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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
The communication of genetic material with biomolecules has been a major interest in cancer biology research for decades. Among its different levels of involvement, DNA is known to be a target of several antitumor agents. Additionally, tissue specific interaction between macromolecules such as proteins and structurally important regions of DNA has been reported to define the onset of certain types of cancers.
Illustrated in Chapter 1 is the general history of research on the interaction of DNA and anticancer drugs, most importantly different congener of bleomycin (BLM). Additionally, several synthetic analogues of bleomycin, including the structural components and functionalities, are discussed.
Chapter 2 describes a new approach to study the double-strand DNA lesion caused by antitumor drug bleomycin. The hairpin DNA library used in this study displays numerous cleavage sites demonstrating the versatility of bleomycin interaction with DNA. Interestingly, some of those cleavage sites suggest a novel mechanism of bleomycin interaction, which has not been reported before.
Cytidine methylation has generally been found to decrease site-specific cleavage of DNA by BLM, possibly due to structural change and subsequent reduced bleomycin-mediated recognition of DNA. As illustrated in Chapter 3, three hairpin DNAs known to be strongly bound by bleomycin, and their methylated counterparts, were used to study the dynamics of bleomycin-induced degradation of DNAs in cancer cells. Interestingly, cytidine methylation on one of the DNAs has also shown a major shift in the intensity of bleomycin induced double-strand DNA cleavage pattern, which is known to be a more potent form of bleomycin induced cleavages.
DNA secondary structures are known to play important roles in gene regulation. Chapter 4 demonstrates a structural change of the BCL2 promoter element as a result of its dynamic interaction with the individual domains of hnRNP LL, which is essential to facilitate the transcription of BCL2. Furthermore, an in vitro protein synthesis technique has been employed to study the dynamic interaction between protein domains and the i-motif DNA within the promoter element. Several constructs were made involving replacement of a single amino acid with a fluorescent analogue, and these were used to study FRET between domain 1 and the i-motif, the later of which harbored a fluorescent acceptor nucleotide analogue.
Illustrated in Chapter 1 is the general history of research on the interaction of DNA and anticancer drugs, most importantly different congener of bleomycin (BLM). Additionally, several synthetic analogues of bleomycin, including the structural components and functionalities, are discussed.
Chapter 2 describes a new approach to study the double-strand DNA lesion caused by antitumor drug bleomycin. The hairpin DNA library used in this study displays numerous cleavage sites demonstrating the versatility of bleomycin interaction with DNA. Interestingly, some of those cleavage sites suggest a novel mechanism of bleomycin interaction, which has not been reported before.
Cytidine methylation has generally been found to decrease site-specific cleavage of DNA by BLM, possibly due to structural change and subsequent reduced bleomycin-mediated recognition of DNA. As illustrated in Chapter 3, three hairpin DNAs known to be strongly bound by bleomycin, and their methylated counterparts, were used to study the dynamics of bleomycin-induced degradation of DNAs in cancer cells. Interestingly, cytidine methylation on one of the DNAs has also shown a major shift in the intensity of bleomycin induced double-strand DNA cleavage pattern, which is known to be a more potent form of bleomycin induced cleavages.
DNA secondary structures are known to play important roles in gene regulation. Chapter 4 demonstrates a structural change of the BCL2 promoter element as a result of its dynamic interaction with the individual domains of hnRNP LL, which is essential to facilitate the transcription of BCL2. Furthermore, an in vitro protein synthesis technique has been employed to study the dynamic interaction between protein domains and the i-motif DNA within the promoter element. Several constructs were made involving replacement of a single amino acid with a fluorescent analogue, and these were used to study FRET between domain 1 and the i-motif, the later of which harbored a fluorescent acceptor nucleotide analogue.
ContributorsRoy, Basab (Author) / Hecht, Sidney M. (Thesis advisor) / Jones, Anne (Committee member) / Levitus, Marcia (Committee member) / Chaput, John (Committee member) / Arizona State University (Publisher)
Created2014
Description
While DNA and protein nanotechnologies are promising avenues for nanotechnology on their own, merging the two could create more diverse and functional structures. In order to create hybrid structures, the protein will have to undergo site-specific modification, such as the incorporation of an unnatural amino, p-azidophenylalanine (AzF), via Shultz amber codon suppression method, which can then participate in click chemistry with modified DNA. These newly synthesized structures will then be able to self-assemble into higher order structures. Thus far, a surface exposed residue on the aldolase protein has been mutated into an amber stop codon. The next steps are to express the protein with the unnatural amino acid, allow it to participate in click chemistry, and visualize the hybrid structure. If the structure is correct, it will be able to self-assemble.
ContributorsAziz, Ann-Marie (Author) / Stephanopoulos, Nicholas (Thesis director) / Mills, Jeremy (Committee member) / School of Social Transformation (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
The two chapters of this thesis focus on different aspects of DNA and the properties of nucleic acids as the whole. Chapter 1 focuses on the structure of DNA and its relationship to enzymatic efficiency. Chapter 2 centers itself on threose nucleic acid and optimization of a step in the path to its synthesis. While Chapter 1 discusses DNA and Uracil-DNA Glycosylase with regards to the base excision repair pathway, Chapter 2 focuses on chemical synthesis of an intermediate in the pathway to the synthesis of TNA, an analogous structure with a different saccharide in the sugar-phosphate backbone.
Chapter 1 covers the research under Dr. Levitus. Four oligonucleotides were reacted for zero, five, and thirty minutes with uracil-DNA glycosylase and subsequent addition of piperidine. These oligonucleotides were chosen based on their torsional rigidities as predicted by past research and predictions. The objective was to better understand the relationship between the sequence of DNA surrounding the incorrect base and the enzyme’s ability to remove said base in order to prepare the DNA for the next step of the base excision repair pathway. The first pair of oligonucleotides showed no statistically significant difference in enzymatic efficiency with p values of 0.24 and 0.42, while the second pair had a p value of 0.01 at the five-minute reaction. The second pair is currently being researched at different reaction times to determine at what point the enzyme seems to equilibrate and react semi-equally with all sequences of DNA.
Chapter 2 covers the research conducted under Dr. Chaput. Along the TNA synthesis pathway, the nitrogenous base must be added to the threofuranose sugar. The objective was to optimize the original protocol of Vorbrüggen glycosylation and determine if there were better conditions for the synthesis of the preferred regioisomer. This research showed that toluene and ortho-xylene were more preferable as solvents than the original anhydrous acetonitrile, as the amount of preferred isomer product far outweighed the amount of side product formed, as well as improving total yield overall. The anhydrous acetonitrile reaction had a final yield of 60.61% while the ortho-xylene system had a final yield of 94.66%, an increase of approximately 32%. The crude ratio of preferred isomer to side product was also improved, as it went from 18% undesired in anhydrous acetonitrile to 4% undesired in ortho-xylene, both values normalized to the preferred regioisomer.
Chapter 1 covers the research under Dr. Levitus. Four oligonucleotides were reacted for zero, five, and thirty minutes with uracil-DNA glycosylase and subsequent addition of piperidine. These oligonucleotides were chosen based on their torsional rigidities as predicted by past research and predictions. The objective was to better understand the relationship between the sequence of DNA surrounding the incorrect base and the enzyme’s ability to remove said base in order to prepare the DNA for the next step of the base excision repair pathway. The first pair of oligonucleotides showed no statistically significant difference in enzymatic efficiency with p values of 0.24 and 0.42, while the second pair had a p value of 0.01 at the five-minute reaction. The second pair is currently being researched at different reaction times to determine at what point the enzyme seems to equilibrate and react semi-equally with all sequences of DNA.
Chapter 2 covers the research conducted under Dr. Chaput. Along the TNA synthesis pathway, the nitrogenous base must be added to the threofuranose sugar. The objective was to optimize the original protocol of Vorbrüggen glycosylation and determine if there were better conditions for the synthesis of the preferred regioisomer. This research showed that toluene and ortho-xylene were more preferable as solvents than the original anhydrous acetonitrile, as the amount of preferred isomer product far outweighed the amount of side product formed, as well as improving total yield overall. The anhydrous acetonitrile reaction had a final yield of 60.61% while the ortho-xylene system had a final yield of 94.66%, an increase of approximately 32%. The crude ratio of preferred isomer to side product was also improved, as it went from 18% undesired in anhydrous acetonitrile to 4% undesired in ortho-xylene, both values normalized to the preferred regioisomer.
ContributorsTamirisa, Ritika Sai (Author) / Levitus, Marcia (Thesis director) / Stephanopoulos, Nicholas (Committee member) / Windman, Todd (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
The honors thesis presented in this document describes an extension to an electrical engineering capstone project whose scope is to develop the receiver electronics for an RF interrogator. The RF interrogator functions by detecting the change in resonant frequency of (i.e, frequency of maximum backscatter from) a target resulting from an environmental input. The general idea of this honors project was to design three frequency selective surfaces that would act as surrogate backscattering or reflecting targets that each contains a distinct frequency response. Using 3-D electromagnetic simulation software, three surrogate targets exhibiting bandpass frequency responses at distinct frequencies were designed and presented in this thesis.
ContributorsSisk, Ryan Derek (Author) / Aberle, James (Thesis director) / Chakraborty, Partha (Committee member) / Electrical Engineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
The natural healing process for bone has multiple signaling cascades where several soluble factors are expressed at specific times to encourage regeneration. Human mesenchymal stromal cells (hMSCs) have three stages of osteogenic differentiation: an increase in cell number (day 1-4), early cell differentiation showing alkaline phosphatase (ALP) expression (day 5-14), and deposition of calcium and phosphate (day 14-28). The first two stages are of particular interest since cell adhesion peptides have been shown to have biological significance during these early stages of bone regeneration. However, far less is known about the temporal dependence of these signals. To mimic these complex systems, developing dynamic biomaterials has become a popular research area over the past decade. Advances in chemistry, materials science, and manufacturing have enabled the development of complex biomaterials that can mimic dynamic cues in the extracellular matrix. One specific area of interest is spatiotemporal control of multiple biomolecules; however, this has generally required diverse chemical approaches making the process difficult and impractical. To circumvent these issues, I developed a novel method that combines a photoresponsive hydrogel with single-stranded DNA to spatiotemporally control multiple biomolecules using a single conjugation scheme.
Here, I describe a detailed protocol to manufacture a fully reversible, spatiotemporal platform using DNA handles. Norbornene-modified hyaluronic acid hydrogels were used to spatially control biomolecule presentation while single-stranded DNA was used to temporally control biomolecule presentation via toehold-mediated strand displacement. This platform was used to orthogonally control the presentation of multiple biomolecules with simple and complex spatial patterning, as well as control the cell morphology of hMSCs by tuning the presentation of the cell adhesion peptide RGDS.
Then, this system was applied to study the temporal presentation of cell adhesion peptides and their effect on early osteogenic differentiation of hMSCs in vitro. The peptides used were RGDS, HAVDI, and OGP. OGP alone expressed higher ALP when presented from day 7-14 than day 0-7 or 0-14. When RGDS, HAVDI, and OGP were combined, there was an increase in ALP activity when HAVDI was presented from day 0-3 indicating that HAVDI plays an important role at earlier time points during osteogenic differentiation.
ContributorsFumasi, Fallon Marie (Author) / Holloway, Julianne L (Thesis advisor) / Stephanopoulos, Nicholas (Committee member) / Green, Matthew D (Committee member) / Stabenfeldt, Sarah E (Committee member) / Acharya, Abhinav (Committee member) / Arizona State University (Publisher)
Created2021
Description
Since the inception of DNA nanotechnology, DNA has found itself poised as one of the most robust self-assembling building blocks due to its well understood double helix structure formed by two anti-parallel strands of DNA held together by hydrogen bond from nucleobases which also provides the material programmability due to the well-understood Watson Crick base pairing rules. These capabilities have led to the exponential increase in publications showing off intricate and remarkable designs alongside ever-expanding applications. However, as the field expands there is an apparent lack of chemical diversity and functionality. To combat this my research focused on creating hybrid peptide oligonucleotide conjugates (POC) where the conjugated peptide could add chemical and structural diversity using the 20 canonical amino acids and various peptide secondary structures. In this work, I conjugate DNA to the self-assembling peptide building block the coiled coil. The coiled coil motif is formed from the self-assembly of two or more α-helical peptides and, like DNA, the coiled coil has well understood programmability. Together as a conjugate, the DNA and coiled coil, create a new self-assembling building block capable of two orthogonal self-assembling modes that can work in tandem. In this work, I used DNA coiled coil conjugates to show the capability to create first of their kind hybrid DNA/coiled coil one-dimensional fibers (chapter 2), integrate proteins (chapter 3), and to create hybrid cage structures (chapter 4). Finally, a POC hydrogel is created using the polypeptide gelatin with DNA crosslinks to create a reversible stiffening gel using toe-hold mediated strand displacement (chapter 5).
ContributorsBuchberger, Alex Richard (Author) / Stephanopoulos, Nicholas (Thesis advisor) / Mills, Jeremy (Committee member) / Van Horn, Wade (Committee member) / Arizona State University (Publisher)
Created2021
Description
The ability of magnetic resonance imaging (MRI) to image any part of the human body without the effects of harmful radiation such as in CAT and PET scans established MRI as a clinical mainstay for a variety of different ailments and maladies. Short wavelengths accompany the high frequencies present in high-field MRI, and are on the same scale as the human body at a static magnetic field strength of 3 T (128 MHz). As a result of these shorter wavelengths, standing wave effects are produced in the MR bore where the patient is located. These standing waves generate bright and dark spots in the resulting MR image, which correspond to irregular regions of high and low clarity. Coil loading is also an inevitable byproduct of subject positioning inside the bore, which decreases the signal that the region of interest (ROI) receives for the same input power. Several remedies have been proposed in the literature to remedy the standing wave effect, including the placement of high permittivity dielectric pads (HPDPs) near the ROI. Despite the success of HPDPs at smoothing out image brightness, these pads are traditionally bulky and take up a large spatial volume inside the already small MR bore. In recent years, artificial periodic structures known as metamaterials have been designed to exhibit specific electromagnetic effects when placed inside the bore. Although typically thinner than HPDPs, many metamaterials in the literature are rigid and cannot conform to the shape of the patient, and some are still too bulky for practical use in clinical settings. The well-known antenna engineering concept of fractalization, or the introduction of self-similar patterns, may be introduced to the metamaterial to display a specific resonance curve as well as increase the metamaterial’s intrinsic capacitance. Proposed in this paper is a flexible fractal-inspired metamaterial for application in 3 T MR head imaging. To demonstrate the advantages of this flexibility, two different metamaterial configurations are compared to determine which produces a higher localized signal-to-noise ratio (SNR) and average signal measured in the image: in the first configuration, the metamaterial is kept rigid underneath a human head phantom to represent metamaterials in the literature (single-sided placement); and in the second, the metamaterial is wrapped around the phantom to utilize its flexibility (double-sided placement). The double-sided metamaterial setup was found to produce an increase in normalized SNR of over 5% increase in five of six chosen ROIs when compared to no metamaterial use and showed a 10.14% increase in the total average signal compared to the single-sided configuration.
ContributorsSokol, Samantha (Author) / Sohn, Sung-Min (Thesis director) / Allee, David (Committee member) / Jones, Anne (Committee member) / Barrett, The Honors College (Contributor) / Electrical Engineering Program (Contributor)
Created2022-05