Role of Density Functional Theory in “Ribocomputing Devices”

Molecular computing devices composed of biological substances, such as nucleic acid and ribonucleic acid plays a key role for the logical processing of a variety of inputs and viable outputs in the cellular machinery of all living organisms. These devices are directly dependent on the advancement in DNA and RNA technology. RNA nanoparticles can be engineered into a programmable and logically acting “Ribocomputing Devices”; a breakthrough at the interface of nanotechnology and synthetic biology. It opens a new path to the synthetic biologists to design reliable synthetic biological circuits which can be useful as the electronic circuits. In this emerging field, a number of challenges persist; as how to translate a variety of nucleic acid based logic gates developed by numerous research laboratories into the realm of silicon-based computing. So in this chapter we will discuss the advances in ribonucleic acid (RNA) based computing and it’s potential to serve as an alternative to revolutionize silicon-based technology by theoretical means. Also the results of the calculated parameters with computational tools using Density functional theory and the designed device circuits will be analyzed

Logic integration of mRNA signals by an RNAi-based molecular computer

Synthetic in vivo molecular ‘computers’ could rewire biological processes by establishing programmable, non-native pathways between molecular signals and biological responses. Multiple molecular computer prototypes have been shown to work in simple buffered solutions. Many of those prototypes were made of DNA strands and performed computations using cycles of annealing-digestion or strand displacement. We have previously introduced RNA interference (RNAi)-based computing as a way of implementing complex molecular logic in vivo. Because it also relies on nucleic acids for its operation, RNAi computing could benefit from the tools developed for DNA systems. However, these tools must be harnessed to produce bioactive components and be adapted for harsh operating environments that reflect in vivo conditions. In a step toward this goal, we report the construction and implementation of biosensors that ‘transduce’ mRNA levels into bioactive, small interfering RNA molecules via RNA strand exchange in a cell-free Drosophila embryo lysate, a step beyond simple buffered environments. We further integrate the sensors with our RNAi ‘computational’ module to evaluate two-input logic functions on mRNA concentrations. Our results show how RNA strand exchange can expand the utility of RNAi computing and point toward the possibility of using strand exchange in a native biological setting

A mechanical Turing machine: blueprint for a biomolecular computer

We describe a working mechanical device that embodies the theoretical computing machine of Alan Turing, and as such is a universal programmable computer. The device operates on three-dimensional building blocks by applying mechanical analogues of polymer elongation, cleavage and ligation, movement along a polymer, and control by molecular recognition unleashing allosteric conformational changes. Logically, the device is not more complicated than biomolecular machines of the living cell, and all its operations are part of the standard repertoire of these machines; hence, a biomolecular embodiment of the device is not infeasible. If implemented, such a biomolecular device may operate in vivo, interacting with its biochemical environment in a program-controlled manner. In particular, it may ‘compute’ synthetic biopolymers and release them into its environment in response to input from the environment, a capability that may have broad pharmaceutical and biological applications

Synthetic biology devices for in vitro and in vivo diagnostics

There is a growing need to enhance our capabilities in medical and environmental diagnostics. Synthetic biologists have begun to focus their biomolecular engineering approaches toward this goal, offering promising results that could lead to the development of new classes of inexpensive, rapidly deployable diagnostics. Many conventional diagnostics rely on antibody-based platforms that, although exquisitely sensitive, are slow and costly to generate and cannot readily confront rapidly emerging pathogens or be applied to orphan diseases. Synthetic biology, with its rational and short design-to-production cycles, has the potential to overcome many of these limitations. Synthetic biology devices, such as engineered gene circuits, bring new capabilities to molecular diagnostics, expanding the molecular detection palette, creating dynamic sensors, and untethering reactions from laboratory equipment. The field is also beginning to move toward in vivo diagnostics, which could provide near real-time surveillance of multiple pathological conditions. Here, we describe current efforts in synthetic biology, focusing on the translation of promising technologies into pragmatic diagnostic tools and platforms.United States. Defense Threat Reduction Agency (Grant HDTRA1-14-1- 0006)United States. Office of Naval Research. Multidisciplinary University Research InitiativeUnited States. Air Force Office of Scientific Research (Grant FA9550-14-1-0060)Wyss Institute for Biologically Inspired EngineeringHoward Hughes Medical Institut

RNA AS A UNIQUE POLYMER TO BUILD CONTROLLABLE NANOSTRUCTURES FOR NANOMEDICINE AND NANOTECHNOLOGY

RNA nanotechnology is an emerging field that involves the design, construction and functionalization of nanostructures composed mainly of RNA for applications in biomedical and material sciences. RNA is a unique polymer with structural simplicity like DNA and functional diversity like proteins. A variety of RNA nanostructures have been reported with different geometrical structures and functionalities. This dissertation describes the design and construction of novel two-dimensional and three-dimensional self-assembled RNA nanostructures with applications in therapeutics delivery, cancer targeting and immunomodulation. Firstly, by using the ultra-stable pRNA three-way junction motif with controllable angles and arm lengths, tetrahedral architectures composed purely of RNA were successfully assembled via one-pot bottom-up assembly with high efficiency and thermal stability. By introducing arm sizes of 22 bp and 55 bp, two RNA tetrahedrons with similar global contour structure but with different sizes of 8 nm and 17 nm were successfully assembled. The RNA tetrahedrons were also highly amenable to functionalization. Fluorogenic RNA aptamers, ribozyme, siRNA, and protein-binding RNA aptamers were integrated into the tetrahedrons by simply fusing the respective sequences with the tetrahedral core modules. Secondly, I reported the design and construction of molecularly defined RNA cages with cube and dodecahedron shapes based on the stable pRNA 3WJ. The RNA cages can be easily self-assembled by single-step annealing. The RNA cages were further characterized by gel electrophoresis, cryo-electron microscopy and atomic force microscopy, confirming the spontaneous formation of the RNA cages. I also demonstrated that the constructed RNA cages could be used to deliver model drugs such as immunomodulatory CpG DNA into cells and elicit enhanced immune responses. Thirdly, by using the modular multi-domain strategy, molecular defined RNA nanowires can be successfully self-assembled via a bottom-up approach. Only four different 44-nucleotide single-stranded RNAs were used to assemble the RNA nanowire. The reported RNA nanowire has the potential to be explored in the future as the carrier for drug delivery or matrix for tissue engineering. Fourthly, the construction of RNA polygons for delivering immunoactive CpG oligonucleotides will be presented. When CpG oligonucleotides were incorporated into the RNA polygons, their immunomodulation effect for cytokine TNF-α and IL-6 induction was greatly enhanced, while RNA polygon controls induced unnoticeable cytokine induction. Moreover, the RNA polygons were delivered to macrophages specifically and the degree of immunostimulation greatly depended on the size, shape, and the number of payload per RNA polygon. Collectively, these findings demonstrated RNA nanotechnology can produce controllable nanostructures with different functionalities and result in potential applications in nanomedicine and nanotechnology

Nucleic Acid Architectures for Therapeutics, Diagnostics, Devices and Materials

Nucleic acids (RNA and DNA) and their chemical analogs have been utilized as building materials due to their biocompatibility and programmability. RNA, which naturally possesses a wide range of different functions, is now being widely investigated for its role as a responsive biomaterial which dynamically reacts to changes in the surrounding environment. It is now evident that artificially designed self-assembling RNAs, that can form programmable nanoparticles and supra-assemblies, will play an increasingly important part in a diverse range of applications, such as macromolecular therapies, drug delivery systems, biosensing, tissue engineering, programmable scaffolds for material organization, logic gates, and soft actuators, to name but a few. The current exciting Special Issue comprises research highlights, short communications, research articles, and reviews that all bring together the leading scientists who are exploring a wide range of the fundamental properties of RNA and DNA nanoassemblies suitable for biomedical applications

Sensing and Regulation from Nucleic Acid Devices

abstract: The highly predictable structural and thermodynamic behavior of deoxynucleic acid (DNA) and ribonucleic acid (RNA) have made them versatile tools for creating artificial nanostructures over broad range. Moreover, DNA and RNA are able to interact with biological ligand as either synthetic aptamers or natural components, conferring direct biological functions to the nucleic acid devices. The applications of nucleic acids greatly relies on the bio-reactivity and specificity when applied to highly complexed biological systems. This dissertation aims to 1) develop new strategy to identify high affinity nucleic acid aptamers against biological ligand; and 2) explore highly orthogonal RNA riboregulators in vivo for constructing multi-input gene circuits with NOT logic. With the aid of a DNA nanoscaffold, pairs of hetero-bivalent aptamers for human alpha thrombin were identified with ultra-high binding affinity in femtomolar range with displaying potent biological modulations for the enzyme activity. The newly identified bivalent aptamers enriched the aptamer tool box for future therapeutic applications in hemostasis, and also the strategy can be potentially developed for other target molecules. Secondly, by employing a three-way junction structure in the riboregulator structure through de-novo design, we identified a family of high-performance RNA-sensing translational repressors that down-regulates gene translation in response to cognate RNAs with remarkable dynamic range and orthogonality. Harnessing the 3WJ repressors as modular parts, we integrate them into biological circuits that execute universal NAND and NOR logic with up to four independent RNA inputs in Escherichia coli.Dissertation/ThesisDoctoral Dissertation Biochemistry 201

New synthetic biology tools for metabolic control

In industrial bioprocesses, microbial metabolism dictates the product yields, and therefore, our capacity to control it has an enormous potential to help us move towards a bio-based economy. The rapid development of multiomics data has accelerated our systematic understanding of complex metabolic regulatory mechanisms, which allow us to develop tools to manipulate them. In the last few years, machine learning-based metabolic modeling, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) derived synthetic biology tools, and synthetic genetic circuits have been widely used to control the metabolism of microorganisms, manipulate gene expression, and build synthetic pathways for bioproduction. This review describes the latest developments for metabolic control, and focuses on the trends and challenges of metabolic engineering strategies

Auf Boolescher Logik basierende Assays für die Analyse verschiedener Bacillus cereus Toxine

Bacillus cereus is a Gram-positive and spore-forming bacterium of the Bacillus cereus group, sharing a closely related phylogenetic similarity with other group members such as Bacillus anthracis and Bacillus thuringiensis. Bacillus cereus is responsible for both gastrointestinal and non-gastrointestinal syndromes. Of the former ones, emesis and diarrhea are caused by either the emetic toxin (cereulide) or different enterotoxins mainly the non-haemolytic enterotoxin (Nhe), haemolysin BL (Hbl) and cytotoxin K (CytK). In addition, other toxins and virulence factors have been reported in the past few years, e.g., haemolysin II (HlyII), Certhrax, vegetative insecticidal proteins (VIPs), immune inhibitor A1 (InhA1) and sphingomyelinase (SMase). Since the relative role of the individual toxins and the other factors in disease is still unknown, there is an urgent demand to detect these potential targets for either diagnostic or food safety purposes. In the first part of the thesis, we present an OR gate based on monoclonal antibodies for the simultaneous detection of multiple toxins in a single tube. To further simplify the operating procedure, the Boolean rule of simplification was used to guide the selection of a marker toxin among the natural toxin profiles. Furthermore, we developed a cellular logic circuit for deciphering the toxin profiles produced by B. cereus, using readout techniques based on pore formation on the cell membrane. This new assay enabled the simultaneous detection of seven biomarkers in pathogenic strains from various sources. Lastly, a cellular logic system capable of combinatorial and sequential logic operations based on bacterial protein-triggered cytotoxicity was constructed. Advanced devices such as a keypad lock, half-adder and several basic Boolean properties were demonstrated on the cells. This represents a first example of a Boolean logic-based system for assaying multiple bacterial toxins. In addition, the results suggest that toxins and other virulence factors of bacteria can be used as toolkits for biocomputing.Bacillus cereus ist ein Gram-positives und Sporen bildendes Bakterium aus der Bacillus cereus Gruppe, welche auch die phylogenetisch nah verwandten Bacillus anthracis und Bacillus thuringiensis beinhaltet. B. cereus ist sowohl für gastrointestinale als auch nicht-gastrointestinale Syndrome verantwortlich. In ersterem Fall werden Erbrechen und Diarrhö entweder durch das emetische Toxin (Cereulid) oder durch verschiedene Enterotoxine, hauptsächlich durch das nicht-hämolytische Enterotoxin (Nhe), das Hämolysin BL (Hbl) und das Cytotoxin K (CytK) verursacht. Des Weiteren wurde in den letzten Jahren auch von anderen Toxinen und Virulenz Faktoren berichtet: Hämolysin II (HlyII), Certhrax, die vegetativ expremierten insektiziden Proteine (VIPs), der Immune inhibitor A1 (InhA1) und die Sphingomyelinase (SMase). Da das Zusammenspiel der einzelnen Toxine und der anderen Faktoren im Krankheitsfall noch viele Rätsel aufweist, gibt es einen dringenden Bedarf diese potentiellen Targets zu detektieren – sowohl für die Diagnostik als auch für die Lebensmittelsicherheit. Im ersten Teil dieser Dissertation wird die mathematische Logikfunktion des ODER-Gatters, basierend auf monoklonalen Antikörpern für die simultane Detektion von verschiedenen Toxinen in einem Teströhrchen benutzt. Um den Arbeitsvorgang weiter zu simplifizieren wurde die Boolesche Regel der Vereinfachung angewandt, um ein Marker Toxin aus dem Spektrum der natürlichen Toxine auszuwählen. Desweitern wurde eine zelluläre Schaltkreislogik entwickelt, um die Toxinprofile von B. cereus zu entschlüsseln. Dafür benutzten wir als Testsignal die Porenbildung auf der Zellmembran. Dieser neuartige Assay ermöglicht in pathogenen Stämmen verschiedenen Ursprungs die simultane Detektion von sieben verschiedenen Biomarkern. Zuletzt wurde ein zellulärer Logikschaltkreis, basierend auf durch bakterielle Proteine induzierte Cytotoxizität konstruiert, der zu kombinatorischen und sequentiellen logischen Verknüpfungen befähigt ist. Auch fortgeschrittene Elemente wie ein Tastaturschloss, Halb-Addierer und verschiedene elementare Booleschen Eigenschaften wurden auf zellulärer Ebene demonstriert. Diese Arbeit repräsentiert ein erstes Beispiel für ein auf Boolescher Logik basierendes System unter Benutzung verschiedener bakterieller Toxine. Die Resultate deuten darauf hin, dass bakterielle Toxine und andere Virulenzfaktoren als Bausteine für Bio-Datenverarbeitung genutzt werden können