Dissertations / Theses on the topic 'Nucleic acid based drug'
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Wong, Frances M. P. "Lipid-based vehicles for nucleic acid drugs." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape4/PQDD_0015/NQ56644.pdf.
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Satyal, Uttam. "Efficient Drug and Nucleic Acid Delivery Systems based on Synthetic Amphiphiles with Tuned Oil/Water Interfaces." Diss., Temple University Libraries, 2018. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/531985.
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Pharmaceutical SciencesPh.D.
Today, drugs are an integral part of healthy human life, with new drug entities being introduced every year in clinic. The advancement of drug development brings complexity and variation, in terms of both physical and chemical properties. Some of these physicochemical characteristics are many times suboptimal, eventually requiring robust delivery systems that can precisely deliver the drugs to the desired tissues. Although many materials have been studied for the generation of drug delivery systems, there is always a need for biomaterials with better properties that can translate into superior delivery systems. In this context, new drug delivery systems that are interface-engineered at materials level for better stability and delivery efficiency in vitro and in vivo are introduced in this dissertation. In the first part of the dissertation, novel oil/water interface-engineered amphiphilic block copolymer micelles that were previously introduced by our lab were assessed for their stability in the presence of various esterase enzymes present in serum and on blood vessel walls, normally encountered by drug delivery systems on route to the targeted tissues. I also assessed the vulnerability of the polymeric micelles in presence of enzymes typically present either inside the tumor cells or secreted in the tumor microenvironment. I revealed the selective stability of empty- and docetaxel-loaded polymeric micelles to enzymatic degradation en route/in tumors and I have correlated this selective stability with polymer structure and interfacial engineering mentioned above. The unique delivery capabilities of interfacial-engineered polymeric micelles were tested in vivo using a mouse model of triple negative breast cancer. We proved that our novel engineered triblock copolymer-based drug delivery systems are superior to similar delivery systems made out of standard diblock copolymer micelles and also to the clinically used Taxotere® formulation towards cancer cell killing and tumor treatment, without displaying any significant toxicity in experimental animals. The second part of the dissertation focuses on the development and assessment of a pyridinium-based pseudo-gemini surfactant that combined the high nucleic acid packaging capacity of pyridinium lipids with the high transfection efficiency of gemini surfactants while displaying a reduced associated cytotoxic effect. I have analyzed the temperature treatment on compaction of nucleic acids into lipoplexes and I have established a high temperature annealing method for this purpose. This novel formulation technique allowed a substantial reduction of the amount of amphiphiles required to compact a specific amount of nucleic acids. This in turn also reduced the cytotoxic effect associated with the use of pyridinium amphiphiles. The effect of inclusion of colipids to lipoplex compaction, the robustness and the transfection efficiency of the lipid/nucleic acid lipoplex systems were assessed in detail, and correlations between formulation composition and biological activity were established. I was also able to show for the first time that pyridinium pseudo-gemini surfactants were able to compact different types of nucleic acids, including pDNA, mRNA and siRNA at lower charge ratios than standard, state-of-the art formulations used for this purposes. I also showed that irrespective to the nucleic acid compacted within the lipoplexes, the novel amphiphiles can efficiently deliver the cargo into the targeted cells even in the presence of very high concentration of serum, a premise for future use of these amphiphiles and formulations in vivo.
Temple University--Theses
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Pel, Joel. "A novel electrophoretic mechanism and separation parameter for selective nucleic acid concentration based on synchronous coefficient of drag alteration (SCODA)." Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/13402.
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Molecular manipulation and separation techniques form the building blocks for
much of fundamental science, yet many separation challenges still remain, in fields as diverse as forensics and metagenomics. This thesis presents SCODA (Synchronous Coefficient of Drag Alteration), a novel and general molecular separation and concentration technique aimed at addressing such challenges. SCODA takes advantage of physical molecular properties associated with the non‐linear response of long, charged polymers to electrophoretic fields, which define a novel parameter for DNA separation. The SCODA method is based on superposition of synchronous, time-varying
electrophoretic fields, which can generate net drift of charged molecules even when the
time-averaged molecule displacement generated by each field individually is zero. Such drift can only occur for molecules, such as DNA, whose motive response to electrophoretic fields is non-linear.
This thesis presents the development of SCODA for extraction of DNA, and
outlines the design of the instrumentation required to achieve the SCODA effect. We
then demonstrate the selectivity, efficiency, and sensitivity of the technique.
Contaminant rejection is also quantified for humic acids and proteins, with SCODA
displaying excellent performance compared to existing technologies. Additionally, the
ability of this technology to extract high molecular weight DNA is demonstrated, as is its inherent fragment length selection capability.
Finally, we demonstrate two applications of this method to metagenomics projects where existing technologies performed poorly or failed altogether. The first is the extraction of high molecular weight DNA from soil, which is limited in length to fragments smaller than 50 kb with current direct extraction methods. SCODA was able
to recover DNA an order of magnitude larger than this. The second application is DNA
extraction from highly contaminated samples originating in the Athabasca tar sands,
where existing technology had failed to recover any usable DNA. SCODA was able to
recover sufficient DNA to enable the discovery of 200 putatively novel organisms.
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Folly, da Silva Constantino Laura. "An effective layered workflow of virtual screening for identification of active ligands of challenging protein targets." Thesis, University of Iowa, 2017. https://ir.uiowa.edu/etd/5754.
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Docking is a computer simulation method used to predict the preferred orientation of two interacting chemical species that has been successfully applied to numerous macromolecules over the years. However, non-traditional targets have inherent difficulties associated with their screening. Large interfaces, lack of obvious binding sites, and transient pockets are some examples. Additionally, most natural ligands of challenging targets are inadequate models for identifying or designing new ligands. Therefore, it is not surprising that customary techniques of structure-based virtual screening are incompatible with these non-traditional targets.
We hypothesized that an integrative virtual screening campaign comprised of docking followed by refinement of best receptor–ligand complexes would effectively identify small-molecule ligands of challenging receptors. We targeted the single-stranded DNA (ssDNA) binding groove of the human RAD52, and a cryptic allosteric pocket of the Helicobacter pylori Glutamate Racemase (GR). In this project, we first determined which docking method was more appropriate for each studied non-traditional target, and then examined how good our two-step docking workflow was in finding novel active ligand scaffolds.
This research developed a powerful layered virtual screening workflow for the discovery of lead compounds against challenging protein targets. Furthermore, we successfully applied a statistical analysis method, which used receiver operating characteristic (ROC) curves, to validate the selected docking protocol that would be used in the screening campaigns. Using the validated workflow, we identified a natural compound that competes with ssDNA to bind to RAD52. The performed screening campaigns also provided new insights into the studied binding pockets, as well as structure-activity relationships (SAR) and binding determinants of the ligands. Our achievements reinforce the power of the ROC curve analysis approach in directing the search for the most appropriate docking protocol and helping to speed up drug discovery in pharmaceutical research.
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BELGIOVINE, GIULIANA. "Delivery of small interfering RNA into airway epithelial cells. Downregulation of the pro-inflammatory cytokine high mobility group BOX 1." Doctoral thesis, Università di Foggia, 2016. http://hdl.handle.net/11369/338921.
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polyplexes with cells. In conclusion, we have found optimal conditions for downregulation
of HMGB1 by siRNA delivery mediated by polyaminoacidic polymers
in airway epithelial cells in the absence of cytotoxicity. Functional and in-vivo
studies are warranted.
In the second part of this thesis, having demonstrated that the PHEA- and CSNAC-
based nanoparticles are able to downregulate HMGB1 in airway epithelial
cells, we sought to determine whether inflammatory conditions could regulate
HMGB1 expression. H441 cells incubated with lipopolysaccharide (LPS) from
Pseudomonas aeruginosa presented higher levels of HMGB1 as compared to
untreated controls both as mRNA and protein as assessed by real time PCR and
western blotting, respectively. This increase was observed both at 3 and 48 hours
post treatment. LPS was also able to induce an increase in the metabolic state of
cells (shown up by the MTT assay) at the same time points, but not in the
proliferation (cell counts), while cell cycle analysis (by cytofluorimetry) showed a
decrease of Go/G1 phase and an increase in G2/M at 6 and 24 h post-treatment.
These data suggest that HMGB1 is involved in the activation of metabolic state of
cells upon induction of an inflammatory condition, and it should further studied
by siRNA downregulation.
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Akhras, Michael S. "Nucleic Acid Based Pathogen Diagnostics." Doctoral thesis, KTH, Skolan för bioteknologi (BIO), 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4684.
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Pathogenic organisms are transmitted to the host organism through all possible connected pathways, and cause a myriad of diseases states. Commonly occurring curable infectious diseases still impose the greatest health impacts on a worldwide perspective. The Bill & Melinda Gates Foundation partnered with RAND Corporation to form the Global Health Diagnostics Forum, with the goal of establishing and interpreting mathematical models for what effects a newly introduced point-of-care pathogen diagnostic would have in developing countries. The results were astonishing, with potentially millions of lives to be saved on an annual basis. Golden standard for diagnostics of pathogenic bacteria has long been cultureable medias. Environmental biologists have estimated that less than 1% of all bacteria are cultureable. Genomic-based approaches offer the potential to identify all microbes from all the biological kingdoms. Nucleic acid based pathogen diagnostics has evolved significantly over the past decades. Novel technologies offer increased potential in sensitivity, specificity, decreased costs and parallel sample management. However, most methods are confined to core laboratory facilities. To construct an ultimate nucleic acid based diagnostic for use in areas of need, potential frontline techniques need to be identified and combined. The research focus of this doctoral thesis work has been to develop and apply nucleic acid based methods for pathogen diagnostics. Methods and assays were applied to the two distinct systems i) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae, and ii) genotype determination of the cancer causative Human Papillomavirus (HPV). The first part of the study included development of rapid, direct and multiplex Pyrosequencing nucleic acid screenings. With improved methodology in the sample preparation process, we could detect an existence of multiple co-infecting HPV genotypes at greater sensitivities than previously described, when using the same type of methodology. The second part of the study focused on multiplex nucleic acid amplification strategies using Molecular Inversion Probes with end-step Pyrosequencing screening. The PathogenMip assay presents a complete detection schematic for virtually any known pathogenic organism. We also introduce the novel Connector Inversion Probe, a padlock probe capable of complete gap-fill reactions for multiplex nucleic acid amplifications.Patogena organismer smittas till värd organismen genom alla möjliga kontaktnätverk och skapar en mångfald olika sjukdomstillstånd. Dock är det fortfarande vanligt förekommande behandlingsbara infektiösa sjukdomar som orsakar den största hälsoförlusten, sett från ett globalt perspektiv. Bill och Melinda Gates Stiftelsen samarbetade med RAND kooperation för att forma “The Global Health Diagnostics Forum”. Deras mål var att etablera och analysera matematiska modeller för vilka effekter en ny diagnostisk metod utrustat för fältarbete skulle ha i utvecklingsländer. Resultaten var häpnadsveckande, med potentiellt miljoner av liv som skulle kunna räddas på en årlig basis. Den etablerade standarden för diagnostik av patogena bakterier har länge varit kultiveringsmedia baserad. Miljö specialiserade biologer har estimerat att mindre än 1 % av alla bakterie arter går att kultivera. Dock erbjuder genetiska analyser potentialen att kunna identifiera alla mikrober från alla de biologiska rikena. Nukleinsyrebaserade diagnostiska metoder har märkbart förbättrats över de senaste årtionden. Nya tekniker erbjuder utökad sensitivitet, selektivitet, sänkta kostnader och parallella analyser av patient prover. Dock är de flesta metoderna begränsade till standardiserade laboratoriemiljöer. För att konstruera en väl fungerande diagnostisk fältutrustning för användning i problem områden, behöver världsledande tekniker identifieras och kombineras. Fokuseringsområdet för denna doktorsavhandling har varit att utveckla och utföra nukleinsyrebaserade metoder för patogen diagnostik. Metoder och experimentella utförande applicerades på två distinkta system i) sökning av antibiotika resistens relaterade mutationer i den patogena bakterien Neisseria gonorrhoeae och ii) genotypning av det cancer orsakande Humana Papillomaviruset (HPV). Den första delen av studien inriktade sig mot utveckling av snabba, direkta och multiplexa Pyrosekvenserings baserade nukleinsyreanalyser. Med förbättrad provprepareringsmetodologi kunde vi detektera multipla HPV infektioner med högre sensitivitet än vad tidigare beskrivits med liknande metodologi. Den andra delen av studien fokuserades på multiplexa nukleinsyre amplifikationer med “Molecular Inversion Probe” tekniken med sista steg Pyrosekvenserings analys. “PathogenMip assay” erbjuder ett komplett detektionsprotokoll för alla kända patogena organismer. Vi introducerar även den nya “Connector Inversion Probe”, en “Padlock Probe” kapabel att genomföra kompletta gap fyllningar för multiplex nukleinsyre amplifiering.
QC 20100624
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Elmén, Joacim. "Nucleic acid based therapeutic approaches /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-047-8/.
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Allsop, Julie Kay. "Development of nucleic acid vaccines for mucosal delivery." Thesis, University of Nottingham, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.263104.
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Zhou, Chenguang. "NANOCARRIERS FOR THERAPEUTIC NUCLEIC ACID DELIVERY." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1336584204.
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O'Daniel, Peter Ivo. "Exploring structural diversity in nucleoside and nucleic acid drug design." Diss., Available online, Georgia Institute of Technology, 2005, 2005. http://etd.gatech.edu/theses/available/etd-08252005-130946/.
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Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2006.Barefield, E. Kent, Committee Member ; Beckham, Haskell W., Committee Member ; Doyle, Donald F., Committee Member ; Weck, Marcus, Committee Member ; Seley, Katherine L., Committee Chair.
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Kedge, Jonathan L. "Synthesis of ferrocene nucleic acid monomers and ferrocene containing drug candidates." Thesis, University of Birmingham, 2017. http://etheses.bham.ac.uk//id/eprint/7424/.
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The first ferrocene nucleic acid (FcNA) was reported by the Tucker group in 2012. Furnished with two nucleobases and two hydroxyl groups, the tetrasubstituted metallocene assumes the position traditionally occupied by the two sugars of a dinucleotide. This thesis describes the successful synthesis of two FcNA monomers; a tetrasubstituted dithyminyl variation and a disubstituted control compound bearing no nucleobases. These monomers were oligomerised, their binding characteristics assessed by thermal melting studies, and compared to other monomers belonging to the group. Through the study of these compounds the Tucker group has demonstrated that FcNA monomers behave similarly to conventional nucleic acids, displaying selective H-bonding and π-stacking interactions within a hybrid duplex. A preliminary methodology for the production of diguaninyl FcNA monomers was also explored. As published in 2014, the corresponding disubstituted systems, in which a hydroxyl and a nucleobase are linked through a sugar-like ferrocene unit, are also being investigated as potential nucleoside analogues. Adding to the groups growing library, a number of related compounds were synthesised in which the hydroxyl linker length, the planar chirality, the substitution pattern of the ferrocene and the nucleobase were varied. The compounds were electrochemically characterised and assessed for their biological activity which revealed interesting structure-activity-relationships involving both the redox potentials and chirality. Following the example of ferroquine and ferrocifen, in which existing pharmaceuticals are modified through the incorporation of ferrocene, the synthesis and preliminary biological activity of novel ferrocenyl β-blockers, in which the metallocene replaces the napthol unit of the prototypical β-blocker propranolol, is reported herein.
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Garner, Mark. "Kinetic and mechanistic studies of Cisplatin derivatives with nucleic acid fragments." Thesis, University of York, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.306475.
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Schaffert, David Henning. "Precise oligoethylenimine-based carriers for nucleic acid delivery." Diss., lmu, 2010. http://nbn-resolving.de/urn:nbn:de:bvb:19-151686.
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O'Meara, Deirdre. "Molecular Tools for Nucleic Acid Analysis." Doctoral thesis, Stockholm : Tekniska högsk, 2001. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3220.
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Perin, Danilo. "Biomaterials for biotechnological applications: synthesis and activity evaluations." Doctoral thesis, Università degli studi di Trieste, 2010. http://hdl.handle.net/10077/3518.
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2008/2009A biomaterial was defined as any non-living material used in a medical device that interacts with biological systems. Many different applications involve the use of biomaterials: pharmacology, controlled drug release, extracorporeal devices (contact lens, emodialysis devices, cardiopulmonary bypass oxygenators), artificial prostheses. One of the most interesting biomaterials application regards the release of nucleic acids and their derivatives as therapeutic agents. These molecules, defined as “nucleic acid base drugs” (NABDs), allows highly targeted cellular metabolism modifications. The aim of this research project concerns the characterization of biomaterials for biotechnological applications and evaluation of their activities. In particular, because of the great therapeutics and commercial interest and the delivery problems that are largely unresolved, the attention is focused on the study of new delivery systems for siRNA, proposed as model system as they represent the most common and best characterized NABD. SiRNA proved to be useful for what concerns the in-stent restenosis, pathology implying the re-occlusion of the artery due to the iper-proliferation of smooth muscle cells induced by the presence of the stent, a metal prosthesis that is applied to avoid the elastic recoil of the artery wall after balloon angioplasty. In this system, the siRNA should act as an anti-proliferative of smooth muscle cells without interfering with endothelial cells. In order to design an appropriate delivery system, it is crucial a precise structural and dimensional characterization of polymeric mesh. This purpose was achieved by the use of various techniques such as Rheology, low field NMR and Cryoporometry. Rheology allows the evaluation of the macroscopic mechanical properties of the system under investigation (Young's and shear modulus for example). Low field NMR, instead, allows evaluating the microscopic properties and, coupled to the rheology, provides an estimation of the polymeric mesh size distribution. Cryoporometry is another method to assess the mesh size distribution. In vivo release tests represent the final step of the experimental process. The polymeric system ability to carry and deliver the liposome-siRNA complexes, was tested in culture models of smooth muscle cells and endothelial cells. The attention has been focused on polymeric hydrogels, whose biocompatibility and biodegradability is well known: • Alginate (polymeric concentration 1% - 2% - 3%), crosslinked by Ca2+ or Cu2+ water solution • Pluronic™ F127 18% in water • Dextran 5% or 30% methacrylate (respectively D40MA5% and D500MA30%; polymeric concentration 5%) crosslinked by UV • Gel systems derived from benzofulvene And polymeric blends: • Pluronic™-alginate hydrogels respectively at 18% and 2% in water • Dextran methacrylate-alginate respectively at 5% and 3% in water (A3D40MA5% or A3D500MA30%)
Un biomateriale è definito come qualsiasi materiale non-vivente utilizzato in un dispositivo medico interagente con i sistemi biologici. Diverse applicazioni comportano l'uso di biomateriali: farmacologia, sistemi di controllato rilascio di farmaci, dispositivi extracorporei (lenti a contatto, dispositivi per emodialisi, ossigenatori, bypass cardiopolmonari, ecc.), protesi artificiali. Una delle applicazioni più interessanti dei biomateriali riguarda il rilascio di acidi nucleici e loro derivati come agenti terapeutici. Queste molecole, definite come " nucleic acid base drugs " (NABDs), consentono modifiche altamente mirate del metabolismo cellulare. L'obiettivo di questo progetto di ricerca riguarda la caratterizzazione dei biomateriali per applicazioni biotecnologiche e la valutazione delle loro attività. In particolare, dato il notevole interesse terapeutico e commerciale, oltre ai problemi di delivery tutt’ora in gran parte irrisolti, l'attenzione è stata focalizzata sullo studio di nuovi sistemi di somministrazione di siRNA, proposti come sistema modello in quanto rappresentano il più comune e meglio caratterizzato NABD. I siRNA si sono rivelati utili per il trattamento della ristenosi in-stent, una patologia che comporta la ri-occlusione dell'arteria in seguito alla iper-proliferazione delle cellule muscolari lisce indotta dalla presenza dello stent, una protesi di metallo applicata per evitare la contrazione elastica della parete arteriosa in seguito ad angioplastica con palloncino. In questo sistema, il siRNA dovrebbe agire come un anti-proliferativo delle cellule muscolari lisce, senza interferire con le cellule endoteliali. Al fine di progettare un adeguato sistema di rilascio, è di fondamentale importanza una precisa caratterizzazione strutturale e dimensionale delle maglie polimeriche. Questo scopo è stato raggiunto mediante l’utilizzo di varie discipline quali la Reologia, l’NMR a basso campo e la Crioporimetria. La Reologia permette una valutazione macroscopica delle proprietà meccaniche del sistema in esame (ad esempio attraverso il modulo di Young ed il modulo di taglio). L’NMR a basso campo, invece, permette di valutare le proprietà microscopiche e, accoppiato alla reologia, fornisce una stima della distribuzione dimensionale delle maglie polimeriche. La Crioporimetria è metodo alternativo per la valutazione della distribuzione dimensionale delle maglie. I test di rilascio in vivo rappresentano l'ultima fase del processo sperimentale. La capacità del sistema polimerico di trasportare e rilasciare liposomi complessati a siRNA, è stata valutata in modelli di cellule muscolari lisce e cellule endoteliali in coltura. L’attenzione e stato focalizzata soprattutto su sistemi idrogel polimerici la cui biocompatibilità e biodegradabilità è ben nota: • Alginato (concentrazione polimero 1% - 2% - 3%), reticolato attraverso soluzioni acquose di Ca2+ o Cu2+ • Pluronico F127 al 18% in acqua • Destrano 5% o 30% metacrilato (rispettivamente D40MA5% e D500MA30% ad una concentrazione polimerica pari al 5% in acqua), reticolati tramite UV • Sistemi gel derivati da benzofulvene E le miscele polimeriche costituite da: • Idrogel di pluronico-alginato rispettivamente al 18% e 2% in acqua • Destrano metacrilato-alginato rispettivamente al 5% e 3% in acqua (A3D40MA5% o A3D500MA30%)
XXII Ciclo
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Goujon, Jennyfer. "Towards the development of a novel colourimetric nucleic acid biosensor based on peptide nucleic acid-functionalised polydiacetylene liposomes." Thesis, Heriot-Watt University, 2009. http://hdl.handle.net/10399/2306.
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The aim of the research project described here was to develop a novel colourimetric nucleic acids biosensor based on polydiacetylene liposomes containing lipophilic peptide nucleic acids. Preliminary investigations in this area have shown that PNA-containing PDA liposomes can be constructed and that they are blue in colour as expected. However, their poor water solubility and the resulting precipitation made it necessary to synthesise and evaluate a second generation. In these, the PNA head-group is separated from the lipid tail by an amino diethylene glycol-type spacer molecule. The first hydrophilic spacer synthesised was 8-(tert-butoxycarbonyl)amino-3,6- dioxaoctanoic acid. Three methods based on the O-alkylation of mono-Boc, di-Boc and dibenzyl 5-amino-3-oxapentanol were devised. The Boc strategy afforded the corresponding ether in low yield of 20% while in the case of the dibenzyl approach a suitable methodology to effectively cleave the protecting groups from the amino function could not be found. Moreover, the model reaction between dibenzyl 8-amino-3,6- dioxaoctanoic acid and thyminyl PNA monomer afforded the corresponding conjugates in only 6%. An alternative type of connection to link the spacer to the PNA headgroup was thus sought. A 1,4-disubstituted [1,2,3]-triazole moiety was obtained in 60% yield by reacting spacer, 8-(tert-butoxycarbonyl)amino-3,6-dioxaoctan-1-azide and N-alkynyl PNA monomers bearing thymine, Cbz protected adenine and Cbz protected cytosine according to the conditions of the ‘Click’ reaction. These ‘spacer-PNA monomer’ intermediates were then functionalised at their N-terminus using 10,12-pentacosadiynoyl fluoride, to afford ‘lipid-spacer-PNA monomer’ models. The saturated conjugates were obtained by first preparing the ‘lipid-spacer’ intermediate, N-(8-azido-3,6- dioxaoctanyl)stearamide which was then connected to the N-alkynyl PNA monomers using the ‘Click’ reaction. Following this last strategy, a homothymine PNA dimer, prepared in solution phase, was also functionalised with the saturated lipid chain. This second generation of lipid functionalised-PNA monomer models were incorporated into PDA-liposomes. Upon photo-polymerisation, the liposome solutions became blue. They were found to be stable in solution, at 4 oC over long period of time. Their colour changed to red upon environmental changes (i.e. pH and temperature).
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Andersson, Sara. "Development of Peptide Nucleic Acid Based Artificial Ribonucleases, PNAzymes." Thesis, Mälardalens högskola, Akademin för hållbar samhälls- och teknikutveckling, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:mdh:diva-12280.
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Since mRNA plays such a vital role in the transfer of genetic code from DNA to proteins, approaches involving interference with mRNA are of great interest for use in therapeutics. Amongst these, peptide nucleic acid based nuclease systems (PNAzymes) are being developed for in vivo gene silencing in future. The PNAzymes hybridize to the RNA, bringing a catalytic group to the phosphodiester bond and inducing the cleavage. A study on PNAzymes has shownhigh site- and sequence-specificity in the cleavage of target RNA sequences and this also occurs at a faster rate than previously reported oligonucleotide-based artificial ribonucleases. However, anincrease in rate is still needed for gene silencing applications to be useful therapeutically.This previous study used 2,9-dimethyl-5-aminophenanthroline conjugated to a PNA strand in order to cleave target RNA. The aim of this project was to synthesize 2,5,9-triaminophenanthroline with tert-butyloxycarbonyl groups (Boc-groups) in the 2- and 9-positions, activate the amino group in the 5-position with phenyl chloroformate and conjugate it to PNA and then compare the cleavage efficiency of the resulting PNAzyme with those carrying the 5-aminophenanthroline and 2,9-dimethyl-5-aminophenanthroline derivatives. In the suggested reaction pathway to produce the Boc-protected 2,5,9-triaminophenanthroline, 5 steps out of 7 were successfully achieved. Cleavage studies on all three phenanthroline derivatives will be performed when the final derivatives are synthesized.
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Xu, Gaolian. "Developing new synthetic tools for nucleic acid based diagnostics." Thesis, University of Glasgow, 2016. http://theses.gla.ac.uk/7704/.
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With increasing globalization, new infectious diseases are being discovered and spread quickly around the world. The traditional ways for the detection and identification of infectious pathogens are time-consuming and usually require specific facilities. This may delay effective treatment and lead to the spread of infectious disease, especially in the early stages of an epidemic. Therefore, accurate and efficient methods for identification of causative pathogens are very important. Here, we take advantages of new synthetic tools and nucleic acid technologies to develop novel infectious disease diagnostic methods that are user-friendly and have high sensitivity and specificity. These include: 1). The development of a rapid ultrasonic DNA isothermal amplification method with multiplexed melting analysis; 2). The development of an origami device based nucleic acid multiplexed detection method; 3). The development of a novel branched Hybridization Chain Reaction (HCR) assay. 1. Rapid ultrasonic isothermal amplification of DNA with multiplexed melting analysis-applications in the clinical diagnosis of sexually transmitted diseases (1) In this project, surface acoustic wave (SAW) signals are generated by interdigitated transducer (IDT) on LiNbO3 and propagated into disposable silicon superstrates on which a droplet of Loop-mediated isothermal amplification (LAMP) reaction mixture has been placed. As SAW interacts with the LAMP mixture, its energy is transferred into the LAMP mixture and causes the temperature of the mixture to increase. By controlling the SAW generation voltage, the temperature of the LAMP mixture could be maintained within a certain range as necessary for the LAMP reaction. During the process of LAMP amplification, a lot of double-strand DNA (dsDNA) is produced; this can incorporate specific fluorescent dyes and result in an exponential increase in fluorescence signal intensity. Also, by gradually increasing the SAW generation voltage, a SAW actuation-based DNA melting method could be used for multiplex detection. Ten-fold serially diluted targets from 105 copies/reaction to 10 copies/reaction were used to quantify the analytical sensitivity of the SAW-LAMP system and measurable signals were found down to 10 copies/reaction. Compared to a Peltier-LAMP system, SAW actuation enables the amplification to be performed more rapidly, about 18.23 % +/- 2.5 faster. Six clinical samples were used to demonstrate the clinical validation of SAW-LAMP by comparing with results from qPCR. The use of a SAW actuation-based DNA melting method distinguished the difference between melting temperatures of C. trachomatis amplicon (79.65 +/- 0.14 °C), and N. gonorrhoea (82.55 +/- 0.53 °C). 2. Development of paper origami device based nucleic acid multiplex detection for infectious diseases diagnostics In this project, a paper-folding origami device to manipulate malaria-infected blood samples was described. Through the simple process of paper folding, the nucleic acid of parasites in the blood sample could be extracted, purified and eluted. The extracted nucleic acid was then amplified with a multiplexed colorimetric LAMP assay in a plastic plate. Finally, the amplification products of multiplexed colorimetric LAMP assay were detected within an array with a low cost hand-held torch by naked eye. The multiplexed colorimetric LAMP assays for Plasmodium pan, Plasmodium falciparum, Plasmodium vivax with an internal control (IC) were investigated. The analytical sensitivity of colorimetric LAMP assays was tested by WHO International Standard DNA, with the limit of detection down to 105 IU/ml. Serially diluted quantified hCMV genomic DNA was used to demonstrate the DNA recovery of our origami device, which was between 60-70%. Serial dilution of a known infected blood samples (from 100 parasites/μl to 1 parasites/μl) were used to study the analytical sensitivity and obtain an LOD of the origami device to 5 parasites/μl. 80 fully characterised fresh malaria infected blood samples were used to assess the clinical validation effect of our origami device through a double blind, randomized controlled, clinical trial. All samples were also tested with commercially available LAMP kit and benchmark real-time PCR assay. The coincidence of our method and benchmark PCR were 88.75% (71/80) for Plasmodium pan, 90% (72/80) for P. falciparum and 93.75% (75/80) for P. vivax. Similarly, the coincidence between our method and the LAMP kit for Plasmodium pan and P. falciparum were 90% (72/80) and 92.50% (74/80) respectively. Using benchmark PCR as a gold standard for the detection of Plasmodium pan, P. ovale, P. falciparum and P. vivax, the sensitivity for our tests was of 85.5%, 92.9%, 61.1% and 81.0%, respectively. While the specificity are 100%, 94.2%, 98.5% and 98.30% respectively. We also established our origami device can diagnose species type from stored samples (either frozen, fixed, or dried). 3. Development of a novel branched Hybridization chain reaction (HCR) By increasing the dimensionality of an HCR system, a novel branched HCR product with complex branched structures instead of linear constructs has been developed. To validate the principle of a transition from a 1D chain to a higher dimension, we adapted a 3-arm branching construct to enable it to form a chain reaction by incorporating hybridization tails onto its sequences. The novel branched HCR reaction can form three-arm junction units with the introduction of a specific initiator. The three-armed units formed not only freed initiators to start of another cycle of HCR, but also bonded to each other to form complex and branched products. We also show that the highly branched polymers produced allow label-free acoustic mass sensing. The product of branched HCR was detected by Love Wave (LW) biosensor with the limit of detection at 2 nM, meaning 0.1% - 0.2% working frequency shift. Based on the branched HCR, we also design a new multiplexed HCR mechanism, where a single reaction is able to detect the presence of different initiators. It is based on designing primers that carry additional hairpin structures, which cross-react specifically upon initiation, yielding branching, thus opening up new applications for this enzyme-free, label-free DNA detection system.
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Daher, Rana. "Recombinase polymerase amplification technology : Assessment for nucleic acid-based acid-based point-of-care diagnostics." Doctoral thesis, Université Laval, 2015. http://hdl.handle.net/20.500.11794/26269.
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Cette thèse de doctorat porte dans l’ensemble une étude approfondie sur une technologie émergente pour l’amplification isotherme des acides nucléiques appelée recombinase polymerase amplification (RPA). L’introduction porte une description détaillée sur la RPA. Cette revue de littérature documente et discute les diverses applications de la RPA en soulignant les connaissances actuelles concernant les applications diagnostiques. Malgré la composition complexe de la RPA (6 à 7 protéines dans le même mélange réactionnel), cette dernière s’avère une technologie rapide (générant des résultats < 20 min), spécifique et sensible (détection de l’ordre de quelques copies de génome), et largement appliquée dans différentes disciplines. Ces avantages nous permettent de croire que la RPA possède la flexibilité nécessaire pour être utilisée comme outil de diagnostic rapide des maladies infectieuses en réduisant le temps d’obtention des résultats à moins d’une heure au lieu de 2 à 3 jours avec les tests de cultures standards. En conséquence, il sera possible d’intégrer la RPA dans des plateformes microfluidiques ou laboratoire sur puce qui permettent la préparation d’échantillons, l’amplification et la détection des acides nucléiques des microbes causant des infections. En premier lieu, les travaux de cette thèse ont généré des lignes directrices additionnelles pour la conception des amorces/sondes RPA. En second lieu, nos travaux ont permis de développer un essai diagnostic RPA pour la détection des streptocoques du groupe B, responsables de la septicémie et la méningite chez les nouveau-nés. Cet essai fut le premier à évaluer la performance de la RPA avec des échantillons cliniques humains. Ce test diagnostic RPA a été comparé à une méthode de référence, la réaction en chaîne par polymérase (PCR). Cette démonstration sur des échantillons cliniques nous à inciter à pousser notre étude pour réaliser le dernier objectif de ce projet qui consistait à automatiser la RPA par intégration dans un système microfluidique miniaturisé centripète. Une collaboration avec des experts en génies et en matériaux a permis de générer un dispositif microfluidique appelé blade ainsi de l’instrument impliqué dans l’opération des différentes tâches mécanistiques. Ces résultats préliminaires suggèrent qu’il sera important d’offrir un système automatisé complet applicable au chevet du patient. Par conséquence, il sera possible d’exécuter une analyse complète des agents infectieux en moins d’une heure sans le besoin des procédures complexes de préparation et de transport des échantillons cliniques ni le recours à du personnel qualifié.This dissertation consists of an exhaustive study on an emerging technology for isothermal amplification of nucleic acids called recombinase polymerase amplification (RPA). The introduction of this thesis is a detailed description of the RPA. This review documents and discusses the various applications of this technology by pointing to the current knowledge about RPA for diagnostic applications. Despite the complex composition of RPA (6 to 7 proteins in the same reaction mixture), the latter was shown to be rapid (generating results in < 20 min), specific and sensitive (detecting few target genome copies), and applied widely in different fields. Based on these advantages, we assume that RPA has a flexibility allowing it to be used for the rapid diagnosis of infectious diseases thus reducing time-to-result to less than an hour. Consequently, it will be possible to integrate RPA in microfluidic platforms providing a lab-on chip system. The first part of this doctoral project generated additional guidelines for RPA primers/probes design to develop specific RPA diagnostic assays. Second, we developed an RPA diagnostic test for the detection of group B streptococci, responsible for sepsis and meningitis in newborns. This assay was the first to evaluate RPA with human clinical samples. This diagnostic test was compared to a reference method, the polymerase chain reaction (PCR). This demonstration with clinical samples served to carry out the final objective of this project that was to automate RPA in a miniaturized microfluidic centripetal system. Collaboration with engineers and experts in materials has generated the microfluidic device called "blade" and the instrument involved in the operation of various mechanistic tasks. These preliminary results suggested that it will be important to provide an automated system applicable at bedside. Consequently, it will be possible to perform a complete analysis of infectious agents in less than an hour without the need for complex procedures for the preparation and transport of clinical specimens or the assistance of qualified personnel.
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Johns, Rachel Elizabeth. "Delivery of anti-inflammatory nucleic acid therapeutics using smart polymeric carriers /." Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/8080.
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Henriksson, Sara. "Application of Padlock Probe Based Nucleic Acid Analysis In Situ." Doctoral thesis, Uppsala universitet, Institutionen för genetik och patologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-128446.
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The great variation displayed by nucleic acid molecules in human cells, and the continuous discovery of their impact on life, consequently require continuous refinements of molecular analysis techniques. Padlock probes and rolling circle amplification offer single nucleotide discrimination in situ, a high signal-to-noise ratio and localized detection within cells and tissues. In this thesis, in situ detection of nucleic acids with padlock probes and rolling circle amplification was applied for detection of DNA in the single cell gel electrophoresis assay to detect nuclear and mitochondrial DNA. This assay is used to measure DNA damage and repair. The behaviour of mitochondrial DNA in the single cell gel electrophoresis assay has earlier been controversial, but it was shown herein that mitochondrial DNA diffuses away early in the assay. In contrast, Alu repeats remain associated with the nuclear matrix throughout the procedure. A new twelve gel approach was also developed with increased throughput of the single cell gel electrophoresis assay. DNA repair of three genes OGG1, XPD and HPRT and of Alu repeats after H2O2 induced damage was further monitored. All three genes and Alu repeats were repaired faster than total DNA. Finally, padlock probes and rolling circle amplification were applied for detection of the single stranded RNA virus Crimean Congo hemorrhagic fever virus. The virus was detected by first reverse transcribing RNA into cDNA.. The virus RNA together with its complementary RNA and the nucleocapsid protein were detected in cultured cells. The work presented here enables studies of gene specific damage and repair as well as viral infections in situ. Detection by ligation offers high specificity and makes it possible to discriminate even between closely related molecules. Therefore, these techniques will be useful for a wide range of applications within research and diagnostics.
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Bamberger, Denise Nadine [Verfasser]. "Polysaccharide-based nanoparticles for nucleic acid delivery / Denise Nadine Bamberger." Mainz : Universitätsbibliothek Mainz, 2018. http://d-nb.info/1160897409/34.
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Schulz, Martin [Verfasser], and Felix von [Akademischer Betreuer] Stetten. "Microfluidic system integration for droplet based digital nucleic acid testing." Freiburg : Universität, 2020. http://d-nb.info/1229349278/34.
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Iemsam-Arng, J. "Poly(ethylene) glycol based delivery systems for nucleic acid therapies." Thesis, University College London (University of London), 2013. http://discovery.ucl.ac.uk/1381001/.
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Our work is aimed at developing a synthetic biocompatible gene and siRNA delivery system for the treatment of primary and metastatic tumours. To facilitate delivery of nucleic acid based drugs into the cell, one strategy is to formulate the naked gene with an amine based non-viral gene delivery system via the counterion interaction. The delivery systems including 4arm-PEG-amine, 4arm-PEG-N-2-ethylamine, 8arm-PEG-amine and 8arm-PEG-N-2-ethylamine were synthesised, characterised and complexed with a reporter gene (β-gal plasmid DNA) in phosphate buffer pH 6.0. The resulting complexes were sized and their zeta potential measured (Malvern Zetasizer 3000HS, Malvern Instruments, UK). The complexes were also imaged using transmission electron microscopy and characterised for DNA binding and DNA protection using gel electrophoresis and the ethidium bromide displacement assay. The in vitro transfection efficiency and cell cytotoxicity of the complexes were determined in the A431 and HeLa cells. Additionally, in vivo therapeutic studies in female nude tumour bearing mice were carried out. A promising DNA-polymer complex of 4arm-PEG-N-2-ethylamine produced a complex of 200-300 nm in diameter (polydispersity < 0.6). Complexes had a zeta potential of +19.8 mV (n=3) and were spherical, fibrillar and toroidal in shape. The new gene delivery complex protected DNA from degradation in serum up to 2 hours and was as efficient as poly(ethylenimine) (PEI) in transfecting the A431 cell line, but it was more than 3 orders of magnitude less cytotoxic than PEI. In vivo a gene medicine, comprising the polymer and the tumour necrosis factor alpha gene, was tumouricidal. When complexed with siRNA, the siRNA polymer complex demonstrated a trend of gene silencing activity. A new synthetic gene delivery polymer of 4arm-PEG-N-2-ethylamine has been synthesised. This polymer is biocompatible to cells and is an efficient in vitro and in vivo gene transfer agent.
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Bacigalupo, Maria Candelaria Rogart. "Locked nucleic acid analogues for novel exciplex-based molecular probes." Thesis, University of Manchester, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.538490.
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Takalkar, Sunitha. "Micro-/Nano- Materials Based Ultrasensitive Lateral Flow Nucleic Acid Biosensors." Diss., North Dakota State University, 2017. https://hdl.handle.net/10365/28376.
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Ultrasensitive detection of nucleic acids plays a very important role in the field of molecular diagnosis for the detection of various diseases. Lateral flow biosensors (LFB) are convenient, easy-to-use, patient friendly forms of detection methods offering rapid and convenient clinical testing in close proximity to the patients thus drawing a lot of attention in different areas of research over the years. In comparison with the traditional immunoassays, the nucleic acid based lateral flow biosensors (NABLFB) has several advantages in terms of stability and interference capabilities. NABLFB utilizes nucleic acid probes as the bio-recognition element. The target analyte typically is the oligonucleotide like the DNA, mRNA, miRNA which are among the nucleic acid secretions by the tumor cells when it comes to detection of cancer. Traditionally gold nanoparticles (GNPs) have been used as labels for conjugating with the detection probes for the qualitative and semi quantitative analysis, the application of GNP-based LFB is limited by its low sensitivity. This dissertation describes the use of different nanomaterials and advanced detection technologies to enhance the sensitivities of the LFB based methods. Silica Nanorods decorated with GNP were synthesized and employed as labels for ultrasensitive detection of miRNA on the LFB. Owing to the biocompatibility and convenience in surface modification of SiNRs, they acted as good carriers to load numerous GNPs. The sensitivity of the GNP-SiNR-based LFSB was enhanced six times compared to the previous GNP-based LFSB. A fluorescent carbon nanoparticle (FCN) was first used as a tag to develop a lateral flow nucleic acid biosensor for ultrasensitive and quantitative detection of nucleic acid samples. Under optimal conditions, the FCN-based LFNAB was capable of detecting minimum 0.4 fM target DNA without complex operations and additional signal amplification. The carbon nanotube was used as a label and carrier of numerous enzyme and DNA molecules simultaneously thus resulting in the enormous amplification of the colorimetric signal. This CNT-enzyme label thus aided the ultra-sensitive detection of pancreatic cancer (PC) biomarker miRNA 210 and PC biomarker panel (miRNA 16, miRNA 21 and miRNA 196a). All these LFBs were also applied in the field of real sample detection.National Institutes of Health (NIH)
DOE-ND EPSCOR
NIH-COBRE (1P20GM09024-01A1)
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Sizovs, Antons. "Development of Carbohydrate-based Diblock Polymers for Nucleic Acid Delivery." Diss., Virginia Tech, 2012. http://hdl.handle.net/10919/77089.
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The delivery of nucleic acids remains the major obstacle for nucleic acid-based therapies such as gene therapy and gene silencing therapies based on RNA interference. In this dissertation we have developed and studied nucleic acid delivery vehicles based on cationic diblock glycopolymers that contain glucosamine and trehalosamine.
Practical procedures were developed to synthesize 2-methacrylamido-2-deoxy glucose and 6-methacrylamido-6-deoxy trehalose starting with commercially available carbohydrates and utilizing trimethylsilyl protecting group chemistry. These monomers were polymerized via reversible addition-fragmentation chain transfer (RAFT) polymerization to yield glycopolymers with the desired lengths and low polydispersity indexes. Glycopolymers were chain-extended with aminoethylmethacrylamide to produce cationic diblock copolymers.
The ability of cationic diblock copolymers to bind nucleic acids was demonstrated with gel electrophoresis and heparin exclusion assays. Complexes of the synthesized polymers with nucleic acids were studied with dynamic light scattering to reveal nanoparticles of 100-250 nm that were stable in the presence of serum proteins. Quartz crystal microbalance experiments showed that serum proteins adsorb on polytrehalose coated gold surfaces and it was suggested that these interactions may help mask the polytrehalose coated nanoparticles from potential actions of the immune system. Polytrehalose was also shown to suppress water crystallization similarly to trehalose by lowering the energies associated with the water/ice phase transition. The property was utilized to freeze-dry siRNA containing polyplexes which could be re-dissolved in water after lyophilization to yield nanoparticles.
The polyplexes formulated with cationic diblock copolymers were shown to efficiently enter cervical cancer cells (HeLa cell line) and glioblastoma cells (U-87 cell line) and to deliver their nucleic acid cargo. Polyglucose-containing polymers were efficient mediators of exogenous gene expression in HeLa cells, and polytrehalose- containing polymers were effective in promoting the target gene down-regulation via RNA interference by delivered siRNA.Ph. D.
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Zhou, Zhun. "Design of polymer motifs for nucleic acid recognition and assembly stabilization." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1437558800.
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Liang, Wanling, and 梁婉玲. "Formulation of nucleic acid with pH-responsive amphipathic peptides for pulmonary delivery." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/207996.
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Falconer, Robert Andrew. "Lipoamino acid based glycolipids for drug delivery." Thesis, University College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.392430.
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Sabatino, David. "Expanding the size and shape of nucleic acids : studies on branched and heptose based nucleic acids." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103291.
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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.
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Roark, Brandon Kyle. "Nucleic Acid-Driven Quantum Dot-Based Lattice Formations for Biomedical Applications." Thesis, The University of North Carolina at Charlotte, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10619578.
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We present a versatile biosensing strategy that uses nucleic acids programmed to undergo an isothermal toehold mediated strand displacement in the presence of analyte. This rearrangement results in a double biotinylated duplex formation that induces the rapid aggregation of streptavidin decorated quantum dots (QDs). As biosensor reporters, QDs are advantageous to organic fluorophores and fluorescent proteins due to their enhanced spectral and fluorescence properties. Moreover, the nanoscale regime aids in an enhanced surface area that increase the number of binding of macromolecules, thus making cross-linking possible. The biosensing transduction response, in the current approach, is dictated by the analysis of the natural single particle phenomenon known as fluorescence intermittency, or blinking is the stochastic switching of fluorescence intensity ON (bright) and OFF (dark) states observed in single QD or other fluorophores. In contrast to binary blinking that is typical for single QDs, aggregated QDs exhibit quasi-continuous emission. This change is used as an output for the novel biosensing techniques developed by us. Analysis of blinking traces that can be measured by laser scanning confocal microscopy revealed improved detection of analytes in the picomolar ranges. Additionally, this unique biosensing approach does not require the analyte to cause any fluorescence intensity or color changes. Lastly, this biosensing method can be coupled with therapeutics, such as RNA interference inducers, that can be conditionally released and thus used as a theranostic probes.
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Planonth, Songsak. "Peptide nucleic acid-encoded libraries for microarray-based high-throughput screening." Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/5862.
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Peptide nucleic acids (PNAs) were used as encoding tags to enable the analysis of peptide libraries by PNA/DNA hybridisation onto DNA microarrays. This allowed entire peptide libraries to be organised and sorted in a two dimensional format whereby all library members could be interrogated and analysed on a one-byone basis. In this thesis, PNA-encoded peptide libraries, generated by split-and-mix library synthesis, were screened for a variety of functions. Peptide sequences identified from the screening of a PNA-encoded library were analysed in detail as the first specific substrates for chymopapain. A new PNAencoded library consisting of D-amino acids was synthesised and screened with a number of proteases in attempts to identify novel/unusual substrates. PNA-encoded libraries were also used in the screening of peptide libraries for other activities. Thus substrates for catalyst-free Hüisgen cycloaddition were identified following the reaction between an alkyne modified peptide library and azidofluorescein, while cell-penetrating peptides were identified by hybridization of an internalized encoded library onto a DNA microarray.
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Slaitas, Andis. "Development of a new PNA analogue as a potential antisense drug and tool for life-science studies /." Stockholm : Karolinska institutet, 2004. http://diss.kib.ki.se/2004/91-7349-642-1/.
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Khater, Mohga Wagdy Yehia Mohamed. "Nanoparticle-based sensors for pathogen nucleic acid detection with interest for agriculture." Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/667373.
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La presente tesis describe el desarrollo de sensibles, bajo coste y portátiles métodos de sensado basados en nanomateriales aplicados en la detección de ADN de patógenos relacionados a plantas. El trabajo se presenta avances significativos en el campo de los biosensores para el diagnóstico de las enfermedades en las plantas. En el Capítulo I se da una visión general de las aplicaciones llevabas a cabo y mejoras aportadas por parte del uso de nanomateriales diferentes en el campo de los biosensores, y las nuevas aplicaciones en la detección de enfermedad de las plantas en lo punto de atención. En las secciones siguientes se presentan tres estrategias de sensado para la detección de secuencias de ADN específicas para el virus del cidro (citrus tristeza virus (CTV)), un virus modelo. Los sistemas están basados en las técnicas electroquímicas y ópticas.
El Capítulo III se presenta el uso de electrodos serigrafiados de carbono (SPCEs) como plataforma para hibridación y detección del ADN mediante impedancia. Esta plataforma se adaptó mediante la disposición de nanopartículas de oro (AuNPs), con el fin de obtener una estrategia más simple, sin marcas, menos costosa y más rápida.
Del mismo modo, en el Capítulo IV se centra en el desarrollo de nuevos métodos para la amplificación y la detección de ADN de CTV en SPCE modificados con AuNPs. Este biosensor opera en modo libre de marcas y con límite de detección (LOD) conseguido está en el rango de 1000 fg μL-1.
Finalmente, en el Capítulo V se presenta la tercera plataforma utilizada, que fue basada en papel y tiene el formato de un inmunoensayo de flujo lateral (LFIA, del inglés lateral flow immunoassays). En esta plataforma, las nanopartículas de oro se usan como marcas para obtener señal de color rojo, con el fin de realizar nuevas aplicaciones en diagnóstico de plantas, más rápido y simpleThis thesis aims at developing sensitive, affordable and portable biosensors based on nanomaterials for the determination of nucleic acid related to plant pathogens. The work strives to contribute to the keeping up in the advancements of biosensing systems relevant to plant infection diagnostics which would be an essential solution in the future to the issues of plant disease monitoring and food security. Following Chapter I, state-of-the-art on the latest trends in the development of advantageous biosensors based on both antibody and DNA receptors for early plant disease detection, as well as the use of different nanomaterials such as nanochannels and metallic nanoparticles for the development of innovative and sensitive biosensing systems for the detection of pathogens (i.e. bacteria and viruses) at the point-of-care is given. The next sections of this dissertation will describe three diagnostic biosensing strategies for the detection of citrus tristeza virus (CTV) related nucleic acid using electrical and optical transducing techniques. The electrical sensing of CTV through DNA hybridization based approach and the in situ amplified nucleic acid method will be achieved on carbon sensing substrate modified with gold nanoparticles, while paper-based sensors will be operated in lateral flow format for the gold nanoparticle-based optical detection of CTV. Furthermore, all aspects of the developed biosensing systems, from the bioassay and biosensor design to their development and optimization are presented in which will be organized in the following manner: Chapter III will present highly specific DNA hybridization sensor based on AuNP-modified SPCE employing label-free impedance for the detection of the CTV-related nucleic acid, together with dedicating emphasis to the study of electrodeposition time of AuNPs, whose precise particle size and shape will be required for the enhancement of DNA hybridization rate. A set of voltammetric studies of deposited AuNPs will be discussed. Particular attention will be paid for assembling the thiolated DNA probe as sensing layer for biosensor construction. The main sensor design aspects such as AuNPs size, probe DNA concentration and immobilization time together with DNA hybridization time will be optimized, in order to precisely select the best working conditions for this diagnostic platform. Chapter IV will cover the whole process undertaken for preparation of in situ nucleic acid amplification on gold nanoparticle-modified sensor for sensitive and quantitative detection of CTV. Plant disease (Citrus tristeza virus (CTV)) diagnostics was selected as relevant target for the demonstration of the proof-of-concept. This chapter will include two parts, the first one focuses on the design of RPA amplification assay, primers design, optimization of all essential bioassay aspects such as amplification temperature, volume and screening primers and finally the electrophoresis analysis for RPA products. The second part of this chapter will demonstrate label-free highly integrated in situ RPA amplification/detection approach at room temperature that takes advantage of the high sensitivity offered by gold nanoparticle-modified sensing substrates and electrochemical impedance spectroscopic (EIS) detection. Chapter V focuses on the application of isothermal nucleic acid amplification technology in simple lateral flow platform. The preparation of AuNP-based LFA for the highly sensitive direct detection of RPA amplified nucleic acid, the assembling of lateral flow step, the conjugation of AuNPs to the antibodies used for colorimetric detection, as well as the optimization of all working conditions and finally the analytical performance of the bioassay in LF will be explored. Moreover, aiming at truly achieving the point of care requirements of simple and affordable diagnostic technologies, the work here will present the possibility of amplifying nucleic acid without heat source and visual color detection. This approach would be of great potential as point of care diagnostics.
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Pouchain, Delphine. "Peptide nucleic acid-encoded libraries for microarray-based enzymatic high-throughput screening." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/3227.
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Protein kinases represent one of the largest families of enzymes. Due to their crucial roles in many cellular processes, protein kinases have become one of the major drug targets for pharmaceutical companies. In order to be able to determine selective substrates, elucidate pathway functionality and design potential kinase inhibitors, it is important to determine kinase substrate specificity. A 10,000-member split and mix peptide nucleic acid (PNA)-encoded peptide library was used to establish the substrate specificity of three different protein tyrosine kinases, using a DNA microarray and a Cy3-fluorescently labelled secondary anti-phosphotyrosine antibody. This approach was proven to be a powerful tool as several peptides (“hits”) were rapidly identified as good substrates for the three kinases. Split and mix combinatorial libraries can generate large peptide libraries consisting of potentially millions of compounds, but the main disadvantage is the deconvolution process required to identify active individual peptides from mixtures. The aim of the second part of the project was to build a PNA-encoded positional scanning library of FRET-based tetrapeptides to carry out microarray-based proteolytic profiling. As proof-of-principle, a 625-member library was first synthesised on solid phase using a split and mix methodology, and successfully used to profile three different proteases. The concept was then extended to the synthesis of a larger positional scanning library containing 50,625 different tetrapeptides encoded for by just 60 PNA tags.
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Takalkar, Sunitha. "Ultrasensitive Lateral Flow Nucleic Acid Biosensors Based on Novel Macro-/Nano-Materials." Diss., North Dakota State University, 2017. https://hdl.handle.net/10365/26493.
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Lin, Lina. "Synthesis, Structure, Function and Biomedical Studies of Nucleic Acid Derivatized with Selenium." Digital Archive @ GSU, 2010. http://digitalarchive.gsu.edu/biology_diss/77.
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Nucleic acids are macromolecules in cells for storing and transferring genetic information. Moreover, nucleic acids, especially RNAs, can fold into well-defined 3D structures and catalyze biochemical reactions. As ubiquitous biological molecules in all living systems, nucleic acids are important drug targets, and they can also be used in diagnostics and therapeutics. Structural information of nucleic acids provides the foundation for DNA and RNA function studies. X-ray crystallography has been a useful tool for structural studies of bio-macromolecules at atomic level. There are two major problems in macromolecular crystal structure determination: phasing and crystallization. Although selenium derivatization is routinely used for solving novel protein structures through the MAD phasing technique, the phase problem is still a critical issue in nucleic acid crystallography. The covalent selenium-derivatization of nucleic acids has been proven to be a useful strategy for solving the phase problem in nucleic acid X-ray crystallography. Besides the facilitation of nucleic acid crystallography, there is also a wide range of other applications for selenium-derivatized nucleic acids (SeNA). The investigation presented in this dissertation mainly focuses on the following research subjects (1) Synthesis and characterization of selenium-derivatized nucleic acids for X-ray crystallography, especially phosphoroselenoate RNAs. They are generated and used for crystallization. (2) Application of selenium-derivatized RNA for RNA interference. Phosphoroselenoate RNAs are tested for RNAi activities. (3) Synthesis and characterization of the uridine 5’-triphosphate modified with selenium at position 4. (4) Facile synthesis and antitumor activities of selenium modified deoxyribonucleosides. MeSe-thymidine nucleosides have shown antitumor activity in cell assays.
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高橋, 洋介. "多足型DNAナノ構造体を利用した核酸医薬の標的指向化および体内動態制御に関する研究." Kyoto University, 2018. http://hdl.handle.net/2433/232325.
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Nip, Lisa. "A nucleic acid-based bacterial message export system for cell-to-cell communication." Thesis, Massachusetts Institute of Technology, 2016. http://hdl.handle.net/1721.1/110882.
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Thesis: S.M., Massachusetts Institute of Technology, School of Architecture and Planning, Program in Media Arts and Sciences, 2016.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 36-38).
Communication within natural systems of eukaryotes and prokaryotes typically entails message transmission between and among cells via small-molecule messengers being funneled from the sender to the receiver cell. Nucleic acids are rarely used as extracellular messengers due to their labile nature and proclivity for enzymatic digestion. Eliminating these obstacles will allow for a larger array of messages to be sent with minimal cellular machinery. Exploiting the bacterial twin-arginine translocation (TAT) pathway and a nucleic-acid binding protein sourced from bacteriophage MS2, we have engineered a message-sending system in Escherichia coli capable of specifically exporting a "pre-written" circularized RNA message to the extracellular environment. This RNA message maintains its integrity over the course of at least four hours in extracellular growth medium, and this system serves as the first demonstration of versatile, stable messaging with nucleic acids, specifically with RNA, in the extracellular environment.
by Lisa Nip.
S.M.
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Ghosh, Sumana. "Design And Synthesis Of Novel Interacalator Based Chemical Nuclease." Thesis, Indian Institute of Science, 2001. http://hdl.handle.net/2005/260.
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Deoxyribonucleic acid and ribonucleic acid under physiological condition are polyanions composed of heterocyclic bases linked through sugar phosphate backbone. Due to Watson-Crick base pairing, DNA exists in double-helical form between two antiparallel strands of nucleic acid. Different conformations of DNA is possible among which the B-DNA form is considered to be the most common, and it is a right-handed double-helix with base pairs stacked at the center. There are two well-defined grooves termed as major and minor grooves, each has characteristic width and depth. Most of the DNA binding proteins generally approach DNA through the major groove, while small molecules such as drugs, antitumor antibiotics,1 their synthetic analogue,2 carcinogens,3 and the transition metal complexes4 interact with DNA through minor groove.
The nucleic acids function in the storage and transfer of genetic information. The function of cell expressions of proteins, synthesis of all bio-materials are directly or indirectly governed by the nucleic acid present in the body. Not only that, the origin of many diseases lie behind the structural modification or alterations in nucleic acids occur beyond our control.5 There are different drugs both natural and synthetic which are important in antibiotic chemotherapy, act against these diseases by interacting with DNA. Now to understand the actual mechanism of many diseases, how drugs interact with DNA and its specificity, binding sites of DNA, we need to develop molecules that modify or interact with biological molecules and such molecules can probe various structural aspects and type of interaction of macromolecular association complexes. One of such probe is the DNA cleaving agent. The potential scope of the utility of these compound is enormous and ranges from the creation of synthetic restriction enzymes for use by molecular biologists to the development of chemotherapeutic agents (Fe(BLM), calicheamicin) that may be effective against a variety of neoplastic diseases. They can also act as a structural probe (e.g. Fe(EDTA)2), drug / protein-DNA footprinting agent and affinity cleaving agent.
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Yang, Xiaojuan. "Development of Nanoparticle Systems for Therapeutic Drug Delivery." The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1248972068.
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Tomlinson, Jennifer A. "Nucleic acid-based methods for on-site detection of plant pathogens : approaches and applications." Thesis, University of Nottingham, 2012. http://eprints.nottingham.ac.uk/12957/.
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The ability to perform nucleic acid-based detection of plant pathogens away from conventional laboratory facilities has the potential to be beneficial in situations where results are required very rapidly or where resources and access to laboratory equipment are limited. Methods for use in such situations must combine sensitivity and specificity with rapid and simple workflows. The aim of this project was to investigate aspects of on-site testing for plant pathogens by developing detection methods for a range of target species. Detection methods based on loop-mediated isothermal amplification (LAMP) exhibit characteristics which make them potentially suitable for on-site testing. LAMP-based methods were developed for detection of plant pathogens with three potential non-laboratory testing scenarios in mind: testing during plant health inspection (assays for Phytophthora ramorum, P. kernoviae and Guignardia citricarpa); testing to assess inoculum levels in the processing of plant products (an assay for Botrytis cinerea); and testing in under-resourced settings (assays for Cassava brown streak virus and Ugandan cassava brown streak virus). In developing these detection methods, attempts were made to address some of the specific requirements of potential end-users of the tests in each case. For testing in the context of inspection, a particular emphasis was placed on the need for simple, rapid methods for nucleic acid extraction. As well as investigating the use of rapid extraction methods in conjunction with LAMP, work was also carried out to investigate how on-site nucleic acid extraction using lateral flow devices could be integrated with current field and laboratory testing for P. ramorum.
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Xiao, Linlin. "Detection of Viable Foodborne Pathogens and Spoilage Microorganisms by Nucleic Acid Amplification Based Platforms." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1308284180.
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Beals, Nathan. "Evaluation of the Delivery and Targeting of Nucleic Acid Based Nanomaterials for Therapeutic Application." Kent State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=kent1533166304898726.
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Canzoneri, Joshua Craig. "Interaction of small molecules with nucleic acid targets: from RNA secondary structure to the riobosome." Diss., Georgia Institute of Technology, 2012. http://hdl.handle.net/1853/45769.
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Nucleic acids have proven to be viable targets for small molecule drugs. While many examples of such drugs are detailed in the literature, only a select few have found practical use in a clinical setting. These currently employed nucleic acid targeting therapies suffer from either debilitating off-target side effects or succumb to a resistance mechanism of the target. The need for new small molecules that target nucleic acids is evident. However, designing a novel drug to bind to DNA or RNA requires a detailed understanding of exactly what binding environments each nucleic acid presents. In an effort to broaden this knowledge, the work presented in this thesis details the binding location and affinity of known and novel nucleic acid binding small molecules with targets ranging from simple RNA secondary structure all the way to the complex structure of ribosomal RNA. Specifically, it is shown that the anthracycline class of antineoplastics prefer to bind at or near mismatch base pairs in both physiologically relevant iron responsive element RNA hairpin constructs as well as DNA hairpin constructs presenting mismatched base pairs. Also characterized in this thesis is a novel class of topoisomerase II / histone deacetylase inhibitor conjugates that display a unique affinity for DNA over RNA. Finally, the novel class of macrolide-peptide conjugates, known as peptolides, are shown to retain potent translation inhibition of the prokaryotic ribosome. The binding pocket of the peptolides, including a crevice previously unreachable by macrolides that extends away from the peptidyl transferase center toward the subunit interface, is confirmed in detail via chemical footprinting of the 70S ribosome. Overall, the identification of a novel binding site for the anthracycline class of drugs and the characterization of the two novel drug designs presented in this thesis will undoubtedly aid in the effort to design and discover new molecules that aim for nucleic acid targets. For example, the anthracycline derivative topoisomerase II / histone deacetylase inhibitor conjugates, with their differential mode of nucleic acid binding, may prove to have a unique side effect profile in a therapeutic application. The peptolide compounds also have the potential to be applied as novel antibiotics as they bind to an area of the prokaryotic ribosome unrelated to known macrolide resistance mutations. Furthermore, as a result of the observation of this thesis work that some peptolides also posses eukaryotic translation inhibition capabilities, they could prove to be useful in preventing the growth of rapidly proliferating eukaryotic cells such as plasmodium, leishmania, or tumor cells. Additionally, different head groups could be utilized in creating new peptolides; for example, an oxazolidinone antibiotic could be employed to sample a different binding area of the ribosome.
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Thompson, Jason Donald. "A syncronous coefficient of drag alteration (SCODA) based technique for sequence specific enrichment of nucleic acids." Thesis, University of British Columbia, 2011. http://hdl.handle.net/2429/33073.
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Sequence based enrichment of nucleic acids is a critical enabling component of future nucleic acid detection methods in many fields including detection of nucleic acid tumor biomarkers in body fluids, non-invasive prenatal detection of fetal genetic abnormalities, and detection of pathogenic microorganisms. In many cases the problem of detecting the nucleic acid biomarker of interest is confounded by the presence of a large excess of nucleic acid sequences that may differ from the sequence of interest by only a single base. Consequently, existing methods are limited in sensitivity and amount of starting material to avoid overwhelming the detection methods with background nucleic acids. This limits their usefulness to a small number of applications. Techniques for enrichment of specific sequences rely on hybridization, and are generally not capable of enriching for low abundance sequences by more than 10 fold, a limit imposed by the thermodynamics of hybridization.
In this dissertation I present a technique for sequence enrichment of nucleic acids based on synchronous coefficient of drag alteration (SCODA), which enables sequence specific enrichment of nucleic acids from sample volumes greater than 100 μL, with concurrent concentration of the nucleic acids to volumes appropriate for PCR detection (<10 μL). We have demonstrated that this technique is capable of at least 10,000 fold enrichment of target sequences with respect to contaminating sequences differing by a single base. We have additionally shown that this technique is capable of at least 100 fold enrichment of a target sequence with a single methylated cytosine residue in a background of unmethylated targets of identical sequence by exploiting the small difference in binding energy of a methylated target to its complementary probe compared to an unmethylated target. To our knowledge this is the most specific hybridization based sequence enrichment scheme in existence, and this is the first demonstration of hybridization based enrichment of unmodified methylated DNA. Although some technical challenges must be overcome before this method will become a tool appropriate for routine laboratory use, we believe that the challenges are not insurmountable, and this method has the potential to enable routine analysis of low abundance nucleic acids.
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LEE, CHEN-CHANG. "Design, Synthesis, and Structure-Bioactivity Characterization of Novel Carbohydrate-Based Polymers as Nucleic Acid Delivery Vectors." University of Cincinnati / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1211919868.
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Mina, James. "Hyaluronic acid based polymer drug conjugates for the treatment of rheumatoid arthritis." Thesis, University of the West of Scotland, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.739391.
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Levy, Maria Patricia Morales. "Development of a DNA vaccine for population control : a zona pellucida based nucleic acid vaccine (ZP-NAV) /." For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2005. http://uclibs.org/PID/11984.
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