A functional RNA-origami as direct thrombin inhibitor with fast-acting and specific single-molecule reversal agents in vivo model.

Injectable anticoagulants are widely used in medical procedures to prevent unwanted blood clotting. However, many lack safe, effective reversal agents. Here, we present new data on a previously described RNA origami-based, direct thrombin inhibitor (HEX01). We describe a new, fast-acting, specific, single-molecule reversal agent (antidote) and present in vivo data for the first time, including efficacy, reversibility, preliminary safety, and initial biodistribution studies. HEX01 contains multiple thrombin-binding aptamers appended on an RNA origami. It exhibits excellent anticoagulation activity in vitro and in vivo. The new single-molecule, DNA antidote (HEX02) reverses anticoagulation activity of HEX01 in human plasma within 30 s in vitro and functions effectively in a murine liver laceration model. Biodistribution studies of HEX01 in whole mice using ex vivo imaging show accumulation mainly in the liver over 24 h and with 10-fold lower concentrations in the kidneys. Additionally, we show that the HEX01/HEX02 system is non-cytotoxic to epithelial cell lines and non-hemolytic in vitro. Furthermore, we found no serum cytokine response to HEX01/HEX02 in a murine model. HEX01 and HEX02 represent a safe and effective coagulation control system with a fast-acting, specific reversal agent showing promise for potential drug development.

Utilizing multiscale engineered biomaterials to examine TGF-ß-mediated myofibroblastic differentiation.

Cells integrate many mechanical and chemical cues to drive cell signalling responses. Because of the complex nature and interdependency of alterations in extracellular matrix (ECM) composition, ligand density, mechanics, and cellular responses it is difficult to tease out individual and combinatorial contributions of these various factors in driving cell behavior in homeostasis and disease. Tuning of material viscous and elastic properties, and ligand densities, in combinatorial fashions would enhance our understanding of how cells process complex signals. For example, it is known that increased ECM mechanics and transforming growth factor beta (TGF-ß) receptor (TGF-ß-R) spacing/clustering independently drive TGF-ß signalling and associated myofibroblastic differentiation. However, it remains unknown how these inputs orthogonally contribute to cellular outcomes. Here, we describe the development of a novel material platform that combines microgel thin films with controllable viscoelastic properties and DNA origami to probe how viscoelastic properties and nanoscale spacing of TGF-ß-Rs contribute to TGF-ß signalling and myofibroblastic differentiation. We found that highly viscous materials with non-fixed TGF-ß-R spacing promoted increased TGF-ß signalling and myofibroblastic differentiation. This is likely due to the ability of cells to better cluster receptors on these surfaces. These results provide insight into the contribution of substrate properties and receptor localisation on downstream signalling. Future studies allow for exploration into other receptor-mediated processes.

Production and Testing of RNA Origami Anticoagulants.

Nucleic acid nanotechnology provides the ability to create unprecedented nanostructures with diverse architectures and functions that can be utilized in myriad fields. A set of self-folding, single-stranded RNA origami structures bearing thrombin RNA aptamers have been demonstrated to act as anticoagulants. Here, we describe the detailed methods of producing and testing of such RNA origami anticoagulants. This method highlights the potential of RNA origami for biomedical applications.

DNA aerogels and DNA-wrapped CNT aerogels for neuromorphic applications.

Nucleic acids are programmable materials that can self-assemble into defined or stochastic three-dimensional network architectures. Various attributes of self-assembled, cross-linked Deoxyribonucleic acid (DNA) hydrogels have recently been investigated, including their mechanical properties and potential biomedical functions. Herein, for the first time, we describe the successful construction of pure DNA aerogels and DNA-wrapped carbon nanotube (CNT) composite (DNA-CNT) aerogels via a single-step freeze-drying of the respective hydrogels. These aerogels reveal highly porous and randomly branched structures with low density. The electrical properties of pure DNA aerogel mimic that of a simple capacitor; in contrast, the DNA-CNT aerogel displays a fascinating resistive switching behavior in response to an applied bias voltage sweep reminiscent of a volatile memristor. We believe these novel aerogels can serve as a platform for developing complex biomimetic devices for a wide range of applications, including real-time computation, neuromorphic computing, biochemical sensing, and biodegradable functional implants. More importantly, insight obtained here on self-assembling DNA to create aerogels will pave the way to construct novel aerogel-based material platforms from DNA coated or wrapped functional entities.

Self-Assembling Nucleic Acid Nanostructures Functionalized with Aptamers.

Researchers have worked for many decades to master the rules of biomolecular design that would allow artificial biopolymer complexes to self-assemble and function similarly to the diverse biochemical constructs displayed in natural biological systems. The rules of nucleic acid assembly (dominated by Watson-Crick base-pairing) have been less difficult to understand and manipulate than the more complicated rules of protein folding. Therefore, nucleic acid nanotechnology has advanced more quickly than de novo protein design, and recent years have seen amazing progress in DNA and RNA design. By combining structural motifs with aptamers that act as affinity handles and add powerful molecular recognition capabilities, nucleic acid-based self-assemblies represent a diverse toolbox for use by bioengineers to create molecules with potentially revolutionary biological activities. In this review, we focus on the development of self-assembling nucleic acid nanostructures that are functionalized with nucleic acid aptamers and their great potential in wide ranging application areas.

Multivalent Aptamer-Functionalized Single-Strand RNA Origami as Effective, Target-Specific Anticoagulants with Corresponding Reversal Agents.

Anticoagulants are commonly utilized during surgeries and to treat thrombotic diseases like stroke and deep vein thrombosis. However, conventional anticoagulants have serious side-effects, narrow therapeutic windows, and lack safe reversal agents (antidotes). Here, an alternative RNA origami displaying RNA aptamers as target-specific anticoagulant is described. Improved design and construction techniques for self-folding, single-molecule RNA origami as a platform for displaying pre-selected RNA aptamers with precise orientational and spatial control are reported. Nuclease resistance is added using 2'-fluoro-modified pyrimidines during in vitro transcription. When four aptamers are displayed on the RNA origami platform, the measured thrombin inhibition and anticoagulation activity is higher than observed for free aptamers, ssRNA-linked RNA aptamers, and RNA origami displaying fewer aptamers. Importantly, thrombin inhibition is immediately switched off by addition of specific reversal agents. Results for single-stranded DNA (ssDNA) and single-stranded peptide nucleic acid (PNA) antidotes show restoration of 63% and 95% coagulation activity, respectively. To demonstrate potential for practical, long-term storage for clinical use, RNA origami is freeze-dried, and stored at room temperature. Freshly produced and freeze-dried RNA show identical levels of activity in coagulation assays. Compared to current commercial intravenous anticoagulants, RNA origami-based molecules show promise as safer alternatives with rapid activity switching for future therapeutic applications.

Genetically Encoded, Functional Single-Strand RNA Origami: Anticoagulant.

Nucleic acid aptamers selected for thrombin binding have been previously shown to possess anticoagulant activity; however, problems with rapid renal clearance and short circulation half-life have prevented translation to clinical usefulness. Here, a family of self-folding, functional RNA origami molecules bearing multiple thrombin-binding RNA aptamers and showing significantly improved anticoagulant activity is described. These constructs may overcome earlier problems preventing clinical use of nucleic acid anticoagulants. RNA origami structures are designed in silico and produced by in vitro transcription from DNA templates. Incorporation of 2'-fluoro-modified C- and U-nucleotides is shown to increase nuclease resistance and stability during long-term storage. Specific binding to human thrombin as well as high stability in the presence of RNase A and in human plasma, comparatively more stable than DNA is demonstrated. The RNA origami constructs show anticoagulant activity sevenfold greater than free aptamer and higher than previous DNA weave tiles decorated with DNA aptamers. Anticoagulation activity is maintained after at least 3 months of storage in buffer at 4 °C. Additionally, inhibition of thrombin is shown to be reversed by addition of single-stranded DNA antidotes. This project paves the way for development of RNA origami for potential therapeutic applications especially as a safer surgical anticoagulant.

Intracellular bacteria engage a STING-TBK1-MVB12b pathway to enable paracrine cGAS-STING signalling.

The innate immune system is crucial for eventual control of infections, but may also contribute to pathology. Listeria monocytogenes is an intracellular Gram-positive bacteria and a major cause of food-borne disease. However, important knowledge on the interactions between L. monocytogenes and the immune system is still missing. Here, we report that Listeria DNA is sorted into extracellular vesicles (EVs) in infected cells and delivered to bystander cells to stimulate the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) pathway. This was also observed during infections with Francisella tularensis and Legionella pneumophila. We identify the multivesicular body protein MVB12b as a target for TANK-binding kinase 1 phosphorylation, which is essential for the sorting of DNA into EVs and stimulation of bystander cells. EVs from Listeria-infected cells inhibited T-cell proliferation, and primed T cells for apoptosis. Collectively, we describe a pathway for EV-mediated delivery of foreign DNA to bystander cells, and suggest that intracellular bacteria exploit this pathway to impair antibacterial defence.

Gene assembly via one-pot chemical ligation of DNA promoted by DNA nanostructures.

Current gene synthesis methods are driven by enzymatic reactions. Here we report the one-pot synthesis of a chemically-ligated gene from 14 oligonucleotides. The chemical ligation benefits from the highly efficient click chemistry approach templated by DNA nanostructures, and produces modified DNA that is compatible with polymerase enzymes.

Controlled aggregation of DNA functionalized poly(phenylene-vinylene).

The morphology of conjugated polymers strongly influences their optical and electronic properties and affects their performance in polymer devices. Using optical spectroscopy and atomic force microscopy, we investigate the fluorescence properties and the aggregation state of DNA-functionalized poly(phenylene-vinylene). We show that polymer aggregation can be controlled in solution through ion and DNA interactions; aggregation is induced in the presence of divalent cations and can be reversed by adding sequence specific DNA. These interactions provide ways to tune polymer aggregation on the timescale of minutes and allows tuning of the polymer's optical properties.

Docking of Antibodies into the Cavities of DNA Origami Structures.

Immobilized antibodies are extensively employed for medical diagnostics, such as in enzyme-linked immunosorbent assays. Despite their widespread use, the ability to control the orientation of immobilized antibodies on surfaces is very limited. Herein, we report a method for the covalent and orientation-selective immobilization of antibodies in designed cavities in 2D and 3D DNA origami structures. Two tris(NTA)-modified strands are inserted into the cavity to form NTA-metal complexes with histidine clusters on the Fc domain. Subsequent covalent linkage to the antibody was achieved by coupling to lysine residues. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) confirmed the efficient immobilization of the antibodies in the origami structures. This increased control over the orientation of antibodies in nanostructures and on surfaces has the potential to direct the interactions between antibodies and targets and to provide more regular surface assemblies of antibodies.

Preparation, Single-Molecule Manipulation, and Energy Transfer Investigation of a Polyfluorene-graft-DNA polymer.

Conjugated polymers have been intensively studied due to their unique optical and electronic properties combined with their physical flexibility and scalable bottom up synthesis. Although the bulk qualities of conjugated polymers have been extensively utilized in research and industry, the ability to handle and manipulate conjugated polymers at the nanoscale lacks significantly behind. Here, the toolbox for controlled manipulation of conjugated polymers was expanded through the synthesis of a polyfluorene-DNA graft-type polymer (poly(F-DNA)). The polymer possesses the characteristics associated with the conjugated polyfluorene backbone, but the protruding single-stranded DNA provides the material with an exceptional addressability. This study demonstrates controlled single-molecule patterning of poly(F-DNA), as well as energy transfer between two different polymer-DNA conjugates. Finally, highly efficient DNA-directed quenching of polyfluorene fluorescence was shown.

Protein patterning by a DNA origami framework.

A spatial arrangement of proteins provides structural and functional advantages in vast technological applications as well as fundamental research. Most protein patterning procedures employ complicated, time consuming and very costly nanofabrication techniques. As an alternative route, we developed a fully biomolecular self-assembly method using DNA Origami Frames (DOF) as a template for both small and large scale protein patterning. We employed a triangular DOF (tDOF) to arrange the Bovine Serum Albumin (BSA) protein. Our in situ protein patterning strategy provides a novel, fully organic platform using a fast and low-cost surface approach with possible utilization in fundamental science and technological applications.

Programmed Switching of Single Polymer Conformation on DNA Origami.

DNA nanotechnology offers precise geometrical control of the positioning of materials, and it is increasingly also being used in the development of nanomechanical devices. Here we describe the development of a nanomechanical device that allows switching of the position of a single-molecule conjugated polymer. The polymer is functionalized with short single-stranded (ss) DNA strands that extend from the backbone of the polymer and serve as handles. The DNA polymer conjugate can be aligned on DNA origami in three well-defined geometries (straight line, left-turned, and right-turned pattern) by DNA hybridization directed by single-stranded guiding strands and ssDNA tracks extending from the origami surface and polymer handle. We demonstrate switching of a conjugated organic polymer conformation between left- and right-turned conformations of the polymer on DNA origami based on toehold-mediated strand displacement. The switching is observed by atomic force microscopy and by Förster resonance energy transfer between the polymer and two different organic dyes positioned in close proximity to the respective patterns. Using this method, the polymer conformation can be switched six times successively. This controlled nanomechanical switching of conjugated organic polymer conformation demonstrates unique control of the shape of a single polymer molecule, and it may constitute a new component for the development of reconfigurable nanophotonic and nanoelectronic devices.

Suspending DNA Origami Between Four Gold Nanodots.

Rectangular DNA origami functionalized with thiols in each of the four corners immobilizes by self-assembly between lithographically patterned gold nanodots on a silicon oxide surface.

Routing of individual polymers in designed patterns.

Synthetic polymers are ubiquitous in the modern world, but our ability to exert control over the molecular conformation of individual polymers is very limited. In particular, although the programmable self-assembly of oligonucleotides and proteins into artificial nanostructures has been demonstrated, we currently lack the tools to handle other types of synthetic polymers individually and thus the ability to utilize and study their single-molecule properties. Here we show that synthetic polymer wires containing short oligonucleotides that extend from each repeat can be made to assemble into arbitrary routings. The wires, which can be more than 200 nm in length, are soft and bendable, and the DNA strands allow individual polymers to self-assemble into predesigned routings on both two- and three-dimensional DNA origami templates. The polymers are conjugated and potentially conducting, and could therefore be used to create molecular-scale electronic or optical wires in arbitrary geometries.

Thermodynamically unfavorable DNA hybridizations can be made to occur by a water to ice phase change.

In an apparent contradiction to Debye-Hückel theory, it was possible to hybridize DNA in solutions of Milli-Q water (resistivity>18MΩcm(-1)) containing no added ions. This was demonstrated by hybridizing four bi-complementary DNA sequences to form an 'X' shape, as indicated by acrylamide gel electrophoresis. The requirement for hybridization was that a water-to-ice phase change should occur. Comparative experiments, using freezing by liquid nitrogen and thawing at different temperatures, showed that hybridization could take place during either the freezing or thawing process provided either was slow enough. We speculate that the low solubility of DNA in ice creates liquid inclusions of extremely high DNA and counter-ion concentration prior to complete freezing, and that hence in these inclusions hybridization was actually in accordance with Debye-Hückel theory.

RGB colour coding of Y-shaped DNA for simultaneous tri-analyte solid phase hybridization detection.

We present a new concept for tri-analyte DNA detection based on the idea of a Y-shaped capture probe which, after tri-target and fluorescently labeled reporter probe binding, becomes colour-coded to generate images in an RGB colour scheme. Hence, the RGB value of the resulting secondary pseudo-colour presented by the hybridized Y-DNA can be related to the ratio of the primary pseudo-colours present in its make-up, and thus to the ratio of the three target concentrations. As a proof of concept we detect sequences from the genes of the pathogenic bacterial strains Escherichia coli O157:H7, Vibrio cholera and Salmonella enteric in a semi-quantitative manner across the range 20-167 nM. The assay was relatively quick, with a time from hybridization to completed data interpretation of approximately 4 h.