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Michalik, Juraj. "Non-redundant sampling in RNA Bioinformatics." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLX009/document.
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Un échantillonnage statistique est central à de nombreuses méthodes algorithmiques pour la bioinformatique structurale des ARNs, où ils sont couramment utilisés pour identifier des modèles structuraux importants, fournir des résumés des espaces de repliement ou approcher des quantités d'intérêt dans l'équilibre thermodynamique. Dans tous ces exemples, la redondance dans l'ensemble échantillonné est non-informative et inefficace, limitant la portée des applications des méthodes existantes. Dans cette thèse, nous introduisons le concept de l'échantillonnage non-redondante et nous explorons ses applications et conséquences en bioinformatique des ARN.Nous commençons par introduire formellement le concept d'échantillonnage non-redondante et nous démontrons que tout algorithme échantillonnant dans la distribution de Boltzmann peut être modifié en une version non-redondante. Son implémentation repose sur une structure de données spécifique et la modification d'une remontée stochastique pour fournir l'ensemble des structures uniques, avec la même complexité.Nous montrons alors une exemple pratique en implémentant le principe d'échantillonnage non-redondant au sein d'un algorithme combinatoire qui échantillonne des structures localement optimales. Nous exploitons cet outil pour étudier la cinétique des ARN, modélisant des espaces de repliement générés à partir des structures localement optimales. Ces structures agissent comme des pièges cinétiques, rendant leur prise en compte essentielle pour analyser la dynamique des ARN. Des résultats empirique montrent que des espaces de repliement générés à partir des échantillons non-redondants sont plus proches de la réalité que ceux obtenus par un échantillonnage classique.Nous considérons ensuite le problème du calcul efficace d'estimateurs statistiques à partir d'échantillons non redondants. L'absence de la redondance signifie que l'estimateur naïf, obtenu en moyennant des quantités observés dans l'échantillon, est erroné. Par contre, nous établissons un estimateur non-trivial non-biaisé spécifique aux échantillons non-redondants suivant la distribution de Boltzmann. Nous montrons que l'estimateur des échantillons non-redondants est plus efficace que l'estimateur naïf, notamment dans les cas où la majorité des l'espace de recherche est échantillonné.Finalement, nous introduisons l'algorithme d'échantillonnage, avec sa contre-partie non-redondante, pour des structures secondaires présentant des pseudonoeuds de type simple. Des pseudonoeuds sont typiquement omis pour des raisons d'efficacité, bien que beaucoup d'entre eux possèdent une grande importance biologique. Nos commençons par proposer une schéma de programmation dynamique qui permet d'énumérer tous les pseudonoeuds composés de deux hélices pouvant contenir des bases non-appariés qui s'entrecroisent. Ce schéma généralise la proposition de Reeders et Giegerich, choisi pour sa base complexité temporelle et spatiale. Par la suite, nous expliquons comment adapter cette décomposition à un algorithme d'échantillonnage statistique pour des pseudonoeuds simples. Finalement, nous présentons des résultats préliminaires et nous discutons sur l'extension de principe non-redondant dnas ce contexte.Le travail présenté dans cette thèse ouvre non seulement la porte à l'analyse cinétique des séquences d'ARN plus longues, mais aussi l'analyse structurale plus détaillée des séquences d'ARN en général. L'échantillonnage non-redondant peut être employé pour analyser des espaces de recherche pour des problèmes combinatoires susceptibles à l'échantillonnage statistique, y inclus virtuellement tous problèmes solvables par la programmation dynamique. Les principes d'échantillonnage non-redondant sont robustes et typiquement faciles à implémenter, comme démontré par l'inclusion d'échantillonnage non-redondant dans les versions récentes de Vienna package populaire<br>Sampling methods are central to many algorithmic methods in structural RNA bioinformatics, where they are routinely used to identify important structural models, provide summarized pictures of the folding landscapes, or approximate quantities of interest at the thermodynamic equilibrium.In all of these examples, redundancy within sampled sets is uninformative and computationally wasteful, limiting the scope of application of existing methods.In this thesis, we introduce the concept of non-redundant sampling, and explore its applications and consequences in RNA bioinformatics.We begin by formally introducing the concept of non-redundant sampling and demonstrate that any algorithm sampling in Boltzmann distribution can be modified into non-redundant variant. Its implementation relies on a specific data structure and a modification of the stochastic backtrack to return the set of unique structures, with the same complexity.We then show a practical example by implementing the non-redundant principle into a combinatorial algorithm that samples locally optimal structures. We use this tool to study the RNA kinetics by modeling the folding landscapes generated from sets of locally optimal structures. These structures act as kinetic traps, influencing the outcome of the RNA kinetics, thus making their presence crucial. Empirical results show that the landscapes generated from the non-redundant samples are closer to the reality than those obtained by classic approaches.We follow by addressing the problem of the efficient computation of the statistical estimates from non-redundant sampling sets. The absence of redundancy means that the naive estimator, obtained by averaging quantities observed in a sample, is erroneous. However we establish a non-trivial unbiased estimator specific to a set of unique Boltzmann distributed secondary structures. We show that the non-redundant sampling estimator performs better than the naive counterpart in most cases, specifically where most of the search space is covered by the sampling.Finally, we introduce a sampling algorithm, along with its non-redundant counterpart, for secondary structures featuring simple-type pseudoknots. Pseudoknots are typically omitted due to complexity reasons, yet many of them have biological relevance. We begin by proposing a dynamic programming scheme that allows to enumerate all recursive pseudoknots consisting of two crossing helices, possibly containing unpaired bases. This scheme generalizes the one proposed by Reeders and Giegerich, chosen for its low time and space complexities. We then explain how to adapt this decomposition into a statistical sampling algorithm for simple pseudoknots. We then present preliminary results, and discuss about extensions of the non-redundant principle in this context.The work presented in this thesis not only opens the door towards kinetics analysis for longer RNA sequences, but also more detailed structural analysis of RNAs in general. Non-redundant sampling can be applied to analyze search spaces for combinatorial problems amenable to statistical sampling, including virtually any problem solved by dynamic programming. Non-redundant sampling principles are robust and typically easy to implement, as demonstrated by the inclusion of non-redundant sampling in recent versions of the popular Vienna package
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Gil, Ley Alejandro. "Enhanced sampling and force field corrections for RNA oligomers." Doctoral thesis, SISSA, 2016. http://hdl.handle.net/20.500.11767/4628.
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The computational study of conformational transitions in nucleic acids still faces many challenges. For example, in the case of single stranded RNA tetranucleotides, agreement between simulations and experiments is not satisfactory due to inaccuracies in the force fields commonly used in molecular dynamics. Improvement of force fields is however hindered by the diiculties of decoupling those errors from the statistical errors caused by insuicient sampling. We here tackle both problems by introducing a novel enhancing sampling method and using experimental data to improve RNA force fields. In this novel method, concurrent well-tempered metadynamics are integrated in a Hamiltonian replica-exchange scheme. The ladder of replicas is built with different strength of the bias potential exploiting the tunability of well-tempered metadynamics. Using this method, free-energy barriers associated to individual collective variables are significantly reduced compared with simple force-field scaling. The introduced methodology is flexible and allows adaptive bias potentials to be self-consistently constructed for a large number of simple collective variables, such as distances and dihedral angles. Additionally, a modified metadynamics algorithm is used to calculate correcting potentials designed to enforce distributions of backbone torsion angles taken from experimental structures. Replica-exchange simulations of tetranucleotides including these correcting potentials show significantly better agreement with independent solution experiments for the oligonucleotides containing pyrimidine bases. Although the proposed corrections do not seem to be portable to generic RNA systems, the simulations reveal the importance of the α and ζ backbone angles for the modulation of the RNA conformational ensemble. The correction protocol presented here suggests a systematic procedure for force-field refinement.
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Buttner, M. "RNA polymerase - DNA interactions in Streptomyces." Thesis, University of Bristol, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.354445.
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Nandakumar, Jayakrishnan. "Discrimination of RNA versus DNA by an RNA ligase and distinct modes of substrate recognition by DNA ligases /." Access full-text from WCMC:, 2007. http://proquest.umi.com/pqdweb?did=1428838891&sid=13&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Gilea, Manuela Aurora. "DNA and RNA synthesis in ionic liquids." Thesis, Queen's University Belfast, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.485198.
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The solid-phase synthesis of oligonucleotide derivatives such as phosphorothioates and phosphoroselenoates was investigated. Some ionic liquids containing the trlbexyl(tetradecyl)phosphonium cation and various anions proved to be very effective in dissolving the chalcogens (sulfur and , selenium) and to prepare oligonucleoside chalcogenophosphates. The suitability ofionic liquid-based chalcogen-transfer mixtures for the synthesis of oligonucleoside chalcogenophosphates on solid-phase was evaluated and subsequently the structure-activity relationship studied in detail. The compatibility of ionic liquid-based chalcogen-transfer mixtures with diverse types of solid supports e.g. controlled-pore glass, poly(vinylacetate) and. different synthetic methods. e.g. phosphoramidite and H-phosphonate method makes them useful as replacement of the more expensive and relatively unstable commerciaily avai1able chalcogen-transfer reagents. The distillation of ionic liquids was also studied.
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Antson, Dan-Oscar. "Genotyping RNA and DNA using padlock probes." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2001. http://publications.uu.se/theses/91-554-5057-1/.
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Zhu, Jian. "The stabilities of RNA and DNA structural elements." Diss., Georgia Institute of Technology, 1998. http://hdl.handle.net/1853/25194.
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Hill, G. R. "NMR studies of DNA and RNA binding proteins." Thesis, University of Cambridge, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604060.
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HMG-D is a 112-residue, non-histone chromosomal protein from <i>Drosophila melanogaster </i>and is a member of the class of non-sequence specific HMGB proteins. The present project was based on the observation that other HMGB complexes that had been solved by NMR had a phenylalanine residue at a key interfacial location (corresponding to position 12 in HMG-D), whereas those like HMG-D that gave few intermolecular NOE cross peaks generally had a tyrosine at this location. This tyrosine was known to be involved in hydrogen-bonding to the DNA in a related complex that had been solved crystallographically. The Y12F mutant of full-length HMG-D was expressed and purified in isotope-labelled form suitable for NMR spectroscopy, and a set of multidimensional triple resonance experiments used to derive assignments for the backbond resonances of the protein both free and in complex with the dA<sub>2 </sub>bulge DNA. Sidechain assignments for the protein were obtained by a combination of “CCH”-transfer-based experiments and NOE spectra, while nearly complete assignments for the DNA in the complex were obtained from a combination of homonuclear 2D NOESY and TOCSY experiments together with filtered NOESY experiments where just cross peaks between protons both of which were not coupled to heteronuclei were selected. Filtered NOESY-based experiments were used to observe intermolecular NOE cross peaks in isolation, and, in contrast to the case of the wild-type complex, these experiments yielded around 50 intermolecular interactions. Together with an extensive set of assigned intramolecular NOE constraints, these formed the basis for a calculation of the structure of the complex starting from random conformations of both protein and DNA chains, which resulted in an NMR structure for the complex that had good precision over the structured region (residues 3-70 of the protein and stem 1 of the DNA).
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O'Hanlon, Karen Ann. "Studies on the enzyme DNA-dependent RNA polymerase." Thesis, University of Reading, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.266340.
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Pritchard, Hannah Louise. "Recognition agents for DNA and RNA quadruplex structures." Thesis, University of Birmingham, 2015. http://etheses.bham.ac.uk//id/eprint/5727/.
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The design and synthesis of a new class of G-quadruplex DNA recognition agents are discussed in this thesis along with their binding abilities to both duplex and G-quadruplex forming DNA. A selection of G-quadruplex binders reported in the literature to date have been reviewed and their interactions with the quadruplex DNA structure analysed. The biisoquinoline ligand used to incorporate into the metal complexes synthesised in this thesis was chosen because of its large aromatic surface area which is ideal for end stacking onto G-quartets. Both the palladium and platinum biisoquinoline complexes bind to quadruplex forming DNA, monitored by UV-vis and circular dichroism. The platinum complex has the most promising DNA binding results showing a selectivity for quadruplex DNA over duplex DNA when examined by gel electrophoresis. Biisoquinoline complex interactions with RNA G-quadruplexes have been investigated to make comparisons of that with DNA. The palladium complex binds less well to parallel quadruplex conformers suggesting its mode of interaction differs from that of the platinum complex. A toxicity assay against two cancer cell lines showed the platinum and palladium complexes to have IC\(_{50}\) values in the nM range. Modifications to the biisoquinoline structure were also attempted in order increase the specificity of the complex to G-quadruplexes by incorporating components that could interact with the quadruplex loops.
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MERONI, ALICE. "RNA IN DNA: FROM STRUCTURE TO GENOME INSTABILITY." Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/570097.
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The presence of RNA in the genome of living cells is one of the emerging topics of the last two decades and has been implicated in many biological processes. I focused my attention on ribonucleotides (rNMPs) embedded into DNA during genome duplication, as a threat to its integrity. In fact, rNMPs have been classified as the most frequent non-canonical nucleotides introduced during genome duplication by DNA polymerases. Such high incorporation frequency has been related to a physiological role in mismatch repair, but it can be easily turned into a source of genomic instability if rNMPs are not removed from DNA. This task is performed by RNase H activities that enable error-free repair of embedded single and multiple ribonucleotides.
I first approached the issue of ribonucleotides incorporation into DNA from a physical point of view. Utilizing Atomic Force Microscopy I studied how ribonucleotides intrusions impact on DNA structure. The results obtained provided new insights on the structural changes imposed by ribonucleotides persistence into DNA. The other part of my Ph.D. project concerned the study of rNMPs incorporation in vivo, using the budding yeast S. cerevisiae as a model organism. The second aim was to unravel the function of the Translesion Synthesis polymerase η (Pol η) when the genome contains residual ribonucleotides and when deoxyribonucleotides (dNTPs) pools are depleted. We found that DNA polymerase η is responsible for the cell lethality observed when dNTPs are scarce and RNase H activities are defective. Therefore, I explored and characterized this unexpected toxic activity. We propose a model where Pol η supports cell survival in low dNTPs conditions by promoting DNA replication using ribonucleotides. While this activity is normally beneficial to wild type cells, it is highly toxic to cells defective for RNase H activities.
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Cramer, Janina. "Funktionelle Charakterisierung der RNA-abhängigen RNA-Polymerase des Hepatitis-C-Virus Untersuchung molekularer Mechanismen der Substratspezifität von DNA-abhängigen DNA-Polymerasen /." [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=971700796.
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Daniel, Laurianne. "Human Ribosomal DNA and RNA Polymerase I Fate during UV-induced DNA Repair." Thesis, Lyon, 2017. http://www.theses.fr/2017LYSE1093/document.
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La réparation par excision de nucléotides (NER) garantit l'intégrité du génome lors de l'exposition aux rayons UV. Après irradiation aux UV, un des premiers problèmes rencontrés par la cellule est l'arrêt général de la transcription dû au blocage de l'ARN polymérase II (ARNP2) au niveau des lésions UV. Pour régler ce problème, le NER possède une voie de réparation spécifiquement couplée à la transcription (TCR). Les connaissances concernant le NER ont été obtenu via des études sur la transcription par l'ARNP2. Cependant, dans les cellules à fort métabolisme, plus de 60% de la transcription correspond à la transcription, dans le nucléole, de l'ADN ribosomique (ADNr) par l'ARN polymérase I (ARNP1). De nombreuses protéines sont absence du nucléole, c'est pourquoi certains processus nucléaires ne peuvent avoir lieu dans cette structure. Afin d ‘être répliqué et réparé, l'ADNr se déplace à la périphérie du nucléole. Malgré l'importance de la transcription par l'ARNP1, la réparation de l'ADNr a été peu étudiée chez l'homme. De plus, à notre connaissance, aucune étude ne s'est penchée sur le mécanisme moléculaire du déplacement de l'ADNr à la périphérie du nucléole. Notre étude démontre l'implication de la TCR dans la réparation de l'ADNr après lésions UV induites. De plus, nos recherches ont démontré que l'ARNP1 reste accrochée à l'ADNr et sont tous les deux délocalisés à la périphérie du nucléole après irradiation aux UV. Enfin, nous avons identifié l'actin et la moysine I nucléaires comme facteurs protéiques nécessaire à cette délocalisation<br>Nucleotide excision repair (NER) guarantees genome integrity and proper cellular functions against UV-induced DNA damage. After UV irradiation, one of the first burden cells have to cope with is a general transcriptional block caused by the stalling of RNA polymerase II (RNAP2) onto distorting UV lesions. To insure UV lesions repair specifically on transcribed genes, NER is coupled with transcription in an extremely organized pathway known as Transcription-Coupled Repair (TCR). Most of the knowledge about TCR has been gathered from RNAP2 transcription. However, in highly metabolic cells, more than 60% of total cellular transcription results from ribosomal DNA (rDNA) transcription, by the RNA polymerase I (RNAP1), which takes place in the nucleolus. Many nuclear proteins are excluded from the nucleolus and because of this some nucleolar processes cannot occur inside this structure. In order to be replicated and repaired rDNAs need to be displaced at the nucleolar periphery. Despite the importance of RNAP1 transcription, repair of the mammalian transcribed rDNA has been scarcely studied. Moreover, to the best of our knowledge no molecular mechanism has been proposed for rDNA displacement. Our study clearly demonstrated that the full TCR machinery is needed to repair UV-damaged rDNA and restart RNAP1 transcription. Our results show that UV lesions block RNAP1 transcription and that RNAP1 is firmly stalled onto rDNAs without being degraded. Our study also describes the displacement of the RNAP1/rDNA complex to the nucleolar periphery after UV irradiation and identifies both nuclear ß-actin and nuclear myosin I as factors required for this displacement
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Girlando, Elanie Michelle. "Sampling for airborne influenza virus using RNA preservation buffer : a new approach." Thesis, University of Iowa, 2014. https://ir.uiowa.edu/etd/1324.
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Characterizing airborne influenza virus exposure is important for infection prevention and exposure control in health care and public settings. Detecting airborne influenza virus is important in assessing infection risk. The virus must also be protected from deterioration during aerosol sampling and long term storage. RNA preservation buffers (RNAPBs) may stabilize influenza virus after sampling and during storage. Bioaerosol samplers are used to collect airborne influenza virus, and many different types of samplers are available.
The objectives of this experiment were to: 1) compare influenza virus concentrations across bioaerosol samplers; 2) compare the efficacy of RNAPB over Hanks Balanced Salt Solution (HBSS) as a sample collection media; and 3) determine whether RNAPB stabilizes viral particles stored over time. In this experiment we aerosolized active influenza virus (H1N1) in a bioaerosol chamber and compared sampling efficiencies using two different samplers: the SKC Biosampler and NIOSH Biosampler, and two different medias: Hanks Balanced Salt Solution (HBSS) and an RNAPB. Ten 15-minute experimental trials were completed. We also compared HBSS and RNAPB in terms of the maintenance of virus RNA integrity during storage at room temperatures. Samples were stored at room temperature for 1, 4, 7, and 14 days. Virus concentrations were measured and compared at each time point.
Significant differences were found between sampler and media type - the SKC Biosampler collected a higher concentration of virus than the NIOSH Biosampler, and HBSS collected a higher concentration of virus than RNAPB. In storage at room temperature conditions, RNAPB maintained virus in concentrations significantly greater than in HBSS. The results of this experiment indicates that the SKC Biosampler should be used to characterize airborne influenza and that RNAPB should not be used as a sampling media but can be used to preserve samples if needed.
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Jia, Yiping. "Mechanistic studies of DNA-dependent transcription initiation and RNA synthesis by bacteriophage T7 RNA polymerase /." The Ohio State University, 1998. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487953204281995.
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Ott, Reina Kristina. "Künstliche Nucleasen - Spaltung von DNA und RNA durch Übergangsmetallkomplexe." [S.l. : s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=962774057.
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Acharya, Sandipta. "Some Aspects of Physicochemical Properties of DNA and RNA." Doctoral thesis, Uppsala University, Department of Bioorganic Chemistry, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6741.
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<p>This thesis is based on nine research publications (<b>I – IX</b>) on structure and reactivity of RNA vis-à-vis DNA. The DNA and RNA are made of flexible pentose sugar units, polyelectrolytic phosphodiester backbone, and heterocyclic nucleobases. DNA stores our genetic code, whereas RNA is involved both in protein biosynthesis and catalysis. Various ligand-binding and recognition properties of DNA/RNA are mediated through inter- and intra-molecular H-bonding and stacking interactions, beside hydration, van der Waal and London dispersion forces. In this work the pH dependant chemical shift, p<i>K</i><sub>a</sub> values of 2'-OH group as well as those the nucleobases in different sequence context, alkaline hydrolysis of the internucleotidic phosphodiester bonds and analysis of NOESY footprints along with NMR constrained molecular dynamics simulation were used as tools to explore and understand the physico-chemical behavior of various nucleic acid sequences, and the forces involved in their self-assembly process. <b>Papers I – II</b> showed that the ionization of 2'-OH group is nucleobase-dependant. <b>Paper III</b> showed that the chemical characters of internucleotidic phosphate are non-identical in RNA compared to that of DNA. <b>Papers IV – VI</b> show that variable intramolecular electrostatic interactions between electronically coupled nearest neighbor nucleobases in a ssRNA can modulate their respective pseudoaromatic character, and result in creation of a unique set of aglycons with unique properties depending on propensity and geometry of nearest neighbor interaction. <b>Paper VII</b> showed that the cross-modulation of the pseudoaromatic character of nucleobases by the nearest neighbor is sequence-dependant in nature in oligonucleotides. <b>Paper VIII</b> showed that the purine-rich hexameric ssDNA and ssRNA retain the right-handed helical structure (B-type in ssDNA and A-type in ssRNA) in the single-stranded form even in absence of intermolecular hydrogen bonding. The directionality of stacking geometry however differs in ssDNA compared to ssRNA. In ssDNA the relatively electron-rich imidazole stacks above the electron-deficient pyrimidine in the 5' to 3' direction, in contradistinction, the pyrimidine stacks above the imidazole in the 5' to 3' direction in ssRNA. <b>Paper IX</b> showed that the p<i>K</i><sub>a</sub> values of the nucleobases in monomeric nucleotides can be used to show that a RNA-RNA duplex is more stable than a DNA-DNA duplex. The dissection of the relative strength of base-pairing and stacking showed that the relative contribution of former compared to that of the latter in an RNA-RNA over the corresponding DNA-DNA duplexes decreases with the increasing content of A-T/U base pairs in a sequence.</p>
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Tetzlaff, Charles N. "Synthesis and evaluation of acylated DNA and RNA oligomers /." Thesis, Connect to Dissertations & Theses @ Tufts University, 2001.
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Thesis (Ph.D.)--Tufts University, 2001.<br>Adviser: Clemens Richert. Submitted to the Dept. of Chemistry. Includes bibliographical references (leaves 228-235). Access restricted to members of the Tufts University community. Also available via the World Wide Web;
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Orban, Mathias. "Die Verteilung transkribierender RNA-Polymerase I auf ribosomaler DNA." Diss., Ludwig-Maximilians-Universität München, 2013. http://nbn-resolving.de/urn:nbn:de:bvb:19-159198.
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Curti, Elena. "Structure function studies of selected RNA and DNA polymerases." Thesis, University of Leeds, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414158.
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Singh, Shivani Shatrughana. "RNA polymerase-DNA interactions at complex gene regulatory regions." Thesis, University of Birmingham, 2014. http://etheses.bham.ac.uk//id/eprint/4877/.
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RNA polymerase (RNAP) \(\sigma\) factor must recognise and bind to specific DNA elements, usually AT-rich, in order to initiate transcription. At AT-rich regulatory regions or with more than one \(\sigma\) factor binding site; RNAP has to distinguish between different targets to initiate transcription correctly. At two regulatory regions: i) cbpA regulatory DNA with overlapping binding sites for \(\sigma\)70 and 38 associated RNAP and ii) regulatory region for ehxCABD operon with AT content of 71 %, I examined how correct RNAP binding is ensured. For cbpA regulatory region it was found that the shared promoter spacer region played a key role. I identified a location in spacer region that differently affected overlapping cbpA promoters. The base change at this position is sensed by \(\sigma\)70 side chain R451. Alterations in spacer sequence modulate conformation, making it easier, or more difficult, for R451-DNA interactions. Using tethered particle motion analysis, DNA compaction properties of cbpA gene product; CbpA was measured. ehxCABD regulatory region contains many sequences resembling \(\sigma\) factor binding elements. RNAP is capable of binding to the correct promoter elements in this region only in the presence of a chromosome folding protein, H-NS which binds AT-rich DNA. H-NS “coats” ehxCABD regulatory region and enables specific RNAP binding. Finally, many intragenic promoters within ehxCABD operon were identified. We thus propose that H-NS plays a role in silencing this pervasive intragenic transcription.
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Taylor, Laura Margaret. "Aspects of RNA directed DNA methylation in Arabidopsis thaliana." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648263.
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Jeynes, Jonathan Charles Gwilym. "Single walled carbon nanotubes functionalised with RNA or DNA." Thesis, University of Surrey, 2007. http://epubs.surrey.ac.uk/843911/.
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Within this thesis, many building blocks that are necessary to fabricate nano-scale biotechnology devices are examined. In this rapidly expanding field, carbon nanotubes (CNTs) have been identified as a key component. In particular, the biomedical applications of CNTs functionalised with DNA or RNA, utilising dielectrophoresis as a nano-manipulating tool, is investigated. The use of RNA and RNase A to generate chemically unmodified and pure single walled CNTs in a simple two step procedure is described. RNA is shown to efficiently wrap and solubilise CNTs while RNase effectively strips the RNA from the CNT, providing a convenient purification technique. The mechanism of binding of DNA to carbon nanotubes (CNTs) is shown to be much more efficient when the DNA is single-stranded rather than a double-stranded helix, while parameters (e.g. pH) are optimised for the most efficient CNT solubilisation with RNA. The compatibility of RNA-CNT composites with mammalian cells in tissue culture is also investigated. Flow cytometry and confocal microscopy show it is highly likely that RNA-CNTs composites are internalised into mammalian cells, while laser heating did not effectively kill cells. This is presumably because the power was too low or not enough CNTs were internalised in the cells. DNA-CNT composites are used to electrically sense the binding of biomolecules which have been trapped between micro-electrodes by dielectrophoresis. In a fluid cell, it is shown that solutions affect the flow of current through the CNTs, as an ionic solution increases the resistance in relation to deionised water, whereas with no CNTs, the opposite is true. Moreover, a rise in the resistance is also seen as proteins bind to CNTs. The nano-manipulation of DNA was studied with dielectrophoresis. It is shown that poly(dG)-poly(dC) (GC) collects at higher frequencies than poly(dA)-poly(dT) (AT) indicating that GC is a better conductor than AT. It was also found that different lengths of DNA polarise at about the same frequency, while shorter lengths need a higher field intensity to trap them.
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Antonopoulos, Ioanna H. "CHARACTERIZING RNA TRANSCRIPTION AND DNA REPLICATION VIA RAMAN CRYSTALLOGRAPHY." Case Western Reserve University School of Graduate Studies / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=case1428076280.
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Mondal, Tanmoy. "Epigenetic Regulation by Noncoding RNA." Doctoral thesis, Uppsala universitet, Genomik, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-160326.
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High throughput transcriptomic analyses have realized us with the fact that eukaryotic genome encodes thousands of noncoding RNAs (ncRNAs) with unknown function. In my thesis, I sought to address epigenetic regulation of transcription by ncRNA using the Kcnq1 imprinted cluster as a model system. Genomic imprinting is an epigenetic phenomenon whereby one of the parental alleles is silenced by epigenetic mechanism in a parent of origin-specific manner. A long ncRNA Kcnq1ot1 regulates imprinting of nearly 8 protein coding genes in the Kcnq1 imprinted cluster. Expression of Kcnq1ot1 is restricted to the paternal chromosome while that of protein-coding genes to the maternal chromosome. Kcnq1ot1 is a 91kb long, moderately stable, nuclear localized and RNAPII encoded transcript. We demonstrated that Kcnq1ot1 RNA itself mediates lineage specific silencing on the paternal chromosome by interacting with chromatin and recruiting the repressive chromatin modifiers to the imprinted gene promoters. Previously we identified an 890bp silencing domain (SD) at the 5´end of the Kcnq1ot1 RNA which is responsible for gene silencing. Targeted deletion of the 890SD in mouse resulted in specific loss of silencing of ubiquitously imprinted genes. We have further shown that Kcnq1ot1 interacts with Dnmt1 and recruit Dnmt1 at the somatic DMRs flanking some of the ubiquitously imprinted genes. We next addressed the stability of the Kcnq1ot1 mediated epigenetic silencing using transgenic mouse where we have conditionally deleted the Kcnq1ot1 RNA at different developmental stages and we found that Kcnq1ot1 RNA is required to maintain the silencing of the ubiquitously imprinted genes. In addition, DNA methylation, which controls imprinting of the ubiquitous genes require Kcnq1ot1 for its maintenance. To characterize the ncRNAs that mediate gene regulation through chromatin interaction we have isolated chromatin associated RNAs (CARs) from sucrose gradient fractioned chromatin. High-throughput sequencing of the CARs resulted in the identification of the 141 intronic and 74 intergenic regions harboring CARs. We characterized one of the intergenic CARs which regulate the transcription of the two neighboring genes by modulating the chromatin marks. In summary current thesis has uncovered unprecedented role of ncRNAs in gene expression via chromatin level regulation.
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Zhang, Zhouwei. "Investigation of DNA and RNA markers by novel technologies demonstrates DNA content intratumoral heterogeneity and long non-coding RNA aberrations in breast tumors." ScholarWorks @ UVM, 2014. https://scholarworks.uvm.edu/graddis/323.
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BACKGROUND:
Breast cancer is the most commonly diagnosed cancer and second leading cancer death cause among females in the U.S.A. About 1 in 8 women in U.S will develop invasive breast cancer over the course of her lifetime. In 2013, 234,580 new invasive breast cancer cases are expected to occur in women within the US and approximately 64,640 non-invasive carcinomas in situ were diagnosed in 2013, most of which were ductal carcinoma in situ (DCIS). Along with technological advances, a wide variety of candidate biomarkers have been proposed for cancer diagnosis and prognosis, including DNA content and non-coding RNA. Current techniques for detecting DNA content abnormalities in formalin-fixed, paraffin-embedded (FFPE) tissue samples by flow cytometric analysis have used cells recovered from ≥50µm whole tissue sections. Here, in our first study, a novel core punch sampling method was investigated for assessing DNA content abnormalities and intratumoral heterogeneity in FFPE specimens. Secondly, long non-coding RNAs (lncRNAs) has been examined. LncRNA participates in a broad spectrum of biological activities by diverse mechanisms and its dysregulation is associated with tumorgenesis. Some lncRNAs may function as oncogenes (O) and others as tumor suppressor genes (TSG). To date, lncRNA has been investigated primarily by qRT-PCR and RNA sequencing. This study has examined the relationship of lncRNA expression patterns to breast tumor pathology by chromogenic in situ hybridization (CISH).
METHODS:
Firstly, FFPE breast carcinoma specimens were selectively targeted using 1.0 mm diameter punch needles. Extracted cores were assayed by flow cytometry using a modified-Headley method. Secondly, the lncRNA expression levels of 6 lncRNAs: HOTAIR, H19, KCNQ1OT1, MEG3, MALAT11 and Zfas1, was examined by RNAscope® CISH using FFPE breast tissue microarrays (TMAs) comprising normal adjacent epithelia (NA), DCIS, and invasive carcinoma (IC) from 46 patients. LncRNA associate polycomb complex protein EZH2 was evaluated by immunohistochemistry (IHC). LncRNA data was also compared to standard breast tumor data including ER, PR, Her2 and Ki67 IHC. SYSTAT version 11 statistical package was used to perform for all the tests.
RESULTS:
Following optimization experiments of the core punch flow cytometric approach, DNA index and percent S-phase fraction intratumoral heterogeneities were detected in 10/23 (44%) and 11/23 (47%) specimens respectively. The lncRNA CISH study utilized a TMA that contained 36 spots of NA breast tissues, 34 DCIS spots and 43 IC spots. HOTAIR CISH staining was significantly stronger in IC than DCIS (p
CONCLUSION:
Core-punching is an effective alternative to whole specimen sectioning and shows that macro-level genomic heterogeneity is common even within a single FFPE block. The interrelationship of DNA content heterogeneity to other forms of heterogeneity requires further study. RNAscope CISH supports bright-field microscopy investigations of lncRNA expression in FFPE tissue specimens. HOTAIR, H19 and KCNQ1OT1 may be potential breast cancer biomarkers, both HOTAIR and H19 may be a marker for DCIS at increased risk of progression to invasive cancer. HOTAIR, in particular, may be a predictor for invasive cancer grade.
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Sfyrakis, Konstantinos. "Computer simulation and advanced visualisation of DNA." Thesis, University of Surrey, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368378.
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Pan, Hu. "Structural and biochemical studies of DNA primase from Bacillus stearothermophilus." Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302122.
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MICHELINI, FLAVIA. "A NEW CLASS OF NON-CODING RNA CONTROLS THE DNA DAMAGE RESPONSE AND DNA REPAIR." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/234137.
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DNA is the most precious molecule in our cells, thus it has to be protected from damage and alterations and, if damaged, it has to be repaired efficiently. The DNA damage response (DDR) is a signaling cascade that follows the generation of a lesion in the DNA double helix and promptly arrests cell proliferation in order to attempt DNA repair. It has been proposed that mammalian genomes are pervasively transcribed, also in non-coding regions. Non-coding RNAs (ncRNAs) have been involved in an increasing number of cellular events and some of them are processed by members of the RNA interference (RNAi) pathway. So far, RNAi and DDR pathways have not been demonstrated to directly interact. During my PhD, I contributed to uncover an unexpected layer of DDR regulation by a new class of DICER- and DROSHA-dependent small non-coding RNA, named DDRNA.
DDR foci stability is sensitive to RNA polymerase II inhibition and to RNase A treatment. Incubation of RNase A-treated cells with DICER- and DROSHA-dependent RNA products restores focal accumulation of DDR factors. DICER and DROSHA are indeed necessary to trigger DDR upon exogenous DNA damage in human cells, and DICER processing activity is necessary to activate DDR. Moreover, DICER and DROSHA knockdown impacts on checkpoint activation and allows senescent cells to re-enter S-phase. Differently, inactivation of GW182, a component of the RNAi machinery involved downstream of DICER and DROSHA in mRNA translational control, does not impact on DDR foci formation and detection. In a mammalian cell system in which a single DNA double-strand break can be generated at a defined exogenous integrated locus, DDR focus formation requires site-specific RNA molecules. RNA deep sequencing confirmed the presence of 22-23-nucleotide sequence-specific transcripts arising from the damaged locus, which are DICER-dependent. These DDR-regulating RNAs (DDRNAs) act at the first steps of the DDR cascade, in an MRN-dependent manner and have an impact also on DNA damage repair. Importantly, DDRNAs, both chemically synthesized or generated in vitro by DICER cleavage, are biologically active and antisense LNA oligonucleotides reduce DDR activation in living cells. Finally, by the use of fluorescently labeled molecules, DDRNAs have been demonstrated to localize at the site-specific damaged locus.
Collectively these results suggest an unanticipated direct role of DICER and DROSHA in the production of small ncRNAs that control DDR activation at sites of DNA damage. Given the known tumor suppressive functions of DDR and the implication of its activation in a number of biological relevant processes, such as senescence, this discovery may have a significant impact on our understanding of ageing and cancer.
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Jarnehammar, Linn. "DNA sampling using different tissues from the butterfly species Vanessa cardui." Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-158611.
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The fundamental challenge to prevent species from going extinct is difficult but of grave importance. Halting species from going extinct minimizes the loss of biodiversity. One way of researching biodiversity is by studying species on a genetic level. This creates a dilemma as studying species genetically often requires using destructive sampling and is not desirable or even allowed when studying threatened species. Thus, there is a necessity for alternate sampling methods. In this study both non-lethal and lethal methods were used to gather tissues from the butterfly species Vanessa cardui. The DNA extractions turned out to give varying amounts of DNA, but it was successfully extracted from all the different tissue types. Amplifiable DNA was successfully gained using PCR and confirmed using gel electrophoresis. Existing and newly designed primers for multi-copy genes were used and several of them gave amplifiable DNA. Even if amplifiable DNA has been obtained in other studies, using various tissues, it turned out to only work with a live butterfly’s body in this study.
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Walter, Heidi-Kristin [Verfasser], and H. A. [Akademischer Betreuer] Wagenknecht. ""DNA/RNA Traffic Lights 2.0" - Entwicklung von wellenlängenverschiebenden DNA- und RNA-Sonden unter Verwendung von "Click"-Modifikationen / Heidi-Kristin Walter ; Betreuer: H.-A. Wagenknecht." Karlsruhe : KIT-Bibliothek, 2016. http://d-nb.info/1122461534/34.
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Henfrey, R. D. "In vitro transcription of exogenous plant DNA." Thesis, University of Hertfordshire, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.381604.
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Cozens, Christopher. "An adaptive path from DNA to RNA and ANA polymerases." Thesis, University of Cambridge, 2012. https://www.repository.cam.ac.uk/handle/1810/252281.
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Mast, Christof. "Polymerization and replication of DNA/RNA in a thermal trap." Diss., Ludwig-Maximilians-Universität München, 2013. http://nbn-resolving.de/urn:nbn:de:bvb:19-175178.
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Sigurgeirsson, Benjamín. "Analysis of RNA and DNA sequencing data : Improved bioinformatics applications." Doctoral thesis, KTH, Genteknologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-184158.
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Massively parallel sequencing has rapidly revolutionized DNA and RNA research. Sample preparations are steadfastly advancing, sequencing costs have plummeted and throughput is ever growing. This progress has resulted in exponential growth in data generation with a corresponding demand for bioinformatic solutions. This thesis addresses methodological aspects of this sequencing revolution and applies it to selected biological topics. Papers I and II are technical in nature and concern sample preparation and data anal- ysis of RNA sequencing data. Paper I is focused on RNA degradation and paper II on generating strand specific RNA-seq libraries. Paper III and IV deal with current biological issues. In paper III, whole exomes of cancer patients undergoing chemotherapy are sequenced and their genetic variants associ- ated to their toxicity induced adverse drug reactions. In paper IV a comprehensive view of the gene expression of the endometrium is assessed from two time points of the menstrual cycle. Together these papers show relevant aspects of contemporary sequencing technologies and how it can be applied to diverse biological topics.<br><p>QC 20160329</p>
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Chen, Cai. "Quantitative studies of RNA editing and nucleosomal DNA-protein interactions." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1417523347.
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Shen, Ying. "Studies on the mechanisms of RNA-driven DNA repair and modification." Diss., Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/45969.
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Our previous studies have demonstrated that RNA can serve as a template for double-strand break (DSB) repair in the yeast Saccharomyces cerevisiae using synthetic RNA-containing oligonucleotides (oligos). Following this initial work, we show that the RNA tract of RNA-containing oligos can be copied into DNA to transfer a genetic change at the chromosomal level also in the bacterium Escherichia coli and in human cells. Exploiting the use of oligos containing ribonucleoside monophosphates (rNMPs), we developed a molecular approach to generate RNA/DNA hybrids of chosen sequence and structure at the chromosomal level in both yeast and E. coli cells. Such technique allows us to study how rNMPs present in the DNA genome of cells are tolerated by cells, what factors recognize and target rNMPs in DNA and to what extent the embedded rNMPs may alter genome integrity. Here we proved that mispaired rNMPs embedded into genomic DNA, if not removed, serve as templates for DNA synthesis during chromosomal replication and produce a genetic change. We discovered that mispaired rNMPs that are embedded in genomic DNA are not only targeted by ribonucleases H (RNases H) but also by the mismatch repair (MMR) system both in yeast and in E. coli. Our data reveal novel substrates for the MMR system, and also uncover an unpredicted competition between RNase H and MMR for the RNA/DNA mispairs.
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Choudury, Sarah G. Choudury. "Identification and characterization of proteins required for RNA-directed DNA Methylation, including the RNA binding protein ALY1." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1543508792612526.
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Nayak, Dhananjaya. "Conformational mechanisms in T7 RNA polymerase transcription a dissertation /." San Antonio : UTHSC, 2008. http://learningobjects.library.uthscsa.edu/cdm4/item_viewer.php?CISOROOT=/theses&CISOPTR=44&CISOBOX=1&REC=11.
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Zhou, Min. "Characterisation of ADAM expression in human myeloma cells." Thesis, University of Sheffield, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312273.
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Yunnan, Jiang. "Testing the occurrence of forward hyper-translocation during the promoter escape transition / Jiang Yunnan." Connect to online version, 2009. http://ada.mtholyoke.edu/setr/websrc/pdfs/www/2009/381.pdf.
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Lee, Sally. "Architecture of RNA polymerase II and RNA polymerase III pre-initiation transcription complexes /." Thesis, Connect to this title online; UW restricted, 1997. http://hdl.handle.net/1773/9213.
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Vystrčilová, Jana. "Tolerance poškození DNA novými, biologicky aktivními komplexy platiny." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2011. http://www.nusl.cz/ntk/nusl-216799.
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The anti-tumor activity of platinum based drugs is mediated by their ability to attack DNA. Platinum complexes can alter the structure of DNA by modifying the bases, mainly guanines. The biological consequnces of such interactions are compromising replication and transcription. RNA polymerase complex can stall at a damaged site in DNA and mark the lesion for repair by proteins that are utilized to execute nucleotide excision repair, a pathway commonly associated with the removal of bulky DNA damage from the genome. This RNA polymerase-induced repair pathway is called transcription-coupled nucleotide excision repair. Main goal of this thesis was to study RNA polymerases tolerance of DNA damage by novel, biologically active platinum (II) complexes involving derivatives of aromatic cytokinines as the ligands; cis-[Pt(2-chloro-6-(4-methoxybenzylamino)-9-isopropylpurin)2Cl2](PR-001), cis-[Pt(2-chloro-6-(benzylamino)-9-isopropylpurin)2Cl2](PR-002 )and cis-[Pt(2-(3-hydroxypropylamino)-6-(benzylamino)-9-isopropylpurin)2Cl2](PR-005). DNA templates (constructs) that contain a single, site-specific DNA lesion and support transcription by human RNA polymerase II and bacteriophage T7 RNA polymerase were prepared. The method is making use of polymerase chain reaction (PCR) and biotin-streptavidin interactions and paramagnetic particles to purify the final product. Synthetic oligomers duplexes (75-mer, 56-mer and 15-mer) are ligated to 5´-biotin pCI-neo-G-lessT7 PCR fragment, the 15-mer is either unmodified or modified with a site-specific lesion of PR-005 and cisplatin. We also studied the inhibition of RNA polymerases activity on globally modified plasmid pCI-neo and pUC 19 by novel platinum complexes and cisplatin. We found that bifunctional adducts of complex PR-005 contrary to adducts of PR-001 and PR-002 effectively decrease amount of full lenght transcripts produced by both, human and bacterial RNA polymerases. This result can be explained by a sterical block, induced to DNA by intrastrand cross-link of PR-005 with bulky aromatic ligands.
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Francis, Rawle Friedman Simon H. "Inhibition of human telomerase by targeting its transitory RNA/DNA heteroduplex." Diss., UMK access, 2005.
Find full textAbstract:
Thesis (Ph. D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2005.<br>"A dissertation in pharmaceutical sciences and chemistry." Advisor: Simon H. Friedman. Typescript. Vita. Description based on contents viewed June 23, 2006; title from "catalog record" of the print edition. Includes bibliographical references (leaves 327-353). Online version of the print edition.
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Fredriksson, Mona. "Using Minisequencing Technology for Analysing Genetic Variation in DNA and RNA." Doctoral thesis, Uppsala University, Department of Medical Sciences, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4789.
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<p>In this thesis, the four-color fluorescence tag-microarray minisequencing system pioneered by our group was further developed and applied for analysing genetic variation in human DNA and RNA. A SNP marker panel representing different chromosomal regions was established and used for identification of informative SNP markers for monitoring chimerism after stem cell transplantation (SCT). The success of SCT was monitored by measuring the allelic ratios of informative SNPs in follow-up samples from nine patients with leukaemia. The results agreed with data obtained using microsatellite markers. Further the same SNP marker panel was used for evaluation of two whole genome amplification methods, primer extension preamplification (PEP) and multiple displacement amplification (MDA) in comparison with genomic DNA with respect to SNP genotyping success and accuracy in tag-array minisequencing. Identical results were obtained from MDA products and genomic DNA.</p><p>The tag-microarray minisequencing system was also established for multiplexed quantification of imbalanced expression of SNP alleles. Two endothelial cell lines and a panel of ten coding SNPs in five genes were used as model system. Six heterozygous SNPs were genotyped in RNA (cDNA) from the cell lines. Comparison of the relative amounts of the SNPs alleles in cDNA to heterozygote SNPs in genomic DNA displayed four SNPs with significant imbalanced expression between the SNP alleles. Finally, the tag-array minisequencing system was modified for detection of splice variants in mRNA from five leukaemia cell lines. A panel of 20 cancer-related genes with 74 alternatively splice variants was screened. Over half of the splice variants were detected in the cell lines, and similar alternative splicing patterns were observed in each cell line. The results were verified by size analysis of the PCR product subjected to the minisequencing primer extension reaction. The data from both methods agreed well, evidencing for a high sensitivity of our system.</p>
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Novoa, Carolina. "RecQ-like helicase SGS1 counteracts DNA : RNA hybrid induced genome instability." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/60964.
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Dividing cells are constantly under threat from both endogenous and exogenous DNA damaging stresses that can lead to mutations and structural variations in DNA. One contributor to genome instability is three-stranded DNA:RNA hybrid structures called R-loops. Though R-loops are known to induce DNA damage and DNA replication stress, it is unclear whether they are recognized and processed by an established DNA repair pathway prior to inducing DNA breaks. Canonically, DNA repair proteins work downstream of R-loop-induced DNA damage to stimulate repair and suppress genome instability. Recently, the possibility that some DNA repair pathways actively destabilize R-loops, thus preventing unscheduled DNA damage has emerged. Here we identify the helicase SGS1 as a suppressor of R-loop stability. Our data reveals that SGS1 depleted cells accumulate R-loops. In addition, we define a role for transcription in genome instability of cells lacking SGS1, which is consistent with an R-loop based mechanism. Hyper-recombination in SGS1 mutants is dependent on transcript length, transcription rate, and active DNA replication. Also, rDNA instability in sgs1Δ can be suppressed by ectopic expression of RNaseH1, a protein that degrades DNA:RNA hybrids. Interestingly, R-loops are known to form at rDNA loci. We favour a model in which SGS1 contributes to the stabilization of stalled replication forks associated with transcription complexes, and unresolved DNA:RNA hybrids. Finally, we showed that knockdown of the human Sgs1 orthologue BLM in HCT116 cells also led to the accumulation of more R-loops than control HCT116 cells. In summary, our data supports the idea that some DNA repair proteins involved in replication fork stabilization might also prevent and process R-loops.<br>Science, Faculty of<br>Graduate
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Tomkuvienė, Miglė. "Methyltransferases as tools for sequence-specific labeling of RNA and DNA." Doctoral thesis, Lithuanian Academic Libraries Network (LABT), 2013. http://vddb.library.lt/obj/LT-eLABa-0001:E.02~2013~D_20131209_091531-59976.
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Investigation of RNA and DNA function often requires sequence-specific incorporation of various reporter and affinity probes. This can be achieved using AdoMet-dependent methyltransferases (MTases) as they can be active with synthetic AdoMet analogues equipped with transferable chains larger than the methyl group. These chains usually carry reactive groups that can be further chemically appended with required reporters. For this, azide-alkyne 1,3-cycloaddition (AAC), also called “click”, reaction is particularly attractive. This work shows that the HhaI cytosine-5 DNA MTase (variant Q82A/Y254S/N204A) catalyzes efficient sequence-specific transfer of hex-2-ynyl side chains containing terminal alkyne or azide groups from synthetic cofactor analogues to DNA. Both the enzymatic transfer and subsequent “click” coupling of a fluorophore can be performed even in cell lysates. For RNA labeling, the activity of an archaeal RNA 2‘-O-MTase C/D ribonucleoprotein complex (RNP) with synthetic cofactors was investigated. It was shown that synthetically reprogrammed guide RNA sequences can be used to direct the C/D RNP-dependent transfer of a prop-2-ynyl group to predetermined nucleotides in substrate RNAs. Followed by AAC this can be used for programmable sequence-specific labeling of a variety of RNA substrates in vitro. These new possibilities for specific labeling of nucleic acids can be adopted in biochemistry, biomedical, nanotechnology, etc. research.<br>Tiriant DNR ir RNR, neretai svarbu prijungti įvairius reporterinius ar giminingumo žymenis griežtai apibrėžtose (sekos) vietose – t.y. specifiškai. Tam galima pasitelkti fermentus metiltransferazes (MTazes). Natūraliai jos naudoja kofaktorių AdoMet, tačiau gali būti aktyvios ir su sintetiniais jo analogais, turinčiais ilgesnes nei metil- pernešamas grandines. Jei šios grandinės turi galines funkcines grupes, prie jų vėliau cheminių reakcijų pagalba galima prijungti norimus žymenis. Tam itin patogi azidų-alkinų cikloprijungimo (AAC), dar vadinama „click“, reakcija.
Šiame darbe parodyta, kad DNR citozino-5 MTazė HhaI (variantas Q82A/Y254S/N204A) efektyviai katalizuoja sekai specifinę heks-2-inil- grandinių, turinčių galines alkinil- arba azido- grupes, pernašą nuo sintetinių kofaktorių ant DNR. Naudojant šią MTazės-kofaktorių sistemą bei AAC, visą specifinio DNR žymėjimo procesą galima atlikti netgi ląstelių lizate. RNR žymėjimui ištirtas archėjų RNR 2‘-O-MTazės C/D ribonukleoproteininio komplekso aktyvumas su sintetiniais kofaktoriais. Parodyta galimybė sintetiškai keičiant kreipiančiąją RNR, prop-2-inilgrupės pernašą nukreipti į norimas įvairių substratinių RNR sekos vietas ir po to AAC reakcijos pagalba prijungti fluoroforą. Taigi, sukurtas naujas molekulinis įrankis, leidžiantis be suvaržymų pasirinkti norimą pažymėti RNR seką. Šios naujos specifinio nukleorūgščių žymėjimo galimybės gali būti pritaikytos biochemijos, biomedicinos, nanotechnologijų ir kitose tyrimų srityse... [toliau žr. visą tekstą]
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Cooke, L. A. "Preparation and evaluation of novel phosphoramidites for labeling DNA and RNA." Thesis, Queen's University Belfast, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.557306.
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Phosphoramidite derivatives of a nucleoside analogue bearing photoswitchable ortho-, meta- and para azobenzene moieties were prepared and used to incorporate the azobenzene groups into DNA.8mers. The photochemical E-Z isomerisation of the azobenzene-appended 8mers was investigated by UV/vis spectroscopy and RP-HPLC. In order to investigate the stabilities of the irradiated-8mers towards thermal Z - E isomerisation, Arrhenius and Eyring parameters for the photoisomerisation were determined. The meta-isomer was found to be the most thermally stable. An initial investigation into the stability of duplexes containing a para-azobenzene-modified 8mer was carried out using melting studies. The duplex-forming activity of the oligonucleotide was modulated by the E- Z photoisomerisation of the para-azobenzene residue. A divergent methodology for the preparation of a novel structural class of photoswitchable oligonucleotide has been described. A novel anthracene methyl phosphoramidite derivative suitable for the preparation of end- labelled oligonucleotides under solid-phase directed-Arbusov conditions was prepared and its reactivity investigated. A comparison of the utility of this anthracene methyl phosphorarnidite with a related benzyl phosphorarnidite in a model reaction with the 5'-hydroxyl of support-bound decathymidylate was made. Directed Michaelis-Arbusov reactions of the putative phosphite triester intermediates with primary and secondary amines in the presence of 0.01 M iodine gave the corresponding phosphoramidate diesters in high yields. This reactivity was also demonstrated using commercially available phosphoramidites for the preparation of inter-nucleotide phosphoramidates bearing terminal primary amines. Derivatisation of these primary amine-functionalised oligomers was accomplished in solution-phase following treatment with the meta-azobenzene NHS ester.
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Firth, Andrew Graeme. "Synthesis and Characterisation of Fluorescent RibonucleotideSubstrates for DNA Dependent RNA Polymerases." Thesis, University of York, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.507614.
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Xiong, Chen. "Enzymatic modification of DNA and RNA 3'-termini for click ligation." Thesis, University of Southampton, 2014. https://eprints.soton.ac.uk/367127/.
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