Design of DNA origami

The generation of arbitrary patterns and shapes at very small scales is at the heart of our effort to miniaturize circuits and is fundamental to the development of nanotechnology. Here I review a recently developed method for folding long single strands of DNA into arbitrary two-dimensional shapes using a raster fill technique - 'scaffolded DNA origami'. Shapes up to 100 nanometers in diameter can be approximated with a resolution of 6 nanometers and decorated with patterns of roughly 200 binary pixels at the same resolution. Experimentally verified by the creation of a dozen shapes and patterns, the method is easy, high yield, and lends itself well to automated design and manufacture. So far, CAD tools for scaffolded DNA origami are simple, require hand-design of the folding path, and are restricted to two dimensional designs. If the method gains wide acceptance, better CAD tools will be required

Construction of a novel phagemid to produce custom DNA origami scaffolds.

DNA origami, a method for constructing nanoscale objects, relies on a long single strand of DNA to act as the 'scaffold' to template assembly of numerous short DNA oligonucleotide 'staples'. The ability to generate custom scaffold sequences can greatly benefit DNA origami design processes. Custom scaffold sequences can provide better control of the overall size of the final object and better control of low-level structural details, such as locations of specific base pairs within an object. Filamentous bacteriophages and related phagemids can work well as sources of custom scaffold DNA. However, scaffolds derived from phages require inclusion of multi-kilobase DNA sequences in order to grow in host bacteria, and those sequences cannot be altered or removed. These fixed-sequence regions constrain the design possibilities of DNA origami. Here, we report the construction of a novel phagemid, pScaf, to produce scaffolds that have a custom sequence with a much smaller fixed region of 393 bases. We used pScaf to generate new scaffolds ranging in size from 1512 to 10 080 bases and demonstrated their use in various DNA origami shapes and assemblies. We anticipate our pScaf phagemid will enhance development of the DNA origami method and its future applications

Modeling technologies and methods for DNA origami

The creation of correctly assembling DNA origami often requires several iterations wherein a researcher tries and troubleshoots an incremental design. In each iteration there exists one or more costly failures that often take immense time or materials to find. These failures occur in part due to a lack of in-depth understanding of how DNA origami self-assembles and functions. To aid researchers in developing correct DNA origami designs, this thesis describes the creation of a DNA origami failure catalog as well as models for elucidating as-of-yet only partially understood properties of DNA origami. The failure catalog helps laboratory scientists gather requirements to preempt failures in their origami designs, and helps laboratory scientists troubleshoot their experiments after the implementation of a design by querying the catalog. Use of the catalog then helps verify the properties of new macro and micro models for DNA origami introduced here. These micro and macro models open up future ways to evaluate DNA origami through a mathematically more rigorous framework. By using both captured knowledge of previous design failures and novel theoretical modeling techniques, this work seeks to reduce the gap in understanding between design and implementation of DNA origami

Connecting DNA Origami Structures Using the Biotin-Streptavidin Specific Binding

This work made use of the strong interaction between biotin and streptavidin to connect designed DNA origami structures. The caDNAno software was used to design a 6 layer 3D origami cross-like structure. Selected DNA strands at the engineered attachment sites on the DNA origami structure were biotinylated. After folding of the origami structures, the biotinylated strands stick out of the attachment sites. Purified samples of origami structures were then mixed with streptavidin and the mixture purified. After characterization, we see that attachment only occurs at the biotinylated sites. Agarose gel electrophoresis, UV-vis spectroscopy and TEM were used to characterize the structure.Key words: DNA Origami, Interaction, Biotin-Streptavidin, Nanomaterials, TEMAbbreviation: EDTA, Ethylenediaminetetraacetic acid; TEM, Transmission electron microscop

DNA 오리가미 구조체의 형상 설계를 위한 기계적 응력 조절 기술

학위논문(박사)--서울대학교 대학원 :공과대학 기계항공공학부,2020. 2. 김도년.In this thesis, we describe two design strategies that engineer mechanical stress to program static or dynamic conformations of the DNA origami structure. DNA origami nanotechnology facilitated the self-assembly of DNA strands into any conceivable shape encoded by their rationally designed sequences. Mechanics-based design approaches have played an important role in improving the structural diversity of the DNA origami structures. Due to low twist controllability and limited reconfiguration mode, however, they have still limitations in achievable diversity or complexity in structural shapes and their reconfigurations and their applications. To this end, first, we developed a design strategy for fine control of twisted DNA origami structures by considering not only amount of geometrical perturbations but also their arrangements within the structures. With the configurational design of geometrical perturbations, we can program various distributions of the mechanical stress enabling a fine control over twist rate of DNA origami structures. Second, we developed a design strategy that transforms a two-dimensional structure into three-dimensional supercoiled one on demand. We employed the topological invariant property to convert a simple twist deformation into complex bending one leading to supercoiling of the DNA origami structure. We expect that our mechanical stress programming strategies can be utilized to design DNA origami structures with desired shapes or reconfiguration motions and enhance the performance of functional structures.본 학위논문은 목표하는 정적 및 동적 형상을 지닌 DNA 오리가미 구조 제작을 위한 기계적 응력 조절 기술에 기반한 설계방법을 제시한다. DNA 오리가미 나노기술은 DNA 가닥들의 자가조립 과정을 통해 기존에 제작이 어려웠던 다양한 형상의 나노구조물을 손쉽게 만들 수 만들 수 있다. 이를 활용해 목표 형상의 나노구조물을 만들기 위해 다양한 설계 방법들이 제시되어 왔다. 이중 역학적 원리에 기반한 설계 방법은 구조 내부에 의도적으로 기계적 스트레스를 발생시켜 구조의 비틀림, 굽힘 등을 정량적으로 조절할 수 있게 만들어, 제작 가능한 형상의 범주를 넓히는데 크게 기여하였다. 하지만 기존 방법들은 세밀한 비틀림 형상 제어가 어렵다는 점 그리고 제한된 종류의 형상변화만이 가능하다는 문제점으로 인해 목표 형상을 지닌 정적 혹은 동적 구조의 제작 및 이러한 구조들의 활용에 어려움이 존재한다. 이에 해결책으로써 본 연구는 다음과 같은 기계적 응력 조절 기술들을 제시한다. 첫째, 구조 내 기하학적 섭동의 분포 설계 통해 DNA 오리가미 구조물의 세밀한 비틀림 형상 조절을 위한 설계 방법을 제시한다. 이를 이용한 구조 내 변형 에너지의 조절을 통해, 미세한 비틀림 형상 조절이 가능해진다. 둘째, 단순한 2차원 구조물을 복잡한 3차원 형상의 구조물로 변환시키는 형상 변환 메커니즘을 제안한다. 양끝이 이어진 닫힌 구조가 지닌 위상학적 불변성을 이용해, 국부적 비틀림을 전역적 굽힘 변형으로 변환시킴으로써, DNA 오리가미 구조의 슈퍼코일링 현상을 제시한다. 이러한 기계적 응력 조절 기술들은 원하는 형상 및 변화 움직임을 지닌 DNA 나노구조물의 설계에 활용되어 기능성 나노구조물들의 성능을 향상시키는데 기여할 것이라고 기대된다.Abstract 1 Table of contents 3 List of tables 5 List of figures 6 Chapter 1. Introduction 9 1.1. Research background 9 1.1.1. DNA origami nanotechnology 9 1.1.2. Self-assembly of DNA origami structure 12 1.1.3. Structural motifs 14 1.1.4. Computational design and analysis tools 16 1.2. Design strategy for DNA Origami structure 18 1.2.1. Lattice-based design 18 1.2.2. Flexible hinge-assisted design 19 1.2.3. Mechanical stress-assisted design 20 1.3. Research motivation 23 1.4. Thesis overview 25 Chapter 2. Methodology 28 2.1. Computational modeling and analysis 28 2.1.1. FE simulation for DNA origami structures 28 2.1.2. MD simulation for DNA origami structures 30 2.2. Fabrication and characterization 31 2.2.1. Self-assembly of DNA origami structures. 31 2.2.2. Agarose gel electrophoresis. 32 2.2.3. AFM imaging 33 2.2.4. TEM imaging 34 Chapter 3. Mechanical stress engineering for fine shape control 35 3.1. Limitation in the design of twisted structures 35 3.2. Configurational design approach 37 3.3. Twist angle variation 41 3.4. Fine control over twist rate 68 3.5. Twist control assisted by mechanical relaxation using gaps 76 3.6. Summary 83 Chapter 4. Mechanical stress engineering for shape reconfiguration 84 4.1. Limitation in the reconfiguration mechanisms 84 4.2. Buckling-induced homeomorphic transformation 86 4.3. Supercoiling of the 6HB ring 91 4.4. Computational analysis of the buckling-induced supercoiling 104 4.5. Reconfiguration control by local defects 110 4.6. Summary 112 Chapter 5. Concluding remark 113 Appendix 115 A1. Calculation of twist angles of 6HB structures 115 A2. Relation between twist angle and trans-ratio (TR) 118 A3. FE simulation of a coiling of a dsDNA ring 120 Bibliography 122 국 문 초 록 132 Acknowledgments 134Docto

Custom-tailored DNA origami mechanics for cellular applications

DNA molecules have been used as the building block for the self-assembly of artificial nanostructures. In particular, the DNA origami method has made the design of DNA nanostructures more robust and approachable. Different design approaches have been created and DNA origami has been used in a variety of fields, from plasmonic, to drug delivery, to biology and biophysics. In recent years, DNA nanotechnology has shown very promising uses in studying forces in biological contexts, both by measuring them and applying them. Mechanosensitive systems in biology are widespread and the study of their complex regulation is increasing in importance, and DNA origami has recently been used as a tool to study them. In paper I, we implement an unsupervised software to simulate wireframe DNA origami structures and evaluate their rigidity. After this evaluation, the software produces mutant structures and then the process is started again, iteratively. In this way the software creates an in-silico evolution towards more rigid wireframe DNA origami. The structures are modified following one of two schemes. In the first one, the individual edges are evaluated and then modified by adding or removing individual bases; in the second scheme, the structures have internal supports, and the software can modify the position of these internal supports to create mutants. We show that these two schemes have different results on the rigidity of the structures, with the internal supports-based scheme increasing the rigidity of structures to up to 50%, after several iterations. In paper II, we compare the mechanical characteristics of a lattice-based DNA origami nanostructure and a wireframe DNA origami nanostructure, exploring how the differences between the two affect their interaction with cancer cells. The wireframe structure showed a higher local flexibility when compared to the lattice-based structure. These physical differences play an important role in the interaction between DNA nanostructures and human cancer cells, in particular thanks to the differences in interaction with scavenger receptors. We show that wireframe origamis are more likely to stay on the cell membrane, while the lattice-based origami are more likely to be internalized. This is also reflected in a deeper penetration of the wireframe structures into cell spheroid tissue models. With these observations, we show that the design method should be considered when applying DNA origami for biological applications. In paper III, we aim to expand the design space of wireframe DNA origami, by designing structures with four-helix bundles (4HB) as edges. This is possible thanks to the addition of additional helices to the edge of the wireframe structures, to create 4HB on a square lattice: this results in increased rigidity of the edges. We developed the software for the design of the new type of structures and then we successfully folded a library of five structures, investigating the rigidity of the new type of structures. In addition, we designed a new type of hybrid structures, presenting more rigid 4HB edges and less rigid single helix edges. We think that the development of new ways of designing DNA origami structures can pave the way for the design of nanostructures more suited for specific applications. In paper IV, we design a DNA origami nanoactuator with the aim of pulling on molecular targets. DNA origami is a promising technology in this field because of its high throughput and the relative simplicity when compared with other force spectroscopy techniques. We designed a barrel-like structure with an internal block connected to ssDNA or dsDNA strands, depending on the activation mode of the mechanism. We estimated that the structure can create forces of up to 40 pN, and coarse-grained molecular dynamics simulations in oxDNA and Förster resonance energy transfer experiments confirm the successful activation of the structure. We also demonstrated that the structure, modified with Cy5, cholesterol, and anti-CD3 aptamer, can interact with T cells. We think that DNA origami can become an important tool in the study of mechanosensitive cellular receptors

Modelling the folding pathway of DNA nanostructures

DNA origami is a robust technique for bottom-up nano-fabrication. It encodes a target shape into uniquely addressable interactions between a set of short 'staple' strands and a long 'scaffold' strand. The mechanisms of self-assembly, particularly regarding kinetics, need to be better understood. Origami design usually relies on optimising the thermodynamic stability of the target structure, and thermal annealing remains the most fool-proof assembly protocol. This work focuses on studying the folding pathway of three types of origami through simulations: a reconfigurable T-junction origami, several traditional origami, and origami with coated scaffolds. The T-junction origami is intended as an economically feasible method of changing the uniqueness of interactions. My contribution to this work is to characterise the basic structural motif through oxDNA, a nucleotide-resolution model of DNA. The thesis then focuses on extending a domain-level model of DNA origami to study several experimental origami designs. We reveal design-dependent free energy barriers using biased simulations and relate this to the observed hysteresis in experiments. We also highlight the role of specific design elements in determining the folding pathway. A novel method of lowering the temperature of error-free assembly using coated scaffolds is then presented, with simulations indicating the existence of an activation barrier. By exposing particular regions of the scaffold, we can lower assembly time and temperature

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

Dimensions and Global Twist of Single-Layer DNA Origami Measured by Small-Angle X-ray Scattering

The rational design of complementary DNA sequences can be used to create nanostructures that self-assemble with nanometer precision. DNA nanostructures have been imaged by atomic force microscopy and electron microscopy. Small-angle X-ray scattering (SAXS) provides complementary structural information on the ensemble-averaged state of DNA nanostructures in solution. Here we demonstrate that SAXS can distinguish between different single-layer DNA origami tiles that look identical when immobilized on a mica surface and imaged with atomic force microscopy. We use SAXS to quantify the magnitude of global twist of DNA origami tiles with different crossover periodicities: these measurements highlight the extreme structural sensitivity of single-layer origami to the location of strand crossovers. We also use SAXS to quantify the distance between pairs of gold nanoparticles tethered to specific locations on a DNA origami tile and use this method to measure the overall dimensions and geometry of the DNA nanostructure in solution. Finally, we use indirect Fourier methods, which have long been used for the interpretation of SAXS data from biomolecules, to measure the distance between DNA helix pairs in a DNA origami nanotube. Together, these results provide important methodological advances in the use of SAXS to analyze DNA nanostructures in solution and insights into the structures of single-layer DNA origami