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- Creators: Barrett, The Honors College
- Creators: Chang, Yung
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
Description
Efforts to quantify the diversity of the T cell repertoire have generally been unsuccessful because not all factors accounting for diversity have been considered. In order to get an accurate representation of the T cell repertoire, one must incorporate analysis of germline gene diversity, diversity from somatic recombination, joining diversity from N- and P- nucleotides, and TCR chain pairing diversity. Because of advances in high-throughput sequencing techniques, estimates have been able to account for diversity from TCR genes. However the ability to account for chain pairing diversity has been more difficult. In order to do so, single cell sorting techniques must be employed. These techniques, though effective, are time consuming and expensive. For this reason, no large-scale analyses have been done on the immune repertoires using these techniques. In this study, we propose a novel method for linking the two TCR chain sequences from an individual cell. DNA origami nanostructure technology is employed to capture and bind the TCRγ and TCRδ chain mRNA inside individual cells using probe strands complementary to the C-region of those sequences. We then use a dual-primer RT and ligation molecular strategy to link the two sequences together. The result is a single amplicon containing the CDR3 region of the TCRγ and TCRδ. This amplicon can then be easily PCR amplified using sequence specific primers, and sequenced. DNA origami nanostructures offer a rapid, cost-effective method alternative to conventional single cell sorting techniques, as both TCR mRNA can be captured on one origami molecule inside a single cell. At present, this study outlines a proof-of-principle analysis of the method to determine its functionality. Using known TCRγ and TCRδ sequences, the DNA origami and RT/PCR method was tested and resulting sequence data proved the effectiveness of the method. The original TCRγ and TCRδ sequences were linked together as a single amplicon containing both CDR3 regions of the genes. Thus, this method can be employed in further research to elucidate the γδ T cell repertoire. This technology is also easily adapted to any gene target or cell type and therefore presents a large opportunity to be used in other immune repertoire analysis and other immunological studies (such as the rapid identification and subsequent production of antibodies).
ContributorsPoindexter, Morgan Elizabeth (Author) / Blattman, Joseph (Thesis director) / Yan, Hao (Committee member) / Schoettle, Louis (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Using DNA nanotechnology a library of structures of various geometries have been built; these structures are modified chemically and/or enzymatically at nanometer precisions. With DNA being chemically very stable, these structures can be functionalized through an abundance of well-established protocols. Additionally, they can be used for various biological and medicinal purposes, such as drug delivery. For in vivo applications, the DNA nanostructures must have a long circulation life in the bloodstream; otherwise, they could be easily excreted shortly after entry. One way of making these nanostructures long lasting in the blood is to cover them with the biocompatible polymer, polyethylene glycol (PEG). Adding DNA to PEG before forming structures has been found to interfere in the hybridization of the DNA in the structure, resulting in formation of deformed structures. In this study we have developed a new methodology based on "click chemistry" (CC) to modify the surface of DNA nanostructures with PEG after they are formed. These structures can then be used for in vivo studies and potential applications in the future.
ContributorsSmith, Eric Lynn (Author) / Yan, Hao (Thesis director) / Liu, Yan (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
Description
The properties of adjuvants to stimulate an immune response to treat cancer has sparked a major area of research in the field of immunotherapy. Given the presence of multiple RNA sensors in mammalian host cells for eliciting innate immunity, synthetic RNA nanostructures present a unique opportunity for adjuvant exploration. While RNA nanostructures are organic and biocompatible in nature than other adjuvants, they could be tailored to have desired structural stability and functional diversity for in vivo application. In this study, a rectangular RNA origami nanostructure was designed to contain double-stranded RNA motifs and possess high structural stability. Using in vitro assays, RNA origami was shown to stimulate the toll-like receptor 3 (TLR3) signaling pathway, which has been reported to activate antigen presenting cells (APCs), natural killer (NK) cells, cluster of differentiation 8 (CD8) T-cells, and the secretion of proinflammatory cytokines. To explore RNA origami as an adjuvant for cancer immunotherapy, intraperitoneal administration of a murine colon cancer cell line (CT26) was used as a model system to mimic peritoneal metastasis (PM), in which RNA origami was investigated for its activities in mitigating PM tumor microenvironment and improving anti-tumor immunity. Given the poor outcome of the patients with PM and urgent need for new interventions, this study aims to translate the adjuvant activities of RNA origami demonstrated in vitro into potent anti-cancer immunotherapeutics. Here, it was shown that multiple intraperitoneal injections of RNA origami could inhibit tumor growth, leading to a significant delay and/or regression of metastatic tumor growth in the peritoneum. Furthermore, tumor-free mice, after being treated with RNA origami, were also resistant to a second challenge of tumor cells, indicating the development of the adaptive anti-tumor immunity. This immunity is dependent on T-cells since nude mice succumbed to tumor growth with or without RNA origami treatment. Thus, RNA-origami can function as an adjuvant to activate the innate immunity and subsequently the adaptive anti-tumor immunity, leading to tumor regression. Conceivably, RNA origami could be explored as an immunotherapeutic agent to improve the disease outcome of patients with peritoneal metastasis and peritoneal carcinogenesis.
ContributorsRodriguez del Villar, Ryan Luis (Author) / Chang, Yung (Thesis advisor) / Liu, Xiaowei (Committee member) / Qi, Xiaodong (Committee member) / Arizona State University (Publisher)
Created2018