Matching Items (7)
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- All Subjects: DNA
- Creators: Ghirlanda, Giovanna
- Member of: Theses and Dissertations
- Status: Published
Description
Telomerase is a unique reverse transcriptase that has evolved specifically to extend the single stranded DNA at the 3' ends of chromosomes. To achieve this, telomerase uses a small section of its integral RNA subunit (TR) to reiteratively copy a short, canonically 6-nt, sequence repeatedly in a processive manner using a complex and currently poorly understood mechanism of template translocation to stop nucleotide addition, regenerate its template, and then synthesize a new repeat. In this study, several novel interactions between the telomerase protein and RNA components along with the DNA substrate are identified and characterized which come together to allow active telomerase repeat addition. First, this study shows that the sequence of the RNA/DNA duplex holds a unique, single nucleotide signal which pauses DNA synthesis at the end of the canonical template sequence. Further characterization of this sequence dependent pause signal reveals that the template sequence alone can produce telomerase products with the characteristic 6-nt pattern, but also works cooperatively with another RNA structural element for proper template boundary definition. Finally, mutational analysis is used on several regions of the protein and RNA components of telomerase to identify crucial determinates of telomerase assembly and processive repeat synthesis. Together, these results shed new light on how telomerase coordinates its complex catalytic cycle.
ContributorsBrown, Andrew F (Author) / Chen, Julian J. L. (Thesis advisor) / Jones, Anne (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2014
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
Natural products that target the DNA of cancer cells have been an important source of knowledge and understanding in the development of anticancer chemotherapeutic agents. Bleomycin (BLM) exemplifies this class of DNA damaging agent. The ability of BLM to chelate metal ions and effect oxidative damage of the deoxyribose sugar moiety of DNA has been studied extensively for four decades. Here, the study of BLM A5 was conducted using a previously isolated library of hairpin DNAs found to bind strongly to metal free BLM. The ability of BLM to effect single-stranded was then extensively characterized on both the 3′ and 5′-arms of the hairpin DNAs. The strongly bound DNAs were found to be efficient substrates for Fe·BLM A5-mediated cleavage. Surprisingly, the most prevalent site of damage by BLM was found to be a 5′-AT-3′ dinucleotide sequence. This dinucleotide sequence and others generally not cleaved by BLM when examined using arbitrarily chosen DNA substrate were found in examining the library of ten hairpin DNAs. In total, 111 sites of DNA damage were found to be produced by exposure of the hairpin DNA library to Fe·BLM A5. Also, an assay was developed with which to test the propensity of the hairpin DNAs to undergo double stranded DNA damage. Adapting methods previously described by the Povirk laboratory, one hairpin was characterized using this method. The results were in accordance with those previously reported.
ContributorsSegerman, Zachary (Author) / Hecht, Sidney M. (Thesis advisor) / Levitus, Marcia (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2011
Description
A major goal of synthetic biology is to recapitulate emergent properties of life. Despite a significant body of work, a longstanding question that remains to be answered is how such a complex system arose? In this dissertation, synthetic nucleic acid molecules with alternative sugar-phosphate backbones were investigated as potential ancestors of DNA and RNA. Threose nucleic acid (TNA) is capable of forming stable helical structures with complementary strands of itself and RNA. This provides a plausible mechanism for genetic information transfer between TNA and RNA. Therefore TNA has been proposed as a potential RNA progenitor. Using molecular evolution, functional sequences were isolated from a pool of random TNA molecules. This implicates a possible chemical framework capable of crosstalk between TNA and RNA. Further, this shows that heredity and evolution are not limited to the natural genetic system based on ribofuranosyl nucleic acids. Another alternative genetic system, glycerol nucleic acid (GNA) undergoes intrasystem pairing with superior thermalstability compared to that of DNA. Inspired by this property, I demonstrated a minimal nanostructure composed of both left- and right-handed mirro image GNA. This work suggested that GNA could be useful as promising orthogonal material in structural DNA nanotechnology.
ContributorsZhang, Su (Author) / Chaut, John C (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2011
Description
In eukaryotes, DNA is packed in a highly condensed and hierarchically organized structure called chromatin, in which DNA tightly wraps around the histone octamer consisting of one histone 3-histone 4 (H3-H4) tetramer and two histone 2A- histone 2B (H2A-H2B) dimers with 147 base pairs in an almost two left handed turns. Almost all DNA dependent cellular processes, such as DNA duplication, transcription, DNA repair and recombination, take place in the chromatin form. Based on the critical importance of appropriate chromatin condensation, this thesis focused on the folding behavior of the nucleosome array reconstituted using different templates with various controllable factors such as histone tail modification, linker DNA length, and DNA binding proteins. Firstly, the folding behaviors of wild type (WT) and nucleosome arrays reconstituted with acetylation on the histone H4 at lysine 16 (H4K16 (Ac)) were studied. In contrast to the sedimentation result, atomic force microscopy (AFM) measurements revealed no apparent difference in the compact nucleosome arrays between WT and H4K16 (Ac) and WT. Instead, an optimal loading of nucleosome along the template was found necessary for the Mg2+ induced nucleosome array compaction. This finding leads to the further study on the role of linker DNA in the nucleosome compaction. A method of constructing DNA templates with varied linker DNA lengths was developed, and uniformly and randomly spaced nucleosome arrays with average linker DNA lengths of 30 bp and 60 bp were constructed. After comprehensive analyses of the nucleosome arrays' structure in mica surface, the lengths of the linker DNA were found playing an important role in controlling the structural geometries of nucleosome arrays in both their extended and compact forms. In addition, higher concentration of the DNA binding domain of the telomere repeat factor 2 (TRF2) was found to stimulate the compaction of the telomeric nucleosome array. Finally, AFM was successfully applied to investigate the nucleosome positioning behaviors on the Mouse Mammary Tumor Virus (MMTV) promoter region, and two highly positioned region corresponded to nucleosome A and B were identified by this method.
ContributorsFu, Qiang (Author) / Lindsay, Stuart M (Thesis advisor) / Yan, Hao (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2010
Description
Due to its difficult nature, organic chemistry is receiving much research attention across the nation to develop more efficient and effective means to teach it. As part of that, Dr. Ian Gould at ASU is developing an online organic chemistry educational website that provides help to students, adapts to their responses, and collects data about their performance. This thesis creative project addresses the design and implementation of an input parser for organic chemistry reagent questions, to appear on his website. After students used the form to submit questions throughout the Spring 2013 semester in Dr. Gould's organic chemistry class, the data gathered from their usage was analyzed, and feedback was collected. The feedback obtained from students was positive, and suggested that the input parser accomplished the educational goals that it sought to meet.
ContributorsBeerman, Eric Christopher (Author) / Gould, Ian (Thesis director) / Wilkerson, Kelly (Committee member) / Mosca, Vince (Committee member) / Barrett, The Honors College (Contributor) / Computer Science and Engineering Program (Contributor)
Created2013-05
Description
Non-canonical amino acids (NCAAs) can be used in protein chemistry to determine their structures. A common method for imaging proteins is cryo-electron microscopy (cryo-EM) which is ideal for imaging proteins that cannot be obtained in large quantities. Proteins with indistinguishable features are difficult to image using this method due to the large size requirements, therefore antibodies designed specifically for binding these proteins have been utilized to better identify the proteins. By using an existing antibody that binds to stilbene, NCAAs containing this molecule can be used as a linker between proteins and an antibody. Stilbene containing amino acids can be integrated into proteins to make this process more access able. In this paper, synthesis methods for various NCAAs containing stilbene were proposed. The resulting successfully synthesized NCAAs were E)-N6-(5-oxo-5-((4-styrylphenyl) amino) pentanoyl) lysine, (R,E)-2-amino-3-(5-oxo-5-((4-styrylphenyl)amino)pentanamido)propanoic acid, (E)-2-amino-5-(5-oxo-5-((4-styrylphenyl) amino) pentanamido) pentanoic acid. A synthesis for three more shorter amino acids, (R,E)-2-amino-3-(3-oxo-3-((4-styrylphenyl) amino) propanamido) propanoic acid, (E)-2-amino-5-(3-oxo-3-((4-styrylphenyl) amino) propanamido) pentanoic acid, and (E)-N6-(3-oxo-3-((4-styrylphenyl) amino) propanoyl) lysine, is also proposed.
ContributorsJenkins, Bryll (Author) / Mills, Jeremy (Thesis director) / Ghirlanda, Giovanna (Committee member) / Nannenga, Brent (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor)
Created2022-05