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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
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
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
Description
The biological and chemical diversity of protein structure and function can be greatly expanded by position-specific incorporation of non-natural amino acids bearing a variety of functional groups. Non-cognate amino acids can be incorporated into proteins at specific sites by using orthogonal aminoacyl-tRNA synthetase/tRNA pairs in conjunction with nonsense, rare, or 4-bp codons. There has been considerable progress in developing new types of amino acids, in identifying novel methods of tRNA aminoacylation, and in expanding the genetic code to direct their position. Chemical aminoacylation of tRNAs is accomplished by acylation and ligation of a dinucleotide (pdCpA) to the 3'-terminus of truncated tRNA. This strategy allows the incorporation of a wide range of natural and unnatural amino acids into pre-determined sites, thereby facilitating the study of structure-function relationships in proteins and allowing the investigation of their biological, biochemical and biophysical properties. Described in Chapter 1 is the current methodology for synthesizing aminoacylated suppressor tRNAs. Aminoacylated suppressor tRNACUAs are typically prepared by linking pre-aminoacylated dinucleotides (aminoacyl-pdCpAs) to 74 nucleotide (nt) truncated tRNAs (tRNA-COH) via a T4 RNA ligase mediated reaction. Alternatively, there is another route outlined in Chapter 1 that utilizes a different pre-aminoacylated dinucleotide, AppA. This dinucleotide has been shown to be a suitable substrate for T4 RNA ligase mediated coupling with abbreviated tRNA-COHs for production of 76 nt aminoacyl-tRNACUAs. The synthesized suppressor tRNAs have been shown to participate in protein synthesis in vitro, in an S30 (E. coli) coupled transcription-translation system in which there is a UAG codon in the mRNA at the position corresponding to Val10. Chapter 2 describes the synthesis of two non-proteinogenic amino acids, L-thiothreonine and L-allo-thiothreonine, and their incorporation into predetermined positions of a catalytically competent dihydrofolate reductase (DHFR) analogue lacking cysteine. Here, the elaborated proteins were site-specifically derivitized with a fluorophore at the thiothreonine residue. The synthesis and incorporation of phosphorotyrosine derivatives into DHFR is illustrated in Chapter 3. Three different phosphorylated tyrosine derivatives were prepared: bis-nitrobenzylphosphoro-L-tyrosine, nitrobenzylphosphoro-L-tyrosine, and phosphoro-L-tyrosine. Their ability to participate in a protein synthesis system was also evaluated.
ContributorsNangreave, Ryan Christopher (Author) / Hecht, Sidney M. (Thesis advisor) / Yan, Hao (Committee member) / Gould, Ian (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
As the genetic information storage vehicle, deoxyribonucleic acid (DNA) molecules are essential to all known living organisms and many viruses. It is amazing that such a large amount of information about how life develops can be stored in these tiny molecules. Countless scientists, especially some biologists, are trying to decipher the genetic information stored in these captivating molecules. Meanwhile, another group of researchers, nanotechnologists in particular, have discovered that the unique and concise structural features of DNA together with its information coding ability can be utilized for nano-construction efforts. This idea culminated in the birth of the field of DNA nanotechnology which is the main topic of this dissertation. The ability of rationally designed DNA strands to self-assemble into arbitrary nanostructures without external direction is the basis of this field. A series of novel design principles for DNA nanotechnology are presented here, from topological DNA nanostructures to complex and curved DNA nanostructures, from pure DNA nanostructures to hybrid RNA/DNA nanostructures. As one of the most important and pioneering fields in controlling the assembly of materials (both DNA and other materials) at the nanoscale, DNA nanotechnology is developing at a dramatic speed and as more and more construction approaches are invented, exciting advances will emerge in ways that we may or may not predict.
ContributorsHan, Dongran (Author) / Yan, Hao (Thesis advisor) / Liu, Yan (Thesis advisor) / Ros, Anexandra (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2012
Description
The ribosome is a ribozyme and central to the biosynthesis of proteins in all organisms. It has a strong bias against non-alpha-L-amino acids, such as alpha-D-amino acids and beta-amino acids. Additionally, the ribosome is only able to incorporate one amino acid in response to one codon. It has been demonstrated that reengineering of the peptidyltransferase center (PTC) of the ribosome enabled the incorporation of both alpha-D-amino acids and beta-amino acids into full length protein. Described in Chapter 2 are five modified ribosomes having modifications in the peptidyltrasnferase center in the 23S rRNA. These modified ribosomes successfully incorporated five different beta-amino acids (2.1 - 2.5) into E. coli dihydrofolate reductase (DHFR). The second project (Chapter 3) focused on the study of the modified ribosomes facilitating the incorporation of the dipeptide glycylphenylalanine (3.25) and fluorescent dipeptidomimetic 3.26 into DHFR. These ribosomes also had modifications in the peptidyltransferase center in the 23S rRNA of the 50S ribosomal subunit. The modified DHFRs having beta-amino acids 2.3 and 2.5, dipeptide glycylphenylalanine (3.25) and dipeptidomimetic 3.26 were successfully characterized by the MALDI-MS analysis of the peptide fragments produced by "in-gel" trypsin digestion of the modified proteins. The fluorescent spectra of the dipeptidomimetic 3.26 and modified DHFR having fluorescent dipeptidomimetic 3.26 were also measured. The type I and II DNA topoisomerases have been firmly established as effective molecular targets for many antitumor drugs. A "classical" topoisomerase I or II poison acts by misaligning the free hydroxyl group of the sugar moiety of DNA and preventing the reverse transesterfication reaction to religate DNA. There have been only two classes of compounds, saintopin and topopyrones, reported as dual topoisomerase I and II poisons. Chapter 4 describes the synthesis and biological evaluation of topopyrones. Compound 4.10, employed at 20 µM, was as efficient as 0.5 uM camptothecin, a potent topoisomerase I poison, in stabilizing the covalent binary complex (~30%). When compared with a known topoisomerase II poison, etoposide (at 0.5 uM), topopyorone 4.10 produced similar levels of stabilized DNA-enzyme binary complex (~34%) at 5 uM concentration.
ContributorsMaini, Rumit (Author) / Hecht, Sidney M. (Thesis advisor) / Gould, Ian (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2013
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