Browsing by Subject "DNA"
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Item A thin film transistor driven microchannel device(Texas A&M University, 2005-02-17) Lee, Hyun HoNovel electrophoresis devices for protein and DNA separation and identification have been presented and studied. The new device utilizes a contact resistance change detection method to identify protein and DNA in situ. The devices were prepared with a microelectronic micromechanical system (MEMS) fabrication method. Three model proteins and six DNA fragments were separated by polyacrylamide gel microchannel electrophoresis and surface electrophoresis. The detection of the proteins or DNA fragments was accomplished using the contact resistance increase of the detection electrode due to adsorption of the separated biomolecules. Key factors for the success of these devices were the optimization of fabrication process and the enhancement of detection efficiency of the devices. Parameters, such as microchannel configuration, size of electrode, and affinity of protein or polyacrylamide gel to the microchannel sidewall and bottom surface were explored in detail. For DNA analysis, the affinity to the bottom surface of the channel was critical. The surface modification method was used to enhance the efficiency of the microchannel surface electrophoresis device. The adsorption of channel separated protein and DNA on the detection electrode was confirmed with the electron spectroscopy for chemical analysis (ESCA) method. The electrical current (I) from the protein microchannel electrophoresis was usually noisy and fluctuated at the early stage of the electrophoresis process. In order to remove the current perturbation, an amorphous silicon (a-Si:H) thin film transistor (TFT) was connected to the microchannel device. The self-aligned a-Si:H TFT was fabricated with a two-photomask process. The result shows that the attachment of the TFT successfully suppressed the current fluctuation of the microchannel electrophoresis process. In summary, protein and DNA samples were effectively separated and detected with the novel TFT-driven or surface microchannel electrophoresis device.Item Amperometric DNA sensing using wired enzyme based electrodes(2003) Zhang, Yongchao; Heller, AdamA water soluble copolymer of acrylamide and 4-vinylpyridine complexed with [Os(bpy)2Cl]+/2+ (bpy = 2,2’-bipyridine), was synthesized. An electrodeposition method of making redox polymer films on electrodes was developed. This method was also shown to be effective in incorporating enzymes and amine-terminated DNA sequences in the redox polymer film. A 38-base DNA sequence was detected at 20 pM concentration in 15-35 μL droplets by an electrochemical enzyme-amplified sandwich-type assay on a mass-manufacturable screen printed carbon electrode with a diameter of 3.5 mm. A DNA-capturing oligonucleotide was attached to the pre-deposited redox polymer film using the electrodeposition method. The electrode was exposed to the droplet containing the tested DNA sample, and was then treated with a droplet containing horseradish peroxidase-labeled detection sequence. Formation of the capture-target-detection sandwich brought the horseradish peroxidase-label of the detection sequence in electrical contact with the redox polymer, making the sandwich an electrocatalyst for the reduction of hydrogen peroxide to water at + 0.2 V (Ag/AgCl). The radial diffusion of electrons through the redox polymer film on the microelectrode allowed the electrodeposition of a thicker film of the redox polymer, an increase in the loading of the capture sequence, and increased the collection efficiency of the electron vacancies originating in the electroreduced H2O2. With a 10-μm diameter carbon fiber microelectrode, as few as 3000 copies of the 38-basse DNA sequence were detected at 0.5 fM concentration in a 10 μL sample. A biofuel cell operating at a power density of 50 μW cm–2 at 0.5 V under physiological conditions (air saturated, pH 7.4, 0.14 M NaCl, 37.5°C, 15 mM glucose) was developed. The cell had a glucose electro-oxidizing anode and an O2 electro-reducing cathode. The anode and the cathode were 7 μm diameter, 2 cm long carbon fibers, on which the catalytic enzyme-redox polymer adducts were cross-linked. When the miniature cell operated at 0.5 V, the power output dropped to about 60% of its initial value after 2 days of continuous operation at 37.5°C.Item An Investigation on Gel Electrophoresis with Quantum Dots End-labeled DNA(2009-05-15) Chen, XiaojiaInvented in the 1950s, gel electrophoresis has now become a routine analytical method to verify the size of nucleic acids and proteins in molecular biology labs. Conventional gel electrophoresis can successfully separate DNA fragments from several base pairs to a few tens of kilo base pairs, beyond which a point is reached that DNA molecules cannot be resolved due to the size independent mobility. In this case, pulsed field gel electrophoresis (PFGE) was introduced to extend the range of DNA fragment sizes that can be effectively separated. But despite the incredible success of PFGE techniques, some important drawbacks remain. First, separation time is extremely long, ranging from several hours to a few days. Second, detection methods still rely on staining the gel after the run. Real time observation and study of band migration behavior is impossible due to the large size of the PFGE device. Finally, many commercial PFGE instruments are relatively expensive, a factor that can limit their accessibility both for routine analytical and preparative use as well as for performing fundamental studies. In this research, a miniaturized PFGE device was constructed with dimension 2cm x 2.6cm, capable of separating DNA fragments ranging from 2.5kb to 32kb within three hours using low voltage. The separation process can be observed in real time under a fluorescence microscope mounted with a cooled CCD camera. Resolution and mobility of the sample were measured to test the efficiency of the device. We also explored manipulating DNA fragments by end labeling DNA molecules with quantum dot nanocrystals. The quantum dot-DNA conjugates can be further modified through binding interactions with biotinylated single-stranded DNA primers. Single molecule visualization was performed during gel electrophoresis and the extension length, entanglement probability and reorientation time of different conjugates were measured to study their effect on DNA migration through the gel. Finally, electrophoresis of DNA conjugates was performed in the miniaturized PFGE device, and shaper bands were observed compared with the non end-labeled sample. Furthermore, by end-labeling DNA with quantum dots, the migration distance of shorter fragments is reduced, providing the possibility of separating a wider range of DNA fragment sizes on the same gel to achieve further device miniaturization.Item Applications of self-assembly : liquid crystalline semiconductors and DNA-conjugated microparticles(2012-12) Tang, Hao, 1985-; Willson, C. Grant, 1939-Self-assembly provides an efficient way to build complex structures with great flexibility in terms of components and properties. This dissertation presents two different forms of self-assembly for technical applications. The first example is the molecular assembly of liquid crystals (LCs). Attaching appropriate side chains on anthracene, oligothiophene, and oligoarenethiophene successfully constructed liquid crystalline organic semiconductors. The phase transitions of the LC semiconductors were analyzed by differential scanning calorimetry (DSC) and polarized optical microscopy (POM). The effect of the LC phase change on charge transport was probed by the space-charge limited current (SCLC) method and the field-effect transistor (FET) method. Mobility in the LC phase rose in anthracenyl esters but decreased in oligothiophenes and oligoarenethiophenes. The different electronic behavior of LC semiconductors may be caused by the difference in domain size and/or the difference in response to electric field. The second example of self-assembly in this dissertation is DNA-guided self-assembly of micrometer-sized particles. Patternable bioconjugation polymers were synthesized to allow for lithographic patterning and DNA conjugation. The base pairing of DNA was then used to drive the self-assembly of DNA-conjugated particles. The DNA conjugation chemistry was studied in detail using a fluorescence-based reaction test platform. The conjugated DNA on the polymer surface retained its ability to hybridize with its complement and was efficient in binding microspheres with complementary strands. Highly specific bead-to-bead assembly was analyzed using imaging flow cytometry, and the fractions of self-assembly products were explained on the basis of chemical equilibrium. The process of particle fabrication using photolithography was successfully developed, and the self-assembly of lithographically-patterned particles was demonstrated. We envision that the technologies described in this dissertation will be useful in a variety of fields ranging from microelectronics to biotechnology.Item Approaching the crystal structure of the polymerase γ catalytic complex(2011-08) Meng, Qingchao, master of arts in cell and molecular biology; Yin, Yuhui Whitney; Molineux, IanIn this thesis, a 4.7Å crystal structure of the human mitochondria DNA polymerase γ catalytic complex is reported. Though the DNA substrate-binding site is not identifiable in the structure, two conformational changes in the enzyme architecture are described: 1) rotation of the distal monomer of the accessory subunit towards the catalytic subunit, and 2) shift of the thumb motif of the polymerase domain towards the active site. Both conformational changes suggest a structure of Pol γ in the DNA-bound state and in its active site “closed” conformation.Item Automation of in vitro selections(2004) Sooter, Letha Jane; Ellington, Andrew D.Automation is a powerful tool, which may be used to increase the throughput of many otherwise laborious manual manipulations. Aptamer and deoxyribozyme selections are prime examples of processes, which require substantial amounts of time at the bench, but which are amenable to automation. Double-stranded DNA binding sites that bound with high affinity to the nuclear factor kappa B (NFκB) p50 homodimer were selected using a Tecan Genesis workstation. This was followed by selections against whole cell lysates. The resultant sequences represented an array of transcription factor binding sites within the E. coli genome. Finally, a Biomek2000 was used to perform a deoxyribozyme ligase selection, which formed an unnatural phosphorothioate linkage.Item A computational model for the diffusion coefficients of DNA with applications(2010-05) Li, Jun, 1977-; Gonzalez, Oscar, 1968-; Demkowicz, Leszek F.; Makarov, Dmitrii E.; Rodin, Gregory J.; van de Geijn, Robert A.The sequence-dependent curvature and flexibility of DNA is critical for many biochemically important processes. However, few experimental methods are available for directly probing these properties at the base-pair level. One promising way to predict these properties as a function of sequence is to model DNA with a set of base-pair parameters that describe the local stacking of the different possible base-pair step combinations. In this dissertation research, we develop and study a computational model for predicting the diffusion coefficients of short, relatively rigid DNA fragments from the sequence and the base-pair parameters. We focus on diffusion coefficients because various experimental methods have been developed to measure them. Moreover, these coefficients can also be computed numerically from the Stokes equations based on the three-dimensional shape of the macromolecule. By comparing the predicted diffusion coefficients with experimental measurements, we can potentially obtain refined estimates of various base-pair parameters for DNA. Our proposed model consists of three sub-models. First, we consider the geometric model of DNA, which is sequence-dependent and controlled by a set of base-pair parameters. We introduce a set of new base-pair parameters, which are convenient for computation and lead to a precise geometric interpretation. Initial estimates for these parameters are adapted from crystallographic data. With these parameters, we can translate a DNA sequence into a curved tube of uniform radius with hemispherical end caps, which approximates the effective hydrated surface of the molecule. Second, we consider the solvent model, which captures the hydrodynamic properties of DNA based on its geometric shape. We show that the Stokes equations are the leading-order, time-averaged equations in the particle body frame assuming that the Reynolds number is small. We propose an efficient boundary element method with a priori error estimates for the solution of the exterior Stokes equations. Lastly, we consider the diffusion model, which relates our computed results from the solvent model to relevant measurements from various experimental methods. We study the diffusive dynamics of rigid particles of arbitrary shape which often involves arbitrary cross- and self-coupling between translational and rotational degrees of freedom. We use scaling and perturbation analysis to characterize the dynamics at time scales relevant to different classic experimental methods and identify the corresponding diffusion coefficients. In the end, we give rigorous proofs for the convergence of our numerical scheme and show numerical evidence to support the validity of our proposed models by making comparisons with experimental data.Item Conformational dynamics of an unfolded biopolymer : theory and simulation(2012-12) Cheng, Ryan; Makarov, Dmitrii E.; Florin, Ernst-Ludwig; Elber, Ron; Henkelman, Graeme; Keatinge-Clay, Adrian T.The conformational dynamics of an unfolded biopolymer such as a polypeptide or DNA has attracted a significant amount of attention in the context of protein folding and the design of biomimetic technologies. To this end, recent advances in single-molecule experiments have allowed for biomolecules to be probed with an unprecedented level of detail, shedding light on their dynamics. Motivated by the need to interpret experimental data and to help guide future studies, we use concepts from polymer physics, computer simulations, and experimental data to study the timescales in which an unfolded biopolymer undergoes conformational rearrangement. First, we examine the end-to-end loop formation time in the experimentally relevant scenario where the dynamics are probed using a fluorescence probe and quencher. We show that the loop formation time in the experimentally relevant case is quantitatively dissimilar from the predictions of previous theoretical studies that neglect the quenching kinetics, which are often used to interpret experimental data. We additionally find that the loop formation times can be re-casted in a simple, universal dependence that is characteristic of random-coils. Furthermore, deviations from this universal dependence can be used as a sensitive tool for detecting structural order in unfolded biopolymers. We also consider a surface-tethered polymer chain and investigate the rate of a reaction between the free end and the surface. We explore this rate in the reaction-controlled limit and the diffusion-controlled limit, providing evidence for near-universal dependences of the rate in the respective limits. Next, we examine the transit time of end-to-end loop formation in a case study. We find that approximating the end-to-end dynamics as diffusion in a 1D potential of mean force fails dramatically to describe the transit time. Furthermore, we find that the transit time is uninfluenced by the average entropic force imposed by the polymer chain and is well described by a simple free-diffusion model. Finally, we explore the role of internal friction in the dynamics of an unfolded protein. Using simple polymer models that incorporate internal friction as an adjustable free parameter, we mimic typical single-molecule experiments that probe the unfolded state dynamics and make several experimentally verifiable predictions.Item Dendrimers as drug and gene delivery vectors : a self consistent field theory study(2013-08) Lewis, Thomas Wade Stakesby; Ganesan, VenkatThis research focuses on the modeling of dendrimer molecules for their application as delivery vectors within drug and gene therapy systems. We examine how the architecture and composition of dendrimers affect their drug and gene binding efficacies along with their interactions with anionic bilayers. We specifically focus on how the weakly basic nature of dendrimer monomers and the addition of neutral grafts to dendrimer surface groups affect their interactions with drugs, linear polyelectrolytes, and bilayers. By using polymer self-consistent field theory (SCFT) to model such systems, we develop a computationally efficient means to provide physical insights into these systems, which are intended to guide dendrimer design for delivery applications.We study the conformational properties of weakly basic (annealed) polyelectrolyte dendrimers by developing a SCFT model that explicitly accounts for the acid-base equilibrium reaction of the weakly basic monomers. We specifically focus on the role of local counterion concentration upon the charge and conformations of the annealed polyelectrolyte dendrimers. We compare our results to existing polymer scaling theories and develop a strong stretching theory for the dendrimer molecules.We extend the previous study to model the interactions between weakly basic dendrimers and weakly acidic, hydrophobic drug molecules. We specifically examine the effects of excluded volume, electrostatic, and enthalpic interactions on the binding efficacies between dendrimers and drugs under a variety of dendrimer generations, solution pOH conditions, drug sizes, and Bjerrum length values.We study the role of neutral dendrimer grafts on the conformations and drug binding efficacies of dendrimers. We then elucidate how the observed conformational changes affect the charge of the dendrimers. Furthermore, we examine how the presence of grafts affects the steric, electrostatic, and hydrophobic interactions between the drugs and dendrimers under a variety of solution conditions. We compare our results with the binding efficacies observed for non-grafted dendrimers to delineate the conditions under which the grafted dendrimers are better suited as drug hosts.We include semi-flexible, anionic linear polyelectrolyte (LPE) molecules in our grafted dendrimer SCFT framework to model the interactions between dendrimers and negatively charged genetic materials. Specifically, we examine how neutral dendrimer grafts, LPE stiffness, and solution pOH affect the interactions between dendrimers and LPEs. We then use our SCFT potential fields as input into Monte Carlo simulations in order to determine the dendrimer-LPE potentials of mean force and the resulting loop and tail statistics of the dendrimer-adsorbed LPE chains.We incorporate a negatively charged bilayer into our grafted dendrimer SCFT framework to model dendrimer interactions with a cellular membrane. We specifically examine the role of dendrimer grafting length, solution pH, and membrane tension on such interactions. By comparing our results with SCFT calculations of fixed dendrimer conformations and hard sphere nanoparticles in the presence of membranes, we delineate the role of dendrimer flexibility and porosity on the interactions between dendrimers and anionic bilayers.Item Design and characterization of convective thermal cyclers for high-speed DNA analysis(2009-05-15) Agrawal, NitinAn ideal polymerase chain reaction (PCR) system should be capable of rapidly amplifying a wide range of targets in both single and multiplex formats. Unfortunately, the timescales and complexities involved in many existing technologies impose significant limitations on achievable throughput. Buoyancy driven PCR is emerging as a simplified version of thermally driven bio-analysis systems. Here, we demonstrate a simplified convectively driven thermocycler capable of performing single and multiplex PCR for amplicons ranging from 191 bp to 1.3 kb within 10 to 50 minutes using 10 to 25 ?L reaction volumes. By positioning two independent thermoelectric heating elements along the perimeter of a flow loop reactor constructed using ordinary plastic tubing, a buoyancy-driven flow is established that continuously circulates reagents through temperature zones associated with the PCR process. Unlike conventional benchtop thermocyclers, this arrangement allows reactions to be performed without the need for dynamic temperature control of inactive hardware components while maintaining comparable product yields and requiring no modifications to standard PCR protocols. We also provide a general correlation that can be applied to design reactor geometries satisfying virtually any combination of reagent volume and cycling time. In addition to offering an attractive combination of cost and performance, this system is readily adaptable for portable battery powered operation, making it feasible to perform PCRbased assays in a broader array of settings.Item Design and development of an injectable, polymer-based immune priming center(2010-05) Singh, Ankur; Roy, Krishnendu; Kwak, Larry W; Peppas, Nicholas A; Schmidt, Christine E; Suggs, Laura J; Williams III, Robert OImmunotherapy, as a strategy to trigger immunity and eradicate a variety of chronic infectious diseases and cancers, has been explored for several decades with significant success in animal models. However, effective translation of these strategies into human clinical settings has proven elusive. Several cell-based anti-tumor therapies have progressed to clinical trials where antigen presenting dendritic cells (DCs) are isolated from patients, loaded with viral or tumor antigens and infused back in the patients. These ex-vivo “trained” DCs then present antigens to naïve T cells (adoptive therapy). However there exist several major limitations to this approach, including morbidity associated with patient cell isolation, high cost of ex vivo cell manipulation, time lag in “training” the immune cells, regulatory concerns, as well as the fact that ~ 90% of transplanted DCs die before they even home to lymph nodes. On the other hand, current immunotherapy approaches using recombinant proteins, synthetic peptides or nucleic acids, which "train" the immune cells in vivo to mount an immune response, have failed to address the tremendous challenge in generating efficient, sustained and protective immunity. There are two major challenges that must be overcome, (a) there exist relatively fewer numbers of immune cells at the sites of vaccine administration and given that these antigens themselves are weakly immunogenic, vaccine formulations must be tailored to attract large number of DCs to the immunization site and (b) immunologically, conventional nucleic acid or protein/peptide based vaccines do not elicit the required T helper type (Th) immunity along with a strong Cytotoxic T Lymphocyte (CTL) response against viral or tumor antigens and therefore new formulations must be able to “direct” the immune response towards a specific Th-type. Our goal was to design polymer-based sustained release formulations to addresses these challenges. Specifically, we have designed and developed delivery systems that can carry multiple biomolecules (nucleic acids, proteins, peptides, and chemoattractants) in a single injectable formulation. The delivery system promoted efficient migration of a large number of DCs to the site of injection and successful delivery of antigen resulting in activation of DCs. The multi-modal delivery system has the ability to bias or switch the immune response to the desired phenotype (e.g. Th1 or Th2) in a controlled manner. Using an infectious disease model against hepatitis B we have shown that co-encapsulation of Interleukin-10 (IL10) cytokine targeted siRNA within polymeric, surface-functionalized microparticles can further enhance DC activation and T cell proliferation in vitro as well as switch the hepatitis-specific immune response towards a strong Th1 phenotype in vivo. Further, in a weakly immunogenic A20 B cell lymphoma mouse model, a combination of microparticles and chemokine releasing in situ crosslinkable hydrogel provided significant Th1 type cellular immune response and delayed the onset of tumor development. Thus, the in situ crosslinkable hydrogel co-delivering microparticles and DC attracting chemokines creates an immune priming center with broad applications in a variety of disease models.Item The Development of an Animated Teaching Module Designed To Increase Understanding Of The Basic Concepts Of DNA, RNA, and Protein Synthesis Among Ninth Grade Biology Students(2011-12-14) Swensen, Jennie; Drumwiede, Kimberly HoggattCan a two-dimensional Flash animation be created to help teach about DNA? The goal of this thesis was to create a teaching module for DNA, RNA, and protein synthesis, designed for ninth grade students. The module contains animations created in Adobe Flash and quizzes after each section. It is to be used as a textbook and lecture supplement for high school students. Quantitative assessment showed an improvement in comprehension. Qualitative assessment showed positive feedback from both students and the teacher.Item Development of wireless DNA microarray sensors(2010-08) Chow, Kwok-Fan; Crooks, Richard M. (Richard McConnell); Bard, Allen J.; Bielawski, Christopher; Manthiram, Arumugam; Stevenson, KeithThe development of wireless DNA microelectrochemical microarray sensors is described. The operational principles of these sensors are based on bipolar electrochemistry. Bipolar electrodes are used to fabricate the wireless microarrays in this work. The systems are configured so that DNA sensing is carried out at the cathodic end of a bipolar electrode (BPE) and the result of the sensing experiment is reported at the anodic end of the BPE. There are two types of reporting platforms developed in this study. The first type relies on the emission of electrogenerated chemiluminescence (ECL). The system is configured so that ECL is emitted at the anodic end of the BPE when the target DNA is hybridized to the capture probe DNA immobilized on the cathodic end of the BPE. However, when there is no hybridization reaction occurs, there is no ECL emission on the electrode surface. The second type of reporting platform developed is based on silver electrodissolution at the anodic end of a BPE. When a reduction reaction occurs at the cathodic end of a BPE, it triggers oxidation and dissolution of silver deposited at the anodic end of the BPE. The loss of silver can easily be detected by the naked eye. This detection principle is used for DNA detection: when the target DNA is hybridized to capture probe DNA on the BPE, the BPE becomes shorter. However, if target DNA does not hybridize to the electrode surface, the length of the BPE remains the same. The BPE microarrays described in this work eliminate the need for complicated microfabrication procedures and instrumentation. For example, as many as 1000 BPEs can be simultaneously controlled using just two driving electrodes and a simple power supply. To fully utilize BPE microarrays for specific sensing tasks, a method based on robotic spotting was developed to modify the cathodic end of each BPE in the array. Because each BPE in a microarray is individually addressable, this development allows each BPE to perform a particular sensing operation.Item DNA threading intercalation: building sequence-specific linear rigidified and cyclic bisintercalators(2007-08) Chu, Yongjun; Iverson, Brent L.The threading polyintercalators are based on a 1,4,5,8-naphthalenetetracarboxylic diimide (NDI) unit that binds DNA via threading intercalation with two imide groups situated in different DNA grooves. In order to extend this strategy to bind DNA with higher affinity and more programmable specificity, it was envisioned that the linkers connecting the NDI units had to be rigidified. Thus a rigid, spiro-cyclic linker was designed and synthesized in the context of a bisintercalator, C1. The new linker has several sites for possible addition of functional groups as recognition elements. DNAse I footprinting results showed that C1 has a higher binding affinity (K[subscript D] ~ 10⁻⁷ M) toward 5'-GGTACC-3' sequence than the previously developed dimer G₃K, which has a flexible linker. NMR structural analysis of the C1-d(CGGTACCG)₂ complex has revealed the versatility of threading polyintercalation based on NDI moieties by verifying the binding of the C1 linker in the minor groove with two NDI units intercalating between GpG steps. The observed binding specificity of C1 is the result of interplay of different factors, such as overall linker length, electrostatic and hydrophobic complementarity to their preferred grooves. The fact that C1 and G₃K can target the same DNA sequence via different grooves, but with different linker structures, prompted us to explore the possibility of creating cyclic bisintercalating molecules. The first example, CBI-1, has been efficiently synthesized through a solid phase synthesis strategy, in which Gly₃Lys, a major groove recognition element for d(GGTACC)₂, was linked at one side and ([Beta]-Ala)₃Lys, a perfect match for a 4-base pair span in the minor groove, was connected on the other side. A dissociation kinetics study on poly(dGdC) indicated a slow dissociation process for CBI-1. Data from DNAse I footprinting and NMR structural studies confirmed that CBI-1 forms a tightly bound complex with DNA d(CG GTAC CG)₂, in which two NDI units intercalate between GC pairs, with linkers interacting with the major and minor grooves simultaneously. CBI-1 also exhibits improved sequence specificity compared to the linear dimer G₃K by only binding 5'-GGTACC-3' sequence. All the results demonstrate that cyclic threading intercalation is a new and effective approach to specifically target DNA sequences.Item DNA-based molecular circuits for diagnostics and therapeutics(2013-08) Codrea, Vlad Alexandru; Ellington, Andrew D.Nucleic acids are a uniquely flexible and multi-faceted class of molecules that fulfill fundamental and defining tasks such as replication and determination of heritable characteristics in every living organism. From the microscopic to the gigantic, from the most primitive to the most complex, life has been both molded and served by nucleic acids. Nucleic acid circuits straddle the realm of nature and technology. The elegance of interaction between nucleic acid molecules invites us to gain a deeper understanding of the naturally occurring systems they compose and to apply our ingenuity and foresight toward developing ever more complex synthetic systems. Nature has provided these basic building blocks, which we can now arrange – and augment – for the purpose of creating molecular-level machinery. Here we describe some ways in which we have rationally harnessed nucleic acids. In preparation for outbreaks of novel and deadly avian influenza viruses, we used quantitative polymerase chain reaction (qPCR) to track the number of flu virus particles surviving in the presence of potential antiviral drugs. We engineered tunable on/off switches that can be used to evaluate a series of conditions for diagnostic applications or to enable ‘smart’ drugs that sense, analyze, and respond to their microenvironment. We optimized the conditions for, and used, a unique set of guanine-rich DNA sequences called G-quadruplexes, whose enzymatic and structural properties make them prime effector candidates in diagnostic platforms. G-quadruplex folding powers isothermal DNA amplification, and the small organic molecules they bind endow G-quadruplexes with expanded catalytic abilities. We genotyped drug resistance mutations in tuberculosis via visually detectable color changes in the reaction buffer. We developed a paper fluidics assay that employs soluble and bead-immobilized nucleic acids to scan for genes in tuberculosis, and upon detection, to generate a readily observable discoloration on the paper strip. Finally, we probed the boundary of nucleic acid circuitry by attempting to expand its language via the incorporation of unnatural nucleobases into oligonucleotide components of a catalytic hairpin assembly (CHA) circuit. We subsequently evaluated the resilience of the unnatural CHA circuit to contamination by random DNA species, such as may be encountered in clinical samples.Item The DNA-binding and DNA endonuclease domains of a group II intron-encoded protein: characterization and application to the engineering of gene-targeting vectors(2003) SanFilippo, Joseph; Lambowitz, AlanItem Ecosystem under Pressure: Examining the Phytoplankton Community in the High Ballast Water Discharge Environment of Galveston Bay, Texas (USA)(2013-01-15) Steichen, Jamie LWith steady growth in global commerce and intensified ship traffic worldwide, comes the increased risk of invasion by non-indigenous organisms. Annually, >7000 vessels traveled across Galveston Bay, Texas from 2005-2010. These vessels discharged ~106 million metric tons of ballast water, equivalent to ~3.4% of the total volume of the Bay. A majority of these discharging vessels originated from around the Gulf of Mexico and the Caribbean Sea. By evaluating the source and frequency of inoculations from various locations, we are striving to assess the invasibility risk to Galveston Bay by way of ballast water. We identified organisms from Galveston Bay, ballast water samples and growout experiments using molecular methods. To our knowledge, this is the first utilization of molecular methods to identify the phytoplankton community within Galveston Bay. Within Galveston Bay, we identified 15 genera of dinoflagellates, 2 of which have previously gone undetected including Takayama and Woloszynskia. Thirteen ballast water samples yielded twenty genera of Protists, Fungi or Animalia from at least ten different phyla. With more than seven genera identified, dinoflagellates were the most diverse group: including the known toxin producer Pfiesteria and Scrippsiella which has not previously been detected in Galveston Bay. The most common diatoms in the ballast water samples were Actinocyclus, Ditylum, Nitzschia, Stephanopyxis and Thalassiosirales. At the termination of the growout experiments eight genera of phytoplankton were identified including: Dinophysis, Gymnodinium, Gyrodinium, Heterocapsa, Peridinium, Scrippsiella, Chaetoceros and Nitzschia. With these findings, Galveston Bay has the potential to be both a recipient and donor region of dinoflagellates. Dinoflagellates, capable of forming harmful algal blooms leading to fish and shellfish kills, are being transported to Galveston Bay via ballast water. Our results suggest that Galveston Bay is at risk for invasive species introductions via ballast water and support the idea that a monitoring system within the ports as well as the bay should be put in place. The actions would help to maintain the current health of this ecosystem and aide in preventing a negative impact in the event of successful establishment of a non-indigenous species of phytoplankton transported to Galveston Bay via ballast water.Item Electrospray ionization tandem mass spectrometry methods for the analysis of DNA and DNA/drug complexes(2010-08) Smith, Suncerae I.; Brodbelt, Jennifer S.; Guziec, Frank S.; Kerwin, Sean M.; Stevenson, Keith J.; Willets, Katherine A.Many anticancer therapies are based on the interaction of small molecule drugs with nucleic acids, particularly DNA. Electrospray ionization tandem mass spectrometry has established itself as an irreplaceable tool for the characterization of DNA adducts produced by alkylating agents, carcinogens, and antitumor drugs, in addition to the characterization of nucleic acid post-transcriptional modifications. ESI-MS was used to assess the non-covalent binding of a novel series of intercalating anthrapyrazoles to duplexes containing different sequences. Relative binding affinities paralleled the shift in melting point of the DNA duplexes measured from a previous study. Upon collisionally induced dissociation of the duplex/anthrapyrazole complexes, different binding strengths were discerned based on the fragmentation patterns. In addition, the interactions of a new series of sulfur-containing acridine ligands, some that functioned as alklyating mustards, with duplex DNA were also evaluated. Non-covalent and covalent binding of each ligand was determined, and the site of adduction (G > A) was revealed for the covalent modifications. The distribution of cross-linked products and mono-adducts by psoralen analogs was also monitored by both LC-UV and IRMPD-MS methods. Reactions at 5’-TA sites were favored over 5’-AT sites. The sites of interstrand cross-linking were determined by fragmentation of the duplex/psoralen complexes by infrared multiphoton dissociation (IRMPD). Ultraviolet photodissociation (UVPD) at 193 nm caused efficient charge reduction of deprotonated oligodeoxynucleotides via electron detachment. Subsequent CID of the charge-reduced oligodeoxynucleotides formed upon electron detachment, in a net process called electron photodetachment dissociation (EPD), resulted in a diverse array of abundant sequence ions which allowed the modification site(s) of three modified oligodeoxynucleotides to be pinpointed to a more specific location than by conventional CID. Electron transfer dissociation (ETD) caused efficient charge reduction of multi-protonated oligonucleotides. Subsequent CAD of the charge-reduced oligonucleotides formed upon electron transfer, in a net process termed electron transfer collision activated dissociation (ETcaD), resulted in rich backbone fragmentation, with a marked decrease in the abundance of base loss ions and internal fragments. ETcaD of an oligonucleotide duplex resulted in specific backbone cleavages, with conservation of weaker non-covalent bonds. In addition, IRMPD and UVPD were used to activate charge-reduced oligonucleotides formed upon electron transfer. ET-IRMPD afforded tunable characterization of the modified DNA and RNA, allowing for modified bases to be directly analyzed. ET-UVPD promoted higher energy backbone fragmentation pathways and created the most diverse MS/MS spectra. The numerous products generated by the hybrid MS/MS techniques (ETcaD, ET-IRMPD, and ET-UVPD) resulted in specific and extensive backbone cleavages which allowed for the modification sites of multiple oligonucleotides to be pinpointed.Item Genomic analysis of sorghum by fluorescence in situ hybridization(Texas A&M University, 2004-11-15) Kim, Jeong-SoonThe reliability of genome analysis and proficiency of genetic manipulation in vivo and in vitro are increased by assignment of linkage groups to specific chromosomes, placement of centromeres, orientation with respect to telomeres, and linear alignment with respect to chromosomal features and dimensions. I undertook five studies aimed at integrating sorghum genomics and cytogenetics at several levels. The results help establish an entirely new "cyto-genomics" resource, impacts of which are likely to be broad. In the first study, I developed a FISH-based karyotyping system for Sorghum bicolor Moench. I used integrated structural genomic resources, including linkage maps and large-insert clonal libraries of sorghum genomic DNA to develop a 17-locus probe cocktail for simultaneous fluorescent in situ hybridization (FISH). This probe enabled facile identification of all chromosome pairs in mitotic chromosome spreads. Perhaps just as important, I established time-efficient means to select sorghum BAC clones for multi-probe FISH. Thus, an integrated cyto-genomics system for sorghum can be constructed without need of chromosome flow sorting or microdissection, both of which are difficult and costly. In the second study, hybridization of DNA clones from 37 different genomic regions enabled the assignment of linkage groups and orientation of linkage maps to chromosomes. Comparisons between genetic and physical distances throughout the genome enabled a new nomenclature for linkage group designation in sorghum. The results provide an integrated nomenclature system of Sorghum bicolor chromosomes and linkage groups. In the third study, I created high-resolution maps by FISH to pachytene bivalents for two linkage groups (B and H), and defined relationships between pericentromeric heterochromatin, centromeres, mapped markers and recombination rates. These relationships will help guide the development and use of sorghum genomics. In the fifth study, I used FISH in two ongoing gene-targeted efforts. For the maturity gene ma5 and fertility restoration gene rfl, I estimated physical lengths between currently available flanking molecular markers. This enables estimation of recombination densities in these regions and assessment of the applicability of map-based and -assisted cloning.Item How trehalose protects DNA in the dry state: a molecular dynamics simulation(Texas A&M University, 2008-10-10) Fu, XuebingMolecular dynamics simulations were conducted on a system consisting of a decamer DNA solvated by trehalose and water (molecular ratio= 1:2), to mimic a relatively dry state for the DNA molecule. Simulations were performed at two different temperatures, 300 K and 450 K. The B-form DNA structure was shown to be stable at both temperatures. The analysis of hydrogen bonds between trehalose/water and DNA revealed that trehalose and backbone DNA formed the largest number of hydrogen bonds and thus constituted the major effect of structural protection for DNA. The number of hydrogen bonds formed by each OH group of trehalose with the backbone DNA was compared. Different types of trehalose-DNA interactions were analyzed, with no prevalent pattern recognized. Diffusion constants for trehalose and water were also calculated, suggesting a glassy/viscose state of the simulation system. It is believed that trehalose protects DNA in the dry state through the network of hydrogen bonds built by the sugars, which reduces the structural fluctuations of DNA and prevents its denaturation.