Short Staple Strands Research Articles

Quantification of ssDNA Scaffold Production by Ion-Pair Reverse Phase Chromatography.

DNA origami is an emerging technology that can be used as a nanoscale platform in numerous applications ranging from drug delivery systems to biosensors. The DNA nanostructures are assembled from large single-stranded DNA (ssDNA) scaffolds, ranging from hundreds to thousands of nucleotides and from short staple strands. Scaffolds are usually obtained by asymmetric PCR (aPCR) or Escherichia coli infection/transformation with phages or phagemids. Scaffold quantification is typically based on agarose gel electrophoresis densitometry for molecules obtained by aPCR, or by UV absorbance, in the case of scaffolds obtained by infection or transformation. Although these methods are well-established and easy-to-apply, the results obtained are often inaccurate due to the lack of selectivity and sensitivity in the presence of impurities. Herein, we present an HPLC method based on ion-pair reversed-phase (IP-RP) chromatography to quantify DNA scaffolds. Using IP-RP chromatography, ssDNA products (449 and 1000 nt) prepared by aPCR were separated from impurities and from the double stranded (ds) DNA byproduct. Additionally, both ss and dsDNA were quantified with high accuracy. The method was used to guide the optimization of the production of ssDNA by aPCR, which targeted the maximization of the ratio of ssDNA to dsDNA obtained. Moreover, ssDNA produced from phage infection of E. coli cells was also quantified by IP-RP using commercial ssDNA from the M13mp18 phage as a standard.

Preparation of DNA Origami Belt and Effect of pH on Its Stability

Abstract The long single-stranded DNA containing multiple copy units prepared by rolling circle amplification technique was used as a scaffold strand. The scaffold strands were hybridized to three short staple strands by molecular self-assembly and produced DNA origami belt. Using the principle of SYBR Green real-time fluorescence quantitative PCR method and atomic force microscopy (AFM) imaging technology, the formation of double-stranded structure was examined by the change of fluorescence signal intensity in the reaction solution while the morphology of DNA belts was observed by AFM intuitively. The effect of pH value on the stability of DNA belt was studied, and its performance of pH-resistant in the wide pH range of 3–11 was obtained. DNA belts exhibited the best stability in neutral pH enviroment (pH = 7), which showed a significantly higher fluorescence signal than that of other groups within 24 h. DNA belts had poor resistance to acid and alkali environment, especially to extreme pH conditions (pH = 3, pH = 11). At extreme pH, even instantaneous treatments (

Complex multicomponent patterns rendered on a 3D DNA-barrel pegboard

DNA origami, in which a long scaffold strand is assembled with a many short staple strands into parallel arrays of double helices, has proven a powerful method for custom nanofabrication. However, currently the design and optimization of custom 3D DNA-origami shapes is a barrier to rapid application to new areas. Here we introduce a modular barrel architecture, and demonstrate hierarchical assembly of a 100 megadalton DNA-origami barrel of ~90 nm diameter and ~250 nm height, that provides a rhombic-lattice canvas of a thousand pixels each, with pitch of ~8 nm, on its inner and outer surfaces. Complex patterns rendered on these surfaces were resolved using up to twelve rounds of Exchange-PAINT super-resolution microscopy. We envision these structures as versatile nanoscale pegboards for applications requiring complex 3D arrangements of matter, which will serve to promote rapid uptake of this technology in diverse fields beyond specialist groups working in DNA nanotechnology.

DNA Block Macromolecules Based on Rolling Circle Amplification Act as Scaffolds to Build Large-Scale Origami Nanostructures.

A simplified origami strategy to create large-scale complex DNA nanostructures based on the rolling circle amplification (RCA) is developed. The long repetitive block single strand DNA (ssDNA) synthesized from RCA with a few staple strands are used to fabricate DNA origami, which can be assembled into the first level assemblies of micro-scale length mono-nanoladders. Depending on base pairing among the sticky ends of short staple strands in nanoladders, the second level assemblies with large-scale and controlled structures such as tri-nanoladder, penta-nanoladder, and even nanobrocade, can be further achieved.

Simplifying DNA origami design

DNA Nanotechnology Many intricate nanostructures have been made with DNA origami. This process occurs when a long DNA scaffold develops a particular shape after hybridization with short staple strands. Most designs, however, require a difficult iterative procedure of refining the base pairing. Veneziano et al. now report algorithms that automate the design of arbitrary DNA wireframe structures. Synthesizing and structurally characterizing a variety of nanostructures allowed for verification of the algorithms' accuracy. Science , this issue p. [1534][1] [1]: http://www.sciencemag.org/content/352/6293/1534

Designer nanoscale DNA assemblies programmed from the top down.

Scaffolded DNA origami is a versatile means of synthesizing complex molecular architectures. However, the approach is limited by the need to forward-design specific Watson-Crick base pairing manually for any given target structure. Here, we report a general, top-down strategy to design nearly arbitrary DNA architectures autonomously based only on target shape. Objects are represented as closed surfaces rendered as polyhedral networks of parallel DNA duplexes, which enables complete DNA scaffold routing with a spanning tree algorithm. The asymmetric polymerase chain reaction is applied to produce stable, monodisperse assemblies with custom scaffold length and sequence that are verified structurally in three dimensions to be high fidelity by single-particle cryo-electron microscopy. Their long-term stability in serum and low-salt buffer confirms their utility for biological as well as nonbiological applications.

Design Techniques for the DNA Cubic-Lattice

We use the Watson-Crick properties of DNA and the principles of graph theory to construct origami folding designs for self-assembling cubic lattices. Our objective is a mathematical design strategy that can be expanded systematically to any size cubic lattice. This design consists of threading a scaffolding strand of DNA through the lattice that is secured in place by short staple strands of DNA. We first add augmenting edges to the cubic lattice to enable a single scaffolding strand threading. We then thread the scaffolding strand through the augmented cube in a way that minimizes the number of different vertex configurations in the structure. Key Words: Watson-Crick, DNA Self-Assembly, Origami folding, Cubic Lattice, Scaffolding Strand, Threading, Staple Strands

Nanomechanical Molecular Devices made of DNA Origami

CONSPECTUS: Eight years have passed since the striking debut of the DNA origami technique ( Rothemund, P. W. K. Nature 2006 , 440 , 297 - 302 ), in which long single-stranded DNA is folded into a designed nanostructure, in either 2D or 3D, with the aid of many short staple strands. The number of proposals for new design principles for DNA origami structures seems to have already reached a peak. It is apparent that DNA origami study is now entering the second phase of creating practical applications. The development of functional nanomechanical molecular devices using the DNA origami technique is one such application attracting significant interest from researchers in the field. Nanomechanical DNA origami devices, which maintain the characteristics of DNA origami structures, have various advantages over conventional DNA nanomachines. Comparatively high assembly yield, relatively large size visible via atomic force microscopy (AFM) or transmission electron microscopy (TEM), and the capability to assemble multiple functional groups with precision using multiple staple strands are some of the advantages of the DNA origami technique for constructing sophisticated molecular devices. This Account describes the recent developments of such nanomechanical DNA origami devices and reviews the emerging target of DNA origami studies. First, simple "dynamic" DNA origami structures with transformation capability, such as DNA origami boxes and a DNA origami hatch with structure control, are briefly summarized. More elaborate nanomechanical DNA origami devices are then reviewed. The first example describes DNA origami pinching devices that can be used as "single-molecule" beacons to detect a variety of biorelated molecules, from metal ions at the size of a few tens of atomic mass number units to relatively gigantic proteins with a molecular mass greater than a hundred kilodaltons, all on a single platform. Clamshell-like DNA nanorobots equipped with logic gates can discriminate different cell lines, open their shell, and bind to their target. An intelligent DNA origami "sheath" can mimic the function of suppressors in a transcription regulation system to control the expression of a loaded gene. DNA origami "rolls" are created to construct precisely arranged plasmonic devices with metal nanoparticles. All of their functions are derived from their nanomechanical movement, which is programmable by designing the DNA sequence or by using the significant repository of technical achievements in nucleic acid chemistry. Finally, some studies on detailed structural parameters of DNA origami or their mechanical properties in nanoscale are discussed, which may be useful and inspiring for readers who intend to design new nanomechanical DNA origami devices.

Building DNA Nanostructures for Molecular Computation, Templated Assembly, and Biological Applications

CONSPECTUS: DNA is a critical biomolecule well-known for its roles in biology and genetics. Moreover, its double-helical structure and the Watson-Crick pairing of its bases make DNA structurally predictable. This predictability enables design and synthesis of artificial DNA nanostructures by suitable programming of the base sequences of DNA strands. Since the advent of the field of DNA nanotechnology in 1982, a variety of DNA nanostructures have been designed and used for numerous applications. In this Account, we discuss the progress made by our lab which has contributed toward the overall advancement of the field. Tile-based DNA nanostructures are an integral part of structural DNA nanotechnology. These structures are formed using several short, chemically synthesized DNA strands by programming their base sequences so that they self-assemble into desired constructs. Design and assembly of several DNA tiles will be discussed in this Account. Tiles include, for example, TX tiles with three parallel, coplanar duplexes, 4 × 4 cross-tiles with four arms, and weave-tiles with weave-like architecture. Another category of tiles we will present involve multiple parallel duplexes that assemble to form closed tubular structures. All of these tile types have been used to form micrometer-scale one- and two-dimensional arrays and lattices. Origami-based structures constitute another category where a long single-stranded DNA scaffold is folded into desired shapes by association with multiple short staple strands. This Account will describe the efforts by our lab in devising new strategies to improve the maximum size of origami structures. The various DNA nanostructures detailed here have been used in a wide variety of different applications. This Account will discuss the use of DNA tiles for logical computation, encoding information as molecular barcodes, and functionalization for patterning of other nanoscale organic and inorganic materials. Consequently, we have used DNA nanostructures for templating metallic nanowires as well as for programmed assembly of proteins and nanoparticles with controlled spacings. Among other applications, we have used DNA nanotechnology in biosensors that detect target DNA sequences and to affect cell surface receptor clustering for communicating with a cell signaling pathway. We used DNA weave-tiles to control the spacing between thrombin-binding aptamers which resulted in very high antithrombin and anticoagulant activity of the construct. We believe that the tremendous progress in DNA nanotechnology over the past three decades will open even more research avenues in the near future for applications in a wide variety of disciplines including electronics, photonics, biomedical engineering, biosensing, therapeutics, and nucleic-acid-based drug delivery.

RCA Strands as Scaffolds To Create Nanoscale Shapes by a Few Staple Strands

Thousands of nucleotide(nt)-long single strand DNAs, generated from rolling-circle-amplification (RCA), were used as scaffolds to create DNA nanoscale wires and plates with a few short staple strands by following the origami design principle with a crossover at 1.5 turns. The core sequence of the circle template, for producing tens and hundreds of tandemly repeated copies of it by RCA, was designed according to Seeman's sequence design principle for nucleic acid structural engineering (Seeman, N. C. J. Biomol. Struct. Dyn. 1990, 8, 573). The significance for folding the RCA products into nanoscale shapes lies in the design flexibility of both staple and scaffold strand codes, simplicity of a few short staple strands to fold the periodic sequence of RCA products, and lower cost.

Nanomechanical DNA origami 'single-molecule beacons' directly imaged by atomic force microscopy

DNA origami involves the folding of long single-stranded DNA into designed structures with the aid of short staple strands; such structures may enable the development of useful nanomechanical DNA devices. Here we develop versatile sensing systems for a variety of chemical and biological targets at molecular resolution. We have designed functional nanomechanical DNA origami devices that can be used as 'single-molecule beacons', and function as pinching devices. Using 'DNA origami pliers' and 'DNA origami forceps', which consist of two levers ~170 nm long connected at a fulcrum, various single-molecule inorganic and organic targets ranging from metal ions to proteins can be visually detected using atomic force microscopy by a shape transition of the origami devices. Any detection mechanism suitable for the target of interest, pinching, zipping or unzipping, can be chosen and used orthogonally with differently shaped origami devices in the same mixture using a single platform.

Single Molecule Atomic Force Microscopy Studies of Photosensitized Singlet Oxygen Behavior on a DNA Origami Template

DNA origami, the folding of a long single-stranded DNA sequence (scaffold strand) by hundreds of short synthetic oligonucleotides (staple strands) into parallel aligned helices, is a highly efficient method to form advanced self-assembled DNA-architectures. Since molecules and various materials can be conjugated to each of the short staple strands, the origami method offers a unique possibility of arranging molecules and materials in well-defined positions on a structured surface. Here we combine the action of light with AFM and DNA nanostructures to study the production of singlet oxygen from a single photosensitizer molecule conjugated to a selected DNA origami staple strand on an origami structure. We demonstrate a distance-dependent oxidation of organic moieties incorporated in specific positions on DNA origami by singlet oxygen produced from a single photosensitizer located at the center of each origami.

DNA origami: Fold, stick, and beyond

DNA origami is the process in which long single-stranded DNA molecules are folded into arbitrary planar nanostructures with the aid of many short staple strands. Since its initial introduction in 2006, DNA origami has dramatically widened the scope of applications of DNA nanotechnology based on the programmed assembly of branched DNA junctions. DNA origami can be used to construct not only arbitrary two-dimensional nanostructures but also nano-sized breadboards for the arraying of nanomaterials or even complicated three-dimensional nano-objects. In this review, we briefly look through the basic designs and applications of DNA origami and discuss the future of this technique.