Functional DNA Nanostructures Research Articles

Tetrahedral DNA nanostructures functionalized by multivalent microRNA132 antisense oligonucleotides promote the differentiation of mouse embryonic stem cells into dopaminergic neurons

MicroRNA132 (miR132) negatively regulates the differentiation of mouse embryonic stem cells (ESCs) into dopaminergic (DAergic) neurons; in contrast, antisense oligonucleotide against miR132 (miR132-ASO) effectively blocks the activity of endogenous miR132 and thereafter promotes the differentiation of DAergic neurons. However, it is difficult for miR132-ASO to enter cells without a suitable delivery system. Tetrahedral DNA nanostructures (TDNs), as a new type of DNA-based nanocarrier, have great potential in biomedical applications and even have been reported to promote stem cell differentiation. In this study, we developed functional multivalent DNA nanostructures by appending miR132-ASO motifs to three-dimensional TDNs (miR132-ASO-TDNs). Our data clearly revealed that miR132-ASO-TDNs exposure can promote the differentiation of ESCs into DAergic neurons as well as elevate DA release from differentiated DAergic neurons. MiR132-ASO-TDNs could serve as a novel biofunctional nanomaterial to improve the efficiency of DAergic neurons differentiation. Our findings may also provide a new approach for stem cell therapy against neurodegenerative diseases.

Recent Advances of DNA Nanostructure-Based Cell Membrane Engineering.

Materials that can regulate the composition and structure of the cell membrane to fabricate engineered cells with defined functions are in high demand. Compared with other biomolecules, DNA has unique advantages in cell membrane engineering due to its excellent programmability and biocompatibility. Especially, the near-atomic scale precision of DNA nanostructures facilitates the investigation of structure-property relations on the cell membrane. In this review, first the state of the art of functional DNA nanostructures is summarized, and then the overview of the use of DNA nanostructures to engineer the cell membrane is presented. Subsequently, applications of DNA nanostructures in modifying cell membrane morphology, controlling ions transport, and synthesizing high precise liposomes are highlighted. Finally, the challenges and outlook on using DNA nanostructures for cell membrane engineering are discussed.

The effects of overhang placement and multivalency on cell labeling by DNA origami.

Through targeted binding to the cell membrane, structural DNA nanotechnology has the potential to guide and affix biomolecules such as drugs, growth factors and nanobiosensors to the surfaces of cells. In this study, we investigated the targeted binding efficiency of three distinct DNA origami shapes to cultured endothelial cells via cholesterol anchors. Our results showed that the labeling efficiency is highly dependent on the shape of the origami as well as the number and the location of the binding overhangs. With a uniform surface spacing of binding overhangs, 3D isotropic nanospheres and 1D anisotropic nanorods labeled cells effectively, and the isotropic nanosphere labeling fit well with an independent binding model. Face-decoration and edge-decoration of the anisotropic nanotile were performed to investigate the effects of binding overhang location on cell labeling, and only the edge-decorated nanotiles were successful at labeling cells. Edge proximity studies demonstrated that the labeling efficiency can be modulated in both nanotiles and nanorods by moving the binding overhangs towards the edges and vertices, respectively. Furthermore, we demonstrated that while double-stranded DNA (dsDNA) bridge tethers can rescue the labeling efficiency of the face-decorated rectangular plate, this effect is also dependent on the proximity of bridge tethers to the edges or vertices of the nanostructures. A final comparison of all three nanoshapes revealed that the end-labeled nanorod and the nanosphere achieved the highest absolute labeling intensities, but the highest signal-to-noise ratio, calculated as the ratio of overall labeling to initiator-free background labeling, was achieved by the end-labeled nanorod, with the edge-labeled nanotile coming in second place slightly ahead of the nanosphere. The findings from this study can help us further understand the factors that affect membrane attachment using cholesterol anchors, thus providing guidelines for the rational design of future functional DNA nanostructures.

Construction of Smart Stimuli-Responsive DNA Nanostructures for Biomedical Applications.

DNA nanostructures have recently attracted increasing interest in biological and biomedical applications by virtue of their unique properties, such as structural programmability, multi-functionality, stimuli-responsive behaviors, and excellent biocompatibility. In particular, the intelligent responsiveness of smart DNA nanostructures to specific stimuli has facilitated their extensive development in the field of high-performance biosensing and controllable drug delivery. This minireview begins with different self-assembly strategies for the construction of various DNA nanostructures, followed by the introduction of a variety of stimuli-responsive functional DNA nanostructures for assembling metastable soft materials and for facilitating amplified biosensing. The recent achievements of smart DNA nanostructures for controllable drug delivery are highlighted. Finally, the current challenges and possible developments of this promising research are discussed in the fields of intelligent nanomedicine.

Protein-Assisted Room-Temperature Assembly of Rigid, Immobile Holliday Junctions and Hierarchical DNA Nanostructures.

Immobile Holliday junctions represent not only the most fundamental building block of structural DNA nanotechnology but are also of tremendous importance for the in vitro investigation of genetic recombination and epigenetics. Here, we present a detailed study on the room-temperature assembly of immobile Holliday junctions with the help of the single-strand annealing protein Redβ. Individual DNA single strands are initially coated with protein monomers and subsequently hybridized to form a rigid blunt-ended four-arm junction. We investigate the efficiency of this approach for different DNA/protein ratios, as well as for different DNA sequence lengths. Furthermore, we also evaluate the potential of Redβ to anneal sticky-end modified Holliday junctions into hierarchical assemblies. We demonstrate the Redβ-mediated annealing of Holliday junction dimers, multimers, and extended networks several microns in size. While these hybrid DNA–protein nanostructures may find applications in the crystallization of DNA–protein complexes, our work shows the great potential of Redβ to aid in the synthesis of functional DNA nanostructures under mild reaction conditions.

A novel fluorescent biosensor based on dendritic DNA nanostructure in combination with ligase reaction for ultrasensitive detection of DNA methylation

BackgroundDNA methylation detection is indispensable for the diagnosis and prognosis of various diseases including malignancies. Hence, it is crucial to develop a simple, sensitive, and specific detection strategy.MethodsA novel fluorescent biosensor was developed based on a simple dual signal amplification strategy using functional dendritic DNA nanostructure and signal-enriching polystyrene microbeads in combination with ligase detection reaction (LDR). Dendritic DNA self-assembled from Y-DNA and X-DNA through enzyme-free DNA catalysis of a hairpin structure, which was prevented from unwinding at high temperature by adding psoralen. Then dendritic DNA polymer labeled with fluorescent dye Cy5 was ligated with reporter probe into a conjugate. Avidin-labeled polystyrene microbeads were specifically bound to biotin-labeled capture probe, and hybridized with target sequence and dendritic DNA. LDR was triggered by adding Taq ligase. When methylated cytosine existed, the capture probe and reporter probe labeled with fluorescent dye perfectly matched the target sequence, forming a stable duplex to generate a fluorescence signal. However, after bisulfite treatment, unmethylated cytosine was converted into uracil, resulting in a single base mismatch. No fluorescence signal was detected due to the absence of duplex.ResultsThe obtained dendritic DNA polymer had a large volume. This method was time-saving and low-cost. Under the optimal experimental conditions using avidin-labeled polystyrene microbeads, the fluorescence signal was amplified more obviously, and DNA methylation was quantified ultrasensitively and selectively. The detection range of this sensor was 10−15 to 10−7 M, and the limit of detection reached as low as 0.4 fM. The constructed biosensor was also successfully used to analyze actual samples.ConclusionThis strategy has ultrasensitivity and high specificity for DNA methylation quantification, without requiring complex processes such as PCR and enzymatic digestion, which is thus of great value in tumor diagnosis and biomedical research.

Target-Triggered Polymerization of Branched DNA Enables Enzyme-free and Fast Discrimination of Single-Base Changes.

SummarySingle-base changes lead to important biological and biomedical implications; however, the discrimination of single-base changes from normal DNA always remains a grand challenge. Herein we developed a DNA recognition and amplification system based on artificial branched DNA, namely, target-triggered polymerization (TTP), to realize enzyme-free and fast discrimination of single-base changes. Branched DNA as monomers rapidly polymerized into DNA nanospheres only with the trigger of specific DNA. Our TTP system worked reliably over a wide range of conditions. Remarkably, our TTP system was capable of discriminating base-changing DNA from normal DNA, including distinguishing 1–4 nucleotide changes and positions of single base, which was attributed to the significant amplification of small differences in hybridization thermodynamics and kinetics. We further proposed a theoretical method for calculating the hybridization probability of nucleic acids, which performed highly consistent with experimental results.

Functionalized DNA nanostructures as scaffolds for guided mineralization.

The field of DNA nanotechnology uses synthetic DNA strands as building blocks for designing complex shapes in one-, two- and three-dimensions. Here, we investigate whether DNA nanostructures are feasible platforms for the precise organization of polyaspartic acid (pAsp), a known mineral carrier, with a goal towards biomimetic mineralization for enamel regeneration. We describe the preparation of DNA-pAsp conjugates and their subsequent assembly into ordered nanostructures. Covalent attachment of pAsp to DNA was noted to hinder DNA nanostructure formation past a certain threshold (50% pAsp) when tested on a previously published DNA system. However, a simplified double stranded DNA system (3sDH system) was more robust and efficient in its pAsp incorporation. In addition, the 3sDH system was successful in organizing mineral inducing groups in one dimension at repeating intervals of 28.7 ± 4.0 nm, as determined by atomic force microscopy. Our results demonstrate that DNA nanostructures can be functionalized with pAsp and act as a platform to investigate guided mineralization.

Detection of Mg2+-dependent, coaxial stacking rearrangements in a bulged three-way DNA junction by single-molecule FRET

Three-way helical junctions (3WJs) arise in genetic processing, and they have architectural and functional roles in structured nucleic acids. An internal bulge at the junction core allows the helical domains to become oriented into two possible, coaxially stacked conformers. Here, the helical stacking arrangements for a series of bulged, DNA 3WJs were examined using ensemble fluorescence resonance energy transfer (FRET) and single-molecule FRET (smFRET) approaches. The 3WJs varied according to the GC content and sequence of the junction core as well as the pyrimidine content of the internal bulge. Mg2+ titration experiments by ensemble FRET show that both stacking conformations have similar Mg2+ requirements for folding. Strikingly, smFRET experiments reveal that a specific junction sequence can populate both conformers and that this junction undergoes continual interconversion between the two stacked conformers. These findings will support the development of folding principles for the rational design of functional DNA nanostructures.

Engineering a responsive DNA triple helix into an octahedral DNA nanostructure for a reversible opening/closing switching mechanism: a computational and experimental integrated study.

We propose an experimental and simulative approach to study the effect of integrating a DNA functional device into a large-sized DNA nanostructure. We selected, as a test bed, a well-known and characterized pH-dependent clamp-switch, based on a parallel DNA triple helix, to be integrated into a truncated octahedral scaffold. We designed, simulated and experimentally characterized two different functionalized DNA nanostructures, with and without the presence of a spacer between the scaffold and the functional elements. The experimental and simulative data agree in validating the need of a spacer for the occurrence of the pH dependent switching mechanism. The system is fully reversible and the switching can be monitored several times without any perturbation, maintaining the same properties of the isolated clamp switch in solution.

Functional-DNA-Driven Dynamic Nanoconstructs for Biomolecule Capture and Drug Delivery.

The discovery of sequence-specific hybridization has allowed the development of DNA nanotechnology, which is divided into two categories: 1) structural DNA nanotechnology, which utilizes DNA as a biopolymer; and 2) dynamic DNA nanotechnology, which focuses on the catalytic reactions or displacement of DNA structures. Recently, numerous attempts have been made to combine DNA nanotechnologies with functional DNAs such as aptamers, DNAzymes, amplified DNA, polymer-conjugated DNA, and DNA loaded on functional nanoparticles for various applications; thus, the new interdisciplinary research field of "functional DNA nanotechnology" is initiated. In particular, a fine-tuned nanostructure composed of functional DNAs has shown immense potential as a programmable nanomachine by controlling DNA dynamics triggered by specific environments. Moreover, the programmability and predictability of functional DNA have enabled the use of DNA nanostructures as nanomedicines for various biomedical applications, such as cargo delivery and molecular drugs via stimuli-mediated dynamic structural changes of functional DNAs. Here, the concepts and recent case studies of functional DNA nanotechnology and nanostructures in nanomedicine are reviewed, and future prospects of functional DNA for nanomedicine are indicated.

Functional and Biomimetic DNA Nanostructures on Lipid Membranes.

Sophisticated and dynamic membrane-anchored DNA nanostructures were developed to mimic a variety of membrane proteins, which play crucial roles in cellular functions. DNA biomimetic constructions bound on membranes are capable of modulating the morphologies, physical properties, and functions of lipid membranes, via mobility on membranes and/or inherent architectural features. This inspired young field of DNA-lipid-based nanobiomimetic systems is on the foundation of DNA nanotechnology. In this review, we highlight key successes in the development of structural DNA nanotechnology and demonstrate some typical static and dynamic complex DNA nanostructures first. Then, we discuss the biophysical properties of lipid membranes. Primary approaches are shown to attach hydrophilic DNA to hydrophobic lipid membranes. With appropriate designs, membrane-floating DNA nanostructures assemble and disassemble on membranes, modulated by external stimuluses. The aggregation of DNA nanostructures could influence the physical properties of lipid membranes. We also summarize artificial nanochannels made of DNA, analogous to transmembrane proteins. Transformations of DNA nanopores might be achieved under certain conditions and realize the transport of small molecules across membranes. Next, we focus on membrane-shaping functions of membrane-anchored DNA nanostructures. Curvature of the membrane is closely related to the rich diversity of cellular functions. Mimicking membrane-sculpting proteins, such as BAR family domains and SNARE proteins etc., DNA biomimetic nanostructures induce the transformations of lipid membranes and modulate membrane adhesion and membrane fusion processes. Although recent studies in DNA nanostructure-lipid membrane biomimetic nanosystems have made great progress, this field is still facing many challenges. In the future, the designs of more elaborated DNA architectures will be explored. Sophisticated dynamic DNA nanostructures inspired by natural membrane machines will be driven by the synergistic effect of multiple interactions, including hydrophobic force, electrostatic force, and ligand-receptor interactions by chemical modifications on bases, to expand their applications in vivo from model membrane to cell membrane to karyotheca.

Differential Effects of Strand Asymmetry on the Energetics and Structural Flexibility of DNA Internal Loops.

Internal loops within structured nucleic acids disrupt local base stacking and destabilize neighboring helical domains; however, these structural motifs also expand the conformational and functional capabilities of structured nucleic acids. Variations in the size, distribution of loop nucleotides on opposing strands (strand asymmetry), and sequence alter their biophysical properties. Here, the thermodynamics and structural flexibility of oligo-T-rich DNA internal loops were systematically investigated in terms of loop size and strand asymmetry. From optical melting experiments, a thermodynamic prediction model is proposed for the energetic penalty of internal loops that accounts for diminishing enthalpic and increasing entropic contributions due to loop size and strand asymmetry for bulges, asymmetric loops, and symmetric loops. These single-stranded domains become less sequence-dependent and more polymeric as the loop size increases. Single-molecule fluorescence resonance energy transfer studies reveal a gradual transition in conformation and structural flexibility from an elongated domain to an increasingly flexible bend that results from increasing strand asymmetry. The findings provide a framework for understanding the thermodynamic and conformational effects of internal loops for the rational design of functional DNA nanostructures.

Self-assembly programming of DNA polyominoes

Fabrication of functional DNA nanostructures operating at a cellular level has been accomplished through molecular programming techniques such as DNA origami and single-stranded tiles (SST). During implementation, restrictive and constraint dependent designs are enforced to ensure conformity is attainable. We propose a concept of DNA polyominoes that promotes flexibility in molecular programming. The fabrication of complex structures is achieved through self-assembly of distinct heterogeneous shapes (i.e., self-organised optimisation among competing DNA basic shapes) with total flexibility during the design and assembly phases. In this study, the plausibility of the approach is validated using the formation of multiple 3×4 DNA network fabricated from five basic DNA shapes with distinct configurations (monomino, tromino and tetrominoes). Computational tools to aid the design of compatible DNA shapes and the structure assembly assessment are presented. The formations of the desired structures were validated using Atomic Force Microscopy (AFM) imagery. Five 3×4 DNA networks were successfully constructed using combinatorics of these five distinct DNA heterogeneous shapes. Our findings revealed that the construction of DNA supra-structures could be achieved using a more natural-like orchestration as compared to the rigid and restrictive conventional approaches adopted previously.

Daisy Chain Rotaxanes Made from Interlocked DNA Nanostructures.

We report the stepwise assembly of supramolecular daisy chain rotaxanes (DCR) made of double‐stranded DNA: Small dsDNA macrocycles bearing an axle assemble into a pseudo‐DCR precursor that was connected to rigid DNA stoppers to form DCR with the macrocycles hybridized to the axles. In presence of release oligodeoxynucleotides (rODNs), the macrocycles are released from their respective hybridization sites on the axles, leading to stable mechanically interlocked DCRs. Besides the expected threaded DCRs, certain amounts of externally hybridized structures were observed, which dissociate into dumbbell structures in presence of rODNs. We show that the genuine DCRs have significantly higher degrees of freedom in their movement along the thread axle than the hybridized DCR precursors. Interlocking of DNA in DCRs might serve as a versatile principle for constructing functional DNA nanostructures where the movement of the subunits is restricted within precisely confined tolerance ranges.

Daisy Chain Rotaxanes Made from Interlocked DNA Nanostructures

AbstractWe report the stepwise assembly of supramolecular daisy chain rotaxanes (DCR) made of double‐stranded DNA: Small dsDNA macrocycles bearing an axle assemble into a pseudo‐DCR precursor that was connected to rigid DNA stoppers to form DCR with the macrocycles hybridized to the axles. In presence of release oligodeoxynucleotides (rODNs), the macrocycles are released from their respective hybridization sites on the axles, leading to stable mechanically interlocked DCRs. Besides the expected threaded DCRs, certain amounts of externally hybridized structures were observed, which dissociate into dumbbell structures in presence of rODNs. We show that the genuine DCRs have significantly higher degrees of freedom in their movement along the thread axle than the hybridized DCR precursors. Interlocking of DNA in DCRs might serve as a versatile principle for constructing functional DNA nanostructures where the movement of the subunits is restricted within precisely confined tolerance ranges.

Multifunctional aptamer-based nanoparticles for targeted drug delivery to circumvent cancer resistance

By its unique advantages over traditional medicine, nanomedicine has offered new strategies for cancer treatment. In particular, the development of drug delivery strategies has focused on nanoscale particles to improve bioavailability. However, many of these nanoparticles are unable to overcome tumor resistance to chemotherapeutic agents. Recently, new opportunities for drug delivery have been provided by oligonucleotides that can self-assemble into three-dimensional nanostructures. In this work, we have designed and developed functional DNA nanostructures to deliver the chemotherapy drug doxorubicin (Dox) to resistant cancer cells. These nanostructures have two components. The first component is a DNA aptamer, which forms a dimeric G-quadruplex nanostructure to target cancer cells by binding with nucleolin. The second component is double-stranded DNA (dsDNA), which is rich in -GC- base pairs that can be applied for Dox delivery. We demonstrated that Dox was able to efficiently intercalate into dsDNA and this intercalation did not affect the aptamer's three-dimensional structure. In addition, the Aptamer-dsDNA (ApS) nanoparticle showed good stability and protected the dsDNA from degradation in bovine serum. More importantly, the ApS&Dox nanoparticle efficiently reversed the resistance of human breast cancer cells to Dox. The mechanism circumventing doxorubicin resistance by ApS&Dox nanoparticles may be predominantly by cell cycle arrest in S phase, effectively increased cell uptake and decreased cell efflux of doxorubicin. Furthermore, the ApS&Dox nanoparticles could effectively inhibit tumor growth, while less cardiotoxicity was observed. Overall, this functional DNA nanostructure provides new insights into the design of nanocarriers to overcome multidrug resistance through targeted drug delivery.

Engineering DNA Three-Way Junction with Multifunctional Moieties: Sensing Platform for Bioanalysis.

Functionalization of DNA nanostructures is critical to the achievement of the application in biosensors. Herein, we demonstrate a novel DNA three-way junction (TWJ) with three functional moieties, which integrates the electrochemical, fluorescent, and colorimetric properties. Upon addition of external stimuli, including DNA, thrombin, and ATP, the specific interactions between targets and sensing elements could induce strand displacement reaction and conformation transformation, resulting in the integration of G-quadruplex/hemin complex as electrochemical probe, lighting up the fluorescence of DNA/Ag nanoclusters and enhancing the catalytic activity of DNAzyme to catalyze the H2O2-TMB system to generate colorimetric signal. Such a functional DNA nanostructure actually serves as a biosensing platform, showing high sensitivity and selectivity for DNA, thrombin, and ATP detection. Furthermore, We also show that this novel sensing platform can be utilized to detect three different kinds of targets independently and simultaneously. Our integrated concept provides a paradigm for exploring the potential of TWJs in analytical applications.

MicroRNA-triggered, cascaded and catalytic self-assembly of functional "DNAzyme ferris wheel" nanostructures for highly sensitive colorimetric detection of cancer cells.

The construction of DNA nanostructures with various sizes and shapes has significantly advanced during the past three decades, yet the application of these DNA nanostructures for solving real problems is still in the early stage. On the basis of microRNA-triggered, catalytic self-assembly formation of the functional "DNAzyme ferris wheel" nanostructures, we show here a new signal amplification platform for highly sensitive, label-free and non-enzyme colorimetric detection of a small number of human prostate cancer cells. The microRNA (miR-141), which is catalytically recycled and reused, triggers isothermal self-assembly of a pre-designed, G-quadruplex sequence containing hairpin DNAs into "DNAzyme ferris wheel"-like nanostructures (in association with hemin) with horseradish peroxidase mimicking activity. These DNAzyme nanostructures catalyze an intensified color transition of the probe solution for highly sensitive detection of miR-141 down to 0.5 pM with the naked eye, and the monitoring of as low as 283 human prostate cancer cells can also, theoretically, be achieved in a colorimetric approach. The work demonstrated here thus offers new opportunities for the construction of functional DNA nanostructures and for the application of these DNA nanostructures as an effective signal amplification means in the sensitive detection of nucleic acid biomarkers.

Layer-by-layer assembled antisense DNA microsponge particles for efficient delivery of cancer therapeutics.

Antisense oligonucleotides can be employed as a potential approach to effectively treat cancer. However, the inherent instability and inefficient systemic delivery methods for antisense therapeutics remain major challenges to their clinical application. Here, we present a polymerized oligonucleotides (ODNs) that self-assemble during their formation through an enzymatic elongation method (rolling circle replication) to generate a composite nucleic acid/magnesium pyrophosphate sponge-like microstructure, or DNA microsponge, yielding high molecular weight nucleic acid product. In addition, this densely packed ODN microsponge structure can be further condensed to generate polyelectrolyte complexes with a favorable size for cellular uptake by displacing magnesium pyrophosphate crystals from the microsponge structure. Additional layers are applied to generate a blood-stable and multifunctional nanoparticle via the layer-by-layer (LbL) assembly technique. By taking advantage of DNA nanotechnology and LbL assembly, functionalized DNA nanostructures were utilized to provide extremely high numbers of repeated ODN copies for efficient antisense therapy. Moreover, we show that this formulation significantly improves nucleic acid drug/carrier stability during in vivo biodistribution. These polymeric ODN systems can be designed to serve as a potent means of delivering stable and large quantities of ODN therapeutics systemically for cancer treatment to tumor cells at significantly lower toxicity than traditional synthetic vectors, thus enabling a therapeutic window suitable for clinical translation.