ASU Electronic Theses and Dissertations
This collection includes most of the ASU Theses and Dissertations from 2011 to present. ASU Theses and Dissertations are available in downloadable PDF format; however, a small percentage of items are under embargo. Information about the dissertations/theses includes degree information, committee members, an abstract, supporting data or media.
In addition to the electronic theses found in the ASU Digital Repository, ASU Theses and Dissertations can be found in the ASU Library Catalog.
Dissertations and Theses granted by Arizona State University are archived and made available through a joint effort of the ASU Graduate College and the ASU Libraries. For more information or questions about this collection contact or visit the Digital Repository ETD Library Guide or contact the ASU Graduate College at gradformat@asu.edu.
Displaying 1 - 2 of 2
Filtering by
- All Subjects: Nanostructures
Description
Proteins and peptides fold into dynamic structures that access a broad functional landscape, however, designing artificial polypeptide systems continues to be a great chal-lenge. Conversely, deoxyribonucleic acid (DNA) engineering is now routinely used to build a wide variety of two dimensional and three dimensional (3D) nanostructures from simple hybridization based rules, and their functional diversity can be significantly ex-panded through site specific incorporation of the appropriate guest molecules. This dis-sertation describes a gentle methodology for using short (8 nucleotide) peptide nucleic acid (PNA) linkers to assemble polypeptides within a 3D DNA nanocage, as a proof of concept for constructing artificial catalytic centers. PNA-polypeptide conjugates were synthesized directly using microwave assisted solid phase synthesis or alternatively PNA linkers were conjugated to biologically expressed proteins using chemical crosslinking. The PNA-polypeptides hybridized to the preassembled DNA nanocage at room tempera-ture or 11 ⁰C and could be assembled in a stepwise fashion. Time resolved fluorescence anisotropy and gel electrophoresis were used to determine that a negatively charged az-urin protein was repelled outside of the negatively charged DNA nanocage, while a posi-tively charged cytochrome c protein was retained inside. Spectroelectrochemistry and an in-gel luminol oxidation assay demonstrated the cytochrome c protein remained active within the DNA nanocage and its redox potential decreased modestly by 10 mV due to the presence of the DNA nanocage. These results demonstrate the benign PNA assembly conditions are ideal for preserving polypeptide structure and function, and will facilitate the polypeptide-based assembly of artificial catalytic centers inside a stable DNA nanocage. A prospective application of assembling multiple cyclic γ-PNA-peptides to mimic the oxygen-evolving complex (OEC) catalytic active site from photosystem II (PSII) is described. In this way, the robust catalytic capacity of PSII could be utilized, without suffering the light-induced damage that occurs by the photoreactions within PSII via triplet state formation, which limits the efficiency of natural photosynthesis. There-fore, this strategy has the potential to revolutionize the process of designing and building robust catalysts by leveraging nature's recipes, and also providing a flexible and con-trolled artificial environment that might even improve them further towards commercial viability.
ContributorsFlory, Justin David (Author) / Fromme, Petra (Thesis advisor) / Yan, Hao (Committee member) / Buttry, Daniel (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2014
Description
There are many biological questions that require single-cell analysis of gene sequences, including analysis of clonally distributed dimeric immunoreceptors on lymphocytes (T cells and B cells) and/or the accumulation of driver/accessory mutations in polyclonal tumors. Lysis of bulk cell populations results in mixing of gene sequences, making it impossible to know which pairs of gene sequences originated from any particular cell and obfuscating analysis of rare sequences within large populations. Although current single-cell sorting technologies can be used to address some of these questions, such approaches are expensive, require specialized equipment, and lack the necessary high-throughput capacity for comprehensive analysis. Water-in-oil emulsion approaches for single cell sorting have been developed but droplet-based single-cell lysis and analysis have proven inefficient and yield high rates of false pairings. Ideally, molecular approaches for linking gene sequences from individual cells could be coupled with next-generation high-throughput sequencing to overcome these obstacles, but conventional approaches for linking gene sequences, such as by transfection with bridging oligonucleotides, result in activation of cellular nucleases that destroy the template, precluding this strategy. Recent advances in the synthesis and fabrication of modular deoxyribonucleic acid (DNA) origami nanostructures have resulted in new possibilities for addressing many current and long-standing scientific and technical challenges in biology and medicine. One exciting application of DNA nanotechnology is the intracellular capture, barcode linkage, and subsequent sequence analysis of multiple messenger RNA (mRNA) targets from individual cells within heterogeneous cell populations. DNA nanostructures can be transfected into individual cells to capture and protect mRNA for specific expressed genes, and incorporation of origami-specific bowtie-barcodes into the origami nanostructure facilitates pairing and analysis of mRNA from individual cells by high-throughput next-generation sequencing. This approach is highly modular and can be adapted to virtually any two (and possibly more) gene target sequences, and therefore has a wide range of potential applications for analysis of diverse cell populations such as understanding the relationship between different immune cell populations, development of novel immunotherapeutic antibodies, or improving the diagnosis or treatment for a wide variety of cancers.
ContributorsSchoettle, Louis (Author) / Blattman, Joseph N (Thesis advisor) / Yan, Hao (Committee member) / Chang, Yung (Committee member) / Lindsay, Stuart (Committee member) / Arizona State University (Publisher)
Created2017