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京都大学 / 新制・課程博士 / 博士(エネルギー科学) / 甲第24854号 / エネ博第463号 / 新制||エネ||87(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 森井, 孝, 教授 片平, 正人, 教授 佐川, 尚 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

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<p dir="ltr">DNA sequence design requires the ability to identify possible tertiary structural defects, secondary structure disruptions, and self-complimentary stretches that will disallow your complimentary strands to come together to form the desired duplex design. However, there is a need for those self-complimentary stretches, especially when designed with the intent for this to homo-oligomerize into the desired building block. With the programmability of nucleic acid hybridization, there is an expanding field wherein this specific, self-complimentary design feature can give new possibility of fine-tuning DNA self-assembly (Chapter 1) or overcome a previously thought limit of DNA ligation (Chapter 2).</p><p dir="ltr">The first chapter will closely look at the branched kissing loop interaction. This interaction was studied as a homo-polymerizable DNA building block that is topologically closed. As such, this paranemic motif has increased stability due to the Watson-Crick base pairing being “protected” by a 3-base adenine branch which close the loop of the sticky-end, meaning no free ends in the binding region. With this, herein we report that the intended higher-level structure could influence the lower-level building block formation. In DNA nanotechnology, this could mean the final higher-level structure would allow for fine-tuning as this would dictate the building blocks that fill in the defected parts of the higher-level structure.</p><p dir="ltr">The second chapter looks at the more finite than broad picture. Whilst the first chapter focusses on the impact the microscale has on the nanoscale through a homo-polymerizable design, the second chapter focusses on the ability to break barriers with homo-polymerizable design. In this chapter, we prove that with our splint strand design, when improved with a hairpin loop on the terminal ends, we can ligate DNA strands enzymatically as short as 16 nucleotides with an efficiency of 97% at high concentrations (100 uM). These hairpins allow for a stable, robust splint strand as they are a self-complimentary region which will maintain its shape throughout the process of joining together the 5’ and 3’ ends of the target strand.</p><p dir="ltr">Overall, this dissertation hopes to prove that homo-polymerizable DNA sequence designs are helping expand upon the DNA nanotechnology toolbox by introducing new possibilities for nanoscale design, as well as push past previously held boundaries through necessary added stability afforded by the self-complimentary strands.</p>

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<p>DNA nanotechnology has provided programming construction of various nanostructures at nanometer-level precision over the last three decades. DNA self-assembly is usually implemented by annealing process in bulk solution. In recent several years, a new method thrives by fabricating two-dimensional (2D) nanostructures on solid surfaces. My researches mainly focus on this field, surface-assisted DNA assembly driven by base stacking. I have developed methods to fabricate DNA 2D networks via isothermal assembly on mica surfaces. I have further explored the applications to realize quasicrystal fabrication and nanoparticles (NPs) patterning.</p><p><br></p> <p>In this dissertation, I have developed a strategy to assemble DNA structures with 1 or 2 pair(s) of blunt ends. Such weak interactions cannot hold DNA motifs together in solution. However, with DNA-surface attractions, DNA motifs can assemble into large nanostructures on solid surface. Further studies reveal that the DNA-surface attractions can be controlled by the variety and concentration of cation in the bulk solution. Moreover, DNA nanostructures can be fabricated at very low motif concentrations, at which traditional solution assembly cannot render large nanostructures. Finally, assembly time course is also studied to reveal a superfast process for surface-assisted method compared with solution assembly.</p><p><br></p> <p>Based on this approach, I have extended my research scope from 1D to 2D structures assembled from various DNA motifs. In my studies, I have successfully realized conformational change regulated by DNA-surface interaction and steric effect. By introduction of DNA duplex “bridges” and unpaired nucleotide (nt) spacers, we can control the flexibility/rigidity of DNA nanomotifs, which helps to fabricate more delicate dodecagonal quasicrystals. The key point is to design the length of spacers. For 6-point-star motif, a rigid structure is required so that only 1-nt spacers are added. On the other hand, 3-nt spacers are incorporated to enable an inter-branch angle change from 60° to 90° for a more flexible 5-point-star motif. By tuning the ratio of 5 and 6 -point-star motifs in solution, we can obtain 2D networks from snub square tiling, dodecagonal tiling, a mixture of dodecagonal tiling and triangular tiling, and triangular tiling.</p><p><br></p> Finally, I have explored the applications of my assembly method for patterning NPs. Tetragonal and hexagonal DNA 2D networks have been fabricated on mica surfaces and served as templates. Then modify the surfaces with positively-charged “glues”, <i>e.g.</i> poly-L-lysine (PLL) or Ni²⁺. After that, various NPs have been patterned into designated lattices, including individual DNA nanomotifs, gold NPs (AuNPs), proteins, and silica complexes. Observed NP lattices and fast Fourier Transform (FFT) patterns have demonstrated the DNA networks’ patterning effect on NPs.

The controlled organization of individual molecules and nanostructures with nanoscale accuracy is of great importance in the investigation of single-molecule events in biological and chemical assays, as well as for the fabrication of the next generation optoelectronic devices. In this regard, the precise patterning of individual molecules into hierarchical structures has attracted substantial research interest in recent years. DNA has been shown to be an ideal structural material for this purpose, due to the specificity of its programmability and outstanding chemical flexibility. DNA origami can display a high degree of positional and precise binding sites, allowing for complex arrangements and the assembly of different nanoscale architectures. In this project, we present a novel platform based on the use of DNA scaffolds for the organization of individual nanomoieties (with nanoscale spatial control), and their selective immobilisation on surfaces for single-molecule investigations. In particular, semiconductor quantum dots (QDs), fluorescence molecules, linear small peptides, and structural proteins were tethered with single-molecule accuracy on DNA origami; their subsequent organization in array configuration on nanopatterned surfaces allowed us to fabricate and test different platforms for single-molecule studies. In particular, we developed a Focused Ion Beam (FIB) nanofabrication strategy and demonstrated its general applicability for the assembly of functionalised DNA nanostructures in highly uniform nanoarrays, with single-molecule control. In addition, we further explored this nanofabricated platform for biological investigations at the single-molecule level, from protein-DNA interactions to cancer cell adhesion studies with single-molecule control. Investigations have been carried out via fluorescence microscopy, scanning electron microscopy (SEM), Focused Ion Beam (FIB) and atomic force microscopy (AFM). By and large, combining the programming ability of DNA as a scaffolding material with a one-step lithographic process, we have developed a platform of general applicability for the fabrication of nanoscale chips that can be employed in a variety of single-molecule investigations.

DNA nanotechnology has been developed in order to construct nanostructures and nanomachines by virtue of the programmable self-assembly properties of DNA molecules. Although DNA nanotechnology initially focused on spatial arrangement of DNA strands, new horizons have been explored owing to the development of the toehold-mediated strand-displacement reaction, conferring new dynamic properties to previously static and rigid structures. A large variety of DNA reconfigurable nanostructures, stepped and autonomous nanomachines and circuits have been operated using the strand-displacement reaction. Biological systems rely on information processing to guide their behaviour and functions. Molecular computation is a branch of DNA nanotechnology that aims to construct and operate programmable computing devices made out of DNA that could interact in a biological context. Similar to conventional computers, the computational processes involved are based on Boolean logic, a propositional language that describes statements as being true or false while connecting them with logic operators. Numerous logic gates and circuits have been built with DNA that demonstrate information processing at the molecular level. However, development of new systems is called for in order to perform new tasks of higher computational complexity and enhanced reliability. The contribution of secondary structure to the vulnerability of a toehold-sequestered device to undesired triggering of inputs was examined, giving new approaches for minimizing leakage of DNA devices. This device was then integrated as a logic component in a DNA-based computer with a retrievable memory, thus implementing two essential biological functions in one synthetic device. Additionally, G-quadruplex logic gates were developed that can be switched between two topological states in a logic fashion. Their individual responses were detected simultaneously, establishing a new approach for parallel biological computing. A new AND-NOT logic circuit based on the seesaw mechanism was constructed that, in combination with the already existing AND and OR gates, form a now complete basis set that could perform any Boolean computation. This work introduces a new mode of kinetic control over the operation of such DNA circuits. Finally, the first example of a transmembrane logic gate being operated at the single-molecule level is described. This could be used as a potential platform for biosensing.

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/Thesis / Doctoral Dissertation Chemistry 2019