Self-assembled DNA Research Articles

Self-Assembled DNA Generated Electric Current Biosensor for HER2 Analysis.

We have developed a new DNA self-assembly amplification technology that generates electric current for electrochemical biosensing. The new technology was used for detection of human epidermal growth factor receptor 2 (HER2). In our technology, an aptamer was utilized both as a ligand for recognition and as a signal generating reporter. The aptasensor is based on a sandwich formatand a DNA primer on a HER2 aptamer initiates auxiliary DNA self-assembled on the electrode to form a long one-dimensional DNA. The resulting DNA is then reacted with molybdate to generate electrochemical current. The sensitivity of the aptasensor with DNA self-assembly was greater than that of the aptasensor without DNA self-assembly due to the extended length of the DNA strand. Aptasensor analysis of HER2 in serum of breast cancer patients and healthy individuals is highly correlated (R2 = 0.9924) with ELISA measurements, with a p value of 1.37 × 10-7. The analysis of HER2 in serum (confirmed by ELISA) suggests that HER2 levels in breast cancer patients are much higher than healthy individuals. For HER2 positive patients, the levels are higher than those of HER2 negative patients. After surgery, there is a drop of HER2 levels in serum, suggesting potential clinical applications of the new self-assembled DNA electric current generating biosensor. Unlike proteins, DNA is easily amplifiable. The DNA signal amplification method presented here enables effective current generation, which can find wide range of biomedical applications for protein detection.

Self-Assembly of Heterogeneously Shaped Nanoparticles into Plasmonic Metamolecules on DNA Origami.

Fabrication of plasmonic metamolecules (PMs) with rationally designed complexity is one of the major goals of nanotechnology. Most self-assembled PMs, however, have been constructed using single-component systems. The corresponding plasmonic assemblies still suffer from the lack of complexity, which is required to achieve a high degree of functionality. Here, we report a general applicable strategy that can realize a series of high-ordered hetero-PMs using bottom-up DNA self-assembly. DNA-functionalized differently shaped nanoparticles were deliberately arranged in prescribed positions on 3D triangular DNA origami frames to form various hetero-PMs. Importantly, we showed that the optical properties of assembled PMs could be facially tuned by selectively regulating the position of each component. This method provides a promising pathway for manufacturing more complex and advanced materials by integrating diverse nanocomponents with particular properties.

Salt Responsive Morphologies of ssDNA-Based Triblock Polyelectrolytes in Semi-Dilute Regime: Effect of Volume Fractions and Polyelectrolyte Length.

A comprehensive study is reported on the effect of salt concentration, polyelectrolyte block length, and polymer concentration on the morphology and structural properties of nanoaggregates self-assembled from BAB single-strand DNA (ssDNA) triblock polynucleotides in which A represents polyelectrolyte blocks and B represents hydrophobic neutral blocks. A morphological phase diagram above the gelation point is developed as a function of solvent ionic strength and polyelectrolyte block length utilizing an implicit solvent ionic strength method for dissipative particle dynamics simulations. As the solvent ionic strength increases, the self-assembled DNA network structures shrinks considerably, leading to a morphological transition from a micellar network to worm-like or hamburger-shape aggregates. This study provides insight into the network morphology and its changes by calculating the aggregation number, number of hydrophobic cores, and percentage of bridge chains in the network. The simulation results are corroborated through cryogenic transmission electron microscopy on the example of the self-assembly of ssDNA triblocks.

Addressable configurations of DNA nanostructures for rewritable memory.

DNA serves as nature's information storage molecule, and has been the primary focus of engineered systems for biological computing and data storage. Here we combine recent efforts in DNA self-assembly and toehold-mediated strand displacement to develop a rewritable multi-bit DNA memory system. The system operates by encoding information in distinct and reversible conformations of a DNA nanoswitch and decoding by gel electrophoresis. We demonstrate a 5-bit system capable of writing, erasing, and rewriting binary representations of alphanumeric symbols, as well as compatibility with ‘OR’ and ‘AND’ logic operations. Our strategy is simple to implement, requiring only a single mixing step at room temperature for each operation and standard gel electrophoresis to read the data. We envision such systems could find use in covert product labeling and barcoding, as well as secure messaging and authentication when combined with previously developed encryption strategies. Ultimately, this type of memory has exciting potential in biomedical sciences as data storage can be coupled to sensing of biological molecules.

Multifunctional quantum dot DNA hydrogels

Biotemplated nanomaterials offer versatile functionality for multimodal imaging, biosensing, and drug delivery. There remains an unmet need for traceable and biocompatible nanomaterials that can be synthesized in a precisely controllable manner. Here, we report self-assembled quantum dot DNA hydrogels that exhibit both size and spectral tunability. We successfully incorporate DNA-templated quantum dots with high quantum yield, long-term photostability, and low cytotoxicity into a hydrogel network in a single step. By leveraging DNA-guided interactions, we introduce multifunctionality for a variety of applications, including enzyme-responsive drug delivery and cell-specific targeting. We report that quantum dot DNA hydrogels can be used for delivery of doxorubicin, an anticancer drug, to increase potency 9-fold against cancer cells. This approach also demonstrated high biocompatibility, trackability, and in vivo therapeutic efficacy in mice bearing xenografted breast cancer tumors. This work paves the way for the development of new tunable biotemplated nanomaterials with multiple synergistic functionalities for biomedical applications.

Tuning the Cavity Size and Chirality of Self-Assembling 3D DNA Crystals.

The foundational goal of structural DNA nanotechnology-the field that uses oligonucleotides as a molecular building block for the programmable self-assembly of nanostructured systems-was to use DNA to construct three-dimensional (3D) lattices for solving macromolecular structures. The programmable nature of DNA makes it an ideal system for rationally constructing self-assembled crystals and immobilizing guest molecules in a repeating 3D array through their specific stereospatial interactions with the scaffold. In this work, we have extended a previously described motif (4 × 5) by expanding the structure to a system that links four double-helical layers; we use a central weaving oligonucleotide containing a sequence of four six-base repeats (4 × 6), forming a matrix of layers that are organized and dictated by a series of Holliday junctions. In addition, we have assembled mirror image crystals (l-DNA) with the identical sequence that are completely resistant to nucleases. Bromine and selenium derivatives were obtained for the l- and d-DNA forms, respectively, allowing phase determination for both forms and solution of the resulting structures to 3.0 and 3.05 Å resolution. Both right- and left-handed forms crystallized in the trigonal space groups with mirror image 3-fold helical screw axes P32 and P31 for each motif, respectively. The structures reveal a highly organized array of discrete and well-defined cavities that are suitable for hosting guest molecules and allow us to dictate a priori the assembly of guest-DNA conjugates with a specified crystalline hand.

DNA Self-assembly Catalyzed by Artificial Agents

Nucleic acids have been shown to be versatile molecules and engineered to produce various nanostructures. However, the poor rate of these uncatalyzed nucleic acid reactions has restricted the development and applications. Herein, we reported a novel finding that DNA self-assembly could be nonenzymatically catalyzed by artificial agents with an increasing dissociation rate constant K2. The catalytic role of several artificial agents in DNA self-assembly was verified by real-time fluorescent detection or agarose gel electrophoresis. We found that 20% PEG 200 could significantly catalyze DNA self-assembly and increase the reaction efficiency, such as linear hybridization chain reaction (HCR) and exponential hairpin assembly (EHA). Therefore, we foresee that a fast and efficient DNA self-assembly in structural DNA nanotechnology will be desirable.

Computational modelling of the kinetic Tile Assembly Model using a rule-based approach

The (abstract) Tile Assembly Model (aTAM), is a mathematical paradigm for the study and algorithmic design of DNA self-assembly systems. It employs the use of so-called DNA-tiles, which are abstractions of experimentally achievable DNA nanostructure complexes with similar inter-matching behaviours. To this day, there are about half-dozen different experimental implementations of DNA tiles and their sub-sequent algorithmic assembly into larger complexes, see e.g. Reif et al. (2012) [29]. In order to provide further insight into the assembly process, the aTAM model has been extended to a kinetic counterpart (kTAM). Although there is a wide abundance of different variants of the abstract model, e.g., stage, step, hierarchical, temperature-k, signal-passing, etc. (see e.g. Patitz (2012) [22]), numerical simulations of the kinetic counterpart have been performed only for a few types of these systems. This might be due to the fact that the numerical models and simulations of kTAM were almost exclusively implemented using classical stochastic simulation algorithms frameworks, which are not designed for capturing models with theoretically un-bounded number of species. In this paper we introduce an agent- and rule-based modelling approach for kTAM, and its implementation on NFsim, one of the available platforms for such type of modelling. We show not only how the modelling of kTAM can be implemented, but we also explore the advantages of this modelling framework for kinetic simulations of kTAM and the easy way such models can be updated and modified. We present numerical comparisons both with classical numerical simulations of kTAM, as well as comparison in between four different kinetic variant of the TAM model, all implemented in NFsim as stand-alone rule-based models.

DNA origami nanorobot fiber optic genosensor to TMV

In the quest of greater sensitivity and specificity of diagnostic systems, one continually searches for alternative DNA hybridization methods, enabling greater versatility and where possible field-enabled detection of target analytes. We present, herein, a hybrid molecular self-assembled scaffolded DNA origami entity, intimately immobilized via capture probes linked to aminopropyltriethoxysilane, onto a glass optical fiber end-face transducer, thus producing a novel biosensor. Immobilized DNA nanorobots with a switchable flap can then be actuated by a specific target DNA present in a sample, by exposing a hemin/G-quadruplex DNAzyme, which then catalyzes the generation of chemiluminescence, once the specific fiber probes are immersed in a luminol-based solution. Integrating organic nanorobots to inorganic fiber optics creates a hybrid system that we demonstrate as a proof-of-principle can be utilized in specific DNA sequence detection. This system has potential applications in a wide range of fields, including point-of-care diagnostics or cellular in vivo biosensing when using ultrathin fiber optic probes for research purposes.

Versatile Self-Assembly and Biosensing Applications of DNA and Carbon Quantum Dots Coordinated Cerium Ions.

Self-assembly exploits noncovalent interactions to offer a facile and effective method for the construction of soft materials with multifunctionalities and diversity. In this work, fluorescence carbon quantum dots coordinated by Ce3+ ions (CQDCe) have been synthesized and exploited as building blocks to generate a series of hierarchical structures through the ionic self-assembly of CQDCe and biomolecules, namely DNA, myoglobin (Mb), and hyaluronic acid (HA). In particular, vesicles can be constructed by the simple mixing of oppositely charged CQDCe and DNA in water. The formation of unusual vesicles can be explained by the self-assembly of CQDCe with a rearranged structure and the rigid DNA biomolecular scaffolds. This facile noncovalent self-assembly method has inspired the innovative use of virgin DNA as a building block to construct vesicles rather than resorting to a sophisticated synthesis. The self-assembly of CQDCe-biopolymers was accompanied by aggregation-induced photoluminescence (PL) quenching. The biosensing platform was designed to detect polypeptides and deoxyribonuclease I through competitive binding of CQDCe and enzymatic hydrolysis of the DNA backbone, respectively. We believe that the integrative self-assembly of CQDCe and DNA will enrich the theoretical study of vesicle formation by DNA molecules and extend the application of fluorescence carbon quantum dots in the biological field.

Two-Dimensional Plasmonic Nanoparticle as a Nanoscale Sensor to Probe Polymer Brush Formation.

Conventional analysis and characterization of polymer brush formation relies on laborious methods that use a quartz crystal microbalance, atomic force microscope, microcantilever, or other tools that measure the concentration change of solutions. Herein we develop a simple and easy method that utilizes intrinsically flat two-dimensional (2D) plasmonic nanoparticles as sensors for unveiling the mechanism of polymer brush formation on surfaces. Via ultraviolet-visible spectroscopy, the plasmonic nanoparticles can be used to determine the amount of polymers near the surface in situ. As the amount of polymers increases near the surface, the nanoparticle characteristic localized surface plasmon resonance wavelength redshifts, and the shift amount corresponds linearly to the polymer density near the surface. By functionalizing the nanoparticles in solutions of thiolated polyethylene glycol (PEG-SH) with or without PEG disulfide (PEG-S-S-PEG), the three-regime kinetics of the polymer brush formation is confirmed. The fast adsorption and slow chain rearrangement in the first regime are found to be the causes of the latent regime. In the latent regime, the adsorbed polymer chains rearrange to anchor their ends onto the surface and contract to liberate space so that other polymer chains can graft onto the surface until saturation. The fundamental understanding gained herein enables the design of surfaces with complex chemistries and properties, which can find broad applications in responsive sensors, films, and coatings. Moreover, the novel analytical method of using 2D plasmonic nanoparticle as a sensor to understand the polymer brush formation is applicable to investigating the grafting of other molecules such as self-assembled monolayers, protein, and DNA.

Application Progress of DNA Nanostructures in Drug Delivery and Smart Drug Carriers

Abstract The nanocarrier systems have been widely used for improving solubility, stability and therapeutic activity of drugs due to their high drug-loading efficiency, good target specificity and long circulation time. To achieve precisely controlled loading and tumor-selective release of drugs, extensive researches have been focused on developing nanocarriers with low toxicity, excellent biocomapatibility and biodegradability. As a type of nano-biomaterials with various functions and applications, self-assembled DNA nanostructures explored new ways to develop drug carriers in smart drug delivery based on their well-defined structures, good biocompatibility and stability, high cell membrane permeability and controlled drug releasing property. In this review, we summarized the developing course and the recent advances of DNA nanostructures for drug delivery, including the application of both static and dynamic DNA nanostructures. The application of dynamic DNA nanostructures for controllable drug release showed great potential in smart drug delivery.

Proton-Fueled, Reversible DNA Hybridization Chain Assembly for pH Sensing and Imaging.

Design of DNA self-assembly with reversible responsiveness to external stimuli is of great interest for diverse applications. We for the first time develop a pH-responsive, fully reversible hybridization chain reaction (HCR) assembly that allows sensitive sensing and imaging of pH in living cells. Our design relies on the triplex forming sequences that form DNA triplex with toehold regions under acidic conditions and then induce a cascade of strand displacement and DNA assembly. The HCR assembly has shown dynamic responses in physiological pH ranges with excellent reversibility and demonstrated the potential for in vitro detection and live-cell imaging of pH. Moreover, this method affords HCR assemblies with highly localized fluorescence responses, offering advantages of improving sensitivity and better selectivity. The proton-fueled, reversible HCR assembly may provide a useful approach for pH-related cell biology study and disease diagnostics.

Sequential self-assembly of DNA functionalized droplets

Complex structures and devices, both natural and manmade, are often constructed sequentially. From crystallization to embryogenesis, a nucleus or seed is formed and built upon. Sequential assembly allows for initiation, signaling, and logical programming, which are necessary for making enclosed, hierarchical structures. Although biology relies on such schemes, they have not been available in materials science. Here, we demonstrate programmed sequential self-assembly of DNA functionalized emulsions. The droplets are initially inert because the grafted DNA strands are pre-hybridized in pairs. Active strands on initiator droplets then displace one of the paired strands and thus release its complement, which in turn activates the next droplet in the sequence, akin to living polymerization. Our strategy provides time and logic control during the self-assembly process, and offers a new perspective on the synthesis of materials.

Precisely Tailored DNA Nanostructures and their Theranostic Applications.

A critical challenge in nanotechnology is the limited precision and controllability of the structural parameters, which brings about concerns in uniformity, reproducibility and performance. Self-assembled DNA nanostructures, as a newly emerged type of nano-biomaterials, possess low-nanometer precision, excellent programmability and addressability. They can precisely arrange various molecules and materials to formspatially ordered complex, resulting in unambiguous physical or chemical properties.Because of these, DNA nanostructures have shown great promise in numerous biomedical theranostic applications. In this account, we briefly review the history and advances on construction of DNA nanoarchitectures and superstructures with accurate structural parameters. We focus on recent progress in exploiting these DNA nanostructures as platforms for quantitative biosensing, intracellular diagnosis, imaging, and smart drug delivery. We also discuss key challenges in practical applications.

Enantiomeric structures of a self-assembling three-dimensional DNA crystal scaffold

Enantiomeric structures of a self-assembling three-dimensional DNA crystal scaffold

Rapid and annealing-free self-assembly of DNA building blocks for 3D hydrogel chaperoned by cationic comb-type copolymers

The construction and self-assembly of DNA building blocks are the foundation of bottom-up development of three-dimensional DNA nanostructures or hydrogels. However, most self-assembly from DNA components is impeded by the mishybridized intermediates or the thermodynamic instability. To enable rapid production of complicated DNA objects with high yields no need for annealing process, herein different DNA building blocks (Y-shaped, L- and L′-shaped units) were assembled in presence of a cationic comb-type copolymer, poly (L-lysine)-graft-dextran (PLL-g-Dex), under physiological conditions. The results demonstrated that PLL-g-Dex not only significantly promoted the self-assembly of DNA blocks with high efficiency, but also stabilized the assembled multi-level structures especially for promoting the complicated 3D DNA hydrogel formation. This study develops a novel strategy for rapid and high-yield production of DNA hydrogel even derived from instable building blocks at relatively low DNA concentrations, which would endow DNA nanotechnology for more practical applications.

Liquid crystal-based detection of DNA hybridization using surface immobilized single-stranded DNA

The authors have investigated (a) the self-assembly of single-stranded DNA (ssDNA) on glass surfaces, and (b) the interaction of DNA with liquid crystals (LCs) on solid surfaces. The results suggest that ssDNA (compared to dsDNA) on the solid interface causes particularly different orientations in LCs. The LC molecules assume a uniform homeotropic orientation on the surface with a typical surface ssDNA coverage of ~2.4 × 1012 molecules per square cm. Once complementary DNA is hybridized on the surface, the homotropic orientation of the LCs becomes disrupted. This orientation transition can be visually observed by using a crossed polarizer. The findings were exploiting to design an assay for target DNA (= analyte DNA) that has an ~0.1 nM detection limit. The assay is highly selective and can easily differentiate target DNA from single-base mismatch and non-complementary DNA. In our perception, it represents a powerful, label-free and portable DNA detection scheme.

Development of DNA Nanostructures for High-Affinity Binding to Human Serum Albumin.

The development of nucleic acid therapeutics has been hampered by issues associated with their stability and in vivo delivery. To address these challenges, we describe a new strategy to engineer DNA structures with strong binding affinity to human serum albumin (HSA). HSA is the most abundant protein in the blood and has a long circulation half-life (19 days). It has been shown to hinder phagocytosis, is retained in tumors, and aids in cellular penetration. Indeed, HSA has already been successfully used for the delivery of small-molecule drugs and nanoparticles. We show that conjugating dendritic alkyl chains to DNA creates amphiphiles that exhibit high-affinity (Kd in low nanomolar range) binding to HSA. Notably, complexation with HSA did not hinder the activity of silencing oligonucleotides inside cells, and the degradation of DNA strands in serum was significantly slowed. We also show that, in a site-specific manner, altering the number and orientation of the amphiphilic ligand on a self-assembled DNA nanocube can modulate the affinity of the DNA cage to HSA. Moreover, the serum half-life of the amphiphile bound to the cage and the protein was shown to reach up to 22 hours, whereas unconjugated single-stranded DNA was degraded within minutes. Therefore, adding protein-specific binding domains to DNA nanostructures can be used to rationally control the interface between synthetic nanostructures and biological systems. A major challenge with nanoparticles delivery is the quick formation of a protein corona (i.e., protein adsorbed on the nanoparticle surface) upon injection to biological media. We foresee such DNA cage-protein complexes as new tools to study the role of this protein adsorption layer with important implications in the efficient delivery of RNAi therapeutics in vitro and in vivo.

Self-Assembled DNA Tetrahedral Scaffolds for the Construction of Electrochemiluminescence Biosensor with Programmable DNA Cyclic Amplification.

A novel DNA tetrahedron-structured electrochemiluminescence (ECL) platform for bioanalysis with programmable DNA cyclic amplification was developed. In this work, glucose oxidase (GOD) was labeled to a DNA sequence (S) as functional conjugation (GOD-S), which could hybridize with other DNA sequences (L and P) to form GOD-S:L:P probe. In the presence of target DNA and a help DNA (A), the programmable DNA cyclic amplification was activated and released GOD-S via toehold-mediated strand displacement. Then, the obtained GOD-S was further immobilized on the DNA tetrahedral scaffolds with a pendant capture DNA and Ru(bpy)32+-conjugated silica nanoparticles (RuSi NPs) decorated on the electrode surface. Thus, the amount of GOD-S assembled on the electrode surface depended on the concentration of target DNA and GOD could catalyze glucose to generate H2O2 in situ. The ECL signal of Ru(bpy)32+-TPrA system was quenched by the presence of H2O2. By integrating the programmable DNA cyclic amplification and in situ generating H2O2 as Ru(bpy)32+ ECL quencher, a sensitive DNA tetrahedron-structured ECL sensing platform was proposed for DNA detection. Under optimized conditions, this biosensor showed a wide linear range from 100 aM to 10 pM with a detection limit of 40 aM, indicating a promising application in DNA analysis. Furthermore, by labeling GOD to different recognition elements, the proposed strategy could be used for the detection of various targets. Thus, this programmable cascade amplification strategy not only retains the high selectivity and good capturing efficiency of tetrahedral-decorated electrode surface but also provides potential applications in the construction of ECL biosensor.