DNA-based Nanostructures Research Articles

Triplex DNA Nanostructures: From Basic Properties to Applications

Triplex nucleic acids have recently attracted interest as part of the rich "toolbox" of structures used to develop DNA-based nanostructures and materials. This Review addresses the use of DNA triplexes to assemble sensing platforms and molecular switches. Furthermore, the pH-induced, switchable assembly and dissociation of triplex-DNA-bridged nanostructures are presented. Specifically, the aggregation/deaggregation of nanoparticles, the reversible oligomerization of origami tiles and DNA circles, and the use of triplex DNA structures as functional units for the assembly of pH-responsive systems and materials are described. Examples include semiconductor-loaded DNA-stabilized microcapsules, DNA-functionalized dye-loaded metal-organic frameworks (MOFs), and the pH-induced release of the loads. Furthermore, the design of stimuli-responsive DNA-based hydrogels undergoing reversible pH-induced hydrogel-to-solution transitions using triplex nucleic acids is introduced, and the use of triplex DNA to assemble shape-memory hydrogels is discussed. An outlook for possible future applications of triplex nucleic acids is also provided.

An Ag+-stabilized triplex DNA molecular switch controlled hybridization chain reaction

We described a new strategy to combine hybridization chain reaction (HCR) process and triplex DNA through Ag+/cysteine, thus to pursue the controllable process of HCR. H1, a specially-designed triplex DNA, with homopyrimidines which can bind to dsDNA through Hoogsteen bonding, forming a cap-like triplex DNA. The presence of Ag+ can play the role as a H1 locker to stop initiator from triggering HCR process, forming Ag+-stabilized triplex DNA molecular switch (denoted as Ag+-STDMS). However, the strong binding ability of cysteine towards Ag+ forming stable Ag+-cysteine can release the locking tail of H1 and realize the retriggering of HCR. This study presented a promising tool to regulate the self-assembly processes of DNA-based nanostructures in neutral environment. Under the optimum conditions, fluorescence intensity (peaking at 582 nm) of HCR is proportional to the concentration of Ag+ in the 0.2–4.0 μM range. Within the presence of Ag+, the fluorescence intensity is also proportional to the concentration of cysteine in the 0.2–3.0 μM range. The method can successfully manipulate the assembly and disassembly of DNA in HCR.

Supramolecular Protein Assemblies Based on DNA Templates.

DNA plays an important role in the process of protein assembly. DNA viruses such as the M13 virus are typical examples in which single DNA genomes behave as templates to induce the assembly of multiple major coat protein (PVIII) monomers. Thus, the design of protein assemblies based on DNA templates attracts much interest in the construction of supramolecular structures and materials. With the development of DNA nanotechnology, precise 1D and 3D protein nanostructures have been designed and constructed by using DNA templates through DNA-protein interactions, protein-ligand interactions, and protein-adapter interactions. These DNA-templated protein assemblies show great potential in catalysis, medicine, light-responsive systems, drug delivery, and signal transduction. Herein, we review the progress on DNA-based protein nanostructures that possess sophisticated nanometer-sized structures with programmable shapes and stimuli-responsive parameters, and we present their great potential in the design of biomaterials and biodevices in the future.

Tunable Fluorescence of a Semiconducting Polythiophene Positioned on DNA Origami.

A novel approach for the integration of π-conjugated polymers (CPs) into DNA-based nanostructures is presented. Using the controlled Kumada catalyst-transfer polycondensation, well-defined thiophene-based polymers with controllable molecular weight, specific end groups, and water-soluble oligoethylene glycol-based side chains were synthesized. The end groups were used for the easy but highly efficient click chemistry-based attachment of end-functionalized oligodeoxynucleotides (ODNs) with predesigned sequences. As demonstrated by surface plasmon resonance spectroscopy, the prepared block copolymers (BCPs), P3(EO)3T-b-ODN, comprising different ODN lengths and specific or repetitive sequences, undergo specific hybridization with complementary, thiol-functionalized ODNs immobilized on a gold surface. Furthermore, the site-specific attachment of the BCPs to DNA origami structures is studied. We demonstrate that a nanoscale object, that is, a single BCP with a single ODN handle, can be directed and bound to the DNA origami with reasonable yield, site-specificity, and high spatial density. On the basis of these results, we are able to demonstrate for the first time that optical properties of CP molecules densely immobilized on DNA origami can be locally fine-tuned by controlling the attractive π-π-stacking interactions between the CPs. In particular, we show that the fluorescence of the immobilized CP molecules can be significantly enhanced by surfactant-induced breakup of π-π-stacking interactions between the CP's backbones. Such molecular control over the emission intensity of the CPs can be valuable for the construction of sophisticated switchable nanophotonic devices and nanoscale biosensors.

Chiroplasmonic DNA-based nanostructures

Chiroplasmonic properties of nanoparticles, organized using DNA-based nanostructures, have attracted both theoretical and experimental interest. Theory suggests that the circular dichroism spectra accompanying chiroplasmonic nanoparticle assemblies are controlled by the sizes, shapes, geometries and interparticle distances of the nanoparticles. In this Review, we present different methods to assemble chiroplasmonic nanoparticle or nanorod systems using DNA scaffolds, and we discuss the operations of dynamically reconfigurable chiroplasmonic nanostructures. The chiroplasmonic properties of the different systems are characterized by circular dichroism and further supported by high-resolution transmission electron microscopy or cryo-transmission electron microscopy imaging and theoretical modelling. We also outline the applications of chiroplasmonic assemblies, including their use as DNA-sensing platforms and as functional systems for information processing and storage. Finally, future perspectives in applying chiroplasmonic nanoparticles as waveguides for selective information transfer and their use as ensembles for chiroselective synthesis are discussed. Specifically, we highlight the upscaling of the systems to device-like configurations. Asymmetric chiroplasmonic structures comprising nanoparticles or nanorods can be assembled using DNA scaffolds by a range of methods. In this Review, these assembly methods, the chiroplasmonic properties of static and dynamically reconfigurable systems, theoretical modelling of the chiroplasmonic structures, their applications for sensing and information processing, and future perspectives are discussed.

Photodynamic Therapy: A Singlet Oxygen Generating Agent by Chirality‐dependent Plasmonic Shell‐Satellite Nanoassembly (Adv. Mater. 18/2017)

Water-dispersible and label-free chiral photodynamic therapy (PDT) agents, through DNA-based self-assembly SS nanostructures have been fabricated, with high reactive oxygen species-generating efficiency, as described in article number 1606864 by Hua Kuang and co-workers. The enantiomer-dependent and structure-induced nanoassemblies play an important role in PDT effects. Moreover, the nanostructures have been well dispersed throughout the cell and show no cytotoxicity, which is important for future biological applications.

Aptasensor with Expanded Nucleotide Using DNA Nanotetrahedra for Electrochemical Detection of Cancerous Exosomes

Exosomes are extracellular vesicles (50-100 nm) circulating in biofluids as intercellular signal transmitters. Although the potential of cancerous exosomes as tumor biomarkers is promising, sensitive and rapid detection of exosomes remains challenging. Herein, we combined the strengths of advanced aptamer technology, DNA-based nanostructure, and portable electrochemical devices to develop a nanotetrahedron (NTH)-assisted aptasensor for direct capture and detection of hepatocellular exosomes. The oriented immobilization of aptamers significantly improved the accessibility of an artificial nucleobase-containing aptamer to suspended exosomes, and the NTH-assisted aptasensor could detect exosomes with 100-fold higher sensitivity when compared to the single-stranded aptamer-functionalized aptasensor. The present study provides a proof-of-concept for sensitive and efficient quantification of tumor-derived exosomes. We thus expect the NTH-assisted electrochemical aptasensor to become a powerful tool for comprehensive exosome studies.

PH-Responsive Reversible Regulation of Enzyme Activity by DNA-Based Nanostructure

Here, we present a novel method for reversibly regulating enzyme activity by caging an enzyme into a pH-responsive DNA-based tetrahedron nanostructure. pH-dependent opening/closing of the DNA cage was verified by measuring fluorescence resonance energy transfer between two vertex corners of the tetrahedron. The position of the covalent enzyme attachment in DNA was carefully chosen such that the attached enzyme faced inward the DNA cage. Both proteinase K protection assay and single-molecule based pull-down assay showed that the encapsulated enzyme were exposed to either proteinase K or target antibody by pH-dependent opening of DNA cage. Remarkably, we found that the caging/uncaging process were reversible, implying that enzyme activity toward relatively larger substrates than DNA cage can be reversibly regulated. Considering that the DNA cage is widely used as a delivery carrier, our method can be further extended to reversibly regulate cell function by pH-dependent activity control of delivered enzyme.

Click-based functionalization of a 2'-O-propargyl-modified branched DNA nanostructure.

DNA has emerged as a versatile building block for programmable self-assembly. DNA-based nanostructures have been widely applied in biosensing, bioimaging, drug delivery, molecular computation and macromolecular scaffolding. A variety of strategies have been developed to functionalize these nanostructures. In this study, we report a facile click-based strategy to incorporate a metal chelating ligand and a fluorescent tag into a three-point-star DNA tile containing 2'-O-propargyl groups. Such a strategy opens up the possibility of functionalizing pre-assembled DNA strands to construct platforms for metal or drug delivery.

DNA Origami Reorganizes upon Interaction with Graphite: Implications for High-Resolution DNA Directed Protein Patterning

Although there is a long history of the study of the interaction of DNA with carbon surfaces, limited information exists regarding the interaction of complex DNA-based nanostructures with the important material graphite, which is closely related to graphene. In view of the capacity of DNA to direct the assembly of proteins and optical and electronic nanoparticles, the potential for combining DNA-based materials with graphite, which is an ultra-flat, conductive carbon substrate, requires evaluation. A series of imaging studies utilizing Atomic Force Microscopy has been applied in order to provide a unified picture of this important interaction of structured DNA and graphite. For the test structure examined, we observe a rapid destabilization of the complex DNA origami structure, consistent with a strong interaction of single-stranded DNA with the carbon surface. This destabilizing interaction can be obscured by an intentional or unintentional primary intervening layer of single-stranded DNA. Because the interaction of origami with graphite is not completely dissociative, and because the frustrated, expanded structure is relatively stable over time in solution, it is demonstrated that organized structures of pairs of the model protein streptavidin can be produced on carbon surfaces using DNA origami as the directing material.

PH-Controlled Assembly of DNA Tiles.

We demonstrate a strategy to trigger and finely control the assembly of supramolecular DNA nanostructures with pH. Control is achieved via a rationally designed strand displacement circuit that responds to pH and activates a downstream DNA tile self-assembly process. We observe that the DNA structures form under neutral/basic conditions, while the self-assembly process is suppressed under acidic conditions. The strategy presented here demonstrates a modular approach toward building systems capable of processing biochemical inputs and finely controlling the assembly of DNA-based nanostructures under isothermal conditions. In particular, the presented architecture is relevant for the development of complex DNA devices able to sense and respond to molecular markers associated with abnormal metabolism.

Metallic Nanostructures Based on DNA Nanoshapes.

Metallic nanostructures have inspired extensive research over several decades, particularly within the field of nanoelectronics and increasingly in plasmonics. Due to the limitations of conventional lithography methods, the development of bottom-up fabricated metallic nanostructures has become more and more in demand. The remarkable development of DNA-based nanostructures has provided many successful methods and realizations for these needs, such as chemical DNA metallization via seeding or ionization, as well as DNA-guided lithography and casting of metallic nanoparticles by DNA molds. These methods offer high resolution, versatility and throughput and could enable the fabrication of arbitrarily-shaped structures with a 10-nm feature size, thus bringing novel applications into view. In this review, we cover the evolution of DNA-based metallic nanostructures, starting from the metallized double-stranded DNA for electronics and progress to sophisticated plasmonic structures based on DNA origami objects.

Understanding the Biomedical Effects of the Self-Assembled Tetrahedral DNA Nanostructure on Living Cells.

Recently, much attention has been paid to DNA again due to the successful synthesis of DNA-based nanostructures that can enter cells via endocytosis and thus have great potential in biomedical fields. However, the impacts of DNA nanostructures on life activities of a living cell are unknown. Herein, the promotion effect of tetrahedral DNA nanostructure (TDN) on cell growth and the underlying molecular mechanisms are reported. Upon exposure to TDN, cell proliferation is significantly enhanced, accompanied by up-regulation of cyclin-dependent kinase like-1 gene, changes in cell cycle distribution, and up-regulation of the Wnt/β-catenin signaling-related proteins (β-catenin, Lef 1 and cyclin D). In contrast, single-stranded DNA (ssDNA) shows no such functions. Furthermore, TDN is able to reverse the inhibition effect of DKK1, a specific inhibitor for Wnt/β-catenin pathway. Hence, the Wnt/β-catenin pathway is the target for TDN to promote cell proliferation. The findings allow TDN to be a novel functional nanomaterial that has great potential in tissue repair and regeneration medicine.

Nucleic Acid Nanostructures for Chemical and Biological Sensing.

The nanoscale features of DNA have made it a useful molecule for bottom-up construction of nanomaterials, for example, two- and three-dimensional lattices, nanomachines, and nanodevices. One of the emerging applications of such DNA-based nanostructures is in chemical and biological sensing, where they have proven to be cost-effective, sensitive and have shown promise as point-of-care diagnostic tools. DNA is an ideal molecule for sensing not only because of its specificity but also because it is robust and can function under a broad range of biologically relevant temperatures and conditions. DNA nanostructure-based sensors provide biocompatibility and highly specific detection based on the molecular recognition properties of DNA. They can be used for the detection of single nucleotide polymorphism and to sense pH both in solution and in cells. They have also been used to detect clinically relevant tumor biomarkers. In this review, recent advances in DNA-based biosensors for pH, nucleic acids, tumor biomarkers and cancer cell detection are introduced. Some challenges that lie ahead for such biosensors to effectively compete with established technologies are also discussed.

Lactose-modified DNA tile nanostructures as drug carriers

Background: DNA hybridization allows the preparation of nanoscale DNA structures with desired shape and size. DNA structures using simple base pairing can be used for the delivery of drug molecules into the cells. Since DNA carries multiple negative charges, their cellular uptake efficiency is low. Thus, the modification of the DNA structures with molecules that may enhance the cellular internalization may be an option.Objective: The objective of this study is to construct DNA-based nanocarrier system and to investigate the cellular uptake of DNA tile with/without lactose modification.Methods: Doxorubicin was intercalated to DNA tile and cellular uptake of drug-loaded DNA-based carrier with/without lactose modification was investigated in vitro. HeLa, BT-474, and MDA-MB-231 cancer cells were used for cellular uptake studies and cytotoxicity assays. Using fluorescence spectroscopy, flow cytometry, and confocal microscopy, cellular uptake behavior of DNA tile was investigated. The cytotoxicity of DNA tile structures was determined with WST-1 assay.Results: The results show that modification with lactose effectively increases the intracellular uptake of doxorubicin loaded DNA tile structure by cancer cells compared with the unmodified DNA tile.Conclusion: The findings of this study suggest that DNA-based nanostructures modified with carbohydrates can be used as suitable multifunctional nanocarriers with simple chemical modifications.

DNA Nanotechnology for Cancer Therapy.

DNA nanotechnology is an emerging and exciting field, and represents a forefront frontier for the biomedical field. The specificity of the interactions between complementary base pairs makes DNA an incredible building material for programmable and very versatile two- and three-dimensional nanostructures called DNA origami. Here, we analyze the DNA origami and DNA-based nanostructures as a drug delivery system. Besides their physical-chemical nature, we dissect the critical factors such as stability, loading capability, release and immunocompatibility, which mainly limit in vivo applications. Special attention was dedicated to highlighting the boundaries to be overcome to bring DNA nanostructures closer to the bedside of patients.

Controlling Hybridization Chain Reactions with pH.

By taking inspiration from nature, where self-organization of biomolecular species into complex systems is finely controlled through different stimuli, we propose here a rational approach by which the assembly and disassembly of DNA-based concatemers can be controlled through pH changes. To do so we used the hybridization chain reaction (HCR), a process that, upon the addition of an initiator strand, allows to create DNA-based concatemers in a controlled fashion. We re-engineered the functional units of HCR through the addition of pH-dependent clamp-like triplex-forming domains that can either inhibit or activate the polymerization reaction at different pHs. This allows to finely regulate the HCR-induced assembly and disassembly of DNA concatemers at either basic or acidic pHs in a reversible way. The strategies we present here appear particularly promising as novel tools to achieve better spatiotemporal control of self-assembly processes of DNA-based nanostructures.

One-Step Formation of "Chain-Armor"-Stabilized DNA Nanostructures.

DNA-based self-assembled nanostructures are widely used to position organic and inorganic objects with nanoscale precision. A particular promising application of DNA structures is their usage as programmable carrier systems for targeted drug delivery. To provide DNA-based templates that are robust against degradation at elevated temperatures, low ion concentrations, adverse pH conditions, and DNases, we built 6-helix DNA tile tubes consisting of 24 oligonucleotides carrying alkyne groups on their 3'-ends and azides on their 5'-ends. By a mild click reaction, the two ends of selected oligonucleotides were covalently connected to form rings and interlocked DNA single strands, so-called DNA catenanes. Strikingly, the structures stayed topologically intact in pure water and even after precipitation from EtOH. The structures even withstood a temperature of 95 °C when all of the 24 strands were chemically interlocked.

Multifunctional DNA Nanomaterials for Biomedical Applications

The rapidly emerging DNA nanotechnology began with pioneer Seeman’s hypothesis that DNA not only can carry genetic information but also can be used as molecular organizer to create well‐designed and controllable nanomaterials for applications in materials science, nanotechnology, and biology. DNA‐based self‐assembly represents a versatile system for nanoscale construction due to the well‐characterized conformation of DNA and its predictability in the formation of base pairs. The structural features of nucleic acids form the basis of constructing a wide variety of DNA nanoarchitectures with well‐defined shapes and sizes, in addition to controllable permeability and flexibility. More importantly, self‐assembled DNA nanostructures can be easily functionalized to construct artificial functional systems with nanometer scale precision for multipurposes. Apparently scientists envision artificial DNA‐based nanostructures as tool for drug loading and in vivo targeted delivery because of their abilities in selective encapsulation and stimuli‐triggered release of cargo. Herein, we summarize the strategies of creating multidimensional self‐assembled DNA nanoarchitectures and review studies investigating their stability, toxicity, delivery efficiency, loading, and control release of cargos in addition to their site‐specific targeting and delivery of drug or cargo molecules to cellular systems.

Cellular Uptake of Tile-Assembled DNA Nanotubes.

DNA-based nanostructures have received great attention as molecular vehicles for cellular delivery of biomolecules and cancer drugs. Here, we report on the cellular uptake of tubule-like DNA tile-assembled nanostructures 27 nm in length and 8 nm in diameter that carry siRNA molecules, folic acid and fluorescent dyes. In our observations, the DNA structures are delivered to the endosome and do not reach the cytosol of the GFP-expressing HeLa cells that were used in the experiments. Consistent with this observation, no elevated silencing of the GFP gene could be detected. Furthermore, the presence of up to six molecules of folic acid on the carrier surface did not alter the uptake behavior and gene silencing. We further observed several challenges that have to be considered when performing in vitro and in vivo experiments with DNA structures: (i) DNA tile tubes consisting of 42 nt-long oligonucleotides and carrying single- or double-stranded extensions degrade within one hour in cell medium at 37 °C, while the same tubes without extensions are stable for up to eight hours. The degradation is caused mainly by the low concentration of divalent ions in the media. The lifetime in cell medium can be increased drastically by employing DNA tiles that are 84 nt long. (ii) Dyes may get cleaved from the oligonucleotides and then accumulate inside the cell close to the mitochondria, which can lead to misinterpretation of data generated by flow cytometry and fluorescence microscopy. (iii) Single-stranded DNA carrying fluorescent dyes are internalized at similar levels as the DNA tile-assembled tubes used here.