DNA origami nanostructures (DOs) are promising tools for applications including drug delivery, biosensing, detecting biomolecules, and probing chromatin substructures. Targeting these nanodevices to mammalian cell nuclei could provide impactful approaches for probing, visualizing, and controlling biomolecular processes within live cells. We present an approach to deliver DOs into live-cell nuclei. We show that these DOs do not undergo detectable structural degradation in cell culture media or cell extracts for 24 hours. To deliver DOs into the nuclei of human U2OS cells, we conjugated 30-nanometer DO nanorods with an antibody raised against a nuclear factor, specifically the largest subunit of RNA polymerase II (Pol II). We find that DOs remain structurally intact in cells for 24 hours, including inside the nucleus. We demonstrate that electroporated anti–Pol II antibody–conjugated DOs are piggybacked into nuclei and exhibit subdiffusive motion inside the nucleus. Our results establish interfacing DOs with a nuclear factor as an effective method to deliver nanodevices into live-cell nuclei.

DNA origami nanotechnology has great potential in multiple fields including biomedical, biophysical, and nanofabrication applications. However, current production pipelines lead to single-use devices incorporating a small fraction of initial reactants, resulting in a wasteful manufacturing process. Here, we introduce two complementary approaches to overcome these limitations by recycling the strand components of DNA origami nanostructures (DONs). We demonstrate reprogramming entire DONs into new devices, reusing scaffold strands. We validate this approach by reprogramming DONs with complex geometries into each other, using their distinct geometries to verify successful scaffold recycling. We reprogram one DON into a dynamic structure and show both pristine and recycled structures display similar properties. Second, we demonstrate the recovery of excess staple strands postassembly and fold DONs with these recycled strands, showing these structures exhibit the expected geometry and dynamic properties. Finally, we demonstrate the combination of both approaches, successfully fabricating DONs solely from recycled DNA components.

Scaffolded DNA origami relies on the programmed interaction between many different single-stranded DNAs (ssDNA). While short oligonucleotides can be readily produced through solid-phase synthesis, production of the scaffold strand is mostly limited to a few available sources. Even though there has been significant effort to expand the available sources for long ssDNAs, sources of ssDNA are still significantly limited compared to widely available sources (e.g. plasmids) of double-stranded DNA (dsDNA).

Chiral materials are essential to perceive photonic devices that control the helicity of light. However, the chirality of natural materials is rather weak, and relatively thick films are needed for noticeable effects. To overcome this limitation, artificial photonic materials were suggested to affect the chiral response in a much more substantial manner. Ideally, a single layer of such a material, a metasurface, should already be sufficient. While various structures fabricated with top-down nanofabrication technologies have already been reported, here we propose to utilize scaffolded DNA origami technology, a scalable bottom-up approach for metamolecule production, to fabricate a chiral metasurface. We introduce a chiral plasmonic metamolecule in the shape of a tripod and simulate its optical properties. By fixing the metamolecule to a rectangular planar origami, the tripods can be assembled into a 2D DNA origami crystal that forms a chiral metasurface. We simulate the optical properties but also fabricate selected devices to assess the experimental feasibility of the suggested approach critically.

Human cells package over two meters of genetic information into the microscopic space of the nucleus in the form of chromatin (a complex between DNA and histone proteins). This packaging or “folding” of the genome is critical for the regulation of gene expression, maintenance of genomic stability, and defining cell fate during development. Change to the spatial organization of chromatin is implicated in a host of neurological disorders as well as in aging and cancer. Remodeling of chromatin via post-translational modifications allows for epigenetic regulation of transcription in healthy and diseased cells. Thus, tools to engineer chromatin epigenetics in a controlled manner are crucial to study the cause-consequence relationship between epigenetics, chromatin organization and gene expression. Previous approaches to chromatin engineering have significant limitations, namely the inability to perform multiple functions (visualize, modify, or detect downstream products) simultaneously. Our work seeks to develop and apply DNA origami nanodevices (DOs) as multi-functional platforms to probe and engineer chromatin epigenetics. We propose to leverage the versatility of DO and its potential as a platform for multiple functional elements to visualize, modify, and sense the transcriptional output of genomic regions in mammalian cell nuclei. We have taken an interdisciplinary approach to develop and test four different DNA origami structures (26-helix bundle, 14-helix bundle, 13-helix bundle, and 8-helix bundle) functionalized with fluorophores as well as an anti-RNA Polymerase II (Pol2) antibody. We show that electroporation enables delivery of these structures into cells and further, 8-helix bundle structures functionalized with RNA Pol2 antibodies are successfully piggybacked into the nucleus. Super-resolution imaging further demonstrates the delivery of single DNA origami structures into the cell nucleus. These results open the door for DNA origami as a multifunctional platform for targeting, modifying, and visualizing the genome.

Recent advances in structural DNA nanotechnology have been facilitated by design tools that continue to push the limits of structural complexity while simplifying an often-tedious design process. We recently introduced the software MagicDNA, which enables design of complex 3D DNA assemblies with many components; however, the design of structures with free-form features like vertices or curvature still required iterative design guided by simulation feedback and user intuition. Here, we present an updated design tool, MagicDNA 2.0, that automates the design of free-form 3D geometries, leveraging design models informed by coarse-grained molecular dynamics simulations. Our GUI-based, stepwise design approach integrates a high level of automation with versatile control over assembly and subcomponent design parameters. We experimentally validated this approach by fabricating a range of DNA origami assemblies with complex free-form geometries, including a 3D Nozzle, G-clef, and Hilbert and Trifolium curves, confirming excellent agreement between design input, simulation, and structure formation.

Control over the mesoscale to microscale patterning of materials is of great interest to the soft matter community. Inspired by DNA origami rotors, we introduce a 2D nearest-neighbor lattice of spinning rotors that exhibit discrete orientational states and interactions with their neighbors. Monte Carlo simulations of rotor lattices reveal that they exhibit a variety of interesting ordering behaviors and morphologies that can be modulated through rotor design parameters. The rotor arrays exhibit diverse patterns including closed loops, radiating loops, and bricklayer structures in their ordered states. They exhibit specific heat peaks at very low temperatures for small system sizes, and some systems exhibit multiple order-disorder transitions depending on inter-rotor interaction design. We devise an energy-based order parameter and show via umbrella sampling and histogram reweighting that this order parameter captures well the order-disorder transitions occurring in these systems. We fabricate real DNA origami rotors which themselves can order via programmable DNA base-pairing interactions and demonstrate both ordered and disordered phases, illustrating how rotor lattices may be realized experimentally and used for responsive organization. This work establishes the feasibility of realizing structural nanomaterials that exhibit locally mediated microscale patterns which could have applications in sensing and precision surface patterning.

DNA nanostructures are a promising tool for delivery of a variety of molecular payloads to cells. DNA origami structures, where 1000’s of bases are folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent-protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR-Cas9 ribonucleoprotein (RNP) binding sites on the DNA nanostructures to increase shuttling of structures into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA (dsDNA) templates in human primary cells. Furthermore, our study validates virus-like particles (VLPs) as an efficient method of DNA nanostructure delivery, opening the possibility of delivering DNA nanostructures in vivo to specific cell types. Together these results provide new approaches to gene delivery with DNA nanostructures and establish their use as large HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as measuring biophysical properties, into cell nuclei. Teaser Sentence CRISPR-Cas9 mediates nuclear transport and integration of nanostructured genes in human primary cells

Enhancing CRISPR-mediated site-specific transgene insertion efficiency by homology-directed repair (HDR) using high concentrations of double-stranded DNA (dsDNA) with Cas9 target sequences (CTSs) can be toxic to primary cells. Here, we develop single-stranded DNA (ssDNA) HDR templates (HDRTs) incorporating CTSs with reduced toxicity that boost knock-in efficiency and yield by an average of around two- to threefold relative to dsDNA CTSs. Using small-molecule combinations that enhance HDR, we could further increase knock-in efficiencies by an additional roughly two- to threefold on average. Our method works across a variety of target loci, knock-in constructs and primary human cell types, reaching HDR efficiencies of >80–90%. We demonstrate application of this approach for both pathogenic gene variant modeling and gene-replacement strategies for IL2RA and CTLA4 mutations associated with Mendelian disorders. Finally, we develop a good manufacturing practice (GMP)-compatible process for nonviral chimeric antigen receptor-T cell manufacturing, with knock-in efficiencies (46–62%) and yields (>1.5 × 109 modified cells) exceeding those of conventional approaches. Combinations of single-stranded DNA repair templates and small molecules markedly enhance genome editing.

Molecular dynamics simulations are often used to provide feedback in the design workflow of DNA nanostructures. However, even with coarse-grained models, the convergence of distributions from unbiased simulation is slow, limiting applications to equilibrium structural properties. Given the increasing interest in dynamic, reconfigurable, and deformable devices, methods that enable efficient quantification of large ranges of motion, conformational transitions, and mechanical deformation are critically needed. Metadynamics is an automated biasing technique that enables the rapid acquisition of molecular conformational distributions by flattening free energy landscapes. Here we leveraged this approach to sample the free energy landscapes of DNA nanostructures whose unbiased dynamics are nonergodic, including bistable Holliday junctions and part of a bistable DNA origami structure. Taking a DNA origami-compliant joint as a case study, we further demonstrate that metadynamics can predict the mechanical response of a full DNA origami device to an applied force, showing good agreement with experiments. Our results exemplify the efficient computation of free energy landscapes and force response in DNA nanodevices, which could be applied for rapid feedback in iterative design workflows and generally facilitate the integration of simulation and experiments. Metadynamics will be particularly useful to guide the design of dynamic devices for nanorobotics, biosensing, or nanomanufacturing applications.

Manipulation of temperature can be used to actuate DNA origami nano-hinges containing gold nanoparticles. We develop a physical model of this system that uses partition function analysis of the interaction between the nano-hinge and nanoparticle to predict the probability that the nano-hinge is open at a given temperature. The model agrees well with experimental data and predicts experimental conditions that allow the actuation temperature of the nano-hinge to be tuned over a range of temperatures from 30 °C to 45 °C. Additionally, the model identifies microscopic interactions that are important to the macroscopic behavior of the system, revealing surprising features of the system. This combination of physical insight and predictive potential is likely to inform future designs that integrate nanoparticles into dynamic DNA origami structures or use strand binding interactions to control dynamic DNA origami behavior. Furthermore, our modeling approach could be expanded to consider the incorporation, stability, and actuation of other types of functional elements or actuation mechanisms integrated into nucleic acid devices.

The self-assembly of a DNA origami structure, although mostly feasible, represents indeed a rather complex folding problem. Entropy-driven folding and nucleation seeds formation may provide possible solutions; however, until now, a unified view of the energetic factors in play is missing. Here, by analyzing the self-assembly of origami domains with identical structure but different nucleobase composition, in function of variable design and experimental parameters, we identify the role played by sequence-dependent forces at the edges of the structure, where topological constraint is higher. Our data show that the degree of mechanical stress experienced by these regions during initial folding reshapes the energy landscape profile, defining the ratio between two possible global conformations. We thus propose a dynamic model of DNA origami assembly that relies on the capability of the system to escape high structural frustration at nucleation sites, eventually resulting in the emergence of a more favorable but previously hidden state.

Many challenges in biosensing originate from the fact that the all-important nanoarchitecture of the biosensor surface, including precise density and orientation of bioreceptors, is not entirely comprehended. Here, we introduced a three-dimensional DNA origami as a bioreceptor carrier to functionalize the fiber optic surface plasmon resonance (FO-SPR) sensor with nanoscale precision. Starting from a 24-helix bundle, two distinct DNA origami structures were designed to position thrombin-specific aptamers with different densities and distances (27 and 113 nm) from the FO-SPR surface. The origami-based biosensors not only proved to be capable of reproducible, label-free thrombin detection but revealed also valuable innovative features: (1) a significantly better performance in the absence of backfilling, known as essential in the biosensing field, suggesting improved bioreceptor orientation and accessibility, and (2) a wider linear range compared to previously reported thrombin biosensors. We envisage that our method will be beneficial for both scientists and clinicians looking for new surface (bio)chemistry and improved diagnostics.

The performance of biosensors strongly depends on the nanoarchitecture of the biosensing surface. In many studies the bioreceptor density, orientation and accessibility are often overlooked, resulting in suboptimal biosensing devices. Here, DNA origami structures were decorated with aptamers and studied as a novel tool to structure the biosensor surface with nanoscale precision, favoring interaction between target and aptamer. Using this novel method to engineer biosensing interfaces of two in-house developed biosensing platforms, we were able to accurately detect the presence of a specific target and to compete with existing biosensors in reproducibility, SNR and LOD, without the need for backfilling.

Natural filaments, such as microtubules and actin filaments, are fundamental components of the cell. Despite their relatively simple linear structure, filaments play a number of crucial roles in living organisms, from scaffolding to cellular adhesion and motility. The mechanical properties of natural filaments mostly rely on the structural features of the component units and on the way they are connected together, thus providing an ideal molecular model for emulation purposes. In this review, we describe the progresses done in this field using DNA for the rational design of synthetic filamentous-like materials with tailored structural and physical characteristics. We firstly survey the strategies that have been adopted until now for the construction of individual DNA building components and their programmable self-assembly into linear oligomeric structures. We then describe the theoretical models of polymer elasticity applied to calculate the bending strength of DNA filaments, expressed in terms of persistence length. Finally, we report some of the most exciting examples of truly biomimetic DNA filaments, which are capable of mimicking not only the sophisticated structural features of their natural counterparts but also their responsiveness to external stimuli, thus resulting in active motion and growing networks between distant loci.

Here, we demonstrate that by integrating multiple DNA nanotechnologies (DNA origami and aptamers), we can design ground-breaking biosensing concepts for sensitive and specific detection of a broad range of targets. DNA origami structures [1, 2] were decorated with aptamers and studied as a novel tool to nanostructure the biosensor surface with nanoscale precision, favoring thus interaction between target and aptamer. Different DNA nanotechnology based bioassays were designed on two in-house developed biosensing platforms.First, a DNA origami-aptamer based bioassay was coupled to a Fiber optic SPR [3, 4] sensor for the detection of thrombin (Figure 1), bypassing the need for backfilling molecules to reduce steric hindrance and unspecific interactions. Furthermore, we demonstrated that DNA origami outperformed basic DNA nanostructures, such as tetrahedrons [7], with respect to stability (Melting temperature:≥ 55 C), flexibility (number/orientation of aptamers per structure) and signal-to-noise ratio (SNR:> 40% increase). Second, DNA nanotechnology was implemented for the first time in a digital detection assay [5-6](Figure 1). An in-house generated aptamer against peanut allergen Ara h1 was favorably positioned on magnetic microparticles using DNA origami, resulting in a digital bioassay to detect Ara h1 with improved SNR (100% increase) and limit of detection (LOD: 40 fM,> 1000x lower than traditional ELISA). In conclusion, using this novel method to engineer biosensing interfaces we were not only able to accurately detect the presence of a specific target, but also to compete with existing biosensors in reproducibility …

Nature uses hierarchical self-assembly to realize fascinating structures with complex architectures from rather simple building blocks. Utilizing this approach, emergent properties arise which are surpassing the sum of properties from the individual components and are inherently coupled to the building blocks and the forces linking them together. Since the advent of structural DNA nanotechnology, scientists strive to realize artificial structures with ever growing complexity and functionality. DNA origami is a method of choice for the bottom-up assembly of biomimetic systems, as it allows the precise and programmable hierarchical self-assembly at the nanoscale, coupled with sub-nanometer spatial resolution. In this work, the hierarchical self-assembly of different macromolecular structures is guided by programmable DNA interactions and used to construct several different homo- and heterooligomeric systems. The emulation of protein filaments is performed by designing and creating a highly modular DNA origami building block, which can undergo dimerization and multimerization reactions, depending on the addressed interfaces, eventually resulting in more than 15 different filamentous structures with distinct ultrastructures and global elastic properties. The synthetic DNA filaments were analyzed by microscopic data, showing the successful realization of artificial, biomimetic structures with persistence lengths similar to – or even larger than – those of natural protein filaments. Furthermore, a nanocage for the compartmentalized spatial confinement of proteins and nanoparticles is designed and realized by using the DNA origami approach. By using hierarchical self-assembly, the pathway to a defined multi-compartments system can be well defined and thus allows a multitude of different final structures to be realized. For this purpose, all fundamental units are pre-assembled into reactive species, dimerized and finally characterized by single-particle and ensemble methods. Both temperature and cation concentration are tuned to precisely control the fate of the system.

DNA Nanotechnology for Bioanalysis, pp. 27-56 (2017) No AccessChapter 2: DNA as Building Material at the Nanoscale: From Concepts to Software-aided DesignWolfgang Pfeifer, Georg Homa, Giuseppe Arrabito and Barbara SaccàWolfgang PfeiferCentre for Medical Biotechnology (ZMB) Universitätstr. 2 of Duisburg-Essen 45117 Essen, Germany, Georg HomaCentre for Medical Biotechnology (ZMB) Universitätstr. 2 of Duisburg-Essen 45117 Essen, Germany, Giuseppe ArrabitoDepartment of Physics and Chemistry University of Palermo Viale delle Scienze, Parco d'Orleans II, 90128 Palermo, Italy and Barbara SaccàCentre for Medical Biotechnology (ZMB) Universitätstr. 2 of Duisburg-Essen 45117 Essen, Germanyhttps://doi.org/10.1142/9781786343802_0002Cited by:0 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: Chapter 2 is the most technical chapter of the book, since it deals with the different approaches and strategies allowing for design of DNA nanostructures such as tile-to-tile assembly or scaffold based design. The reader will also find information about DNA aptamers design for applications in analytical chemistry. FiguresReferencesRelatedDetails DNA Nanotechnology for BioanalysisMetrics History PDF download