DNA-Nanostructure-Guided Assembly of Proteins into Programmable Shapes.

The development of methods to synthesize artificial protein complexes with precisely controlled configurations will enable diverse biological and medical applications. Using DNA to link proteins provides programmability that can be difficult to achieve with other methods. Here, we use DNA origami as an "assembler" to guide the linking of protein-DNA conjugates using a series of oligonucleotide hybridization and displacement operations. We constructed several isomeric protein nanostructures, including a dimer, two types of trimer structures, and three types of tetramer assemblies, on a DNA origami platform by using a C3-symmetric building block composed of a protein trimer modified with DNA handles. Our approach expands the scope for the precise assembly of protein-based nanostructures and will enable the formulation of functional protein complexes with stoichiometric and geometric control.

A rhythmically pulsing leaf-spring DNA-origami nanoengine that drives a passive follower.

Molecular engineering seeks to create functional entities for modular use in the bottom-up design of nanoassemblies that can perform complex tasks. Such systems require fuel-consuming nanomotors that can actively drive downstream passive followers. Most artificial molecular motors are driven by Brownian motion, in which, with few exceptions, the generated forces are non-directed and insufficient for efficient transfer to passive second-level components. Consequently, efficient chemical-fuel-driven nanoscale driver-follower systems have not yet been realized. Here we present a DNA nanomachine (70 nm × 70 nm × 12 nm) driven by the chemical energy of DNA-templated RNA-transcription-consuming nucleoside triphosphates as fuel to generate a rhythmic pulsating motion of two rigid DNA-origami arms. Furthermore, we demonstrate actuation control and the simple coupling of the active nanomachine with a passive follower, to which it then transmits its motion, forming a true driver-follower pair.

High-affinity binding to the SARS-CoV-2 spike trimer by a nanostructured, trivalent protein-DNA synthetic antibody.

Multivalency enables nanostructures to bind molecular targets with high affinity. Although antibodies can be generated against a wide range of antigens, their shape and size cannot be tuned to match a given target. DNA nanotechnology provides an attractive approach for designing customized multivalent scaffolds due to the addressability and programmability of the nanostructure shape and size. Here, we design a nanoscale synthetic antibody ("nano-synbody") based on a three-helix bundle DNA nanostructure with one, two, or three identical arms terminating in a mini-binder protein that targets the SARS-CoV-2 spike protein. The nano-synbody was designed to match the valence and distance between the three receptor binding domains (RBDs) in the spike trimer, in order to enhance affinity. The protein-DNA nano-synbody shows tight binding to the wild-type, Delta, and several Omicron variants of the SARS-CoV-2 spike trimer, with affinity increasing as the number of arms increases from one to three. The effectiveness of the nano-synbody was also verified using a pseudovirus neutralization assay, with the three-arm nanostructure inhibiting two Omicron variants against which the structures with only one or two arms are ineffective. The structure of the three-arm nano-synbody bound to the Omicron variant spike trimer was solved by negative-stain transmission electron microscopy reconstruction, and shows the protein-DNA nanostructure with all three arms attached to the RBD domains, confirming the intended trivalent attachment. The ability to tune the size and shape of the nano-synbody, as well as its potential ability to attach two or more different binding ligands, will enable the high-affinity targeting of a range of proteins not possible with traditional antibodies.

RNA origami: design, simulation and application.

Design strategies for DNA and RNA nanostructures have developed along parallel lines for the past 30 years, from small structural motifs derived from biology to large 'origami' structures with thousands to tens of thousands of bases. With the recent publication of numerous RNA origami structures and improved design methods-even permitting co-transcriptional folding of kilobase-sized structures - the RNA nanotechnolgy field is at an inflection point. Here, we review the key achievements which inspired and enabled RNA origami design and draw comparisons with the development and applications of DNA origami structures. We further present the available computational tools for the design and the simulation, which will be key to the growth of the RNA origami community. Finally, we portray the transition from RNA origami structure to function. Several functional RNA origami structures exist already, their expression in cells has been demonstrated and first applications in cell biology have already been realized. Overall, we foresee that the fast-paced RNA origami field will provide new molecular hardware for biophysics, synthetic biology and biomedicine, complementing the DNA origami toolbox.

The oxDNA Coarse-Grained Model as a Tool to Simulate DNA Origami.

This chapter introduces how to run molecular dynamics simulations for DNA origami using the oxDNA coarse-grained model.

Coarse-Grained Simulations for the Characterization and Optimization of Hybrid Protein-DNA Nanostructures.

We present here the combination of experimental and computational modeling tools for the design and characterization of protein-DNA hybrid nanostructures. Our work incorporates several features in the design of these nanostructures: (1) modeling of the protein-DNA linker identity and length; (2) optimizing the design of protein-DNA cages to account for mechanical stresses; (3) probing the incorporation efficiency of protein-DNA conjugates into DNA nanostructures. The modeling tools were experimentally validated using structural characterization methods like cryo-TEM and AFM. Our method can be used for fitting low-resolution electron density maps when structural insights cannot be deciphered from experiments, as well as enable in-silico validation of nanostructured systems before their experimental realization. These tools will facilitate the design of complex hybrid protein-DNA nanostructures that seamlessly integrate the two different biomolecules.

Design and simulation of DNA, RNA and hybrid protein-nucleic acid nanostructures with oxView.

Molecular simulation has become an integral part of the DNA/RNA nanotechnology research pipeline. In particular, understanding the dynamics of structures and single-molecule events has improved the precision of nanoscaffolds and diagnostic tools. Here we present oxView, a design tool for visualization, design, editing and analysis of simulations of DNA, RNA and nucleic acid-protein nanostructures. oxView provides an accessible software platform for designing novel structures, tweaking existing designs, preparing them for simulation in the oxDNA/RNA molecular simulation engine and creating visualizations of simulation results. In several examples, we present procedures for using the tool, including its advanced features that couple the design capabilities with a coarse-grained simulation engine and scripting interface that can programmatically edit structures and facilitate design of complex structures from multiple substructures. These procedures provide a practical basis from which researchers, including experimentalists with limited computational experience, can integrate simulation and 3D visualization into their existing research programs.

Nanobase.org: a repository for DNA and RNA nanostructures.

We introduce a new online database of nucleic acid nanostructures for the field of DNA and RNA nanotechnology. The database implements an upload interface, searching and database browsing. Each deposited nanostructures includes an image of the nanostructure, design file, an optional 3D view, and additional metadata such as experimental data, protocol or literature reference. The database accepts nanostructures in any preferred format used by the uploader for the nanostructure design. We further provide a set of conversion tools that encourage design file conversion into common formats (oxDNA and PDB) that can be used for setting up simulations, interactive editing or 3D visualization. The aim of the repository is to provide to the DNA/RNA nanotechnology community a resource for sharing their designs for further reuse in other systems and also to function as an archive of the designs that have been achieved in the field so far. Nanobase.org is available at https://nanobase.org/.

OxDNA.org: a public webserver for coarse-grained simulations of DNA and RNA nanostructures.

OxDNA and oxRNA are popular coarse-grained models used by the DNA/RNA nanotechnology community to prototype, analyze and rationalize designed DNA and RNA nanostructures. Here, we present oxDNA.org, a graphical web interface for running, visualizing and analyzing oxDNA and oxRNA molecular dynamics simulations on a GPU-enabled high performance computing server. OxDNA.org automatically generates simulation files, including a multi-step relaxation protocol for structures exported in non-physical states from DNA/RNA design tools. Once the simulation is complete, oxDNA.org provides an interactive visualization and analysis interface using the browser-based visualizer oxView to facilitate the understanding of simulation results for a user's specific structure. This online tool significantly lowers the entry barrier of integrating simulations in the nanostructure design pipeline for users who are not experts in the technical aspects of molecular simulation. The webserver is freely available at oxdna.org.

Coarse-grained nucleic acid-protein model for hybrid nanotechnology.

The emerging field of hybrid DNA-protein nanotechnology brings with it the potential for many novel materials which combine the addressability of DNA nanotechnology with the versatility of protein interactions. However, the design and computational study of these hybrid structures is difficult due to the system sizes involved. To aid in the design and in silico analysis process, we introduce here a coarse-grained DNA/RNA-protein model that extends the oxDNA/oxRNA models of DNA/RNA with a coarse-grained model of proteins based on an anisotropic network model representation. Fully equipped with analysis scripts and visualization, our model aims to facilitate hybrid nanomaterial design towards eventual experimental realization, as well as enabling study of biological complexes. We further demonstrate its usage by simulating DNA-protein nanocage, DNA wrapped around histones, and a nascent RNA in polymerase.

Unified Nanotechnology Format: One Way to Store Them All.

The domains of DNA and RNA nanotechnology are steadily gaining in popularity while proving their value with various successful results, including biosensing robots and drug delivery cages. Nowadays, the nanotechnology design pipeline usually relies on computer-based design (CAD) approaches to design and simulate the desired structure before the wet lab assembly. To aid with these tasks, various software tools exist and are often used in conjunction. However, their interoperability is hindered by a lack of a common file format that is fully descriptive of the many design paradigms. Therefore, in this paper, we propose a Unified Nanotechnology Format (UNF) designed specifically for the biomimetic nanotechnology field. UNF allows storage of both design and simulation data in a single file, including free-form and lattice-based DNA structures. By defining a logical and versatile format, we hope it will become a widely accepted and used file format for the nucleic acid nanotechnology community, facilitating the future work of researchers and software developers. Together with the format description and publicly available documentation, we provide a set of converters from existing file formats to simplify the transition. Finally, we present several use cases visualizing example structures stored in UNF, showcasing the various types of data UNF can handle.

Meta-DNA structures.

DNA origami has emerged as a highly programmable method to construct customized objects and functional devices in the 10-100 nm scale. Scaling up the size of the DNA origami would enable many potential applications, which include metamaterial construction and surface-based biophysical assays. Here we demonstrate that a six-helix bundle DNA origami nanostructure in the submicrometre scale (meta-DNA) could be used as a magnified analogue of single-stranded DNA, and that two meta-DNAs that contain complementary 'meta-base pairs' can form double helices with programmed handedness and helical pitches. By mimicking the molecular behaviours of DNA strands and their assembly strategies, we used meta-DNA building blocks to form diverse and complex structures on the micrometre scale. Using meta-DNA building blocks, we constructed a series of DNA architectures on a submicrometre-to-micrometre scale, which include meta-multi-arm junctions, three-dimensional (3D) polyhedrons, and various 2D/3D lattices. We also demonstrated a hierarchical strand-displacement reaction on meta-DNA to transfer the dynamic features of DNA into the meta-DNA. This meta-DNA self-assembly concept may transform the microscopic world of structural DNA nanotechnology.

Design, optimization and analysis of large DNA and RNA nanostructures through interactive visualization, editing and molecular simulation.

This work seeks to remedy two deficiencies in the current nucleic acid nanotechnology software environment: the lack of both a fast and user-friendly visualization tool and a standard for structural analyses of simulated systems. We introduce here oxView, a web browser-based visualizer that can load structures with over 1 million nucleotides, create videos from simulation trajectories, and allow users to perform basic edits to DNA and RNA designs. We additionally introduce open-source software tools for extracting common structural parameters to characterize large DNA/RNA nanostructures simulated using the coarse-grained modeling tool, oxDNA, which has grown in popularity in recent years and is frequently used to prototype new nucleic acid nanostructural designs, model biophysics of DNA/RNA processes, and rationalize experimental results. The newly introduced software tools facilitate the computational characterization of DNA/RNA designs by providing multiple analysis scripts, including mean structures and structure flexibility characterization, hydrogen bond fraying, and interduplex angles. The output of these tools can be loaded into oxView, allowing users to interact with the simulated structure in a 3D graphical environment and modify the structures to achieve the required properties. We demonstrate these newly developed tools by applying them to design and analysis of a range of DNA/RNA nanostructures.

TacoxDNA: A user-friendly web server for simulations of complex DNA structures, from single strands to origami.

Simulations of nucleic acids at different levels of structural details are increasingly used to complement and interpret experiments in different fields, from biophysics to medicine and materials science. However, the various structural models currently available for DNA and RNA and their accompanying suites of computational tools can be very rarely used in a synergistic fashion. The tacoxDNA webserver and standalone software package presented here are a step toward a long-sought interoperability of nucleic acids models. The webserver offers a simple interface for converting various common input formats of DNA structures and setting up molecular dynamics (MD) simulations. Users can, for instance, design DNA rings with different topologies, such as knots, with and without supercoiling, by simply providing an XYZ coordinate file of the DNA centre-line. More complex DNA geometries, as designable in the cadnano, CanDo and Tiamat tools, can also be converted to all-atom or oxDNA representations, which can then be used to run MD simulations. Though the latter are currently geared toward the native and LAMMPS oxDNA representations, the open-source package is designed to be further expandable. TacoxDNA is available at http://tacoxdna.sissa.it. © 2019 Wiley Periodicals, Inc.

Triangulated Wireframe Structures Assembled Using Single-Stranded DNA Tiles.

The field of structural DNA nanotechnology offers a wide range of design strategies with which to build structures with a desired aspect ratio, size, and shape. Compared with traditional close-packed DNA structures, triangulated wireframe structures require less material per surface or volume unit and improve the stability in biologically relevant conditions due to the reduced electrostatic repulsion. Herein, we expand the design space of the DNA single-stranded tile method to cover a range of anisotropic, finite, triangulated wireframe structures as well as a number of one-dimensional crystalline assemblies. These structures are composed of six-arm junctions with a single double helix as connecting edges that assemble in physiologically relevant salinities. For a reliable folding of the structures, single-stranded spacers 2-4 nucleotides long have to be introduced in the junction connecting neighboring arms. Coarse-grained molecular dynamics simulations using the oxDNA model suggests that the spacers prevent the stacking of DNA helices, thereby facilitating the assembly of planar geometries.

Enzymatic Phosphorylation of Ser in a Type I Collagen Peptide.

Phosphoproteomics studies have reported phosphorylation at multiple sites within collagen, raising the possibility that these post-translational modifications regulate the physical or biological properties of collagen. In this study, molecular dynamics simulations and experimental studies were carried out on model peptides to establish foundational principles of phosphorylation of Ser residues in collagen. A (Gly-Xaa-Yaa)11 peptide was designed to include a Ser-containing sequence from type I collagen that was reported to be phosphorylated. The physiological kinase involved in collagen phosphorylation is not known. In vitro studies showed that a model kinase ERK1 (extracellular signal-regulated protein kinase 1) would phosphorylate Ser within the consensus sequence if the collagen-like peptide is in the denatured state but not in the triple-helical state. The peptide was not a substrate for FAM20C, a kinase present in the secretory pathway, which has been shown to phosphorylate many extracellular matrix proteins. The unfolded single chain (Gly-Xaa-Yaa)11 peptide containing phosphoSer was able to refold to form a stable triple helix but at a reduced folding rate and with a small decrease in thermal stability relative to the nonphosphorylated peptide at neutral pH. These biophysical studies on model peptides provide a basis for investigations into the physiological consequences of collagen phosphorylation and the application of phosphorylation to regulate the properties of collagen biomaterials.

Effects of flexibility of the α2 chain of type I collagen on collagenase cleavage.

Cleavage of collagen by collagenases such as matrix metalloproteinase 1 (MMP-1) is a key step in development, tissue remodeling, and tumor proliferation. The abundant heterotrimeric type I collagen composed of two α1(I) chains and one α2(I) chain is efficiently cleaved by MMP-1 at a unique site in the triple helix, a process which may be initiated by local unfolding within the peptide chains. Atypical homotrimers of the α1(I) chain, found in embryonic and cancer tissues, are very resistant to MMP cleavage. To investigate MMP-1 cleavage, recombinant homotrimers were constructed with sequences from the MMP cleavage regions of human collagen chains inserted into a host bacterial collagen protein system. All triple-helical constructs were cleaved by MMP-1, with α2(I) homotrimers cleaved efficiently at a rate similar to that seen for α1(II) and α1(III) homotrimers, while α1(I) homotrimers were cleaved at a much slower rate. The introduction of destabilizing Gly to Ser mutations within the human collagenase susceptible region of the α2(I) chain did not interfere with MMP-1 cleavage. Molecular dynamics simulations indicated a greater degree of transient hydrogen bond breaking in α2(I) homotrimers compared with α1(I) homotrimers at the MMP-1 cleavage site, and showed an extensive disruption of hydrogen bonding in the presence of a Gly to Ser mutation, consistent with chymotrypsin digestion results. This study indicates that α2(I) homotrimers are susceptible to MMP-1, proves that the presence of an α1(I) chain is not a requirement for α2(I) cleavage, and supports the importance of local unfolding of α2(I) in collagenase cleavage.