Effect of Ionic Strength on the Thermal Stability of DNA Origami Nanostructures

The stability of DNA origami nanostructures in aqueous media is closely tied to the presence of cations that screen electrostatic inter-helix repulsion. Here, the thermal melting behavior of different DNA origami nanostructures is investigated in dependence on Mg²⁺ concentration and compared to calculated ensemble melting temperatures of the staple strands used in DNA origami folding. Strong deviations of the measured DNA origami melting temperatures from the calculated ones are observed, in particular at high ionic strength where the melting temperature saturates and becomes independent of ionic strength. The degree of deviation between the measured and calculated melting temperatures further depends on the superstructure and in particular the mechanical properties of the DNA origami nanostructures. This indicates that thermal stability of a given DNA origami design at high ionic strength is governed predominantly not by electrostatic inter-helix repulsion but mostly by mechanical strain.     

Salting-Out of DNA Origami Nanostructures by Ammonium Sulfate

DNA origami technology enables the folding of DNA strands into complex nanoscale shapes whose properties and interactions with molecular species often deviate significantly from that of genomic DNA. Here, we investigate the salting-out of different DNA origami shapes by the kosmotropic salt ammonium sulfate that is routinely employed in protein precipitation. We find that centrifugation in the presence of 3 M ammonium sulfate results in notable precipitation of DNA origami nanostructures but not of double-stranded genomic DNA. The precipitated DNA origami nanostructures can be resuspended in ammonium sulfate-free buffer without apparent formation of aggregates or loss of structural integrity. Even though quasi-1D six-helix bundle DNA origami are slightly less susceptible toward salting-out than more compact DNA origami triangles and 24-helix bundles, precipitation and recovery yields appear to be mostly independent of DNA origami shape and superstructure. Exploiting the specificity of ammonium sulfate salting-out for DNA origami nanostructures, we further apply this method to separate DNA origami triangles from genomic DNA fragments in a complex mixture. Our results thus demonstrate the possibility of concentrating and purifying DNA origami nanostructures by ammonium sulfate-induced salting-out.     

E. coli K-12琥珀酸脱氢酶sdhC基因纳米锥的设计及自组装

采用DAEDALUS软件，以E. coli K-12 MG1655的琥珀酸脱氢酶复合体C亚基编码酶基因sdhC的双链DNA为脚手架链，进行一个边长为17.68 nm的正四面锥设计，并最终通过核酸自组装反应获得了sdhC基因的纳米锥聚合体。利用琼脂糖凝胶电泳、扫描电子显微镜和透射电子显微镜对多组样品进行化学组分和形貌分析，同时利用原子力显微镜液下成像法测定了核酸聚合体的三维尺度。结果表明：DAEDALUS软件设计出的16条订书钉链，确实将624 bp的sdhC双链DNA中的一条链折叠成了平均边长为19.05nm的正四面锥，与预设模型仅相差1.37nm，证明普通基因的双链DNA可以替代M13mp18单链核酸作为DNA折纸的核酸材料，为构建核酸纳米材料提供了一种新方法。     

可控式citZ基因纳米盒的设计与研究

DNA分子具有自我识别的特殊能力，DNA折纸术就是利用这一特性进行核酸纳米材料精准设计和组装的一种新技术。研究者可以利用与DNA脚手架链互补的订书钉链，将长链核酸折叠成与预设模型一致的纳米结构。DNA折纸术最早是2006年由Rothemund提出，一直以来，人们利用M13mp18单链线性DNA进行各种纳米图形的自组装。为了寻找更多的核酸材料进行DNA折纸研究，本文以枯草芽孢杆菌（Bacillus. subtilis 168）citZ基因序列为研究对象，采用改进的DAEDALUS软件，引入“锁钥”结构设计，利用“从下向上”的方法使DNA分子进行自组装，设计出三维体积为50.71nm×50.71nm×50.71nm的citZ基因纳米盒，只有遇到可识别的基因和匹配的“钥匙”时，才可能打开盖子，释放盒中的内容物。这种核酸纳米材料还可以通过调节DNA序列长度调节盒子的内部空间，有望成为一种新型的靶向药物运送载体。     

Structural DNA nanotechnology: from design to applications

The exploitation of DNA for the production of nanoscale architectures presents a young yet paradigm breaking approach, which addresses many of the barriers to the self-assembly of small molecules into highly-ordered nanostructures via construct addressability. There are two major methods to construct DNA nanostructures, and in the current review we will discuss the principles and some examples of applications of both the tile-based and DNA origami methods. The tile-based approach is an older method that provides a good tool to construct small and simple structures, usually with multiply repeated domains. In contrast, the origami method, at this time, would appear to be more appropriate for the construction of bigger, more sophisticated and exactly defined structures.     

Construction of a Holliday Junction in Small Circular DNA Molecules for Stable Motifs and Two‐Dimensional Lattices

Design rules for DNA nanotechnology have been mostly learnt from using linear single‐stranded (ss) DNA as the source material. For example, the core structure of a typical DAO (double crossover, antiparallel, odd half‐turns) tile for assembling 2D lattices is constructed from only two linear ss‐oligonucleotide scaffold strands, similar to two ropes making a square knot. Herein, a new type of coupled DAO (cDAO) tile and 2D lattices of small circular ss‐oligonucleotides as scaffold strands and linear ss‐oligonucleotides as staple strands are reported. A cDAO tile of cDAO‐c64nt (c64nt: circular 64 nucleotides), shaped as a solid parallelogram, is constructed with a Holliday junction (HJ) at the center and two HJs at both poles of a c64nt; similarly, cDAO‐c84nt, shaped as a crossed quadrilateral composed of two congruent triangles, is formed with a HJ at the center and four three‐way junctions at the corners of a c84nt. Perfect 2D lattices were assembled from cDAO tiles: infinite nanostructures of nanoribbons, nanotubes, and nanorings, and finite nanostructures. The structural relationship between the visible lattices imaged by AFM and the corresponding invisible secondary and tertiary molecular structures of HJs, inclination angle of hydrogen bonds against the double‐helix axis, and the chirality of the tile can be interpreted very well. This work could shed new light on DNA nanotechnology with unique circular tiles.     

Surfactant-Assisted Purification of Hydrophobic DNA Nanostructures

Purification of functional DNA nanostructures is an essential step in achieving intended functions because misfolded structures and the remaining free DNA strands in a solution can interact and affect their behavior. However, due to hydrophobicity-mediated aggregation, it is difficult to purify DNA nanostructures modified with hydrophobic molecules by conventional methods. Herein, we report the purification of cholesterol-modified DNA nanostructures by using a novel surfactant-assisted gel extraction. The addition of sodium cholate (SC) to the sample solution before structure folding prevented aggregation; this was confirmed by gel electrophoresis. We also found that adding sodium dodecyl sulfate (SDS) to the sample inhibited structural folding. The cholesterol-modified DNA nanostructures prepared with SC were successfully purified by gel extraction, and their ability to bind to the lipid membrane surfaces was maintained. This method will facilitate the purification of DNA nanostructures modified with hydrophobic molecules and expand their applicability in the construction of artificial cell-like systems.     

DNA Origami-Based Protein Manipulation Systems: From Function Regulation to Biological Application

Proteins directly participate in tremendous physiological processes and mediate a variety of cellular functions. However, precise manipulation of proteins with predefined relative position and stoichiometry for understanding protein-protein interactions and guiding cellular behaviors is still challenging. With superior programmability of DNA molecules, DNA origami technology is able to construct arbitrary nanostructures that can accurately control the arrangement of proteins with various functionalities to solve these problems. Herein, starting from the classification of DNA origami nanostructures and the category of assembled proteins, we summarize the existing DNA origami-based protein manipulation systems (PMSs), review the advances on the regulation of their functions, and discuss their applications in cellular behavior modulation and disease therapy. Moreover, the limitations and potential directions of DNA origami-based PMSs are also presented, which may offer guidance for rational construction and ingenious application.     

DNA origami and biotechnology applications: a perspective

The use of DNA as a material has opened up new possibilities in the field of nanobiotechnology. Specifically, DNA origami – a technique in which one long single‐stranded DNA scaffold is folded into nanoscale shapes and structures using hundreds of short 'staple' oligonucleotides – has contributed to new innovations within this field. Nanostructures created using DNA origami have found use in applications such as biosensing, triggered drug delivery, enzyme cascades and biomolecular analysis platforms. The unmatched features of DNA nanostructures such as cell permeability, biocompatibility, and spatial positioning have contributed to DNA origami playing an important role in the development of materials for biotechnology applications. © 2015 Society of Chemical Industry     

DNA Nanoarchitectures: Steps towards Biological Applications

DNA's remarkable molecular recognition properties, flexibility, and structural features make it one of the most promising scaffolds to design a variety of nanostructures. During recent decades, two major methods have been developed for the construction of DNA nanomaterials in a programmable way; both generate nanostructures in one, two, and three dimensions. The tile‐based assembly process is a useful tool to construct large and simple structures; the DNA origami method is suitable for the production of smaller, more sophisticated and well‐defined structures. Proteins, nanoparticles and other functional elements have been specifically positioned into designed patterns on these structures. They can also act as templates to study chemical reactions, help in the structural determination of proteins, and be used as platform for genomic and drug delivery applications. In this review we examine recent progresses towards the potential use of DNA nanostructures in molecular and cellular biology.     

Time-Dependent DNA Origami Denaturation by Guanidinium Chloride,Guanidinium Sulfate,and Guanidinium Thiocyanate

Guanidinium (Gdm) undergoes interactions with both hydrophilic and hydrophobic groups and, thus, is a highly potent denaturant of biomolecular structure. However, our molecular understanding of the interaction of Gdm with proteins and DNA is still rather limited. Here, we investigated the denaturation of DNA origami nanostructures by three Gdm salts, i.e., guanidinium chloride (GdmCl), guanidinium sulfate (Gdm₂SO₄), and guanidinium thiocyanate (GdmSCN), at different temperatures and in dependence of incubation time. Using DNA origami nanostructures as sensors that translate small molecular transitions into nanostructural changes, the denaturing effects of the Gdm salts were directly visualized by atomic force microscopy. GdmSCN was the most potent DNA denaturant, which caused complete DNA origami denaturation at 50 °C already at a concentration of 2 M. Under such harsh conditions, denaturation occurred within the first 15 min of Gdm exposure, whereas much slower kinetics were observed for the more weakly denaturing salt Gdm₂SO₄ at 25 °C. Lastly, we observed a novel non-monotonous temperature dependence of DNA origami denaturation in Gdm₂SO₄ with the fraction of intact nanostructures having an intermediate minimum at about 40 °C. Our results, thus, provide further insights into the highly complex Gdm–DNA interaction and underscore the importance of the counteranion species.     

Chemical Approaches to DNA Nanotechnology

Due to its self‐assembling nature, DNA is undoubtedly an excellent molecule for the creation of various multidimensional nanostructures and the placement of functional molecules and materials. DNA molecules behave according to the programs of their sequences. Mixtures of numbers of DNA molecules can be placed precisely and organized into single structures to form nanoarchitectures. Once the appropriate sequences for the target nanostructure are established, the predesigned structure can be built up by self‐assembly of the designed DNA strands. DNA nanotechnology has already reached the stage at which the organization of desired functional molecules and nanomaterials can be programmed on a defined DNA scaffold. In this review, we will focus on DNA nanotechnology and describe the potential of synthetic chemistry to contribute to the further development of DNA nanomaterials.     

Strategies for Stabilizing DNA Nanostructures to Biological Conditions

DNA is one of the most promising building blocks for creating functional nanostructures for applications in biology and medicine. However, these highly programmable nanomaterials (e.g., DNA origami) often require supraphysiological salt concentrations for stability, are degraded by nuclease enzymes, and can elicit an inflammatory response. Herein, three key strategies for stabilizing DNA nanostructures to conditions required for biological applications are outlined: 1) tuning the buffer conditions or nanostructure design; 2) covalently crosslinking the strands that make up the structures; and 3) coating the structures with polymers, proteins, or lipid bilayers. Taken together, these approaches greatly expand the chemical diversity and future applicability of DNA nanotechnology both in vitro and in vivo.     

Nanorings to Probe Mechanical Stress of Single-Stranded DNA Mediated by the DNA Duplex

The interplay between the mechanical properties of double-stranded and single-stranded DNA is a phenomenon that contributes to various genetic processes in which both types of DNA structures coexist. Highly stiff DNA duplexes can stretch single-stranded DNA (ssDNA) segments between the duplexes in a topologically constrained domain. To evaluate such an effect, we designed short DNA nanorings in which a DNA duplex with 160 bp is connected by a 30 nt single-stranded DNA segment. The stretching effect of the duplex in such a DNA construct can lead to the elongation of ssDNA, and this effect can be measured directly using atomic force microscopy (AFM) imaging. In AFM images of the nanorings, the ssDNA regions were identified, and the end-to-end distance of ssDNA was measured. The data revealed a stretching of the ssDNA segment with a median end-to-end distance which was 16% higher compared with the control. These data are in line with theoretical estimates of the stretching of ssDNA by the rigid DNA duplex holding the ssDNA segment within the nanoring construct. Time-lapse AFM data revealed substantial dynamics of the DNA rings, allowing for the formation of transient crossed nanoring formations with end-to-end distances as much as 30% larger than those of the longer-lived morphologies. The generated nanorings are an attractive model system for investigation of the effects of mechanical stretching of ssDNA on its biochemical properties, including interaction with proteins.     

Enhancement of Stability and Activity of siRNA by Terminal Substitution with Serinol Nucleic Acid (SNA)

RNA interference (RNAi ), sequence‐specific gene silencing triggered by double‐stranded, small interfering RNA (siRNA), has become a facile and effective tool for biological research and holds potential for therapeutic applications. However, the application of siRNA is hindered by susceptibility to nucleases and off‐target effects. In this study, we introduced artificial nucleotides, serinol nucleic acid (SNA), with an acyclic scaffold, at the termini of siRNA strands. Our aim was appropriately to accommodate the antisense strand in an RNA‐induced silencing complex (RISC) by inhibiting sense‐strand incorporation and thus improve resistance to nuclease‐mediated degradation. Substitution of SNA into siRNA at both termini of the sense strand and at the 3′ terminus of the antisense strand improved antisense strand selectivity remarkably in the formation of RISC, RNAi activity, and nuclease resistance.     

N-Glycosylation as a Tool to Study Antithrombin Secretion,Conformation, and Function

N-linked glycosylation is a crucial post-translational modification involved in protein folding, function, and clearance. N-linked glycosylation is also used therapeutically to enhance the half-lives of many proteins. Antithrombin, a serpin with four potential N-glycosylation sites, plays a pivotal role in hemostasis, wherein its deficiency significantly increases thrombotic risk. In this study, we used the introduction of N-glycosylation sites as a tool to explore what effect this glycosylation has on the protein folding, secretion, and function of this key anticoagulant. To accomplish this task, we introduced an additional N-glycosylation sequence in each strand. Interestingly, all regions that likely fold rapidly or were surrounded by lysines were not glycosylated even though an N-glycosylation sequon was present. The new sequon in the strands of the A- and B-sheets reduced secretion, and the B-sheet was more sensitive to these changes. However, the mutations in the strands of the C-sheet allowed correct folding and secretion, which resulted in functional variants. Therefore, our study revealed crucial regions for antithrombin secretion and could potentially apply to all serpins. These results could also help us understand the functional effects of natural variants causing type-I deficiencies.     

Functionalized DNA Nanostructures for Nanomedicine

DNA nanotechnology utilizes synthetic DNA strands as the building material to construct nanoscale devices, and the field has developed rapidly over the past decade. Recently, the use of DNA nanostructures for various applications, particularly biomedical ones, has drawn great interest. This review focuses on the most recent research directed at utilizing functionalized DNA devices for nanomedical applications and presents representative research progress in disease diagnosis, treatment and prevention. In addition, the safety and future clinical applications of DNA nanostructures are discussed.     

Guanine-rich DNA nanocircles for the synthesis and characterization of long cytosine-rich telomeric DNAs

Short synthetic oligonucleotides derived from the human telomeric repeat have been studied recently for their ability to fold into four-stranded structures that are thought to be important to their biological function. Because telomeric DNAs are several kilobases in length, however, their folding might well be affected by cooperative or high-order interactions in these long sequences. Here, we present a new molecular system that allows for easy synthesis of very long stretches of the cytosine-rich strand of human telomeric DNA. Small circular DNAs composed of the G-rich sequence of human telomeres were prepared and used as templates in a rolling-circle replication mechanism. To facilitate the synthesis of the repetitive G-rich circles, an orthogonal base-protection strategy that made use of dimethylformamidine-protected guanine nucleobases was developed. Nanometer-scale circles ranging in size from 42 to 54 nucleotides were prepared. Subsequently, we tested the action of various DNA polymerases on these circular templates, and identified DNA Pol I (Klenow fragment) and T7 DNA polymerase as enzymes that are able to generate very long, C-rich telomeric DNA strands. Purification and initial structural examination of these C-rich polymeric products revealed evidence of a folded structure in the polymer.     

DNA Origami as Scaffolds for Self-Assembly of Lipids and Proteins

Since first being reported in 2006, the DNA origami approach has attracted increasing attention due to programmable shapes, structural stability, biocompatibility, and fantastic addressability. Herein, we provide an account of recent developments of DNA origami as scaffolds for templating the selfassembly of distinct biocomponents, essentially proteins and lipids, into a diverse spectrum of integrated supramolecular architectures. First, the historical development of the DNA origami concept is briefly reviewed. Next, various applications of DNA origami constructs in controllable directed assembly of soluble proteins are discussed. The manipulation and self-assembly of lipid membranes and membrane proteins by using DNA origami as scaffolds are also addressed. Furthermore, recent progress in applying DNA origami in cryoelectron microscopy analysis is discussed. These advances collectively emphasize that the DNA origami approach is a highly versatile, fast evolving tool that may be integrated with lipids and proteins in a way that meets future challenges in molecular biology and nanomedicine.     

Sequence-Independent Logical Regulation of the Assembly and Disassembly of DNA Polyhedra

Controlling the self-assembly of DNA nanostructures using rationally designed logic gates is a major goal of dynamic DNA nanotechnology, which could facilitate the development of biomedicine, molecular computation, et al. In previous works, the regulations mostly relied on either toehold-mediated strand displacement or stimuli-driven conformational switch, requiring elaborately-designed or specific DNA sequences. Herein, we reported a facile, base-sequence-independent strategy for logically controlling DNA self-assembly through external molecules. The INHIBIT and XOR logic controls over the assembly/disassembly of DNA polyhedra were realized through cystamine ( Cyst ) and ethylenediamine ( EN ) respectively, which were further integrated into a half subtractor circuit thanks to the sharing of the same inputs. Our work provides a sequence-independent strategy in logically controlling DNA self-assembly, which may open up new possibilities for dynamic DNA nanotechnology.