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 Beyond allostery: Catalytic regulation of a deoxyribozyme through an entropy-driven DNA amplifier(Journal of Systems Chemistry, 2010-10-01) Eckhoff, Grace; Codrea, Vlad; Ellington, Andrew D.; Chen, XiThe programmability and replicability of RNA and DNA have respectively enabled the design and selection of a number of allosteric ribozymes and deoxyribozymes. These catalysts have been adapted to function as signal transducers in biosensors and biochemical reaction networks both in vitro and in vivo. However, allosteric control of nucleic acid catalysts is currently limited by the fact that one molecule of effector (input) generally regulates at most one molecule of ribozyme or deoxyribozyme (output). In consequence, allosteric control is usually inefficient when the concentration of input molecules is low. In contrast, catalytic regulation of protein enzymes, as in protein phosphorylation cascades, generally allows one input molecule (e.g., one kinase molecule) to regulate multiple output molecules (e.g., kinase substrates). Achieving such catalytic signal amplification would also be of great utility for nucleic acid circuits. Here we show that allosteric regulation of nucleic acid enzymes can be coupled to signal amplification in an entropy-driven DNA circuit. In this circuit, kinetically trapped DNA logic gates are triggered by a specific sequence, and upon execution generate a peroxidase deoxyribozyme that converts a colorless substrate (ABTS) into a green product (ABTS•+). This scheme provides a new paradigm for the design of enzyme-free biosensors for point-of-care diagnostics.Item Characterization of factors involved in 3' to 5' mRNA degradation in yeast(2005) Wang, Lingna; Johnson, Arlen W.The pathways for eukaryotic mRNA degradation are composed of numerous interconnected elements. I have characterized the general roles of factors involved in 3’ to 5’ mRNA degradation pathway in the budding yeast Saccharomyces cerevisiae. The Ski2/3/8 complex [composed of a DEVH ATPase—Ski2p, a TPR (Tetratricopeptide Repeat) protein—Ski3p and a WD protein—Ski8p] and Ski7p (a putative GTPase) are essential to 3’ mRNA degradation. To better understand their role in mRNA decay, first, I explored the domain interactions within the Ski2/3/8 complex and between the Ski2/3/8 complex and Ski7p using a directed two hybrid approach combined with IP (immunoprecipitation) experiments. A model that describes all the interactions identified is presented. Since Ski2p belongs to the DEVH RNA helicase family, I then demonstrated the biochemical activity of the Ski2/3/8 complex in vitro. My results indicate that the Ski2/3/8 is a hetrotrimeric complex with the stoichiometry of 1:1:1. The complex contains ATPase activity, which is specific to Ski2p. The Ski2/3/8 complex also possesses 3’ to 5’ RNA helicase activity. The duplex unwinding activity is intrinsic to Ski2p and requires the ATPase activity of Ski2p. More importantly, the helicase acitivity of Ski2p is stimulated by the presence of a short poly(A) tail (A12). Interestingly, mutations in Ski2p changing DEVH to AEVA cause this protein to be dominant negative. The dominant negative phenotype is caused by outcompeting wildtype Ski2p for incorporation into the Ski2/3/8 complex and forming a large inactive complex with the exosome. To better understand the role of GTPase Ski7p in 3’ mRNA decay, a dominant negative screen of SKI7 was carried out. Further characterization of the identified dominant negative mutants of SKI7 revealed that these mutants altered the interaction with either the Ski2/3/8 complex or the exosome, supporting the idea that Ski7p functions as a mediator between the Ski2/3/8 complex and the exosome in 3’ to 5’ mRNA degradation.Item Computational and experimental studies of biomolecules(2018-10-08) Cheng, Sara Yuengee; Florin, Ernst-Ludwig; Ren, Pengyu; Gordon, Vernita; Marder, Michael; Russell, RickIntegrating experiments and computational modeling is critical for understanding the structure and dynamics of biomolecules. Beyond providing validation for experimental results, computational modeling, that incorporates accurate physical models and enhanced sampling methods, can provide insight into the mechanisms underlying experimental observations. I will present four projects where experiments and computational modeling were used together, to understand mechanisms underlying the structure and dynamics of biomolecules. The first project involves using enhanced sampling to improve the efficiency of calculating the hydration free energies of small molecules using a polarizable force field. These predictions are compared with a conventional free energy method, and excellent agreement is found between the methods. The second project involves using atomic molecular dynamics simulations to determine the molecular mechanism underlying the ability of nanosensor to detect point-mutations in a DNA sequence. By analyzing the nearest-neighbor hydrogen bonding profile, from simulations of the nanosensor, a molecular mechanism was proposed to explain the experimental data. The third project involves the incorporation of non-canonical hydrogen bonding in a RNA coarse-grained model in order to improve 3D structure prediction. This new model is applied to study the sequence-dependent stability of several RNAs including RNA G-quadruplexes. The final project involves the development of a new single-molecule assay to measure local transitions in nucleic acid structures using ultrashort DNA tethers. This project involves collaboration with an experimental biochemistry group to design the DNA tethers and to prepare single-molecule samples. All projects involve the development of new methods to understand the 3D structure and dynamics of biomoleculesItem Computational investigation of thermocycling as a means of improving folding success rates of single-stranded RNA(2013-04) Porter, Andrew; Barrick, JeffreyIn vitro selection has proven itself as a great tool for isolating functional single stranded RNA sequences from large random pools. However, the multitude of available folding pathways leave many potentially functional sequences in misfolded states. This research aimed to use kinetic folding simulations of single-stranded RNA to test thermocycling as a means of overcoming energy barriers that prevent sequences from reaching a functional secondary structure. In this pursuit, I tested the negative effects of adding a number of random bases to each end of a known sequence, and showed that additional bases can drastically diminish the folding success of sequences that otherwise fold correctly at a very high rate. I then simulated thermocycling of a typically unsuccessful sequence and demonstrated that high temperatures break most or all base-pairs within a sequence, allowing refolding to occur.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 Exploring Variability in Reads from Next Generation RNA-Sequencing Data(2015-05) Butler, Andrew; Wilke, ClausNext generation RNA-sequencing (RNA-seq) technologies have revolutionized the study of the transcriptome, allowing for estimation of RNA levels in a given population of cells or even in individual cells. The levels of mRNAs are most commonly examined as they are generally a good proxy for gene expression, an important variable in many studies. Much less commonly studied are the levels of tRNA. We investigated whether information about the levels of tRNA present in Escherichia coli provide useful information with regards to amino acid levels in the context of a larger metabolic characterization study. To this end, we found very little correlation but did observe several distinct patterns in tRNA read coverage that may suggest flaws in read mapping or sequencing techniques when dealing with this type of RNA. Further work should be pursued to determine whether these patterns are biologically relevant or simply an artifact of the current sequencing and processing techniques.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 Identification of human biomarkers for cocaine addiction diagnosis(2008) Romack, Rebecca; Mayfield, R. DayneAddiction is a result of long-lasting behavioral changes including tolerance (the need for escalating doses to achieve the same effect) and dependence (physical symptoms manifested during abstinence). It is estimated that 35.3 million Americans have tried cocaine at least once, 6.1 million have used in the past year, and 2.4 million have used it in the past month according to the National Household Survey on Drug Abuse report in 2006, indicating the need for accurate diagnosis of dependence among a large portion of the population. It is important to diagnose cocaine use and dependence by finding definitive markers, termed biomarkers, of such a disease state. For comparison, blood tests for the presence of cocaine are only effective for the 20 minutes to several hours that the drug remains in the body. At present diagnosis of cocaine dependence requires subjective psychological and physical testing and interviews. The goal is to use microarrays to identify a specific set of biomarkers which can reliably determine a diagnosis of cocaine addiction. The impact of this research includes development of novel pharmacotherapies to prevent addiction and relapse and to improve the quality of life and productivity of addicted and recovering individuals and their communities. This experiment uses global gene expression analysis to identify changes in the RNA of cocaine addicts versus non-addicted individuals. Total RNA is extracted from whole blood samples, purified, hemoglobin transcripts are removed, and RNA is quantified with Nanodrop spectroscopy. The RNA is the hybridized to oligonucleotide microarrays containing 49,000 genes. This project will ultimately produce useful data on diagnostic biomarkers of cocaine addiction. It can help clinicians to diagnose addicts and to determine what drugs a patient has been using long term. The project is a collaboration that will identify correlations between gene expression patterns and subject behavior as reported by UTMB Galveston and UTHSC-Houston, Page 3 thus enabling unambiguous diagnosis of addiction. My role in the project is to obtain high quality, ultra pure RNA from whole blood samples, with the goal of discovering biomarkers of cocaine addiction.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 Messenger RNA Technology Takeaways(2021-02-22) Maurice, SydneyItem 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 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.Item Porous silicon nanoneedles for intracellular delivery of small interfering RNA(2011-05) Chiappini Dottore, Ciro; Liu, Xuewu; Ferrari, Mauro, 1959-; Tasciotti, Ennio; Markey, Mia; Ruoff, Rodney; Zhang, XiaojingThe rational and directed delivery of genetic material to the cell is a formidable tool to investigate the phenotypic effects of gene expression regulation and a promising therapeutic strategy for genetic defects. RNA interference constitutes a versatile approach to gene silencing. Despite the development of numerous strategies the transfection of small interfering RNA (siRNA) is highly dependent on cell type and conditions. Direct physical access to the intracellular compartment is a promising path for high efficiency delivery independently of cell type and conditions. Silicon nanowires grant such access with minimal toxic effects, and allow intracellular delivery of DNA when actuated by atomic force microscope. These findings reveal the potential for porous silicon nanostructures to serve as delivery vectors for nucleic acids due to their porous nature, elevated biocompatibility, and biodegradability. This dissertation illustrates the development a novel platform for efficient siRNA transfection based on an array of porous silicon nanoneedles. The synthesis of biodegradable and biocompatible porous nanowires was accomplished by a novel strategy for electroless etch of silicon that allows anisotropic etch simultaneously with porosification. An ordered array of cone shaped porous silicon nanoneedles with tunable tip size, array density and aspect ratio was obtained coupling this strategy with patterned metal deposition and selective reactive ion etch. This process also granted control over porosity, nanopore size and flexural modulus. The combination of these parameters was appropriately optimized to ensure cell penetration, maximize siRNA loading and minimize cytotoxic effects. Loading of the negatively charged siRNA molecules was optimized by applying an external electric field to the nanoneedles under appropriate voltage conditions to obtain a tenfold increase over open circuit loading, and efficient penetration of the siRNA within the porous volume of the needles. Alternative surface chemistry modification provided a means for effective siRNA loading and sustained release. siRNA transfection was achieved by either imprinting the nanoneedles array chip over a culture of MDA-MB-231 cells or allowing the cells to self-impale over the needles. The procedures allowed the needles to penetrate across the cell membrane without influencing cell proliferation. siRNA was successfully transfected and was effective at suppressing gene expression.