Self-assembled DNA Nanostructures Research Articles

Zeta potential of Z-DNA: A new signature to study B-Z transition in linear and branched DNA

Zeta potential is commonly referred as surface charge density and is a key factor in modulating the structural and functional properties of nucleic acids. Although the negative charge density of B-DNA is well understood, there is no prior description of the zeta potential measurement of Z-DNA. In this study, for the first time we discover the zeta potential difference between B-DNA and lanthanum chloride-induced Z-DNA. A series of linear repeat i.e. (CG)n and (GC)n DNA as well as branched DNA (bDNA) structures was used for the B-to-Z DNA transition. Herein, the positive zeta potential of Z-DNA has been demonstrated as a powerful tool to discriminate between B-form and Z-form of DNA. The generality of the approach has been validated both in linear and bDNA nanostructures. Thus, we suggest zeta potential can be used as an ideal signature for the left-handed Z-DNA.

Self-assembly of DNA nanostructures in different cations

Self-assembly of DNA nanostructures in different cations

Metal-mediated DNA strand displacement and molecular device operations based on base-pair switching of 5-hydroxyuracil nucleobases

Rational design of self-assembled DNA nanostructures has become one of the fastest-growing research areas in molecular science. Particular attention is focused on the development of dynamic DNA nanodevices whose configuration and function are regulated by specific chemical inputs. Herein, we demonstrate the concept of metal-mediated base-pair switching to induce inter- and intramolecular DNA strand displacement in a metal-responsive manner. The 5-hydroxyuracil (UOH) nucleobase is employed as a metal-responsive unit, forming both a hydrogen-bonded UOH–A base pair and a metal-mediated UOH–GdIII–UOH base pair. Metal-mediated strand displacement reactions are demonstrated under isothermal conditions based on the base-pair switching between UOH–A and UOH–GdIII–UOH. Furthermore, metal-responsive DNA tweezers and allosteric DNAzymes are developed as typical models for DNA nanodevices simply by incorporating UOH bases into the sequence. The metal-mediated base-pair switching will become a versatile strategy for constructing stimuli-responsive DNA nanostructures, expanding the scope of dynamic DNA nanotechnology.

Label-Free, Ultralow Detection Limit DNA Biosensor Employing Tilted Fiber Bragg Grating Functionalized by 3-D Nanostructure Probes

A label-free, ultra-low detection limit DNA biosensor based on tilted fiber Bragg grating (TFBG) is proposed and demonstrated. Optical sensing interface of the TFBG is functioned by self-assembled 3D tetrahedral DNA nanostructure (TDN) probes with size of 17 base pairs to greatly enhance the sensing performance for DNA detection. DNA hybridization processes in various concentration of target DNA solutions are monitored according to the transmission spectral evolution of the TFBG in real-time, interrogated by narrow-linewidth laser source. Experimental results show that the detection response of the proposed DNA biosensor is ranged from 1 fM to 10 pM, with an ultra-low detection limit of ~3.4 aM. Single nucleotide mismatch recognition is also achieved. As compared to the sensing performance using single-strand DNA (ssDNA) probes, the detection limit is about 1000 times lower for using the TDN probes. The proposed DNA biosensor possesses desirable merits as label-free method, ultra-low detection limit, high specificity and fast response, making it a promising candidate for sensing applications in bioengineering, disease diagnosis and environmental science.

Self-Assembly of DNA Nanostructures in Different Cations.

The programmable nature of DNA allows the construction of custom-designed static and dynamic nanostructures, and assembly conditions typically require high concentrations of magnesium ions that restricts their applications. In other solution conditions tested for DNA nanostructure assembly, only a limited set of divalent and monovalent ions are used so far (typically Mg2+ and Na+ ). Here, weinvestigate the assembly of DNA nanostructures in a wide variety of ions using nanostructures of different sizes: a double-crossover motif (76 bp), a three-point-star motif (~134 bp), a DNA tetrahedron (534 bp) and a DNA origami triangle (7221 bp). Weshow successful assembly of a majority of these structures in Ca2+ , Ba2+ , Na+ , K+ and Li+ and provide quantified assembly yields using gel electrophoresis and visual confirmation of a DNA origami triangle using atomic force microscopy. Wefurther show that structures assembled in monovalent ions (Na+ , K+ and Li+ ) exhibit up to a 10-fold higher nuclease resistance compared to those assembled in divalent ions (Mg2+ , Ca2+ and Ba2+ ). Ourwork presents new assembly conditions for a wide range of DNA nanostructures with enhanced biostability.

Molecular-electromechanical system for unamplified detection of trace analytes in biofluids.

Biological research and diagnostic applications normally require analysis of trace analytes in biofluids. Although considerable advancements have been made in developing precise molecular assays, the trade-off between sensitivity and ability to resist non-specific adsorption remains a challenge. Here, we describe the implementation of a testing platform based on a molecular-electromechanical system (MolEMS) immobilized on graphene field-effect transistors. A MolEMS is a self-assembled DNA nanostructure, containing a stiff tetrahedral base and a flexible single-stranded DNA cantilever. Electromechanical actuation of the cantilever modulates sensing events close to the transistor channel, improving signal-transduction efficiency, while the stiff base prevents non-specific adsorption of background molecules present in biofluids. A MolEMS realizes unamplified detection of proteins, ions, small molecules and nucleic acids within minutes and has a limit of detection of several copies in 100 μl of testing solution, offering an assay methodology with wide-ranging applications. In this protocol, we provide step-by-step procedures for MolEMS design and assemblage, sensor manufacture and operation of a MolEMS in several applications. We also describe adaptations to construct a portable detection platform. It takes ~18 h to construct the device and ~4 min to finish the testing from sample addition to result.

Framework Nucleic Acids: A Promising Vehicle for Small Molecular Cargos.

Framework nucleic acids (FNAs), which are a series of self-assembled DNA nanostructures, are highly versatile tools for engineering intelligent molecular delivery vehicles. Owing to their precise and controllable design and construction, excellent programmability and functionality, as well as favorable intercalation between DNA and small molecules, FNAs provide a promising approach for small molecule delivery. This review discusses the advantages, applications, and current challenges of FNAs for the delivery of small molecular cargo. First, the physicochemical and biological properties that make FNAs favorable for the transport of small molecules are introduced. Thereafter, the classification of loaded cargos and the mechanism of combination between small molecules and FNAs are summarized in detail, and recent research on FNA-based delivery systems and their applications are highlighted. Finally, the challenges and prospects of FNA nanocarriers are discussed to advance their exploitation and clinical adoption.

Structural DNA tetrahedra and its electrochemical-related surface sensing

Structural DNA tetrahedra, one of the most popular self-assembled DNA nanostructures, show significant potential in the engineering of surface bioanalysis. The structural and functional information encoded in DNA tetrahedra enables various configuration and modification of the tetrahedra. These configurations could be achieved by functionalizing the tetrahedra vertices with different motifs or readout labels, or regulating the tetrahedra edges by integrating different DNA motifs, or controlling the size of the tetrahedra by changing the length of its edges. This review particularly introduces the DNA tetrahedra that shows specific physical features on the electrode interface owing to the size-controllability and programmability, and it summerizes recent advances of DNA tetrahedra on the surface sensing application of electrochemistry, electrochemiliminescence, photochemiluminescence, surface-enhanced Raman scattering. The research field of DNA tetrahedra-based sensing surfaces appears to be at the top of an exciting and potential explosion of the application of bioanalysis, nanomedicine, and environmental and food analysis.

Regulating the Polymerization of DNA Structures via Allosteric Control of Monomers.

Regulation of self-assembly is crucial in constructing structural biomaterials, such as tunable DNA nanostructures. Traditional tuning of self-assembled DNA nanostructures was mainly conducted by introducing external stimuli after the assembly process. Here, we explored the allosteric assembly of DNA structures via introducing external stimuli during the assembly process to produce structurally heterogeneous polymerization products. We demonstrated that ethidium bromide (EB), a DNA intercalator, could increase the left-handed out-of-plane chirality of curved DNA structures. Then, EB and double strands were introduced as competing stimuli to transform monomers into allosteric conformations, leading to three different polymerization products. The steric trap between different polymerization products promoted the polymerized structures to keep their geometric properties, like chirality, under varying intensity of external stimuli. Our strategy harnesses allosteric effects for assembly of DNA-based materials and is expected to expand the design space for advanced control in synthetic materials.

Attenuating endothelial leakiness with self-assembled DNA nanostructures for pulmonary arterial hypertension.

Vascular endothelium dysfunction plays an important role in oncological and pulmonary diseases. Endothelial barrier dysfunction is the initial step of pulmonary vascular remodeling (PVR) and pulmonary arterial hypertension. Upregulation of a pro-autophagy protein Atg101 in the endothelial cells triggered a cascade of intracellular events that leads to endothelial dysfunction through apoptosis. Herein, we proposed a strategy that used endothelial targeting DNA nanostructures to deliver Atg101 siRNA (siAtg101) as a safe, biocompatible "band-aid" to restore pulmonary arterial endothelial barrier integrity within the intricate milieu of pulmonary cells and the pulmonary vasculature. The siAtg101 and aptamer conjugated DNA nanostructures were found to attenuate hypoxia-induced pulmonary endothelial leakiness with surprisingly high selectivity and efficacy. Further in vivo study revealed that functionalized DNA nanostructures likewise attenuated the vascular remodeling in a monocrotaline-induced PVR mouse model. Mechanistically, functionalized DNA nanostructures suppressed PVR by knocking down Atg101, which in turn, downregulated Beclin-1 and subsequently upregulated VE-cadherin to restore endothelial cells' adherin junctions. This work opened a new window for future nanomaterial design that directly addresses the interfacial endothelial cell layer that often stands between the blood and many diseased sites of nanotherapeutic interest.

Self-assembled DNA nanostructure containing oncogenic miRNA-mediated cell proliferation by downregulation of FOXO1 expression

FOXO1 transcription factor not only limits the cell cycle progression but also promotes cell death as a tumor suppressor protein. Though the expression of FOXO1 is largely examined in breast cancer, the regulation of FOXO1 by miRNA is yet to be explored. In the current study, self-assembled branched DNA (bDNA) nanostructures containing oncogenic miRNAs were designed and transfected to the MCF7 cell line to decipher the FOXO1 expression. bDNA containing oncogenic miRNAs 27a, 96, and 182 synergistically downregulate the expression of FOXO1 in MCF7 cells. The down-regulation is evident both in mRNA and protein levels suggesting that bDNA having miRNA sequences can selectively bind to mRNA and inhibit translation. Secondly, the downstream gene expression of p21 and p27 was also significantly downregulated in presence of miR-bDNA nanostructures. The cell proliferation activity was progressively increased in presence of miR-bDNA nanostructures which confirms the reduced tumor suppression activity of FOXO1 and the downstream gene expression. This finding can be explored to design novel bDNA structures which can downregulate the tumor suppressor proteins in normal cells and induce cell proliferation activity to identify early-phase markers of cancer.

A bistable and reconfigurable molecular system with encodable bonds.

Molecular systems with ability to controllably transform between different conformations play pivotal roles in regulating biochemical functions. Here, we report the design of a bistable DNA origami four-way junction (DOJ) molecular system that adopts two distinct stable conformations with controllable reconfigurability by using conformation-controlled base stacking. Exquisite control over DOJ's conformation and transformation is realized by programming the stacking bonds (quasi-blunt-ends) within the junction to induce prescribed coaxial stacking of neighboring junction arms. A specific DOJ conformation may be achieved by encoding the stacking bonds with binary stacking sequences based on thermodynamic calculations. Dynamic transformations of DOJ between various conformations are achieved by using specific environmental and molecular stimulations to reprogram the stacking codes. This work provides a useful platform for constructing self-assembled DNA nanostructures and nanomachines and insights for future design of artificial molecular systems with increasing complexity and reconfigurability.

DNA Nanostructure-Guided Plasmon Coupling Architectures

DNA Nanostructure-Guided Plasmon Coupling Architectures

A label-free impedance-based electrochemical sensor based on self-assembled dendritic DNA nanostructures for Pb2+ detection

Here, a label-free impedance-based electrochemical sensor was developed for the quantitative detection of Pb2+. Using conductive gold nanomaterials as electrode substrate materials can provide sensors with larger specific surface area, action sites and excellent conductivity. DNA nanostructures are used for the determination of biomolecules due to their good properties. The Y-DNA structure is formed by the annealing of three DNA sequences, which acts as a stable structure and forms a dendritic structure in combination with the hybrid chain reaction. In the presence of the target Pb2+, it induces the conversion of specific aptamers into G-quadruplexes, resulting in HCR and Y-DNA loading on the electrodes and a significant change in the impedance value signal. Therefore, the proposed biosensor realizes the quantitative detection of Pb2+. Under the optimal experimental conditions, the concentration of Pb2+ exhibited a linear correlation range from 0.5 to1000 nmol/L with a limit of detection (LOD) of 0.38 nmol/L. The designed sensors have good recoveries in real samples (tap water and tea). This flexible experimental protocol has broad application prospects.

Actuating tension-loaded DNA clamps drives membrane tubulation.

Membrane dynamics in living organisms can arise from proteins adhering to, assembling on, and exerting force on cell membranes. Programmable synthetic materials, such as self-assembled DNA nanostructures, offer the capability to drive membrane-remodeling events that resemble protein-mediated dynamics but with user-defined outcomes. An illustrative example is the tubular deformation of liposomes by DNA nanostructures with purposely designed shapes, surface modifications, and self-assembling properties. However, stimulus-responsive membrane tubulation mediated by DNA reconfiguration remains challenging. Here, we present the triggered formation of membrane tubes in response to specific DNA signals that actuate membrane-bound DNA clamps from an open state to various predefined closed states, releasing prestored energy to activate membrane deformation. We show that the timing and efficiency of vesicle tubulation, as well as the membrane tube widths, are modulated by the conformational change of DNA clamps, marking a solid step toward spatiotemporal control of membrane dynamics in an artificial system.

Assembling defined DNA nanostructures with anticancer drugs: a metformin/DNA complex nanoplatform with a synergistic antitumor effect for KRAS-mutated lung cancer therapy

Herein, a strategy is proposed to simultaneously deliver the small-molecule drug metformin and siRNA with self-assembled DNA nanostructures. The biomedical application of DNA nanostructures is highly promising but still in its infancy. DNA nanostructures as drug delivery vehicles are conventionally synthesized in a magnesium-containing buffer. We propose using an anticancer drug to assemble DNA nanostructures and deliver them with siRNA for synergistic anticancer therapy. The metformin cargo induces DNA self-assembly into well-defined, uniform nanostructures, producing a drug–DNA nanocomplex with multiple functionalities for cancer therapy. Both tile-based and DNA origami structures can be assembled with metformin. The as-prepared metformin/DNA nanocomplex showed high structural and thermal stability and enzymatic resistance in physiological settings. The metformin in the nanocomplex and the KRASG12C siRNA exerted a strong, synergistic antitumor effect against KRAS-mutated non-small cell lung cancer (NSCLC) both in vitro and in vivo by suppressing the RAS/AKT/mTOR and AMPK/AKT/mTOR signaling pathways. The current study suggests that the assembly of complex DNA nanomaterials with carefully chosen small molecules is key to endowing DNA nanostructures with new functionalities and subsequently expanding their applications in multidisciplinary research fields.

Engineering the Surface Properties of DNA Nanostructures by Tuning the Valency of Assembling Species for Biomedical Applications.

Self-assembled DNA nanostructures hold great potentials in biomedical applications. Nevertheless, the negatively charged DNA backbone and susceptivity to enzyme degradation pose challenges to this regard. Engineering the surface properties of DNA nanostructures by assembling DNA with guest molecules in magnesium-free system is promising to solve these issues. In this study, the polyamines-mediated DNA self-assembly with an emphasis on the valency of polyamines is investigated. Both spermine, spermidine, and putrescine can assemble DNA tetrahedron under appropriate concentrations. The cytotoxicity and cellular uptake efficiencies vary with the polyamine valency. Compared with magnesium-assembled DNA tetrahedron, polyamine-assembled DNA tetrahedron exhibits higher cellular uptake efficiency and serum stability. Circular dichroism spectrum results indicate that polyamines induce DNA conformation slightly shifting from B form to A form. The improved performances of polyamine-assembled DNA tetrahedrons under physiological settings are attributed to the surface properties that altered by guest molecules polyamine. The current study suggests that engineering the surface properties of DNA nanostructures by assembling them with guest cationic species is promising to further their biomedical applications.

Self-assembled tetrahedral DNA nanostructures-based ultrasensitive label-free detection of ampicillin.

Antibiotics are widely used for improving the living conditions of livestock. However, residual antibiotics present in animal products induce several human diseases. Therefore, a simple, rapid, and cost-effective system for detecting and monitoring the presence of antibiotics in foods is in great demand to alleviate safety concerns. In this study, a highly sensitive and selective aptameric electrochemical sensing platform was designed based on nanomaterial modification and DNA nanotechnology. Electrochemically reduced graphene oxide and gold nanoparticles were used to modify the working surface of a screen-printed electrode to enhance electrical conductivity and biocompatibility. The electrode surface was further modified with self-assembled tetrahedral DNA nanostructures (TDN) to improve the detection sensitivity. The TDN allowed controlling the nano-spacing of aptamers immobilized on the electrode surface and placing aptamers in a solution-phase-like detecting environment to improve the target-binding efficiency without signal amplification modules. Differential pulse voltammetry was employed to measure electrical signals in proportion to the amount of ampicillin, the target antibiotic, present in buffer and spiked milk samples. The designed aptasensor was able to detect and measure the target ampicillin in less than 30min over a wide concentration range of 10 pM to 1mM, with a limit of detection of 1 pM, which is 100 times better than when using the same sensing probe without TDN modification. The aptasensor was reusable by simply rinsing with deionized water, remained stable during 15-day storage, and yielded reproducible results.

A label-free and selective electrochemical aptasensor for ultrasensitive detection of Di(2-ethylhexyl) phthalate based on self-assembled DNA nanostructure amplification

• An electrochemical aptasensor was constructed to detect Di(2-ethylhexyl) phthalate. • The aptasensor has the advantages of easy operation, label-free and low cost. • Utilizing self-assembly of DNA nanostructures for amplifying detection signal. • The aptasensor shows wide linear range and low detection limit of 0.04 ng mL −1 . • The aptasensor shows good reproducibility and high selectivity for DEHP assay. Di(2-ethylhexyl) phthalate (DEHP) is an endocrine disrupting compound that is often used as a plasticizer and tends to migrate in the environment and accumulates in the human body causing harm to humans. Thus, a simple, label-free, enzyme-free, high selective and ultrasensitive electrochemical aptasensor has been constructed for detection of DEHP based on the self-assembly of DNA nanostructure signal amplification strategy in this paper. The DNA-junction nanostructure is made of hairpin 1 (HP1), hairpin 2 (HP2), hairpin 3 (HP3) and quarter sequence (QS). Without DEHP, the electrochemical detection response is very low. Only when the aptamer binds to the target and detaches from the electrode surface, the DNA-junction is trapped on the electrode surface, enhancing the electrochemical signal. This is because the DNA-junction nanostructure is rich in guanine, which is able to adsorb more methylene blue (MB) for the purpose of enhancing the detection signal. The linear detection range of the obtained aptasensor is 0.1 ng mL −1 to 5000 ng mL −1 and the limit detection is 0.04 ng mL −1 . Finally, the proposed aptasensor has been applied to analyze DEHP in real samples, laying the foundation for its utilization in environmental water samples and food safety.

Stress in DNA Gridiron Facilitates the Formation of Two-Dimensional Crystalline Structures.

Programmable DNA nanotechnology has generated some of the most intricate self-assembled nanostructures and has been employed in a growing number of applications, including functional nanomaterials, nanofabrication, biophysics, photonics, molecular machines, and drug delivery. An important design rule for DNA nanostructures is to minimize the mechanical stress to reduce the potential energy in these nanostructures whenever it is possible. This work revisits the DNA gridiron design consisting of Holliday junctions and compares the self-assembly of the canonical DNA gridiron with a new design of DNA gridiron, which has a higher degree of mechanical stress because of the interweaving of DNA helices. While the interweaving DNA gridiron indeed exhibits lower yield, compared to its canonical counterpart of a similar size, we discover that the mechanical stress within the interweaving gridiron can promote the formation of the two-dimensional crystalline lattice instead of nanotubes. Furthermore, tuning the design of interweaving gridiron leads to the change of overall crystal size and regularity of geometry. Interweaving DNA double helices represents a new design strategy in the self-assembly of DNA nanostructures. Furthermore, the discovery of the new role of mechanical stress in the self-assembly of DNA nanostructures provides useful knowledge to DNA nanotechnology practitioners: a more balanced view regarding mechanical stress can be considered when designing future DNA nanostructures.