Self-assembled DNA Research Articles

Self-Assembled DNA Nanostructures-Based Nanocarriers Enabled Functional Nucleic Acids Delivery.

Nucleic acids are recognized as the blueprints of life and the repositories of genetic information, which play essential roles in the "central dogma of molecular biology". As classical and specific nucleic acids, apart from carrying genetic information, functional nucleic acids (FNAs) perform many intriguing functions such as targeted recognition, therapy and enzymatic catalysis. FNAs with excellent biocompatibility provide great promise for future applications in bioanalysis, material science, and nanotechnology. Nevertheless, effective delivery of FNAs to the targeted location still suffers from one or more challenges, especially in the field of biomedicine. Encouragingly, DNA nanotechnology shows great potential for applications in bioimaging, biosensing, and molecules delivery. Importantly, it provides an unprecedented opportunity to design and synthesize a series of self-assembled DNA nanostructures with well-defined size, shape, surface chemistry, and function. Through Watson-Crick base-pairing rules, FNAs, including aptamers, DNAzymes, small interfere RNA (siRNA), antisense oligonucleotides (ASOs), and unmethylated cytosine-phosphate-guanine dinucleotide oligonucleotides (CpG ODNS), are successfully decorated with self-assembled DNA nanostructures for delivery. In this progress report, we focus on self-assembled DNA nanostructures-based nanocarriers to deliver FNAs for applications in cellular analysis and cancer theranostics. First, we briefly summarize the superior properties of the prospective nanocarriers and introduce the self-assembled DNA nanostructures. Then we mainly highlight the most recent achievements of self-assembled DNA nanostructures as comers for the delivery of FNAs. Finally, this review points to some of the current challenges in the applications of self-assembled DNA nanostructures as FNAs nanocarriers and provides an insight into the future perspectives of self-assembled DNA nanostructures-based nanocarriers for FNAs delivery.

Tailoring DNA Self-assembly to Build Hydrogels.

DNA hydrogels are crosslinked polymeric networks in which DNA is used as the backbone or the crosslinker. These hydrogels are novel biofunctional materials that possess the biological character of DNA and the framed structure of hydrogels. Compared with other kinds of hydrogels, DNA hydrogels exhibit not only high mechanical strength and controllable morphologies but also good recognition ability, designable responsiveness, and programmability. The DNA used in this type of hydrogel acts as a building block for self-assembly or as a responsive element due to its sequence recognition ability and switchable structural transitions, respectively. In this review, we describe recent developments in the field of DNA hydrogels and discuss the role played by DNA in these hydrogels. Various synthetic strategies for and a range of applications of DNA hydrogels are detailed.

Enhanced Immunostimulatory Activity of a Cytosine-Phosphate-Guanosine Immunomodulator by the Assembly of Polymer DNA Wires and Spheres.

Unmethylated cytosine-phosphate-guanosine (CpG) oligodeoxynucleotides are immunostimulatory nucleic acids wildly utilized as adjuvants or for vaccines to treat diseases. However, there is a lack of simple and efficient vectors for CpG oligodeoxynucleotide delivery with long-lasting immune stimulation. Herein, self-assembled polymer wires consisting of CpG motifs by hybridization chain reaction were constructed with excellent biocompatibility and immunostimulatory activity. The designed polymer DNA wires acted as programmable multivalent immunoadjuvants and triggered immune response, stimulated pro-inflammatory cytokine secretion, and induced the apoptosis of cancer cells. More strikingly, polymer nanospheres assembled from the polymer DNA wires and cationic poly-l-lysine further improved cellular uptake and continuously stimulate the lysosomal Toll-like receptor 9 of immune cells, thereby remarkably enhancing the activation of immune cells. These results demonstrated that self-assembled polymer DNA nanoassemblies with multivalent CpG could trigger strong immune response and further induce cancer cell death.

The poly-thymine based DNA photolithography onto electrostatic coupling substrates.

In order to develop a rapid and high fidelity process for DNA self-assembly with patterning, the pattern of thymine dimerization is presented onto electrostatically bound DNA substrate by photolithography. The ability of binding for the process, which is attenuated conditions such as contact with photomask and washing by solution buffer is evaluated by X-ray photoelectron spectroscopy (XPS). Through thymine dimerization and hybridization chain reaction (HCR), DNA patterns, including multi-patterns, are demonstrated. For expansion to protein molecular patterning, the target DNA is tethered to biotin, allowing patterning with streptavidin linked fluorophores such as Cy3-streptavidin and phyecocayine-streptavidin.

<p>Targeted Therapy of Colon Cancer by Aptamer-Guided Holliday Junctions Loaded with Doxorubicin</p>

PurposeChemotherapy is the primary treatment for advanced colon cancer, but its efficacy is often limited by severe toxicities. Targeted therapy in the form of selectively drug delivery system (SDDS) is an important strategy to reduce adverse effects. Here, we aim to design a novel SDDS with potential for practical application using biocompatible components and scalable production process, for targeted delivery of doxorubicin (Dox) to colon cancer cells.MethodsThe SDDS was made of a self-assembled DNA nano-cross (Holliday junction, or HJ) functionalized by four AS1411 aptamers (Apt-HJ) and loaded with Dox.ResultsApt-HJ had an average size of 12.45 nm and a zeta potential of −11.6 mV. Compared with the monovalent AS1411 aptamer, the quadrivalent Apt-HJ showed stronger binding to target cancer cells (CT26). A complex of Apt-HJ and doxorubicin (Apt-HJ-Dox) was formed by intercalating Dox into the DNA structure of Apt-HJ, with each complex carrying approximately 17 Dox molecules. Confocal microscopy revealed that Apt-HJ-Dox selectively delivered Dox into CT26 colon cancer cells but not the control cells. Moreover, Apt-HJ-Dox achieved targeted killing of CT26 cancer cells in vitro and reduced the damage to control cells. Importantly, compared with free Dox, Apt-HJ-Dox significantly enhanced the antitumor efficacy in vivo without boosting the adverse effects.ConclusionThese results suggest that Apt-HJ-Dox has application potential in targeted treatment of colon cancer.

Scaling Up DNA Self-Assembly

Recent advances in structural DNA nanotechnology, including DNA origami and DNA bricks, have enabled arbitrarily complexed nanopatterns. However, most of these DNA structures are limited with sub-100 nm dimensions because of the limits from the length of scaffold strand, as well as the sequence library. This review will focus on different strategies for scaling-up DNA self-assembly, including the hierarchical assembly of the preformed DNA building blocks both in solution and on surface, the scaffolded assembly of finite sized DNA structures, the nonhierarchical assembly of single-stranded DNA bricks, and the seed-mediated algorithmic assembly. The design criteria, the building blocks, and the key assembly conditions for each assembly strategy are described. In addition, the future challenges, as well as application potentials of large-area DNA structures, are discussed.

Chiral Ru(ii) complexes act as a potential non-viral gene carrier for directional transportation to the nucleus and cytoplasm.

Guanine-rich DNA sequences can spontaneously fold into four-stranded structures called G-quadruplexes (G4s). G4s have been identified extensively in the promoter regions of several proto-oncogenes, including c-myc, as well as telomeres. G4s have attracted an increasing amount of attention in the field of nanotechnology because of their use as versatile building blocks of DNA-based nanostructures. In this study, we report the self-assembly of c-myc G-quadruplex DNA controlled by a pair of chiral ruthenium(ii) complexes coordinated by 2-(4-phenyacetylenephenyl)-1H-imidazo[4,5f][1,10]phenanthroline (PBEPIP), Λ-[Ru(bpy)2(PBEPIP)](ClO4)2 (Λ-RM0627, bpy = bipyridine) and Δ-[Ru(bpy)2(PBEPIP)](ClO4)2 (Δ-RM0627). Λ-RM0627 could promote the high-order self-assembly of c-myc G-quadruplex DNA into a nanowire structure, whereas Δ-RM0627 could induce DNA condensation into G-quadruplex aggregates. Moreover, in vitro studies on human liver carcinoma HepG2 cells showed that the nanowire of c-myc G-quadruplex DNA promoted by Λ-RM0627 could be localized in the nuclei of cells, whereas the nanoparticle of c-myc G-quadruplex DNA generated by Δ-RM0627 was taken up and localized in the cytoplasm. This study provides examples of the enantioselective self-assembly of G4 DNA molecules controlled by chiral ruthenium(ii) complexes and suggests the potential applications of assembled nanostructures as non-viral DNA vectors for gene therapy.

Elucidation of leak-resistance DNA hybridization chain reaction with universality and extensibility.

Hybridization chain reaction (HCR) was a significant discovery for the development of nanoscale materials and devices. One key challenge for HCR is the vulnerability to background leakage in the absence of the initiator. Here, we systematically analyze the sources of leakage and refine leak-resistant rule by using molecular thermodynamics and dynamics, biochemical and biophysical methods. Transient melting of DNA hairpin is revealed to be the underlying cause of leakage and that this can be mitigated through careful consideration of the sequence thermodynamics. The transition threshold of the energy barrier is proposed as a testing benchmark of leak-resistance DNA hairpins. The universal design of DNA hairpins is illustrated by the analysis of hsa-miR-21-5p as biomarker when used in conjunction with surface-enhanced Raman spectroscopy. We further extend the strategy for specific signal amplification of miRNA homologs. Significantly, it possibly provides a practical route to improve the accuracy of DNA self-assembly for signal amplification, and that could facilitate the development of sensors for the sensitive detection of interest molecules in biotechnology and clinical medicine.

Dynamic Imaging of Moving Cells Based on DNA Nanotechnology

Nanostructures based on DNA self-assembly have made great progress in recent years, and have been widely used. Therefore, the research method of dynamic imaging of moving cells based on DNA nanotechnology is proposed. The dynamic imaging process of moving cells is studied from two kinds of DNA Nanotechnologies: DNA gene locus marker and DNA nano-fluorescence probe. Among them, crispr-cas9 system is used to mark DNA gene sites through in vitro fluorescent labeling of different sgRNAs, which can mark different gene sites in cells in multi-color and track gene sites in living cells dynamically; based on self-assembled CdTe quantum dot @ AnnexinV-DNA nanowire fluorescent probe, based on the DNA fitness of moving cells, the system is designed to be specific to target moving cells. The specific detection of motor cells can be realized by combining the sequences. The key point is to use the structural characteristics of the probe to realize the indication of the physiological changes of the drug receiving cells. The results show that: the polychromatic labeling of different gene sites in the same cell can be realized by using different fluorescent labeled sgRNA. By using fluorescent labeled telomere specific sgRNA to track the movement of telomeres in living cells, it can be seen from the movement track that in living cells, Ma SAT is more flexible than that in living cells. About 84.5% of Ma SAT is not mobile, but only 12.9% for telomere. Ma sat and the direction of telomere movement in the cell nucleus are random and in all directions. CdTe QD @ AnnexinV-DNA nanowire fluorescent probe can not only detect the moving cells, but also indicate the physiological state of normal and apoptotic cells at the same time. A large number of S7 ends of DNA allow the probe to firmly bind to the surface of Ramos cells in the way of "twine ball." The probe can indicate the physiological state of cells, and the corresponding characteristic emission wavelength changes with the change of target cell movement state. It shows that DNA nanotechnology can be used to analyze the dynamic imaging of moving cells in all directions and from multiple angles.

Coating and Stabilization of Liposomes by Clathrin-Inspired DNA Self-Assembly.

The self-assembly of the protein clathrin on biological membranes facilitates essential processes of endocytosis and has provided a source of inspiration for materials design by the highly ordered structural appearance. By mimicking the architecture of the protein building blocks and clathrin self-assemblies to coat liposomes with biomaterials, advanced hybrid carriers can be derived. Here, we present a method for fabricating DNA-coated liposomes by hydrophobically anchoring and subsequently connecting DNA-based triskelion structures on the liposome surface inspired by the assembly of the protein clathrin. Dynamic light scattering, ζ-potential, confocal microscopy, and cryo-electron microscopy measurements independently demonstrate successful DNA coating. Nanomechanical measurements conducted with atomic force microscopy show that the DNA coating enhances the mechanical stability of the liposomes relative to uncoated ones. Furthermore, we provide the possibility to reverse the coating process by triggering the disassembly of the DNA coats through a toehold-mediated displacement reaction. Our results describe a straightforward, versatile, and reversible approach for coating and stabilizing lipid vesicles through the assembly of rationally designed DNA structures. This method has potential for further development toward the ordered arrangement of tailored functionalities on the surface of liposomes and for applications as hybrid nanocarriers.

PH-Responsive Reversible DNA Self-assembly Mediated by Zwitterion

pH-Responsive DNA assembles have drawn growing attentions owing to their great potential in diverse areas. However, pH-responsive motifs are limited to specific DNA sequences and annealing is usually needed for DNA assemblies; therefore, sequence-independent pH-responsive DNA assembly at room temperature is highly desired as a more general way. Here, we propose a reversible pH-responsive DNA assembly strategy at room-temperature using zwitterion, glycine betaine(GB), as charge-regulation molecules. The reversible assembly and disassembly of DNA nanostructures could be achieved by alternatively regulating the acidic and basic environments in the presence of GB, respectively. In an acidic environment, carboxylate group in GB was protonated and GB was positively charged, which facilitated to shield the inherent electrostatic repulsion of DNA strands. Molecular simulation showed that the newly formed carboxyl group in protonated GB could form hydrogen bonds with bases in DNA to promote the assembly of DNA strands. In a basic solution, carboxylate group in GB was deprotonated and GB was neutral, thus inducing the dissociation of DNA assembly.

Engineering a label- and enzyme-free detection of HIV-DNA on a cyclic DNA self-assembling strategy using G-triplexes as the signal reporter

An efficient amplification strategy based on the G-triplex DNA structure was developed for label- and enzyme-free fluorescence detection of human immunodeficiency virus (HIV) DNA. In this system, three hairpin molecules (H1, H2, and H3) were used to detect the target and amplify the signal. Fluorescence enhancement was achieved by producing more G-triplexes to bind with Thioflavine T. A detection limit of 33 pM with good linearity in the range from 400 pM to 110 nM was observed. This method avoided using of enzyme and cumbersome labeling procedures and was relatively superior to previous G-quadruplex-based approaches, which often encounter some difficulties in control and excitation. Moreover, the application for detecting HIV-DNA in biological fluids indicates that further applications for clinical diagnosis are expected.

Shape-controllable Synthesis of Functional Nanomaterials on DNA Templates

DNA nanotechnology enables precise organization of nanoscale objects with extraordinarily structural programmability. Self-assembled DNA nanostructures possess a lot of interesting features, such as designable size and shape, and structural addressability at nanometer scale. Taking advantage of these properties, DNA nanostructures could work as templates or molds for the controllable synthesis of functional nanomaterials, such as organic macromolecules, metallic or inorganic nonmetallic nanomaterials. In this review, we summarize the recent progress in the shape-controllable synthesis of functional nanomaterials on DNA templates. The potential application fields of these nanomaterials are also discussed.

Precision-Guided Missile-Like DNA Nanostructure Containing Warhead and Guidance Control for Aptamer-Based Targeted Drug Delivery into Cancer Cells in Vitro and in Vivo.

It is crucial to deliver anticancer drugs to target cells with high precision and efficiency. While nanomaterials have been shown to enhance the delivery efficiency once they reach the target, it remains challenging for precise drug delivery to overcome the nonspecific adsorption and off-target effect. To meet this challenge, we report herein the design of a novel DNA nanostructure to act as a DNA nanoscale precision-guided missile (D-PGM) for highly efficient loading and precise delivery of chemotherapeutic agents to specific target cells. The D-PGM consists of two parts: a warhead (WH) and a guidance/control (GC). The WH is a rod-like DNA nanostructure as a drug carrier, whose trunk is a three-dimensionally self-assembled DNA nanoscale architecture from the programmed hybridization among two palindromic DNA sequences in the x-y dimension and two common DNA oligonucleotides in the z direction, making the WH possess a high payload capacity of drugs. The GC is an aptamer-based logic gate assembled in a highly organized fashion capable of performing cell-subtype-specific recognition via the sequential disassembly, mediated by cell-anchored aptamers. Because of the cooperative effects between the WH and the GC, the GC logic gates operate like the guidance and control system in a precision-guided missile to steer the doxorubicin (DOX)-loaded DNA WH toward target cancer cells, leading to selective and enhanced therapeutic efficacy. Moreover, fluorophores attached to different locations of D-PGM and DOX fluorescence dequenching upon release enable intracellular tracing of the DNA nanostructures and drugs. The results demonstrate that by mimicking the functionalities of a military precision-guided missile to design the sequential disassembly of the GC system in multistimuli-responsive fashion, our intrinsically biocompatible and degradable D-PGM can accurately identify target cancer cells in complex biological milieu and achieve active targeted drug delivery. The success of this strategy paves the way for specific cell identity and targeted cancer therapy.

DNA Framework-Encoded Mineralization of Calcium Phosphate

Summary Nature has evolved unique strategies to generate delicate biominerals at various length scales with genetically encoded protein scaffolds. Nevertheless, the ability to design programmable biomimetic mineralized nanostructures remains limited. Here, we report that self-assembled DNA frameworks can encode the nanoscale mineralization of calcium phosphate (CaP) with precision and versatility. We establish that the size and shape of DNA-CaP nanostructures are programmed by the structural information encoded within DNA sequences and the electrostatic interactions between DNA phosphate backbone and mineral counterparts. The generality of this strategy is verified by using two- and three-dimensional DNA framework ranging from ∼10 to ∼100 nm. We further find that CaP mineralization-solidified DNA framework functions as a sustainable nanoagent for live-cell delivery of drugs. These findings open a new avenue for nanoscale mineralization with unprecedented sophisticated architectures for versatile applications.

A DNA tetrahedron nanoprobe-based fluorescence resonance energy transfer sensing platform for intracellular tumor-related miRNA detection.

Accurate and sensitive detection of disease-related microRNAs (miRNAs) is of great significance for early disease diagnosis. In this work, a DNA tetrahedron nanoprobe (DTNP)-based fluorescence resonance energy transfer (FRET) sensing platform (termed DTNP sensor) was constructed for sensitive detection of tumor-related miRNA (e.g., hsa-miR-146b-5p) with DNA assisted cyclic amplification. DTNP was synthesized by DNA self-assembly. In the absence of hsa-miR-146b-5p, the fluorescence DNA (HP) modified with FAM at the 5' terminal and TAMRA at the 3' terminal cannot form the hairpin structure because of the hybridization with the extended DNA strand of the DNA tetrahedron, resulting in a low FRET effect. In the presence of hsa-miR-146b-5p, it would complementarily hybridize with the extended DNA strand of the DNA tetrahedron, leading to the release of HP and occurrence of strong FRET. Thus, the concentration of hsa-miR-146b-5p can be revealed by the change in the fluorescence intensity. Moreover, an assistant DNA was employed to replace hsa-miR-146b-5p for cyclic signal amplification, which can further enhance the detection sensitivity. Under the optimal experimental conditions, the limit of detection for hsa-miR-146b-5p was as low as 6 pM (S/N = 3). Furthermore, the DTNP sensor was successfully applied to evaluate the hsa-miR-146b-5p expression levels in different cell lines. The inhibition of hsa-miR-146b-5p expression in different cells was also investigated and a satisfactory result was obtained.

A novel self-assembled Na{Cu12Zn4} multifunctional material: first report of a discrete coordination compound for detection of Ca2+ ions and selective adsorption of cationic dyes in water.

A novel metal directed discrete self-assembled compound, Na2O[Cu12Zn4(μ-OH)8(H4btp)8](ClO4)8·26H2O (1), was synthesized employing a flexible aminoalcohol ligand, 2,2'-(propane-1,3-diyldiimino)bis-[2-(hydroxylmethyl)propane-1,3-diol] (H6btp), and mixed metal ions (CuII and ZnII) in order to explore it as a multifunctional material. The molecular cluster is characterized by spectral and analytical techniques, single crystal X-ray diffraction and magnetic studies. Crystallography reveals the presence of eight peripheral CuII centers in octahedral environments, while four interior CuII and ZnII centers are present in tetrahedral systems. The ligand is coordinated in the dianionic form (H4btp2-) with copper and zinc metal ions through various bridging modes. Magnetic data suggest the presence of strong antiferromagnetic coupling between the twelve Cu(ii) centers (C = 4.31 cm3 K mol-1 and θ = -120.44 K). Interestingly, the eight ligands present in the system have eight free hydroxyl groups at the periphery of the cluster, which could be employed in molecular recognition for the analytes under investigation. One of the sodium cations of the lattice sodium oxide [Na2O or Na+(Na+O2-)] is encapsulated in the {Cu12Zn4} cavity and held through electrostatic interactions with the oxygen atoms, forming a Na{Cu12Zn4} core. Taking advantage of this entrapped cation, we have used the present assembly in the sensing of various s-block cations. Interestingly, the cluster shows a sensitive and selective sensing ability towards Ca2+ ions through cation exchange. The selectivity towards Ca2+ can be rationalized in terms of the identical size of the sodium and calcium ions and the HSAB principle. Hard sodium is exchanged with the hard Ca2+ ion, which effectively interacts with the hard oxygen donor, giving rise to a stable hard-hard interaction. Moreover the Na{Cu12Zn4} cluster shows excellent selectivity towards the adsorption of cationic dyes, specifically methylene blue (MB), via non-covalent interactions. An abundance of free O-H groups in the cluster gives rise to the existence of O-Hπ interactions with the dye for enhanced adsorption. Thus, the present report is the first example of any heterometallic coordination compound (in contrast to complex systems like polymeric frameworks/nanocomposites) with discriminating sensing of Ca2+ ions with ultra-low detection limit (4 nM) and with high efficiency in the selective adsorption and separation of methylene blue.

Programmable 3D rigid clathrate hydrogels based on self-assembly of tetrahedral DNA and linker PCR products.

A clathrate tetrahedral DNA gel was assembled by combining tetrahedral DNA and rigid linker PCR products to achieve visible detection of Salmonella spp. This method overcame the shortcomings of AuNPs in coloration and enriched the use of tetrahedral DNA for the visible detection of virtually any target concerned with pathogens.

A tetrahedral DNA nanostructure-decorated electrochemical platform for simple and ultrasensitive EGFR genotyping of plasma ctDNA.

Genotyping of the epidermal growth factor receptor (EGFR) mutation status is of great importance in the screening of appropriate patients with advanced non-small cell lung carcinoma (NSCLC) to receive superior tyrosine kinase inhibitor (TKIs) therapy. Yet conventional assays are generally costly with a relatively long turnaround time for obtaining results, which can lead to a bottleneck for immediately starting TKI therapy in late-staged patients. In this study, we propose an on-site electrochemical platform for sensitive simultaneous genotyping of the two major EGFR mutations (19del and L858R) through plasma ctDNA based on tetrahedral DNA nanostructure decorated screen-printed electrodes (SPE). Linear-after-the-exponential (LATE)-PCR combined with the amplification refractory mutation system (ARMS) was adopted to produce abundant biotin-labeled single-stranded DNA with high amplification efficiency and specificity. Disposable SPE decorated with self-assembled tetrahedral nanostructured DNA probes that showed ordered orientation and good target accessibility enabled the highly efficient hybridization of the specific amplicons through a sandwich-type and quantitatively translated the interfacial hybridization event into electrochemical signals via enzymatic amplification. Taking advantage of the ARMS-based LATE-PCR and the tetrahedral nanostructure-decorated SPE platform, we achieved the accurate detection of around 30 pg DNA of 19del or L858R, or as low as 0.1% of them in the presence of wild-type DNA. Moreover, the EGFR mutation profiles of 13 NSCLC patients we enlisted were accurately genotyped by our electrochemical platform, the results of which were in good agreement with those of commercial genetic detection methods.

A self-delivery DNA nanoprobe for reliable microRNA imaging in live cells by aggregation induced red-shift-emission.

A new DNA nanoprobe based on a Y-shape and pyrene-modified DNA self-assembly is developed to sensitively and specifically detect microRNA through a pyrene excimer-monomer switch. Exhibiting the capability of self-delivery and resistance to nuclease degradation, the nanoprobe has been successfully applied for microRNA imaging in live cells.