Matching Items (17)
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- All Subjects: Biochemistry
- Creators: Chen, Julian
- Member of: Theses and Dissertations
Description
Since Darwin popularized the evolution theory in 1895, it has been completed and studied through the years. Starting in 1990s, evolution at molecular level has been used to discover functional molecules while studying the origin of functional molecules in nature by mimicing the natural selection process in laboratory. Along this line, my Ph.D. dissertation focuses on the in vitro selection of two important biomolecules, deoxynucleotide acid (DNA) and protein with binding properties. Chapter two focuses on in vitro selection of DNA. Aptamers are single-stranded nucleic acids that generated from a random pool and fold into stable three-dimensional structures with ligand binding sites that are complementary in shape and charge to a desired target. While aptamers have been selected to bind a wide range of targets, it is generally thought that these molecules are incapable of discriminating strongly alkaline proteins due to the attractive forces that govern oppositely charged polymers. By employing negative selection step to eliminate aptamers that bind with off-target through charge unselectively, an aptamer that binds with histone H4 protein with high specificity (>100 fold)was generated. Chapter four focuses on another functional molecule: protein. It is long believed that complex molecules with different function originated from simple progenitor proteins, but very little is known about this process. By employing a previously selected protein that binds and catalyzes ATP, which is the first and only protein that was evolved completely from random pool and has a unique α/β-fold protein scaffold, I fused random library to the C-terminus of this protein and evolved a multi-domain protein with decent properties. Also, in chapter 3, a unique bivalent molecule was generated by conjugating peptides that bind different sites on the protein with nucleic acids. By using the ligand interactions by nucleotide conjugates technique, off-the shelf peptide was transferred into high affinity protein capture reagents that mimic the recognition properties of natural antibodies. The designer synthetic antibody amplifies the binding affinity of the individual peptides by ∼1000-fold to bind Grb2 with a Kd of 2 nM, and functions with high selectivity in conventional pull-down assays from HeLa cell lysates.
ContributorsJiang, Bing (Author) / Chaput, John C (Thesis advisor) / Chen, Julian (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2013
Description
The principle of Darwinian evolution has been applied in the laboratory to nucleic acid molecules since 1990, and led to the emergence of in vitro evolution technique. The methodology of in vitro evolution surveys a large number of different molecules simultaneously for a pre-defined chemical property, and enrich for molecules with the particular property. DNA and RNA sequences with versatile functions have been identified by in vitro selection experiments, but many basic questions remain to be answered about how these molecules achieve their functions. This dissertation first focuses on addressing a fundamental question regarding the molecular recognition properties of in vitro selected DNA sequences, namely whether negatively charged DNA sequences can be evolved to bind alkaline proteins with high specificity. We showed that DNA binders could be made, through carefully designed stringent in vitro selection, to discriminate different alkaline proteins. The focus of this dissertation is then shifted to in vitro evolution of an artificial genetic polymer called threose nucleic acid (TNA). TNA has been considered a potential RNA progenitor during early evolution of life on Earth. However, further experimental evidence to support TNA as a primordial genetic material is lacking. In this dissertation we demonstrated the capacity of TNA to form stable tertiary structure with specific ligand binding property, which suggests a possible role of TNA as a pre-RNA genetic polymer. Additionally, we discussed the challenges in in vitro evolution for TNA enzymes and developed the necessary methodology for future TNA enzyme evolution.
ContributorsYu, Hanyang (Author) / Chaput, John C (Thesis advisor) / Chen, Julian (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2013
Description
DNA has recently emerged as an extremely promising material to organize molecules on nanoscale. The reliability of base recognition, self-assembling behavior, and attractive structural properties of DNA are of unparalleled value in systems of this size. DNA scaffolds have already been used to organize a variety of molecules including nanoparticles and proteins. New protein-DNA bio-conjugation chemistries make it possible to precisely position proteins and other biomolecules on underlying DNA scaffolds, generating multi-biomolecule pathways with the ability to modulate inter-molecular interactions and the local environment. This dissertation focuses on studying the application of using DNA nanostructure to direct the self-assembly of other biomolecular networks to translate biochemical pathways to non-cellular environments. Presented here are a series of studies toward this application. First, a novel strategy utilized DNA origami as a scaffold to arrange spherical virus capsids into one-dimensional arrays with precise nanoscale positioning. This hierarchical self-assembly allows us to position the virus particles with unprecedented control and allows the future construction of integrated multi-component systems from biological scaffolds using the power of rationally engineered DNA nanostructures. Next, discrete glucose oxidase (GOx)/ horseradish peroxidase (HRP) enzyme pairs were organized on DNA origami tiles with controlled interenzyme spacing and position. This study revealed two different distance-dependent kinetic processes associated with the assembled enzyme pairs. Finally, a tweezer-like DNA nanodevice was designed and constructed to actuate the activity of an enzyme/cofactor pair. Using this approach, several cycles of externally controlled enzyme inhibition and activation were successfully demonstrated. This principle of responsive enzyme nanodevices may be used to regulate other types of enzymes and to introduce feedback or feed-forward control loops.
ContributorsLiu, Minghui (Author) / Yan, Hao (Thesis advisor) / Liu, Yan (Thesis advisor) / Chen, Julian (Committee member) / Zhang, Peiming (Committee member) / Arizona State University (Publisher)
Created2013
Description
The need for a renewable and sustainable light-driven energy source is the motivation for this work, which utilizes a challenging, yet practical and attainable bio-inspired approach to develop an artificial oxygen evolving complex, which builds upon the principles of the natural water splitting mechanism in oxygenic photosynthesis. In this work, a stable framework consisting of a three-dimensional DNA tetrahedron has been used for the design of a bio-mimic of the Oxygen-Evolving Complex (OEC) found in natural Photosystem II (PSII). PSII is a large protein complex that evolves all the oxygen in the atmosphere, but it cannot be used directly in artificial systems, as the light reactions lead to damage of one of Photosystem II's core proteins, D1, which has to be replaced every half hour in the presence of sunlight. The final goal of the project aims to build the catalytic center of the OEC, including the Mn4CaCl metal cluster and its protein environment in the stable DNA framework of a tetrahedron, which can subsequently be connected to a photo-stable artificial reaction center that performs light-induced charge separation. Regions of the peptide sequences containing Mn4CaCl ligation sites are implemented in the design of the aOEC (artificial oxygen-evolving complex) and are attached to sites within the tetrahedron to facilitate assembly. Crystals of the tetrahedron have been obtained, and X-ray crystallography has been used for characterization. As a proof of concept, metal-binding peptides have been coupled to the DNA tetrahedron which allowed metal-containing porphyrins, specifically Fe(III) meso-Tetra(4-sulfonatophenyl) porphyrin chloride, to be encapsulated inside the DNA-tetrahedron. The porphyrins were successfully assembled inside the tetrahedron through coordination of two terminal histidines from the orthogonally oriented peptides covalently attached to the DNA. The assembly has been characterized using Electron Paramagnetic Resonance (EPR), optical spectroscopy, Dynamic Light Scattering (DLS), and x-ray crystallography. The results reveal that the spin state of the metal, iron (III), switches during assembly from the high-spin state to low-spin state.
ContributorsRendek, Kimberly Nicole (Author) / Fromme, Petra (Thesis advisor) / Chen, Julian (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2012
Description
DNA is a unique, highly programmable and addressable biomolecule. Due to its reliable and predictable base recognition behavior, uniform structural properties, and extraordinary stability, DNA molecules are desirable substrates for biological computation and nanotechnology. The field of DNA computation has gained considerable attention due to the possibility of exploiting the massive parallelism that is inherent in natural systems to solve computational problems. This dissertation focuses on building novel types of computational DNA systems based on both DNA reaction networks and DNA nanotechnology. A series of related research projects are presented here. First, a novel, three-input majority logic gate based on DNA strand displacement reactions was constructed. Here, the three inputs in the majority gate have equal priority, and the output will be true if any two of the inputs are true. We subsequently designed and realized a complex, 5-input majority logic gate. By controlling two of the five inputs, the complex gate is capable of realizing every combination of OR and AND gates of the other 3 inputs. Next, we constructed a half adder, which is a basic arithmetic unit, from DNA strand operated XOR and AND gates. The aim of these two projects was to develop novel types of DNA logic gates to enrich the DNA computation toolbox, and to examine plausible ways to implement large scale DNA logic circuits. The third project utilized a two dimensional DNA origami frame shaped structure with a hollow interior where DNA hybridization seeds were selectively positioned to control the assembly of small DNA tile building blocks. The small DNA tiles were directed to fill the hollow interior of the DNA origami frame, guided through sticky end interactions at prescribed positions. This research shed light on the fundamental behavior of DNA based self-assembling systems, and provided the information necessary to build programmed nanodisplays based on the self-assembly of DNA.
ContributorsLi, Wei (Author) / Yan, Hao (Thesis advisor) / Liu, Yan (Thesis advisor) / Chen, Julian (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2014
Description
DNA nanotechnology is one of the most flourishing interdisciplinary research fields. Through the features of programmability and predictability, DNA nanostructures can be designed to self-assemble into a variety of periodic or aperiodic patterns of different shapes and length scales, and more importantly, they can be used as scaffolds for organizing other nanoparticles, proteins and chemical groups. By leveraging these molecules, DNA nanostructures can be used to direct the organization of complex bio-inspired materials that may serve as smart drug delivery systems and in vitro or in vivo bio-molecular computing and diagnostic devices. In this dissertation I describe a systematic study of the thermodynamic properties of complex DNA nanostructures, including 2D and 3D DNA origami, in order to understand their assembly, stability and functionality and inform future design endeavors. It is conceivable that a more thorough understanding of DNA self-assembly can be used to guide the structural design process and optimize the conditions for assembly, manipulation, and functionalization, thus benefiting both upstream design and downstream applications. As a biocompatible nanoscale motif, the successful integration, stabilization and separation of DNA nanostructures from cells/cell lysate suggests its potential to serve as a diagnostic platform at the cellular level. Here, DNA origami was used to capture and identify multiple T cell receptor mRNA species from single cells within a mixed cell population. This demonstrates the potential of DNA nanostructure as an ideal nano scale tool for biological applications.
ContributorsWei, Xixi (Author) / Liu, Yan (Thesis advisor) / Yan, Hao (Thesis advisor) / Chen, Julian (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2014
Description
Telomerase enzyme is a truly remarkable enzyme specialized for the addition of short, highly repetitive DNA sequences onto linear eukaryotic chromosome ends. The telomerase enzyme functions as a ribonucleoprotein, minimally composed of the highly conserved catalytic telomerase reverse transcriptase and essential telomerase RNA component containing an internalized short template region within the vastly larger non-coding RNA. Even among closely related groups of species, telomerase RNA is astonishingly divergent in sequence, length, and secondary structure. This massive disparity is highly prohibitive for telomerase RNA identification from previously unexplored groups of species, which is fundamental for secondary structure determination. Combined biochemical enrichment and computational screening methods were employed for the discovery of numerous telomerase RNAs from the poorly characterized echinoderm lineage. This resulted in the revelation that--while closely related to the vertebrate lineage and grossly resembling vertebrate telomerase RNA--the echinoderm telomerase RNA central domain varies extensively in structure and sequence, diverging even within echinoderms amongst sea urchins and brittle stars. Furthermore, the origins of telomerase RNA within the eukaryotic lineage have remained a persistent mystery. The ancient Trypanosoma telomerase RNA was previously identified, however, a functionally verified secondary structure remained elusive. Synthetic Trypanosoma telomerase was generated for molecular dissection of Trypanosoma telomerase RNA revealing two RNA domains functionally equivalent to those found in known telomerase RNAs, yet structurally distinct. This work demonstrates that telomerase RNA is uncommonly divergent in gross architecture, while retaining critical universal elements.
ContributorsPodlevsky, Joshua (Author) / Chen, Julian (Thesis advisor) / Mangone, Marco (Committee member) / Kusumi, Kenro (Committee member) / Wilson-Rawls, Norma (Committee member) / Arizona State University (Publisher)
Created2015
Description
In the 1970s James Watson recognized the inability of conventional DNA replication machinery to replicate the extreme termini of chromosomes known as telomeres. This inability is due to the requirement of a building block primer and was termed the end replication problem. Telomerase is nature's answer to the end replication problem. Telomerase is a ribonucleoprotein which extends telomeres through reverse transcriptase activity by reiteratively copying a short intrinsic RNA sequence to generate 3' telomeric extensions. Telomeres protect chromosomes from erosion of coding genes during replication, as well as differentiate native chromosome ends from double stranded breaks. However, controlled erosion of telomeres functions as a naturally occurring molecular clock limiting the replicative capacity of cells. Telomerase is over activated in many cancers, while inactivation leads to multiple lifespan limiting human diseases. In order to further study the interaction between telomerase RNA (TR) and telomerase reverse transcriptase protein (TERT), vertebrate TERT fragments were screened for solubility and purity following bacterial expression. Soluble fragments of medaka TERT including the RNA binding domain (TRBD) were identified. Recombinant medaka TRBD binds specifically to telomerase RNA CR4/CR5 region. Ribonucleotide and amino acid pairs in close proximity within the medaka telomerase RNA-protein complex were identified using photo-activated cross-linking in conjunction with mass spectrometry. The identified cross-linking amino acids were mapped on known crystal structures of TERTs to reveal the RNA interaction interface of TRBD. The identification of this RNA TERT interaction interface furthers the understanding of the telomerase complex at a molecular level and could be used for the targeted interruption of the telomerase complex as a potential cancer treatment.
ContributorsBley, Christopher James (Author) / Chen, Julian (Thesis advisor) / Allen, James (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2011
Description
Membrane protein structure is continuing to be a topic of interest across the scientific community. However, high resolution structural data of these proteins is difficult to obtain. The amino acid transport protein, Outer Envelope Protein, 16kDa (OEP16) is a transmembrane protein channel that allows the passive diffusion of amino acids across the outer chloroplast membrane, and is used as a model protein in order to establish methods that ultimately reveal structural details about membrane proteins using nuclear magnetic resonance (NMR) spectroscopy. Methods include recombinant expression of isotope enriched inclusion bodies, purification and reconstitution in detergent micelles, and pre-characterization techniques including circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), and high pressure liquid chromatography (HPLC). High resolution NMR spectroscopy was able to assign 99% of the amide backbone and the chemical shifts provided detailed secondary structure of OEP16 on a per residue basis using the software TALOS+. Relaxation studies explored the intramolecular dynamics of OEP16 and results strongly support the resonance assignments. Successful titration studies were able to locate residues important for amino acid binding for import into the chloroplast as well as provide information on how the transmembrane helices of OEP16 are packed together. For the first time there is experimental evidence that can assign the location of secondary structure in OEP16 and creates a foundation for a future three dimensional structure.
ContributorsZook, James Duncan (Author) / Fromme, Petra (Thesis advisor) / Chen, Julian (Committee member) / Wachter, Rebekka (Committee member) / Arizona State University (Publisher)
Created2012
Description
Telomerase ribonucleoprotein is a unique reverse transcriptase that adds telomeric DNA repeats to chromosome ends. Telomerase RNA (TER) is extremely divergent in size, sequence and has to date only been identified in vertebrate, yeast, ciliate and plant species. Herein, the identification and characterization of TERs from an evolutionarily distinct group, filamentous fungi, is presented. Based on phylogenetic analysis of 69 TER sequences and mutagenesis analysis of in vitro reconstituted Neurospora telomerase, we discovered a conserved functional core in filamentous fungal TERs sharing homologous structural features with vertebrate TERs. This core contains the template-pseudoknot and P6/P6.1 domains, essential for enzymatic activity, which retain function in trans. The in vitro reconstituted Neurospora telomerase is highly processive, synthesizing canonical TTAGGG repeats. Similar to Schizosaccharomycetes pombe, filamentous fungal TERs utilize the spliceosomal splicing machinery for 3' processing. Neurospora telomerase, while associating with the Est1 protein in vivo, does not bind homologous Ku or Sm proteins found in both budding and fission yeast telomerase holoenzyme, suggesting a unique biogenesis pathway. The development of Neurospora as a model organism to study telomeres and telomerase may shed light upon the evolution of the canonical TTAGGG telomeric repeat and telomerase processivity within fungal species.
ContributorsQi, Xiaodong (Author) / Chen, Julian (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Chaput, John (Committee member) / Arizona State University (Publisher)
Created2011