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Solid-state nanopores are a versatile class single-molecule sensors to electrically characterize a range of biological molecules. Nanopores operate on the simple premise that when a voltage is applied across a pore immersed in a salt solution, the passage of a biomolecule results in a transient blockage in the ionic current that provide information about the translocating molecule. This thesis presents studies employing various DNA nanostructures with solid-state nanopore electrical readout for the development of high sensitivity digital single-molecule assays to detect low-abundance biomarkers. Toward this ultimate goal, work presented in this thesis use nanopores to probe DNA nanostructures, their assembly, mechanical properties, and monitor their dynamics with time and temperature. DNA nanostructures are self-assembled via specific base pairing of DNA, their programmability make them particularly useful for applications including drug delivery, molecular computation and biosensing. Here, I first show results of translocation profiles and discuss folding characteristics, mobility, and molecular configuration during passage for different DNA nanostructures such as the short star-shaped DNA nanostructures and large helix-bundle DNA origami structures under various experimental conditions in an effort to understand the passage characteristics through nanopores of these structures before using them in biological assays. I conclude by presenting a magnetic bead-based immunoassay scheme using a digital solid-state nanopore readout. Nanopore has the ability to count molecules one at a time, this allows accurate and precise determination of the concentration of a biomarker in solution. Coupled with the use of specific choice of DNA nanostructures, as proxy labels for proteins of interest, I establish that nanopores sensors can reliably quantify the concentration of a protein biomarker from complex biofluids and overcome the traditional challenges associated with nanopore-based protein sensing, such as specificity, sensitivity, and consistency. I demonstrate the quantification of thyroid stimulating hormone (TSH) with a high degree of precision down to the femtomolar range by using a nanoparticle-based signal amplification strategy. The proposed assay scheme is generalizable to a framework for the detection and quantification of a wide range of target proteins, and given that its performance can further be improved with the use of parallelization, preconcentration, or miniaturization, it opens up exciting opportunities for the development of ultra-sensitive digital assay in a format that is compatible for point-of-care.

<p> DNA origami is a powerful method to construct DNA nanostructures. It requires long, single-stranded DNAs. The preparation of such long DNA strands is often quite tedious and has a limited production yield. In contrast, duplex DNAs can be easily prepared via enzymatic reactions in large quantities. Thus, we ask a question: can we design DNA nanostructures in such a way that the two complementary strands can simultaneously fold into the designed structures in the same solution instead of hybridizing with each other to form a DNA duplex? By engineering DNA interaction kinetics, herein, we are able to provide multiple examples to concretely demonstrate a positive answer to this question. The resulting DNA nanostructures have been thoroughly characterized by electrophoresis and atomic force microscopy imaging. The reported strategy is compatible with the DNA cloning method; thus, would provide a convenient way for large-scale production of the designed DNA nanostructures. </p>

<p>  </p> <p>This chapter introduces geometry as a means to program the tile-based DNA self-assembly in two dimensions. This strategy complements the sequence-focused programmable assembly. DNA crystal assembly critically relies on intermotif, sticky-end cohesion, which requires complementarity not only in sequence but also in geometry. For DNA motifs to assemble into 2D crystals, they must be associated with each other in the proper geometry and orientation to ensure that geometric hindrance does not prevent sticky ends from associating. For DNA motifs with exactly the same pair of sticky-end sequences, by adjusting the length (thus, helical twisting phase) of the motif branches, it is possible to program the assembly of these distinct motifs to either mix with one another, to self-sort and consequently separate from one another, or to be alternatingly arranged. We demonstrate the ability to program homogeneous crystals, DNA “alloy” crystals, and definable grain boundaries through self-assembly. We believe that the integration of this strategy and conventional sequence-focused assembly strategy could further expand the programming versatility of DNA self-assembly.</p>

No description available.

<p dir="ltr">Deoxyribonucleic acid or DNA is an essential component in cells and organisms for genetic information storage and transduction. The base paring chemistry offers excellent programmability and structural predictability. This gives rise to the field of DNA nanotechnology, which uses DNA to design nanostructures and nanomachines with unprecedented designability and controllability. With the development of DNA nanotechnology, numerous chemical tools have been introduced for designing complex molecular mechanisms with DNA molecules. Various nanostructures of arbitrary shapes have been demonstrated, which shows the immense potential of DNA-based engineering. Dynamic nanodevices and their programmable actuations have also been successfully realized using DNA strand displacement and/or enzymatic reactions.</p><p dir="ltr">With controllable interactions with various biomolecules, it is possible to implement DNA in synthetic biological systems to program their behaviors. Two systems with programmed behaviors are introduced in this dissertation. The first system is a lipid-based protocell that can perform programmed migration with DNA-based mechanisms. This model system extracts chemical energy from fuel strands via enzymatic reaction and converts it into autonomous translocation on a surface. A mechanistic model is proposed to understand the migration dynamics. Furthermore, a path-tracking behavior between synthetic vesicles is demonstrated, which mimics cellular chemotaxis for the first time.</p><p dir="ltr">The second synthetic biological system explored is DNA origami structures capable of programmable auxetic reconfiguration. Auxetic materials are artificial systems with a negative Poisson’s ratio, which show great promise in various applications including space engineering and flexible/wearable electronics. With DNA-based sliding mechanisms, the proposed auxetic architecture can switch between two conformations with different Poisson’s ratios. The proposed strategy may be applied to designing adaptive materials or biochemical sensors with mechanical responses. The DNA-programmed behaviors demonstrated in this dissertation show unprecedented versatility and programmability, thus opening new opportunities for using molecular mechanisms to control synthetic biological systems with complex functions in diverse areas including biology, biomedicine, and material sciences.</p>

One common limitation of enzymatic reactions is the diffusion of a substrate to the enzyme active site and/or the release of the reaction products. These reactions are known as diffusion –controlled. Overcoming this limitation may enable faster catalytic rates, which in the case of catalytic biosensors can potentially lower limits of detection of specific analyte. Here we created an artificial system to enable deoxyribozyme (Dz) 10-23 based biosensor to overcome its diffusion limit. The sensor consists of the two probe strands, which bind to the analyzed nucleic acid by Watson-Crick base pairs and, upon binding re-form the catalytic core of Dz 10-23. The activated Dz 10-23 cleaves the fluorophore and quencher-labeled DNA-RNA substrate which separates the fluorophore from the quencher thus producing high fluorescent signal. This system uses a Dz 10-23 biosensor strand associated to a DNA antenna tile, which captures the fluorogenic substrate and channels it to the reaction center where the Dz 10-23 cleaves the substrate. DNA antenna tile captures fluorogenic substrate and delivers it to the activated Dz 10-23 core. This allows for lower levels of analyte to be detected without compromising the specificity of the biosensor. The results of this experiment demonstrated that using DNA antenna, we can create a synthetic environment around the Dz 10-23 biosensor to increase its efficiency and allow for lower levels of analyte to be detected without using amplification techniques like PCR.

Hybridization of two or more DNA or RNA strands is well documented for the process taking place with all strands free in solution or when one strand is immobilized on a substrate. This study contributes to the investigation of the hybridization process when two single DNA strands (ssDNA) are in close proximity. We took advantage of an X sensor in which hybridization of four DNA strands enables the formation of a DNA four-way junction (crossover or X) structure. We immobilized multiple layers of crossover structures to study its hybridization being triggered by short ssDNA coming from solution and further investigate how many layers of these structures can hybridize by the addition of only one ssDNA (called input). Using a molecular beacon as reporter, we combined crossover DNA strands that recognize the reporter sequence at one side and at the other, the sequence of its input or downward crossover layer. Fluorescent signal was detected by separation of the molecular beacon’s fluorophore and quencher, as it hybridizes with the system of layers. Immobilization of the X structures into the scaffold proved to increase their communication, in comparison to being free in solution. This evidence gives us significant information for the communication of hybridized layers in a localized system, showing a promising standard for development of multilayered logic gates. The potential of these crossover DNA strands using X structure include applications in the future of biological systems, nanotechnology, and target DNA recognition for its ability to quickly recognize a signal and propagate it through extended DNA nanostructure in a controlled manner.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第23724号 / 理博第4814号 / 新制||理||1689(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 深井 周也, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(理学) / 甲第23726号 / 理博第4816号 / 新制||理||1689(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 深井 周也, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM