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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

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.
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

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

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
Description
Nucleic acid nanotechnology is a field of nanoscale engineering where the sequences of deoxyribonucleicacid (DNA) and ribonucleic acid (RNA) molecules are carefully designed to create self–assembled nanostructures with higher spatial resolution than is available to top–down fabrication methods. In the 40 year history of the field, the structures created have scaled

Nucleic acid nanotechnology is a field of nanoscale engineering where the sequences of deoxyribonucleicacid (DNA) and ribonucleic acid (RNA) molecules are carefully designed to create self–assembled nanostructures with higher spatial resolution than is available to top–down fabrication methods. In the 40 year history of the field, the structures created have scaled from small tile–like structures constructed from a few hundred individual nucleotides to micron–scale structures assembled from millions of nucleotides using the technique of “DNA origami”. One of the key drivers of advancement in any modern engineering field is the parallel development of software which facilitates the design of components and performs in silico simulation of the target structure to determine its structural properties, dynamic behavior, and identify defects. For nucleic acid nanotechnology, the design software CaDNAno and simulation software oxDNA are the most popular choices for design and simulation, respectively. In this dissertation I will present my work on the oxDNA software ecosystem, including an analysis toolkit, a web–based graphical interface, and a new molecular visualization tool which doubles as a free–form design editor that covers some of the weaknesses of CaDNAno’s lattice–based design paradigm. Finally, as a demonstration of the utility of these new tools I show oxDNA simulation and subsequent analysis of a nanoscale leaf–spring engine capable of converting chemical energy into dynamic motion. OxDNA simulations were used to investigate the effects of design choices on the behavior of the system and rationalize experimental results.
ContributorsPoppleton, Erik (Author) / Sulc, Petr (Thesis advisor) / Yan, Hao (Committee member) / Forrest, Stephanie (Committee member) / Stephanopoulos, Nicholas (Committee member) / Arizona State University (Publisher)
Created2022
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G protein-coupled receptors (GPCRs) are a large family of proteins involved in the cell signaling and regulation of many biological and pathological processes in the human body. To fully understand their functions, various approaches are needed. This work combines several techniques to advance the study of GPCRs with the overarching

G protein-coupled receptors (GPCRs) are a large family of proteins involved in the cell signaling and regulation of many biological and pathological processes in the human body. To fully understand their functions, various approaches are needed. This work combines several techniques to advance the study of GPCRs with the overarching goal of pursuing X-ray crystallization using lipidic cubic phase (LCP). In meso, or LCP crystallization method involves imbedding the GPCR into a lipid membrane-mimetic material which spontaneously forms when monoacylglycerols (MAGs) are mixed at the correct hydration level and temperature. It provides a stable environment for GPCRs and has been established as the most common method to resolve structural details of GPCRs (Chapter 2). Yet, before crystallization, GPCRs need to be put through several rounds of optimization of the construct design, including truncation of N- and C- termini, fusing different soluble proteins, and mutating the receptor (Chapter 3). Other methods were also used to gain structural insights into GPCR interactions, such as coarse-grained molecular dynamic simulations, which showed the specific regions of interactions with cholesterol molecules imbedded in the membranes (Chapter 4). This study demonstrated β2-adrenergic receptor (β2AR), a GPCR, as a model of a cholesterol-sensitive receptor. Mutations were made to test the effect of removing specific residues of interest on cholesterol stabilization through the LCP-Tm assay, producing results that align with the simulation data. Finally, the goal of the last study is to provide a guide to identify which host lipids form stable LCP phases for different applications (Chapter 5). Small angle X-ray scattering is used to identify phases in hundreds of different precipitant conditions in the search of suitable host lipid for LCP studies. The results present a systematic overview of the compatibility of common MAGs by screening them against different precipitant solutions including varying salts and polyethylene glycol (PEG) concentrations, different PEG sizes, the presence of detergent or protein in the sample, and the addition of cholesterol. Together, these studies present a variety of methods to advance the structural studies of GPCRs using LCP
ContributorsAL-SAHOURI, ZINA (Author) / Liu, Wei (Thesis advisor) / Stephanopoulos, Nicholas (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2021
Description
Membrane proteins act as sensors, gatekeepers and information carriers in the cell membranes. Functional engineering of these proteins is important for the development of molecular tools for biosensing, therapeutics and as components of artificial cells. However, using protein engineering to modify existing protein structures is challenging due to the limitations

Membrane proteins act as sensors, gatekeepers and information carriers in the cell membranes. Functional engineering of these proteins is important for the development of molecular tools for biosensing, therapeutics and as components of artificial cells. However, using protein engineering to modify existing protein structures is challenging due to the limitations of structural changes and difficulty in folding polypeptides into defined protein structures. Recent studies have shown that nanoscale architectures created by DNA nanotechnology can be used to mimic various protein functions, including some membrane proteins. However, mimicking the highly sophisticated structural dynamics of membrane proteins by DNA nanostructures is still in its infancy, mainly due to lack of transmembrane DNA nanostructures that can mimic the dynamic behavior, ubiquitous to membrane proteins. Here, I demonstrate design of dynamic DNA nanostructures to mimic two important class of membrane proteins. First, I describe a DNA nanostructure that inserts through lipid membrane and dynamically reconfigures upon sensing a membrane-enclosed DNA or RNA target, thereby transducing biomolecular information across the lipid membrane similar to G-protein coupled receptors (GPCR’s). I use the non-destructive sensing property of our GPCR-mimetic nanodevice to sense cancer associated micro-RNA biomarkers inside exosomes without the need of RNA extraction and amplification. Second, I demonstrate a fully reversibly gated DNA nanopore that mimics the ligand mediated gating of ion channel proteins. The 20.4 X 20.4 nm-wide channel of the DNA nanopore allows timed delivery of folded proteins across synthetic and biological membranes. These studies represent early examples of dynamic DNA nanostructures in mimicking membrane protein functions. I envision that they will be used in synthetic biology to create artificial cells containing GPCR-like and ion channel-like receptors, in site-specific drug or vaccine delivery and highly sensitive biosensing applications.
ContributorsDey, Swarup (Author) / Yan, Hao (Thesis advisor) / Hariadi, Rizal F (Thesis advisor) / Liu, Yan (Committee member) / Stephanopoulos, Nicholas (Committee member) / Arizona State University (Publisher)
Created2021