DNA Chemical Reaction Network Design Synthesis and Compilation

The advantages of biomolecular computing include 1) the ability to interface with, monitor, and intelligently protect and maintain the functionality of living systems, 2) the ability to create computational devices with minimal energy needs and hazardous waste production during manufacture and lifecycle, 3) the ability to store large amounts of information for extremely long time periods, and 4) the ability to create computation analogous to human brain function. To realize these advantages over electronics, biomolecular computing is at a watershed moment in its evolution. Computing with entire molecules presents different challenges and requirements than computing just with electric charge. These challenges have led to ad-hoc design and programming methods with high development costs and limited device performance. At the present time, device building entails complete low-level detail immersion. We address these shortcomings by creation of a systems engineering process for building and programming DNA-based computing devices. Contributions of this thesis include numeric abstractions for nucleic acid sequence and secondary structure, and a set of algorithms which employ these abstractions. The abstractions and algorithms have been implemented into three artifacts: DNADL, a design description language; Pyxis, a molecular compiler and design toolset; and KCA, a simulation of DNA kinetics using a cellular automaton discretization. Our methods are applicable to other DNA nanotechnology constructions and may serve in the development of a full DNA computing model

In vitro evolution of artificial enzymes: method development and applications

University of Minnesota Ph.D. dissertation. September 2014. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Burckhard Seelig. 1 computer file (PDF); x, 176 pages.Artificial enzymes have the potential to aid in the production of pharmaceuticals and facilitate basic biomedical research. There are two methods for making artificial enzymes: rational design and de novo selection. Rational design utilizes detailed knowledge of enzyme catalysis to design an enzyme active site, and then introduces this active site into a protein. However, due to the limited understanding of protein folding and structure-function relationships this approach is still extremely challenging and far from routine. In contrast, we utilize a directed evolution approach to isolate de novo artificial enzymes from a large library of protein variants by in vitro selection. Each of the trillions of proteins in a library are tested in a single experiment to determine if any have the desired activity. The artificial enzymes are created when the library is made so a high quality library is important for success. My thesis research focuses on two goals: (1) Construct a library built on the robust (alpha/beta)8 barrel enzyme scaffold for future enzyme selections and (2) Characterize a thermostable artificial RNA ligase and develop an application for this enzyme. The (alpha/beta)8 fold is used to catalyze a wide range of chemical reactions in nature. We used this fold to create a library containing > 1014 unique proteins by replacing loops of the catalytic face with randomized codons via PCR. Small sub-libraries were subjected to a protease-based folding selection to improve library quality by enriching for folded sequences. The final folding-enriched library contained > 1012 folded proteins representing an up to 50-fold improvement relative to a control library. These libraries will provide a valuable source of new enzymes for future in vitro selections. The previously generated artificial RNA ligases join 5'-triphosphate RNA to the 3'-hydroxyl of a second RNA substrate; a reaction not observed in nature. However the enzymes were also highly dynamic, which prevented the solving of the protein structure by NMR or X-ray crystallography. A more structured enzyme, called ligase 10C, was isolated by performing the ligase selection at 65°C and its structure was solved revealing a novel primordial fold. Here, we describe the detailed biochemical characterization of ligase 10C. Using a variety of RNA substrates, we also determined how ligation rates change with sequence composition revealing an enzyme with broad sequence specificity. We developed a method for the specific ligation and sequencing of 5'-triphosphorylated RNA. These results highlight ligase 10C as an attractive tool for the selective isolation of 5'-triphosphate RNA from a complex mixture, something which is difficult with current methods

Lipid carriers for endothelial-specific delivery of siRNA:From particle development to attenuation of inflammation

Nanotechnologie heeft inmiddels een belangrijke invloed op veel aspecten van de wetenschap en van ons leven, onder andere in de ontwikkeling van veelbelovende therapieën de gezondheidszorg. Het doel van dit onderzoek was de ontwikkeling van nano-carrier systemen om in proefdier modellen, in bloedvatwand cellen (zogenaamde endotheelcellen) specifiek “short interfering” RNAs (siRNAs) af te leveren om de therapie voor ontstekingsziekten te verbeteren. SiRNAs zijn heel kleine dubbelstrengs RNA molekulen (20-25 basenparen) die in de natuur voorkomen, maar ook kunnen worden toegepast om tijdelijk de expressie van specifieke genen uit te zetten. Ons uiteindelijke doel, siRNA therapieën voor humane ziekten, kan alleen bereikt worden wanneer de veiligheid, efficiëntie en de specificiteit van siRNA afgifte systemen verbeterd worden. Bloedvatwand cellen (endotheelcellen) zijn aantrekkelijke doelcellen voor anti-inflammatoire therapie omdat ze betrokken zijn bij ontstekingsprocessen en een centrale rol spelen in de pathologie van (chronische) ontstekingsziekten. In de onderzoeken die in dit proefschrift zijn beschreven hebben wij siRNA-carriers ontwikkeld die siRNA kunnen ”inpakken”. Door gebruik te maken van deze nano-carriers, die we SAINT-O-Somen noemen, konden we siRNA gericht tegen het ontsteking gen NFκB afleveren in endotheelcellen die het ontsteking specifieke molecuul VCAM-1 aan de buitenkant van de cel tot expressie brachten. Bovendien hebben we laten zien dat remming van NFκB door siRNA in deze cellen leidde tot vermindering van de ontsteking reactie in muizen. De in dit proefschrift gepresenteerde afgifte van siRNA aan ontstoken endotheelcellen, biedt mogelijkheden om in de toekomst ontstekingsziekten beter te behandelen

Rakennemuunnosten ja kantajien käyttö oligonukleotidien saattamisessa vaikutuskohteeseensa : Synteesi, analyysi ja biologinen testaus

Several serious diseases remain without non-toxic curative treatments. To fill this void, one of the promising groups of medicines is that of oligonucleotides, encompassing aptamers, transcription factor decoys, and antisense therapeutics such as short interfering RNA and splice-correcting oligonucleotides. These short strands of DNA or RNA can bind to specific cellular nucleic acids or proteins and thereby inhibit or correct the function of disease-causing molecules. Extensive enzymatic degradation and poor cellular uptake are the most important obstacles for systemic oligonucleotide therapy. Numerous chemical modifications have been introduced to improve enzymatic stability, but they must be carefully optimized to avoid toxicity and to maintain target affinity. One solution is to design topological modifications, such as looped or circular oligonucleotides, which conserve the natural phosphodiester backbone but cannot be attacked by exonucleases. Cellular uptake has proven to be even more challenging. Oligonucleotides are internalized into cells by endocytosis, after which they often remain trapped in endosomes. Therefore, it would be advantageous to develop delivery vectors capable of bypassing endocytic routes of uptake or enhancing endosomal escape. Cell-penetrating peptides, for example, exploit several mechanisms of uptake, some of which lead to rapid entry without endosomal localization. In addition, encouraging results have been achieved using liposomes, gold nanoparticles, and other nanocarriers, which also shield the oligonucleotide from degrading enzymes. The aim of this work was to improve the in vitro delivery of oligonucleotides by employing chemical modifications and nanoparticle carriers. The synthesis of the compounds, their characterization by various analytical methods, and the evaluation of biological effects are described. Antisense oligonucleotides covalently linked to cell-penetrating peptides via convergent conjugation displayed improved cellular uptake but failed to inhibit reporter genes due to endosomal entrapment in cells. Circular oligonucleotides exhibited enhanced selectivity of mismatch detection and increased stability in biological fluids compared to linear oligonucleotides. Altogether 44 compounds were analyzed by electrospray ionization mass spectrometry and liquid chromatography mass spectrometry, which were found to be excellent methods for the characterization of modified oligonucleotides. Finally, we synthesized cationic gold nanoparticles modified with a Tat-related peptide, which did not adversely affect cell viability and effectively delivered short interfering RNA into cells as non-covalent complex.Monet parantumattomat sairaudet johtuvat haitallisten proteiinien syntymisestä elimistössä. Näiden proteiinien toimintaa pyritään estämään yleisimmin perinteisillä pienimolekyylisillä lääkeaineilla. Vielä tehokkaampi ja turvallisempi vaikutus voidaan saada estämällä haitallisten proteiinien syntyminen jo RNA-tasolla esimerkiksi oligonukleotidilääkkeillä. Nämä lyhyet nukleiinihappomolekyylit sitoutuvat spesifisesti kohde-RNA:han ja voivat hajottaa sen useilla eri mekanismeilla. Ne voivat myös korjata RNA:n viallista prosessointia tai sitoutua suoraan kohdeproteiiniin. Oligonukleotideja tutkitaan muun muassa syöpien ja parantumattomien geneettisten sairauksien hoitoa varten. Tähän mennessä vain yksi ei-paikallinen oligonukleotidilääke on hyväksytty kliiniseen käyttöön Yhdysvalloissa, mutta toksisuuden takia sen käyttö on tarkoin rajattu eikä sitä ole hyväksytty Euroopassa. Ongelmana hoitojen kehittämisessä on oligonukleotidien entsymaattinen hajoaminen elimistössä sekä niiden huono pääsy solujen sisään. Oligonukleotideja on muunneltu entsyyminkestäviksi muokkaamalla niiden kemiallista rakennetta, mutta tällöin toksisuus voi lisääntyä ja tehokkuus heikentyä. Oligonukleotidi voidaan myös naamioida entsyymeiltä muuttamatta luonnollista nukleotidirankaa, jos se syklisoidaan liittämällä nukleotidiketjujen päät toisiinsa. Kolmas vaihtoehto on liittää oligonukleotidi sopivaan kantajamolekyyliin, jonka läsnäolo suojaa oligonukleotidia entsyymeiltä ja lisäksi auttaa sen vaikutuspaikalleen solun sisään. Lupaavia kantajia ovat muun muassa liposomit, polymeeri- ja kultananopartikkelit sekä soluun penetroituvat peptidit, joista viimeksi mainitut on löydetty tutkittaessa virusten tehokasta pääsyä soluihin. Näiden peptidien toivotaan pystyvän ohittamaan endosytoosireitin, jonka kautta soluun kulkiessaan oligonukleotidi voi jäädä loukkuun endosomiin. Tässä väitöskirjatyössä tutkittiin kemiallisten rakennemuunnosten ja nanopartikkelikantajien mahdollisuuksia oligonukleotidien saattamisessa soluihin. Osatöissä paitsi tutkittiin muunnosten ja kantajien biologisia vaikutuksia, myös optimoitiin synteesi- ja analyysimenetelmiä. Kovalentisti liitetyt soluun penetroituvat peptidit paransivat oligonukleotidien soluunottoa, mutta antisense-vaikutusta ei silti nähty soluissa yhdisteiden juututtua endosomeihin. DNA-diagnostiikkaa varten valmistetut sykliset oligonukleotidit osoittautuivat huomattavasti lineaarisia oligonukleotideja selektiivisemmiksi emäsmutaatioiden havaitsemisessa, ja myös niiden entsyyminkestävyys oli parempi. Yhteensä 44 yhdistettä analysoitiin sähkösumutusmassaspektrometrialla ja nestekromatografia massaspektrometrialla, jotka sopivat erinomaisesti muunneltujen oligonukleotidien analysointiin. Viimeisessä osatyössä syntetisoitiin kationisia kultananopartikkeleita, joihin lisättiin peptidianalogi. Tähän kantajaan kompleksoidulla pienellä häiritsevällä RNA:lla (siRNA:lla) hiljennettiin rekombinanttisolujen ilmentämä reportterigeeni heikentämättä solujen elävyyttä

Étude du chemin réactionnel du ribozyme de l'hépatite D humaine

Du fait de la complexité du repliement de l'ARN en général et des mécanismes impliqués dans ce processus, il est actuellement impossible de prédire la structure tridimensionnelle et encore moins le chemin réactionnel d'un ARN en se basant uniquement sur sa séquence primaire. Ces deux questions fondamentales doivent être adressées pour comprendre ce qui se passe dans une cellule et pouvoir un jour être capable de créer de novo des molécules d'ARN avec une structure et une activité précise. L'objectif de ce travail est de mieux comprendre les forces permettant d'obtenir une structure tridimensionnelle et les mécanismes impliqués dans le chemin réactionnel d'un ARN modèle hautement structuré: le ribozyme de l'hépatite D humaine (ribozyme HDV). L'idée est que s'il est possible de comprendre dans les moindres détails une structure complexe, ces informations pourront être utilisées pour la prédiction de structures plus simples. Premièrement, il s'agit de trouver quels sont les changements conformationnels du chemin réactionnel et dans quel ordre ils ont lieu. Une fois le chemin réactionnel connu, il devient possible de générer des mutants formant des intermédiaires stables entre chaque changement conformationnel. L'étude thermodynamique de ces mutants permet de dresser le profil énergétique du chemin réactionnel. Finalement, la modélisation permet de suivre l'évolution de la structure tertiaire et ainsi vérifier différentes hypothèses. Ensemble, ces approches ont permis de comprendre comment le ribozyme HDV atteint sa structure tridimensionnelle, quel chemin réactionnel il emprunte et quelle est l'étape limitante

T cell targeted nanoparticles for pulmonary siRNA delivery as novel asthma therapy

The aim of this work was to develop and optimize a T cell targeted delivery system for pulmonary delivery of siRNA directed against GATA3, the central transcription factor of Th2 cytokines, as a novel therapy for asthma. Therefore, an existing carrier system on the basis of polycationic polymer polyethylenimine (PEI) and targeting Ligand transferrin (Tf), resulting in the so-called Tf-PEI, was chosen and fully characterized concerning relevant siRNA polyplex characteristics such as size, zeta potential, siRNA encapsulation efficiency and gene silencing capability in vitro and in vivo. Subsequently, Tf-PEI was blended with a second conjugate, Tf-Mel, containing the lysosomal Peptide melittin, in order to increase endosomal escape of the polyplexes. Resulting Tf-Mel-PEI blends were characterized and optimized to achieve siRNA polyplexes combining specific targeting of activated T cells and efficient cytoplasmic siRNA release, resulting in successful gene knockdown. For GATA3 silencing, a suitable siRNA sequence combination was found and applied within the Tf-Mel-PEI blend polyplexes to investigate down-stream effects of the gene knockdown on cytokine levels. These were concludingly tested in an optimized model for activated T cells as a first step for evaluation of relevant therapeutic effects in an inflammatory environment

Arteriogenesis and Therapeutic Angiogenesis

Although advances in therapeutic interventions improved outcomes, vascular occlusive diseases are still challenging not only afflicted but also attending physicians, requiring novel therapeutic strategies. Arteriogenesis, sometimes also called therapeutic angiogenesis, refers to the body’s own capacity to create a natural bypass around a narrowing or occluded arterial vessel. This book gives an insight into current knowledge and advances in vascular sciences and future prospects of therapeutic options. The utility and relevance of circulating biomarkers together with the potential of machine learning methods are discussed as well as the challenges and prospects of novel therapies such as protein- gene-, and stem cell therapy along with multicistronic multigene vectors and the use of microRNAs, exosomes, and secretomes. Vascular smooth muscle phenotype switch as a target to promote arteriogenesis is critically addressed, highlighting the problem of promoting atherosclerosis in parallel. Two articles even deal with cold-inducible RNA-binding protein CIRP/CIRPB presenting it as promising target to promote vascularization concomitant the reduction in ischemic tissue damage. BMPR kinase inhibition is introduced to improve tissue repair in a hereditary form of vascular disorder, and the role of the AP-1 transcription factor JunB in blood vessel formation is described. Some more experimental oriented articles deal with the relevance of choosing the appropriate mouse strain for investigations as well as in vitro Matrigel plug assay as a potent method to investigate angiogenesis. Last but not least, two-photon intravital microscopy is presented as suitable tool to assess plaque angiogenesis in atherosclerotic lesions

Applications of Optical Control of Oligonucleotide and Protein Function

Optical regulation using light as an external trigger was applied to the control of biological processes with high spatio-temporal resolution. Photoremovable caging groups were site-specifically incorporated onto oligonucleotides and proteins to optically regulate their function in biological environments, typically for the photochemical control of gene expression. These caging group modifications enabled both OFF → ON and ON → OFF optochemical switches for important chemical biology tools. Oligonucleotides containing caging group modifications were synthesized to regulate nucleic acid function with light. Specifically, photocaged triplex-forming oligonucleotides were developed to optochemically control transcription in cell culture. Light-activated antagomirs were designed for the optical inhibition of miR-21 and miR-122 function in the regulation of endogenous microRNA activity. This technology was then applied to the study of miR-22 and miR-124 function in cortical neuron migration during cerebral corticogenesis. Splice-switching oligonucleotides were engineered to optically control mRNA splicing pathways in both human cells and zebrafish. The optical control of plasmid-based gene expression was demonstrated with a caged promoter, and applied to the photochemical activation of transcription in a live animal model. The caging of oligonucleotides was also applied to DNA computation in the production of optically controlled logic gates and amplification cycles, providing spatio-temporal control over hybridization cascades to add new functionality to DNA computation modules. These studies in DNA computation led to the development of novel biosensors for logic gate-based detection of specific micro RNA signatures in live cells. In addition, proteins were optically controlled through the site-specific installation of caging groups on amino acid side chains that are essential for protein function using unnatural amino acid mutagenesis in mammalian cells with an expanded genetic code. A caged lysine analogue was incorporated into T7 RNA polymerase to photochemically regulate transcription in the development of a light-activated synthetic gene network and light-triggered RNA interference. A light-activated Cas9 endonuclease was engineered through the installation of a caged lysine analogue to optically control CRISPR/Cas9 editing of both exogenous and endogenous genes. Lastly, a system for the incorporation of unnatural amino acids in zebrafish was studied in efforts to produce the first vertebrate species with an expanded genetic code

Bio-Nano Robo-Mofos : Design and Synthesis of DNA Origami Nanostructures and Assembly of Nanobot Superstructures

In the field of bio-nanotechnology, molecules like DNA are repurposed as building materials for the construction of self-assembling nanostructures. The DNA origami method involves rationally coding many short synthetic DNA strands which ‘fold’ longer scaffold strands into precise, addressable structures for applications in areas like medicine, structural biology and molecular biophysics. DNA origami subunits are also used to explore fundamental principles of self-assembly, revealing insights into biology and expanding our control of matter at the nanoscale. But despite the usefulness of the method, DNA origami designs are limited in size by the length of scaffold strands, and in scope by the available tools needed to navigate the complex geometries of DNA nanostructures. My thesis addresses this in two ways: First, I present a set of principles for the design of DNA origami nanotubes, a class of strained structure with many applications. I parametrised variables related to nanotube design and created a computational tool to convert desired geometries into DNA strand layouts. I validated this via synthesis of various designs, including novel nanotubes with pleated walls, reconfigurable twist and varying diameter, characterising them with TEM, SAXS and MD simulations. This revealed insights into how design variables affect properties such as diameter and rigidity, and how global strain affects DNA nanostructures. Next, I present two schemes for assembling DNA origami subunits into self-limiting, open superstructures, exploring fundamental principles to control self-assembly while also overcoming DNA origami’s size limitations. The first is a strain accumulation scheme, which was explored theoretically and then embodied in a modular subunit with allosteric binding domains. With simulation and synthesis, I demonstrated that the subunit could structurally encode the extent of its own polymerisation. The second scheme is Vernier assembly, in which I showed that the combined geometries of two DNA origami subunits could determine the size of a superstructure and explored parameters important to maximise yield. Both studies provide guidance for future studies and applications which may require finite superstructures made from small numbers of unique components. Combined, the works in this thesis expand the design space for DNA-nanotechnology and fields beyond, enabling a range of biologically-inspired nanoscale autonomous modular formations, or ‘Bio-Nano Robo-Mofos’