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Description
Since the conception of DNA nanotechnology, the field has evolved towards the development of complex, dynamic 3D structures. The predictability of Watson-Crick base pairing makes DNA an unparalleled building block, and enables exceptional programmability in nanostructure shape and size. The work presented in this dissertation focuses on expanding two facets of the field: (1) introducing functionality through the incorporation of peptides to create DNA-peptide hybrid materials, and (2) the development of self-assembling DNA crystal lattices for scaffolding biomolecules. DNA nanostructures have long been proposed as drug delivery vehicles; however, they are not biocompatible because of their low stability in low salt environments and entrapment within the endosome. To address these issues, a functionalized peptide coating was designed to act as a counterion to a six-helix bundle, while simultaneously displaying numerous copies of an endosomal escape peptide to enable cytosolic delivery. This functionalized peptide coating creates a DNA-peptide hybrid material, but does not allow specific positioning or orientation of the peptides. The ability to control those aspects required the synthesis of DNA-peptide or DNA-peptide-DNA conjugates that can be incorporated into the nanostructure. The approach was utilized to produce a synbody where three peptides that bind transferrin with micromolar affinity, which were presented for multivalent binding to optimize affinity. Additionally, two DNA handle was attached to an enzymatically cleavable peptide to link two unique nanostructures. The second DNA handle was also used to constrain the peptide in a cyclic fashion to mimic the cell-adhesive conformations of RGD and PHSRN in fibronectin.
The original goal of DNA nanotechnology was to use a crystalline lattice made of DNA to host proteins for their structural determination using X-ray crystallography. The work presented here takes significant steps towards achieving this goal, including elucidating design rules to control cavity size within the scaffold for accommodating guest molecules of unique sizes, approaches to improve the atomic detail of the scaffold, and strategies to modulate the symmetry of each unique lattice. Finally, this work surveys methodologies towards the incorporation of several guest molecules, with promising preliminary results that constitute a significant advancement towards the ultimate goal of the field.
ContributorsMacCulloch, Tara Lynn (Author) / Stephanopoulos, Nicholas (Thesis advisor) / Borges, Chad (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2021
Description
Over the past four decades, DNA nanotechnology has grown exponentially from a field focused on simple structures to one capable of synthesizing complex nano-machines capable of drug delivery, nano-robotics, digital data storage, logic gated circuitry, nano-photonics, and other applications. The construction of these nanostructures is possible because of the predictable and programmable Watson-Crick base pairing of DNA. However, there is an increasing need for the incorporation of chemical diversity and functionality into these nanostructures. To overcome this challenge, this work explored creating hybrid DNA nanostructures by making self-assembling small molecule/protein-DNA conjugates.In one direction, well studied host-guest interactions (i.e. cucurbituril[7]-adamantane) were used as the choice of self-assembling species. Binding studies using these small molecule-DNA conjugates were performed and thereafter they were used to assemble larger DNA origami nanostructures. Finally, a stimulus responsive DNA nano-box that opens and closes based on these interactions was also demonstrated. In another direction, a trimeric KDPG aldolase protein-DNA conjugate was probed as a structural building block by assembling it into a DNA origami tetrahedron with four cavities. This hybrid building block was thereafter characterized by single particle cryo-EM and the resulting electron density map was best fit by simulating origami cages with varying number of proteins (ranging from 0 to 4).
Next, to increase access and for larger democratization of the field, an automation designer software tool capable of making DNA nanostructures was made. In this work, the focus was on making curved 3D DNA nanostructures. The last direction probed in this work was to make optical metamaterials based on complex 3D DNA architectures. Realization of a self-assembled 3D tetrastack geometry is still an unachieved dream in the field of DNA self-assembly. Thus, this direction was probed using DNA origami icosahedrons. Finally, the work covered in my thesis probes multiple directions for advancing DNA nanotechnology, both fundamentally and for potential applications.
ContributorsNarayanan Pradeep, Raghu (Author) / Yan, Hao HY (Thesis advisor) / Stephanopoulos, Nicholas NS (Thesis advisor) / Liu, Yan YL (Committee member) / Mills, Jeremy JHM (Committee member) / Arizona State University (Publisher)
Created2021
Description
I studied the molecular mechanisms of ultraviolet radiation mitigation (UVR) in the terrestrial cyanobacterium Nostoc punctiforme ATCC 29133, which produces the indole-alkaloid sunscreen scytonemin and differentiates into motile filaments (hormogonia). While the early stages of scytonemin biosynthesis were known, the late stages were not. Gene deletion mutants were interrogated by metabolite analyses and confocal microscopy, demonstrating that the ebo gene cluster, was not only required for scytonemin biosynthesis, but was involved in the export of scytonemin monomers to the periplasm. Further, the product of gene scyE was also exported to the periplasm where it was responsible for terminal oxidative dimerization of the monomers. These results opened questions regarding the functional universality of the ebo cluster. To probe if it could play a similar role in organisms other than scytonemin producing cyanobacteria, I developed a bioinformatic pipeline (Functional Landscape And Neighbor Determining gEnomic Region Search; FLANDERS) and used it to scrutinize the neighboring regions of the ebo gene cluster in 90 different bacterial genomes for potentially informational features. Aside from the scytonemin operon and the edb cluster of Pseudomonas spp., responsible for nematode repellence, no known clusters were identified in genomic ebo neighbors, but many of the ebo adjacent regions were enriched in signal peptides for export, indicating a general functional connection between the ebo cluster and biosynthetic compartmentalization. Lastly, I investigated the regulatory span of the two-component regulator of the scytonemin operon (scyTCR) using RNAseq of scyTCR deletion mutants under UV induction. Surprisingly, the knockouts had decreased expression levels in many of the genes involved in hormogonia differentiation and in a putative multigene regulatory element, hcyA-D. This suggested that UV could be a cue for developmental motility responses in Nostoc, which I could confirm phenotypically. In fact, UV-A simultaneously elicited hormogonia differentiation and scytonemin production throughout a genetically homogenous population. I show through mutant analyses that the partner-switching mechanism coded for by hcyA-D acts as a hinge between the scytonemin and hormogonia based responses. Collectively, this dissertation contributes to the understanding of microbial adaptive responses to environmental stressors at the genetic and regulatory level, highlighting their phenomenological and mechanistic complexity.
ContributorsKlicki, Kevin (Author) / Garcia-Pichel, Ferran (Thesis advisor) / Wilson, Melissa (Committee member) / Mukhopadhyay, Aindrila (Committee member) / Misra, Rajeev (Committee member) / Arizona State University (Publisher)
Created2021
Description
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
Exerting bias on a diverse pool of random short single stranded oligonucleotides (ODNs) by favoring binding to a specific target has led to the identification of countless high affinity aptamers with specificity to a single target. By exerting this same bias without prior knowledge of targets generates libraries to capture the complex network of molecular interactions presented in various biological states such as disease or cancer. Aptamers and enriched libraries have vast applications in bio-sensing, therapeutics, targeted drug delivery, biomarker discovery, and assay development. Here I describe a novel method of computational biophysical characterization of molecular interactions between a single aptamer and its cognate target as well as an alternative to next generation sequencing (NGS) as a readout for a SELEX-based assay. I demonstrate the capability of an artificial neural network (ANN) trained on the results of screening an aptamer against a random sampling of a combinatorial library of short synthetic 11mer peptides to accurately predict the binding intensities of that aptamer to the remainder of the combinatorial space originally sampled. This machine learned comprehensive non-linear relationship between amino acid sequence and aptamer binding to synthetic peptides can also make biologically relevant predictions for probable molecular interactions between the aptamer and its cognate target. Results of SELEX-based assays are determined by quantifying the presence and frequency of informative species after probing patient specimen. Here I show the potential of DNA microarrays to simultaneously monitor a pool of informative sequences within a diverse library with similar variability and reproducibility as NGS.
ContributorsLevenberg, Symon (Author) / Woodbury, Neal (Thesis advisor) / Borges, Chad (Committee member) / Ghirlanda, Giovanna (Committee member) / Redding, Kevin (Committee member) / Arizona State University (Publisher)
Created2021
Description
Particulate Guanylyl Cyclase Receptor A (pGC-A) is an atrial natriuretic peptide receptor, which plays a vital role in controlling cardiovascular, renal, and endocrine functions. The extracellular domain of pGC-A interacts with natriuretic peptides and triggers the intracellular guanylyl cyclase domain to convert GTP to cGMP. To effectively develop a method that can regulate pGC-A, structural information regarding its intact form is necessary. Currently, only the extracellular domain structure of rat pGC-A has been determined. However, structural data regarding the transmembrane domain, as well as functional intracellular domain regions, need to be elucidated.This dissertation presents detailed information regarding pGC-A expression and optimization in the baculovirus expression vector system, along with the first purification method for purifying functional intact human pGC-A. The first in vitro evidence of a purified intact human pGC-A tetramer was detected in detergent micellar solution. Intact pGC-A is currently proposed to function as a homodimer. Upon analyzing my findings and acknowledging that dimer formation is required for pGC-A functionality, I proposed the first tetramer complex model composed of two functional subunits (homodimer). Forming tetramer complexes on the cell membrane increases pGC-A binding efficiency and ligand sensitivity.
Currently, a two-step mechanism has been proposed for ATP-dependent pGC-A signal transduction. Based on cGMP functional assay results, it can be suggested that the binding ligand also moderately activates pGC-A, and that ATP is not crucial for the activation of guanylyl cyclase. Instead, three modulators can regulate different activation levels in intact pGC-A.
Crystallization of purified intact pGC-A was performed to determine its structure. During the crystallization condition screening process, I successfully selected seven promising initial crystallization conditions for intact human pGC-A crystallization. One selected condition led to the formation of excellent needle-shaped crystals. During the serial crystallography diffraction experiment, five diffraction patterns were detected. The highest diffraction resolution spot reached 3 Å.
This work will allow the determination of the intact human pGC-A structure while also providing structural information on the protein signal transduction mechanism. Further structural knowledge may potentially lead to improved drug design. More precise mutation experiments could help verify the current pGC-A signal transduction and activation mechanism.
ContributorsZhang, Shangji (Author) / Fromme, Petra (Thesis advisor) / Johnston, Stephen (Committee member) / Mazor, Yuval (Committee member) / Arizona State University (Publisher)
Created2021
Description
Osteocalcin (Oc) is the most abundant non-collagen protein found in the bone, but its precise function is still not completely understood. Three glutamic acid (Glu) residues within its sequence are sites for vitamin K-dependent post-translational modification, replacing a hydrogen with a carboxylate located at the γ-carbon position, converting these to γ-carboxyglutamic acid (Gla) residues. This modification confers increased binding of Oc to Ca2+ and hydroxyapatite matrix. Presented here, novel metal binding partners Mn2+, Fe3+, and Cr3+ of human Oc were determined, while the previously identified binders to (generally) non-human Oc, Ca2+, Mg2+, Pb2+ and Al3+ were validated as binders to human Oc by direct infusion mass spectrometry with all metals binding with higher affinity to the post-translationally modified form (Gla-Oc) compared to the unmodified form (Glu-Oc). Oc was also found to form pentamer (Gla-Oc) and pentamer and tetramer (Glu-Oc) homomeric self-assemblies in the absence of NaCl, which disassembled to monomers in the presence of near physiological Na+ concentrations. Additionally, Oc was found to form filamentous structures in vitro by negative stain TEM in the presence of increased Ca2+ titrations in a Gla- and pH-dependent manner. Finally, by combining circular dichroism spectroscopy to determine the fraction of Gla-Oc bound, and inductively-coupled plasma mass spectrometry to quantify total Al concentrations, the data were fit to a single-site binding model and the equilibrium dissociation constant for Al3+ binding to human Gla-Oc was determined (Kd = 1.0 ± 0.12 nM). Including citrate, a known competitive binder of Al3+, maintained Al in solution and enabled calculation of free Al3+ concentrations using a Matlab script to solve the complex set of linear equations. To further improve Al solubility limits, the pH of the system was lowered to 4.5, the pH during bone resorption. Complementary binding experiments with Glu-Oc were not possible due to the observed precipitation of Glu-Oc at pH 4.5, although qualitatively if Glu-Oc binds Al3+, it is with much lower affinity compared to Gla-Oc. Taken together, the results presented here further support the importance of post-translational modification, and thus adequate nutritional intake of vitamin K, on the binding and self-assembly properties of human Oc.
ContributorsThibert, Stephanie (Author) / Borges, Chad R (Thesis advisor) / LaBaer, Joshua (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2021
Description
In vitro selection technologies allow for the identification of novel biomolecules endowed with desired functions. Successful selection methodologies share the same fundamental requirements. First, they must establish a strong link between the enzymatic function being selected (phenotype) and the genetic information responsible for the function (genotype). Second, they must enable partitioning of active from inactive variants, often capturing only a small number of positive hits from a large population of variants. These principles have been applied to the selection of natural, modified, and even unnatural nucleic acids, peptides, and proteins. The ability to select for and characterize new functional molecules has significant implications for all aspects of research spanning the basic understanding of biomolecules to the development of new therapeutics. Presented here are four projects that highlight the ability to select for and characterize functional biomolecules through in vitro selection.
Chapter one outlines the development of a new characterization tool for in vitro selected binding peptides. The approach enables rapid screening of peptide candidates in small sample volumes using cell-free translated peptides. This strategy has the potential to accelerate the pace of peptide characterization and help advance the development of peptide-based affinity reagents.
Chapter two details an in vitro selection strategy for searching entire genomes for RNA sequences that enhance cap-independent initiation of translation. A pool of sequences derived from the human genome was enriched for members that function to enhance the translation of a downstream coding region. Thousands of translation enhancing elements from the human genome are identified and the function of a subset is validated in vitro and in cells.
Chapter three discusses the characterization of a translation enhancing element that promotes rapid and high transgene expression in mammalian cells. Using this ribonucleic acid sequence, a series of full length human proteins is expressed in a matter of only hours. This advance provides a versatile platform for protein synthesis and is espcially useful in situations where prokaryotic and cell-free systems fail to produce protein or when post-translationally modified protein is essential for biological analysis.
Chapter four outlines a new selection strategy for the identification of novel polymerases using emulsion droplet microfluidics technology. With the aid of a fluorescence-based activity assay, libraries of polymerase variants are assayed in picoliter sized droplets to select for variants with improved function. Using this strategy a variant of the 9°N DNA polymerase is identified that displays an enhanced ability to synthesize threose nucleic acid polymers.
Chapter one outlines the development of a new characterization tool for in vitro selected binding peptides. The approach enables rapid screening of peptide candidates in small sample volumes using cell-free translated peptides. This strategy has the potential to accelerate the pace of peptide characterization and help advance the development of peptide-based affinity reagents.
Chapter two details an in vitro selection strategy for searching entire genomes for RNA sequences that enhance cap-independent initiation of translation. A pool of sequences derived from the human genome was enriched for members that function to enhance the translation of a downstream coding region. Thousands of translation enhancing elements from the human genome are identified and the function of a subset is validated in vitro and in cells.
Chapter three discusses the characterization of a translation enhancing element that promotes rapid and high transgene expression in mammalian cells. Using this ribonucleic acid sequence, a series of full length human proteins is expressed in a matter of only hours. This advance provides a versatile platform for protein synthesis and is espcially useful in situations where prokaryotic and cell-free systems fail to produce protein or when post-translationally modified protein is essential for biological analysis.
Chapter four outlines a new selection strategy for the identification of novel polymerases using emulsion droplet microfluidics technology. With the aid of a fluorescence-based activity assay, libraries of polymerase variants are assayed in picoliter sized droplets to select for variants with improved function. Using this strategy a variant of the 9°N DNA polymerase is identified that displays an enhanced ability to synthesize threose nucleic acid polymers.
ContributorsLarsen, Andrew Carl (Author) / Chaput, John C (Thesis advisor) / Jacobs, Bertram L (Committee member) / Karr, Timothy L. (Committee member) / Arizona State University (Publisher)
Created2015
Description
Transient receptor potential (TRP) channels are a superfamily of ion channels found in plasma membranes of both single-celled and multicellular organisms. TRP channels all share the common aspect of having six transmembrane helices and a TRP domain. These structures tetramerize to form a receptor-activated non-selective ion channel. The specific protein being investigated in this thesis is the human transient receptor potential melastatin 8 (hTRPM8), a channel activated by the chemical ligand menthol and temperatures below 25 °C. TRPM8 is responsible for cold sensing and is related to pain relief associated with cooling compounds. TRPM8 has also been found to play a role in the regulation of various types of tumors. The structure of TRPM8 has been obtained through cryo-electron microscopy, but the functional contribution of individual portions of the protein to the overall protein function is unknown.
To gain more information about the function of the transmembrane region of hTRPM8, it was expressed in Escherichia coli (E. coli) and purified in detergent membrane mimics for experimentation. The construct contains the S4-S5 linker, pore domain (S5 and S6 transmembrane helices), pore helix, and TRP box. hTRPM8-PD+ was purified in the detergents n-Dodecyl-B-D-Maltoside (DDM), 16:0 Lyso PG, 1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphoglycerol (LPPG), and 14:0 Lyso PG, 1-Myristoyl-2-hydroxy-sn-glycero-3-phosphoglycerol (LMPG) to determine which detergent resulted in a hTRPM8-PD+ sample of the most stability, purity, and highest concentrations. Following bacterial expression and protein purification, hTRPM8-PD+ was studied and characterized with circular dichroism (CD) spectroscopy to learn more about the secondary structures and thermodynamic properties of the construct. Further studies can be done with more circular dichroism (CD) spectroscopy, planar lipid bilayer (BLM) electrophysiology, and nuclear magnetic resonance spectroscopy (NMR) to gain more understanding of how the pore domain plus contributes to the activity of the whole protein construct.
To gain more information about the function of the transmembrane region of hTRPM8, it was expressed in Escherichia coli (E. coli) and purified in detergent membrane mimics for experimentation. The construct contains the S4-S5 linker, pore domain (S5 and S6 transmembrane helices), pore helix, and TRP box. hTRPM8-PD+ was purified in the detergents n-Dodecyl-B-D-Maltoside (DDM), 16:0 Lyso PG, 1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphoglycerol (LPPG), and 14:0 Lyso PG, 1-Myristoyl-2-hydroxy-sn-glycero-3-phosphoglycerol (LMPG) to determine which detergent resulted in a hTRPM8-PD+ sample of the most stability, purity, and highest concentrations. Following bacterial expression and protein purification, hTRPM8-PD+ was studied and characterized with circular dichroism (CD) spectroscopy to learn more about the secondary structures and thermodynamic properties of the construct. Further studies can be done with more circular dichroism (CD) spectroscopy, planar lipid bilayer (BLM) electrophysiology, and nuclear magnetic resonance spectroscopy (NMR) to gain more understanding of how the pore domain plus contributes to the activity of the whole protein construct.
ContributorsMorelan, Danielle Taylor (Co-author) / Morelan, Danielle (Co-author) / Van Horn, Wade (Thesis director) / Chen, Julian (Committee member) / Luu, Dustin (Committee member) / Dean, W.P. Carey School of Business (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-12
Description
Enzyme Replacement Therapy (ERT) is a treatment often used for patients with disorders that affect the production of various enzymes within the body, such as Cystic Fibrosis and Fabry Disease. ERT involves the use of artificially-produced enzymes, which can be derived from humans, pigs, and bacteria. Generally, enzymes derived from porcine and bacterial sources are much less expensive and more accessible than those derived from a human source. This, and the ethical implications that porcine enzymes carry, make the decision of choosing treatment simple to some and complex to others. Ethically, human-derived enzymes are often considered more ethical, while not conflicting with religious beliefs and practices as porcine-derived enzymes do.
In order to further compare porcine and human-derived enzymes, a determination of the enzyme effectiveness was done via digestion simulation. The digestion for both the human and porcine-derived enzymes consisted of three steps: oral, gastric, and intestinal. After the digestion, the absorbance for each enzyme class as well as a dilution curve of the formula used was read and recorded. Using the standard dilution curve and the absorbance values for each unknown, the formula and thus enzyme concentration that was lost through the reaction was able to be calculated.
The effectiveness of both the human and porcine enzymes, determined by the percent of formula lost, was 18.2% and 19.7%, respectively, with an error of 0.6% from the spectrophotometer, and an error of about 10% from the scale used for measuring the enzymes. This error was likely due to the small mass required of the enzymes and can be prevented in the future by performing the experiment at a larger scale.
In order to further compare porcine and human-derived enzymes, a determination of the enzyme effectiveness was done via digestion simulation. The digestion for both the human and porcine-derived enzymes consisted of three steps: oral, gastric, and intestinal. After the digestion, the absorbance for each enzyme class as well as a dilution curve of the formula used was read and recorded. Using the standard dilution curve and the absorbance values for each unknown, the formula and thus enzyme concentration that was lost through the reaction was able to be calculated.
The effectiveness of both the human and porcine enzymes, determined by the percent of formula lost, was 18.2% and 19.7%, respectively, with an error of 0.6% from the spectrophotometer, and an error of about 10% from the scale used for measuring the enzymes. This error was likely due to the small mass required of the enzymes and can be prevented in the future by performing the experiment at a larger scale.
ContributorsBlevins, Brianna R (Author) / Martin, Thomas (Thesis director) / McILwraith, Heide (Committee member) / College of Integrative Sciences and Arts (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05