Matching Items (17)
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- All Subjects: DNA nanotechnology
- All Subjects: RNA
- Creators: Yan, Hao
Description
Telomerase is a specialized enzyme that adds telomeric DNA repeats to the chromosome ends to counterbalance the progressive telomere shortening over cell divisions. It has two essential core components, a catalytic telomerase reverse transcriptase protein (TERT), and a telomerase RNA (TR). TERT synthesizes telomeric DNA by reverse transcribing a short template sequence in TR. Unlike TERT, TR is extremely divergent in size, sequence and structure and has only been identified in three evolutionarily distant groups. The lack of knowledge on TR from important model organisms has been a roadblock for vigorous studies on telomerase regulation. To address this issue, a novel in vitro system combining deep-sequencing and bioinformatics search was developed to discover TR from new phylogenetic groups. The system has been validated by the successful identification of TR from echinoderm purple sea urchin Strongylocentrotus purpuratus. The sea urchin TR (spTR) is the first invertebrate TR that has been identified and can serve as a model for understanding how the vertebrate TR evolved with vertebrate-specific traits. By using phylogenetic comparative analysis, the secondary structure of spTR was determined. The spTR secondary structure reveals unique sea urchin specific structure elements as well as homologous structural features shared by TR from other organisms. This study enhanced the understanding of telomerase mechanism and the evolution of telomerase RNP. The system that was used to identity telomerase RNA can be employed for the discovery of other TR as well as the discovery of novel RNA from other RNP complex.
ContributorsLi, Yang (Author) / Chen, Julian Jl (Thesis advisor) / Yan, Hao (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2011
Description
ABSTRACT The unique structural features of deoxyribonucleic acid (DNA) that are of considerable biological interest also make it a valuable engineering material. Perhaps the most useful property of DNA for molecular engineering is its ability to self-assemble into predictable, double helical secondary structures. These interactions are exploited to design a variety of DNA nanostructures, which can be organized into both discrete and periodic structures. This dissertation focuses on studying the dynamic behavior of DNA nanostructure recognition processes. The thermodynamics and kinetics of nanostructure binding are evaluated, with the intention of improving our ability to understand and control their assembly. Presented here are a series of studies toward this goal. First, multi-helical DNA nanostructures were used to investigate how the valency and arrangement of the connections between DNA nanostructures affect super-structure formation. The study revealed that both the number and the relative position of connections play a significant role in the stability of the final assembly. Next, several DNA nanostructures were designed to gain insight into how small changes to the nanostructure scaffolds, intended to vary their conformational flexibility, would affect their association equilibrium. This approach yielded quantitative information about the roles of enthalpy and entropy in the affinity of polyvalent DNA nanostructure interactions, which exhibit an intriguing compensating effect. Finally, a multi-helical DNA nanostructure was used as a model `chip' for the detection of a single stranded DNA target. The results revealed that the rate constant of hybridization is strongly dominated by a rate-limiting nucleation step.
ContributorsNangreave, Jeanette (Author) / Yan, Hao (Thesis advisor) / Liu, Yan (Thesis advisor) / Chen, Julian J.-L. (Committee member) / Seo, Dong Kyun (Committee member) / Arizona State University (Publisher)
Created2011
Description
A major goal of synthetic biology is to recapitulate emergent properties of life. Despite a significant body of work, a longstanding question that remains to be answered is how such a complex system arose? In this dissertation, synthetic nucleic acid molecules with alternative sugar-phosphate backbones were investigated as potential ancestors of DNA and RNA. Threose nucleic acid (TNA) is capable of forming stable helical structures with complementary strands of itself and RNA. This provides a plausible mechanism for genetic information transfer between TNA and RNA. Therefore TNA has been proposed as a potential RNA progenitor. Using molecular evolution, functional sequences were isolated from a pool of random TNA molecules. This implicates a possible chemical framework capable of crosstalk between TNA and RNA. Further, this shows that heredity and evolution are not limited to the natural genetic system based on ribofuranosyl nucleic acids. Another alternative genetic system, glycerol nucleic acid (GNA) undergoes intrasystem pairing with superior thermalstability compared to that of DNA. Inspired by this property, I demonstrated a minimal nanostructure composed of both left- and right-handed mirro image GNA. This work suggested that GNA could be useful as promising orthogonal material in structural DNA nanotechnology.
ContributorsZhang, Su (Author) / Chaut, John C (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2011
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
Deoxyribonucleic acid (DNA), a biopolymer well known for its role in preserving genetic information in biology, is now drawing great deal of interest from material scientists. Ease of synthesis, predictable molecular recognition via Watson-Crick base pairing, vast numbers of available chemical modifications, and intrinsic nanoscale size makes DNA a suitable material for the construction of a plethora of nanostructures that can be used as scaffold to organize functional molecules with nanometer precision. This dissertation focuses on DNA-directed organization of metallic nanoparticles into well-defined, discrete structures and using them to study photonic interaction between fluorophore and metal particle. Presented here are a series of studies toward this goal. First, a novel and robust strategy of DNA functionalized silver nanoparticles (AgNPs) was developed and DNA functionalized AgNPs were employed for the organization of discrete well-defined dimeric and trimeric structures using a DNA triangular origami scaffold. Assembly of 1:1 silver nanoparticle and gold nanoparticle heterodimer has also been demonstrated using the same approach. Next, the triangular origami structures were used to co-assemble gold nanoparticles (AuNPs) and fluorophores to study the distance dependent and nanogap dependencies of the photonic interactions between them. These interactions were found to be consistent with the full electrodynamic simulations. Further, a gold nanorod (AuNR), an anisotropic nanoparticle was assembled into well-defined dimeric structures with predefined inter-rod angles. These dimeric structures exhibited unique optical properties compared to single AuNR that was consistent with the theoretical calculations. Fabrication of otherwise difficult to achieve 1:1 AuNP- AuNR hetero dimer, where the AuNP can be selectively placed at the end-on or side-on positions of anisotropic AuNR has also been shown. Finally, a click chemistry based approach was developed to organize sugar modified DNA on a particular arm of a DNA origami triangle and used them for site-selective immobilization of small AgNPs.
ContributorsPal, Suchetan (Author) / Liu, Yan (Thesis advisor) / Yan, Hao (Thesis advisor) / Lindsay, Stuart (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2012
Description
Telomerase is a special reverse transcriptase that extends the linear chromosome termini in eukaryotes. Telomerase is also a unique ribonucleoprotein complex which is composed of the protein component called Telomerase Reverse Transcriptase (TERT) and a telomerase RNA component (TR). The enzyme from most vertebrate species is able to utilize a short template sequence within TR to synthesize a long stretch of telomeric DNA, an ability termed "repeat addition processivity". By using human telomerase reconstituted both in vitro (Rabbit Reticulocyte Lysate) and in vivo (293FT cells), I have demonstrated that a conserved motif in the reverse transcriptase domain of the telomerase protein is crucial for telomerase repeat addition processivity and rate. Furthermore, I have designed a "template-free" telomerase to show that RNA/DNA duplex binding is a critical step for telomere repeat synthesis. In an attempt to expand the understanding of vertebrate telomerase, I have studied RNA-protein interactions of telomerase from teleost fish. The teleost fish telomerase RNA (TR) is by far the smallest vertebrate TR identified, providing a valuable model for structural research.
ContributorsXie, Mingyi (Author) / Chen, Julian J.L. (Thesis advisor) / Yan, Hao (Committee member) / Wachter, Rebekka M. (Committee member) / Arizona State University (Publisher)
Created2010
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 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
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 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
Description
Cell immunotherapies have revolutionized clinical oncology. While CAR T cell therapy has been very effective in clinical studies, off-target immune toxicity limits eligible patients. Thus, NK cells have been approached with the same therapy design since NK cells have a more favorable safety profile. Therefore, the purpose of this research project is to explore DNA nanotech-based NK cell engagers (NKCEs) that force an immunological synapse between the NK cell and the cancer cell, leading to cancer death. DNA tetrabody (TB) and DNA tetrahedron (TDN) are fabricated and armed with HER2 affibody for tight adhesion to HER2+ cancer cell lines like SKBR3. Overall, relationship between TB-NK treatment and cancer cell apoptosis is still unclear. TB-NK treatment induces an apoptotic profile similar to PMA/IO stimulation. Pilot cell assay needs to be replicated with additional controls and a shortened treatment window. For DNA TDN fabrication, HER2 affibody polishing with Ni-NTA affinity chromatography achieves high purity with 20% to 100% high-imidazole elution gradient. ssDNA-HER2 affibody conjugation is optimal when ssDNA is treated with 40-fold excess sulfo-SMCC for 4 hours. In conclusion, the manufacturing of DNA-based NKCEs is rapid and streamlined, which gives these NKCEs the potential to become a ready to use immunotherapy.
ContributorsLuca, Michael (Author) / Yan, Hao (Thesis director) / Stephanopoulos, Nicholas (Committee member) / Blattman, Joseph (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / School of International Letters and Cultures (Contributor) / School of Molecular Sciences (Contributor)
Created2024-05
Description
As a rapidly evolving field, nucleic acid nanotechnology focuses on creating functional nanostructures or dynamic devices through harnessing the programmbility of nucleic acids including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), enabled by the predictable Watson-Crick base pairing. The precise control over the sequence and structure, along with the development of simulation softwares for the prediction of the experimental implementation provides the base of designing structures or devices with arbitrary topology and operational logic at nanoscale. Over the past 40 years, the thriving field has pushed the boundaries of nucleic acids, from originally biological macromolecules to functional building blocks with applications in biomedicine, molecular diagnostics and imaging, material science, electronics, crystallography, and more have emerged through programming the sequences and generating the various structures or devices. The underlying logic of nucleic acid programming is the base pairing rule, straightforward and robust. While for the complicated design of sequences and quantitative understanding of the programmed results, computational tools will markedly reduced the level of difficulty and even meet the challenge not available with manual effort. With this thesis three individual projects are presented, with all of them interweaving theory/computation and experiments. In a higher level abstraction, this dissertation covers the topic of biophysical understanding of the dynamic reactions, designing and realizing complex self-assembly systems and finally super-resolutional imaging. More specifically, Chapter 2 describes the study of RNA strand displacement kinetics with dedicated model extracting the reaction rates, providing guidelines for the rational design and regulation of the strand displacement reactions and eventually biochemical processes. In chapter 3 the platform for the design of complex symmetry of the self-assembly target and first experimental implementation of the assembly of pyrochlore lattices with DNA origamis are presented, which potentially can be applied to manipulate lights as optical materials. Chapter 4 focuses on the in solution characterization of the periodicity of DNA origami lattices with super-resolutional microscopy, with algorithms in development for three dimensional structural reconstruction.
ContributorsLiu, Hao (Author) / Yan, Hao (Thesis advisor) / Sulc, Petr (Thesis advisor) / Guo, Jia (Committee member) / Heyden, Matthias (Committee member) / Arizona State University (Publisher)
Created2023