Stabilization and structural changes of 2D DNA origami by enzymatic ligation

The low thermal stability of DNA nanostructures is the major drawback in their practical applications. Most of the DNA nanotubes/tiles and the DNA origami structures melt below 60°C due to the presence of discontinuities in the phosphate backbone (i.e., nicks) of the staple strands. In molecular biology, enzymatic ligation is commonly used to seal the nicks in the duplex DNA. However, in DNA nanotechnology, the ligation procedures are neither optimized for the DNA origami nor routinely applied to link the nicks in it. Here, we report a detailed analysis and optimization of the conditions for the enzymatic ligation of the staple strands in four types of 2D square lattice DNA origami. Our results indicated that the ligation takes overnight, efficient at 37°C rather than the usual 16°C or room temperature, and typically requires much higher concentration of T4 DNA ligase. Under the optimized conditions, up to 10 staples ligation with a maximum ligation efficiency of 55% was achieved. Also, the ligation is found to increase the thermal stability of the origami as low as 5°C to as high as 20°C, depending on the structure. Further, our studies indicated that the ligation of the staple strands influences the globular structure/planarity of the DNA origami, and the origami is more compact when the staples are ligated. The globular structure of the native and ligated origami was also found to be altered dynamically and progressively upon ethidium bromide intercalation in a concentration-dependent manner.     

Enzyme‐Free Ligation of 5′‐Phosphorylated Oligodeoxynucleotides in a DNA Nanostructure

Multicomponent reactions are difficult synthetic transformations. For DNA, there is a special opportunity to align multiple strands in a folded nanostructure, so that they are preorganized to give a specific sequence. Multistrand reactions in DNA origami structures have previously been performed using photochemical crosslinking, 1,3‐diploar cycloadditions or phosphoramidate‐forming reactions. Here we report carbodiimide‐driven phosphodiester formation in a small origami sheet that produces DNA strands up to 600 nucleotides in length in a single step. The method uses otherwise unmodified oligodeoxynucleotides with a 5′‐terminal phosphate as starting materials. Compared to an enzymatic multistrand ligation involving linear duplexes, the carbodiimide‐driven ligation gave fewer side products, as detected by gel electrophoresis. The full‐length 600mer product was successfully amplified by polymerase chain reaction.     

Functional patterning of DNA origami by parallel enzymatic modification

We demonstrate here a rapid and cost-effective technique for nanoscale patterning of functional molecules on the surface of a DNA origami. The pattern is created enzymatically by transferring a functionalized dideoxynucleotide to the 3'-end of an arbitrary selected set of synthetic DNA oligonucleotides positioned approximately 6 nm apart in a 70 × 100 nm(2) rectangular DNA origami. The modifications, which are performed in a single-tube reaction, provide an origami surface modified with a variety of functional groups including chemical handles, fluorescent dyes, or ligands for subsequent binding of proteins. Efficient labeling and patterning was demonstrated by gel electrophoresis shift assays, reverse-phase HPLC, mass spectrometry, atomic force microscopy (AFM) analysis, and fluorescence measurements. The results show a very high yield of oligonucleotide labeling and incorporation in the DNA origami. This method expands the toolbox for constructing several different modified DNA origami from the same set of staple strands.     

A label-free light-scattering method to resolve assembly and disassembly of DNA nanostructures

DNA self-assembly, and in particular DNA origami, has evolved into a reliable workhorse for organizing organic and inorganic materials with nanometer precision and with exactly controlled stoichiometry. To ensure the intended performance of a given DNA structure, it is beneficial to determine its folding temperature, which in turn yields the best possible assembly of all DNA strands. Here, we show that temperature-controlled sample holders and standard fluorescence spectrometers or dynamic light-scattering setups in a static light-scattering configuration allow for monitoring the assembly progress in real time. With this robust label-free technique, we determine the folding and melting temperatures of a set of different DNA origami structures without the need for more tedious protocols. In addition, we use the method to follow digestion of DNA structures in the presence of DNase I and find strikingly different resistances toward enzymatic degradation depending on the structural design of the DNA object.     

DNA origami as biocompatible surface to match single-molecule and ensemble experiments

Single-molecule experiments on immobilized molecules allow unique insights into the dynamics of molecular machines and enzymes as well as their interactions. The immobilization, however, can invoke perturbation to the activity of biomolecules causing incongruities between single molecule and ensemble measurements. Here we introduce the recently developed DNA origami as a platform to transfer ensemble assays to the immobilized single molecule level without changing the nano-environment of the biomolecules. The idea is a stepwise transfer of common functional assays first to the surface of a DNA origami, which can be checked at the ensemble level, and then to the microscope glass slide for single-molecule inquiry using the DNA origami as a transfer platform. We studied the structural flexibility of a DNA Holliday junction and the TATA-binding protein (TBP)-induced bending of DNA both on freely diffusing molecules and attached to the origami structure by fluorescence resonance energy transfer. This resulted in highly congruent data sets demonstrating that the DNA origami does not influence the functionality of the biomolecule. Single-molecule data collected from surface-immobilized biomolecule-loaded DNA origami are in very good agreement with data from solution measurements supporting the fact that the DNA origami can be used as biocompatible surface in many fluorescence-based measurements.     

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.     

Nanopores formed by DNA origami: A review

Nanopores have emerged over the past two decades to become an important technique in single molecule experimental physics and biomolecule sensing. Recently DNA nanotechnology, in particular DNA origami, has been used for the formation of nanopores in insulating materials. DNA origami is a very attractive technique for the formation of nanopores since it enables the construction of 3D shapes with precise control over geometry and surface functionality. DNA origami has been applied to nanopore research by forming hybrid architectures with solid state nanopores and by direct insertion into lipid bilayers. This review discusses recent experimental work in this area and provides an outlook for future avenues and challenges.     

Genotype-phenotype mapping with polyominos made from DNA origami tiles

The correlation between genetic information and characteristics of a living cell—its genotype and its phenotype—constitutes the basis of genetics. Here, we experimentally realize a primitive form of genotype-phenotype mapping with DNA origami. The DNA origami can polymerize into two-dimensional lattices (phenotype) via blunt-end stacking facilitated by edge staples at the seam of the planar DNA origami. There are 80 binding positions for edge staples, which allow us to translate an 80-bit long binary code (genotype) onto the DNA origami. The presence of an edge staple thus corresponds to a “1” and its absence to a “0.” The interactions of our DNA-based system can be reproduced by a polyomino model. Polyomino growth simulations qualitatively reproduce our experimental results. We show that not only the absolute number of base stacks but also their sequence position determine the cluster size and correlation length of the orientation of single DNA origami within the cluster. Importantly, the mutation of a few bits can result in major morphology changes of the DNA origami cluster, while more often, major sequence changes have no impact. Our experimental realization of a correlation between binary information (“genotype”) and cluster morphology (“phenotype”) thus reproduces key properties of genotype-phenotype maps known from living systems.     

DNA折纸纳米载体在药物递送中的应用

DNA纳米技术是基于沃森克里克碱基配对原则产生可编程核酸结构的技术。因其具有高精度的工程设计、前所未有的可编程性和内在的生物相容性等特点，运用该技术合成的纳米结构不仅可以与小分子、核酸、蛋白质、病毒和癌细胞相互作用，还可以作为纳米载体，递送不同的治疗药物。DNA折纸作为一种有效的、多功能的方法来构建二维和三维可编程的纳米结构，是DNA纳米技术发展的一个里程碑。由于其高度可控的几何形状、空间寻址性、易于化学修饰，DNA折纸在许多领域具有巨大的应用潜力。本文通过介绍DNA折纸的起源、基本原理和目前进展，归纳总结了运用DNA折纸进行药物装载和释放的方式，并基于此技术，展望了今后的发展趋势以及所面临的机遇和挑战。     

Modulating the chemo-mechanical response of structured DNA assemblies through binding molecules

Recent advances in DNA nanotechnology led the fabrication and utilization of various DNA assemblies, but the development of a method to control their global shapes and mechanical flexibilities with high efficiency and repeatability is one of the remaining challenges for the realization of the molecular machines with on-demand functionalities. DNA-binding molecules with intercalation and groove binding modes are known to induce the perturbation on the geometrical and mechanical characteristics of DNA at the strand level, which might be effective in structured DNA assemblies as well. Here, we demonstrate that the chemo-mechanical response of DNA strands with binding ligands can change the global shape and stiffness of DNA origami nanostructures, thereby enabling the systematic modulation of them by selecting a proper ligand and its concentration. Multiple DNA-binding drugs and fluorophores were applied to straight and curved DNA origami bundles, which demonstrated a fast, recoverable, and controllable alteration of the bending persistence length and the radius of curvature of DNA nanostructures. This chemo-mechanical modulation of DNA nanostructures would provide a powerful tool for reconfigurable and dynamic actuation of DNA machineries.     

EGNAS: an exhaustive DNA sequence design algorithm

ABSTRACT: BACKGROUND: The molecular recognition based on the complementary base pairing of deoxyribonucleicacid (DNA) is the fundamental principle in the fields of genetics, DNA nanotechnologyand DNA computing. We present an exhaustive DNA sequence design algorithm thatallows to generate sets containing a maximum number of sequences with definedproperties. EGNAS (Exhaustive Generation of Nucleic Acid Sequences) offers thepossibility of controlling both interstrand and intrastrand properties. The guanine-cytosinecontent can be adjusted. Sequences can be forced to start and end with guanine orcytosine. This option reduces the risk of "fraying" of DNA strands. It is possible to limitcross hybridizations of a defined length, and to adjust the uniqueness of sequences.Self-complementarity and hairpin structures of certain length can be avoided. Sequencesand subsequences can optionally be forbidden. Furthermore, sequences can be designed tohave minimum interactions with predefined strands and neighboring sequences. RESULTS: The algorithm is realized in a C++ program. TAG sequences can be generated andcombined with primers for single-base extension reactions, which were described formultiplexed genotyping of single nucleotide polymorphisms. Thereby, possible foldbackthrough intrastrand interaction of TAG-primer pairs can be limited. The design ofsequences for specific attachment of molecular constructs to DNA origami is presented. CONCLUSIONS: We developed a new software tool called EGNAS for the design of unique nucleic acidsequences. The presented exhaustive algorithm allows to generate greater sets ofsequences than with previous software and equal constraints. EGNAS is freely availablefor noncommercial use at http://www.chm.tu-dresden.de/pc6/EGNAS.     

Probing tethered targets of a single biomolecular complex with atomic force microscopy

DNA origami shows tremendous promise as templates for the assembly of nano‐components and detection of molecular recognition events. So far, the method of choice for evaluating these structures has been atomic force microscopy (AFM), a powerful tool for imaging nanoscale objects. In most cases, tethered targets on DNA origami have proven to be highly effective samples for investigation. Still, while maximal assembly of the nanostructures might benefit from the greatest flexibility in the tether, AFM imaging requires a sufficient stability of the adsorbed components. The balance between the tether flexibility and sample stability is a major, poorly understood, concern in such studies. Here, we investigated the dependence of the tethering length on molecular capture events monitored by AFM. In our experiments, single biotin molecules were attached to DNA origami templates with various linker lengths of thymidine nucleotides, and their interaction with streptavidin was observed with AFM. Our results show that the streptavidin‐biotin complexes are easily detected with short tethered lengths, and that their morphological features clearly change with the tethering length. We identify the functionally useful tether lengths for these investigations, which are also expected to prove useful in the construction and further application of DNA origami in bio‐nanotechnology studies. Copyright © 2013 John Wiley & Sons, Ltd.     

A primer to scaffolded DNA origami

Molecular self-assembly with scaffolded DNA origami enables building custom-shaped nanometer-scale objects with molecular weights in the megadalton regime. Here we provide a practical guide for design and assembly of scaffolded DNA origami objects. We also introduce a computational tool for predicting the structure of DNA origami objects and provide information on the conditions under which DNA origami objects can be expected to maintain their structure.     

Beyond DNA origami: the unfolding prospects of nucleic acid nanotechnology

Nucleic acid nanotechnology exploits the programmable molecular recognition properties of natural and synthetic nucleic acids to assemble structures with nanometer-scale precision. In 2006, DNA origami transformed the field by providing a versatile platform for self-assembly of arbitrary shapes from one long DNA strand held in place by hundreds of short, site-specific (spatially addressable) DNA 'staples'. This revolutionary approach has led to the creation of a multitude of two-dimensional and three-dimensional scaffolds that form the basis for functional nanodevices. Not limited to nucleic acids, these nanodevices can incorporate other structural and functional materials, such as proteins and nanoparticles, making them broadly useful for current and future applications in emerging fields such as nanomedicine, nanoelectronics, and alternative energy.     

Sequence-specific 1H-NMR assignments and folding topology of human CD59.

CD59 is a recently discovered cell-surface glycoprotein that restricts lysis by homologous complement and has limited sequence similarity to snake venom neurotoxins. This paper describes the first results of a two-dimensional NMR study of CD59 prepared from human urine. Nearly complete 1H-NMR assignments were obtained for the 77 amino acid residues and partial assignments for the N-glycan and the glycosylphosphatidylinositol (GPI) anchor. These results together confirm that the C-terminal residue of the mature protein is Asn 77 and that the urine-derived form retains the nonlipid part of the GPI anchor. The data further indicate that the GPI anchor and possibly the N-glycan are structurally inhomogeneous and suggest that the phospholipid present in the intact GPI anchor was removed by phosphatidylinositol-specific phospholipase-D. The folding topology of the protein was determined from NOE enhancements and slowly exchanging backbone amide protons and consists primarily of five extended strands (denoted beta 1-beta 5 in sequence order), arranged into separate two-stranded (beta 1 and beta 2) and three-stranded (beta 3-beta 5) antiparallel beta-sheets. The same folding topology is found in all of the snake venom neurotoxins whose structures have been determined. The region between the beta 4 and beta 5 strands has helical character, a feature that is not present in the neurotoxins but that is seen in the topologically similar wheat germ agglutinin.     

135 Arranging enzymes and receptors with nanometer-scale precision on molecular switchboards

DNA is a useful material for constructing nanoscale structures in nearly any three-dimensional (3D) shape desired. The DNA nanostructure can also be equipped with specific docking sites for proteins. Cellular processes and chemical transformations take place in several reaction steps. Multiple enzymes cooperate in specific fashion to catalyze the sequential chemical transformation steps. Such natural systems are effectively reconstructed in vitro if the individual enzymes locate in the correct relative orientations. DNA-origami structures can be used as “molecular switchboards” to arrange enzymes and other proteins with nanometer-scale precision. A new method was developed for locating the proteins by means of special “adapters” known as zinc-finger proteins based only on proteins. Zinc fingers are suitable site-selective adapters for targeting specific locations within DNA-origami structures. Several different adapters carrying different proteins can independently bind at defined locations on this type of nanostructure. A basic leucine zipper (bZIP) protein is also a candidate for the site-selective adaptor. A well-characterized bZIP protein GCN4 was chosen as an adaptor for specific addresses. Analyses by atomic force microscopy and gel electrophoreses demonstrate specific binding of GCN4 adaptor to the addresses containing the GCN4 binding sites on DNA origami. The adaptor derived from GCN4 and that form a zinc-finger protein zif268, for which we have reported previously, acted as orthogonal adaptors to the respective addresses on DNA origami. Therefore, these orthogonal adaptors would be useful to place multiple engineered proteins at different addresses on DNA origami. Especially, the homodimeric nature of GCN4 adaptor is indispensable for constructing the assembly of the naturally abundant dimeric proteins and/or enzymes to efficiently carry out chemical reactions and signal transductions in vitro on DNA origami.     

The in vivo replication origin of the yeast 2 microns plasmid

We have used two-dimensional neutral/alkaline agarose gel electrophoresis to separate the nascent strands of replicating yeast 2 micron plasmid DNA molecules according to extent of replication, away from nonreplicating molecules and parental strands. Analysis of the lengths of nascent strands by sequential hybridization with short probes shows that replication proceeds bidirectionally from a single origin at map position 3700 +/- 100, coincident with the genetically mapped ARS element. The two recombinational isomers of 2 microns plasmid (forms A and B) replicate with equal efficiency. These results suggest that ARS elements may prove to be replication origins for chromosomal DNA.     

Designing a Bio-responsive Robot from DNA Origami

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information¹, they can be utilized in parallel computing^2,3 , recognize and bind molecular or cellular targets^4,5 , catalyze chemical reactions^6,7 , and generate calculated responses in a biological system^8,9. Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures^10-12, enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect.Creating shapes from nucleic acids was first proposed by Seeman¹³, and several variations on this theme have since been realized using various techniques^11,12,14,15 . However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami¹⁶. In this technique, the folding of a long (>7,000 bases) single-stranded DNA ''scaffold'' is directed to a desired shape by hundreds of short complementary strands termed ''staples''. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well^17,18 .The current paper will focus on the caDNAno 2.0 software¹⁹ developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient.In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described²⁰. This robot is ''robotic'' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando²¹ to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings.     

Controlling the stoichiometry and strand polarity of a tetramolecular G-quadruplex structure by using a DNA origami frame

Guanine-rich oligonucleotides often show a strong tendency to form supramolecular architecture, the so-called G-quadruplex structure. Because of the biological significance, it is now considered to be one of the most important conformations of DNA. Here, we describe the direct visualization and single-molecule analysis of the formation of a tetramolecular G-quadruplex in KCl solution. The conformational changes were carried out by incorporating two duplex DNAs, with G–G mismatch repeats in the middle, inside a DNA origami frame and monitoring the topology change of the strands. In the absence of KCl, incorporated duplexes had no interaction and laid parallel to each other. Addition of KCl induced the formation of a G-quadruplex structure by stably binding the duplexes to each other in the middle. Such a quadruplex formation allowed the DNA synapsis without disturbing the duplex regions of the participating sequences, and resulted in an X-shaped structure that was monitored by atomic force microscopy. Further, the G-quadruplex formation in KCl solution and its disruption in KCl-free buffer were analyzed in real-time. The orientation of the G-quadruplex is often difficult to control and investigate using traditional biochemical methods. However, our method using DNA origami could successfully control the strand orientations, topology and stoichiometry of the G-quadruplex.