Heat-resistant DNA tile arrays constructed by template-directed photoligation through 5-carboxyvinyl-2'-deoxyuridine

Template-directed DNA photoligation has been applied to a method to construct heat-resistant two-dimensional (2D) DNA arrays that can work as scaffolds in bottom-up assembly of functional biomolecules and nano-electronic components. DNA double-crossover AB-staggered (DXAB) tiles were covalently connected by enzyme-free template-directed photoligation, which enables a specific ligation reaction in an extremely tight space and under buffer conditions where no enzymes work efficiently. DNA nanostructures created by self-assembly of the DXAB tiles before and after photoligation have been visualized by high-resolution, tapping mode atomic force microscopy in buffer. The improvement of the heat tolerance of 2D DNA arrays was confirmed by heating and visualizing the DNA nanostructures. The heat-resistant DNA arrays may expand the potential of DNA as functional materials in biotechnology and nanotechnology.     

Binding of DNA origami to lipids: maximizing yield and switching via strand displacement

Liposomes are widely used as synthetic analogues of cell membranes and for drug delivery. Lipid-binding DNA nanostructures can modify the shape, porosity and reactivity of liposomes, mediated by cholesterol modifications. DNA nanostructures can also be designed to switch conformations by DNA strand displacement. However, the optimal conditions to facilitate stable, high-yield DNA–lipid binding while allowing controlled switching by strand displacement are not known. Here, we characterized the effect of cholesterol arrangement, DNA structure, buffer and lipid composition on DNA–lipid binding and strand displacement. We observed that binding was inhibited below pH 4, and above 200 mM NaCl or 40 mM MgCl₂, was independent of lipid type, and increased with membrane cholesterol content. For simple motifs, binding yield was slightly higher for double-stranded DNA than single-stranded DNA. For larger DNA origami tiles, four to eight cholesterol modifications were optimal, while edge positions and longer spacers increased yield of lipid binding. Strand displacement achieved controlled removal of DNA tiles from membranes, but was inhibited by overhang domains, which are used to prevent cholesterol aggregation. These findings provide design guidelines for integrating strand displacement switching with lipid-binding DNA nanostructures. This paves the way for achieving dynamic control of membrane morphology, enabling broader applications in nanomedicine and biophysics.     

Nanopore fingerprinting of supramolecular DNA nanostructures

DNA nanotechnology has paved the way for new generations of programmable nanomaterials. Utilizing the DNA origami technique, various DNA constructs can be designed, ranging from single tiles to the self-assembly of large-scale, complex, multi-tile arrays. This technique relies on the binding of hundreds of short DNA staple strands to a long single-stranded DNA scaffold that drives the folding of well-defined nanostructures. Such DNA nanostructures have enabled new applications in biosensing, drug delivery, and other multifunctional materials. In this study, we take advantage of the enhanced sensitivity of a solid-state nanopore that employs a poly-ethylene glycol enriched electrolyte to deliver real-time, non-destructive, and label-free fingerprinting of higher-order assemblies of DNA origami nanostructures with single-entity resolution. This approach enables the quantification of the assembly yields for complex DNA origami nanostructures using the nanostructure-induced equivalent charge surplus as a discriminant. We compare the assembly yield of four supramolecular DNA nanostructures obtained with the nanopore with agarose gel electrophoresis and atomic force microscopy imaging. We demonstrate that the nanopore system can provide analytical quantification of the complex supramolecular nanostructures within minutes, without any need for labeling and with single-molecule resolution. We envision that the nanopore detection platform can be applied to a range of nanomaterial designs and enable the analysis and manipulation of large DNA assemblies in real time.     

An Overview of Structural DNA Nanotechnology

Structural DNA Nanotechnology uses unusual DNA motifs to build target shapes and arrangements. These unusual motifs are generated by reciprocal exchange of DNA backbones, leading to branched systems with many strands and multiple helical domains. The motifs may be combined by sticky ended cohesion, involving hydrogen bonding or covalent interactions. Other forms of cohesion involve edge-sharing or paranemic interactions of double helices. A large number of individual species have been developed by this approach, including polyhedral catenanes, a variety of single-stranded knots, and Borromean rings. In addition to these static species, DNA-based nanomechanical devices have been produced that are ultimately targeted to lead to nanorobotics. Many of the key goals of structural DNA nanotechnology entail the use of periodic arrays. A variety of 2D DNA arrays have been produced with tunable features, such as patterns and cavities. DNA molecules have be used successfully in DNA-based computation as molecular representations of Wang tiles, whose self-assembly can be programmed to perform a calculation. About 4 years ago, on the fiftieth anniversary of the double helix, the area appeared to be at the cusp of a truly exciting explosion of applications; this was a correct assessment, and much progress has been made in the intervening period.     

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.     

DNA nanotubes as combinatorial vehicles for cellular delivery

This work explores using self-assembled DNA nanostructures as carriers for drug delivery. We have recently developed an organic nanotube system that is assembled from a single component: a 52-base-long DNA single strand. In this work, functional agents (folate as a cancer cell target agent and Cy3 as a fluorescence imaging agent) are conjugated with the DNA strands; the conjugates self-assemble into micrometers-long nanotubes (NTs). The conjugated DNA-NTs can be effectively taken by cancer cells as demonstrated by fluorescence imaging and fluorescence-activated cell sorting. No obvious toxicity has been observed under current experimental conditions.     

Chemical modification of amine groups on PS II protein(s) retards photoassembly of the photosynthetic water-oxidizing complex

Four Mn atoms function as catalysts in the water-oxidizing complex located on the oxidizing side of PS II. We have studied the involvement of amine groups of the PS II proteins in photoligation of Mn2+ to the apo water-oxidizing complex, using the combined techniques of photoactivation and chemical modification with the modifiers methyl acetimidate (MAI), acetic acid N-hydroxysuccinimide ester (NHS), and 2,4,6-trinitrobenzenesulfonic acid (TNBS). Chemical modification of hydroxylamine-treated PS II core complexes decreased their capacity for restoration of oxygen evolution and photoligation of Mn2+ to the apo water-oxidizing complex (WOC), but did not affect their electron transfer activity in the vicinity of PS II. The number of functional high-affinity Mn-binding sites, but not of low-affinity sites, was significantly modulated by chemical modification. Kinetic analysis of photoactivation with the repetitive flashes revealed that the intermediate generated during a photoactivation process was destabilized by the chemical modification. To identify which proteins possess the amine groups involved in ligation of functional Mn, we examined the difference in NHS biotinylation between PS II core complexes with and without the Mn cluster. NHS biotinylation resulting in altered ligation of functional Mn apparently occurred on three proteins: an antenna chlorophyll binding protein (CP47), a light-harvesting chlorophyll protein (CP29), and another chlorophyll binding protein (PS II-S). Of these proteins, only the Mn-dependent biotinylation of CP47 was found to occur independently of the application of an NHS-masking concentration before removal of the functional Mn. These results suggest that lysyl residues of CP47, and perhaps also CP29 and PS II-S, function in direct photoligation of Mn2+ to the apo WOC.     

Unraveling the interaction between doxorubicin and DNA origami nanostructures for customizable chemotherapeutic drug release

Doxorubicin (DOX) is a common drug in cancer chemotherapy, and its high DNA-binding affinity can be harnessed in preparing DOX-loaded DNA nanostructures for targeted delivery and therapeutics. Although DOX has been widely studied, the existing literature of DOX-loaded DNA-carriers remains limited and incoherent. Here, based on an in-depth spectroscopic analysis, we characterize and optimize the DOX loading into different 2D and 3D scaffolded DNA origami nanostructures (DONs). In our experimental conditions, all DONs show similar DOX binding capacities (one DOX molecule per two to three base pairs), and the binding equilibrium is reached within seconds, remarkably faster than previously acknowledged. To characterize drug release profiles, DON degradation and DOX release from the complexes upon DNase I digestion was studied. For the employed DONs, the relative doses (DOX molecules released per unit time) may vary by two orders of magnitude depending on the DON superstructure. In addition, we identify DOX aggregation mechanisms and spectral changes linked to pH, magnesium, and DOX concentration. These features have been largely ignored in experimenting with DNA nanostructures, but are probably the major sources of the incoherence of the experimental results so far. Therefore, we believe this work can act as a guide to tailoring the release profiles and developing better drug delivery systems based on DNA-carriers.     

Rational Design of Metal‐Organic Framework Derived Hollow NiCo2O4 Arrays for Flexible Supercapacitor and Electrocatalysis

Metal‐organic frameworks (MOFs) are promising porous precursors for the construction of various functional materials for high‐performance electrochemical energy storage and conversion. Herein, a facile two‐step solution method to rational design of a novel electrode of hollow NiCo₂O₄ nanowall arrays on flexible carbon cloth substrate is reported. Uniform 2D cobalt‐based wall‐like MOFs are first synthesized via a solution reaction, and then the 2D solid nanowall arrays are converted into hollow and porous NiCo₂O₄ nanostructures through an ion‐exchange and etching process with an additional annealing treatment. The as‐obtained NiCo₂O₄ nanostructure arrays can provide rich reaction sites and short ion diffusion path. When evaluated as a flexible electrode material for supercapacitor, the as‐fabricated NiCo₂O₄ nanowall electrode shows remarkable electrochemical performance with excellent rate capability and long cycle life. In addition, the hollow NiCo₂O₄ nanowall electrode exhibits promising electrocatalytic activity for oxygen evolution reaction. This work provides an example of rational design of hollow nanostructured metal oxide arrays with high electrochemical performance and mechanical flexibility, holding great potential for future flexible multifunctional electronic devices.     

Biochemistry and structural DNA nanotechnology: an evolving symbiotic relationship

Structural DNA nanotechnology is derived from naturally occurring structures and phenomena in cellular biochemistry. Motifs based on branched DNA molecules are linked together by sticky ends to produce objects, periodic arrays, and nanomechanical devices. The motifs include Holliday junction analogues, double and triple crossover molecules, knots, and parallelograms. Polyhedral catenanes, such as a cube or a truncated octahedron, have been assembled from branched junctions. Stiff motifs have been used to produce periodic arrays, containing topographic features visible in atomic force microscopy; these include deliberately striped patterns and cavities whose sizes can be tuned by design. Deliberately knotted molecules have been assembled. Aperiodic arrangements of DNA tiles can be used to produce assemblies corresponding to logical computation. Both DNA structural transitions and branch migration have been used as the basis for the operation of DNA nanomechanical devices. Structural DNA nanotechnology has been used in a number of applications in biochemistry. An RNA knot has been used to establish the existence of RNA topoisomerase activity. The sequence dependence of crossover isomerization and branch migration at symmetric sites has been established through the use of symmetric immobile junctions. DNA parallelogram arrays have been used to determine the interhelical angles for a variety of DNA branched junctions. The relationship between biochemistry and structural DNA nanotechnology continues to grow.     

Physical map of the centromeric region of human chromosome 7: relationship between two distinct alpha satellite arrays.

A long-range physical map of the centromeric region of human chromosome 7 has been constructed in order to define the region containing sequences with potential involvement in centromere function. The map is centered around alpha satellite DNA, a family of tandemly repeated DNA forming arrays of hundreds to thousands of kilobasepairs at the primary constriction of every human chromosome. Two distinct alpha satellite arrays (the loci D7Z1 and D7Z2) have previously been localized to chromosome 7. Detailed one- and two- locus maps of the chromosome 7 centromere have been constructed. Our data indicate that D7Z1 and D7Z2 arrays are not interspersed with each other but are both present on a common Mlu I restriction fragment estimated to be 3500 kb and 5500 kb on two different chromosome 7's investigated. These long-range maps, combined with previous measurements of the D7Z1 and D7Z2 array lengths, are used to construct a consensus map of the centromere of chromosome 7. The analysis used to construct the map provides, by extension, a framework for analysis of the structure of DNA in the centromeric regions of other human and mammalian chromosomes.     

Studies of Thermal Stability of Multivalent DNA Hybridization in a Nanostructured System

A fundamental understanding of molecular self-assembly processes is important for improving the design and construction of higher-order supramolecular structures. DNA tile based self-assembly has recently been used to generate periodic and aperiodic nanostructures of different geometries, but there have been very few studies that focus on the thermodynamic properties of the inter-tile interactions. Here we demonstrate that fluorescently-labeled multihelical DNA tiles can be used as a model platform to systematically investigate multivalent DNA hybridization. Real-time monitoring of DNA tile assembly using fluorescence resonance energy transfer revealed that both the number and the relative position of DNA sticky-ends play a significant role in the stability of the final assembly. As multivalent interactions are important factors in nature's delicate macromolecular systems, our quantitative analysis of the stability and cooperativity of a network of DNA sticky-end associations could lead to greater control over hierarchical nanostructure formation and algorithmic self-assembly.     

Surface-Enhanced Resonance Raman Scattering (SERRS) Using Au Nanohole Arrays on Optical Fiber Tips

Circular and bow tie-shaped Au nanoholes arrays were fabricated on gold films deposited on the tips of single-mode optical fibers. The nanostructures were milled using focused ion beam with a high quality control of their shapes and sizes. The optical fiber devices were used for surface-enhanced resonance Raman scattering (SERRS) measurements in both back- and forward-scattering geometries, yielding promising performance in both detection arrangements. The effect of the hole shape on the SERRS performance was explored with the bow tie nanostructures presenting a better SERRS performance than the circular holes arrays. The results present here are another step towards the development of optical fiber tips modified with plasmonic nanostructures for SERRS applications.

A fourier tool for the analysis of coherent light scattering by bio-optical nanostructures

The fundamental dichotomy between incoherent (phase independent) and coherent (phase dependent) light scattering provides the best criterion for a classification of biological structural color production mechanisms. Incoherent scattering includes Rayleigh, Tyndall, and Mie scattering. Coherent scattering encompasses interference, reinforcement, thin-film reflection, and diffraction. There are three main classes of coherently scattering nanostructures-laminar, crystal-like, and quasi-ordered. Laminar and crystal-like nanostructures commonly produce iridescence, which is absent or less conspicuous in quasi-ordered nanostructures. Laminar and crystal-like arrays have been analyzed with methods from thin-film optics and Bragg's Law, respectively, but no traditional methods were available for the analysis of color production by quasi-ordered arrays. We have developed a tool using two-dimensional (2D) Fourier analysis of transmission electron micrographs (TEMs) that analyzes the spatial variation in refractive index (available from the authors). This Fourier tool can examine whether light scatterers are spatially independent, and test whether light scattering can be characterized as predominantly incoherent or coherent. The tool also provides a coherent scattering prediction of the back scattering reflectance spectrum of a biological nanostructure. Our applications of the Fourier tool have falsified the century old hypothesis that the non-iridescent structural colors of avian feather barbs and skin are produced by incoherent Rayleigh or Tyndall scattering. 2D Fourier analysis of these quasi-ordered arrays in bird feathers and skin demonstrate that these non-iridescent colors are produced by coherent scattering. No other previous examples of biological structural color production by incoherent scattering have been tested critically with either analysis of scatterer spatial independence or spectrophotometry. The Fourier tool is applied here for the first time to coherent scattering by a laminar array from iridescent bird feather barbules (Nectarinia) to demonstrate the efficacy of the technique on thin films. Unlike previous physical methods, the Fourier tool provides a single method for the analysis of coherent scattering by a diversity of nanostructural classes. This advance will facilitate the study of the evolution of nanostructural classes from one another and the evolution of nanostructure itself. The article concludes with comments on the emerging role of photonics in research on biological structural colors, and the future directions in development of the tool.     

Two-dimensional (2D) DNA crystals assembled from two DNA strands

Sequence symmetry has been used to simplify the design of a DNA double-crossover (DX) molecule. The resulting DX molecule can self-assemble into two-dimensional (2D) crystalline arrays, but only requires two instead of otherwise four different DNA strands.     

Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this article, we describe detailed protocols on how to fabricate sophisticated 2D shapes through the self-assembly of uniquely addressable single-stranded DNA tiles which act as molecular pixels on a molecular canvas. Each single-stranded tile (SST) is a 42-nucleotide DNA strand composed of four concatenated modular domains which bind to four neighbors during self-assembly. The molecular canvas is a rectangle structure self-assembled from SSTs. A prescribed complex 2D shape is formed by selecting the constituent molecular pixels (SSTs) from a 310-pixel molecular canvas and then subjecting the corresponding strands to one-pot annealing. Due to the modular nature of the SST approach we demonstrate the scalability, versatility and robustness of this method. Compared with alternative methods, the SST method enables a wider selection of information polymers and sequences through the use of de novo designed and synthesized short DNA strands.     

Template-directed photoligation of oligodeoxyribonucleotides via 4-thiothymidine.

Non-enzymatic, template-directed ligation of oligonucleotides in aqueous solution has been of great interest because of its potential synthetic and biomedical utility and implications for the origin of life. Though there are many methods for template-directed chemical ligation of oligonucleotides, there are only three reported photochemical methods. In the first report, template-directed photoligation was effected by cyclobutane dimer formation between the 5'- and 3'-terminal thymidines of two oligonucleotides with >290 nm light, which also damages DNA itself. To make the photochemistry of native DNA more selective, we have replaced the thymidine at the 5'-end of one oligonucleotide with 4-thiothymidine (s4T) and show that it photoreacts at 366 nm with a T at the 3'-endof another oligonucleotide in the presence of a complementary template. When a single mismatch is introduced opposite either the s4T or its adjoining T, the ligation efficiency drops by a factor of five or more. We also show that by linking the two ends of the oligonucleotides together, photoligation can be used to form circular DNA molecules and to 'photopadlock' circular DNA templates. Thus, s4T-mediated photo-ligation may have applications to phototriggered antisense-based or antigene-based genetic tools, diagnostic agents and drugs, especially for those situations in which chemical or enzyme-mediated ligation isundesirable or impossible, for example inside a cell.     

Switchable DNA-origami nanostructures that respond to their environment and their applications

Structural DNA nanotechnology, in which Watson-Crick base pairing drives the formation of self-assembling nanostructures, has rapidly expanded in complexity and functionality since its inception in 1981. DNA nanostructures can now be made in arbitrary three-dimensional shapes and used to scaffold many other functional molecules such as proteins, metallic nanoparticles, polymers, fluorescent dyes and small molecules. In parallel, the field of dynamic DNA nanotechnology has built DNA circuits, motors and switches. More recently, these two areas have begun to merge—to produce switchable DNA nanostructures, which change state in response to their environment. In this review, we summarise switchable DNA nanostructures into two major classes based on response type: molecular actuation triggered by local chemical changes such as pH or concentration and external actuation driven by light, electric or magnetic fields. While molecular actuation has been well explored, external actuation of DNA nanostructures is a relatively new area that allows for the remote control of nanoscale devices. We discuss recent applications for DNA nanostructures where switching is used to perform specific functions—such as opening a capsule to deliver a molecular payload to a target cell. We then discuss challenges and future directions towards achieving synthetic nanomachines with complexity on the level of the protein machinery in living cells.     

DNA Tetrominoes: The Construction of DNA Nanostructures Using Self-Organised Heterogeneous Deoxyribonucleic Acids Shapes

The unique programmability of nucleic acids offers alternative in constructing excitable and functional nanostructures. This work introduces an autonomous protocol to construct DNA Tetris shapes (L-Shape, B-Shape, T-Shape and I-Shape) using modular DNA blocks. The protocol exploits the rich number of sequence combinations available from the nucleic acid alphabets, thus allowing for diversity to be applied in designing various DNA nanostructures. Instead of a deterministic set of sequences corresponding to a particular design, the protocol promotes a large pool of DNA shapes that can assemble to conform to any desired structures. By utilising evolutionary programming in the design stage, DNA blocks are subjected to processes such as sequence insertion, deletion and base shifting in order to enrich the diversity of the resulting shapes based on a set of cascading filters. The optimisation algorithm allows mutation to be exerted indefinitely on the candidate sequences until these sequences complied with all the four fitness criteria. Generated candidates from the protocol are in agreement with the filter cascades and thermodynamic simulation. Further validation using gel electrophoresis indicated the formation of the designed shapes. Thus, supporting the plausibility of constructing DNA nanostructures in a more hierarchical, modular, and interchangeable manner.