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- All Subjects: Chemistry
- Creators: Yan, Hao
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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
Nucleosomes are the basic repetitive unit of eukaryotic chromatin and are responsible for packing DNA inside the nucleus of the cell. They consist of a complex of eight histone proteins (two copies of four proteins H2A, H2B, H3 and H4) around which 147 base pairs of DNA are wrapped in ~1.67 superhelical turns. Although the nucleosomes are stable protein-DNA complexes, they undergo spontaneous conformational changes that occur in an asynchronous fashion. This conformational dynamics, defined by the "site-exposure" model, involves the DNA unwrapping from the protein core and exposing itself transiently before wrapping back. Physiologically, this allows regulatory proteins to bind to their target DNA sites during cellular processes like replication, DNA repair and transcription. Traditional biochemical assays have stablished the equilibrium constants for the accessibility to various sites along the length of the nucleosomal DNA, from its end to the middle of the dyad axis. Using fluorescence correlation spectroscopy (FCS), we have established the position dependent rewrapping rates for nucleosomes. We have also used Monte Carlo simulation methods to analyze the applicability of FRET fluctuation spectroscopy towards conformational dynamics, specifically motivated by nucleosome dynamics. Another important conformational change that is involved in cellular processes is the disassembly of nucleosome into its constituent particles. The exact pathway adopted by nucleosomes is still not clear. We used dual color fluorescence correlation spectroscopy to study the intermediates during nucleosome disassembly induced by changing ionic strength. Studying the nature of nucleosome conformational change and the kinetics is very important in understanding gene expression. The results from this thesis give a quantitative description to the basic unit of the chromatin.
ContributorsGurunathan, Kaushik (Author) / Levitus, Marcia (Thesis advisor) / Lindsay, Stuart (Committee member) / Woodbury, Neal (Committee member) / Yan, Hao (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
Though DNA nanostructures (DNs) have become interesting subjects of drug delivery, in vivo imaging and biosensor research, however, for real biological applications, they should be ‘long circulating’ in blood. One of the crucial requirements for DN stability is high salt concentration (like ~5–20 mM Mg2+) that is unavailable in a cell culture medium or in blood. Hence DNs denature promptly when injected into living systems. Another important factor is the presence of nucleases that cause fast degradation of unprotected DNs. The third factor is ‘opsonization’ which is the immune process by which phagocytes target foreign particles introduced into the bloodstream. The primary aim of this thesis is to design strategies that can improve the in vivo stability of DNs, thus improving their pharmacodynamics and biodistribution.
Several strategies were investigated to address the three previously mentioned limitations. The first attempt was to study the effect length and conformation of polyethylene glycol (PEG) on DN stability. DNs were also coated with PEG-lipid and human serum albumin (HSA) and their stealth efficiencies were compared. The findings reveal that both PEGylation and albumin coating enhance low salt stability, increase resistance towards nuclease action and reduce uptake of DNs by macrophages. Any protective coating around a DN increases its hydrodynamic radius, which is a crucial parameter influencing their clearance. Keeping this in mind, intrinsically stable DNs that can survive low salt concentration without any polymer coating were built. Several DNA compaction agents and DNA binders were screened to stabilize DNs in low magnesium conditions. Among them arginine, lysine, bis-lysine and hexamine cobalt showed the potential to enhance DN stability.
This thesis also presents a sensitive assay, the Proximity Ligation Assay (PLA), for the estimation of DN stability with time. It requires very simple modifications on the DNs and it can yield precise results from a very small amount of sample. The applicability of PLA was successfully tested on several DNs ranging from a simple wireframe tetrahedron to a 3D origami and the protocol to collect in vivo samples, isolate the DNs and measure their stability was developed.
Several strategies were investigated to address the three previously mentioned limitations. The first attempt was to study the effect length and conformation of polyethylene glycol (PEG) on DN stability. DNs were also coated with PEG-lipid and human serum albumin (HSA) and their stealth efficiencies were compared. The findings reveal that both PEGylation and albumin coating enhance low salt stability, increase resistance towards nuclease action and reduce uptake of DNs by macrophages. Any protective coating around a DN increases its hydrodynamic radius, which is a crucial parameter influencing their clearance. Keeping this in mind, intrinsically stable DNs that can survive low salt concentration without any polymer coating were built. Several DNA compaction agents and DNA binders were screened to stabilize DNs in low magnesium conditions. Among them arginine, lysine, bis-lysine and hexamine cobalt showed the potential to enhance DN stability.
This thesis also presents a sensitive assay, the Proximity Ligation Assay (PLA), for the estimation of DN stability with time. It requires very simple modifications on the DNs and it can yield precise results from a very small amount of sample. The applicability of PLA was successfully tested on several DNs ranging from a simple wireframe tetrahedron to a 3D origami and the protocol to collect in vivo samples, isolate the DNs and measure their stability was developed.
ContributorsBanerjee, Saswata (Author) / Yan, Hao (Thesis advisor) / Angell, Austen (Committee member) / Woodbury, Neal (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2018
Description
DNA and RNA are generally regarded as one of the central molecules in molecular biology. Recent advancements in the field of DNA/RNA nanotechnology witnessed the success of usage of DNA/RNA as programmable molecules to construct nano-objects with predefined shapes and dynamic molecular machines for various functions. From the perspective of structural design with nucleic acid, there are basically two types of assembly method, DNA tile based assembly and DNA origami based assembly, used to construct infinite-sized crystal structures and finite-sized molecular structures. The assembled structure can be used for arrangement of other molecules or nanoparticles with the resolution of nanometers to create new type of materials. The dynamic nucleic acid machine is based on the DNA strand displacement, which allows two nucleic acid strands to hybridize with each other to displace one or more prehybridized strands in the process. Strand displacement reaction has been implemented to construct a variety of dynamic molecular systems, such as molecular computer, oscillators, in vivo devices for gene expression control.
This thesis will focus on the computational design of structural and dynamic nucleic acid systems, particularly for new type of DNA structure design and high precision control of gene expression in vivo. Firstly, a new type of fundamental DNA structural motif, the layered-crossover motif, will be introduced. The layered-crossover allow non-parallel alignment of DNA helices with precisely controlled angle. By using the layered-crossover motif, the scaffold can go through the 3D framework DNA origami structures. The properties of precise angle control of the layered-crossover tiles can also be used to assemble 2D and 3D crystals. One the dynamic control part, a de-novo-designed riboregulator is developed that can recognize single nucleotide variation. The riboregulators can also be used to develop paper-based diagnostic devices.
This thesis will focus on the computational design of structural and dynamic nucleic acid systems, particularly for new type of DNA structure design and high precision control of gene expression in vivo. Firstly, a new type of fundamental DNA structural motif, the layered-crossover motif, will be introduced. The layered-crossover allow non-parallel alignment of DNA helices with precisely controlled angle. By using the layered-crossover motif, the scaffold can go through the 3D framework DNA origami structures. The properties of precise angle control of the layered-crossover tiles can also be used to assemble 2D and 3D crystals. One the dynamic control part, a de-novo-designed riboregulator is developed that can recognize single nucleotide variation. The riboregulators can also be used to develop paper-based diagnostic devices.
ContributorsHong, Fan, Ph. D (Author) / Yan, Hao (Thesis advisor) / Liu, Yan (Thesis advisor) / Green, Alexander A. (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2019
Description
Deoxyribonucleic acid (DNA) has emerged as an attractive building material for creating complex architectures at the nanometer scale that simultaneously affords versatility and modularity. Particularly, the programmability of DNA enables the assembly of basic building units into increasingly complex, arbitrary shapes or patterns. With the expanding complexity and functionality of DNA toolboxes, a quantitative understanding of DNA self-assembly in terms of thermodynamics and kinetics, will provide researchers with more subtle design guidelines that facilitate more precise spatial and temporal control. This dissertation focuses on studying the physicochemical properties of DNA tile-based self-assembly process by recapitulating representative scenarios and intermediate states with unique assembly pathways.
First, DNA double-helical tiles with increasing flexibility were designed to investigate the dimerization kinetics. The higher dimerization rates of more rigid tiles result from the opposing effects of higher activation energies and higher pre-exponential factors from the Arrhenius equation, where the pre-exponential factor dominates. Next, the thermodynamics and kinetics of single tile attachment to preformed “multitile” arrays were investigated to test the fundamental assumptions of tile assembly models. The results offer experimental evidences that double crossover tile attachment is determined by the electrostatic environment and the steric hindrance at the binding site. Finally, the assembly of double crossover tiles within a rhombic DNA origami frame was employed as the model system to investigate the competition between unseeded, facet and seeded nucleation. The results revealed that preference of nucleation types can be tuned by controlling the rate-limiting nucleation step.
The works presented in this dissertation will be helpful for refining the DNA tile assembly model for future designs and simulations. Moreover, The works presented here could also be helpful in understanding how individual molecules interact and more complex cooperative bindings in chemistry and biology. The future direction will focus on the characterization of tile assembly at single molecule level and the development of error-free tile assembly systems.
First, DNA double-helical tiles with increasing flexibility were designed to investigate the dimerization kinetics. The higher dimerization rates of more rigid tiles result from the opposing effects of higher activation energies and higher pre-exponential factors from the Arrhenius equation, where the pre-exponential factor dominates. Next, the thermodynamics and kinetics of single tile attachment to preformed “multitile” arrays were investigated to test the fundamental assumptions of tile assembly models. The results offer experimental evidences that double crossover tile attachment is determined by the electrostatic environment and the steric hindrance at the binding site. Finally, the assembly of double crossover tiles within a rhombic DNA origami frame was employed as the model system to investigate the competition between unseeded, facet and seeded nucleation. The results revealed that preference of nucleation types can be tuned by controlling the rate-limiting nucleation step.
The works presented in this dissertation will be helpful for refining the DNA tile assembly model for future designs and simulations. Moreover, The works presented here could also be helpful in understanding how individual molecules interact and more complex cooperative bindings in chemistry and biology. The future direction will focus on the characterization of tile assembly at single molecule level and the development of error-free tile assembly systems.
ContributorsJiang, Shuoxing (Author) / Yan, Hao (Thesis advisor) / Liu, Yan (Thesis advisor) / Hayes, Mark (Committee member) / Wang, Xu (Committee member) / Arizona State University (Publisher)
Created2016
Description
Nature is a master at organizing biomolecules in all intracellular processes, and researchers have conducted extensive research to understand the way enzymes interact with each other through spatial and orientation positioning, substrate channeling, compartmentalization, and more.
DNA nanostructures of high programmability and complexity provide excellent scaffolds to arrange multiple molecular/macromolecular components at nanometer scale to construct interactive biomolecular complexes and networks. Due to the sequence specificity at different positions of the DNA origami nanostructures, spatially addressable molecular pegboard with a resolution of several nm (less than 10 nm) can be achieved. So far, DNA nanostructures can be used to build nanodevices ranging from in vitro small molecule biosensing to sophisticated in vivo therapeutic drug delivery systems and multi-enzyme networks.
This thesis focuses on how to use DNA nanostructures as programmable biomolecular scaffolds to arranges enzymatic systems. Presented here are a series of studies toward this goal. First, we survey approaches used to generate protein-DNA conjugates and the use of structural DNA nanotechnology to engineer rationally designed nanostructures. Second, novel strategies for positioning enzymes on DNA nanoscaffolds has been developed and optimized, including site-specific/ non site-specific protein-DNA conjugation, purification and characterization. Third, an artificial swinging arm enzyme-DNA complex has been developed to mimic substrate channeling process. Finally, we extended to build a artificial 2D multi-enzyme network.
DNA nanostructures of high programmability and complexity provide excellent scaffolds to arrange multiple molecular/macromolecular components at nanometer scale to construct interactive biomolecular complexes and networks. Due to the sequence specificity at different positions of the DNA origami nanostructures, spatially addressable molecular pegboard with a resolution of several nm (less than 10 nm) can be achieved. So far, DNA nanostructures can be used to build nanodevices ranging from in vitro small molecule biosensing to sophisticated in vivo therapeutic drug delivery systems and multi-enzyme networks.
This thesis focuses on how to use DNA nanostructures as programmable biomolecular scaffolds to arranges enzymatic systems. Presented here are a series of studies toward this goal. First, we survey approaches used to generate protein-DNA conjugates and the use of structural DNA nanotechnology to engineer rationally designed nanostructures. Second, novel strategies for positioning enzymes on DNA nanoscaffolds has been developed and optimized, including site-specific/ non site-specific protein-DNA conjugation, purification and characterization. Third, an artificial swinging arm enzyme-DNA complex has been developed to mimic substrate channeling process. Finally, we extended to build a artificial 2D multi-enzyme network.
ContributorsYang, Yuhe Renee (Author) / Yan, Hao (Thesis advisor) / Liu, Yan (Thesis advisor) / Chen, Julian (Committee member) / Hayes, Mark (Committee member) / Arizona State University (Publisher)
Created2016
Description
In eukaryotes, DNA is packed in a highly condensed and hierarchically organized structure called chromatin, in which DNA tightly wraps around the histone octamer consisting of one histone 3-histone 4 (H3-H4) tetramer and two histone 2A- histone 2B (H2A-H2B) dimers with 147 base pairs in an almost two left handed turns. Almost all DNA dependent cellular processes, such as DNA duplication, transcription, DNA repair and recombination, take place in the chromatin form. Based on the critical importance of appropriate chromatin condensation, this thesis focused on the folding behavior of the nucleosome array reconstituted using different templates with various controllable factors such as histone tail modification, linker DNA length, and DNA binding proteins. Firstly, the folding behaviors of wild type (WT) and nucleosome arrays reconstituted with acetylation on the histone H4 at lysine 16 (H4K16 (Ac)) were studied. In contrast to the sedimentation result, atomic force microscopy (AFM) measurements revealed no apparent difference in the compact nucleosome arrays between WT and H4K16 (Ac) and WT. Instead, an optimal loading of nucleosome along the template was found necessary for the Mg2+ induced nucleosome array compaction. This finding leads to the further study on the role of linker DNA in the nucleosome compaction. A method of constructing DNA templates with varied linker DNA lengths was developed, and uniformly and randomly spaced nucleosome arrays with average linker DNA lengths of 30 bp and 60 bp were constructed. After comprehensive analyses of the nucleosome arrays' structure in mica surface, the lengths of the linker DNA were found playing an important role in controlling the structural geometries of nucleosome arrays in both their extended and compact forms. In addition, higher concentration of the DNA binding domain of the telomere repeat factor 2 (TRF2) was found to stimulate the compaction of the telomeric nucleosome array. Finally, AFM was successfully applied to investigate the nucleosome positioning behaviors on the Mouse Mammary Tumor Virus (MMTV) promoter region, and two highly positioned region corresponded to nucleosome A and B were identified by this method.
ContributorsFu, Qiang (Author) / Lindsay, Stuart M (Thesis advisor) / Yan, Hao (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2010
Description
Molecular tessellation research aims to elucidate the underlying principles that govern intricate patterns in nature and to leverage these principles to create precise and ordered structures across multiple scales, thereby facilitating the emergence of novel functionalities. DNA origami technology enables the fabrication of nearly arbitrary DNA architectures with nanoscale precision, which can serve as excellent building blocks for the construction of tessellation patterns. However, the size and complexity of DNA origami tessellation systems are currently limited by several unexplored factors relevant to the accuracy of essential design parameters, the applicability of design strategies, and the compatibility between different tiles. Here, a general design and assembly method are described for creating DNA origami tiles that grow into tessellation patterns with micrometer-scale order and nanometer-scale precision. A critical design parameter, interhelical distance (D), was identified, which determined the conformation of monomer tiles and the outcome of tessellation. Finely tuned D facilitated the accurate geometric design of monomer tiles with minimized curvature and improved tessellation capability. To demonstrate the generality of the design method, 9 tile geometries and 15 unique tile designs were generated. The designed tiles were assembled into single-crystalline lattices ranging from tens to hundreds of square micrometers with micrometer-scale, nearly defect-free areas readily visualized by atomic force microscopy. Two strategies were applied to further increase the complexity of DNA origami tessellation, including reducing the symmetry of monomer tiles and co-assembling tiles of various geometries. The designed 6 complex tilings that includes 5 Archimedean tilings and a 12-fold quasicrystal tiling yielded various tiling patterns that great in size and quality, indicating the robustness of the optimized tessellation system. The described design and assembly approach can also be employed to create square DNA origami units for algorithmic self-assembly. As the square units assembled and expanded, they executed the binary function XOR, which generated the Sierpinski triangular pattern according to the predetermined instructions. This study will promote DNA-templated, programmable molecular and material patterning and open up new opportunities for applications in metamaterial engineering, nanoelectronics, and nanolithography.
ContributorsTang, Yue (Author) / Yan, Hao (Thesis advisor) / Guo, Jia (Committee member) / Stephanopoulos, Nicholas (Committee member) / Arizona State University (Publisher)
Created2023
Description
The discovery of DNA helical structure opened the door of modern molecular biology. Ned Seeman utilized DNA as building block to construct different nanoscale materials, and introduced a new field, know as DNA nanotechnology. After several decades of development, different DNA structures had been created, with different dimension, different morphology and even with complex curvatures. In addition, after construction of enough amounts DNA structure candidates, DNA structure template, with excellent spatial addressability, had been used to direct the assembly of different nanomaterials, including nanoparticles and proteins, to produce different functional nanomaterials. However there are still many challenges to fabricate functional DNA nanostructures. The first difficulty is that the present finite sized template dimension is still very small, usually smaller than 100nm, which will limit the application for large amount of nanomaterials assembly or large sized nanomaterials assembly. Here we tried to solve this problem through developing a new method, superorigami, to construct finite sized DNA structure with much larger dimension, which can be as large as 500nm. The second problem will be explored the ability of DNA structure to assemble inorganic nanomaterials for novel photonic or electronic properties. Here we tried to utilize DNA Origami method to assemble AuNPs with controlled 3D spacial position for possible chiral photonic complex. We also tried to assemble SWNT with discrete length for possible field effect transistor device. In addition, we tried to mimic in vivo compartment with DNA structure to study internalized enzyme behavior. From our results, constructed DNA cage origami can protect encapsulated enzyme from degradation, and internalized enzyme activity can be boosted for up to 10 folds. In summary, DNA structure can serve as an ideal template for construction of functional nanomaterials with lots of possibilities to be explored.
ContributorsZhao, Zhao (Author) / Yan, Hao (Thesis advisor) / Liu, Yan (Thesis advisor) / Chen, Julian (Committee member) / Seo, Dong-Kyun (Committee member) / Arizona State University (Publisher)
Created2013