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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.

 

In this work, we describe the development of a computational model for arrays of rotary DNA origami elements which can self-organize on a large scale and explore the interesting morphologies and order–disorder transition behavior of these systems.

 

Structural DNA nanotechnology has enabled the design and construction of complex nanoscale structures with precise geometry and programmable dynamic and mechanical properties. Recent efforts have led to major advances in the capacity to actuate shape changes of DNA origami devices and incorporate DNA origami into larger assemblies, which open the prospect of using DNA to design shape-morphing assemblies as components of micro-scale reconfigurable or sensing materials. Indeed, a few studies have constructed higher order assemblies with reconfigurable devices; however, these demonstrations have utilized structures with relatively simple motion, primarily hinges that open and close. To advance the shape changing capabilities of DNA origami assemblies, we developed a multi-component DNA origami 6-bar mechanism that can be reconfigured into various shapes and can be incorporated into larger assemblies while maintaining capabilities for a variety of shape transformations. We demonstrate the folding of the 6-bar mechanism into four different shapes and demonstrate multiple transitions between these shapes. We also studied the shape preferences of the 6-bar mechanism in competitive folding reactions to gain insight into the relative free energies of the shapes. Furthermore, we polymerized the 6-bar mechanism into tubes with various cross-sections, defined by the shape of the individual mechanism, and we demonstrate the ability to change the shape of the tube cross-section. This expansion of current single-device reconfiguration to higher order scales provides a foundation for nano to micron scale DNA nanotechnology applications such as biosensing or materials with tunable properties. 

Abstract Single molecule force spectroscopy is a powerful approach to probe the structure, conformational changes, and kinetic properties of biological and synthetic macromolecules. However, common approaches to apply forces to biomolecules require expensive and cumbersome equipment and relatively large probes such as beads or cantilevers, which limits their use for many environments and makes integrating with other methods challenging. Furthermore, existing methods have key limitations such as an inability to apply compressive forces on single molecules. We report a nanoscale DNA force spectrometer (nDFS), which is based on a DNA origami hinge with tunable mechanical and dynamic properties. The angular free energy landscape of the nDFS can be engineered across a wide range through substitution of less than 5% of the strand components. We further incorporate a removable strut that enables reversible toggling of the nDFS between open and closed states to allow for actuated application of tensile and compressive forces. We demonstrate the ability to apply compressive forces by inducing a large bend in a 249bp DNA molecule, and tensile forces by inducing DNA unwrapping of a nucleosome sample. These results establish a versatile tool for force spectroscopy and robust methods for designing nanoscale mechanical devices with tunable force application. 

DNA nanostructures are a promising tool to deliver molecular payloads to cells. DNA origami structures, where long single-stranded DNA is 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 binding sites on DNA nanostructures to increase shuttling 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 templates in human primary cells. Furthermore, our study validates virus-like particles as an efficient method of DNA nanostructure delivery, opening the possibility of delivering nanostructures in vivo to specific cell types. Together, these results provide new approaches to gene delivery with DNA nanostructures and establish their use as 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 biosensing, into cell nuclei.

 

Dynamic DNA nanotechnology involves the creation of nanoscale devices made of DNA whose primary function arises from their ability to undergo controlled motion or reconfiguration. In the past two decades, dynamic DNA nanotechnology has evolved to the point where it is now being employed in devices intended for applications in sensing, drug delivery, computation, nanorobotics, and more. In this review article, we discuss the design of dynamic DNA nanodevices and the characterization and prediction of device behavior. We also identify a number of continuing challenges in dynamic DNA nanotechnology and discuss potential solutions to those challenges. 

Significant progress in DNA nanotechnology has accelerated the development of molecular machines with functions like macroscale machines. However, the mobility of DNA self‐assembled nanorobots is still dramatically limited due to challenges with designing and controlling nanoscale systems with many degrees of freedom. Here, an origami‐inspired method to design transformable DNA nanomachines is presented. This approach integrates stiff panels formed by bundles of double‐stranded DNA connected with foldable creases formed by single‐stranded DNA. To demonstrate the method, a DNA version of the paper origami mechanism called a waterbomb base (WBB) consisting of six panels connected by six joints is constructed. This nanoscale WBB can follow four distinct motion paths to transform between five distinct configurations including a flat square, two triangles, a rectangle, and a fully compacted trapezoidal shape. To achieve this, the sequence specificity of DNA base‐pairing is leveraged for the selective actuation of joints and the ion‐sensitivity of base‐stacking interactions is employed for the flattening of joints. In addition, higher‐order assembly of DNA WBBs into reconfigurable arrays is achieved. This work establishes a foundation for origami‐inspired design for next generation synthetic molecular robots and reconfigurable nanomaterials enabling more complex and controllable motion.