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1
Fritz, Sarah E. "Molecular basis of the DExH-box RNA helicase RNA helicase A (RHA/DHX9) in eukaryotic protein synthesis." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1437413252.
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Johnston, Julie Catherine. "In vitro translation of cucumber necrosis virus RNA." Thesis, University of British Columbia, 1989. http://hdl.handle.net/2429/28969.
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The in vitro translation products directed by cucumber necrosis virus (CNV) RNA were analyzed in both rabbit reticulocyte lysate and wheat germ extract cell-free translation systems. In rabbit reticulocyte lysates, one major protein of ca. 33 Mr was produced. In wheat germ extracts, four proteins of ca. 41, 33, 21 and 20 Mr were produced. Hybrid-arrested translation (HART) studies using synthetic CNV antisense RNA corresponding to the entire CNV genome demonstrated that the four major proteins synthesized from CNV virion RNA in wheat germ extracts are virus-specific translation products. The genomic locations of the CNV in vitro translation products were determined using a number of experimental approaches including: (1) HART using antisense RNA corresponding to selected regions of the CNV genome; (2) in vitro translation of synthetic messenger-sense CNV transcripts; (3) immunoprecipitation of in vitro translation products with CNV polyclonal antisera and (4) in vitro translation of size-fractionated CNV virion RNA. Together, these experiments demonstrated that the ca. 33 Mr protein is derived from the 5' proximal coding region, the ca. 41 Mr protein is derived from an internal coding region, and that at least one but probably both of the ca. 20 and 21 Mr proteins are derived from the 3' terminal coding region(s) of the CNV genome. In addition, immunoprecipitation experiments provided further evidence that the ca. 41 Mr protein is the viral coat protein. The size, number, and genomic locations of the CNV in vitro translation products reported here are in agreement with those predicted from nucleotide sequence data (Rochon & Tremaine, 1989). The natural template for the expression of downstream cistrons in the CNV genome was investigated by in vitro translation of sucrose fractionated CNV virion RNA as well as in vitro translation of messenger-sense synthetic transcripts. These studies indicate that in vitro, both subgenomic and genomic-length CNV RNA molecules may act as templates for the synthesis of the ca. 41,21 and 20 Mr proteins as well as the ca. 33 Mr protein.Land and Food Systems, Faculty of
Graduate
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Peters, D. W. "RNA synthesis in Candida albicans." Thesis, University of Warwick, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.373051.
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Lackey, Jeremy. "New methods for the synthesis of RNA, novel RNA pro-drugs and RNA microarrays." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=92290.
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The demand for synthetic oligonucleotides has grown exponentially over the past decades as genome sequencing, functional genomics, polymerase chain reaction (PCR)-based detection methods, and gene silencing via RNA interference (RNAi) consume enormous numbers of DNA and RNA oligonucleotides. Although various RNA synthesis chemistries now allow oligoribonucleotides to be produced routinely, the higher complexity and cost of RNA (over DNA) has somewhat limited its availability.A major goal of this thesis work was aimed at finding ribonucleoside synthons that potentially benefit two critical aspects of RNA manufacturing: yield and ease of post-synthesis processing. Towards these goals, we developed methods for the synthesis of RNA using 2'-O-Lv and 2'-O-acetal Lv (ALE) ribonucleoside derivatives. Deprotection of the RNA chains consisted of a three-step deprotection scheme, which eliminated the need for any harsh basic hydrolytic steps, generally composed of: (1) treatment with anhydrous NEt3 (r.t., 1 h) to deblock the phosphate's cyanoethyl groups; (2) hydrazinolysis (r.t., 30 min 4 h) to simultaneously deprotect the nucleobases and 2'-OH positions, and (3) fluoride treatment (r.t., 30 min) to effect cleavage from the controlled pore glass solid support. Significantly, the rather mild conditions to remove 2'-O-Lv or 2'-O-ALE protecting groups did not lead to RNA strand scission. Furthermore, in the case of 2'-O-ALE protection, higher step-wise monomer coupling yields (~98.7%) was possible, since the ALE protection is less bulky than conventional silyl protection, i.e. TBDMS. Furthermore, both 2'-O-Lv or 2'O-ALE chemistries are completely compatible with the synthesis cycles used by all automated gene synthesizers.
With adjustments in protecting group strategies for the 5'-OH, exocyclic amino nucleobase groups and the development of a light-labile solid support, two other major goals were achieved: (1) the first in situ synthesis of RNA on microarrays, and (2) synthesis of chemically modified RNA strands with 2'-O-acetal ester and 2'-O-acetal ester pyrrolidines in order to increase lipophilicity and cellular permeability over native RNA. When RNA synthesis was carried out with 5'-O-NPPOC 2'-O-ALE monomers on a microarray ("chip"), deprotection typically involved (1) cleavage of the photolabile 5'-protecting group; (2) treatment with anhydrous NEt3 (r.t., 1 h) to deblock the phosphate's cyanoethyl groups; (3) hydrazinolysis (r.t., 30 min 4 h) to simultaneously deprotect bases and 2'-OH positions. The latter step could also be accomplished with ethylenediamine at room temperature. An RNase A assay was performed as "proof-of-principle" to demonstrate the value of a DNA-RNA microarray for studying enzyme kinetics and specificity on oligonucleotide based libraries. We showed that RNase A acts effectively on a DNA-RNA substrate with measurable kinetics analogous to those of the reference substrates.
The novel 2'-O-modified RNA were tested as short interfering RNA pro-drugs ("pro-siRNA") that would cross the cell membrane and be hydrolyzed (at the 2'-O-ester groups) by ubiquitous esterases to release the active (siRNA) molecules. Indeed, both siRNA and pro-siRNA prepared via 2'-O-ALE chemistry were shown to be active in an RNAi luciferase gene knockdown assay, confirming the integrity of the synthesized RNA strands and the promise of the pro-siRNA approach.
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Brown, Michael Dean. "Genetic analysis of RNA splicing in the thymidylate synthase gene of bacteriophage T4." Diss., Georgia Institute of Technology, 1990. http://hdl.handle.net/1853/25390.
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Attwater, James. "Ice as a medium for RNA-catalysed RNA synthesis and evolution." Thesis, University of Cambridge, 2011. https://www.repository.cam.ac.uk/handle/1810/246525.
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A critical event in the origin of life is thought to have been the emergence of a molecule capable of self-replication and evolution. According to the RNA World hypothesis, this could have been an RNA polymerase ribozyme capable of generating copies of itself from simple nucleotide precursors. In vitro evolution experiments have provided modern examples of such ribozymes, such as the R18 RNA polymerase ribozyme, exhibiting basic levels of this crucial catalytic activity; R18’s activity, however, falls far short of that required of an RNA replicase, leaving unanswered the question of whether RNA can catalyse its self-replication. This thesis describes the development and use of a novel in vitro selection system, Compartmentalised Bead-Tagging (CBT), to isolate variants of the R18 ribozyme with improved sequence generality and extension capabilities. CBT evolution and engineering of polymerase ribozymes, together with RNA template evolution, allowed the synthesis of RNA molecules over 100 nucleotides long, as well as the RNA-catalysed transcription of a catalytic hammerhead ribozyme. This demonstrates the catalytic capabilities of ribozyme polymerases. The R18 ribozyme was also exploited as an analogue of a primordial replicase, to determine replicase behaviour in different reaction environments. Substantial ribozyme polymerisation occurred at −7˚C in the liquid eutectic phase of water-ice; increased ribozyme stability at these low temperatures allowed longer extension products to be generated than at ambient temperatures. The concentration effect of eutectic phase formation could also yield RNA synthesis from dilute solutions of substrates, and provide quasicellular compartmentalisation of ribozymes. These beneficial physicochemical features of ice make it a potential protocellular medium for the emergence of primordial replicases. Ice also could serve as a medium for CBT, allowing the isolation of a polymerase ribozyme adapted to the low temperatures in the ice phase, demonstrating the primordial potential and modern feasibility of ribozyme evolution in ice.
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Collis, Alana E. C. "The synthesis of vinylphosphonate-linked RNA." Thesis, University of Nottingham, 2008. http://eprints.nottingham.ac.uk/10541/.
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An introductory chapter discusses the steric block, RNase H and RNA interference antisense mechanisms and the application of antisense nucleic acids as therapeutic agents. Examples of existing chemical modifications of the sugar and backbone regions of nucleic acids are given, followed by the introduction of the vinylphosphonate modification. The vinylphosphonate has previously been examined in DNA and has been synthesised by either Pd(0) catalysed cross-coupling of an H-phosphonate with a vinyl bromide, or by the cross-metathesis of a vinylphosphonate with a terminal olefin. This thesis details the first examples of the vinylphosphonate modification in RNA. The initial aim of this project was the synthesis of a range of nucleosides where the 5'-C-O was replaced by a vinyl bromide carbon-carbon double bond. Starting from alpha-D-glucose, acid catalysed formation of the 1,2:5,6-diisopropylidene alpha-D-glucofuranose was carried out followed by protection of the 3-OH as an acetate. The 5,6-isopropylidene was then subjected to H5IO6 mediated one-pot hydrolysis-oxidative cleavage to obtain the 5-aldehyde. Wittig olefination using CBr4 and Ph3P led to the dibromo olefin which was then stereoselectively reduced using dimethyl phosphite and diisopropylamine to obtain the pure trans-vinyl bromide. Following hydrolysis of the acetate, the stereochemistry of the 3-OH was then inverted by sequential oxidation and reduction. With the correct stereochemistry, the 3-OH was protected as the 2-methylnaphthyl ether. The 1,2-isopropylidene moiety was then hydrolysed and acetylated to the bis-acetate which was subjected to Vorbruggen conditions obtaining the uridine (93%), adenosine (77%), cytidine (30) and guanosine (63%) vinyl bromide nucleosides. The 2'-OAc of the nucleosides were hydrolysed to the 2'-OH in yields of 74-92%. The uridine 2'-OH was protected as the 2'-OTBS ether (98%), analogous to the commercially available phosphoramidites used in automated oligonucleotide synthesis. Similarly, the adenosine and uridine nucleosides could also be blocked as the 2'-OMe (59% and 73% respectively). In the case of the uridine vinyl bromide, the 3'-O-(2-methylnaphthyl) protecting group was cleaved using DDQ, this then enabled the vinylphosphonate-linked uridine dinucleotides to be functionalised at the 3'-OH as the cyanoethyl phosphoramidite using N,N-diisopropyl-2-cyanoethyl-chlorophosphoramidite, DIPEA and DMAP in dichloromethane (2'-OTBS 74%, 2'-OMe 41%). These could then be used in automated solid phase oligonucleotide synthesis. The H-phosphonates were prepared in a single step form the commercially available phosphoramidites using a tetrazole. These were then coupled to the vinyl bromide nucleosides using standard conditions of Pd(OAc)2 (0.2 eq.), dppf (0.4 eq.) and propylene oxide (20 eq.) in THF at 70 oC in a sealed vial for 6 hours. A range of vinylphosphonate-linked dinucleotides were accessed in yields of 61-99%. A detailed experimental section at the end of this thesis describes the procedures used in the synthesis and the analysis of the structures obtained.
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Liu, Qi. "Synthesis of small molecules targeting RNA /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2004. http://wwwlib.umi.com/cr/ucsd/fullcit?p3142456.
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Finnegan, Patrick Michael. "RNA synthesis in maize mitochondria : the identification of autonomously replicating RNA species and a kinetic analysis of transcript accumulation." Thesis, McGill University, 1989. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=75931.
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Transcription in mammalian and yeast mitochondria proceeds from a few well defined promoters, with processing of polycistronic transcripts producing the mature RNAs. The levels of different sequences in the steady-state RNA populations depend on differential promoter strengths, transcription attenuation and/or selective termination, and differential RNA stabilities. To gain insights into the processes governing transcription and RNA levels in plant mitochondria, a system using isolated maize mitochondria, which synthesize bona fide mitochondrial RNAs, was developed and partially characterized with respect to exogenous requirements and sensitivity to inhibitors of DNA-dependent RNA synthesis.Although initiation and processing probably occur at reduced levels in isolated maize mitochondria, endogenous DNA templates are extensively transcribed at the same relative rates as in vivo. Isolated maize mitochondria were used to demonstrate that differential rates of both synthesis and turnover help determine the steady-state abundances of various mitochondrial RNA sequences and that mitochondria from certain lines possess an autonomously-replicating, RNA-based genetic system.
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D'Abramo, Claudia M. "Biochemical characterization of the BVDV RNA-dependent RNA polymerase during initiation and elongation of RNA synthesis." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111870.
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The RNA-dependent RNA polymerase (RdRp) of viruses belonging to the Flaviviridae family, including the hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) is critical for viral replication. The major goal of this PhD study was to biochemically characterize the role of the polymerase during initiation and elongation of RNA synthesis, utilizing the BVDV RdRp as a model system. We showed that the BVDV polymerase efficiently incorporates chain-terminating nucleoside analogues, which ultimately arrest RNA synthesis. The incorporated chain-terminators, however, can be removed from the primer terminus in the presence of pyrophosphate (PPi). These results suggest that the phosphorolytic excision of incorporated chain-terminators is a possible mechanism that can diminish the efficiency of this class of compounds against viral RdRps. The chain-terminators then served as valuable tools in subsequent experiments to analyze the functional role(s) of the RdRp-associated GTP-specific binding site (G-site) and the consequences of GTP binding during the initiation of RNA synthesis. The results provide biochemical evidence for the existence of a G-site in the BVDV enzyme, and suggest that GTP binding controls template positioning during de novo initiation. Finally, through the development of a novel ribonuclease-based footprinting assay, it was determined that catalytically active complexes contact the newly synthesized RNA during elongation of RNA synthesis with approximately 6-7 base pairs. The polymerase moves along the template according to the position where RNA synthesis is arrested. Taken together, this study provides novel insight into mechanisms involved during initiation and elongation of RNA replication of viruses belonging to the Flaviviridae family. The ability of RdRps to excise incorporated chain-terminators points to possible shortcomings of nucleoside analogue inhibitors that are under development as antiviral agents for the treatment of infection with HCV.
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Matsuura, Satoshi. "Synthetic RNA-based logic computation in mammalian cells." Kyoto University, 2019. http://hdl.handle.net/2433/242426.
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Repass, John F. "Studies of murine coronavirus cis-acting RNA elements that affect RNA synthesis /." Digital version accessible at:, 2000. http://wwwlib.umi.com/cr/utexas/main.
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Webb, Vera Ann B. "In vivo in vitro synthesis of ribosomal RNA in bacillus subtilis." Thesis, University of British Columbia, 1988. http://hdl.handle.net/2429/29448.
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The work presented explored the in vivo and in vitro synthesis of ribosomal RNA in the Gram positive, spore-forming bacterium Bacillus subtilis. The investigation began with a study of rRNA synthesis in B. subtilis during steady state growth and under nutritional shift-up conditions. The percent of transcription which is ribosomal RNA was measured by hybridization of pulse labeled RNA to a specific DNA probe carrying the 3' end of the 23S RNA gene. The fractional rate of ribosomal RNA synthesis increased with cellular growth rate, and showed a rapid increase after a nutritional shift up. RNA synthesis during infection with an amber mutant of bacteriophage SP01 was also examined. Infected cells continued to synthesize rRNA at the preinfection rate, but could not respond to media enrichment by increasing the percent rRNA-synthesis. The latter study suggested the existence of a specific RNA polymerase that transcribed ribosomal RNA genes.
The conclusions from the in vivo study led to an analysis of rRNA transcription in vitro. The isolation of the putative ribosomal RNA specific RNA polymerase was attempted by affinity chromatography on cellulose complexed with plasmid DNA containing the promoter region of the B. subtilis rrnB rRNA operon, and by sedimentation through a glycerol gradient. No difference in activity profile was observed when transcription
activity at the rRNA tandem promoters was compared to activity at a non-ribosomal promoter. Since in vivo analysis of the control of rRNA synthesis in Escherichia coli suggested that regulation occurs at the level of transcription initiation, in vitro transcription initiation at the B. subtilis rRNA promoters was investigated using the single round transcription assay. Initial rates of transcription were different at each of the two tandem promoters of the B. subtilis rrnB operon: the upstream promoter, PI, initiated slowly, while the downstream promoter, P2, initiated faster. In addition, transcription initiation at the two promoters appeared to be linked. The formation of a heparin resistant complex at the PI promoter affected the stability of the heparin resistant complex formed at the P2 promoter. The kinetics of transcription initiation at the tandem rRNA promoters were examined using the tau plot analysis. RNA polymerase had a high affinity for both rRNA promoters, but the rate of initiation at these promoters was relatively slow when compared to non-ribosomal promoters. Finally, transcription initiation on two artificial tandem promoter constructs was compared with initiation on the native tandem promoter construct. In general, PI was shown to have a positive effect on transcription from downstream promoters, but had specific effects on different promoters.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
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Wang, Yang. "Chemical synthesis of DNA and RNA containing thio-substituted bases and their post-synthetic modifications." Thesis, Aston University, 2003. http://publications.aston.ac.uk/11013/.
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Modified oligonucleotides containing sulphur group have been useful tools for studies of carcinogenesis, protein or nucleic acid structures and functions, protein-nucleic acid interactions, and for antisense modulation of gene expression. One successful example has been the synthesis and study of oligodeoxynucleotides containing 6-thio-2'-deoxyguanine. 6-Thio-2'deoxyguanosine was first discovered as metabolic compound of 6- mercaptopurine (6-MP). Later, it was applied as drug to cure leukaemia. During the research of its toxicity, a method was developed to use the sulphur group as a versatile position for post-synthetic modification. The advantage of application of post-synthetic modification lies in its convenience. Synthesis of oligomers with normal sequences has become routine work in most laboratories. However, design and synthesis of a proper phosphoramidite monomer for a new modified nucleoside are always difficult tasks even for a skilful chemist. Thus an alternative method (post-synthetic method) has been invented to overcome the difficulties. This was achieved by incorporation of versatile nucleotides into oligomers which contain a leaving group, that is sufficiently stable to withstand the conditions of synthesis but can be substituted by nucleophiles after synthesis, to produce, a series of oligomers each containing a different modified base. In the current project, a phosphoramidite monomer with 6-thioguanine has been successfully synthesised and incorporated into RNA. A deprotection procedure, which is specific for RNA was designed for oligomers containing 6-thioguanosine. The results were validated by various methods (UV, HPLC, enzymatic digestion). Pioneer work in utilization of the versatile sulphur group for post-synthetic modification was also tested. Post-synthetic modification was also carried out on DNA with 6- deoxythioguanosine. Electrophilic reagents with various functional groups (alphatic, aromatic, fluorescent) and bi-functional groups have been attached with the oliogmers.
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Usman, Nassim. "Nucleoside phosphoramidites in the automated, solid phase synthesis of oligoribonucleotides and their analogues : the chemical synthesis of an E. Coli N-Formyl-Methionine tRNA." Thesis, McGill University, 1986. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=75344.
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The use of ribonucleoside phosphoramidites protected at the 2$ sp prime$-hydroxyl with alkylsilyl ethers was developed for the solid phase synthesis of oligoribonucleotides. A series of pentadecameric oligomers of various base composition was synthesized using this methodology and a complete protocol for the deprotection of synthetic oligoribonucleotides was developed. The use of $ beta$-cyanoethyl phosphate protection was compared to that of methyl protection in the synthesis of pentadeca-uridylic acid sequences and no significant difference between the use of the two protecting groups was found. Concurrently a procedure for the derivatization of long chain alkylamine controlled pore glass beads with ribonucleosides was used to provide an improved solid phase support for the efficient automated synthesis of long oligomers. A side reaction on this support was identified and a method for its prevention developed.A reaction occurring at the O$ sp6$-position of guanosine residues, which leads to chain cleavage, was identified. The basis of this reaction was characterized by both $ sp{31}$P nuclear magnetic resonance and the synthesis of oligoriboguanylate sequences. Three methods involving: protection of the O$ sp6$-position, the use of an alternate coupling cycle, and the use of a different phosphoramidite activator, were successfully applied to the resolution of this problem. The aforementioned procedures were utilized in the synthesis of a half E. Coli N- f - met tRNA molecule 43 units in length.
Finally the cumulative knowledge gained from the above studies was applied to the synthesis of an entire E. Coli N- f - met analogue 77 units in length along with a number of other very long sequences. Methods for the deprotection of these oligomers were investigated culminating in the isolation of oligoribonucleotides which were successfully characterized by polyacrylamide gel electrophoresis, enzyme degradation, enzymatic and chemical sequencing, terminal nucleotide analysis, and, in the case of the E. Coli f - met tRNA analogue, an aminoacylation assay.
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Hamilton, Andrew John. "Inhibiting gene expression with anti-sense RNA." Thesis, University of Nottingham, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316938.
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Akhtar, Y. "Studies on the maturation pathway of ribosomal precursor RNA : Analysis of Xenopus ribosomal RNA synthesised by transcription in vitro." Thesis, University of Liverpool, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.382054.
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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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Schulz, Daniel. "Transcriptome surveillance in S. cerevisiae by RNA synthesis and degradation coupling and selective termination of non-coding RNAs." Diss., Ludwig-Maximilians-Universität München, 2013. http://nbn-resolving.de/urn:nbn:de:bvb:19-163595.
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Eukaryotic gene transcription is highly complex and regulation occurs at multiple stages. RNA Polymerase II (Pol II) is recruited to promoter regions of the DNA to initiate transcription.
Shortly after initiation, Pol II exchanges initiation factors for elongation factors. After Pol II passes termination signals, the RNA is cleaved and Pol II eventually released from the DNA template. pre-mRNAs are polyadenylated and exported to the cytosol for translation and ultimately degradation. Mechanisms regulating transcription have been studied extensively, but mechanisms of mRNA degradation are less well understood. To monitor mRNA synthesis and degradation, we developed the comparative dynamic transcriptome analysis (cDTA). cDTA provides absolute rates of mRNA synthesis and decay in Saccharomyces cerevisiae Sc cells with the use of Schizosaccharomyces pombe Sp cells as internal standard. We show that Sc mutants can buffer mRNA levels and that impaired transcription causes decreased mRNA synthesis rates compensated by decreased decay rates. Conversely, impairing mRNA degradation causes decreased decay rates, but also
decreased synthesis rates. Thus, although separated by the nuclear membrane, transcription
and mRNA degradation are coupled. In addition to regulated mRNA synthesis, pervasive transcription can be found throughout the genome, governed by an intrinsic affinity of Pol II for DNA. These divergent noncoding RNAs (ncRNAs) stem to a large extent from bidirectional promoters. However,
global mechanisms for the termination of ncRNA synthesis that could act as a transcriptome
surveillance mechanism are not known. It is also unclear if such a surveillance system protects the transcriptome from deregulation. Here we show that ncRNA transcription in Sc is globally restricted by early termination which relies on the essential RNA-binding factor Nrd1. Depletion from the nucleus results in Nrd1-unterminated transcripts (NUTs) that originate from nucleosome-depleted regions (NDRs) throughout the genome and can deregulate mRNA synthesis by antisense repression and transcription interference.
Transcriptome-wide Nrd1-binding maps reveal divergent NUTs at essentially all promoters and antisense NUTs in most 3’-regions of genes. Nrd1 preferentially binds RNA motifs which are enriched in ncRNAs and depleted in mRNAs except in some mRNAs whose synthesis is controlled by transcription attenuation. These results describe a mechanism for transcriptome surveillance that selectively terminates ncRNA synthesis to provide promoter directionality and prevent transcriptome deregulation
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Garcia, Martin Juan Antonio. "RNA inverse folding and synthetic design." Thesis, Boston College, 2016. http://hdl.handle.net/2345/bc-ir:106989.
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Thesis advisor: Welkin E. JohnsonThesis advisor: Peter G. Clote
Synthetic biology currently is a rapidly emerging discipline, where innovative and interdisciplinary work has led to promising results. Synthetic design of RNA requires novel methods to study and analyze known functional molecules, as well as to generate design candidates that have a high likelihood of being functional. This thesis is primarily focused on the development of novel algorithms for the design of synthetic RNAs. Previous strategies, such as RNAinverse, NUPACK-DESIGN, etc. use heuristic methods, such as adaptive walk, ensemble defect optimization (a form of simulated annealing), genetic algorithms, etc. to generate sequences that minimize specific measures (probability of the target structure, ensemble defect). In contrast, our approach is to generate a large number of sequences whose minimum free energy structure is identical to the target design structure, and subsequently filter with respect to different criteria in order to select the most promising candidates for biochemical validation. In addition, our software must be made accessible and user-friendly, thus allowing researchers from different backgrounds to use our software in their work. Therefore, the work presented in this thesis concerns three areas: Create a potent, versatile and user friendly RNA inverse folding algorithm suitable for the specific requirements of each project, implement tools to analyze the properties that differentiate known functional RNA structures, and use these methods for synthetic design of de-novo functional RNA molecules
Thesis (PhD) — Boston College, 2016
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology
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Wesselhoeft, R. Alexander(Robert Alexander). "Synthetic circular RNA for protein expression." Thesis, Massachusetts Institute of Technology, 2019. https://hdl.handle.net/1721.1/122710.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2019Cataloged from PDF version of thesis.
Includes bibliographical references (pages 111-126).
Messenger RNA (mRNA) has broad potential for therapeutic and engineering applications. One fundamental limitation of mRNA is its relatively short half-life in biological systems, effected in part by rapid exonuclease-mediated degradation upon delivery. Circular RNA (circRNA), a type of single-stranded RNA with a contiguous structure that lacks the end motifs necessary for exonuclease recognition, may be resistant to this mechanism of degradation and therefore may exhibit superior stability. However, challenges in circularization, purification, and protein expression have impeded a thorough investigation of exogenous circRNA. By rationally designing ubiquitous accessory sequences to facilitate circularization, we engineered a permuted self-splicing intron that efficiently circularized RNAs up to 5kb in length in vitro.
With the addition of these accessory sequences, we were able to demonstrate nearly complete circularization of precursor RNAs containing an internal ribosome entry site (IRES) for translation initiation and a coding region such as erythropoietin or eGFP. We found that translation from optimized circRNA was robust, and circRNA protein expression stability far exceeded that of both unmodified and nucleoside modified linear mRNA in some cellular contexts. We monitored cytokine release and antiviral defense induction in sensitive cells transfected with circRNA purified by different methods and found that the immunogenicity and stability of circRNA preparations was dependent on the degree of purity, with small amounts of contaminating linear RNA leading to robust cellular immune responses.
In contrast to purified unmodified linear mRNA, purified unmodified circRNA was invisible to several RNA sensors including RIG-i and endosomai toil-like receptors (TLRs) and did not provoke a significant cytokine response upon transfection. Using purified circRNA, we finally provided the first demonstration to our knowledge of exogenous circRNA delivery and translation in vivo, and showed that the duration of circRNA translation was extended in adipose tissue in comparison to unmodified and uridine-modified linear mRNAs. In total, this work suggests that circRNA is a promising alternative to linear mRNA for therapeutic applications.
by R. Alexander Wesselhoeft.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biology
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Wang, Muhan. "Studies of piRNA synthesis." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:1e134b7b-4e9d-4d0e-bfd8-e4696f960ad6.
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RNA silencing is a form of post-transcriptional gene regulation, in which a small RNA guides a member of the Argonaute protein family in an effector complex to repress target gene expression. piRNAs, found in germ cells, are the most recently discovered major subset of small RNAs. A key known function of piRNA is to repress the transposable elements in the germline and maintain the germline genome integrity. The defining features of the piRNAs are 1) they are ubiquitously methylated at the 3’-end of the 2’-OH group by methyltransferase Hen1; 2) they associate exclusively with the Piwi subfamily Argonaute proteins. Much is not understood about the biogenesis and the regulation of the piRNA pathway. One of the fundamental questions is how the 3’-end of the piRNA is generated and recognised specifically by Piwi but not by Ago subfamily Argonaute proteins. In this thesis, the high resolution crystal structure of the Aubergine PAZ domain, a domain from a Piwi subfamily Argonaute, bound to a 7 mer single-stranded methylated piRNA ‘mimic’, reveals the mode of recognition for the 3’-end of piRNAs by Piwi subfamily Argonautes. The structure provides the molecular basis for why Piwi but not Ago PAZ domains preferentially bind to RNAs with 2’-O-methylation at the 3’-end, thus conferring substrate specificity. The structural results are confirmed by biochemical studies. Biochemical and biophysical studies on the methyltransferase Hen1 have provided insights into substrate specificity for piRNA 3’-end methylation and revealed a potential regulatory role for the C-terminal region of the protein. Extensive biochemical analysis defined a minimal active Hen1/short RNA complex, though crystallisation screening yielded no crystals for structure determination. Overall, this study provides insights into the generation and molecular recognition of the piRNA in animals.
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Johnson, Moira A. "Kinetics of RNA synthesis in Rotavirus infected cells." Thesis, University of Warwick, 1988. http://wrap.warwick.ac.uk/98142/.
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A method has been developed to allow the independent quantification of the level of synthesis of each strand of different viral genes during a synchronous infection of tissue culture cells. It was developed and applied to the UK tissue culture adapted bovine Rotavirus whose genome consists of 11 discrete segments of monocistronic double stranded RNA (dsRNA). Cloned copies of the 11 genes of the UKtc Bovine Rotavirus have been sub-cloned into the polylinker region of the pGEM1 and pGEM2 transcription vectors. Using the T7 and SP6 promotors flanking this region, high specific activity RNA probes to either strand of the gene have been generated and used in Solution Hybridisations to quantitate the transcription and replication of each gene. Quantification of the plus-strand (m-RNA) synthesis at hourly intervals throughout the growth cycle provided evidence for both quantitative and qualitative regulation of transcription. Quantitative control of transcription was demonstrated by the accumulation of much higher levels of m-RNA for some viral genes (eg:-2 and 7) than others (eg:-4 and 6). The relative molar amounts of the different viral proteins was measured at 6.5 hours post-infection and showed that they did not directly reflect the accumulation of their encoding mRNAs, providing evidence for the existence of translational control of gene expression. For example the high levels of VP6 and VP10 produced was due primarily to high translational frequency, whereas the high level of VP8 could be correlated to a high level of accumulation of its encoding m-RNA. The existence of qualitative control of viral transcription was demonstrated by the finding that the transcription of 4 genes (5,6,7 and 9) occurred independently of protein synthesis in infected cells. These data suggested that the proteins encoded by these genes may have a regulatory role in the replication cycle. Study of the minus-strand synthesis for each viral gene indicated that the RNA replication for Rotaviruses is probably the same as that of mammalian Reo viruses. The method described in this thesis is a generic one since with the availability of suitable c-DNA clones it could be used to achieve qualitative and quantitative analysis of viral gene expression in any viral system.
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Whitfield, Julie Nicole. "Studies on a potentially prebiotic synthesis of RNA." Thesis, University of Oxford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.320683.
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Cook, Stephen D. "Studies on a potentially prebiotic synthesis of RNA." Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.267951.
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Takeuchi, Akiko. "RNA-protein interaction in the selenoprotein synthesis machinery." Strasbourg, 2009. http://www.theses.fr/2009STRA6054.
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La sélénocystéine est incorporée co-traductionnellement dans les sélénoprotéines en réponse à un codon UGA habituellement l’un des 3 codons stop. La protéine SBP2 joue un rôle majeur dans ce mécanisme de recodage en se liant à une structure en tige-boucle (SECIS) située dans la région 3’UTR de l’ARNm des sélénoprotéines. Nous avons isolé et caractérisé fonctionnellement SBP2 de Drosophila melanogaster. Par comparison avec SBP2 humaine, nous avons identifié un domaine de liaison à l’ARN additionnel essentiel à la liaison au SECIS et à la sous-unité ribosomique 60S et permettant une sélectivité structurale du SECIS. Des prédictions structurales et des analyses biophysiques ont établi que SBP2 était une protéine globalement désordonnée ou “Intrinsically Disordered Protein” qui ne se replie qu’en présence de partenaires. Enfin, nous avons établi que l’assemblage des mRNP de sélénoprotéines faisait appel à des facteurs communs et présentait de multiples similarités avec celui des sn/snoRNPThe 21st amino acid selenocysteine is encoded by a UGA codon that usually signifies translational termination. Selenoprotein synthesis therefore requires specialized factors. Among these is SBP2 that binds the SECIS, a stem-loop structure in the 3’UTR of selenoprotein mRNAs. In structural analyses of SBP2, we isolated and functionally characterized Drosophila melanogaster SBP2. By comparing it with human SBP2, we identified an additional RNA binding domain that is essential for SECIS and 60S ribosomal subunit binding, and also enables SECIS structure selectivity. In addition, computational and biophysical analyses established that SBP2 is globally unfolded, supporting our hypothesis that SBP2 is an Intrinsically Disordered Protein and becomes folded in the presence of partners yet to be identified. Finally, we searched for potential partners of SBP2 and our results showed that the molecular assembly of selenoprotein mRNPs has many similarities with that of sn/snoRNPs
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Chen, Yuanyuan. "Characterizing RNA Structure and synthesis by Raman Microscopy." Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1277761094.
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Yunus, Muhammad Amir. "Studies on the mechanism of norovirus RNA synthesis." Thesis, Imperial College London, 2012. http://hdl.handle.net/10044/1/9811.
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Noroviruses are common pathogens associated with gastroenteritis in humans. As single-stranded positive-sense RNA viruses, noroviruses achieve their genome replication through the synthesis of negative strand RNA intermediates that template the generation of new positive strand RNA genomes. The murine norovirus (MNV) RNA-dependent RNA polymerase (NS7) protein which is a key player in catalysing this process, has been characterised in this study. Tandem affinity purification of MNV NS7 was performed in order to identify host cell factors which interact with the NS7 protein. Proteomic analysis demonstrated that guanosine monophosphate reductase and N(2)- dimethylguanosine tRNA methyltransferase could potentially interact with the MNV NS7. Furthermore, affinity selection of small peptides which specifically bind to the NS7 was carried out by using the phage display technique in an effort to generate peptide inhibitors. Peptide phage with two different conserved motifs and several peptide phage pools with binding activity to the NS7 were successfully identified. However, further cross-binding analysis using ELISA demonstrated that these peptide phage possibly bound non-specifically to the MNV NS7. An optimised RNA based reverse genetics system and reporter-tagged replicon system for MNV were also successfully developed and used to quantify the effects of specific mutations in the MNV genome on viral replication in tissue culture. Using these newly developed systems, the functional role of a small stem loop structure located upstream of the start site of the subgenomic RNA was characterised. This was identified as the potential viral RNA polymerase promoter responsible for subgenomic RNA synthesis. Furthermore, identification and characterisation of this stem loop using mutations suggested the potential involvement of long range RNA-RNA interactions (on negative strand RNA) in regulating the norovirus subgenomic RNA synthesis. Overall, this study has unveiled the importance of protein-protein interactions and RNA-protein interactions in regulating norovirus replication. These interactions could provide interesting targets for antiviral therapeutic intervention in the future.
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Wang, Hai. "Design, synthesis and RNA binding of aminoglycoside antibiotics /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 1998. http://wwwlib.umi.com/cr/ucsd/fullcit?p9820848.
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Takeuchi, Akiko Krol Alain Allmang-Cura Christine. "RNA-protein interaction in the selenoprotein synthesis machinery." Strasbourg : Université de Strasbourg, 2009. http://eprints-scd-ulp.u-strasbg.fr:8080/1133/01/TAKEUCHI_Akiko_2009.pdf.
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Thèse de doctorat : Sciences du Vivant. Aspects moléculaire et cellulaire de la Biologie : Strasbourg : 2009.Thèse soutenue sur un ensemble de travaux. Titre provenant de l'écran-titre. Bibliogr. 11 p.
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York, Ashley D. "A study of viral and cellular factors in the regulation of the influenza virus RNA-dependent RNA polymerase." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:5958fafd-4c91-4434-910e-29e2dd0539b9.
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The segmented negative-sense vRNA genome of influenza A virus is replicated via a complementary RNA (cRNA) intermediate by the viral RNA-dependent RNA polymerase (RdRP). The replicative intermediate has been thought to exist as a complementary ribonucleoprotein (cRNP) complex. Development of an RNA-based affinity-purification strategy for the isolation of viral ribonucleoprotein complexes enabled the structural and functional characterisation of the previously uncharacterised cRNP complex. The cRNP exhibits a filamentous double-helical organisation with defined termini, containing the viral RdRP at one end and a loop structure at the other end. In vitro characterisation of cRNP activity led to a proposal of a model of vRNA synthesis that relies on a trans-activating RdRP. The viral RdRP is an important host range determinant indicating that its function is affected by interactions with cellular factors. However, the identities and the roles of most of these factors remain unknown. Affinity-purification followed by mass spectrometry was performed to identify cellular proteins that interact with the viral RdRP. 171 cellular proteins were found to co-purify with the viral RdRP, the most abundant of which were chaperones, cytoskeletal proteins, importins, proteins involved in ubiquitination, kinases and phosphatases, mitochondrial and ribosomal proteins. Among the phosphatases, three subunits of the cellular serine/threonine-protein phosphatase 6 (PP6) were identified. PP6 was found to interact directly with the PB1 and PB2 subunits of the viral RdRP, and siRNA-mediated knockdown of the catalytic subunit of PP6 in infected cells resulted in the reduction of viral RNA accumulation and the attenuation of virus growth. Taken together, these results suggest that PP6 interacts with and positively regulates the activity of the influenza virus RdRP.
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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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Bhutia, Pema choden. "Accuracy of TransferRNA Selection in Protein synthesis." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-162811.
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ACCURACY OF TRANSFER RNA SELECTION IN PROTEIN SY The ribosome is a rapid magnificent molecular machine that plays an important role in proteinsynthesis and it consists of RNA and protein. The 70S bacterial ribosome comprises twosubunits, 30S and 50S. The 30S small subunit of the bacterial ribosome contains a protein calledS12, encoded by the rpsL gene. The function of this S12 protein is to help arrange the mRNAcorrectly to the ribosome and to interact with transfer RNA (tRNA) to initiate translation.Mutations in the rpsL gene generate phenotypes like resistance, dependence or pseudodependenceto the antibiotic streptomycin in bacteria. It is believed that mutations in the rpsLgene can increase the accuracy of tRNA selection in protein synthesis.The ribosome conducts the selection of tRNA in two steps: the initial selection and theproofreading step. During these multiple steps, the ribosome chooses the cognate aminoacyltRNAsin a ternary complex with EF-Tu and GTP and accommodates in the A site of ribosome.Therefore, the accuracy of the ribosome in selection of cognate aminoacyl-tRNA is crucial for the production of functional polypeptide sequences. Here, three different Escherichia coli strains; wild type MG1655, streptomycin restrictive (SmR) strain res222, and a streptomycin pseudo-dependent (SmP) strain w3110 are used, for studying the accuracy of tRNA selection inprotein synthesis. The mutant SmR shows hyper-accurate phenotype, which means, it has lowerpeptide bond formation efficiency and higher accuracy than the wild type. SmP shows pseudodependentto streptomycin phenotype which means it has higher peptide bond formation efficiency in the presence of antibiotic streptomycin. I have estimated the accuracy of tRNA selection in protein synthesis with enzyme kinetics. The kinetics data of these experiments display that mutant streptomycin restrictive is hyper-accurate and lower peptide bond formation efficiency than the wild type. SmP for the near cognate reaction in presence of antibiotic streptomycin has higher peptide bond formation efficiency than the SmP in absence of antibiotic streptomycin. SmP in presence antibiotic streptomycin has lower accuracy than the SmP in absence of antibiotic streptomycin.
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Formanová, Nataša. "A complex synthesizing the maize mitochondrial plasmid RNA b /." Thesis, McGill University, 1993. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=68173.
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RNA b is the most abundant member of a family of autonomously replicating single- and double-stranded RNA plasmids found in mitochondria of several maize races. The extent to which this molecule associates in vivo with proteins was investigated by both rate zonal and CsCl equilibrium density gradient centrifugations of clarified lysate of mitochondria from maize plants with the S-type cytoplasm. A soluble endogenous complex of RNA b, responsible for synthesis of the more abundant (+) RNA b strand in in vitro conditions (in mitochondrial lysate), was identified. The complex had a density of 1.49 g/cm$ sp3$ but a surprisingly low sedimentation coefficient, only slightly larger than the naked RNA b. Only a minor fraction of RNA b molecules were bound in the complex; the majority of RNA b sedimented as naked RNA molecules. Complexes synthesizing other, less abundant, maize RNA plasmids were not identified. However, in vitro synthesis of all RNA plasmid species in mitochondrial lysate was resistant to heparin, suggesting that in all cases preformed RdRp-RNA template complexes, capable of elongating in vivo preinitiated RNA plasmid strands, were present.
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Lee, Hung-chiu. "Synthetic RNA interference against influenza A virus." Click to view the E-thesis via HKUTO, 2005. http://sunzi.lib.hku.hk/hkuto/record/B35537814.
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Lee, Hung-chiu, and 李洪釗. "Synthetic RNA interference against influenza A virus." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B35537814.
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Che, Austin 1979. "Engineering RNA logic with synthetic splicing ribozymes." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/47786.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2008.Includes bibliographical references (p. 169-185).
Reusable components, such as logic gates and code libraries, simplify the design and implementation of electronic circuits and computer programs. The engineering of biological systems would benefit also from reusable components. In this thesis, I show the utility of splicing ribozymes for the biological engineer. Ribozymes allow the engineer to manipulate existing biological systems and to program self-modifying RNA systems. In addition, splicing ribozymes are easy to engineer, malleable, modular, and scalable. I used the model ribozyme from Tetrahymena to explore the principles behind engineering biological splicing systems in vivo. I show that the core ribozyme is modular and functions properly in many different contexts. Simple base pairing rules and computational RNA folding can predict splicing efficiency in bacterial cells. To test our understanding of the ribozyme, I generated synthetic ribozymes by manipulating the primary sequence while maintaining the secondary structure. Results indicate that our biochemical understanding of the ribozyme is accurate enough to support engineering. Splicing ribozymes can form core components in an all-RNA logic system. I developed biological transzystors, switches analogous to electrical transistors. Transzystors can use any trans-RNA as input and any RNA as output, allowing the genetic reading of RNA levels. I also show the ribozyme can write RNA using the trans-splicing reaction.
(cont.) Trans-splicing provides an easy mechanism to hook into an existing biological system and patch its operation. The generality of these ribozymes for a wide set of applications makes them promising tools for synthetic biology. Keywords: synthetic biology, RNA, Tetrahymena, ribozyme, splicing, transzystor.
by Austin J. Che.
Ph.D.
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Thomas, Gregory Stuart. "Targeting prostate cancer with synthetic RNA ligands." Diss., University of Iowa, 2012. https://ir.uiowa.edu/etd/1508.
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Prostate cancer represents a serious health concern as the most diagnosed form of cancer in men and the second leading cause of cancer death in the Western world. Current treatments for prostate cancer are non-targeted and result in a number of undesirable, non-specific effects, highlighting the need for novel, targeted therapeutics in the treatment of prostate cancer.
Prostate Specific Membrane Antigen (PSMA) offers great promise in the targeting of prostate cancer for imaging and therapy. PSMA is a transmembrane carboxypeptidase with cell surface expression several orders of magnitude higher in cancerous prostatic epithelia than found in other tissue and PSMA is constitutively internalized into cells. The unique expression profile of PSMA and its constitutive internalization offer great value in the targeted delivery of therapeutics to prostate cancer cell.
In 2002, two synthetic RNA ligands, aptamers, were selected for their ability to inhibit the enzymatic activity of PSMA. In 2006, the utility of these aptamers in the delivery of cytotoxic siRNA across the cell membrane was demonstrated in vivo using aptamer-siRNA chimeras. However, those experiments were performed by intratumoral injection, and systemic administration will be necessary for use in the clinic.
In this thesis, we improve PSMA targeted chimeras to serve as more powerful therapeutics in the treatment of prostate cancer. We optimize existing aptamer-siRNA chimeras for increased potency and stability and improved pharmacokinetics to enable systemic administration. We truncate the PSMA binding aptamers for amenability to large-scale chemical synthesis. With emerging roles for PSMA enzymatic activity in the prostate cancer disease we identify aptamers that are suitable for chemical synthesis and retain inhibitory properties against PSMA. Finally, we assess the use of aptamers as synthetic ligands in the functional inhibition of PSMA mediated motility in prostate cancer.
Our results demonstrate the ability of aptamer-siRNA chimeras to specifically kill PSMA-expressing cells with cytotoxic siRNA upon systemic injection. We confirm a newly reported role for PSMA in the promotion of cell motility and demonstrate the ability of aptamers to effectively neutralize PSMA-mediated motility. The results presented within argue strongly for the functional utility of aptamers in the treatment of prostate cancer.
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Yu, Hongchuan. "Novel Cyclo Deoxynucleoside: Synthesis and Evaluation." Thesis, Boston College, 2012. http://hdl.handle.net/2345/2751.
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Thesis advisor: Larry W. McLaughlinThesis advisor: Mary F. Roberts
Nucleic acids are essential biological molecules for life. For example, deoxyribonucleic acid (DNA) is the main genetic information carrier; ribonucleic acid (RNA) plays a critical role in translation and transcription. These characteristics place nucleic acids as the fundamental genetic materials of a living system. Since over a century ago, intensive attempts have been made by researchers to study the nucleic acid properties. For chemists, it is particularly interesting and important to understand the relationship between structures and properties of nucleic acids. For instance chemical modifications can alter stability of nucleic acids, and consequently influence their biochemical behaviors. In this work, we began by investigation of a 5',6-cyclo-modified nucleic acid resembling the product of DNA oxidation, and then developed a library of cyclomodifications. Our research on their structures and properties indicated that by installing cyclo-modifications we might be able to add some properties, that were not observed in nature to nucleic acids
Thesis (PhD) — Boston College, 2012
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
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Steger, Courtney Long. "Determinants of Core Shell Dependent Rotavirus Polymerase Activity." Diss., Virginia Tech, 2019. http://hdl.handle.net/10919/87755.
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Rotaviruses (RVs) are medically significant gastrointestinal pathogens and are a leading cause of childhood mortality in many countries. The RV RNA-dependent RNA polymerase, VP1, synthesizes RNA during viral replication only in the presence of another RV protein, VP2, which comprises the innermost core shell layer of the virion. Though these VP1-VP2 interactions are essential for RV replication, the mechanism by which the core shell regulates polymerase activity remains incompletely understood. Here, we sought to identify and characterize specific regions of both VP1 and VP2 that are required for core shell dependent polymerase activity. First, we used bioinformatics approaches to analyze VP1 and VP2 sequence diversity across many RV strains and identify positional locations of critical amino acid changes within the context of known structural domains and motifs. We next tested how the identified sequence differences influenced VP2-dependent VP1 activity in vitro. These data revealed that VP1 and VP2 protein diversity correlates with functional differences between avian and mammalian RV strains. Then, we used these sequential and functional incompatibilities to map key regions of VP1 important for mediating RNA synthesis. To pinpoint critical interacting regions of VP1 and VP2, we used site directed mutagenesis to engineer several modified VP1 and VP2 proteins. Then, we employed an in vitro RNA synthesis assay to test how the introduced mutations influenced VP2-dependent VP1 activity. Altogether, our results revealed several functionally important VP1 residues critical for in vitro VP2-dependent VP1 activity, either individually or in combination with neighboring residues, including E265/L267, R614, and D971/S978/I980. Structural analyses show VP2 interactions at these surface-exposed VP1 sites, which altogether supports a direct contact model of core shell dependent RV polymerase activity. Moreover, recombinant VP1 proteins containing multiple mutations at buried residues were incapable of facilitating RNA synthesis in vitro under the assay conditions, indicating that an extensive intramolecular signaling network exists to mediate VP1 activity. Taken together, these results suggest that VP2 binding at the VP1 surface may induce intramolecular interactions critical for VP1 activity. Overall, results from these studies provide important insight into VP1-VP2 binding interface(s) that are necessary for RV replication.Ph. D.
Rotaviruses (RVs) are clinically-significant gastrointestinal pathogens that cause severe diarrhea and dehydration in children. RVs encode a specialized polymerase enzyme, called VP1, which functions to synthesize RNA during viral replication. RNA synthesis activities of VP1 are tightly regulated by another RV protein, VP2, which comprises the innermost core shell layer of the virion. Though these VP1-VP2 interactions are essential for viral replication, the mechanism by which the core shell supports polymerase activity remains poorly understood. Here, we sought to identify and characterize specific regions of both VP1 and VP2 that are essential for polymerase activity in a test tube (i.e., in vitro). First, we analyzed VP1 and VP2 sequence diversity across many RV strains. Then, we tested how the identified sequence differences influenced VP2-dependent VP1 activation in vitro. To pinpoint critical regions of VP1 and VP2, we next engineered and assayed several mutant proteins. Altogether, our results revealed several functionally important residues of VP1 and VP2, which raises new ideas about VP1-VP2 binding interface(s) that are important for viral replication. Moreover, results from these studies may provide a scientific platform for the rational design of next-generation RV vaccines or antiviral therapeutics.
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Hannoush, Rami Nabil. "RNA folding : synthesis, structure and biological properties of hairpins based on 2',5'-linked RNA loops." Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=82890.
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Ribonucleic acids (RNA) are polymers of ribonucleotides linked together by 3',5'-phosphodiester linkages and play a prominent role in protein biosynthesis. Much less common are ribonucleic acids based on 2',5'-phosphodiester linkages (2',5'-RNA). In this study, the synthesis, physical and biological properties of several 2' ,5'-linked oligoribonucleotides are described.One aspect of this thesis deals with the synthesis and structural studies of 2',5'-dodecaribonucleotides. Their base sequence was designed to promote intramolecular folding by base pairing leading to the formation of "hairpin loops". The hairpins consist of a tetranucleotide loop and a 4 base-pair duplex (stem). The thermodynamic stability and conformation of these hairpins were assessed by UV, CD and NMR spectroscopy. Melting experiments (UV) revealed that with a few exceptions, hairpins comprised of 2',5'-(UUCG) loops exhibit greater thermodynamic stability (e.g. Tm) than the corresponding hairpins with 3' ,5'-linked loops. The 'extra' stability imparted by the 2',5'-UUCG) loop was found to be virtually independent of the sugar composition of the stem. For example, the 2',5'-tetraloop stabilizes hairpins whether their duplex stem is based on double-stranded DNA or RNA. In contrast, the 3',5-tetraloop stabilizes hairpins only when their stem duplex is double-stranded RNA. NMR studies revealed that the 2',5'-tetraloop (UUCG) is self-stabilized by a wobble U·G base pair, extensive base stacking and sugar-base contacts. A more striking feature is the protrusion of the cytosine residue into the solvent without participating in any of the loop stabilizing interactions. This identifies the 2',5 '-linked (UUCG) loop as a novel structural motif and provides the first demonstration that 2',5' can fold back on itself to form a hairpin structure of unusual thermodynamic stability.
The ability of hairpin structures to inhibit human immunodeficiency virus (HIV-1) reverse transcriptase (RT) was also evaluated. Four potent hairpins based on 3',5'- and 2' ,5'-RNA were able to inhibit the RNase H activity of HIV-1 reverse transcriptase, a key enzyme for the proliferation of the human immunodeficiency virus (HIV-1). The polymerase activity of HIV-1 RT was not affected by this class of oligonucleotides. The hairpins were identified from a nucleic acid library synthesized via diversity-oriented solid-phase synthesis. These compounds represent the first examples of hairpins, 12 nucleotides in length, that inhibit specifically the RNase H activity of HIV-1 RT without affecting its polymerase activity.
Another study in this work dealt with yeast RNase III (Rnt1p), an enzyme implicated in the mechanism of action of RNA interference (RNAi). Through these investigations, it was discovered that Rntlp cleaves the DNA strand of DNA:RNA hybrids. This was totally unexpected since Rntlp, like other RNase III enzymes, was thought to be specific only towards the cleavage of double-stranded RNA. These studies also increased our knowledge about the mechanism by which the enzyme cleaves the target RNA (or DNA) strand and suggest that the vicinal 2'-hydroxyl group on the ribose sugar does not participate directly in the cleavage reaction.
Finally, the formation of C-tetrad structures (i-motif) was induced through the design and synthesis of extra-stable hairpin loops containing deoxycytidine rich stems. The corresponding hairpins containing ribocytidine folded into duplexes rather than C-tetrad structures. These studies lead to the first detection and identification of antiparallel 2',5 '-RNA duplexes based on hemiprotonated C·C+ base pairs.
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Tetzlaff, Charles N. "Synthesis and evaluation of acylated DNA and RNA oligomers /." Thesis, Connect to Dissertations & Theses @ Tufts University, 2001.
Find full textAbstract:
Thesis (Ph.D.)--Tufts University, 2001.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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Roberts, Lisa Oriel. "Internal initiation of protein synthesis on picorna virus RNA." Thesis, University of Kent, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.245654.
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Zhang, Ze. "The control of ribosomal RNA synthesis in mammalian cells." Thesis, University of Southampton, 2013. https://eprints.soton.ac.uk/350477/.
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The biogenesis of ribosomes is a fundamental process that occurs in all living cells. In mammalian cells, it is a highly complex process consisting of the coordinated synthesis and assembly of four ribosomal RNAs (rRNAs) with about 80 ribosomal proteins (RPs). More than 150 non-ribosomal proteins are involved in the processing of rRNAs. The main focus of this project is to use adult rat ventricular cardiomyocytes (ARVCs) as a model to address how mTOR complex 1 (mTORC1) and other signalling pathways regulate the synthesis of rRNAs. A new technique has been developed to monitor the synthesis of new rRNAs using 4-thiouridine (4-SU) and I have applied it in both HeLa cells and heart muscle cells to study the control of ribosome synthesis. HeLa cells were treated with different mTOR inhibitors to identify effects on the transcription and/or decay of rRNA. We analysed both the synthesis rate and the decay rate of new RNAs made by Pol I and Pol III using real-time RT-PCR. Interestingly, rapamycin not only blocked the synthesis of 18S, 28S and 5S rRNA, but also induced the decay of newly synthesized rRNAs. This demonstrates that mTORC1 regulates Pol I and Pol III transcription, as well as the decay of rRNA. In cardiomyocytes, hypertrophic agents such as phenylephrine (PE) strongly activate protein synthesis and lead to heart cell growth. The boost of protein synthesis drives the increase of cell size and leads to hypertrophy. Cardiac hypertrophy (CH) is a major risk factor for heart failure. Therefore, it is important to understand the mechanisms that how hypertrophic agents which cause the overgrowth of heart muscle increase ribosome production. Although it is known that inhibiting mTORC1 largely blocks the rapid activation of protein synthesis by PE, here it did not affect the synthesis of new 18S rRNAs. However, inhibitors of the MEK/Erk pathway and p90RSK each block the new rRNA synthesis. These data reveal that, in contrast to many other types of cell, ribosome biogenesis is controlled by MEK/ERK/p90RSK signalling, not mTORC1, in cardiomyocytes. Taken together, the data presented here may provide cues for potential valuable therapy of cardiac left ventricular hypertrophy.
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Dingwall, C. "The accumulation of proteins in the Xenopus oocyte nucleus." Thesis, Open University, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.354967.
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The ability of proteins to accumulate in the nucleus has been studied by injecting nucleoplasmin and calf thymus histone H1 into the cytoplasm of Xenopus oocytes. Nucleoplasmin, the most abundant protein in the Xenopus oocyte nucleus is pentameric and proteolysis of the nuceloplasmin pentamer produces a relatively protease resistant 'core' molecule that cannot enter the nucleus after microinjection into the cytoplasm. The polypeptide domain ('lq tail') of each subunit removed by proteolysis was obtained as a discrete fragment and has the ability to accumulate in the nucleus. Partially cleaved pentameric molecules with a single intact sub unit can still accumulate in the nucleus. Therefore a polypeptide domain of nucleoplasmin has been found that is both necessary and sufficient for accumulation in the nucleus. When the `core' molecule was injected directly into the oocyte nucleus it remained there, indicating that the 'tail' region confers selective entry rather than selective retention. In the case of histone H1 a proteolytic fragment encompassing the carboxyterminal domain can accumulate in the nucleus. The amino acids lysine, proline and alanine comprise 75 of the 89 amino acids in this fragment. Since the remaining 14 amino acids are scattered throughout the fragment and not clustered any primary sequence specifying entry into the nucleus would seem necessarily to involve the amino acids lysine, proline and alanine. Positive charge alone cannot explain the accumulation of this gragment since poly L-lysine does not accumulate after microinjection into the cytoplasm. Fragments encompassing other domains of the molecule are so unstable in the oocyte that their ability to accumulate in the oocyte nucleus cannot be assayed. The gene for nucleoplasmin has been cloned and sequences have been found in the 'tail' region of nuceloplasmin that show homology to sequences identified in other nuclear proteins that appear to constitute a signal specifying nuclear localisation.
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Greenhalgh, Duncan Alan. "Effects of 5-fluorouracil on RNA metabolism in human tumour cells." Thesis, University of Leeds, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.236315.
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Pertile, Jack James. "Synthesis of Fluorescent Promazines and Evaluation of Photophysical Properties." OpenSIUC, 2019. https://opensiuc.lib.siu.edu/theses/2524.
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A new series of fluorescent prorin (promazine-coumarin hybrid) derivatives were synthesized via piperidine catalyzed cyclization with 3-formyl-2-hydroxypromazine and ethyl acetoacetate or Meldrum’s acid. The synthesis of these new compounds focused on the introduction of electron withdrawing carbonyl groups on the 3-position of the coumarin lactone ring in order to increase polarization of the molecule which results in a longer absorbance wavelength by stabilizing intramolecular charge transfer that occurs in the excited state. The 3-acetyl derivative was further subjected to an aldol condensation with either benzaldehyde or cinnamaldehyde in order to further extend the conjugation of the molecule. The four new prorin compounds exhibited excellent photophysical properties and had Stokes shifts ranging from 160-190 nm. These properties are ideal for use as fluorescent probes to detect RNA binding ligands. The optical properties of the new prorin compounds were compared with a previous series of prorins as well as theoretical calculations in order to gain a more comprehensive understanding of how the photophysical properties of the molecule can be tuned by introducing different substituents. Although increasing polarization of the molecule with electron withdrawing substituents does red-shift the absorbance wavelength, it has little effect on the emission wavelength and can actually cause a blue-shift in some cases. It was observed that introducing aromatic substituents has the greatest effect on red-shifting the emission wavelength and increasing Stokes shift.
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Li, Tin-wai Olive, and 李天慧. "Influenza polymerase subunit compatibility between human H1 and H5 viruses." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B41896890.
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Martin, Alarcon Daniel Alberto. "Tools for RNA and cell-free synthetic biology." Thesis, Massachusetts Institute of Technology, 2016. http://hdl.handle.net/1721.1/104124.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016.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 (pages 58-63).
Amid the myriad recent developments in synthetic biology, progress has been fastest in the areas with the most versatile tools for understanding and engineering biological systems. RNA synthetic biology and synthetic minimal cells are areas where design is limited by the availability of tools to observe, program, and manipulate the systems in question. In this work I present expanded toolsets to achieve these goals. The ability to monitor and perturb RNAs in living cells would benefit greatly from a modular, programmable protein architecture for targeting unmodified RNA sequences. I report that the RNA-binding protein PumHD (Pumilio homology domain), which has been widely used in native and modified form for targeting RNA, can be engineered to yield a set of four canonical protein modules, each of which targets one RNA base. These modules (which I call Pumby, for Pumilio-based assembly) can be concatenated in chains of varying composition and length, to bind desired target RNAs. I validate that the Pumby architecture can perform RNA-directed protein assembly and enhancement of translation of RNAs. I further demonstrate a new use of such RNA-binding proteins, measurement of RNA translation in living cells. Pumby may prove useful for many applications in the measurement, manipulation, and biotechnological utilization of unmodified RNAs in intact cells and systems. Genetic circuits are a fundamental tool in synthetic biology; an open question is how to maximize the modularity of their design, to facilitate their integrity, scalability, and flexibility. Liposome encapsulation enables chemical reactions to proceed in well-isolated environments. I here adapt liposome encapsulation to enable the modular, controlled compartmentalization of genetic circuits and cascades. I demonstrate that it is possible to engineer genetic circuit-containing synthetic minimal cells (synells) so that they contain multiple-part genetic cascades, that these cascades can be controlled by external as well as inter-liposomal communication without cross-talk, and that these cascades can also be fused in a controlled way so that the products of incompatible reactions can be brought together. Synells thus enable more modular creation of synthetic biology cascades, an essential step towards their ultimate programmability.
by Daniel Alberto Martin Alarcon.
Ph. D.
50
Smith, Richard Wilson. "RNA metabolism and the control of protein synthesis in fish." Thesis, University of Aberdeen, 1996. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU089891.
Full textAbstract:
This thesis examines the regulation of protein synthesis by pre - translational events; with particular reference to the means by which RNA is able to reduce the energetic cost of protein synthesis. Protein synthesis was measured by the application of a "flooding dose" of 3H-phenylalnine. Protein synthesis rates are then calculated from the "free intracellular" and "protein bound" specific radioactivity (dpm nmole-1 phenylalanine). A similar approach was used to investigate RNA synthesis: i.e. a flooding dose of 3H-uridine. As with protein synthesis RNA synthesis rates was assessed by the relating precursor and product (uridine nucleotide and RNA) radioactivity. Oxygen consumption was measured by monitoring the decline in partial pressure in calibrated respirometery chambers. In fish cells protein synthesis was regulated in terms of the amount (ie the "capacity" for protein synthesis) and the translational efficiency of the RNA. Translationally efficient RNA equated to RNA with an increased turnover. In order to minimise RNA production costs, rapidly synthesised RNA places more reliance on the salvage of exogenous nucleosides, as opposed to the relatively expensive alternative of intracellular synthesis. During yolk sac larval development of the African wels (Clarius gariepinus) protein synthesis rates decline whilst oxygen consumption and the amount of RNA (relative to protein) remains constant. Thus the increasing protein synthesis costs resulted from a reduction in RNA translational efficiency. This was mirrored by a declining RNA synthesis rate. Larval fish growth is primarily due to the repartitioning of yolk sac proteins since early life history stages are thought unable to sustain rapid rates of protein turnover. This pre - translational strategy optimises growth and regulates protein synthesis; whilst, at the same time maintaining the capacity for protein synthesis in anticipation of exogenous feeding.