3D DNA Nanostructures Research Articles

A highly versatile platform based on geometrically well-defined 3D DNA nanostructures for selective recognition and positioning of multiplex targets.

We develop a versatile recognition system based on 3D triangular-shaped DNA nanotubes by integrating three different aptamer sequences along the three edges. This would allow multiple binding activities to be combined into a single system. The versatility of this nanotube platform can also provide a framework for spatial orientation and positioning of different aptamer-binding ligands in a 'pea-pod' architecture.

Addressing the instability of DNA nanostructures in tissue culture.

DNA nanotechnology is an advanced technique that could contribute diagnostic, therapeutic, and biomedical research devices to nanomedicine. Although such devices are often developed and demonstrated using in vitro tissue culture models, these conditions may not be compatible with DNA nanostructure integrity and function. The purpose of this study was to characterize the sensitivity of 3D DNA nanostructures produced via the origami method to the in vitro tissue culture environment and identify solutions to prevent loss of nanostructure integrity. We examined whether the physiological cation concentrations of cell culture medium and the nucleases present in fetal bovine serum (FBS) used as a medium supplement result in denaturation and digestion, respectively. DNA nanostructure denaturation due to cation depletion was design- and time-dependent, with one of four tested designs remaining intact after 24 h at 37 °C. Adjustment of medium by addition of MgSO4 prevented denaturation. Digestion of nanostructures by FBS nucleases in Mg2+-adjusted medium did not appear design-dependent and became significant within 24 h and when medium was supplemented with greater than 5% FBS. We estimated that medium supplemented with 10% FBS contains greater than 256 U/L equivalent of DNase I activity in digestion of DNA nanostructures. Heat inactivation at 75 °C and inclusion of actin protein in medium inactivated and inhibited nuclease activity, respectively. We examined the impact of medium adjustments on cell growth, viability, and phenotype. Adjustment of Mg2+ to 6 mM did not appear to have a detrimental impact on cells. Heat inactivation was found to be incompatible with in vitro tissue culture, whereas inclusion of actin had no observable effect on growth and viability. In two in vitro assays, immune cell activation and nanoparticle endocytosis, we show that using conditions compatible with cell phenotype and nanostructure integrity is critical for obtaining reliable experimental data. Our study thus describes considerations that are vital for researchers undertaking in vitro tissue culture studies with DNA nanostructures and some potential solutions for ensuring that nanostructure integrity and functions are maintained during experiments.

Imparting biomolecules to a metal-organic framework material by controlled DNA tetrahedron encapsulation.

Recently, the incorporation of biomolecules in Metal-organic frameworks (MOFs) attracts many attentions because of controlling the functions, properties and stability of trapped molecules. Although there are few reports on protein/MOFs composites and their applications, none of DNA/MOFs composite is reported, as far as we know. Here, we report a new composite material which is self-assembled from 3D DNA (guest) and pre-synthesized MOFs (host) by electrostatic interactions and hydrophilic interactions in a well-dispersed fashion. Its biophysical characterization is well analyzed by fluorescence spectroscopy, quartz crystal microbalance (QCM) and transmission electron microscopy (TEM). This new composite material keeps 3D DNA nanostructure more stable than only 3D DNA nanostructure in DI water at room temperature, and stores amounts of genetic information. It will make DNA as a guest for MOFs and MOFs become a new platform for the development of DNA nanotechnology.

Electrochemical switching with 3D DNA tetrahedral nanostructures self-assembled at gold electrodes.

Nanomechanical switching of functional three-dimensional (3D) DNA nanostructures is crucial for nanobiotechnological applications such as nanorobotics or self-regulating sensor and actuator devices. Here, DNA tetrahedral nanostructures self-assembled onto gold electrodes were shown to undergo the electronically addressable nanoswitching due to their mechanical reconfiguration upon external chemical stimuli. That enables construction of robust surface-tethered electronic nanodevices based on 3D DNA tetrahedra. One edge of the tetrahedron contained a partially self-complementary region with a stem-loop hairpin structure, reconfigurable upon hybridization to a complementary DNA (stimulus DNA) sequence. A non-intercalative ferrocene (Fc) redox label was attached to the reconfigurable tetrahedron edge in such a way that reconfiguration of this edge changed the distance between the electrode and Fc.

Ultrasensitive electrochemical cocaine biosensor based on reversible DNA nanostructure

We proposed an ultrasensitive electrochemical cocaine biosensor based on the three-dimensional (3D) DNA structure conversion of nanostructure from Triangular Pyramid Frustum (TPFDNA) to Equilateral Triangle (ETDNA). The presence of cocaine triggered the aptamer-composed DNA nanostructure change from “Close” to “Open”, leading to obvious faradaic impedance changes. The unique properties with excellent stability and specific rigid structure of the 3D DNA nanostructure made the biosensing functions stable, sensitive, and regenerable. The Faradaic impedance responses were linearly related to cocaine concentration between 1.0nM and 2.0μM with a correlation coefficient of 0.993. The limit of detection was calculated to be 0.21nM following IUPAC recommendations (3Sb/b). It is expected that the distinctive features of DNA nanostructure would make it potentially advantageous for a broad range of biosensing, bionanoelectronics, and therapeutic applications.

Drug delivery systems based on nucleic acid nanostructures

The field of DNA nanotechnology has progressed rapidly in recent years and hence a large variety of 1D-, 2D- and 3D DNA nanostructures with various sizes, geometries and shapes is readily accessible. DNA-based nanoobjects are fabricated by straight forward design and self-assembly processes allowing the exact positioning of functional moieties and the integration of other materials. At the same time some of these nanosystems are characterized by a low toxicity profile. As a consequence, the use of these architectures in a biomedical context has been explored. In this review the progress and possibilities of pristine nucleic acid nanostructures and DNA hybrid materials for drug delivery will be discussed. For the latter class of structures, a distinction is made between carriers with an inorganic core composed of gold or silica and amphiphilic DNA block copolymers that exhibit a soft hydrophobic interior.

Fractal Networks of Real Worlds of Fluorescing DNA in Complete Set of Chromosomes inside Blood Cells for Medical Diagnostics

We analyze fluorescence due to oxidizing activity of DNA in neutrophils of peripheral blood in the large populations ~104 - 105 of cells. Fluorescence is registered by flow cytometry method. Spatial resolution is about a few nanometers for varied complex three-dimensional (3D) DNA nanostructures of all non-coding and coding parts of DNA. It’s shown that oxidative activity of all 3D DNA in the full set of chromosomes inside cells is defined by new standards for complex networks of “exponentially small worlds”, with more dense packing than in the well known networks of “small worlds”. Analysis of various blood samples in vivo and during medical treatment shown that only two classes of Good and Bad Networks of DNA for a good and a bad health existed. This division is defined by any network to one from two classes of “n” or “s” shaped curves for typical deviations and from straight line in perfect networks of “exponentially small worlds”, as for two types of hysteresis curves at phase transitions or at switching of bistability. These deviations coincide with two types of positive and negative trends of changing fractal dimension by changing the scales of multi-scale networks of fluorescing DNA. These trends give the overall assessments of human immunity, including hidden and unidentified diseases, and as a sum of all kinds of health and illness of given person, from the point of view the inner life of neutrophils, living in different parts of human body in given time. Characteristics of deviations associated with type, level and complexity of illness in the dependence on the

Reversible switches of DNA nanostructures between “Closed” and “Open” states and their biosensing applications

A novel and versatile biosensing platform based on the structural conversion of 3D DNA nanostructures from ETDNA (Equilateral Triangle) to TPFDNA (Triangular Pyramid Frustum) was proposed for the first time. The inputs of aptamers and their relative targets made the DNA structure change from the "Open" to the "Closed" state, leading to the faradaic impedance changes as the output signals. The specific properties of excellent stability and specific rigid structure of 3D DNA nanostructures made the biosensor function as a regenerable, reusable and intelligent platform. The sensor exhibited excellent selectivity for IFN-γ detection with a wide linear range of 1.0 × 10(-9) to 2.0 × 10(-6) M and a low detection limit of 5.2 × 10(-10) M. The distinctive features of DNA nanostructures make them potentially advantageous for a broad range of biosensing, bionanoelectronics, and therapeutic applications.

Three-Dimensional Structures Self-Assembled from DNA Bricks

We describe a simple and robust method to construct complex three-dimensional (3D) structures by using short synthetic DNA strands that we call "DNA bricks." In one-step annealing reactions, bricks with hundreds of distinct sequences self-assemble into prescribed 3D shapes. Each 32-nucleotide brick is a modular component; it binds to four local neighbors and can be removed or added independently. Each 8-base pair interaction between bricks defines a voxel with dimensions of 2.5 by 2.5 by 2.7 nanometers, and a master brick collection defines a "molecular canvas" with dimensions of 10 by 10 by 10 voxels. By selecting subsets of bricks from this canvas, we constructed a panel of 102 distinct shapes exhibiting sophisticated surface features, as well as intricate interior cavities and tunnels.

Fabrication of ssDNA/Oligo(ethylene glycol) Monolayers and Complex Nanostructures by an Irradiation‐Promoted Exchange Reaction

Creative design: An approach to preparing mixed monolayers of thiolated single-stranded DNA (ssDNA) and oligo(ethylene glycol)s (OEG-AT) in a broad range of compositions as well as ssDNA/OEG-AT patterns of any required shape (see top figure) has been shown. A combination of this approach with surface-initiated enzymatic polymerization allows complex 3D DNA nanostructures to be sculpted with high spatial precision (bottom).

Charge Transport within a Three-Dimensional DNA Nanostructure Framework

Three-dimensional (3D) DNA nanostructures have shown great promise for various applications including molecular sensing and therapeutics. Here we report kinetic studies of DNA-mediated charge transport (CT) within a 3D DNA nanostructure framework. A tetrahedral DNA nanostructure was used to investigate the through-duplex and through-space CT of small redox molecules (methylene blue (MB) and ferrocene (Fc)) that were bound to specific positions above the surface of the gold electrode. CT rate measurements provide unambiguous evidence that the intercalative MB probe undergoes efficient mediated CT over longer distances along the duplex, whereas the nonintercalative Fc probe tunnels electrons through the space. This study sheds new light on DNA-based molecular electronics and on designing high-performance biosensor devices.

DNA Origami: Synthesis and Self‐Assembly

DNA origami is an emerging technology for designing defined two- and three-dimensional (2D and 3D) DNA nanostructures. Here, we report an introductory practical guide with step-by-step experimental details for the design and synthesis of origami structures, and their size expansion in 1D and 2D space by means of self-assembly.

DNA‐Directed Three‐Dimensional Protein Organization

All bound together: self-assembled symmetric DNA polyhedra were used to organize proteins in 3D space. Biotin moieties were incorporated into the self-assembled symmetric DNA polyhedra. Upon incubation with streptavidin (STV) protein, an STV protein became bound to each polyhedral face, thus resulting in well-structured DNA polyhedra/STV complexes. This strategy was also applied to different 3D DNA nanostructures and different proteins.

Single molecule microscopy methods for the study of DNA origami structures

Single molecule microscopy techniques play an important role in the investigation of advanced DNA structures such as those created by the DNA origami method. Three single molecule microscopy techniques are particularly interesting for the investigation of complex self-assembled three-dimensional (3D) DNA nanostructures, namely single molecule fluorescence microscopy, atomic force microscopy (AFM), and cryogenic transmission electron microscopy (cryo-EM). Here we discuss the strengths of these three techniques and demonstrate how their interplay can yield very important and unique new insights into the structure and conformation of advanced biological nanostructures. The applications of the three single molecule microscopy techniques are illustrated by focusing on a self-assembled DNA origami 3D box nanostructure. Its size and structure were studied by AFM and cryo-EM, while the lid opening, which can be controlled by the addition of oligonucleotide keys, was recorded by Förster/fluorescence resonance energy transfer (FRET) spectroscopy.

Organizing protein–DNA hybrids as nanostructures with programmed functionalities

The structural and functional information encoded in the base sequence of nucleic acids provides a means to organize hybrid protein-DNA nanostructures with pre-designed, programmed functionality. This review discusses the activation of enzyme cascades in supramolecular DNA-protein hybrid structures, the bioelectrocatalytic activation of redox enzymes on DNA scaffolds, and the programmed positioning of enzymes on 1D, 2D and 3D DNA nanostructures. These systems provide starting points towards the design of interconnected enzyme networks. Substantial progress in the tailoring of functional protein-DNA nanostructures has been accomplished in recent years, and advances in this field warrant a comprehensive discussion. The application of these systems for the control of biocatalytic transformations, for amplified biosensing, and for the synthesis of metallic nanostructures are addressed, and future prospects for these systems are highlighted.

Self-assembly of three-dimensional DNA nanostructures and potential biological applications

A current challenge in nanoscience is to achieve controlled organization in three-dimensions, to provide tools for biophysics, molecular sensors, enzymatic cascades, drug delivery, tissue engineering, and device fabrication. DNA displays some of the most predictable and programmable interactions of any molecule, natural or synthetic. As a result, 3D-DNA nanostructures have emerged as promising tools for biology and materials science. In this review, strategies for 3D-DNA assembly are discussed. DNA cages, nanotubes, dendritic networks, and crystals are formed, with deliberate variation of their size, shape, persistence length, and porosities. They can exhibit dynamic character, allowing their selective switching with external stimuli. They can encapsulate and position materials into arbitrarily designed patterns, and show promise for numerous biological and materials applications.

DNA self-assembly: from 2D to 3D

This paper describes our recent efforts on the self-assembly of three-dimensional (3D) DNA nanostructures from DNA star motifs (tiles). DNA star motifs are a family of DNA nanostructures with 3, 4, 5, or 6 branches; they are named as 3-, 4-, 5-, 6-point-star motifs, respectively. Such motifs are programmed to further assemble into nanocages (regular polyhedra or irregular nanocapsules) with diameters ranging from 20 nm to 2 microm. Among them, DNA nanocages derived from 3-point-star motif consists of a group of regular polyhedra: tetrahedra, hexahedra (or cubes), dodecahedra and buckyballs (containing 4, 8, 20, and 60 units of the 3-point-star motif, respectively). An icosahedron consists of twelve 5-point-star motifs and is similar to the shapes of spherical viruses. 6-point-star motifs can not assemble into regular polyhedra; instead, some sphere-like or irregular cages with diameters about 1-2 microm will form. Similar large cages can also assemble from the 5-point-star motif when the DNA concentrations are higher than those for assembling regular icosahedra. In our study, we have identified several important factors for assembly of well-defined 3D nanostructures, including the concentration, the flexibility, and the arm length of the DNA tiles and the association strength between the DNA tiles.