DNA Tetrahedron Research Articles

DNA-Tetrahedral-Nanostructure-Based Entropy-Driven Amplifier for High-Performance Photoelectrochemical Biosensing.

In virtue of the inherent molecular recognition and programmability, DNA has recently become the most promising for high-performance biosensors. The rationally engineered nucleic acid architecture will be very advantageous to hybridization efficiency, specificity, and sensitivity. Herein, a robust and split-mode photoelectrochemical (PEC) biosensor for miRNA-196a was developed based on an entropy-driven tetrahedral DNA (EDTD) amplifier coupled with superparamagnetic nanostructures. The DNA tetrahedron structure features in rigidity and structural stability that contribute to obtain precise identification units and specific orientations, improving the hybridization efficiency, sensitivity, and selectivity of the as-designed PEC biosensor. Further, superparamagnetic Fe3O4@SiO2@CdS particles integrated with DNA nanostructures are beneficial for the construction of a split-mode, highly selective, and reliable PEC biosensor. Particularly, the enzyme- and hairpin-free EDTD amplifier eliminates unnecessary interference from the complex secondary structure of pseudoknots or kissing loops in typical hairpin DNAs, significantly lowers the background noise, and improves the detection sensitivity. This PEC biosensor is capable of monitoring miRNA-196a in practical settings with additional advantages of efficient electrode fabrication, stability, and reproducibility. This strategy can be extended to various miRNA assays in complex biological systems with excellent performance.

Ratiometric and amplified fluorescence nanosensor based on a DNA tetrahedron for miRNA imaging in living cells.

Enzyme-free signal amplification approaches have attracted considerable attention in the field of intracellular miRNA analysis. However, the application of nucleic acid amplification has been limited by intracellular delivery of multiple oligonucleotide components with precise stoichiometry. In this work, we propose a new DNA tetrahedron (DTN)-based sensing platform addressing the delivery and stoichiometric control of nucleic components for enzyme-free amplification. The nanosensor is composed of two DTN probes; DTN-F served as the target recognition and signal output unit, and DTN-H served as the signal amplification unit. DTNs could facilitate the cell internalization of the nucleic acid probes and protect them from nuclease degradation. In the absence of target miRNA, the fluorescent strands (F) hybridize with the hanging sequences of DTN, and FAM and TAMRA labeled on F will be separated, blocking fluorescence resonance energy transfer (FRET). In the presence of the target miRNA, F will be displaced by the target and the hairpin structure will be restored, bringing the FRET pair into close proximity and inducing a FRET signal. Moreover, the helper strands (H) on DTN-H could liberate target miRNA through strand displacement, which will initiate a new round of reaction, generating an amplified FRET signal. The DTN nanosensor realized sensitive and selective detection of let-7a in buffer solution and 10% FBS solution. In addition, imaging of miRNA in the different cell lines and monitoring of intracellular miRNA fluctuations were carried out The developed method offers a new tool for bioanalytical and biomedical research.

A multicolor DNA tetrahedron nanoprobe for analyzing human telomerase in living cells.

Herein, we report the in situ analysis of human telomerase by a multicolor DNA tetrahedron nanoprobe. The elongated telomeric repeats can hybridize with settled molecular beacons in order, accompanied by sequentially lighted up fluorescence. Imaging telomerase activity, real-time monitoring telomerase action and determining product length distribution in living cells are realized. It detects multiple information of intracellular telomerase and provides deeper insights into the function of telomerase.

Accelerated DNA tetrahedron-based molecular beacon for efficient microRNA imaging in living cells

Herein, we develop an accelerated DNA tetrahedron based molecular beacon for efficient detection and imaging of miRNA in living cells. Our study provides an improved strategy for fluorescence amplified RNA imaging in early clinical diagnosis.

Aptamer-modified DNA tetrahedra-gated metal-organic framework nanoparticle carriers for enhanced chemotherapy or photodynamic therapy.

UiO-66 metal–organic framework nanoparticles (NMOFs) gated by aptamer-functionalized DNA tetrahedra provide superior biomarker-responsive hybrid nano-carriers for biomedical applications. Hybrid nano-carriers consisting of ATP-aptamer or VEGF-aptamer functionalized tetrahedra-gated NMOFs are loaded with the chemotherapeutic drug, doxorubicin (DOX). In the presence of ATP or VEGF, both abundant in cancer cells, the tetrahedra-gated NMOFs are unlocked to release the drug. Enhanced and selective permeation of the DOX-loaded ATP/VEGF-responsive tetrahedra-gated NMOFs into MDA-MB-231 breast cancer cells as compared to the reference ATP/VEGF-responsive duplex-gated NMOFs or non-malignant MCF-10A epithelial breast cells is observed. This results in enhanced and selective cytotoxicity of the tetrahedra-gated DOX-loaded NMOFs toward the malignant cells. Additional nano-carriers, consisting of photosensitizer Zn(ii) protoporphyrin IX (Zn(ii)-PPIX)-loaded VEGF-responsive tetrahedra-gated NMOFs, are introduced. The VEGF-triggered unlocking of the NMOFs yields separated G-quadruplex-VEGF aptamer complexes conjugated to the tetrahedra, resulting in the release of loaded Zn(ii)-PPIX. Association of the released Zn(ii)-PPIX to the G-quadruplex structures generates highly fluorescent supramolecular Zn(ii)-PPIX/G-quadruplex VEGF aptamer-tetrahedra structures. The efficient and selective generation of the highly fluorescent Zn(ii)-PPIX/G-quadruplex VEGF aptamer-tetrahedra nanostructures in malignant cells allows the light-induced photosensitized generation of reactive oxygen species (ROS), leading to high-efficacy PDT treatment of the malignant cells.

Reversible assembly/disassembly of DNA frames and applications in logic design, ratiometric sensing and bioimaging

Molecular computing holds great promise for intelligent diagnosis and treatment of diseases at molecular level, thus designing molecular logic gates to operate programmably and autonomously for molecular analysis is urgently needed. In this work, controllable assembly/disassembly of DNA tetrahedron, a typical DNA frame structure, was achieved by alternate addition of two stimulating factors including nucleic acid and antisense nucleic acid, Hg2+ and cysteine, or H+ and OH−. Such a DNA frame structure assembly/disassembly behavior had been proved to be reversible and could be cycled for several times, which was clearly reflected by repeated switch-on and switch-off of the fluorescence resonance energy transfer signal. Based on this principle, three binary INHIBIT logic gates were constructed by using different input combinations. Such a principle was also successfully used to develop various sensing systems for the ratiometric detection of microRNA, Hg2+, cysteine and pH. Thanks to the highly efficient internalization of DNA nanostructures by cells, the proposed sensing systems were demonstrated to work well for the ratiometric imaging of microRNA, Hg2+ and pH value in living cells.

Carbon Nanomaze for Biomolecular Detection with Zeptomolar Sensitivity

AbstractDespite numerous efforts, the accurate determination of trace biomolecules with zeptomolar sensitivity remains elusive. Here, a 3D carbon nanomaze (CAM) electrode for the ultrasensitive detection of trace biomolecules such as nucleic acids, proteins, and extracellular vesicles is reported. The CAM electrode consists of an interlaced carbon fiber array on which intercrossed graphene sheets are vertically tethered in situ, permitting local confinement of trace molecules to increase molecular hybridization efficiency. Furthermore, a self‐assembled DNA tetrahedron array adopts a rigid spatial conformation to guarantee the controllable arrangement of immobilized biological probes, facilitating analytical sensitivity and reproducibility. In a proof‐of‐concept experiment on detecting microRNA‐155, a linearity of 0.1 aM to 100 nM and a sensitivity of 0.023 aM (23 zM) are achieved. With the optimal parameters, the proposed nanoelectrode demonstrates encouraging consistency with quantitative real‐time polymerase chain reaction during clinical sample detection. Through simple functionalization by appending various biomolecular probes of interest, the developed CAM platform with ultrahigh sensitivity can be exploited as a versatile tool in environmental, chemistry, biology, and healthcare fields.

A Carbon-Based DNA Framework Nano-Bio Interface for Biosensing with High Sensitivity and a High Signal-to-Noise Ratio.

Biosensing interface based on screen-printed carbon electrodes (SPCE) has been widely used for electrochemical biosensors in the field of medical diagnostics, food safety, and environmental monitoring. Nevertheless, SPCE always has a rough surface, which is easy to result in the disorder of nucleic acid capture probes, the nonspecific adsorption of signaling probes, the steric hindrance of target binding, and decrease in the signal-to-noise ratio and sensitivity of biosensors. So far, it still remains extremely challenging to develop high-efficiency carbon-based biosensing interfaces, especially for DNA probe-based assembly and functionalization. In this paper, we first used a specific DNA framework, DNA tetrahedron to solve the defects of the carbon interface, improving the biosensing ability of SPCE. With covalent coupling, the DNA tetrahedron could be immobilized on the carbon surface. Biosensing probe sequences extending from the DNA tetrahedron can be changed for different target molecules. We demonstrated that the improved SPCE could be applied for the detection of a variety of bioactive molecules. Typically, we designed gap hybridization, aptamer "sandwich" and aptamer competition reduction strategy for the detection of miRNA-141, thrombin, and ATP, respectively. High signal-to-noise ratio, sensitivity, and specificity were obtained for all of these kinds. Especially, the DNA tetrahedron-modified SPCE can work well with serum samples. The carbon-based DNA framework nano-bio interface would expand the use of SPCE and make electrochemical biosensors more available and valuable in clinical diagnosis.

Frontispiece: O6‐Alkylguanine DNA Alkyltransferase Mediated Disassembly of a DNA Tetrahedron

DNA nanostructures show promise for drug delivery with the ability to enter cells and can release cargo in response to stimuli. In this work, the authors show that a DNA tetrahedron can self-assemble containing an alkylene cross-link connecting the O6-atoms of adjacent 2′-deoxyguanosines at the vertices’ three-way junctions. These cross-links are unhooked by the human DNA repair protein O6-alkylguanine DNA alkyltransferase leading to tetrahedron disassembly. For more details see the Communication by C. J. Wilds et al. on page 14802 ff.

Kidney-Targeted Cytosolic Delivery of siRNA Using a Small-Sized Mirror DNA Tetrahedron for Enhanced Potency.

A proper intracellular delivery method with target tissue specificity is critical to utilize the full potential of therapeutic molecules including siRNAs while minimizing their side effects. Herein, we prepare four small-sized DNA tetrahedrons (sTds) by self-assembly of different sugar backbone-modified oligonucleotides and screened them to develop a platform for kidney-targeted cytosolic delivery of siRNA. An in vivo biodistribution study revealed the kidney-specific accumulation of mirror DNA tetrahedron (L-sTd). Low opsonization of L-sTd in serum appeared to avoid liver clearance and keep its size small enough to be filtered through the glomerular basement membrane (GBM). After GBM filtration, L-sTd could be delivered into tubular cells by endocytosis. The kidney preference and the tubular cell uptake property of the mirror DNA nanostructure could be successfully harnessed for kidney-targeted intracellular delivery of p53 siRNA to treat acute kidney injury (AKI) in mice. Therefore, L-sTd could be a promising platform for kidney-targeted cytosolic delivery of siRNA to treat renal diseases.

Zippering DNA Tetrahedral Hyperlink for Ultrasensitive Electrochemical MicroRNA Detection.

Pluripotency of a DNA tetrahedron (DNATT) has made the iconic framework a compelling keystone in biosensors and biodevices. Herein, distinct from the well-tapped applications in substrate fabrication, we focus on exploring their tracing and signaling potentials. A homologous family of four isostructural DNATT, i.e., DNATTα/β/γ/δ, was engineered to form a sensor circuitry, in which a target-specific monolayer of thiolated DNATTγ pinned down the analyte jointly with the reciprocal DNATTδ into a sandwich complex; the latter further rallied an in situ interdigital relay of biotinylated DNATTα/β into a microsized hyperlink dubbed polyDNATT. Its scale and growth factors were illuminated rudimentarily in transmission electron microscopy and confocal laser scanning microscopy. Using a nonsmall-cell lung cancer-related microRNA (hsa-miR-193a-3p) as the subject, a compound DNA-backboned construct was synthesized, fusing all building blocks together. Its superb tacticity and stereochemical conformality avail the templating of a horseradish peroxidase train, which boosted the paralleled catalytic surge of proton donors, resulting in an attomolar detection limit and a broad calibration range of more than seven orders of magnitude. Such oligomerization bested the conventional hybridization chain reaction laddering at both biomechanical stability and stoichiometric congruency. More significantly, it demonstrates the flexibility of DNA architectures and their multitasking ability in biosensing.

O6 -Alkylguanine DNA Alkyltransferase Mediated Disassembly of a DNA Tetrahedron.

Tetrahedron DNA structures were formed by the assembly of three-way junction (TWJ) oligonucleotides containing O6 -2'-deoxyguanosine-alkylene-O6 -2'-deoxyguanosine (butylene and heptylene linked) intrastrand cross-links (IaCLs) lacking a phosphodiester group between the 2'-deoxyribose residues. The DNA tetrahedra containing TWJs were shown to undergo an unhooking reaction by the human DNA repair protein O6 -alkylguanine DNA alkyltransferase (hAGT) resulting in structure disassembly. The unhooking reaction of hAGT towards the DNA tetrahedra was observed to be moderate to virtually complete depending on the protein equivalents. DNA tetrahedron structures have been explored as drug delivery platforms that release their payload in response to triggers, such as light, chemical agents or hybridization of release strands. The dismantling of DNA tetrahedron structures by a DNA repair protein contributes to the armamentarium of approaches for drug release employing DNA nanostructures.

Inducing Endoplasmic Reticulum Stress to Expose Immunogens: A DNA Tetrahedron Nanoregulator for Enhanced Immunotherapy

AbstractImmunogenic cell death (ICD) is an important modulation type for stimulating anticancer immune responses and amplifying immunotherapy efficacy. When ICD occurs, endoplasmic reticulum (ER) stress plays a vital role for exposing immunogens. Herein, a functionalized DNA tetrahedron nanoregulator to specifically trigger ER stress for enhancing cancer immunotherapy is designed. The nanoregulator can target ER organelles by binding to the sulfonamide receptor of cancer cells. Then glucose depletion and reactive oxygen species generation cause a strong ER stress response to induce ICD to expose tumor immunogens. Thereafter, the dendritic cell (DC) maturation is promoted, and T cell proliferation and infiltration are stimulated to advance cancer immunotherapy. Combined with immune checkpoint inhibitor (α‐PD‐1), the ER stress triggered nanoregulator exhibits significant suppression for breast cancer and melanoma.

Loop-armed DNA tetrahedron nanoparticles for delivering antisense oligos into bacteria

BackgroundAntisense oligonucleotides (ASOs) based technology is considered a potential strategy against antibiotic-resistant bacteria; however, a major obstacle to the application of ASOs is how to deliver them into bacteria effectively. DNA tetrahedra (Td) is an emerging carrier for delivering ASOs into eukaryotes, but there is limited information about Td used for bacteria. In this research, we investigated the uptake features of Td and the impact of linkage modes between ASOs and Td on gene-inhibition efficiency in bacteria.ResultsTd was more likely to adhere to bacterial membranes, with moderate ability to penetrate into the bacteria. Strikingly, Td could penetrate into bacteria more effectively with the help of Lipofectamine 2000 (LP2000) at a 0.125 μL/μg ratio to Td, but the same concentration of LP2000 had no apparent effect on linear DNA. Furthermore, linkage modes between ASOs and Td influenced gene-knockdown efficiency. Looped structure of ASOs linked to one side of the Td exhibited better gene-knockdown efficiency than the overhung structure.ConclusionsThis study established an effective antisense delivery system based on loop-armed Td, which opens opportunities for developing antisense antibiotics.

Grafting homogenous electrochemical biosensing strategy based on reverse proximity ligation and Exo III assisted target circulation for multiplexed communicable disease DNA assay

Rapid and effective diagnosis of communicable disease is one of the critical issues of the modern society, especially for detecting different targets at the same time. In this work, a grafting homogenous electrochemical biosensing strategy is proposed by integrating of reverse proximity ligation and exonuclease III (Exo III) assisted target circulation to analyze hepatitis B (HBV) and human immunodeficiency (HIV). Specially, a two-wing nanodevice (TWD) with two detection paths is elaborately designed based on analogous proximity ligation assay. The reverse proximity ligation process provides a new way of signal conversion and amplification, what accomplished by demolishing the TWD in the presence of targets. Meanwhile, a vast number of signal probes are released via Exo III assisted target circulation. Then the signal probes are grafted on the universal sensing interface, which is decorated with graftable tetrahedron DNA (GTD). These lead to a highly amplified electrochemical signal. Compared with the conventional strategies, the grafting homogenous electrochemical biosensing strategy not only achieves convenient sensitive detection of multiple communicable diseases DNA simultaneously, but also performs well in the detection of sole target. This strategy effectively decreases the background, homogenizes the distribution of probes, and avoids the complex and time-consuming modification process of the working electrode, which holds great potential application in early diagnosis for communicable disease in the future.

DNA Hydrogelation-Enhanced Imaging Ellipsometry for Sensing Exosomal microRNAs with a Tunable Detection Range.

Conventional imaging ellipsometry-based biosensing faces the challenges of poor sensitivity and narrow dynamic range, especially for some small molecules such as microRNA. Given that detection of various exosomal miRNAs with tunable range could provide high-precision disease information and improve the accuracy of diagnosis, a sensitive imaging ellipsometry sensor was introduced to improve sensitivity with a tunable detection range by terminus-regulated DNA hydrogelation. Tetrahedron DNA probes with complementary sequence to the target miRNA were used as biorecognition elements to form DNA hydrogelation. This DNA hydrogelation was formed by template-independent and isothermal amplification on the Au film. Due to its high dielectric constant, DNA hydrogelation structure could be used for improving the sensitivity of imaging ellipsometry significantly. Importantly, by changing the cycle of the DNA hydrogelation amplification, this strategy showed a tunable detection range from fM to nM for miRNA with a limit of detection of 0.2 fM for let-7a, 10 fM for miR-375, and 40 pM for miR-21. Furthermore, it also performed satisfactorily for the miRNA sensing in 50% human serum and 50% human plasma. This DNA hydrogelation-enhanced imaging ellipsometry could broaden the applications of conventional imaging ellipsometry in biosensing and provide a sensitive method for sensing miRNAs at different abundances.

A Nongenetic Proximity-Induced FRET Strategy Based on DNA Tetrahedron for Visualizing the Receptor Dimerization.

Protein dimerization or oligomerization is a key factor in signal transduction processes. It is of great significance to develop novel strategies for investigating protein dimerization in cells and in vivo. Herein, we report a nongenetic proximity-induced fluorescence resonance energy transfer (FRET) strategy based on DNA tetrahedron for visualizing the receptor dimerization. In this nanostrategy, the aptamer serves as the recognition sequence for specifically binding to the receptor monomer. As long as the receptor dimers exist, the proximity switch probes hybridize with each other to induce the FRET between Cy3 and Cy5. Good stability of the DNA tetrahedron in this approach ensures the application value in multiple biological samples. This work provides a new way for developing novel protein dimerization visualization nanostrategies.

Target-triggered configuration change of DNA tetrahedron for SERS assay of microRNA 122.

A surface-enhanced Raman scattering (SERS) method is proposed for the assay of microRNA 122 based on configuration change of DNA tetrahedron. Firstly, a DNA tetrahedron was self-assembled with one vertex labeled with toluidine blue (TB). Then, it was immobilized on the porous Ni/SiO2@PEI@Au as a SERS platform, which was characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). At this time, the DNA tetrahedron was contracted; so, the TB is close to AuNPs and the Raman signal is high. When target microRNA 122 existed, with the nicking enzyme amplification strategy, a great deal of DNA signal chains (S5) was obtained, which can extend the contracted DNA tetrahedron and change it into a three-dimensional DNA tetrahedron. In this case, the TB was far from AuNPs, resulting in a lower Raman signal. Due to the configuration change of DNA tetrahedron, the Raman signal at 1624cm-1 (with the excitation wavelength of 633nm) has a linear relationship with the logarithm concentration of microRNA 122. This SERS assay has high sensitivity for microRNA 122 with a determination range from 0.01aM to 10fM and a detection limit of 0.009aM. The recoveries from spiked samples were in the range 95 to 109%. This SERS strategy is designed based on the target-triggered configuration change of DNA tetrahedron, which can give new insight for DNA structures in bioanalysis. Graphical abstract A sensitive surface-enhanced Raman scattering (SERS) biosensor was developed to detect microRNA 122 using the configuration change of DNA tetrahedron to indirectly control the position of TB and hot spot.

Multifunctional Double-Bundle DNA Tetrahedron for Efficient Regulation of Gene Expression.

DNA nanostructures have garnered considerable interest as research tools in the field of cell biology and pathology. Herein, we develop an addressable double-bundle DNA tetrahedron with distinct modification sites to load multiple functional components for efficient regulation of gene expression. In our tailored nanoplatform, nucleic acid drugs (antisense for gene therapy) and protein drugs (KillerRed for photodynamic therapy) are precisely organized in the chemically well-defined DNA tetrahedron. With the attachment of active targeting groups, this functional DNA nanocarrier can efficiently penetrate into the cell membrane and subsequently transport drugs to the target subcellular organelles (mitochondrion and nucleus) for inducing synergistic cell behavior regulation to start the endogenous apoptotic process. This tailored DNA nanocarrier provides unprecedented opportunities for intelligent drug delivery and cell biology research.

Sensitive electrochemical detection of aflatoxin B1 using DNA tetrahedron-nanostructure as substrate of antibody ordered assembly and template of aniline polymerization.

A novel strategy for AFB1 detection in grains was proposed based on DNA tetrahedron-structured probe (DTP) and horseradish peroxidase (HRP) triggered polyaniline (PANI) deposition. Briefly, the DNA tetrahedron nanostructures were assembled on the gold electrode, with carboxylic group designed on top vertex of them. The carboxylic group was conjugated with the AFB1 monoclonal antibody (mAb) to form DTP. The test sample and a known fixed concentration of HRP-labeled AFB1 were mixed and they compete for binding to DTP. The HRP assembled on the gold electrode catalyzed the polymerization of aniline on DTP. AFB1 in grains could be determined by using PANI as electrochemical signal molecules. Interestingly, DNA tetrahedron-structure, which has mechanical rigidity and structural stability, can improve antigen-antibody specific recognition and binding efficiency through the use of mAb ordered assembly. Meanwhile, nucleic acid backbone with a large amount of negative charge is good template for aniline polymerization under mild conditions.