral DNA nanostructure. First, a stable and rigid three-dimensional tetrahedral DNA nanostructure (TDN) was modified on a gold electrode, which was used as a capture element. The TDN improves the accuracy and sensitivity of this biosensor. In addition, silver nanoparticles (Ag NPs) with excellent electrical conductivity were introduced on the edges of TDNs to enhance the ECL intensity of the metal-organic framework (MOF) nanomaterial. Apart from imparting conductivity, the Ag NPs can also act as co-reactant accelerators to enhance the ECL intensity. A Eu3+ doped Zr-MOF material (Eu@MOF) was proposed and used as an ECL material for the first time. Terminal phosphate-modified MUC1 aptamer DNA bonded with the exposed Zr nodes on the surface of MOFs through Zr-O-P bonds to construct the DNA-Eu@MOF signal element. Based on the Ag NP-modified TDN capture element and the DNA-Eu@MOF signal element, a hypersensitive biosensor was established. Under the optimal conditions, this biosensor exhibited a wide detection range from 1.135 fg mL-1 to 0.1135 ng mL-1 and a low detection limit of 0.37 fg mL-1 (S/N = 3) toward the target MUC1. The biosensor also showed excellent stability and high selectivity for MUC1 detection.

Mitochondria-targeted tetrahedral DNA nanostructures for doxorubicin delivery and enhancement of apoptosis.

Mitochondria-targeted nanoparticles, such as liposomes, polymers and inorganic particles, suffer from heterogeneity, low biocompatibility and low drug loading efficiency. Here, we present a novel delivery platform based on tetrahedral DNA nanostructures (TDNs) that enable the mitochondrial transportation of the anticancer drug doxorubicin (DOX) for cancer therapy. In our design, DOX was intercalated into TDNs, which executed the cell-killing function inside the tumor cells. Various numbers of d-(KLAKLAK)2 (KLA) were conjugated to TDNs to achieve the mitochondria targeting effect. The mean size of the KLA-modified TDNs was about 15 nm, and the TDNs were stable in FBS. The DOX loading efficiency of the TDNs was up to around 77%. The 3KLA-modified TDNs exhibited the most efficient DOX accumulation in mitochondria, leading to an effective release of cytochrome c, and the upregulated expression levels of caspase-9, caspase-3, p21 and p53. Meanwhile, 3KLA-TDNs/DOX elevated the pro-apoptotic Bax, reduced the anti-apoptotic Bcl-2 protein expression and increased the Bax/Bcl-2 ratio, which finally activated the mitochondria-mediated, programmed apoptosis pathway to enhance the anticancer efficacy in vitro. This 3KLA-TDN and DOX co-assembling strategy can be further developed to transport other anthracyclines and chemotherapeutic agents for enhanced apoptosis effects.

A label-free homogeneous electrochemical cytosensor for the ultrasensitive detection of cancer cells based on multiaptamer-functionalized DNA tetrahedral nanostructures.

We developed a label-free homogeneous electrochemical cytosensor for ultrasensitive detection of cancer cells based on multiaptamer-functionalized DNA tetrahedral nanostructures, which avoided expensive labeling and sophisticated immobilization procedures, providing opportunities for precisely detecting cancer cells in clinical applications.

Simultaneous fluorescent detection of multiplexed miRNA of liver cancer based on DNA tetrahedron nanotags

The level of miRNA-21, miRNA-122, and miRNA-223 are always elevated when liver cancer is present at an early stage. In this paper, a novel assay to simultaneous detect miRNA-21, miRNA-122, and miRNA-223 was proposed based on DNA tetrahedron nanotags and fluorescence resonance energy transfer (FRET), which used a single laser stimulate wavelengh from one nucleic acid stain TOTO-1 to three diverse organic dyes (Cy3, Cy3.5, Cy5). In brief, a DNA tetrahedral nanostructure (DTN) was designed with three adaptor oligos on its vertices. TOTO-1, as a fluorescent donor, can imbed into native nucleic acid backbone of DTN. Three organic dye-functionalized strands (FRET oligos) are fluorescent receptors. In the presence of target miRNAs, they can be hybridized with FRET oligos and adaptor oligos on the vertices of DTN and the stable DNA tetrahedron nanotags are formed. As a result, the confinement of TOTO-1 is in close proximity to three fluorescence dyes, the FRET between TOTO-1 and three fluorescence dyes is generate efficiently in DNA tetrahedron nanotags. Point-of-care clinical applicability is demonstrated by sensitive multiplexed quantification of three miRNAs in 10% human serum samples.

A tetrahedral DNA nanoflare for fluorometric determination of nucleic acids and imaging of microRNA using toehold strands.

The authors describe a tetrahedral DNA nanostructure loaded with SYBR Green (SG-TDN) for fluorometric determination of nucleic acids. After intercalating into the TDN, fluorescence of SG is enhanced by 260-fold (exc 480nm, em 524nm), and the resulting SG-TDN nanoflare displays >7-fold stronger fluorescence than that of FAM-labeled TDN. The SG-TDNs were coupled to magnetic microparticles and polydopamine nanoparticles to construct multi-functional nanoprobes through sequence hybridization using a toehold strand. The method was applied to detect a stretch of microRNA sequence (20bp) in buffer and in undiluted serum with excellent selectivity, over awide linear range and with alow limit of detection (0.2nM). The probe was also applied for visualization of tumor-related microRNA in living cells via fluorescence imaging. Graphical abstract Schematic representation of tetrahedron-based DNA nanoflare for fluorometricnucleic acid determination in undiluted blood serum and living cells.

Size-Independent Transmembrane Transporting of Single Tetrahedral DNA Nanostructures.

DNA nanostructures have attracted considerable attention as drug delivery carriers. However, the transmembrane kinetics of DNA nanostructures remains less explored. Herein, the dynamic process of transporting single tetrahedral DNA nanostructures (TDNs) is monitored in real time using a force‐tracing technique based on atomic force microscopy. The results show that transporting single TDNs into living HeLa cells need ≈53 pN force and ≈25 ms duration with the average speed of ≈0.6 µm s−1. Interestingly, the dynamic parameters are irrelevant to the size of TDNs, while the larger TDNs rotated slightly during the transporting process. Meanwhile, both the results from single‐molecule force tracing and ensemble fluorescence imaging demonstrate that the different size TDNs transmembrane transporting depends on caveolin‐mediated endocytosis.

Ultrasensitive biosensor for microRNA-155 using synergistically catalytic nanoprobe coupled with improved cascade strand displacement reaction

MicroRNAs, essential for gene expression and physiological regulation, are considered to be reliable biomarkers for the early diagnosis and treatment of cancers. Herein, a sensitive biosensor that uses a synergistically catalytic nanoprobe and improved toehold strand displacement reaction (TSDR) has been fabricated, and successfully applied to microRNA-155 (miR-155) detection. A nanoscale copper-based metal organic framework assembled by Pt nanoparticles and horseradish peroxidase (Cu-NMOF@PtNPs/HRP) served as a co-catalytic nanoprobe and was coupled with improved TSDR to achieve multiple amplifications. In the absence of miR-155, the tetrahedral DNA nanostructures (TDNs) immobilized on the gold electrode were independent of the TSDR system because of the binding of the shielding region of the locked probe (LP) with the template probe (TP). Instead, the target would initiate the TSDR system, leading to the conformational change of TDNs and hybridization of the nanoprobe. Cu-NMOF@PtNPs/HRP exhibited extraordinary catalytic property towards the hydroquinone-hydrogen peroxide system, demonstrating that the nanoprobe exerted a concerted effect on the electrochemical performance of the biosensor. Under optimal conditions, the cathodic current exhibited a logarithmic relation over 0.50–1.0 × 105fM miR-155, with a detection limit of 0.13 fM, indicating that the constructed biosensor has considerable potential in the field of clinical disease diagnostics for miR-155.