Chemistry & Biochemistry Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2752
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Item EXPANDING THE TOOLKIT: STRUCTURE, DYNAMICS, AND DRUG INTERACTIONS OF THE “PRIMING LOOP” FROM HEPATITIS B VIRUS PRE-GENOMIC RNA BY SOLUTION NMR SPECTROSCOPY(2022) Olenginski, Lukasz Tyler; Dayie, Theodore K; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)RNAs are dynamic macromolecules that function as essential components of biological pathways that result in human disease, making them attractive therapeutic targets. Yet, RNA structural biology lags significantly behind that of proteins, limiting mechanistic understanding of RNA chemical biology. Fortunately, solution NMR spectroscopy can probe the structure, dynamics, and interactions of RNA in solution at atomic resolution, opening the door to their functional understanding. However, NMR analysis of RNA – with only four unique ribonucleotide building blocks – suffers from spectral crowding and broad linewidths, especially as RNAs grow in size. One effective strategy to overcome these challenges is to introduce NMR-active stable isotopes into RNA in an atom- and position-specific manner. Here, we outline the development of labeling technologies, their use in benefiting RNA dynamics measurements, and applications to study the structure, dynamics, and interactions of a conserved regulatory RNA stem-loop from hepatitis B virus that is critical for viral replication.Item ISOTOPIC LABELING STRATEGIES AND NMR METHODOLOGIES TO FACILITATE RNA STRUCTURAL AND DYNAMICS STUDIES: APPLICATIONS TO A LONG NON-CODING RNA FROM KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS(2020) Becette, Owen; Dayie, Kwaku T; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)RNAs are essential components of biological pathways that result in human disease making them attractive therapeutic targets. Currently, NMR spectroscopy is the only high-resolution technique capable of probing RNA interactions in solution. Although NMR spectroscopy is well-suited to characterize macromolecular interactions at atomic-level detail, the currently available isotopic labeling strategies and NMR methodologies are limited to relatively small RNAs (~ 30 nts, ~ 10 kDa). This size limitation is due to poor sensitivity and limited spectral resolution both of which worsen with increasing size. Here I present novel isotopic labeling schemes and NMR experiments to help expand the size limitations of NMR. These new technologies are then applied to characterize the structure and dynamics of a non-coding RNA from Kaposi’s sarcoma-associated herpesvirus (KSHV) that causes cancer in AIDS patients.Item THE DEVELOPMENT AND APPLICATION OF SITE-SELECTIVELY ISOTOPICALLY LABELED NUCLEOTIDES TO PROBLEMS IN NMR SPECTROSCOPY:STRUCTURAL INSIGHTS INTO THE EPSILON RNA AND TARGET COMPOUND INTERACTIONS(2017) Longhini, Andrew Paul; Dayie, Theodore K; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)RNA plays a central role in a multitude of cellular processes. Understanding the complex interplay between its structure and function is a requisite for understanding these cellular roles mechanisms of action. Herein we describe technologies that we have developed to help better study RNA structure and function via NMR. Our development of site-selectively isotopically labeled pyrimidine and purine nucleotides has reduced spectral crowding, eliminated problems associated with scalar coupling, and led to novel assignment protocols. We have applied these labels to a 61-nucleotide viral RNA element, HBV-ε. This RNA is central Hepatitis B’s viral life cycle. Its NMR resonances have been assigned and initial structure calculations have begun to show how it interacts with compounds screened to bind to an internal six nucleotide bulge.Item Analysis of Genetic Regulatory Mechanisms that Control Ethanolamine Utilization in Enterococcus faecalis(2017) Gebbie, Margo Page; Winkler, Wade C; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In this project, we studied the genetic regulatory mechanisms that affect utilization of ethanolamine, an abundant compound in the gastrointestinal environment. In Enterococcus faecalis, the ethanolamine utilization (eut) gene cluster encodes for a two-component regulatory system (TCS), comprised of a histidine kinase, EutW, which autophosphorylates upon sensing EA, and a cognate response regulator, EutV, which dimerizes upon receiving the phosphoryl group from EutW and binds the nascent transcript to prevent premature transcription termination. This TCS is responsible for coupling sensing of ethanolamine to production of eut transcripts. However, clues from other organisms had previously suggested that adenosylcobalamin (AdoCbl) might also be an important genetic regulatory signal for the E. faecalis eut genes. Indeed, we discovered a novel trans-acting noncoding RNA (EutX) that contained an AdoCbl-responsive riboswitch. Our data demonstrated that the riboswitch promotes a shortened form of EutX when cellular AdoCbl levels are replete. In contrast, a longer form is synthesized when AdoCbl levels are depleted. We demonstrated that structural motifs contained in the longer form of EutX act to sequester the EutV protein, preventing it from promoting transcription elongation of eut transcripts. These unexpected data revealed an important new type of regulatory mechanism for riboswitch RNAs. In support of this overall genetic regulatory model, we recapitulated the full genetic circuitry in a heterologous host. Using this system, we employed extensive site-directed mutagenesis to examine the functional importance of highly conserved EutV residues. This led to the identification of a cluster of positively charged residues, which we speculated are important determinants for RNA-binding activity. Consistent with this hypothesis, mutations of these residues resulted in loss of RNA-binding activity. Furthermore, we also explored whether the eut gene cluster was affected by additional genetic regulatory mechanisms. From these efforts, we concluded that oxygen is not a genetic regulatory feature of eut genes, in contrast to previously published speculation. However, we did find that it is likely to be repressed under conditions of high glucose. Therefore, these aggregate studies revealed new mechanisms of post-initiation genetic regulation, and showed how E. faecalis specifically controls expression of ethanolamine catabolism genes.Item QUANTITATIVE ANALYSIS OF INTACT PROTEINS AND RNAS CARRIED BY IMMUNOSUPPRESSIVE EXOSOMES(2016) Geis Asteggiante, Lucia Giorgina; Fenselau, Catherine; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Myeloid-derived suppressor cells (MDSC) are immature myeloid cells that accumulate in the tumor microenvironment of most cancer patients. They are a major obstacle to immunotherapy because they suppress both adaptive and innate immune responses. MDSCs collected from tumor-bearing mice release nano-sized vesicles, called exosomes, which carry biologically active molecules and participate in intercellular communication. Exosomes released by MDSC stimulate migration of other MDSC towards the tumor microenvironment and convert macrophages to a tumor-promoting phenotype. Among the proteins identified in MDSC-released exosomes, S100A8 and S100A9 are low-mass, highly abundant, pro-inflammatory mediators already known to contribute directly to the immune suppressive functions of MDSC. The aim of this work was to successfully interrogate the exosomal intact protein cargo using top-down proteomics, a strategy for protein analysis that has not previously been applied to exosomes of any kind. Several protein forms (proteoforms) were fully characterized, which is critical as post-translational modifications regulate protein functions, cellular location and protein interactions. Additionally, since the tumor promoting activity of MDSC is enhanced by inflammation, we focused on evaluating the effect of increased inflammation on the proteoforms relative abundance using current top-down label-free quantitation techniques (peak intensities and peak areas), and comparing them to our recently validated spectral counting approach. Using spectral counting we were able to estimate differences in abundances of both S100A8 and S100A9 proteoforms. Furthermore, it has been previously reported that exosomes can carry micro RNAs and messenger RNAs. In order to investigate if MDSC-derived exosomes also contain RNAs, a collaborative study was carried out entailing the qualitative and quantitative analysis of miRNAs, mRNA and proteins present in MDSC and their exosomes, and evaluate their changes due to heightened inflammation. The MDSC and exosome protein cargo was analysed by bottom-up proteomics in this case, and the RNA cargo by next generation sequencing. A large number of mRNA and miRNA species were found to be carried by MDSC-derived exosomes and, strikingly, their putative functions were associated to MDSC expansion and suppressive function, and cancer development.Item The conformational landscape of RNA translational regulators and their potential as drug discovery targets(2016) LeBlanc, Regan Michael; Dayie, Theodore K; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)RNA is an underutilized target for drug discovery. Once thought to be a passive carrier of genetic information, RNA is now known to play a critical role in essentially all aspects of biology including signaling, gene regulation, catalysis, and retroviral infection. It is now well-established that RNA does not exist as a single static structure, but instead populates an ensemble of energetic minima along a free-energy landscape. Knowledge of this structural landscape has become an important goal for understanding its diverse biological functions. In this case, NMR spectroscopy has emerged as an important player in the characterization of RNA structural ensembles, with solution-state techniques accounting for almost half of deposited RNA structures in the PDB, yet the rate of RNA structure publication has been stagnant over the past decade. Several bottlenecks limit the pace of RNA structure determination by NMR: the high cost of isotopic labeling, tedious and ambiguous resonance assignment methods, and a limited database of RNA optimized pulse programs. We have addressed some of these challenges to NMR characterization of RNA structure with applications to various RNA-drug targets. These approaches will increasingly become integral to designing new therapeutics targeting RNA.Item NEW CHEMICAL TOOLS TO INVESTIGATE RNA FUNCTIONS(2013) Luo, Yiling; Dayie, Theodore K; Sintim, Herman O; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ribonucleic acid (RNA), as one of the essential macromolecules of life, plays an active role in gene regulation, catalysis, and signaling because of its ability to adopt complex 3D structures that can exist in multiple conformations. Until now, RNA preparation methods devised by most investigators utilized partially denatured the RNA. The mis-folding caused by denaturing - renaturing can seriously affect RNA structure and functional activity. To test this hypothesis, in PART I of this dissertation, we presented a simple strategy using `click' chemistry to couple biotin to a `caged' photocleavable guanosine monophosphate to synthesize native RNAs that are properly folded. We demonstrated that RNA ribozymes, ranging in size from 27 to 527 nt, prepared by our non-denaturing method form a homogenous population with superior catalytic activity than those prepared by traditional refolding methods. Having developed a method for in vitro RNA synthesis, we studied the riboswitch family of RNAs that require remolding their structure for function in PART II. C-di-GMP riboswitch, as the only currently known secondary messenger riboswitch, senses c-di-GMP using its aptamer domain to modulate the expression of genes which affects biofilm formation and virulence factors production in bacteria. To specifically target pathogenic bacteria in polymicrobial systems that control the RNA-mediated c-di-GMP signaling pathway through riboswitch regulation, different c-di-GMP analogs have been used as chemical tools to investigate the structure-activity relationship (SAR) of c-di-GMP binding to different c-di-GMP riboswitches. We demonstrated that different 2'-position modified c-di-GMP analogs could differentiate between two different classes of c-di-GMP riboswitches and even bind to one particular riboswitch in class I with different affinities. Specifically, Clostridium tetani (Ct-E88) RNA in class I c-di-GMP riboswitch was used for (structural dynamic) study to understand how ligand binding drives the conformational change to regulate the downstream genes within the expression platform. Our preliminary data obtained via different biochemical and biophysical tools - such as selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE), small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy - demonstrated that Mg2+ ions accelerate ligand recognition by pre-organizing the RNA, and then rapid ligand binding folds the RNA into a compact structure for likely downstream gene regulation.