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.

Molecular Robots Obeying Asimov's Three Laws of Robotics.

Asimov's three laws of robotics, which were shaped in the literary work of Isaac Asimov (1920-1992) and others, define a crucial code of behavior that fictional autonomous robots must obey as a condition for their integration into human society. While, general implementation of these laws in robots is widely considered impractical, limited-scope versions have been demonstrated and have proven useful in spurring scientific debate on aspects of safety and autonomy in robots and intelligent systems. In this work, we use Asimov's laws to examine these notions in molecular robots fabricated from DNA origami. We successfully programmed these robots to obey, by means of interactions between individual robots in a large population, an appropriately scoped variant of Asimov's laws, and even emulate the key scenario from Asimov's story "Runaround," in which a fictional robot gets into trouble despite adhering to the laws. Our findings show that abstract, complex notions can be encoded and implemented at the molecular scale, when we understand robots on this scale on the basis of their interactions.

Thought-Controlled Nanoscale Robots in a Living Host.

We report a new type of brain-machine interface enabling a human operator to control nanometer-size robots inside a living animal by brain activity. Recorded EEG patterns are recognized online by an algorithm, which in turn controls the state of an electromagnetic field. The field induces the local heating of billions of mechanically-actuating DNA origami robots tethered to metal nanoparticles, leading to their reversible activation and subsequent exposure of a bioactive payload. As a proof of principle we demonstrate activation of DNA robots to cause a cellular effect inside the insect Blaberus discoidalis, by a cognitively straining task. This technology enables the online switching of a bioactive molecule on and off in response to a subject's cognitive state, with potential implications to therapeutic control in disorders such as schizophrenia, depression, and attention deficits, which are among the most challenging conditions to diagnose and treat.

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.

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.

The influence of chemical chaperones on enzymatic activity under thermal and chemical stresses: common features and variation among diverse chemical families.

Molecular and chemical chaperones are key components of the two main mechanisms that ensure structural stability and activity under environmental stresses. Yet, chemical chaperones are often regarded only as osmolytes and their role beyond osmotic regulation is not fully understood. Here, we systematically studied a large group of chemical chaperones, representatives of diverse chemical families, for their protective influence under either thermal or chemical stresses. Consistent with previous studies, we observed that in spite of the structural similarity between sugars and sugar alcohols, they have an apparent difference in their protective potential. Our results support the notion that the protective activity is mediated by the solvent and the presence of water is essential. In the current work we revealed that i) polyols and sugars have a completely different profile of protective activity toward trifluoroethanol and thermal stress; ii) minor changes in solvent composition that do not affect enzyme activity, yet have a great effect on the ability of osmolytes to act as protectants and iii) increasing the number of active groups of carbohydrates makes them better protectants while increasing the number of active groups of methylamines does not, as revealed by attempts to synthesize de novo designed methylamines with multiple functional groups.

Quantitative structure-activity relationship analysis of ß-amyloid aggregation inhibitors.

Inhibiting the aggregation process of the ß-amyloid peptide is a promising strategy in treating Alzheimer's disease. In this work, we have collected a dataset of 80 small molecules with known inhibition levels and utilized them to develop two comprehensive quantitative structure-activity relationship models: a Bayesian model and a decision tree model. These models have exhibited high predictive accuracy: 87% of the training and test sets using the Bayesian model and 89 and 93% of the training and test sets, respectively, by the decision tree model. Subsequently these models were used to predict the activities of several new potential ß-amyloid aggregation inhibitors and these predictions were indeed validated by in vitro experiments. Key chemical features correlated with the inhibition ability were identified. These include the electro-topological state of carbonyl groups, AlogP and the number of hydrogen bond donor groups. The results demonstrate the feasibility of the developed models as tools for rapid screening, which could help in the design of novel potential drug candidates for Alzheimer's disease.

The proto-oncogene Int6 is essential for neddylation of Cul1 and Cul3 in Drosophila.

Int6 is a proto-oncogene implicated in various types of cancer, but the mechanisms underlying its activity are not clear. Int6 encodes a subunit of the eukaryotic translation initiation factor 3, and interacts with two related complexes, the proteasome, whose activity is regulated by Int6 in S. pombe, and the COP9 signalosome. The COP9 signalosome regulates the activity of Cullin-Ring Ubiquitin Ligases via deneddylation of their cullin subunit. We report here the generation and analysis of two Drosophila mutants in Int6. The mutants are lethal demonstrating that Int6 is an essential gene. The mutant larvae accumulate high levels of non-neddylated Cul1, suggesting that Int6 is a positive regulator of cullin neddylation. Overexpression in Int6 in cell culture leads to accumulation of neddylated cullins, further supporting a positive role for Int6 in regulating neddylation. Thus Int6 and the COP9 signalosome play opposing roles in regulation of cullin neddylation.