Matching Items (21)
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- All Subjects: Biochemistry
- Creators: Stephanopoulos, Nicholas
Description
Extensive efforts have been made to develop efficient and low-cost methods for diagnostics to identify molecular biomarkers that are linked to a wide array of conditions, including cancer. A highly developed method includes utilizing the gene-editing enzyme CRISPR-Cas12a (Cpf1), which demonstrates double-stranded DNase activity with RuvC catalytic domain with high sensitivity and specificity. This DNase activity is RNA-guided and requires a T-rich PAM site on the target sequence for functional cleavage. There have been recent efforts to utilize this DNase activity of Cas12a by combining it with isothermal amplification and analysis by lateral strip tests. This project examined CRISPR-based early detection of microRNA biomarkers. MicroRNA are short RNA molecules that have large roles in post-transcriptional gene regulation. However, due the short length of microRNA and its single-stranded nature, it is challenging to use Cas12a for microRNA detection using existing methods. Thus, this project investigated the potential of two microRNA detection strategies for recognition by CRISPR-Cas12a. These methods were microRNA-splinted ligation with polymerase chain reaction (PCR) and MicroRNA-specific reverse transcriptase PCR (RT-PCR). Gel imaging demonstrated effective amplification of ligated DNA through microRNA-splinted ligation with PCR/RPA. In addition, lateral strips tests showed effective cleavage of the target sequences by Cas12a. However, RT-PCR method demonstrated low amplification by PCR and inefficient poly(A) elongation. This project paves the way for the detection of an extensive range of microRNA biomarkers that are linked to an array of diseases. Future directions include analysis and modifications of RT-PCR method to improve experimental results, extending these detection methods to a larger range of microRNA sequences, and eventually utilizing them for detection in human samples.
ContributorsStaren, Michael Steven (Author) / Green, Alexander (Thesis director) / Stephanopoulos, Nicholas (Committee member) / Diehnelt, Chris (Committee member) / School of Life Sciences (Contributor) / College of Health Solutions (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
The main objective of this project is to create a hydrogel based material system to capture and release CCRF-CEM Leukemia cancer cells via chemo-mechanical modulation. This system is composed of an aptamer-functionalized hydrogel thin film at the bottom of a microfluidic channel, which changes its film thickness as the temperature of the fluid in the system changes. The functionalized hydrogel film has been created as the primary steps to creating the microfluidic device that could capture and release leukemia cells by turning the temperature of the fluid and length of exposure. Circulating tumor cells have recently become a highly studied area since they have become associated with the likelihood of patient survival. Further, circulating tumor cells can be used to determine changes in the genome of the cancer leading to targeted treatment. First, the aptamers were attached onto the hydrogel through an EDC/NHS reaction. The aptamers were verified to be attached onto the hydrogel through FTIR spectroscopy. The cell capture experiments were completed by exposing the hydrogel to a solution of leukemia cells for 10 minutes at room temperature. The cell release experiments were completed by exposing the hydrogel to a 40°C solution. Several capture and release experiments were completed to measure how many cells could be captured, how quickly, and how many cells captured were released. The aptamers were chemically attached to the hydrogel. 300 cells per square millimeter could be captured at a time in a 10 minute time period and released in a 5 minute period. Of the cells captured, 96% of them were alive once caught. 99% of cells caught were released once exposed to elevated temperature. The project opens the possibility to quickly and efficiently capture and release tumor cells using only changes in temperature. Further, most of the cells that were captured were alive and nearly all of those were released leading to high survival and capture efficiency.
ContributorsPaxton, Rebecca Joanne (Author) / Stephanopoulos, Nicholas (Thesis director) / He, Ximin (Committee member) / Gould, Ian (Committee member) / Materials Science and Engineering Program (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Identifying disease biomarkers may aid in the early detection of breast cancer and improve patient outcomes. Recent evidence suggests that tumors are immunogenic and therefore patients may launch an autoantibody response to tumor associated antigens. Single-chain variable fragments of autoantibodies derived from regional lymph node B cells of breast cancer patients were used to discover these tumor associated biomarkers on protein microarrays. Six candidate biomarkers were discovered from 22 heavy chain-only variable region antibody fragments screened. Validation tests are necessary to confirm the tumorgenicity of these antigens. However, the use of single-chain variable autoantibody fragments presents a novel platform for diagnostics and cancer therapeutics.
ContributorsSharman, M. Camila (Author) / Magee, Dewey (Mitch) (Thesis director) / Wallstrom, Garrick (Committee member) / Petritis, Brianne (Committee member) / Barrett, The Honors College (Contributor) / College of Liberal Arts and Sciences (Contributor) / Virginia G. Piper Center for Personalized Diagnostics (Contributor) / Biodesign Institute (Contributor)
Created2012-12
Description
Time spent alone is a topic that has been studied in great detail, particularly the manner in which it is spent and the effect it has during the adolescent stage of life. Similarly, stress levels in adolescents have always been a topic of interest because of the effects they could have on the individual later in adulthood. Oddly enough however, the two areas of study have never been looked at in relation to one another. This study will look at different types of alone time as possible stressors in a community sample (N=82) of adolescents transitioning to college. The data on time alone and stress levels was collected through diary reports over a period of 3 days. The analysis only yielded significant effects for females and only for specific categories. It was found that females experience the lowest amount of perceived stress when they are alone and want to be alone, they have more negative affect when their desired environment differs from their current situation, and more positive affect in both the alone incongruence and not alone congruence situations. These results indicate that only women experience stress and affect changes when they encounter different congruent and incongruent environments.
ContributorsVanderwerf, Jennifer (Author) / Doane, Leah (Thesis director) / Knight, George (Committee member) / Arbona, P. Anita (Committee member) / Barrett, The Honors College (Contributor) / College of Liberal Arts and Sciences (Contributor)
Created2012-12
Description
The purpose of my honors thesis project was to generate the tools needed for in vivo imaging by determining the optimal plasmid-fluorophore combination. To determine the optimal plasmid and fluorophore, asd plasmids were constructed with various promoters, origins of replications, and red fluorophores. The optimal asd plasmid for fluorescent in vivo imaging was determined by the plasmid stability, growth rate, and growth phase dependence on fluorescent intensity. The end goal is to be able to use the asd plasmid in vaccine strains for the purpose of in vivo imaging of the recombinant attenuated Salmonella vaccine (RASV).
ContributorsEudy, L. Adam (Author) / Curtiss, Roy (Thesis director) / Roland, Kenneth (Committee member) / Forbes, Stephen (Committee member) / Barrett, The Honors College (Contributor) / College of Liberal Arts and Sciences (Contributor)
Created2012-12
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
DNA nanotechnology is ideally suited for numerous applications from the crystallization and solution of macromolecular structures to the targeted delivery of therapeutic molecules. The foundational goal of structural DNA nanotechnology was the development of a lattice to host proteins for crystal structure solution. To further progress towards this goal, 36 unique four-armed DNA junctions were designed and crystallized for eventual solution of their 3D structures. While most of these junctions produced macroscale crystals which diffracted successfully, several prevented crystallization. Previous results used a fixed isomer and subsequent investigations adopted an alternate isomer to investigate the impact of these small sequence changes on the stability and structural properties of these crystals. DNA nanotechnology has also shown promise for a variety biomedical applications. In particular, DNA origami has been demonstrated as a promising tool for targeted and efficient delivery of drugs and vaccines due to their programmability and addressability to suit a variety of therapeutic cargo and biological functions. To this end, a previously designed DNA barrel nanostructure with a unique multimerizable pegboard architecture has been constructed and characterized via TEM for later evaluation of its stability under biological conditions for use in the targeted delivery of cargo, including CRISPR-containing adeno-associated viruses (AAVs) and mRNA.
ContributorsHostal, Anna Elizabeth (Author) / Anderson, Karen (Thesis director) / Stephanopoulos, Nicholas (Committee member) / Yan, Hao (Committee member) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / School of International Letters and Cultures (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
Structural-based drug discovery is becoming the essential tool for drug development withlower cost and higher efficiency compared to the conventional method. Knowledge of the
three-dimensional structure of protein targets has the potential to accelerate the process
for screening drug candidates. X-ray crystallography has proven to be the most used and
indispensable technology in structural-based drug discovery. The provided
comprehensive structural information about the interaction between the disease-related
protein target and ligand can guide the chemical modification on the ligand to improve
potency and selectivity. X-ray crystallography has been upgraded from traditional
synchrotron to the third generation, which enabled the surge of the structural
determination of macromolecular. The introduction of X-ray free electron laser further
alleviated the uncertain and time-consuming crystal size optimization process and
extenuated the radiation damage by “diffraction before destruction”. EV-D68 2A
protease was proved to be an important pharmaceutical target for acute flaccid myelitis.
This thesis reports the first atomic structure of the EV-D68 2A protease and the structuresof its two mutants, revealing it adopting N-terminal four-stranded sheets and C-terminal
six-stranded ß-barrels structure, with a tightly bound zinc atom. These structures will
guide the chemical modification on its inhibitor, Telaprevir. Integrin ⍺Mβ2 is an integrin
with the α I-domain, related to many immunological functions including cell
extravasation, phagocytosis, and immune synapse formation, so studying the molecular
ligand-binding mechanism and activation mechanism of ⍺Mβ2 is of importance. This
thesis uncovers the preliminary crystallization condition of ⍺Mβ2-I domain in complex
with its ligand Pleiotrophin and the initial structural model. The structural model shows consistency with the previous hypothesis that the primary binding sites are metal iondependent
adhesion sites on ⍺Mβ2-I domain and the thrombospondin type-1 repeat (TSR)
domains of Pleiotrophin. Drug molecules with high potency and selectivity can be
designed based on the reported structures of the EV-D68 2A protease and ⍺Mβ2-I domain
in the future.
ContributorsLiu, Chang (Author) / Liu, Wei (Thesis advisor) / Stephanopoulos, Nicholas (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2021
Description
Transient protein-protein and protein-molecule interactions fluctuate between associated and dissociated states. They are widespread in nature and mediate most biological processes. These interactions are complex and are strongly influenced by factors such as concentration, structure, and environment. Understanding and utilizing these types of interactions is useful from both a fundamental and design perspective. In this dissertation, transient protein interactions are used as the sensing element of a biosensor for small molecule detection. This is done by using a transcription factor-small molecule pair that mediates the activation of a CRISPR/Cas12a complex. Activation of the Cas12a enzyme results in an amplified readout mechanism that is either fluorescence or paper based. This biosensor can successfully detect 9 different small molecules including antibiotics with a tuneable detection limit ranging from low µM to low nM. By combining protein and nucleic acid-based systems, this biosensor has the potential to report on almost any protein-molecule interaction, linking this to the intrinsic amplification that is possible when working with nucleic acid-based technologies. The second part of this dissertation focuses on understanding protein-molecule interactions at a more fundamental level, and, in so doing, exploring design rules required to generalize sensors like the ones described above. This is done by training a neural network algorithm with binding data from high density peptide micro arrays incubated with specific protein targets. Because the peptide sequences were chosen simply to evenly, though sparsely, represent all sequence space, the resulting network provides a comprehensive sequence/binding relationship for a given target protein. While past work had shown that this works well on the arrays, here I have explored how well the neural networks thus trained, predict sequence-dependent binding in the context of protein-protein and peptide-protein interactions. Amino acid sequences, either free in solution or embedded in protein structure, will display somewhat different binding properties than sequences affixed to the surface of a high-density array. However, the neural network trained on array sequences was able to both identify binding regions in between proteins and predict surface plasmon resonance-based binding propensities for peptides with statistically significant levels of accuracy.
ContributorsSwingle, Kirstie Lynn (Author) / Woodbury, Neal W (Thesis advisor) / Green, Alexander A (Thesis advisor) / Stephanopoulos, Nicholas (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2022
Description
Molecular engineering is an emerging field that aims to create functional devices for modular purposes, particularly bottom-up design of nano-assemblies using mechanical and chemical methods to perform complex tasks. In this study, we present a novel method for constructing an RNA clamp using circularized RNA and a broccoli aptamer for fluorescence sensing. By designing a circular RNA with the broccoli aptamer and a complementary DNA strand, we created a molecular clamp that can stabilize the aptamer. The broccoli aptamer displays enhanced fluorescence when bound to its ligand, DFHBI-1T. Upon induction with this small molecule, the clamp can exhibit or destroy fluorescence. We demonstrated that we could control the fluorescence of the RNA clamp by introducing different complementary DNA strands, which regulate the level of fluorescence. Additionally, we designed allosteric control by introducing new DNA strands, making the system reversible. We explored the use of mechanical tension to regulate RNA function by attaching a spring-like activity through the RNA clamp to two points on the RNA surface. By adjusting the stiffness of the spring, we could control the tension between the two points and induce reversible conformational changes, effectively turning RNA function on and off. Our approach offers a simple and versatile method for creating RNA clamps with various applications, including RNA detection, regulation, and future nanodevice design. Our findings highlight the crucial role of mechanical forces in regulating RNA function, paving the way for developing new strategies for RNA manipulation, and potentially advancing molecular engineering. Although the current work is ongoing, we provide current progress of both theoretical and experimental calculations based on our findings.
ContributorsJoseph, Joel (Author) / Yan, Hao (Thesis director) / Stephanopoulos, Nicholas (Committee member) / Lapinaite, Audrone (Committee member) / Barrett, The Honors College (Contributor) / Historical, Philosophical & Religious Studies, Sch (Contributor) / School of Molecular Sciences (Contributor)
Created2023-05