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Thesis advisor: Michelle M. Meyer / Thesis advisor: Jeffery A. Byers / Riboswitches are a class of regulatory structures located in the 5’ untranslated region of many bacterial mRNAs. Validating riboswitch-ligand interactions has historically been costly and low-throughput. Recently, graphene field-effect transistors (G-FETs) have emerged as effective biosensors in detecting interactions of such regulators with charged, high molecular weight analytes. However, a bottleneck still exists in detecting relatively neutral small molecules. The Bacillus subtilis guanine riboswitch (Xpt) within the xpt-pbuX operon contains a purine-responsive aptamer region with affinity for guanine, hypoxanthine, and other purine analogs. The G-FET sensor revealed successful detection of Xpt-hypoxanthine interactions at saturating concentrations. The specificity of Xpt was also demonstrated by a lack of signal detection when incubated with adenine. Therefore, such G-FET devices are effective in detecting aptamer binding to small, electrically-neutral molecules, which will allow for rapid screening of potential therapeutic ligands. Further, different electrical observations of n-doping upon aptamer functionalization and p-doping upon ligand binding reveal unique interactions at the graphene surface. Molecular dynamics simulations were carried out to interpret experimental results and to determine if another well characterized aptamer (FMN) is a suitable candidate for G-FET studies. Trajectory data from the Xpt aptamer domain complexed with hypoxanthine (PDBID: 4FE5) and guanine (PDBID: 1Y27) showed significant differences in root mean square deviation (RMSD) and radius of gyration (Rg) from their respective non-binding mutants. These findings provide evidence that compaction of the RNA phosphodiester backbone is responsible for graphene detection. RMSD and Rg differences from FMN (PDBID: 3F4E) indicate that this aptamer may not show a significant change in G-FET signal. These findings suggest that G-FET biosensors can provide an avenue for the discovery of novel antibiotics for aptamer targets to combat burgeoning antibiotic resistance. / Thesis (BS) — Boston College, 2021. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biochemistry.

Antibiotic resistance has become a major problem in the United States. Approximately 2 million people are affected by hospital-acquired infections. Each year about 90,000 people are killed from them. Of the infections 70% of them are resistant to at least one drug. In order to trigger antibiotic resistance in bacteria, the antibiotics need to be detected by sensors in the bacteria. Riboswitches may act as toxin sensors in bacteria. Riboswitches are RNA aptamers that regulate gene expression via allosteric structural changes triggered by binding of a small molecule. Most identified riboswitches specifically recognize the metabolic product of the gene to be regulated. When the concentration of the metabolite reaches its threshold it binds to the riboswitch causing a structural change that in most cases turns off transcription or translation of the metabolite-producing gene. The ykkCD riboswitch appears to recognize the antibiotic, tetracycline to up-regulate expression of an efflux pump (also called ykkCD) that exports toxic drugs from the bacterial cell. In this work we present initial characterization of the previously uncharacterized ykkCD riboswitch. With the help of tetracycline derivatives and mutagenesis studies on the riboswitch we will (1) determine the substrate specificity of this riboswitch; (2) assess the importance of aromatic character and/or functional groups in antibiotic recognition. To achieve this goal we have developed a fluorescent binding assays. The binding assays will measure the binding affinity (Kd) of the riboswitch-antibiotic complex. Since substrates of the efflux pump are toxic to the bacterial cell, we posit that the ykkCD riboswitch might work as a toxin sensor and could serve as a target in the fight against bacterial pathogens. Afterwards we will evaluate how the ykkCD riboswitch sensor works in vivo. In order to do this we will have to quantify the amount of protein produced in the presence of tetracycline derivatives and mutant sensors. However quantifying the level of a particular protein in the cell is difficult so instead we replace the sequence of the efflux pump with the B-gal gene in B subtilis cell and quantify B-gal enzymatic activity using a colorimetric assay. This is a widely used technique in which the fluorescence corresponds to how much protein is produced. / Department of Chemistry

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Riboswitches are naturally occurring RNA aptamers that form a precise three-­dimensional structure and selectively bind to cellular target molecules. Binding of the target molecule initiates an allosteric structural change in the riboswitch that in turn regulates expression of a relevant target gene. Most riboswitches specifically recognize the metabolic product of the gene that is being regulated. Expression may be regulated at either transcription or translation stage of gene expression. Most riboswitches are off switches meaning they turn off expression of metabolite producing gene when metabolite concentration is high enough. The ykkCD putative riboswitch appears to increases production of an efflux pump that expels toxic drugs from the cell by binding to the antibiotic tetracycline. Based on previous data collected the ykkCD putative riboswitch seems to regulate the efflux pump at the transcriptional level. To confirm this hypothesis we want to map the structural change that takes place upon binding of the antibiotic tetracycline to the mRNA. Nucleic acid footprinting studies will be used to map the binding site of tetracycline and the allosteric change that takes place upon tetracycline binding. / Department of Chemistry

Natural products have long furnished medical science playing a significant role in drug discovery and development. Their importance notwithstanding, it is estimated that less than 1% of microorganisms can be cultivated from environmental sources using standard laboratory techniques. It is therefore necessary to develop biochemical and genetic techniques to access these uncultivable genomes. Here as a point of departure toward this goal, two cDNA libraries of two microorganisms were constructed along with five fosmid libraries with DNA isolated from marine environmental samples. We describe the construction of cDNA libraries from marine microbial species and detail experiments to exploit these libraries for their natural product biosynthetic pathways and other metabolic enzymes they harbor. However, no useful biosynthetic pathways were detected within the cDNA libraries. Genetic selection by complementation was additionally explored as a method to identify and localize biosynthetic gene clusters within marine microbial DNA libraries. Genetic selection is a fast and economic method which utilizes selection of a part of a pathway to represent the presence of an entire pathway for the complementation of known mutant strains. We describe genetic selection to localize biotin biosynthetic pathways of Hon6 (Chromohalobacter sp.) as a proof of principle experiment for the identification and localization of biosynthetic pathways in general. Instead of developing purification methods or manipulating cultivation conditions, large fragments of non-culturable bacterial genomes can be cloned and expressed using recombinant DNA technology. A strong transcriptional promoter to control high-level gene expression is required in recombinant expression plasmids. We aimed to develop new tools to control gene expression through the use of riboswitches. Riboswitches are metabolite-sensing ribonucleic acid (RNA) elements that possess the remarkable ability to control gene expression. The thiamine pyrophosphate (TPP) riboswitch system was chosen as it will enable use of E. coli as a suitable host strain. This system is particularly attractive because it has one of the simplest structures among the riboswitches elucidated to date. The use of the TPP riboswitch will also enable modulation of pathway gene expression by varying the TPP coccentration as many gene products are toxic. The violacein gene cluster from Chromobacterium violaceum was selected and placed under the control of this riboswitch. We describe modulation of heterologous gene expression by the ThiC/Riboswitch and detail experiments to investigate the expression and manipulation of the gene cluster under various promoters.

Multi-drug resistance among bacterial pathogens can be mediated by a number of mechanisms, including multidrug efflux pumps. One such pump in Bacillus spp. is ykkCD, a heterodimer of the SMR family consisting of C and D subunits. Previous studies suggest that the expression of ykkCD is controlled by a putative riboswitch and that the antibiotic tetracycline binds to the riboswitch in vitro. Additional studies have shown that two derivatives of tetracycline also bind to the putative riboswitch. These findings now need to be validated by an in vivo study. In this study, the effects that tetracycline and its commercially available derivatives—doxycycline, minocycline, anhydrotetracycline, and oxytetracycline—have on the expression levels of the ykkCD gene in Bacillus subtilis were explored. The level of ykkCD expression was quantified using two different methods: (1) ykkCD protein levels was determined using a ykkCD RNA--galactosidase reporter gene construct and (2) ykkCD mRNA levels was quantified by quantitative RT-PCR. Although the findings from method (1) were inconclusive, upregulation was observed for tetracycline and minocycline, in agreement with the results of the previous binding studies. / Department of Chemistry

Les riboswitches sont des modules structurés dans les régions 5’ (voire 3’) UTR des ARNm. Chaque riboswitch reconnaît spécifiquement un petit métabolite, ce qui provoque un changement de conformation et permet de moduler au niveau transcriptionnel ou traductionnel (voire par épissage alternatif) l’expression d’un gène impliqué dans la synthèse du métabolite.Ce travail portait sur la cinétique de repliement des domaines aptamères de deux riboswitches homologues liant la thiamine pyrophosphate (TPP), un régulant la transcription du gène thiC (E. coli) et l’autre régulant l’épissage alternatif du gène THIC (A. thaliana).La première approche utilisée (méthode cinétique classique par quenched-flow) consistait à sonder la structure des aptamères par des radicaux hydroxyles au cours du repliement initié par l’addition de TPP. Nous avons également développé (coll. avec A.Griffiths), une approche microfluidique visant à remplacer l’appareillage classique et, à terme, de le dépasser en permettant d’augmenter le nombre d’entrées pour l’étude de systèmes complexes. Nous avons aussi utilisé une méthode (kinITC) récemment développée au laboratoire qui permet d’obtenir des informations thermodynamiques et cinétiques inédites par microcalorimétrie isotherme.Nos résultats ont montré que l’aptamère bactérien se replie beaucoup plus vite que celui d’A. thaliana. Cependant, l’aptamère d’A. thaliana étudié dérivait de la forme sauvage (raccourcissement de l’hélice P3). Par comparaison avec d’autres travaux récents, nos résultats soulignent le rôle fondamental de P3 dans la cinétique de repliement. La méthode kinITC a aussi mis en évidence que le régime cinétique du fonctionnement global du riboswitch de E. coli n’est pas contradictoire avec une première étape de fixation du TPP sous régime thermodynamique.Les résultats obtenus avec la nouvelle méthode de microfluidique sont mitigés. Si nous avons pu reproduire le schéma de coupure de l’ARN lié au TPP obtenu par sondage chimique classique (valide ainsi la première étape de ce développement), la cinétique de repliement observée est plus rapide sans que nous en ayons pour le moment une explication satisfaisante. / Riboswitches are RNA modules found in the 5’-UTR of bacterial mRNA where they control gene expression at the transcriptional or translational level. They are occasionally found in the 3’-UTR where they control alternative splicing. Each riboswitch-controlled gene is involved in the biosynthetic pathway of a metabolite and a feedback loop is ensured by the specific binding of the metabolite.To study the folding kinetics of TPP-binding riboswitch aptamer domains from E.coli (regulating transcription of thiC) and from A. thaliana, (regulating alternative splicing of THIC) two different approaches were used. First, each aptamer structure was probed by hydroxyl radical footprinting during RNA folding triggered by TPP . We also developed (coll. with A. Griffiths) a microfluidic approach aimed at replacing the classical quenched-flow apparatus and, eventually, superseding it by giving access to more entries. We also used a method recently developed in our lab (kinITC), which gives access to rich kinetic and thermodynamic information from isothermal titration calorimetry.Our results showed that the E. coli aptamer folds much faster than its A. thaliana counterpart. However, the form that we used for the latter had a helix P3 shorter than that of the wt and, when compared with recent results, our results highlight the fundamental role of this helix in the kinetics of folding. It was also clear that, globally, the E. coli riboswitch is kinetically controlled but the kinITC method allowed us to show that this is not in contradiction with a thermodynamic control of the first TPP binding step.The results obtained with the microfluidic device are mitigated. We were indeed able to make the proof of concept of hydroxyl RNA probing on a microfluidic chip, but the kinetics of RNA folding appeared to be faster than that observed with the quenched flow. We are not yet able to propose an explanation for this strange fact.

<p>Small molecule metabolites have important and diverse roles in every major cellular function. To study the activities of metabolites and the biological processes in which they are involved, it is important to be able to detect their levels within cells. Technologies that measure the concentrations of small molecules within the context of living, growing cells are highly advantageous but are challenging to produce. In this thesis, a novel class of intracellular small molecule sensors is produced, characterized and applied to address novel and relevant research questions. These sensors detect a specific target molecule within bacterial cells using RNA regulatory elements known as riboswitches and one of many possible reporter proteins. In addition to a project that yielded new methodology to create custom riboswitches, two projects that assess the capabilities of sensors that detect an active form of vitamin B12 are described. These projects present an abundance of data that provide novel insights into the transport and metabolism of vitamin B12 in <em>E. coli</em> cells. Overall, the results presented indicate that riboswitch-based sensors represent valuable and unique tools for the study of microbial biology. The thesis is concluded with a discussion that describes design strategies and several exciting potential applications for future riboswitch sensors.</p> / Doctor of Philosophy (PhD)

RNAs are biomolecules ubiquitous in all living cells. Usually, they fold into complex molecular structures, which often mediate their biological function. In this work, models of RNA folding have been studied in detail. One can distinguish two fundamentally different approaches to RNA folding. The first one is the thermodynamic approach, which yields information about the distribution of structures in the ensemble in its equilibrium. The second approach, which is required to study the dynamics of folding during the course of time, is the kinetic folding analysis. It is much more computationally expensive, but allows to incorporate changing environmental parameters as well as time-dependent effects into the analysis. Building on these methods, the BarMap framework (Hofacker, Flamm, et al., 2010) allows to chain several pre-computed models and thus simulate folding reactions in a dynamically changing environment, e. g., to model co- transcriptional folding. However, there is no obvious way to identify spurious output, let alone assessing the quality of the simulation results. As a remedy, BarMap-QA, a semi-automatic software pipeline for the analysis of cotranscriptional folding, has been developed. For a given input sequence, it automatically generates the models for every step of the RNA elongation, applies BarMap to link them together, and runs the simulation. Post-processing scripts, visualizations, and an integrated viewer are provided to facilitate the evaluation of the unwieldy BarMap output. Three novel, complementary quality measures are computed on-the-fly, allowing the analyst to evaluate the coverage of the computed models, the exactness of the computed mapping between the individual states of each model, and the fraction of correctly mapped population during the simulation run. In case of deficiencies, the output is automatically re-rendered after parameter adjustment. Statistical evidence is presented that, even when coarse graining the ensemble, kinetic simulations quickly become infeasible for longer RNAs. However, within the individual gradient basins, most high-energy structures only have a marginal probability and could safely be excluded from the analysis. To tell relevant and irrelevant structures apart, a precise knowledge of the distribution of probability mass within a basin is necessary. Both a theoretical result concerning the shape of its density, and possible applications like the prediction of a basin’s partition function are given. To demonstrate the applicability of computational folding simulations to a real-world task of the life sciences, we conducted an in silico design process for a synthetic, transcriptional riboswitch responding to the ligand neomycin. The designed constructs were then transfected into the bacterium Escherichia coli by a collaborative partner and could successfully regulate a fluorescent reporter gene depending on the presence of its ligand. Additionally, it was shown that the sequence context of the riboswitch could have detrimental effects on its functionality, but also that RNA folding simulations are often capable to predict these interactions and provide solutions in the form of decoupling spacer elements. Taken together, this thesis offers the reader deep insights into the world of RNA folding and its models, and how these can be applied to design novel biomolecules.