Browsing by Subject "RNA"
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Item A Lattice Path Algorithm for RNA Secondary Structure Prediction: an Application to a Yellow Fever Virus Molecule(2018) Kutbi, Ayat; Nkwanta, Asamoah; Computer Science and Bioinformatics Program; Master of ScienceBioinformatics algorithms and tools have been established within explicit contexts for experimental modeling and data analysis. In this thesis, a lattice path algorithm based on RNA combinatorics is proposed for RNA secondary structure prediction. The developed lattice path algorithm graphically represents a certain subset of lattice path, a type of combinatorical objects enumerated by the RNA numbers, that are related to RNA secondary sequences which are composed of the RNA alphabet {A,U,G,C}. The algorithm supports visualizing all possible predicted foldings of an RNA sequence. The algorithm is applied to an RNA subsequence of a conserved structure of the 3' untranslated region of the yellow fever virus. The thermodynamic algorithm, RNAfold, is utilized to obtain minimum free energy and base pair probabilities of the predicted folds. Constraints are applied to enforce particular RNA secondary structure foldings. By applying the lattice path algorithm and RNAfold, the results show additional possible folds of the target RNA sequence. Results illustrate new predicted secondary sequences for the conserved structure of the 3' untranslated region of the yellow fever virus. The lattice path algorithm correctly predicted the secondary structure of an RNA subsequence derived from the peptidyl transferase center region of a ribosomal RNA. Subject to further validation, the algorithm could be applied to the prediction of mucleic acids aptamers for different medical applications. With a precise computational approach and a rigorous model of bioinformatics algorithms, RNA secondary structure prediction can be achieved with this approach, which can be valuable for understanding RNA structure and function.Item Assigning NMR spectra of RNA, peptides and small organic molecules using molecular network visualization software(Springer Netherlands, 2019-07-19) Marchant, Jan; Summers, Michael F.; Johnson, Bruce A.NMR assignment typically involves analysis of peaks across multiple NMR spectra. Chemical shifts of peaks are measured before being assigned to atoms using a variety of methods. These approaches quickly become complicated by overlap, ambiguity, and the complexity of correlating assignments among multiple spectra. Here we propose an alternative approach in which a network of linked peak-boxes is generated at the predicted positions of peaks across all spectra. These peak-boxes correlate known relationships and can be matched to the observed spectra. The method is illustrated with RNA, but a variety of molecular types should be readily tractable with this approach.Item CHARACTERIZATION OF THE MONOMERIC CONFORMATION OF THE HIV-1 5' LEADER(2015-01-01) Monti, Sarah Ann; Summers, Michael F.; Chemistry & Biochemistry; BiochemistryThe structured 5' leader (5'-L) of the HIV genome regulates several important steps in the HIV life cycle and is the most highly conserved region of the genome. Previous work identified that the 5'-L exists in two mutually exclusive conformations: a monomeric and a dimeric conformation. This work focuses on enhancing our understanding of the monomeric conformation of the 5'-L, and its role in the HIV life cycle. Structural studies of the 5'-L are hindered by its large size: at 356 nucleotides it is approximately thirteen times larger than the average nuclear magnetic resonance (NMR) derived RNA structure of 27 nucleotides. To overcome these difficulties, a novel NMR strategy, long-range probing by Adenosine Interaction Detection (lr-AID), was utilized to identify the interactions in the 5'-L that stabilize the monomeric conformation. In contrast to previous predictions, we discovered that the monomeric conformation of the 5'-L is stabilized by sequestration of the dimerization initiation site (DIS) in base pairing with residues 105 through 109 of the unique 5' region. Identification of the base pairing interactions that stabilize the monomeric conformation allowed the structure of the monomeric 5'-L to be probed by NMR. Two-dimensional proton-proton NOESY spectra of predicted secondary structure elements were compared to the spectrum of the monomeric 5'-L. The presence of a number of these elements including the top of the TAR stemloop, the intact DIS stem, the splice-donor stemloop, and an extended ? stemloop were confirmed by NMR. These findings map out large portions of the monomeric 5'-L secondary structure, allowing development of a smaller construct for structural studies of a monomeric core. Additionally, inconsistencies in the reported 5' transcription start site (TSS) of the HIV genomic RNA were identified. As some of the differences were reported to result from the presence of the 5'-7-methylguanosine cap, the capped 5'-L corresponding to each TSS was synthesized and compared. Studies of the monomer-dimer equilibrium of the capped 5'-L corresponding to the different TSSs revealed that the TSS affects the dimerization propensity of the 5'-L. Our increased understanding of the structure of the monomeric 5'-L provides insights into how this region regulates important events in the HIV life cycle.Item Developing a Quantitative Framework for Designing Responsive RNA Electrochemical Aptamer-Based Sensors and Applications(2015-01-01) Schoukroun-Barnes, Lauren R.; White, Ryan J; Chemistry & Biochemistry; ChemistryElectrochemical aptamer-based sensors utilizing structure-switching aptamers are specific, selective, sensitive, and widely applicable to the detection of a variety of targets. The specificity is achieved by the binding properties of an electrode-bound RNA or DNA aptamer biorecognition element that is a single-strand of DNA or RNA selected for in vitro to bind to a specific target molecule. Signaling in this class of sensors arises from changes in electron transfer efficiency upon target-induced changes in the conformation/flexibility of the aptamer probe. The changes in aptamer flexibility can be readily monitored electrochemically. The signaling mechanism enables several approaches to maximize the analytical attributes (i.e., sensitivity, limit of detection, and observed binding affinity) of the aptamer sensor. The work in this dissertations describes the quantitative effects of two different approaches to control sensor signaling in order to rationally tune sensor performance. The first part of this dissertations describes the effects of nucleic acid sequence and structure on the signaling of a representative small molecule aptamer-based sensor for the detection of aminoglycoside antibiotics. Modifying aptamer sequences to undergo large conformation changes upon target addition improves and maximizes E-AB sensor signaling because the collisional frequency and electron transfer rate of the 3?-attached redox molecule exhibits strong distance dependence. This dissertations also discusses the effects of stabilizing a folded structure of the aptamer to conserve the signal change, but reduce the binding affinity in order to shift the functional region of the sensor towards the therapeutic window of aminoglycoside antibiotics. Finally, with a newly developed family of aptamer sequences, tunable and predictable sensor responses achieved by employing different ratios of two aptamers with different affinities for the same target molecule on one sensor surface. The studies here were performed on a test bed aminoglycoside E-AB sensor, however the design criteria and framework established to tune sensor responses are generally applicable to any aptamer-based sensor. The second part of this dissertations explains the use of hydrogels to protect RNA E-AB sensors to enable use in complex media, such as whole blood, serum, plasma, etc.. The motivation is to bring the promising attributes of E-AB sensors to the clinic or bedside for real-time therapeutic drug monitoring. However, RNA E-AB sensor application has been limited as a result of degradation of the RNA sensing element in biological samples. To improve E-AB sensor function in complex samples, this work describes the development of a biocompatible hydrogel material to protect the oligonucleotides from degradation and inhibit non-specific absorption of proteins to the sensor surface ? both of which impede sensor function. Specifically, RNA sensors for aminoglycoside antibiotics were coated with a polyacrylamide hydrogel and tested in serum. Coating the RNA sensors with the hydrogel enabled sensor stability for a period of 3h in serum, which is a significant improvement from the uncoated sensors. The hydrogel coating also did not significantly affect E-AB sensor function based on the comparable titration curves of the uncoated and coated E-AB sensors. While sensor function and stability were tested specifically with aminoglycoside targets the technique employed to coat sensors with a hydrogel should be generally applicable to any small molecule E-AB sensor.Item Genome-Scale Analyses of Escherichia coli and Salmonella enterica AraC Reveal Noncanonical Targets and an Expanded Core Regulon(American Society for Microbiology, 2013-11-22) Stringer, Anne M.; Currenti, Salvatore; Bonocora, Richard P.; Baranowski, Catherine; Petrone, Brianna L.; Palumbo, Michael J.; Reilly, Andrew A.; Zhang, Zhen; Erill, Ivan; Wade, Joseph T.Escherichia coli AraC is a well-described transcription activator of genes involved in arabinose metabolism. Using complementary genomic approaches, chromatin immunoprecipitation (ChIP)-chip, and transcription profiling, we identify direct regulatory targets of AraC, including five novel target genes: ytfQ, ydeN, ydeM, ygeA, and polB. Strikingly, only ytfQ has an established connection to arabinose metabolism, suggesting that AraC has a broader function than previously described. We demonstrate arabinose-dependent repression of ydeNM by AraC, in contrast to the well-described arabinose-dependent activation of other target genes. We also demonstrate unexpected read-through of transcription at the Rho-independent terminators downstream of araD and araE, leading to significant increases in the expression of polB and ygeA, respectively. AraC is highly conserved in the related species Salmonella enterica. We use ChIP sequencing (ChIP-seq) and RNA sequencing (RNA-seq) to map the AraC regulon in S. enterica. A comparison of the E. coli and S. enterica AraC regulons, coupled with a bioinformatic analysis of other related species, reveals a conserved regulatory network across the family Enterobacteriaceae comprised of 10 genes associated with arabinose transport and metabolism.Item Glyceraldehyde-3-phosphate dehydrogenase as a model to investigate protein-protein interactions, non-canonical protein-RNA interactions, and post-translational modifications(2016-01-01) White, Michael R.; Garcin, Elsa D; Chemistry & Biochemistry; ChemistryThe topic of this dissertations is a highly collaborative and multifaceted look into clinically relevant aspects of the interactions of the homotetrameric enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This work details the importance of basic and translational research centered on GAPDH structure and function as the enzyme has been linked to several cardiovascular, diabetic, neurologic, and tumorigenic disorders. The project consists of three components, outlined below. (1) Developing a model system based on GAPDH to study biomolecular interactions using coherent X-ray scattering (BICXS). BICXS is a technique based on Small Angle X-ray Scattering (SAXS) signatures of gold nanoparticle (GNP) reporter agents that are indicative of an interaction between target biomolecules. This is of importance as there is a need for innovative methodologies to identify and characterize biomolecular interactions in vivo to complement those currently in use. Detailed here is a proof-of-concept study describing the utility of GNPs as reporter agents, demonstrating the process by which the fraction of interacting species within a sample can be determined. Also discussed is an analysis of the development of antibody conjugated GNPs targeted against GAPDH to test the ability of the technique in the identification and characterization of protein-protein interactions. (2) Elucidating the binding site and regulatory mechanisms of TNF-? mRNA in GAPDH for therapeutic intervention. GAPDH binds many clinically relevant mRNAs and alters their stability and/or translation. Most of these interactions occur via GAPDH binding to adenine-uridine rich elements (AREs) within their 3? untranslated regions (UTRs). The exact site and mechanism of binding, though, remain elusive. Through a collaborative approach, we found that GAPDH binds to the core AREs of the tumor necrosis factor-? mRNA 3? UTR via a sequential two-step mechanism. A single point mutation at the GAPDH dimer interface resulted in reduced binding affinity and structural alteration of the bound RNA. These results lead to the novel hypothesis that the dimer interface may be a crucial region for RNA binding. (3) Dveloping a high-throughput assay for the identification of small molecules that modulate GAPDH oxidoreductase activity. Due to the multifunctional nature of GAPDH and its crucial role in metabolism, it is a promising target for cancer therapy. Dithiolethione compounds possess chemopreventive, cytoprotective, and antiproliferative properties. In collaboration with researchers at the National Cancer Institute (NIH-NCI) we have determined that the dithiolethione, 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ACS1), disrupts glycolysis in cancer cells and modifies GAPDH. However, while ACS1 may modify GAPDH in cells, we definitively show that it does not affect GAPDH oxidoreductase activity in vitro. Accordingly, we have repurposed our high-throughput kinetic assay, and applied it to the identification of novel GAPDH modulators using the commercially available compound library LOPAC (library of pharmacologically active compounds) with the National Center for Advancing Translational Sciences (NIH-NCATS). From our initial screen we identified eight high efficacy inhibitors of GAPDH oxidoreductase activity that can be further validated and used in secondary assays.Item Investigation of HIV-1 Conserved Components for Genomic Recognition(2020-01-01) Swanson, Canessa Jordan; Summers, Michael F; Chemistry & Biochemistry; BiochemistryGenomic recognition for HIV-1 is an intricate process that requires the coordination of distinct intermolecular interactions between the 5'-leader (5'-L) of the viral genome and the RNA-binding nucleocapsid (NC) domain of the Gag polyprotein. Structural and biophysical studies in combination with in vivo packaging experiments identified the minimal packaging unit within the 5'-L, termed the ?CES (core encapsidation signal), which adopts a unique tandem three-way junction structure and is predicted to function as a nucleation site for Gag multimerization. Identification of the initial high affinity binding sites within the lower three-way junction of the ?CES, coined the ?3WJ-1 revealed a potential mechanism of selective RNA packaging. However, these findings, along with the vast majority of HIV-1 structural biology, stem from investigating the widely utilized laboratory strain known as NL4-3. As a means of interpreting the possibility of a structure-function relationship for the conserved 5'- L the present studies investigate another variant of HIV-1, MAL. Sequence alignment of the ?CES region of the two strains indicates a ninety-one percent sequence homology,while additional evaluation of the ?3WJ-1 illustrated higher sequence identity at ninety- five percent. Isothermal titration calorimetry (ITC) experiments identified that the binding isotherms for both strains are similar, indicating the sequence variations between MAL and NL4-3 do not perturb the mechanism of action for NC binding. Through the utilization of solution-state nuclear magnetic resonance (NMR), nucleotide-specific 2H-labeling, and residual dipolar coupling (RDC) measurements the three-dimensional structure of the MAL_?3WJ-1 was characterized, leading to a more comprehensive understanding of the complexities behind selective packaging of the viral genome.Item STRUCTURES AND FUNCTIONS OF THE HIV-1 RNA GENOME(2018-01-01) Brown, Joshua D; Summers, Michael F; Chemistry & Biochemistry; BiochemistryThe function and fate of the HIV-1 RNA genome is decided by its monomer-dimer equilibrium, which is regulated by the highly conserved 5' leader region. Recently, it was found that all lentiviral genomes are transcribed in infected cells from an integrated proviral DNA that contains a stretch of three sequential guanosines, any of which could potentially serve as the transcription start site. The 5�-capped genomes beginning with one guanosine (1G) favored dimerization and were selected efficiently for packaging. The 5�-capped 2G and 3G genomes favored the monomeric conformation and were enriched on polysomes, apparently preferred for translation and possibly for splicing. Using a nuclear magnetic resonance (NMR) approach and a variety of unique 2H-labeling schemes, we analyzed the start site region of the native 5' leader in its dimeric and previously elusive monomeric conformation. The additional guanosine(s) enables the disruption of the lower stem of the adjacent polyA stem loop, freeing up residues to base pair with and sequester the palindromic loop of the dimer-promoting DIS hairpin, thereby stabilizing the monomeric form of the RNA. We have confirmed multiple secondary structures within the native 5�-L monomeric structure and discovered an end-to-end hairpin stacking structure in the dimeric conformation that sequesters the cap and may attenuate translation due to its inability to bind to the translation promoting protein, eIF4E. We have also solved the three-dimensional structure of a 42 kDa HIV-1 capped RNA representing the start site of the dimeric 5�-L.