Browsing by Subject "RNA"
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Item The application of aptamer microarraying techniques to the detection of HIV-1 reverse transcriptase and its mutant variants(2010-08) Syrett, Heather Angel; Ellington, Andrew D.; Kitto, George B.; Willets, Katherine A.; Iyer, Vishwanath R.; Yin, Yuhui W.The work described here details the experimental progress toward an improved means of HIV-1 diagnosis and an explanation of the experimental approaches taken to advance a previously developed HIV-1 reverse transcriptase detection assay using RNA aptamers for protein capture. After characterization of the identity and function of the aptamer samples to be used, we first set about clarifying the nature of the assay and pinning down sources of variability inherent in the original Aptamer Antibody Sandwich Assay (AASA) such that through the course of this work we might bring the assay to a point of high reproducibility. In doing so, we devised a set of criteria for data analysis and filtration and established a process to examine whether modifications to the method resulted in measurable improvement. Two new methods were tested in the hope that they might later be extended to our ultimate project goal of distinguishing binding affinity variations among HIV-1 reverse transcriptase protein and its mutant variants. Both method modifications involved the addition of a fluorescently labeled Cy5 probe to the immobilized aptamer construct. The addition of a fluorescent label to each printed aptamer allowed for detection of aptamer presence in addition to protein binding, essentially serving as a simple internal control for aptamer-protein binding. After optimizing the AASA aptamer construct and experimental procedure, the AASA was extended to a multiplexed array format. Using four groups of aptamers selected against two HIV-1 RT variants (wild-type and mutant 3) we tested the hypothesis that immobilized anti-HIV-1 aptamers might be capable of binding HIV-1 RT variants and regardless of their selective target. The experiments described here are the first example of these aptamers being used in a multiplexed array format, and the results are not only a clear exemplification of the capacity of RNA aptamers for detection in this novel, immobilized assay format, but also an indicator of the utility and flexibility of RNA aptamer functionality. The promising results described in these preliminary studies are the starting block from which several interesting aptamer-protein interaction and drug-competition studies have begun.Item Aptamers as cross-reactive receptors : using binding patterns to discriminate biomolecules(2013-05) Stewart, Sara, 1980-; Anslyn, Eric V., 1960-; Ellington, Andrew D.Exploration into the use of aptamers as cross-reactive receptors was the focus of this work. Cross-reactivity is of interest for developing assays to identify complex targets and solutions. By exploiting the simple chemistries of aptamers, we hope to introduce a new class of receptors to the science of molecular discrimination. This manuscript first addresses the use designed aptamers for the identification of variants of HIV-1 reverse transcriptase. In this research aptamers were immobilized on a platform and were used to discriminate four variants of HIV-1 reverse transcriptase. It was found that not only could the array discriminate HIV-1 reverse transcriptase variants for which aptamers were designed, it would also discriminate variants for which no aptamers exist. A panel of aptamers was used to discriminate four separate cell lines, which were chosen as examples of complex targets. This aptamer panel was used to further explore the use of aptamers as cross-reactive sensors. Forty-six aptamers were selected from the literature that were designed to be specific to cells or molecules expected to be in the surface of cells. This panel showed differential binding patterns to each of the cell types, displaying cross-reactive behavior. During the course of this research, we also developed a novel ratiometric method of using aptamer count derived from next-generation sequencing as a method for discrimination. This is in lieu of the more commonly used fluorescent signals. Finally the use of multiple signals for pattern recognition routines was further explored by running various models using artificial data. Various situations were applied to replicate different possible situation which might arise when working with macromolecular interactions. The purpose of this was to advance the communities understanding and ability to interpret results from the pattern recognition methods of PCA and LDA.Item Cell-Free Formation of RNA Granules(2012-07-16) Han, Tina Wei; Mcknight, Steven L.Asymmetric RNA localization is a mechanism by which a cell can spatially and temporally regulate the translation of RNAs. This mechanism is essential for many developmental processes such as germ cell formation in Drosophila embryos, as well as establishment of cell polarity and synaptic plasticity in the brain. In many instances, asymmetric RNA localization is achieved through transport and sequestration by RNA granules. RNA granules are large, non-membrane bound ribonucleoparticles that have been observed in various biological contexts. Unfortunately, because RNA granules are highly heterogeneous and weakly associating aggregates, they can be difficult to study biochemically, which constitutes a major impediment for gaining a more detailed understanding of the mechanisms governing RNA granule assembly. Here we describe two in vitro models for studying RNA granule assembly. The first method is based on the precipitation activity of a 3,5-disubstituted isoxazole compound that can be used as a quick and efficient pharmacological tool to probe the function and regulation of RNA granules. The second method utilizes a three-dimensional protein-retaining hydrogel formed from a recombinant protein. Polypeptides of low amino acid complexity were found to be the sequence determinants of isoxazole precipitation and hydrogel retention. Next generation sequencing was used to identify RNAs that partitioned with granule components in both isoxazole and hydrogel models and were found to be enriched in mRNAs known to be constituents of neuronal transport granules for dendritic localization. The overrepresented gene ontology categories for these RNAs included cell adhesion, extracellular matrix, and synaptic proteins. The average length of the 3’UTR of these RNAs was found to be longer than the 3’UTRs of RNAs excluded from the cell-free RNA granule preparations. These two in vitro models for studying RNA granule assembly offer a novel approach to identify candidate targets recruited to RNA granules by specific RNA-binding proteins. [Keywords: RNA granules, low complexity sequence, hydrogel, isoxazole]Item Characterization of an Orphan Riboswitch: Identification of a Metal-Sensing Regulatory RNA(2011-09-30T18:58:50Z) Wakeman, Catherine Ann; Winkler, Wade C.Riboswitches are RNA-based genetic control elements found in untranslated regions of the mRNA transcript that they regulate. These RNA motifs are highly structured and bind metabolites to elicit control of gene expression. Typically, the metabolite sensed by these RNAs is a component of the metabolic pathway in which the regulated gene product resides. The focus of this project has been the identification of the ligand for a riboswitch that was discovered using bioinformatics-based search methods. This riboswitch was designated the ykoK RNA element due to its location in the 5' UTR of the B. subtilis ykoK (mgtE) gene, which appears to be a magnesium transporter. Therefore, the possibility that this RNA senses magnesium levels was explored. The data revealed that the RNA element imparts magnesium-responsive regulation to the ykoK gene. These data also indicated which portions of the RNA are essential for genetic regulation. The results of a battery of biochemical tests demonstrated that magnesium triggers a concerted conformational change in the RNA such that it adopts a compacted tertiary structure. Resolution of the three-dimensional structure of the RNA in the magnesium bound state revealed the basis of this metal-induced tertiary conformation and how this relates to genetic control. Intriguingly, this structure revealed the presence of six magnesium ions, making this the first example of multiple ligands binding to a single riboswitch aptamer. When individual metal-binding sites were eliminated using phosphorothioate substitutions, it became evident that all six of these magnesium-binding sites and up to three additional metal-binding sites are required for function of this RNA. Therefore, these data demonstrate that the ykoK RNA element, now designated the M-box RNA, directly senses intracellular magnesium levels for the purposes of genetic control. These findings should have broad implications given that this RNA element is wide spread among Gram-positive bacteria and appears to regulate many additional gene categories such as ABC transporters, cell division proteins, and proteins of unknown function. The exploration of the connection between magnesium concentration and the expression levels of these proteins might provide insights into previously undefined functional roles. [Keywords: regulatory RNA; metal homeostasis; RNA structure; magnesium; riboswitch]Item Characterization of factors involved in 3' to 5' mRNA degradation in yeast(2005) Wang, Lingna; Johnson, Arlen W.Item Computational identification and evolutionaty enalysis of metazoan micrornas(2009-05-15) Anzola Lagos, Juan ManuelMicroRNAs are a large family of 21-26 nucleotide non-coding RNAs with a role in the post-transcriptional regulation of gene expression. In recent years, microRNAs have been proposed to play a significant role in the expansion of organism complexity. MicroRNAs are expressed in a cell or tissue-specific manner during embryonic development, suggesting a role in cellular differentiation. For example, Let-7 is a metazoan microRNA that acts as developmental timer between larval stages in C. elegans. We conducted a comparative study that determined the distribution of microRNA families among metazoans, including the identification of new family members for several species. MicroRNA families appear to have evolved in bursts of evolution that correlate with the advent of major metazoan groups such as vertebrates, eutherians, primates and hominids. Most microRNA families identified in these organisms appeared with or after the advent of vertebrates. Only a few of them appear to be shared between vertebrates and invertebrates. The distribution of these microRNA families supports the idea that at least one whole genome duplication event (WGS) predates the advent of vertebrates. Gene ontology analyses of the genes these microRNA families regulate show enrichments for functions related to cell differentiation and morphogenesis. MicroRNA genes appear to be under great selective constraints. Identification of conserved regions by comparative genomics allows for the computational identification of microRNAs. We have identified and characterized ultraconserved regions between the genomes of the honey bee (Apis mellifera) and the parasitic wasp (Nasonia vitripennis), and developed a strategy for the identification of microRNAs based on regions of ultraconservation. Ultraconserved regions preferentially localize within introns and intergenic regions, and are enriched in functions related to neural development. Introns harboring ultraconserved elements appear to be under negative selection and under a level of constraint that is higher than in their exonic counterparts. This level of constraint suggests functional roles yet to be discovered and suggests that introns are major players in the regulation of biological processes. Our computational strategy was able to identify new microRNA genes shared between honey bee and wasp. We recovered 41 of 45 previously validated microRNAs for these organisms, and we identified several new ones. A significant fraction of these microRNA candidates are located in introns and intergenic regions and are organized in genomic clusters. Expression of 13 of these new candidates was verified by 454 sequencing.Item Data mining techniques for classifying RNA folding structures(2016-08) Kim, Vince; Garg, Vijay K. (Vijay Kumar), 1963-; Gutell, Robin RRNA is a crucial biological molecule that is critical for protein synthesis. Significant research has been done on folding algorithms for RNA, in particular the 16S rRNA of bacteria and archaea. Rather than modifying current works on these folding algorithms, this report ventures into the pioneering works for data mining the same 16S rRNA. Initial works were based on a single complex helix across seven organisms. However, classification analysis proved to be inaccurate due to severe multicollinearity in the data set. A secondary data mining analysis was done on the entire RNA sequence of the same seven organisms, and was successfully used to sequentially categorically predict the characteristic of a given nucleotide in the RNA sequence.Item Design and evolution of synthetic biological systems(2006-08) Tabor, Jeffrey Jay; Ellington, Andrew D.The study of biology has undergone a fundamental change due to advancements in genetic engineering, DNA synthesis and DNA sequencing technologies. As opposed to the traditional dissective mentality of discovering genes via genetics, describing genetic behaviors through biochemistry, and then drawing diagrams of functional networks, researchers now have the potential (albeit limited) to construct novel biological molecules, networks, and even whole organisms with user-defined specifications. We have engineered novel catalytic DNAs (deoxyribozymes) with the ability to 'read' an input DNA sequence and then 'write' (by ligation) a separate DNA sequence which can in turn be detected sensitively. In addition, the deoxyribozymes can read unnatural (synthetic) nucleotides and write natural sequence information. Such simple nanomachines could find use in a variety of applications, including the detection of single nucleotide polymorphisms in genomic DNA or the identification of difficult to detect (short) nucleic acids such as microRNAs. As an extension of in vitro biological engineering efforts, we aimed to construct novel signal transduction systems in vivo. To this end, we used directed evolution to generate a catalytic RNA (ribozyme) capable of creating genetic memory in E. coli. In the end we evolved an RNA which satisfied the conditions of our genetic screen. Rather than maintaining genetic memory, however, the RNA increased relative cellular gene expression by minimizing the translational burden it imposed on the host cell. Interestingly, detailed mutational analysis of the evolved RNA led us to new studies on the relationship between ribosome availability and stochasticity in cellular gene expression, an effect that had frequently been alluded to in the literature, yet never examined. We have also taken a more canonical approach to the forward engineering of biological systems with unnatural behaviors. To this end, we designed a protein-based synthetic genetic circuit that allows a community of E. coli to function as biological film, capable of capturing and recapitulating a projected light pattern at high resolution (theoretically 100 mexapixels). The ability to control bacterial gene expression at high resolution could be used to ‘print’ complex bio-materials or deconvolute signaling pathways through precise spatial and temporal control of regulatory states.Item Discovery and design of an optimal microRNA loop substrate(2013-05) Hwang, Tony Weiyang; Ellington, Andrew D.RNA interference, or RNAi, is a cellular mechanism that describes the sequence-specific post transcriptional gene silencing observed in plants, fungi, and metazoans, facilitated by short double-stranded RNAs and microRNAs (miRNAs) with sequence complementarity to target mRNAs. Many of the regulatory mechanisms of the RNAi pathway by which these small miRNAs are first processed, from primary transcripts to precursor miRNA stemloops and then to mature miRNAs, by the multiprotein complexes Drosha and Dicer, respectively, still remain unknown. Within the miRNA biogenesis pathway, there is strong evidence pointing to the terminal loop region as an important regulatory determinant of miRNA maturation. To further elucidate the terminal loop's exerted control over miRNA processing, we propose a combined in vitro / in vivo selection experiment of a randomized pri-miRNA terminal loop library in search of an optimally processed pre-miRNA substrate. Here, we report the isolation of a premiRNA terminal loop sequence that is favorably processed by Drosha in vivo but also functions as an effective cis-inhibitor of further pre-miRNA processing by downstream Dicer. This terminal loop also demonstrated modular properties of Dicer inhibition in two different miRNAs, and should prove useful in further elucidating the mechanisms of miRNA processing in context of a newly proposed Dicer cleavage model (Gu et al. 2012). In combination, these findings may have important implications in both Drosha and Dicer's direct role in gene expression and miRNA biogenesis, the regulatory proteins that modulate their respective functions, as well as the potential development of new design rules for the more efficient processing and targeting of miRNA-based technology and RNAi therapeutics.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 The evolution of RNA and the actin protein family(2012-05) Keller, Thomas E.; Bull, James J.; Wilke, C. (Claus); Juenger, Thomas; Meyers, Lauren A; Sawyer, Sara LIn my dissertation I have broadly studied the evolution of RNA as well as the phylogenetic history of the actin protein family. In the first chapter I examined how various evolutionary processes interact at high mutation rates, which led to simple prediction based on the strength of selection. In the second chapter, I tested mRNA secondary structure stability at the beginning of genes as a way of identifying whether putative genes might be functional or not. Finally, I reconstructed the phylogenetic history of the actin protein family in vertebrates, revealing that a novel isoform is actively evolving in contrast to the remaining protein isoforms.Item Heme-Based Oxygen Sensors of Commensal, Symbiotic, and Pathogenic Bacteria(2012-08-15) Tuckerman, Jason Robert; Gilles-Gonzalez, Marie-AldaDirect oxygen sensors are proteins that serve as "on-off switches" to cause reversible and adaptive changes in the activities of other proteins or genes, with great specificity in response to fluctuations in oxygen concentration. The heme-based oxygen sensors are a large class of direct oxygen sensors that feature direct binding of oxygen to a sensory heme-containing domain. This heme-binding region couples to a regulatory domain within the same polypeptide. The types of functionalities controlled by these oxygen-specific switches are diverse, and include the regulation of protein activities, gene expression, and second messenger elaboration. A primary focus of this work was the biochemical characterization of a pair of heme-based oxygen sensors involved in the control of the bacterial second messenger cyclic diguanylic acid (c-di-GMP) in Escherichia coli. We discovered that these enzymes, designated DosC and DosP, serve as a diguanylate cyclase and c-di-GMP phosphodiesterase pair that associate with components of the E. coli RNA degradosome in vivo. Importantly, one member of these degradosomes, PNPase, is a direct, high-affinity target of c-di-GMP. These findings imply that specialized oxygen-sensing degradosomes exist in E. coli. In these oxygen-sensing degradosomes cellular oxygen levels regulate PNPase processing of specific RNA transcripts via c-di-GMP. A secondary focus of this work was the characterization of a novel two-component system in M. tuberculosis involved in the non-replicating persistent phase of this bacterium in a typical TB infection. Here, the activities of two heme-containing histidine kinases, DosT and DevS, were discovered to be inhibited specifically by oxygen. As DosT and DevS are the primary regulators of the dormancy survival regulator (DosR/DevR) transcription factor, these results contributed a molecular explanation for the numerous observations linking oxygen and DevR to the dormancy phenotype of M. tuberculosis seen both in vitro and in vivo. [Keywords: oxygen sensor; c-di-GMP; histidine kinase; signal transduction; Escherichia coli; Bordetella pertussis; Mycobacterium tuberculosis]Item Identification and application of functional RNAs(2003) Hesselberth, Jay Richard; Ellington, Andrew D.RNA assumes many roles in nature. As a structural entity, it is responsible for proxying genetic information, ferrying amino acids and organizing ribosome architecture. RNA also participates in several genetic regulatory mechanisms. Catalytic RNAs are responsible for the faithful modification of unprocessed transfer, ribosomal and messeger RNAs, and the assembly of proteins from amino acid monomers may be performed by RNA-derived catalytic residues in the ribosome. Previously researchers have developed methods for the isolation of unnatural RNA ligands and enzymes from random sequence pools. We have used these techniques in order to develop novel biosensors and biosensor components. RNA ligands (aptamers) were isolated from a random sequence pool that bind and inhibit ricin A-chain, a ribosome-inactivating protein and proposed biological warfare agent. In addition, ribozymes that were previously engineered to respond to a set of ligands were incorporated into a sensor platform for the first demonstration of an RNA-based detection system for the simultaneous detection of diverse analyte classes including DNA, proteins, peptides and small-molecules. We have also developed a set of computational tools in order to identify functional RNAs from both random sequence pools and genomic sequence databases. A computationally-accessible model was conceived in order to describe and predict the structural rearrangements undergone by allosteric ribozymes upon ligand binding, and the model was in turn used to select novel allosteric ribozymes from random sequence pools. In addition, we have used a structure-based search strategy in order to characterize well-folded regions in mRNAs with the goal of discovering additional examples of a novel class of genetic regulatory element, the riboswitches.Item Improving RNA folding prediction algorithms with enhanced interactive visualization software(2016-08) Grant, Kevin Marcus; Markey, Mia Kathleen; Gutell, RobinSoftware improvements from this project will enable new algorithms for RNA folding prediction to be explored. Issues with capacity, extensibility, multi-tasking, usability, efficiency, accuracy and testing in the original program have been addressed, and the corresponding software architecture changes are discussed herein. Previously limited to just hundreds of helices, the software can now display and manipulate million-helix RNAs. Actions on large data sets are now feasible, such as continuous zooming. A new scripting interface adds flexibility and is especially useful for repetitive tasks and software testing. Structural analysis of RNA can be streamlined using the new mechanisms for organizing experiments, running other programs and displaying results (helices, or arbitrary text and images such as statistics). Finally, usability has been enhanced with more documentation, controls and settings.Item Mechanism of MDA5 Recognition of Short RNA Ligands and Crystal Structure of PepQ(2013-05-16) Watts, Tylan AubreyThe innate immune pathways that stimulate the expression of cytokines and proapoptotic factors in response to infection are triggered by the activation of the cytosolic receptors retinoic acid-inducible gene I (RIG-I) and melanoma differentiationassociated gene 5 (MDA5). Activation of both receptors occurs as a result of binding to RNA. MDA5 only recognizes double stranded forms of RNA, whereas RIG-I is capable of recognizing both single and double stranded RNA. In vivo, MDA5 is known to be stimulated by long (>1 kb) strands of RNA, forming filaments along the phosphate backbone. However, the manner in which MDA5 can recognize the terminal end of its RNA ligand is uncertain. I have examined the mechanism of binding of the MDA5 protein by comparing MDA5 binding to short (<18 bp) blunt RNA, 5? triphosphate RNA, and RNA with a 3? or 5? overhang. It is shown that while the MDA5 protein regulatory domain (RD) is essential for RNA recognition, the MDA5 RD only weakly recognizes short double stranded RNA ligands with overhangs or a 5? triphosphate group. The Cys951 residue was shown to disrupt stability of the MDA5 RD-RNA complex. Binding analyses were performed using a combination of SDS-PAGE, gel filtration analysis, and nondenaturing gel electrophoresis. In addition, structural data was gathered by crystallization of the MDA5 RD-RNA complex using X-ray crystallography. These results help to establish the manner in which MDA5 is regulated predominantly to the binding of long RNA ligands. Also included in this document is structural data on the dimer form of the PepQ protein from E. coli. PepQ is a highly conserved proline peptidase that has a secondary activity of hydrolyzing organophosphorus triesters, toxic compounds found in many pesticides. The PepQ protein was crystallized and analyzed by X-ray diffraction. The dimer interface was clearly defined within the structure and provides insight into how the active dimer forms from the PepQ monomer.Item Metal interactions and activities of truncated and extended hammerhead ribozyme constructs(Texas A&M University, 2007-04-25) Osborne, Edith MarieThe hammerhead ribozyme (HHRz) catalyzes a site-specific phosphodiester bond cleavage reaction that is enhanced by the presence of metal cations. Metal cations are thought to aid in the folding and possibly the catalytic mechanism of this ribozyme. The goal of this research is to characterize the activities and metal interactions of minimal and extended HHRz constructs using kinetic and spectroscopic studies. Metal binding to the cleavage site of the HHRz was probed using 31P NMR to monitor Cd2+ titrations of HHRzs with a phosphorothioate modification at the cleavage site. Either a 2'-F or a 2'-NH2 substitution at the nucleophile position was used to block cleavage. With a 2'-F, no metal binding to the cleavage site phosphate was observed. However, with a 2'-NH2 substitution, a large change in 31P chemical shift of the phosphorothioate peak suggests Cd2+ binding. A 2'-NH2 is a potential metal ligand, but a 2'-F is not. This suggests that a metal ion binds to the cleavage site phosphate when the 2' nucleophile position also provides a ligand. Minimal HHRzs with only one stem loop structure show little activity in presence of physiologically relevant concentrations of divalent cations. A kinetic and thermodynamic characterization of an extended HHRz derived from Schistosoma mansoni with loops in stems I and II was performed. High catalytic activity was observed with low concentrations of divalent cations, and loss of potential loop-loop interactions resulted in a large decrease in activity. An electrostatic surface plot of a HHRz crystal structure revealed an area of high negative electrostatic potential in the cleft between stems I and II with contributions from nucleotides U7, A6, and C17 of the HHRz that could serve to trap metal ions. To probe this putative metal site, kinetic studies of HHRz constructs with phosphorothioate substitutions 5' to U7 or C17 or with an A6 2'- OMe substitution were performed. Results of these studies suggest that a metal interaction at this site would include direct coordination to A6 2'-OH, but indirect interactions with the phosphates.Item Modeling the structure, dynamics, and interactions of biological molecules(2013-05) Xia, Zhen, active 2013; Ren, PengyuBiological molecules are essential parts of organisms and participate in a variety of biological processes within cells. Understanding the relationship between sequence, structure, and function of biological molecules are of fundamental importance in life science and the health care industry. In this dissertation, a multi-scale approach was utilized to develop coarse-grained molecular models for protein and RNA simulations. By simplifying the atomistic representation of a biomolecular system, the coarse-grained approach enables the molecular dynamics simulations to reveal the biological processes, which occur on the time and length scales that are inaccessible to the all-atom models. For RNA, an "intermediate" coarse-grained model was proposed to provide both accuracy and efficiency for RNA 3D structure modeling and prediction. The overall potential parameters were derived based on structural statistics sampled from experimental structures. For protein, a general, transferable coarse-grain framework based on the Gay-Berne potential and electrostatic point multipole expansion was developed for polypeptide simulations. Next, an advanced atomistic model was developed to model electrostatic interaction with high resolution and incorporates electronic polarization effect that is ignored in conventional atomistic models. The last part of my thesis work involves applying all-atom molecular simulations to address important questions and problems in biophysics and structural biology. For example, the interaction between protein and miRNA, the recognition mechanism of antigen and antibody, and the structure dynamics of protein in mixed denaturants.Item Models of RNA folding in planetary environments(2011-08) Sluder, Alan; Scalo, John M.; Milosavljevic, MilosMultiple lines of evidence suggest that RNA performed all of the biological functions in the first life forms on earth. These functions included cleavage, ligation, polymerization, recognition, binding, and replication. In order to perform these functions, populations of RNA molecules with unevolved sequences must have been able to fold into compact three dimensional shapes, in unregulated environments, and without the help of proteins. Folding into compact tertiary structures is difficult because of the high charge density of RNA. Consequently, the ranges of temperature, salinity, pH, and pressure that allow RNA to fold into functional shapes is very restricted. We use thermodynamic arguments and Brownian dynamics simulations to compute the range of these environmental parameters that will allow RNA to fold. This is a non-trivial calculation due to the formation of an ion atmosphere around RNA that reduces its electric field. The results can be used to clarify the environments in which the transition to life is possible. Our preliminary calculations suggest that environments with low temperatures ($0-50^\circ C$) and high salt concentrations (greater than 100mM) are the most favorable for unassisted RNA folding and thus the transition to RNA-based life. Applications of our results include determining the environments on early earth where life formed, assesing the habitability of Europa, Titan, and (using modeled parameters) extrasolar planets.Item Monitoring folding pathways for large RNAs using site-directed spin-labeling techniques(Texas A&M University, 2007-04-25) Zalma, Carre AlisonThe function of biomolecules is very sensitive to structure. Folding in proteins and nucleic acids is a hierarchical process progressing from primary to secondary, then tertiary, and finally, quaternary structures. RNA in its folded form performs a variety of biological activities. Obtaining intramolecular distance measurements makes it possible to generate structural models along the folding pathway that may be related to the overall function of the molecule. Distances can be measured by Site-Directed Spin-Labeling (SDSL), in which nitroxyl spin-label probes are attached and observed by EPR spectroscopy. Spin-labels can provide information concerning structure and conformational changes because they are particularly sensitive to molecular motion and interspin distances. Continuous-wave EPR spectroscopy has been commonly applied to detect and monitor nitroxide spin-label probes within biological systems. A previous published SDSL study from this laboratory investigated a 10-mer RNA duplex model system with spin-label probe succinimdyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-carboxylate; however, an increased spin-labeling efficiency was observed with an isocyanate derivative of tetramethylpiperidyl-N-oxy (TEMPO). In this thesis, a 4-isocyano TEMPO spin-label probe replaced the previously used succinimdyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-carboxylate in 10-mer SDSL studies. The influence of labeling with the 4-iscocyano TEMPO spin-label in a 10-mer RNA model system was investigated with thermal denaturation, Matrix Assisted Laser Desorption Time of Flight Mass Spectrometry (MALDI-TOF-MS), Electron Paramagnetic Resonance (EPR) spectroscopy, and reverse phase high performance liquid chromatography (RP-HPLC). In the 10-mer RNA duplex model system a 4-isocyano TEMPO spin-label is individually attached to one strand and two strands are annealed to measure distances. This methodology is limited to systems in which two oligonucleotides are annealed together. To circumvent this limitation and also to explore single-strand dynamics a new methodology was implemented, double spin-labeling. Double spin-labeled single-stranded RNA was investigated as a single-strand and within a duplex via MALDI-TOF-MS, EPR spectroscopy and RP-HPLC. A double spin-labeling strategy in this work will be applicable to large complex RNAs like Group I intron of Tetrahymena thermophilia.Item Mutation: lessons from RNA models(2008-05) Cowperthwaite, Matthew Cranston, 1973-; Meyers, Lauren AncelMutation is a fundamental process in evolution because affects the amount of genetic variation in evolving populations. Molecular-structure models offer significant advantages over traditional population-genetics models for studying mutation, mainly because such models incorporate simple, tractable genotype-to-phenotype maps. Here, I use RNA secondary structure models to study four basic properties of mutation. The first section of this thesis studies the statistical properties of beneficial mutations. According to population genetics theory, the fitness effects of new beneficial mutations will be exponentially distributed. I show that in RNA there is sufficient correlation between a genotype and its point mutant neighbors to produce non-exponential distributions of fitness effects of beneficial mutations. These results suggest that more sophisticated statistical models may be necessary to adequately describe the distribution of fitness effects of new beneficial mutations. The second section of this thesis addresses the dynamics of deleterious mutations in evolving populations. There is a vast body of theoretical work addressing deleterious mutations that almost universally assumes that the fitness effects of deleterious mutations are static. I use an RNA simulation model to show that, at moderately high mutation rates, initially deleterious mutations may ultimately confer beneficial effects to the individuals harboring them. This result suggests that deleterious mutations may play a more important role in evolution than previously thought. The third section of this thesis studies the global patterns of mutations connecting phenotypes in fitness landscapes. I developed a network model to describe global characteristics of the relationship between sequence and structure in RNA fitness landscapes. I show that phenotype abundance varies in a predictable manner and critically influences evolutionary dynamics. A study of naturally occurring functional RNA molecules using a new structural statistic suggests that these molecules are biased towards abundant phenotypes. These results are consistent with an "ascent of the abundant" hypothesis, in which evolution yields abundant phenotypes even when they are not the most fit. The final section of this thesis addresses the evolution of mutation rates infinite asexual populations. I developed an RNA-based simulation model in which each individual's mutation rate is controlled by a neutral modifier locus. Using this model, I show that smaller populations maintain higher mutation rates than larger populations. I also show that genome length and shape of the fitness function do not significantly determine the evolved mutation rate. Lastly, I show that intermediate rates of environmental change favor evolution of the largest mutation rates.