<p>When developing applications for self-assembling nanostructures, a challenge is to organize the self assembling components within integrated nano-microsystems. One approach is to impart nanostructure recognition properties to patterned surfaces, such that nanostructure placement could be thermodynamically driven. This research focuses upon self assembling nanostructures composed of DNA and their reversible specific assembly upon functionalized planar surfaces. Assembly strategies that have been developed for solution phase assembly are herein demonstrated as potentially appropriate for heterogeneous nanosystem integration.</p><p>The assembly of DNA nanostructures relies upon unique base pair interactions between single strands. While DNA hybridization that involves many base pairs results in structures that are strongly bound, an assembly strategy that underlies much DNA nanostructure engineering is formation of nanostructures at temperatures at which the interactions are weak. Here, DNA specific nanostructure immobilization is driven by weak forces. Association is characterized using surface sensitive surface plasmon resonance and quartz crystal microbalance methods. The results suggest that future strategies for nanostructure - system integration that require precise nanostructure placement may be accomplished using specific molecular recognition under thermodynamic control.</p><p>Several methods of solution phase nanostructure characterization are explored. The diffusive properties of DNA nanostructures are examined using dynamic light scattering. Effective hydrodynamic radii are found to be large relative to the nanostructure geometric size. The temperature dependence of light scattering from nanostructures is investigated using both resonance light scattering and nonresonant laser light scattering. Additionally, DNA nanostructure building block and superstructure geometry are interrogated in solution using small angle x-ray scattering. Results derived from comparison of small angle data with simulations of scattering from coarse-grained models are compared with structural information derived from imaging immobilized nanostructures with atomic force microscopy. </p><p>Finally, plasmon coupling in systems comprised of metal particles of unlike composition is described. Through simulation, three phenomena that contribute to interparticle coupling are explored. Off resonant metal particles positioned in between pairs of particles near resonance are found to promote optical coupling in a manner similar to that provided by bulk dielectric media.</p> / Dissertation

abstract: DNA nanotechnology has been a rapidly growing research field in the recent decades, and there have been extensive efforts to construct various types of highly programmable and robust DNA nanostructures. Due to the advantage that DNA nanostructure can be used to organize biochemical molecules with precisely controlled spatial resolution, herein we used DNA nanostructure as a scaffold for biological applications. Targeted cell-cell interaction was reconstituted through a DNA scaffolded multivalent bispecific aptamer, which may lead to promising potentials in tumor therapeutics. In addition a synthetic vaccine was constructed using DNA nanostructure as a platform to assemble both model antigen and immunoadjuvant together, and strong antibody response was demonstrated in vivo, highlighting the potential of DNA nanostructures to serve as a new platform for vaccine construction, and therefore a DNA scaffolded hapten vaccine is further constructed and tested for its antibody response. Taken together, my research demonstrated the potential of DNA nanostructure to serve as a general platform for immunological applications. / Dissertation/Thesis / Movie 4.1-a / Movie 4.1-b / Movie 4.1-c / Ph.D. Biochemistry 2012

Human respiratory syncytial virus (RSV) is the primary cause of acute lower respiratory tract infections in children and the elderly worldwide, for which neither a vaccine nor an effective therapy is approved. The entry of RSV into the host cell is mediated by stepwise structural changes in the surface RSV fusion (RSV-F) glycoprotein. Recent progress in structural and functional studies of RSV-F glycoprotein revealed conformation-dependent neutralizing epitopes which have become attractive targets for vaccine and therapeutic development. As RSV-F is present on viral surface in a trimeric form, a trivalent binding interaction between a candidate fusion inhibitor and the respective epitopes on each of the three monomers is expected to prevent viral infection at higher potency than a monovalent or bivalent inhibitor. Here we demonstrate a novel RSV entry inhibitory approach by implementing a trimeric DNA nanostructure as a template to display up to three linear peptide moieties that simultaneously target an epitope on the surface of the prefusion RSV-F protein. In order to design synthetic binding peptides that can be coupled to the DNA nanostructure, the prefusion RSV-F-specific monoclonal antibody (D25) was selected. Complementarity-determining region 3 (CDR3) derived peptides underwent truncation and alanine-scanning mutagenesis analysis, followed by systematic sequence modifications using non-canonical amino acids. The most effective peptide candidate was used as a binding moiety to functionalize the DNA nanostructure. The designed DNA-peptide construct was able to block RSV infection on cells more efficiently than the monomeric peptides, however a more moderate reduction of viral load was observed in the lungs of infected mice upon intranasal application, likely due to dissociation or absorption of the underlying DNA structure by cells in the lungs.Taken together, our results point towards the inhibitory potential of a novel trimeric DNA-peptide based approach against RSV and open the possibility to apply this platform to target other viral infections.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第22397号 / 薬科博第119号 / 新制||薬科||13(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 髙倉 喜信, 教授 山下 富義, 教授 小野 正博 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第22087号 / エネ博第395号 / 新制||エネ||76(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 木下 正弘, 教授 片平 正人 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19992号 / 工博第4236号 / 新制||工||1655(附属図書館) / 33088 / 京都大学大学院工学研究科マイクロエンジニアリング専攻 / (主査)教授 田畑 修, 教授 北條 正樹, 教授 山田 啓文 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

DNA nanostructures enable the attachment of functional molecules to nearly any unique location on their underlying structure. Due to their single-base-pair structural resolution, several ligands can be spatially arranged and closely controlled according to the geometry of their desired target, resulting in optimized binding and/or signaling interactions. Here, the efficacy of SWL, an ephrin-mimicking peptide that binds specifically to EphrinA2 (EphA2) receptors, increased by presenting up to three of these peptides on small DNA nanostructures in an oligovalent manner. Ephrin signaling pathways play crucial roles in tumor development and progression. Moreover, Eph receptors are potential targets in cancer diagnosis and treatment. Here, the quantitative impact of SWL valency on binding, phosphorylation (key player for activation) and phenotype regulation in EphA2-expressing prostate cancer cells was demonstrated. EphA2 phosphorylation was significantly increased by DNA trimers carrying three SWL peptides compared to monovalent SWL. In comparison to one of EphA2’s natural ligands ephrin-A1, which is known to bind promiscuously to multiple receptors, pinpointed targeting of EphA2 by oligovalent DNA-SWL constructs showed enhanced cell retraction. Overall, we show that DNA scaffolds can increase the potency of weak signaling peptides through oligovalent presentation and serve as potential tools for examination of complex signaling pathways.