Universal computing by DNA origami robots in a living animal

Biological systems are collections of discrete molecular objects that move around and collide with each other. Cells carry out elaborate processes by precisely controlling these collisions, but developing artificial machines that can interface with and control such interactions remains a significant challenge. DNA is a natural substrate for computing and has been used to implement a diverse set of mathematical problems1-3, logic circuits4-6 and robotics7-9. The molecule also naturally interfaces with living systems, and different forms of DNA-based biocomputing have previously been demonstrated10-13. Here we show that DNA origami14-16 can be used to fabricate nanoscale robots that are capable of dynamically interacting with each other17-18 in a living animal. The interactions generate logical outputs, which are relayed to switch molecular payloads on or off. As a proof-of-principle, we use the system to create architectures that emulate various logic gates (AND, OR, XOR, NAND, NOT, CNOT, and a half adder). Following an ex vivo prototyping phase, we successfully employed the DNA origami robots in living cockroaches (Blaberus discoidalis) to control a molecule that targets the cells of the animal

A new cell-sized support for 3D cell cultures based on recombinant spider silk fibers

It is now generally accepted that 2D cultures cannot accurately replicate the rich environment and complex tissue architecture that exists in vivo, and that classically cultured cells tend to lose their original function. Growth of spheroids as opposed to 2D cultures on plastic has now been hailed as an efficient method to produce quantities of high-quality cells for cancer research, drug discovery, neuroscience, and regenerative medicine. We have developed a new recombinant protein that mimics dragline spidersilk and that self-assembles into cell-sized coils. These have high thermal and shelf-life stability and can be readily sterilized and stored for an extended period of time. The fibers are flexible, elastic, and biocompatible and can serve as cell-sized scaffold for the formation of 3D cell spheroids. As a proof of concept, recombinant spidersilk was integrated as a scaffold in spheroids of three cell types: primary rat hepatocytes, human mesenchymal stem cells, and mouse L929 cells. The scaffolds significantly reduced spheroid shrinkage and unlike scaffold-free spheroids, spheroids did not disintegrate over the course of long-term culture. Cells in recombinant spidersilk spheroids showed increased viability, and the cell lines continued to proliferate for longer than control cultures without spidersilk. The spidersilk also supported biological functions. Recombinant spidersilk primary hepatocyte spheroids exhibited 2.7-fold higher levels of adenosine triphosphate (ATP) continued to express and secrete albumin and exhibited significantly higher basal and induced CYP3A activity for at least 6 weeks in culture, while control spheroids without fibers stopped producing albumin after 27 days and CPY3A activity was barely detectable after 44 days. These results indicate that recombinant spidersilk can serve as a useful tool for long-term cell culture of 3D cell spheroids and specifically that primary hepatocytes can remain active in culture for an extended period of time which could be of great use in toxicology testing. </jats:p

Novel Assembly Properties of Recombinant Spider Dragline Silk Proteins

AbstractSpider dragline silk, which exhibits extraordinary strength and toughness, is primarily composed of two related proteins that largely consist of repetitive sequences. In most spiders, the repetitive region of one of these proteins is rich in prolines, which are not present in the repetitive region of the other [1]. The absence of prolines in one component was previously speculated to be essential for the thread structure [2]. Here, we analyzed dragline proteins of the garden spider Araneus diadematus, ADF-3 and ADF-4, which are both proline rich, by employing the baculovirus expression system. Whereas ADF-3 represented an intrinsically soluble protein, ADF-4 was insoluble in vitro and self-assembled into filaments in the cytosol of the host insect cells. These ADF-4 filaments displayed the exceptional chemical stability of authentic silk threads. We provide evidence that the observed properties of ADF-3 and ADF-4 strongly depend on intrinsic characteristics such as hydropathicity, which differs dramatically between the two proteins, as in most other pairs of dragline silk proteins from other Araneoidea species, but not on their proline content. Our findings shed new light on the structural components of spider dragline silk, allowing further elucidation of their assembly properties, which may open the door for commercial applications

Universal computing by DNA origami robots in a living animal

These authors contributed equally to this work. Biological systems are collections of discrete molecular objects that move around and collide with each other. Cells carry out elaborate processes by precisely controlling these collisions, but developing artificial machines that can interface with and control such interactions remains a significant challenge. DNA is a natural substrate for computing and has been used to implement a diverse set of mathematical problems1-3, logic circuits4-6 and robotics7-9. The molecule also naturally interfaces with living systems, and different forms of DNA-based biocomputing have previously been demonstrated10-13. Here we show that DNA origami14-16 can be used to fabricate nanoscale robots that are capable of dynamically interacting with each other17-18 in a living animal. The interactions generate logical outputs, which are relayed to switch molecular payloads on or off. As a proof-of-principle, we use the system to create architectures that emulate various logic gates (AND, OR, XOR, NAND, NOT, CNOT, and a half adder). Following an ex vivo prototyping phase, we successfully employed the DNA origami robots in living cockroaches (Blaberus discoidalis) to control a molecule that targets the cells of the animal

An antiviral self-replicating molecular heterotroph

AbstractWe report the synthesis of a molecular machine, fabricated from nucleic acids, which is capable of digesting viral RNA and utilizing it to assemble additional copies of itself inside living cells. The machine’s body plan combines several parts that build upon the target RNA, assembling an immobile, DNA:RNA 4-way junction, which contains a single gene encoding a hammerhead ribozyme (HHR). Full assembly of the machine’s body from its parts enables the subsequent elongation of the gene and transcription of HHR molecules, followed by HHR-mediated digestion of the target molecule. This digestion converts the target to a building block suitable for participation in the assembly of more copies of the machine, mimicking biological heterotrophy. In this work we describe the general design of a prototypical machine, characterize its activity cycle and kinetics, and show that it can be efficiently and safely delivered into live cells. As a proof of principle, we constructed a machine that targets the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) GP64 gene, and show that it effectively suppresses viral propagation in a cell population, exhibiting predator/prey-like dynamics with the infecting virus. In addition, the machine significantly reduced viral infection, stress signaling, and innate immune activation inside virus-infected animals. This preliminary design could control the behavior of antisense therapies for a range of applications, particularly against dynamic targets such as viruses and cancer.</jats:p