DNA Tetrahedron Research Articles

Plasmon-Enhanced Electrochemiluminescence for Nucleic Acid Detection Based on Gold Nanodendrites.

Gold nanodendrites (Au NDs) exhibit extremely strong electromagnetic field located around multiple tip branches due to a plasmon coupling effect. In this work, a novel LSPR-enhanced ECL emission from CdTe nanocrystals (NCs) by Au NDs for the detection of nucleic acid is reported. This system is composed of a thin film of CdTe NCs on glassy carbon electrode (GCE) as anodic ECL emitter and Au NDs as plasmon enhancer. DNA tetrahedron embedded with a stem-loop hairpin structure on one edge was applied as a switch to regulate the distance between CdTe NCs and Au NDs. At original state, the hairpin structure was closed and DNA tetrahedron played in a relaxed state on CdTe NCs film. The ECL emission of CdTe NCs was quenched by proximal Au NDs due to Förster resonance energy transfer (FRET), which was defined as the "turn-off" mode. After the complementary hybridization with target DNA, the hairpin structure changed to a rodlike configuration, resulting in an increased distance between CdTe NCs and Au NDs, and a significant enhancement of ECL induced by LSPR of Au NDs, which was defined as a "turn-on" mode. Along with the asymmetric modification method, a controllable and versatile pathway for modifying nanomaterials, the ECL sensor performed well with great stability and repeatability for nucleic acid detection in the range from 1.0 to 500 fM. Considering the high sensitivity and selectivity in the serum sample assay, this proposed method indicates a great potential for bioassay application.

A dual-targeting DNA tetrahedron nanocarrier for breast cancer cell imaging and drug delivery

To enhance efficacy of chemotherapy and achieve real-time imaging of cancer cells, it is crucial to develop nanocarriers with targeted drug delivery capacity and fluorescence property for cancer theranostics. Herein, a dual-targeting DNA tetrahedron nanocarrier (MUC1-Td-AS1411) was constructed for breast cancer cell imaging and targeted drug delivery. This nanocarrier consisted of three components: (i) DNA tetrahedron core for multivalent conjugation of function ligands and loading doxorubicin (Dox); (ii) activatable MUC1 aptamer probe (MUC1-probe), formed by the hybridization of MUC1 aptamer sequence with fluorophore extended from one vertex and complementary sequence with quencher, for targeting and imaging MUC1 protein on cytomembrane; (iii) AS1411 aptamer, which was hybridized to the overhang on three vertexes via prolonged sequence, for binding to nucleolin. Firstly, MUC1-probe of this nanocarrier targeted MUC1 protein of MUC1-positive cells, causing a conformational reorganization of MUC1 aptamer, releasing complementary sequence with quencher and leading to fluorescence recovery. Subsequently, after internalizing into cells, AS1411 aptamer moiety of nanocarrier bound to nucleolin selectively, then the whole nanocarrier targeted nucleus and released Dox into nucleus. MUC1-positive cells and MUC1-negative cells could be differentiated by means of fluorescence imaging. Versus free Dox, Dox-loaded MUC1-Td-AS1411 showed lower cytotoxicity to MUC1-negative HL-7702 cells (P < 0.01), approximately equal lethality to sensitive MCF-7 cells (P > 0.05) whereas more effective to doxorubicin-resistant MCF-7 cells (P < 0.01). Therefore, this nanocarrier could be used as a promising candidate for cancer theranostics.

An electrochemical approach capable of prostate specific antigen assay in human serum based on exonuclease-aided target recycling amplification

In this report, an ultrasensitive electrochemical aptasensor for the detection of prostate specific antigen (PSA) in human serum is presented. DNA tetrahedron molecular layer is designed on the interface of a gold working electrode, which provides the recognition element for PSA capture. Next, exonuclease-aided target recycling amplification is performed, which determines the amount of attached silver nanoparticles (AgNPs) via silver-amino chemistry. By recording electrochemical responses from AgNPs, initial concentration of PSA can be easily evaluated. The limit of detection is pushed down to 0.11pgmL−1, which is attributed to the cyclic digestion effect of exonuclease and highly effective electrochemical reporters of AgNPs. The efficiency and robustness of this approach are further studied using human serum samples. The achieved results are satisfactory for future clinical applications.

Real-Time Study of the Interaction between G-Rich DNA Oligonucleotides and Lead Ion on DNA Tetrahedron-Functionalized Sensing Platform by Dual Polarization Interferometry.

G-quadruplex plays roles in numerous physiological and pathological processes of organisms. Due to the unique properties of G-quadruplex (e.g., forming G4/hemin complexes with catalytic activity and electron acceptability, binding with metal ions, proteins, fluorescent ligands, and so on), it has been widely applied in biosensing. But the formation process of G-quadruplex is not yet fully understood. Here, a DNA tetrahedron platform with higher reproducibility, regenerative ability, and time-saving building process was coupled with dual polarization interferometry technique for the real-time and label-free investigation of the specific interaction process of guanine-rich singled-stranded DNA (G-rich ssDNA) and Pb2+. The oriented immobilization of probes greatly decreased the spatial hindrance effect and improved the accessibility of the probes to the Pb2+ ions. Through real-time monitoring of the whole formation process of the G-quadruplex, we speculated that the probes on the tetrahedron platform initially stood on the sensing surface with a random coil conformation, then the G-rich ssDNA preliminarily formed unstable G-quartets by H-bonding and cation binding, subsequently forming a completely folded and stable quadruplex structure through relatively slow strand rearrangements. On the basis of these studies, we also developed a novel sensing platform for the specific and sensitive determination of Pb2+ and its chelating agent ethylenediaminetetraacetic acid. This study not only provides a proof-of-concept for conformational dynamics of G-quadruplex-related drugs and pathogenes, but also enriches the biosensor tools by combining nanomaterial with interfaces technique.

Collapse of DNA Tetrahedron Nanostructure for "Off-On" Fluorescence Detection of DNA Methyltransferase Activity.

As a potential detection technique, highly rigid and versatile functionality of DNA tetrahedron nanostructures is often used in biosensing systems. In this work, a novel multifunctional nanostructure has been developed as an "off-on" fluorescent probe for detection of target methyltransferase by integrating the elements of DNA tetrahedron, target recognition, and dual-labeled reporter. This sensing system is initially in an "OFF" state owing to the close proximity of fluorophores and quenchers. After the substrate is recognized by target methyltransferase, the DNA tetrahedron can be methylated to produce methylated DNA sites. These sites can be recognized and cut by the restriction endonuclease DpnI to bring about the collapse of the DNA tetrahedron, which leads to the separation of the dual-labeled reporters from the quenchers, and thus the recovery of fluorescence signal to produce an "ON" state. The proposed DNA tetrahedron-based sensing method can detect Dam methyltransferase in the range of 0.1-90 U mL-1 with a detection limit of 0.045 U mL-1 and shows good specificity and reproducibility for detection of Dam methyltransferase in a real sample. It has been successfully applied for screening various methylation inhibitors. Thus, this work possesses a promising prospect for detection of DNA methyltransfrase in the field of clinical diagnostics.

A highly sensitive gold nanoparticle-based electrochemical aptasensor for theophylline detection

Theophylline is a common bronchodilator for the treatment of diseases like asthma, bronchitis and emphysema. However, it should be strictly used and monitored due to its toxicity when the concentration is above certain levels. In this work, an electrochemical biosensor for theophylline detection is proposed by recognition of RNA aptamer and gold nanoparticle (AuNP)-based amplification technique. First, RNA aptamer is splitted into two single-stranded RNA probes. One is hybridized with DNA tetrahedron and the resulted nanostructure is then immobilized onto a gold electrode; the other is modified on the surface of AuNPs which is also labeled with methylene blue (MB) as electrochemical species. The recognition process between the two RNA probes and theophylline causes the localization of AuNPs and the enrichment of MB on the electrode interface. A significant electrochemical response is thus generated which is related to the concentration of initial theophylline. This proposed aptasensor shows excellent sensitivity and selectivity which could also be applied in quantitatively detection of theophylline in serums samples.

Fabrication of circular assemblies with DNA tetrahedrons: from static structures to a dynamic rotary motor.

DNA tetrahedron as the simplest 3D DNA nanostructure has been applied widely in biomedicine and biosensing. Herein, we design and fabricate a series of circular assemblies of DNA tetrahedron with high purity and decent yields. These circular nanostructures are confirmed by endonuclease digestion, gel electrophoresis and atomic force microscopy. Inspired by rotary protein motor, we demonstrate these circular architectures can serve as a stator for a rotary DNA motor to achieve the circular rotation. The DNA motor can rotate on the stators for several cycles, and the locomotion of the motor is monitored by the real-time fluorescent measurements.

Scallop-Inspired DNA Nanomachine: A Ratiometric Nanothermometer for Intracellular Temperature Sensing.

Accurate measurement of intracellular temperature is of great significance in biology and medicine. With use of DNA nanotechnology and inspiration by nature's examples of "protective and reversible responses" exoskeletons, a scallop-inspired DNA nanomachine (SDN) is desgined as a ratiometric nanothermometer for intracellular temperature sensing. The SDN is composed of a rigid DNA tetrahedron, where a thermal-sensitive molecular beacon (MB) is embedded in one edge of the DNA tetrahedron. Relying on the thermal-sensitive MB and fluorescence resonance energy transfer (FRET) signaling mechanism, the "On" to "Off" signal is reversibly responding to "below" and "over" the melting temperature. Mimicking the functional anatomy of a scallop, the SDN exhibits high cellular permeability and resistance to enzymatic degradation, good reversibility, and tunable response range. Furthermore, FRET ratiometric signal that allows the simultaneous recording of two emission intensities at different wavelengths can provide a feasible approach for precise detection, minimizing the effect of system fluctuations.

Hand-in-hand RNA nanowire-based aptasensor for the detection of theophylline

Theophylline is a popular drug for many respiratory diseases. However, certain toxic side effects may be developed and the narrow safety range raises the demand for sensitive methods to constantly monitor theophylline levels. This study presents an electrochemical approach towards theophylline detection based on the recognition by split RNA aptamers. Target induced construction of hand-in-hand RNA nanowire on the electrode surface could further absorb silver nanoparticles (Ag NPs) as electrochemical species. When theophylline is not present, RNA probes are stable and their conformations remain unchanged. In contrast, theophylline is able to trigger the hairpin opening of RNA probe and subsequent self-assembly of RNA nanowire, which could be captured by DNA tetrahedron on the electrode interface. After further decorating Ag NPs on the nanowire, silver stripping current is measured to reveal initial theophylline concentration. The developed sensing strategy shows excellent specificity and sensitivity with the limit of detection of 50nM. Its practical utility is demonstrated by quantitative determination of theophylline levels in complex biological samples.

An ultrasensitive electrochemical biosensor for the detection of mecA gene in methicillin-resistant Staphylococcus aureus

Electrochemical DNA biosensor has unique advantages for on-site pathogenic microorganism detection, yet the detection of long DNA towards genome DNA (gDNA) analysis remains challenge. In this work, we report a novel electrochemical biosensor for the ultrasensitive analysis of mecA DNA on methicillin-resistant Staphylococcus aureus (MRSA) genome, using a multi-signal probes (MSP) system. The MSP consists of 7 biotin-labelled signal probes that will combine to the target DNA in a prehybridization step, and then the complex will be captured by a DNA tetrahedron structure probe (TSP) on the electrode surface. Then, after the introduction of the streptavidin-labelled HRP enzyme, a catalysis current signal is detected that is found to be corresponding to the concentration of the target DNA. MSP in this work plays a critical role not only for the signal amplification through bringing 7 biotins, but also dramatically improves the accessibility of the target sequence embedded in the double-strand DNA molecules and complex second structures. The 3-D DNA TSP here provides steady support and optimized surface density for the very "large" complex of MSP system and gDNA, as a base of the capture probe. Finally, as low as 10fM synthetic target DNA was successfully detected, which is at least 3 magnitudes lower than that using single signal probe. Most importantly, we demonstrated the practicability of our analysis method by analyzing a 57fM MRSA gDNA sample showing excellent selectivity, and the reliability of the analysis was also demonstrated by digital PCR.

Simultaneous Imaging of Three Tumor-Related mRNAs in Living Cells with a DNA Tetrahedron-Based Multicolor Nanoprobe.

We constructed a DNA tetrahedron based multicolor nanoprobe, which could simultaneously imaging of three tumor-related mRNAs in living cells through fluorescence restoration caused by competitive chain replacement reaction. The oligonucleotides used to construct the tetrahedron were extended by adding three 21-base recognition sequences modified with different fluorophores (FAM, Cy3, and Cy5) in the 5' end. Three 11-base complementary sequences modified with quencher (BHQ1 for FAM and BHQ2 for Cy3 and Cy5) were hybridized with the recognition sequences to quench the fluorescence. In the presence of the specific mRNA targets, the recognition sequences hybridized with the targets to form longer duplexes and the fluorescence was restored. Compared with previously reported nanoprobes based on DNA tetrahedron, the multicolor nanoprobe can effectively avoid false positive results.

Clinically Relevant Detection of Streptococcus pneumoniae with DNA-Antibody Nanostructures.

Streptococcus pneumoniae (SP) is a pathogenic bacterium and a major cause of community-acquired pneumonia that could be fatal if left untreated. Therefore, rapid and sensitive detection of SP is crucial to enable targeted treatment during SP infections. In this study, DNA tetrahedron (DNA TH) with a hollow structure is anchored on gold electrodes to construct an electrochemical immunosensor for rapid detection of pneumococcal surface protein A (PspA) peptide and SP lysate from synthetic and actual human samples. This DNA nanostructure-based immunosensor displays excellent electrochemical activity toward PspA with a sensitive linear region from 0 to 8 ng/mL of PspA peptide and a low limit of detection (LOD) of 0.218 ng/mL. In addition, this DNA-TH-based immunosensor exhibits good sensing performance toward SP lysate in a clinically relevant linear range from 5 to 100 CFU/mL with a LOD of 0.093 CFU/mL. Along with these attractive features, this electrochemical immunosensor is able to specifically recognize and detect the PspA peptide mixed with other physiologically relevant components like bovine serum albumin (BSA) and lipopolysaccharide. In addition, our sensor could detect SP lysate even when dispersed in BSA or Escherichia coli lysate. Lastly, uncultured samples from the nasal cavity, mouth, and axilla of a human subject could be successfully determined by this well-designed electrochemical immunosensor.

A DNA Tetrahedron Nanoprobe with Controlled Distance of Dyes for Multiple Detection in Living Cells and in Vivo.

Multicomponent quantitative detection in living samples is becoming increasingly important; however, the current detection strategy may cause fluorescence self-quenching and reduce the sensitivity of detection. To solve the problem, we develop a DNA tetrahedral nanoprobe to control the dyes distance for simultaneous detection of multiple analytes. Compared to mesoporous silica nanoparticles based nanoprobes, the DNA tetrahedral nanoprobes display enhanced fluorescence intensities due to partially avoiding the fluorescence resonance energy transfer. Confocal fluorescence images show that the nanoprobes are capable of detecting and visualizing pH and O2•- in living cells under a single wavelength excitation. In an inflammation model for mice, the nanoprobes simultaneously image the down-regulation of pH and up-regulation of O2•-. We expect that the current strategy can provide new opportunities in designing probes for multiplexed detection with reduced self-quenching and enhanced sensitivity.

Exonuclease and Nicking Endonuclease‐Assisted Amplified Electrochemical Detection of Coralyne

AbstractBased on the catalysis of exonuclease I (Exo I) and nicking endonuclease (NEase), a novel electrochemical biosensor for the detection of coralyne is fabricated in this work. Specifically, DNA probe 1 can be protected from Exo I‐catalyzed digestion upon interacting with coralyne, the most representative homo‐adenine binding molecule. Subsequently, DNA probe 1 can further hybridize with a pendant sequence on top of the DNA tetrahedron, which is previously modified on the gold electrode surface. As the formed duplex contains the recognition site of Nb.BssSI, a type of NEase, the pendant sequence is thus cleaved, releasing the electrochemical probe on the 5’‐terminus from the electrode surface. DNA probe 1 is also released, which could then initiate the subsequent cycles of NEase‐catalyzed cleavage and amplify the declined electrochemical signals. We can determine coralyne in a linear range from 0.1 to 100 nM with the detection limit of 0.07 nM by using the proposed method. Experiments in real samples also demonstrate good results, showing excellent practical utility of this method. Moreover, it could be extended to assay other analytes by modifying certain DNA sequences.

SL2B aptamer and folic acid dual-targeting DNA nanostructures for synergic biological effect with chemotherapy to combat colorectal cancer.

DNA nanostructures prepared by self-assembly possess good stability, high biocompatibility, and low immunogenicity as drug delivery vehicles. In this work, DNA tetrahedron (TD) was constructed and modified with SL2B aptamer (S) and folic acid (F). TD possessed a small diameter (~6 nm) and entered into the nucleus quickly. SL2B aptamer can inhibit cancer cell growth by disturbing vascular endothelial growth factor/Notch signaling pathways. To explore the effect of SL2B number on colorectal cancer inhibition, SL2B multimers (dimer, trimer, and tetramer) were constructed by functionalization of TD with different numbers of SL2B. One SL2B per TD was the most efficient anticancer strategy and showed significantly better anticancer efficacy than SL2B, probably due to the enhanced stability of SL2B by TD. Doxorubicin (DOX) is a potent anticancer agent that can intercalate into DNA double strands. Results showed that TD could facilitate DOX entrance into the nucleus and the intracellular delivery of DOX was further enhanced by functionalization of SL2B and F. DOX-intercalated TD modified with two F and two S (DOX@TD-2F2S) could cause sufficient HT-29 cell inhibition at a much lower DOX concentration. In sum, DOX@TD-2F2S exhibited a synergic anticancer biological effect with chemotherapy and can be a promising strategy for treating colorectal cancer.

Fluorescence Resonance Energy Transfer-Based DNA Tetrahedron Nanotweezer for Highly Reliable Detection of Tumor-Related mRNA in Living Cells.

Accurate detection and imaging of tumor-related mRNA in living cells hold great promise for early cancer detection. However, currently, most probes designed to image intracellular mRNA confront intrinsic interferences arising from complex biological matrices and resulting in inevitable false-positive signals. To circumvent this problem, an intracellular DNA nanoprobe, termed DNA tetrahedron nanotweezer (DTNT), was developed to reliably image tumor-related mRNA in living cells based on the FRET (fluorescence resonance energy transfer) "off" to "on" signal readout mode. DTNT was self-assembled from four single-stranded DNAs. In the absence of target mRNA, the respectively labeled donor and acceptor fluorophores are separated, thus inducing low FRET efficiency. However, in the presence of target mRNA, DTNT alters its structure from the open to closed state, thus bringing the dual fluorophores into close proximity for high FRET efficiency. The DTNT exhibited high cellular permeability, fast response and excellent biocompatibility. Moreover, intracellular imaging experiments showed that DTNT could effectively distinguish cancer cells from normal cells and, moreover, distinguish among changes of mRNA expression levels in living cells. The DTNT nanoprobe also exhibits minimal effect of probe concentration, distribution and laser power as other ratiometric probe. More importantly, as a result of the FRET "off" to "on" signal readout mode, the DTNT nanoprobe almost entirely avoids false-positive signals due to intrinsic interferences, such as nuclease digestion, protein binding and thermodynamic fluctuations in complex biological matrices. This design blueprint can be applied to the development of powerful DNA nanomachines for biomedical research and clinical early diagnosis.

Tetrahedral DNA Nanoparticle Vector for Intracellular Delivery of Targeted Peptide Nucleic Acid Antisense Agents to Restore Antibiotic Sensitivity in Cefotaxime-Resistant Escherichia coli.

The bacterial cell wall presents a barrier to the uptake of unmodified synthetic antisense oligonucleotides, such as peptide nucleic acids, and so is one of the greatest obstacles to the development of their use as therapeutic anti-bacterial agents. Cell-penetrating peptides have been covalently attached to antisense agents, to facilitate penetration of the bacterial cell wall and deliver their cargo into the cytoplasm. Although they are an effective vector for antisense oligonucleotides, they are not specific for bacterial cells and can exhibit growth inhibitory properties at higher doses. Using a bacterial cell growth assay in the presence of cefotaxime (CTX 16 mg/L), we have developed and evaluated a self-assembling non-toxic DNA tetrahedron nanoparticle vector incorporating a targeted anti-blaCTX-M-group 1 antisense peptide nucleic acid (PNA4) in its structure for penetration of the bacterial cell wall. A dose-dependent CTX potentiating effect was observed when PNA4 (0-40 μM) was incorporated into the structure of a DNA tetrahedron vector. The minimum inhibitory concentration (to CTX) of an Escherichia coli field isolate harboring a plasmid carrying blaCTX-M-3 was reduced from 35 to 16 mg/L in the presence of PNA4 carried by the DNA tetrahedron vector (40 μM), contrasting with no reduction in MIC in the presence of PNA4 alone. No growth inhibitory effects of the DNA tetrahedron vector alone were observed.

DNA tetrahedron nanostructures for biological applications: biosensors and drug delivery.

With the rapid development of DNA nanotechnology, various DNA nanostructures with different shapes and sizes have been self-assembled using "bottom-up" fabrication strategies and applied to a wide range of fields such as biosensors, drug delivery and tools for molecular biology. As a classical and simple polyhedron, DNA tetrahedron can be easily synthesised by a one-step assembly. Due to the excellent biocompatibility and cellular permeability, it provides a universal and promising platform to construct a series of biosensors and drug delivery systems for living cells studies. Moreover, the high programmability of DNA tetrahedron determines its capability to perform artful design and combine with other materials. Herein, we review and summarise the development and applications of DNA tetrahedron in living cell studies. We mainly focus on two parts, cellular biosensors for the detection of nucleic acids, proteins, small molecules and cancer cells and drug delivery systems for chemotherapy, immunotherapy, photodynamic therapy and gene silencing. With the rapid progress in DNA tetrahedron as well as DNA nanotechnology, new avenues and opportunities have opened up in analytical chemistry, molecular biology and medicine.

A Duo-toehold-mediated displacement amplification on DNA tetrahedron for RNA detection of dengue virus

A Duo-toehold-mediated displacement amplification on DNA tetrahedron for RNA detection of dengue virus

DNA-affibody nanoparticles for inhibiting breast cancer cells overexpressing HER2.

In this study, we have prepared a DNA-affibody nanoparticle which mimics a antibody in its ability to specifically target the HER2 receptor. This nanoparticle has a smaller size (95 kDa) than the monoclonal antibody, trastuzumab (150 kDa) and at least two-fold greater activity toward BT474 cells than trastuzumab. The DNA in this nanoparticle structure has two functions, namely as a support to anchor two affibody molecules and as a vehicle to non-covalently bind multiple copies of a small molecule drug for drug delivery. Each DNA-affibody nanoparticle can bind ∼53 molecules of doxorubicin (DOX) to form a complex, which exhibits greater selectivity toward and inhibition of breast cancer cells overexpressing HER2 than doxorubicin does. As expected, the nanoparticle exhibits lesser inhibition of cells expressing HER2 at a low level. Thus, the nanoparticle represents a highly efficacious agent for inhibiting cancer cells which overexpress HER2, but with low toxicity toward normal cells.