Dissertations / Theses on the topic 'Branched RNA'

RNA is essential for the transfer of genetic information, as the central dogma of biology dictates. The role of RNA, however, is not limited to serving as an information shuttle between DNA and fully functional protein. Indeed, RNA has experienced a surge of interest in the field of chemical biology for its other critical roles in biology including those in control of transcription, translation, splicing, genetic replication, and catalysis. RNA has proven to be a difficult and complex target for the design of small molecular ligands because of its structural heterogeneity and conformational flexibility. Yet, the highly folded tertiary structures of these oligomers present unique scaffolds which designed ligands should be able to selectively target. To that end, two branched peptide libraries ranging in size from 4,096–46,656 unique sequences were screened for their ability to bind HIV-1 related RNA structures, the transactivation response element (TAR) and the Rev response element (RRE). In addition to discovering a mid-nanomolar branched peptide ligand for TAR, the first branched boronic acid peptide library designed to target RNA was screened for binding to RRE. Each of these efforts resulted in the identification of selective binders to their respective RNA targets, and the unnatural branching of these compounds was demonstrated to provide a multivalent binding interaction with the RNA. Furthermore, these compounds were shown to be cell permeable and displayed little to no cytotoxicity in HeLa and A2780 cells.
Ph. D.

RNAs have gained significant attention in recent years because they can fold into well-defined secondary or tertiary structures. These three dimensional architectures provide interfaces for specific RNA-RNA or RNA-protein interactions that are essential for biological processes in a living system. These discoveries greatly increased interest in RNA as a potential drug target for the treatment of diseases. Two of the most studied RNA based regulatory systems are HIV-1 trans-activating response element (TAR)/Tat replication pathway and Rev response element (RRE)/Rev export pathway. To efficiently target TAR and RRE RNA, we designed and synthesized three generations of branched peptide libraries that resulted in medium sized molecules. The first generation of BPs were discovered from screening a one-bead one-compound library (4,096 compounds) against HIV-1 TAR RNA. One peptide FL4 displayed a binding affinity of 600 nM to TAR RNA, which is tighter than its native protein counterpart, Tat. Biophysical characterization of these BP demonstrated that "branches" in BPs impart multivalency, and they are cell permeable and non-toxic. The second generation peptides were discovered from an on-bead high-throughput screening of a 3.3.4 branched peptide boronic acids (BPBAs) library that bind selectively to the tertiary structure of RRE IIB. The library comprised of 46,656 unique sequences. We demonstrate that our highest affinity BPBA (BPBA1) selectively binds RRE IIB in the presence of competitor tRNAs as well as against six RRE IIB structural variants. Further, we show that the boronic acid moieties afford a novel binding mode towards RNA that is tunable; their Lewis acidity has critical effects on binding affinity. In addition, biophysical characterizations provide evidence that "branching" in these peptides is a key structural motif for multivalent interactions with the target RNA. Finally, RNA footprinting studies revealed that the BPBA1 binding site encompasses a large surface area that spans both the upper stem as well as the internal loop regions of RRE IIB. BPBA1 is cell permeable and non-toxic. In the next generation of branched peptides, a 3.3.4 branched peptide library composed of 4,096 unique sequences that featured boronic acid and acridine moieties was designed. We chose acridine as the amino acid side chain due to its potential for π-stacking interaction that provides high binding affinity to RNA target. The library was screened against HIV-1 RRE IIB RNA. Fifteen peptides were sequenced and four contained acridine alone and/or in conjunction with boronic acid moieties displayed dissociation constants lower than 100 nM. The ribonuclease protection assays of A7, a sequence that contains both boronic acid and acridine residues, showed a similar protection pattern compared to previous peptide BPBA1, suggesting that the 3.3.4 branched peptides shared similar structural elements and contacted comparable regions of the RRE IIB RNA. The results from this research indicated that "branching" in peptides imparts multivalent interactions to the RNA, and that functional groups such as boronic acid and acridine are key structural features for efficient binding and selectivity for the folded RNA target. We demonstrated that the branched peptides are cell permeable and non-toxic.
Ph. D.

The interaction of the protein Rev with Rev Response Element (RRE) RNA is critical to the HIV-1 life cycle as this complex is required for the export of singly-spliced and unspliced mRNAs from the nucleus to the cytoplasm. Disruption of this interaction is considered to be a powerful strategy towards the development of HIV-1 therapeutics. Therefore, we have developed several branched peptide libraries containing unnatural amino acids to target the high-affinity binding site of RRE RNA (RRE IIB), with the idea that branching in peptides can provide multivalent contacts with folded RNA structures and boost binding affinity and selectivity for the target. Unnatural amino acids were incorporated into the library design to encourage non-canonical interactions with the RNA and to improve proteolytic stability. The on-bead high-throughput screening of our first branched peptide library (46,656 sequences) against HIV-1 RRE RNA generated hit peptides with binding affinities in the low micromolar range. We demonstrated that branching in the peptide is required for efficient binding and selectivity towards the RNA, and that the peptides bind a large surface area of RRE IIB. Introduction of boronic acids into branched peptides boosted selectivity of the peptides for RRE IIB, and proved to be a novel and tunable mode of binding towards RNA. Additionally, we revealed that these branched peptide boronic acids (BPBAs) were cell permeable and non-toxic. One BPBA (BPBA3) bound RRE IIB selectively and was able to inhibit HIV-1 replication in vitro, revealing enzymatic cleavage of the RNA upon binding. A second generation BPBA library that introduced acridinyl lysine as an intercalator (4,096 sequences) was screened against RRE IIB. Several hit compounds bound in the low nanomolar regime, and a significant number of compounds inhibited HIV-1 replication in vitro. These BPBAs were also found to severely inhibit the microbial growth of bacteria and fungus, with MICs as low as 1 µg/mL against Staphylococcus aureus, Candida albicans, and Escherichia coli. These compounds were also found to significantly inhibit biofilm formation and growth, and were non-hemolytic. High-throughput screening of a third generation BPBA library containing all unnatural amino acids (46,656 sequences) revealed several hits that bound RRE IIB RNA in the nanomolar range. Sequence motifs present in the hit peptides suggested that the location and composition of amino acids within the branched peptide structure were important for recognizing the RNA target. In particular, lead compounds 2C5 and 4B3 demonstrated selectivity towards RRE, and footprinting experiments combined with SHAPE experiments revealed different interactions of the peptides with the RNA Toxicity assays revealed no impact on cell viability for the majority of hit sequences tested up to 100 µM, and several compounds also demonstrated inhibition of HIV-1 replication.
Ph. D.

The generation of synthetic oligonucleotides is dependent on an efficient solid-phase synthesis methodology. This thesis evaluates the 2'-O -levulinyl (Lv) and 2'-O-monomethoxytrityl (MMT) ribonucleosides, as possible synthons for RNA and branched RNA synthesis. A key feature of this RNA and bRNA synthesis procedure is their removal while still attached to the solid support and under conditions that prevent isomerization or cleavage of the nascent strands. For the first time, the stability of 3'-5'-internucleotide phosphate triesters (and diesters) adjacent to a ribose 2'-hydroxyl group was determined on a solid support. These studies are not only relevant to the proper assembly of branched and linear RNA species, but also to the stability of an unusual branched RNA species ("RNA X") proposed to form during the pre-mRNA splicing reactions in vitro. These studies are also important to the development of large quantities of native and chemically modified short interfering RNA (siRNA) for animal and human studies.
The 2'-O-Lv and 2'-O-MMT ribonucleoside monomers served as building blocks for the assembly of a series of branched nucleic acid species (bRNA, bDNA, msDNA and hyperbranched or "dendritic" DNA/RNA) with discrete length, base composition and structure. These structures were synthesized via an iterative divergent-growth strategy, which facilitates the regioselective branching, deblocking and chain lengthening steps from a branchpoint core. These structures served as useful materials (bio-probes) as demonstrated by the biological studies performed with E. coli RNaseH and the yeast lariat RNA debranching enzyme (yDBr1). These studies not only led to the identification of novel branched nucleic acid inhibitors of yDBR1 and RNase H, but also provided new insights about the substrate specificity of these important enzymes.
This thesis also describes the synthesis of a new nucleic acid form, the so-called "oxepane nucleic acids" (ONAs), in which the pentofuranose ring of DNA and RNA was replaced with a 7-membered heptose sugar ring. ONA were found to be much more resistant towards nuclease degradation than natural DNA, an important feature if these analogues are to be used in biological media. Furthermore, ONAs exhibited cross-pairing with complementary RNA and were found to elicit E. coli RNaseH mediated degradation of the RNA strand. These finding are significant because oligonucleotide-directed RNase H degradation of the target RNA is a key determinant for the gene-specific inhibitory potency of antisense oligonucleotides. When comparing the rates of RNase H-mediated degradation induced by 5 (furanose), 6 (2'-ene-pyranose) and 7 (oxepane) membered ring oligonucleotides, the following trend was observed: DNA > 2'-ene-pyranose NA > ONA. The implications of these results are discussed in the context of our current understanding of the catalytic mechanism of the enzyme, particularly with regard to the required flexibility of the oligonucleotide strands that bind to the RNA target. Hence, ONAs are useful tools for biological studies and provide new insights into the structure/function of natural and alternative genetic systems.

A method to synthesize a diastereomeric mixture of 2',5 '-phophorothioate A(psG) dimers in solution was developed. The sulfurizing reagent "EDITH" allowed for the synthesis of the diastereomeric mixture of dimers with minimal formation of oxidized side products. The separation of the two isomers was carried out using silica gel flash chromatography to afford the stereoisomerically pure Rp and S p dimers. The orthogonal solution-phase coupling of the individual dimers to the appropriately protected monomers allowed for the creation of the corresponding branched trimers bearing vicinal 2',5'-phophorothioate and 3',5'-phosphodiester linkages.
Conversely, a convergent solid-phase strategy applicable to the synthesis of branched oligonucleotides was employed to construct a symmetrical branched phosphodiester trimer, A(pG)pG, using an adenosine bisphosphoramidite synthon. This compound served as a positive control substrate, relative to both the Sp and Rp-phophorothioate V-trimers, in the investigation of the stereochemical requirements of the yeast debranching enzyme (yDBR) at the 2',5'-phosphodiester linkage. (Abstract shortened by UMI.)

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2015.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references.
Pre-mRNA splicing is a ubiquitous process necessary for the production of functional eukaryotic mRNAs. The branch point (BP) sequence is one of three key nucleotide sequences required for pre-mRNA splicing, however, in metazoa it has been less comprehensively studied than the 5' splice site (5'SS) and 3' splice site (3'SS) due to the relative difficulty of identifying each sequence element. 5'SS and 3'SS are readily identified by aligning spliced cDNAs, ESTs, or RNA-Seq reads to the genome, while lower throughput techniques such as primer extension are usually required to map BPs, with some exceptions. To understand how the BP affects splicing outcomes, we developed an experimental method to locate BPs on a genome-wide scale. Applying our method to Saccharomyces cerevisiae (S. cerevisiae), one of the only eukaryotes for which most BPs are known, allowed us to assess the sensitivity and specificity of our method. We enriched for RNA lariats by isolating RNA from debranching enzyme null yeast and purified circular RNAs (including lariats) from linear RNAs using a 2D PAGE gel. This was followed by a custom library preparation protocol that produced insert ends that identified the BP and 5'SS of individual lariats. Using this method, we located known BPs and discovered a substantial number of novel BPs both in annotated introns and other genomic regions. We attempted to verify these novel introns using RNA-seq and Lariat-seq and surprisingly observed considerable amounts of alternative splicing (AS) in S. cerevisiae beyond the previously known stress-regulated intron retention events and handful of alterative splice sites. Additionally, we observed several introns with 2 BPs and one intron with 3 BPs. In the LSM2 transcript, we showed alternative BP usage was associated with alternative splice site usage, where one of the mRNA isoforms contains a premature termination codon and leads to nonsense-mediated mRNA decay of the transcript. This suggests AS may control gene expression levels in yeast as is known to be the case in metazoans. Preliminary application of our method to Drosophila melanogaster showed recursive splicing, a phenomenon known only to occur in introns larger than 10Kb, to occur in a 383nt intron.
by Genevieve Michelle Gould.
Ph. D.

The intronic lariat RNA generated during pre-mRNA splicing includes a branch-point adenosine residue that is linked through the 2'-O position to the 5'-end of the RNA sequence. This 2'-5'-phosphodiester linkage in the lariat RNA backbone is cleaved (debranched) by the lariat debranching enzyme (Dbr1p). Following this debranching reaction, some introns can participate in highly important biological processes like snoRNA biogenesis, microRNA pathways etc. Although Dbr1p has structural resemblance to some non-specific nucleases, it still remains elusive how Dbr1p can selectively cleave the 2'-5' phosphodiester bond in lariat RNAs without affecting the 3'-5' phosphodiester bonds. The major roadblock towards understanding Dbr1p mechanism has been easy access to its substrate. We developed a branching phosphoramidite with 3'-photoprotecing group which gives us facile access to 2'-5' phosphodiester linked backbone branched RNAs (bbRNAs) which are mimics of the lariat RNAs. Our method of bbRNA synthesis is versatile and allows incorporation of both internal and terminal modifications. To demonstrate this, several bbRNAs with modified 2'-branch were synthesized using this method which showed different debranching activity. To examine the kinetics of the debranching reaction, we synthesized a dual-fluorescently-labeled bbRNA with Cy3 and Cy5 dyes that acts as donor and acceptor in a FRET based assay. By following the increase in the donor emission with time for this dual-labeled substrate, the debranching reaction can be followed in real time. Using this assay we have been able to find the kinetics parameters of the Dbr1p enzyme for the first time. We have also synthesized a non-cleavable analogue of the native bbRNA substrate where the 2'-5' phosphodiester bond is replaced by a triazole (click) linkage. Using the FRET based kinetics we show that this ‘click’ branched RNA (cbRNA) is a competitive inhibitor of Dbr1p. This suggests that the cbRNA binds the Dbr1p enzyme in a similar fashion as the native substrate. The unique structure of 2'-5' phosphodiester linked bbRNAs could play an important role in the selective cleavage of bbRNA substrates by Dbr1p. While synthetic access to bbRNAs has enabled biochemical and kinetics studies of the debranching reaction, these studies do not inform regarding the structure and dynamics of the bbRNAs. Towards this goal, we performed single molecule FRET (smFRET) experiments using dual-labeled (Alexa488 Alexa594) bbRNA and its non-cleavable analogue, cbRNA. SmFRET experiments using confocal microscopy revealed a single broad distribution of cbRNA conformations, whereas the native bbRNAs showed two distinct populations. For both species, addition of divalent metal ions (Mn2+ and Mg2+) induces a high FRET population indicating that the stem and the branch strands of the bbRNA are in close proximity. Additionally, conversion of the stem to a duplex gives rise to a single narrow FRET distribution meaning that both the native bbRNA and cbRNA lose conformational flexibility. Finally, using TIRF microscopy, we showed that cbRNAs undergo a conformational switch between a high and a medium FRET state. However the importance of these conformations and their fluctuations, for the debranching reaction remains to be investigated. Together this two-pronged approach of biochemical and single molecule studies will help the reaction mechanism of the elusive lariat debranching enzyme to be elucidated in the future.

In 1985 Richard Preston identified three Royal Navy (RN) traditions (recruitment of officers at an early age, selection of officers from an elite social group, and insistence on sea service) which had shaped the Royal Canadian Navy (RCN). These traditions, he argued, ensured a high level of professionalism amongst officers in the infant RCN, as well as complete interoperability between the two navies, but failed to recognise the distinct needs of Canadian society. Consequently, from the Second World War onwards the RCN chose to move away from the British model and to ???Canadianise??? its officer corps. The Royal Australian Navy (RAN) also adopted these traditions, and they are examined here in the context of the social backgrounds, development and character of the permanent executive branch officers of the RAN between 1913 and 1950. This thesis argues that while the British model ensured a high level of professionalism within the RAN officer corps, in many other areas the system proved to be of doubtful utility for Australia. Although the Australian government tried to ensure that its naval officers maintained an Australian character and identity, the selection, training and operational policies of the RAN meant that its officers were, to all intents and purposes, virtually indistinguishable from their RN colleagues. While RAN officers were highly disciplined and professional men with excellent seamanship skills, unfortunately a wide social gulf developed between the Navy???s officers and its sailors. Further, the essentially scientific and practical education and indoctrination that naval officers received in their early years, combined with their narrow professional development, meant that they were, at best, only average higher level administrators and often performed poorly in dealings with their Australian political masters. The system produced a conservative type of officer, suspicious of political activity and intellectual effort, bound to the tradition of ???the Silent Service???, who felt that his country did not understand his work or sacrifices but who had not the capacity to change such community perceptions. Lacking highly educated and politically aware senior officers, the RAN found it difficult to cope with social changes after the Second World War. Consequently, the ???Australianisation??? of the naval officer corps was a slow and painful process and the profession of naval officer in Australia was to be even more marginal than numbers alone dictated.

O presente estudo enfoca o papel das redes de agências de avaliação da qualidade e Acreditação da Educação Superior na América Latina a partir das redes RANA E RIACES. O tema da Acreditação tem alcançado ressonância nas sociedades científicas internacionais, em especial na União Europeia, USA, e atualmente Caribe e América Latina. A temática de Redes de Avaliação da qualidade e Acreditação da Educação Superior na América Latina ainda é pouco explorada, do ponto de vista da pesquisa, mas é tema de discussões diversas em muitos países. O estudo traz o cenário que propiciou o surgimento das redes de cooperação e intercâmbio para o desenvolvimento de conhecimentos em matéria de Acreditação e Avaliação da qualidade da Educação Superior. Considerações sobre as reformas da Educação Superior na América Latina são discutidas, além dos “apelos” da globalização à Universidade Latina do século XXI. Apresentam-se evidências de um cenário da Educação Superior na América Latina marcado por desigualdades de desenvolvimento e de características em seus sistemas de Educação Superior. No âmbito das reformas da Educação Superior há a preocupação de marcar o momento da inserção de processo de formação de sistemas de asseguramento da qualidade da Educação Superior. Duas redes da América Latina são estudadas a RANA e a RIACES. O método escolhido foi o estudo de caso, que propiciou conhecer a dinâmica de uma política de cooperação e integração no contexto de Redes. O estudo baseia-se em várias fontes, como a pesquisa documental, coleta de dados em fontes primárias e secundárias, entrevistas e visita às sedes das redes de agências.
The present study focuses on the role played by Quality Evaluation Agencies and Accreditation in Higher Education Agencies in Latin America from RANA and RIACES point of view. Accreditation is a theme that has, lately, reached great propagation in International Scientific Societies, mainly in the European Union, USA and more recently in the Caribbean and Latin America. The idea of Quality Evaluation and Accreditation Networks in Higher Education in Latin America is not very explored from a research point of view, but is a theme of great discussion in many countries. This study brings the scenario that made possible the upcoming cooperation and exchange networks to the development of knowledge in terms of Accreditation and Quality Evaluation in Higher Education. Considerations about the reforms in Higher Education in Latin America are discussed as well as the “calls” for globalization that the Latin University has received in the 21st century. Evidences of a scenario of inequalities in development in Higher Education in Latin America are presented as well as the characteristics of Higher Education systems. As for the reforms scope, there seems to be a certain concern about emphasizing the exact moment in which the process of formation of quality reassurance in Higher Education is inserted. Two Latin American agencies are studied: RANA and RIACES. The method chosen was case study, which enabled the acknowledgement of a cooperation and integration policy in these networks context. The study is based in many different resources such as documental research, data collection in primary and secondary sources, interviews and onsite visits to the agencies headquarters.
En esta investigación se enfoca el rol de las Redes de Agencias de Evaluación de la Calidad y de las Redes de Acreditación de la Educación Superior en América Latina, a partir del estudio de RANA y RIACES. El tema de la Acreditación ha alcanzado resonancia internacional en las sociedades científicas, en especial en la Unión Europea, América Latina y el Caribe. El temático de redes de la evaluación de la calidad y de la acreditación de la educación superior en América Latina todavía poco se explora del punto de vista de la investigación, pero es tema de controversias en muchos países. El estudio trae a la escena el brote de las redes de la cooperación y del intercambio para desarrollo del conocimiento en la acreditación y la evaluación de la calidad de la educación superior. Consideraciones sobre las reformas de la educación superior en América Latina son discutidas, en el encuadre de las reformas de la educación superior en el siglo XX, más allá de los “llamados” de la globalización a la universidad latina del siglo XXI. Evidencias de una escena de la educación superior enmarcada por la desigualdad y características diversificadas de desarrollo en sus sistemas de la educación superior, se superponen a los requisitos del mercado en líneas de dirección para su eficacia. Es en el momento de las reformas de la educación superior que se tiene la preocupación para enmarcar la inserción del proceso de la formación de sistemas de aseguramiento de la calidad de la educación superior. Dos agencias latinoamericanas son estudiadas: RANA y RIACES. El método elegido fue el estudio de caso, para comprehender la dinámica de una política de cooperación e integración en el contexto de redes. El estudio se basa en investigación documental, fuentes primarias y secundarias, recoge datos en entrevistas y visitas a las sedes de las redes de agencias.

碩士
長庚大學
生物醫學研究所
101
Hepatitis delta virus (HDV) is a subviral satellite of hepatitis B virus. HDV is unique in having an unbranched rod-like circular RNA with ribozyme activity, and a double rolling-circle replication strategy utilizing host RNA polymerases. HDV RNA recombination has been observed in patients with mixed genotype infections and in cultured cells co-transfected with two cloned HDV sequences. The recombination maps obtained from cells co-transfected with genotype I and IV HDV as well as the one from cells co-trasfected with two genotype I clones have been established in the lab. Parallels of these two recombination maps revealed several recombination hot spots. Analyses of the potential RNA structures around these hot spots indicated that a stem-loop structure covering nt 435~455 and two asymmetric bubbles appearing on two sides of HDV rod-like structure (nt 535 and nt 1055~1059 as well as nt 305 and nt 1287~1290) might promote HDV RNA recombination. These observations prompted me to hypothesize that the above-mentioned RNA structures might cause polymerase pausing, a step which is crucial for the replication-dependent template-switch mechanism for RNA recombination. Another structure feature for causing polymerase pausing and then promoting HDV RNA recombination is the prolonged double-stranded regions on HDV rod-like RNA genome. Therefore, a series of mutants affecting the above-mentioned structures have been constructed to investigate their effect on not only HDV RNA recombination but also replication. These studies will contribute to the understanding of the underlined molecular mechanism of HDV RNA replication and recombination.

Thesis (Ph. D.)--Florida State University, 2006.
Advisor: Nancy L. Greenbaum, Florida State University, College of Arts and Sciences, Dept. of Chemistry and Biochemistry. Title and description from dissertation home page (viewed Jan. 2, 2007). Document formatted into pages; contains xix, 173 pages. Includes bibliographical references.

Thesis (Ph. D.)--University of Illinois at Urbana-Champaign, 2006.
Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3784. Adviser: Scott K. Silverman. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

碩士
國立中央大學
土木工程學系碩士在職專班
99
The fire department carries out its three missions of “fire prevention”, “rescue work”, and “emergency first-aid” in accordance with fire services related laws. As time goes by, there is an increasing number of emergency medical services need to be accommodated. For example, the Fire Bureau of Taoyuan County had around sixty-three thousand emergency medical services in 2009. Although the mentioned bureau has been always in the first rank of fire service satisfaction survey, there are still some extraordinary cases, mainly related to the response interval, which might cause disputes. The main reason of an extraordinary case is usually concluded of some colleagues’ error. However, if we consider Reason’s “Swiss Cheese Theory” (1990), the error might only be the active failure, not the latent failure. In order to find out the potential failures, this research uses Root Cause Analysis (RCA) to filter out the root cause and design relevant action plans to eliminate similar risk factors.