Folding molecular origami from ribosomal RNA.

Approximately 80 percent of the total RNA in cells is ribosomal RNA (rRNA), making it an abundant and inexpensive natural source of long, single-stranded nucleic acid, which could be used as raw material for the fabrication of molecular origami. In this study, we demonstrate efficient and robust construction of 2D and 3D origami nanostructures utilizing cellular rRNA as a scaffold and DNA oligonucleotide staples. We present calibrated protocols for the robust folding of contiguous shapes from one or two rRNA subunits that are efficient to allow folding using crude extracts of total RNA. We also show that RNA maintains stability within the folded structure. Lastly, we present a novel and comprehensive analysis and insights into the stability of RNA:DNA origami nanostructures and demonstrate their enhanced stability when coated with polylysine-polyethylene glycol in different temperatures, low Mg2+ concentrations, human serum, and in the presence of nucleases (DNase I or RNase H). Thus, laying the foundation for their potential implementation in emerging biomedical applications, where folding rRNA into stable structures outside and inside cells would be desired.

Biodistribution and function of coupled polymer-DNA origami nanostructures.

Spatial control over the distribution of therapeutics is a highly desired feature, which could limit the side effects of many drugs. Here we describe a nanoscale agent, fabricated from a coupled polymer-DNA origami hybrid that exhibits stability in serum and slow diffusion through tissues, in a manner correlating with shape and aspect ratio. Coupling to fragments of polyethylene glycol (PEG) through polyamine electrostatic interactions resulted in marked stability of the agents in-vivo, with > 90% of the agents maintaining structural integrity 5 days following subcutaneous injection. An agent functionalized with aptamers specific for human tumor necrosis factor TNF-alpha, significantly abrogated the inflammatory response in a delayed-type hypersensitivity model in humanized TNF-alpha mice. These findings highlight polymer-DNA hybrid nanostructures as a programmable and pharmacologically viable update to mainstream technologies such as monoclonal antibodies, capable of exerting an additional layer of control across the spatial dimension of drug activity.

Visualizing the structure and motion of the long noncoding RNA HOTAIR.

Long noncoding RNA molecules (lncRNAs) are estimated to account for the majority of eukaryotic genomic transcripts, and have been associated with multiple diseases in humans. However, our understanding of their structure-function relationships is scarce, with structural evidence coming mostly from indirect biochemical approaches or computational predictions. Here we describe direct visualization of the lncRNA HOTAIR (HOx Transcript AntIsense RNA) using atomic force microscopy (AFM) in nucleus-like conditions at 37°. Our observations reveal that HOTAIR has a discernible, although flexible, shape. Fast AFM scanning enabled the quantification of the motion of HOTAIR, and provided visual evidence of physical interactions with genomic DNA segments. Our report provides a biologically plausible description of the anatomy and intrinsic properties of HOTAIR, and presents a framework for studying the structural biology of lncRNAs.

Discovery of tumoricidal DNA oligonucleotides by response-directed in vitro evolution.

Drug discovery is challenged by ineffectiveness of drugs against variable and evolving diseases, and adverse effects due to poor selectivity. We describe a robust platform which potentially addresses these limitations. The platform enables rapid discovery of DNA oligonucleotides evolved in vitro for exerting specific and selective biological responses in target cells. The process operates without a priori target knowledge (mutations, biomarkers, etc). We report the discovery of oligonucleotides with direct, selective cytotoxicity towards cell lines, as well as patient-derived solid and hematological tumors. A specific oligonucleotide termed E8, induced selective apoptosis in triple-negative breast cancer (TNBC) cells. Polyethylene glycol-modified E8 exhibited favorable biodistribution in animals, persisting in tumors up to 48-hours after injection. E8 inhibited tumors by 50% within 10 days of treatment in patient-derived xenograft mice, and was effective in ex vivo organ cultures from chemotherapy-resistant TNBC patients. These findings highlight a drug discovery model which is target-tailored and on-demand.

Nanoscale Robots Exhibiting Quorum Sensing.

Multi-agent systems demonstrate the ability to collectively perform complex tasks (e.g., construction, search, and locomotion) with greater speed, efficiency, or effectiveness than could a single agent alone. Direct and indirect coordination methods allow agents to collaborate to share information and adapt their activity to fit dynamic situations. A well-studied example is quorum sensing (QS), a mechanism allowing bacterial communities to coordinate and optimize various phenotypes in response to population density. Here we implement, for the first time, bio-inspired QS in robots fabricated from DNA origami, which communicate by transmitting and receiving diffusing signals. The mechanism we describe includes features such as programmable response thresholds and quorum quenching, and is capable of being triggered by proximity of a specific target cell. Nanoscale robots with swarm intelligence could carry out tasks that have been so far unachievable in diverse fields such as industry, manufacturing, and medicine.

A Non-Newtonian Fluid Robot.

New types of robots inspired by biological principles of assembly, locomotion, and behavior have been recently described. In this work we explored the concept of robots that are based on more fundamental physical phenomena, such as fluid dynamics, and their potential capabilities. We report a robot made entirely of non-Newtonian fluid, driven by shear strains created by spatial patterns of audio waves. We demonstrate various robotic primitives such as locomotion and transport of metallic loads-up to 6-fold heavier than the robot itself-between points on a surface, splitting and merging, shapeshifting, percolation through gratings, and counting to 3. We also utilized interactions between multiple robots carrying chemical loads to drive a bulk chemical synthesis reaction. Free of constraints such as skin or obligatory structural integrity, fluid robots represent a radically different design that could adapt more easily to unfamiliar, hostile, or chaotic environments and carry out tasks that neither living organisms nor conventional machines are capable of.

Folding and Characterization of a Bio-responsive Robot from DNA Origami.

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both stimuli and cargo can be tailored according to specific needs. Here we describe the DNA nanorobot fabrication protocol, with the use of the DNA origami technique. The procedure initiates by mixing short single-strand DNA staples into a stock mixture which is then added to a long, circular, single-strand DNA scaffold in presence of a folding buffer. A standard thermo cycler is programmed to gradually lower the mixing reaction temperature to facilitate the staples-to-scaffold annealing, which is the guiding force behind the folding of the nanorobot. Once the 60 hr folding reaction is complete, excess staples are discarded using a centrifugal filter, followed by visualization via agarose-gel electrophoresis (AGE). Finally, successful fabrication of the nanorobot is verified by transmission electron microscopy (TEM), with the use of uranyl-formate as negative stain.

Meshing complex macro-scale objects into self-assembling bricks.

Self-assembly provides an information-economical route to the fabrication of objects at virtually all scales. However, there is no known algorithm to program self-assembly in macro-scale, solid, complex 3D objects. Here such an algorithm is described, which is inspired by the molecular assembly of DNA, and based on bricks designed by tetrahedral meshing of arbitrary objects. Assembly rules are encoded by topographic cues imprinted on brick faces while attraction between bricks is provided by embedded magnets. The bricks can then be mixed in a container and agitated, leading to properly assembled objects at high yields and zero errors. The system and its assembly dynamics were characterized by video and audio analysis, enabling the precise time- and space-resolved characterization of its performance and accuracy. Improved designs inspired by our system could lead to successful implementation of self-assembly at the macro-scale, allowing rapid, on-demand fabrication of objects without the need for assembly lines.

Cooperativity-based modeling of heterotypic DNA nanostructure assembly.

DNA origami is a robust method for the fabrication of nanoscale 2D and 3D objects with complex features and geometries. The process of DNA origami folding has been recently studied, however quantitative understanding of it is still elusive. Here, we describe a systematic quantification of the assembly process of DNA nanostructures, focusing on the heterotypic DNA junction-in which arms are unequal-as their basic building block. Using bulk fluorescence studies we tracked this process and identified multiple levels of cooperativity from the arms in a single junction to neighboring junctions in a large DNA origami object, demonstrating that cooperativity is a central underlying mechanism in the process of DNA nanostructure assembly. We show that the assembly of junctions in which the arms are consecutively ordered is more efficient than junctions with randomly-ordered components, with the latter showing assembly through several alternative trajectories as a potential mechanism explaining the lower efficiency. This highlights consecutiveness as a new design consideration that could be implemented in DNA nanotechnology CAD tools to produce more efficient and high-yield designs. Altogether, our experimental findings allowed us to devise a quantitative, cooperativity-based heuristic model for the assembly of DNA nanostructures, which is highly consistent with experimental observations.

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 problems, logic circuits and robotics. The molecule also interfaces naturally with living systems, and different forms of DNA-based biocomputing have already been demonstrated. Here, we show that DNA origami can be used to fabricate nanoscale robots that are capable of dynamically interacting with each other 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 used the DNA origami robots in living cockroaches (Blaberus discoidalis) to control a molecule that targets their cells.

Designing a bio-responsive robot from DNA origami.

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information(1), they can be utilized in parallel computing(2,3) , recognize and bind molecular or cellular targets(4,5) , catalyze chemical reactions(6,7) , and generate calculated responses in a biological system(8,9). Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures(10-12), enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect. Creating shapes from nucleic acids was first proposed by Seeman(13), and several variations on this theme have since been realized using various techniques(11,12,14,15) . However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami(16). In this technique, the folding of a long (>7,000 bases) single-stranded DNA 'scaffold' is directed to a desired shape by hundreds of short complementary strands termed 'staples'. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well(17,18) . The current paper will focus on the caDNAno 2.0 software(19) developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient. In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described(20). This robot is 'robotic' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando(21) to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings.

Fundus albipunctatus: novel mutations and phenotypic description of Israeli patients.

PURPOSE: To characterize the genetic defects associated with fundus albipunctatus (FAP) in patients in Israel. METHODS: Twenty patients with FAP from diverse ethnicities underwent ophthalmic and electroretinogram tests following the International Society for Clinical Electrophysiology of Vision protocol. Genomic DNA was extracted from peripheral blood. Mutation analysis of the 11-cis retinol dehydrogenase (RDH5) gene was performed with direct sequencing of PCR-amplified exons. RESULTS: Four novel RDH5 gene mutations were identified. Of them, the null mutations c.343C>T (p.R54X) and c.242delTGCC were most prevalent. Macular involvement was present in two patients who carry different mutation types. CONCLUSIONS: Mutation analysis of the RDH5 gene in the present series revealed four novel mutations and a previously reported one. No significant genotype-phenotype correlation was found.