Optimization of supply diversity for the self-assembly of simple objects in two and three dimensions

The field of algorithmic self-assembly is concerned with the design and analysis of self-assembly systems from a computational perspective, that is, from the perspective of mathematical problems whose study may give insight into the natural processes through which elementary objects self-assemble into more complex ones. One of the main problems of algorithmic self-assembly is the minimum tile set problem (MTSP), which asks for a collection of types of elementary objects (called tiles) to be found for the self-assembly of an object having a pre-established shape. Such a collection is to be as concise as possible, thus minimizing supply diversity, while satisfying a set of stringent constraints having to do with the termination and other properties of the self-assembly process from its tile types. We present a study of what we think is the first practical approach to MTSP. Our study starts with the introduction of an evolutionary heuristic to tackle MTSP and includes results from extensive experimentation with the heuristic on the self-assembly of simple objects in two and three dimensions. The heuristic we introduce combines classic elements from the field of evolutionary computation with a problem-specific variant of Pareto dominance into a multi-objective approach to MTSP.Comment: Minor typos correcte

Hairygami: Analysis of DNA Nanostructures' Conformational Change Driven by Functionalizable Overhangs

DNA origami is a widely used method to construct nanostructures by self-assembling designed DNA strands. These structures are often used as "breadboards" for templated assembly of proteins, gold nanoparticles, aptamers, and other molecules, with applications ranging from therapeutics and diagnostics to plasmonics and photonics. Imaging these structures using AFM or TEM is not capable to capture their full conformation ensemble as they only show their structure flattened on a surface. However, certain conformations of the nanostructure can position guest molecules into distances unaccounted for in their intended design, thus leading to spurious interactions between guest molecules that are designed to be separated. Here, we use molecular dynamics simulations to capture conformational ensemble of 2D DNA origami tiles and show that introducing single-stranded overhangs, which are typically used for functionalization of the origami with guest molecules, induces a curvature of the tile structure in the bulk. We show that the shape deformation is of entropic origin, with implications for design of robust DNA origami breadboards as well as potential approach to modulate structure shape by introducing overhangs. We then verify experimentally that the overhangs introduce curvature into the DNA origami tiles

Design, Synthesis and Analysis of Self-Assembling Triangulated Wireframe DNA Structures

The field of DNA nanotechnology offers a wide range of design strategies with which nanometer-sized structures with a desired shape, size and aspect ratio can be built. The most established techniques in the field rely on close-packed 'solid' DNA nanostructures produced with either the DNA origami or the single-stranded tile techniques. These structures depend on high-salt buffer solutions and require more material than comparable size hollow wireframe structures. This dissertation explores the construction of hollow wireframe DNA nanostructures composed of equilateral triangles. To achieve maximal material efficiency the design is restricted to use a single DNA double helix per triangle edge. As a proof of principle, the DNA origami technique is extended to produce a series of truss structures including the flat, tetrahedral, octahedral, or irregular dodecahedral truss designs. In contrast to close packed DNA origami designs these structures fold at low-salt buffer conditions. These structures have defined cavities that may in the future be used to precisely position functional elements such as metallic nanoparticles or enzymes. The design process of these structures is simplified by a custom design software. Next, the triangulated construction motif is extended to the single-stranded DNA tile technique. A collection of finite structures, as well as one-dimensional crystalline assemblies is explored. The ideal assembly conditions are determined experimentally and using molecular dynamics simulations. A custom design software is presented to simplify the design and handling of these structures. At last, the cost-effective prototyping of triangulated wireframe DNA origami structures is explored. This is achieved through the introduction of single-stranded “gap” regions along the triangle edges. These gap regions are then filled using a DNA polymerase rather than by synthetic oligonucleotides. This technique also allows the mechanical transformation of these structures, which is exemplified by the transition of a bent into a straight structure upon completion of the gap filling.:Abstract v Publications vii Acknowledgements ix Contents xi Chapter 1 A short introduction into DNA nanotechnology 1 1.1 Nanotechnology 1 1.1.1 Top down 1 1.1.2 Bottom up 3 1.2 Deoxyribonucleic acid (DNA) 4 1.3 DNA Nanotechnology 6 1.3.1 Tile based assembly 9 1.3.2 DNA origami and single-stranded tiles 10 1.3.3 Some applications of DNA nanotechnology 12 1.3.4 Wireframe structures 15 1.3.5 Computational tools and DNA nanotechnology. 17 Chapter 2 Motivation and objectives 19 Chapter 3 Design and Synthesis of Triangulated DNA Origami Trusses 20 3.1 Introduction 20 3.2 Results and Discussion 21 3.2.1 Design 21 3.2.2 Nomenclature and parameters of the tube structures 23 3.2.3 Gel electrophoreses analysis 25 3.2.4 Imaging of the purified structures 26 3.2.5 Optimizing the folding conditions 28 3.2.6 Comparison to vHelix 29 3.3 Conclusions 29 3.4 Methods 30 3.4.1 Standard DNA origami assembly reaction. 30 3.4.2 Gel purification. 30 3.4.3 AFM sample preparation. 31 3.4.4 TEM sample preparation. 31 3.4.5 Instructions for mixing the staple sets. 31 Chapter 4 Triangulated wireframe structures assembled using single-stranded DNA tiles 33 4.1 Introduction 33 4.2 Results and Discussion 35 4.2.1 Designing the structures 35 4.2.2 Synthesis of test structures 37 4.2.3 Molecular dynamics simulations of 6-arm junctions 38 4.2.4 Assembly of the finite structures 40 4.2.5 Influence of salt concentration and folding times 42 4.2.6 Molecular dynamics simulations of the rhombus structure 43 4.2.7 1D SST crystals 44 4.2.8 Controlling the crystal growth 46 4.3 Conclusions 48 4.4 Methods 49 4.4.1 SST Folding 49 4.4.2 Agarose Gel Electrophoresis 49 4.4.3 tSEM Characterization 49 4.4.4 AFM Imaging 49 4.4.5 AGE-Based Folding-Yield Estimation 49 4.4.6 Molecular Dynamics Simulations 50 Chapter 5 Structural transformation of wireframe DNA origami via DNA polymerase assisted gap-filling 52 5.1 Introduction 52 5.2 Results and Discussion 54 5.2.1 Design of the Structures 54 5.2.2 Folding of Gap-Structures 56 5.2.3 Inactivation of Polymerase. 57 5.2.4 Secondary Structures. 58 5.2.5 Folding Kinetics of Gap Origami. 60 5.3 Conclusions 61 5.4 Methods 62 5.4.1 DNA origami folding 62 5.4.2 Gap filling of the wireframe DNA origami structures 63 5.4.3 Agarose gel electrophoresis 63 5.4.4 PAGE gel analysis 63 5.4.5 tSEM characterization 64 5.4.6 AFM imaging 64 5.4.7 AGE based folding-yield estimation 64 5.4.8 Gibbs free energy simulation using mfold 65 5.4.9 List of sequence for folding the DNA origami triangulated structures 65 Chapter 6 Summary and outlook 67 Appendix 69 A.1 Additional figures from chapter 369 A.2 Additional figures from chapter 4 77 A.3 Additional figures from chapter 5 111 Bibliography 127 Erklärung 13

Computational Design and Study of Structural and Dynamic Nucleic Acid Systems

abstract: DNA and RNA are generally regarded as one of the central molecules in molecular biology. Recent advancements in the field of DNA/RNA nanotechnology witnessed the success of usage of DNA/RNA as programmable molecules to construct nano-objects with predefined shapes and dynamic molecular machines for various functions. From the perspective of structural design with nucleic acid, there are basically two types of assembly method, DNA tile based assembly and DNA origami based assembly, used to construct infinite-sized crystal structures and finite-sized molecular structures. The assembled structure can be used for arrangement of other molecules or nanoparticles with the resolution of nanometers to create new type of materials. The dynamic nucleic acid machine is based on the DNA strand displacement, which allows two nucleic acid strands to hybridize with each other to displace one or more prehybridized strands in the process. Strand displacement reaction has been implemented to construct a variety of dynamic molecular systems, such as molecular computer, oscillators, in vivo devices for gene expression control. This thesis will focus on the computational design of structural and dynamic nucleic acid systems, particularly for new type of DNA structure design and high precision control of gene expression in vivo. Firstly, a new type of fundamental DNA structural motif, the layered-crossover motif, will be introduced. The layered-crossover allow non-parallel alignment of DNA helices with precisely controlled angle. By using the layered-crossover motif, the scaffold can go through the 3D framework DNA origami structures. The properties of precise angle control of the layered-crossover tiles can also be used to assemble 2D and 3D crystals. One the dynamic control part, a de-novo-designed riboregulator is developed that can recognize single nucleotide variation. The riboregulators can also be used to develop paper-based diagnostic devices.Dissertation/ThesisDoctoral Dissertation Chemistry 201

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

DNA origami structures as calibration standards for nanometrology

Peer reviewe

Multiplexed single molecule observation and manipulation of engineered biomolecules

Molecular processes in organisms are often enabled by structural elements resilient to mechanical forces. For instance, the microbial and hierarchical cellulosome protein system comprises enzymes and the receptor-ligand complexes Cohesin-Dockerin (Coh-Doc), that act in concert for the efficient hydrolysis of plant polysaccharides. The Coh-Doc complexes can withstand remarkably high forces to keep host cells and enzymes bound to their substrates in the extreme environmental conditions the microorganisms frequently live in. This work focuses on the investigation of mechanical stability of such biomolecules on the single-molecule level. The highly symmetric binding interface of the Coh-Doc type I complex from Clostridium thermocellum, enables two different binding conformations withcomparable affinity and similar strength. I was able to show that both conformations exist in the wild-type molecules and are occupied under native conditions. I further characterized one of the strongest non-covalent protein complexes known, Coh-Doc type III from Ruminococcus flavefaciens by elucidating the pivotal role of the adjacent xModule domain for the mechanical stabilization of the whole complex and the role of the bimodal rupture force distribution. Such large forces impair accuracy of measured contour length increments in unfolding studies by inducing conformational changes in poly-ethylene glycol (PEG) linkers in aqueous buffer systems. This problemwas solved by introducing elastin-like polypeptides (ELP) as surface tethers. Having a peptide backbone similar to that of unfolded proteins, ELP linkers do not alter accuracy of the single-molecule force spectroscopy (SMFS) assay. To provide high throughput and precise comparability, I worked on a microfluidic platform for the in vitro protein synthesis and immobilization. The Coh-Doc system was hereby integrated as a binding handle for multiplexed measurements of mechanostability. Employing a single AFM probe to measure multiple different molecules facilitates force precision required to shed light onto molecular mechanisms down to the level of single amino acids. I also applied the Coh-Doc complex to a purely protein based single-molecule cut and paste assay for the bottom-up assembly of molecular systems for quick phenotyping of spatial arrangements. With this system, interactions in enzymatic synergies can be studied by defined positioning patterns on the single molecule level. To understand and design force responses of complex systems, I complemented the investigation of protein systems with SMFS studies on DNA Origami structures. The results of SMFS on DNA were compared to a simulation framework. Despite their difference in force loading rates, both methods agree well within their results, enabling better fundamental understanding of complex molecular superstructures.Molekulare Prozesse in Organismenwerden oft von Strukturelementen ermöglicht, die mechanischen Kräften standhalten können. Ein Beispiel hierfür ist das mikrobielle und hierarchisch aufgebaute Proteinsystem des Zellulosoms. Enzyme und die Rezeptor-Liganden Komplexe Cohesin-Dockerin (Coh-Doc) arbeiten hierbei für die effiziente Hydrolyse von pflanzlichen Polysacchariden zusammen. Die Coh-Doc Komplexe können bemerkenswerten Kräften standhalten, um in den extremen Umweltbedingungen, in denen die Mikroorganismen teilweise leben, die Wirtszellen und Enzyme an ihre Substrate binden zu können. Die vorliegende Arbeit untersucht den Einfluss von mechanischer Kraft auf solche Biomoleküle mittels Einzelmolekülmessungen. Die hohe Symmetrie des Bindeinterfaces des Coh-Doc Typ I Komplexes aus Clostridium thermocellum ermöglicht zwei verschiedene Konformationen, die vergleichbare Affinität und Stärke aufweisen. Im Rahmen dieser Arbeit konnte ich beide in denWildtyp-Molekülen und unter nativen Bedingungen nachweisen. Eines der stärksten bekannten nicht-kovalenten Rezeptor-Liganden Systeme, Coh- Doc Typ III aus Ruminococcus flavefaciens wurde charakterisiert, und die Kernrolle des benachbarten xModuls für die Stabilität des gesamten Komplexes sowie die Rolle der bimodalen Kraftverteilung untersucht. Solch hohe Kräfte vermindern die Genauigkeit der gemessenenKonturlängeninkremente von Proteinentfaltungen, indem sie Konformationsänderungen der Poly- Ethylenglykol (PEG) Oberflächenanker in wässrigen Puffersystemen verursachen. Mit Elastin-ähnlichen Polypeptiden (ELP) als Anker wurde dieses Problem gelöst: durch die Ähnlichkeit des Peptid-Rückgrates von ELPs mit dem entfaltener Proteine beeinflussen diese die Genauigkeit des Experiments nicht. Für die Optimierung von Messdurchsatz und Vergleichbarkeit entwickelte ich an einer Mikrofluidik-Plattform zur in vitro Proteinsynthese und -immobilisierung. Das Coh-Doc System wurde hierbei als Binde-Molekül für gemultiplexte Messungen integriert. Die dadurch ermöglichte Nutzung einer einzigen AFM Messsonde für die Messung verschiedener Moleküle erlaubt die nötige Kraftpräzision, um molekulare Mechanismen bis auf die Ebene einzelner Aminosäuren aufzuklären. Des weiteren habe ich den Coh-Doc Komplex in einem rein auf Proteininteraktionen basierten ’Cut and Paste’ Assay für den modularen Aufbau molekularer Systeme implementiert. Dieses ermöglicht schnelle Phänotypisierung geometrischer Anordnunungen und die Untersuchung von Wechselwirkung zwischen Enzymen mittels definierter Positionierung auf Einzelmolekülebene. Um die Kraftantwort komplexer Systeme besser verstehen und letztendlich gestalten zu können, ergänzte ich die Untersuchung von Proteinsystemen mit derer von DNA-Origami Strukturen. Die Ergebnisse der Kraftspektroskopie an DNA wurden mit Computersimulationen verglichen, und trotz des großen Unterschieds ihrer Ladungsraten stimmen beide Methoden gut überein. Dadruch legen sie die Grundlagen für ein besseres Verständnis komplexer molekularer Superstrukturen

DNA-nanorakenteiden ja biomakromolekyylien elektrostaattinen kompleksointi

DNA origami folding technique allows the construction of nanoscale structures with almost any arbitrary shape or pattern. The self-assembly and the addressable surface makes the DNA origami method interesting in engineering novel functional materials and molecular-scale devices. However, the susceptibility to nuclease digestion in physiological conditions and poor transfection rates compromises their use in cellular applications, such as drug delivery vehicles. The aim of this thesis is to coat DNA origamis to improve their structural integrity and transfection rate. Two separate coating systems were studied, both based on the electrostatic and multivalent interactions between a negatively charged origami and positively charged biomacromolecules. The complexation of DNA origamis with the biomacromolecules was studied with agarose gel electrophoretic mobility shift assay and verified with transmission electron microscopy. The structural integrity of the coated origamis was studied by subjecting the origamis to enzymatic digestion by DNase I endonuclease. Finally, the transfection efficiency was studied using confocal microscopy and quantified with fluorescence-activated cell sorting. The results revealed successful coating of DNA origamis with the protein-dendron conjugate coating system. Two different proteins were used: bovine serum albumin (BSA) and hydrophobic I (HFBI). The proteins were attached to dendrons that served as the synthetic binding unit that attaches to the origami. BSA-coating conferred protection against nuclease digestion and enhanced the transfection efficiency into HEK293 cells. This work proposes a novel coating strategy that could find applications in sophisticated drug delivery and in other nucleotide-based bionanotechnological devices.DNA-origamien valmistustekniikka mahdollistaa lähes mielivaltaisen muotoisten nanokokoisten rakenteiden valmistuksen. DNA-origamien itsejärjestäytyminen sekä mahdollisuus niiden pinnan funktionalisointiin tekee niistä mielenkiintoisen menetelmän valmistattaessa uusia toiminnallisia materiaaleja sekä molekyylikoon laitteita. Fysiologisissa olosuhteissa DNA-origamit ovat kuitenkin alttiita hajottaville nukleaasientsyymeille. Lisäksi DNA-origamit läpäisevät heikosti solukalvon, mikä vaikeuttaa niiden käyttöä esimerkiksi lääkeainekuljettimina. Tämän diplomityön tarkoituksena on päällystää DNA-origameja niiden solukalvon läpäisevyyden sekä rakenteellisen kestävyyden parantamiseksi. Työssä tutkittiin kahta eri päällystysmenetelmää, jotka molemmat perustuvat elektrostaattiseen vuorovaikutukseen negatiivisen origamin sekä positiivisten biomakromolekyylien välillä. DNA-origamien ja biomakromolekyylien sitoutumista tutkittiin agaroosigeelielektroforeesilla sekä läpäisyelektronimikroskopialla. Rakenteellista stabiilisuutta tutkittiin altistamalla päällystetyt DNA-origamit DNase I -endonukleaasin hajottamiselle. Lopuksi päällystettyjen origamien soluläpäisevyyttä HEK293-soluihin tutkittiin konfokaalimikroskopialla sekä kvantitatiivisesti FACScan-virtaussytometrialla. Tulokset osoittivat DNA-origamien päällystämisen onnistuvan proteiini-dendroni -konjugaatteihin perustuvalla päällystysmenetelmällä. Konjugaatissa dendronit muodostivat synteettisen DNA:han sitoutuvan osan, johon voitiin edelleen kiinnittää proteiineja. Kaksi käytettyä proteiinia olivat naudan seerumialbumiini (BSA) sekä hydrofobiini I (HFBI). Näistä BSA-päällystyksen todettiin suojelevan origameja nukleaasien hajottamiselta sekä parantavan origamien soluläpäisevyyttä HEK293-soluihin. Tämä työ esittelee uuden DNA-origamien päällystysmenetelmän, jota voitaisiin hyödyntää lääkeainekuljetussovelluksissa sekä muissa nukleotidirakenteisissa bionanoteknologian sovelluksissa

Elektrostatisk självorganisation av DNA origami och guldnanopartiklar

Spatially well-ordered structures of gold nanoparticles(AuNPs) and other metal nanoparticles have unique electronic, magnetic and optical properties, and hence there is ever-increasing interest towards these kinds of nanomaterials. DNA and DNA nanostructures have successfully been used to direct the higher-ordered arrangement of AuNPs, but the programmable arrangement of them into larger, well-defined structures is still challenging. The objective of this thesis is to establish a self-assembly method based on electrostatic interactions in which DNA origami nanostructures can be used to guide the higher ordered arrangement of cationic AuNPs in a controlled and programmable manner. The AuNP binding properties of different DNA origami structures was studied with UV/Vis spectroscopy and agarose gel electrophoretic mobility shift assay. DNA origami-AuNP assemblies were formed during dialysis against decreasing ionic strength, and the formed assemblies were characterized using small-angle X-ray scattering, transmission electron microscopy and cryogenic electron tomography. Electrostatic self-assembly of DNA origami 6HB nanostructures and small AuNPs (D_core = 2.5 nm, D_hydrodynamic_diameter = 8.5 nm) yielded highly ordered superlattice structures with a 3D tetragonal lattice structure, whereas other studied combinations of DNA origami structures and AuNPs resulted in amorphous aggregates. These results suggest that both shape and charge complementarity between the building blocks are needed for well-ordered structures to be formed through electrostatic self-assembly. According to the results, electrostatic self-assembly guided by DNA origami structures seems promising for construction of novel, well-ordered structures with unique properties, such as lattice geometry, designed specifically for the chosen application.Guldnanopartiklar och andra metallnanopartiklar organiserade i välordnade strukturer har unika elektroniska, magnetiska och optiska egenskaper och därför finns det ett ständigt växande intresse för dessa typer av nanomaterial. DNA och nanostrukturer av DNA har framgångsrikt använts för att framställa välordnade, förutbestämda tredimensionella guldnanopartikelstrukturer, men det finns fortfarande utmaningar att tackla. Målet med detta diplomarbete är att utveckla en metod för självorganisation baserat på elektrostatiska interaktioner i vilken DNA-origaminanostrukturer på ett programmerbart och kontrollerat sätt kan användas för att styra hurudana strukturer som byggs upp av katjoniska guldnanopartiklar. De olika DNA-origamistrukturernas förmåga att binda guldnanopartiklar studerades med UV/Vis-spektroskopi och agarosgelelektrofores. DNA-origami-guldnanopartikelsystem byggdes upp genom dialys mot stegvis minskade jonkoncentrationer och de uppkomna strukturerna karaktäriserades med lågvinkelspridning, transmissionselektronmikroskopi och kryelektrontomografi. Elektrostatisk självorganisation av DNA-origami 6HB nanostrukturer och små guldnanopartiklar (D_kärna = 2.5 nm, D_hydrodynamisk_diameter = 8.5 nm) gav välordnade tredimensionella tetragonala kristallstrukturer, medan andra undersökta kombinationer av DNA origami strukturer och guldpartiklar endast resulterade i amorfa strukturer. Detta indikerar att de enskilda byggstenarna behöver kompletterande form och laddning för att välordnade strukturer skall kunna byggas upp genom elektrostatik självorganisation. Det förefaller dock finnas goda framtidsutsikter för elektrostatisk självorganisation som en metod att framställa välordnade strukturer med egenskaper, så som typ av kristallstruktur, lämpliga just för det önskade användningsområdet