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.

Engineered DNA-binding proteins enable targeted manipulation of the genome. Zinc fingers are the most well characterized DNA-binding domain and for many years research has focused on understanding and manipulating the sequence-specificities of these proteins. Recently, major advances in the ability to engineer zinc finger proteins, as well as the discovery of a new class of DNA-binding domains - transcription activator-like effectors (TALEs), have made it possible to rapidly and reliably engineer proteins targeted to any sequence of interest. With this capability, focus has shifted to exploring the applications of this powerful technology. In this dissertation I explore three important applications of engineered DNA-binding proteins.

The human genome encodes information that instructs human development, physiology, medicine, and evolution. Massive amount of genomic data has generated an ever-growing pool of hypothesis. Genome editing, broadly defined as targeted changes to the genome, posits to deliver the promise of genomic revolution to transform basic science and personalized medicine. This thesis aims to contribute to this scientific endeavor with a particular focus on the development of effective human genome engineering tools.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 4 uppsatser.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Físicas e Matemáticas. Programa de Pós-Graduação em Química. / Made available in DSpace on 2012-10-23T11:17:23Z (GMT). No. of bitstreams: 1 239805.pdf: 1384835 bytes, checksum: c2736cc980bf3b78a9263b52d8d6bfcb (MD5) / Muitas reações importantes para a manutenção da vida na Terra são catalisadas por metaloenzimas (enzimas com pelo menos um íon metálico em seu sítio ativo). Dentre as metaloenzimas pode-se citar as catecol oxidases, que catalisam a oxidação de o-catecóis às correspondentes o-quinonas e as hidrolases/nucleases sintéticas, que catalisam a clivagem de ésteres de fosfato. Diversos complexos metálicos que visam modelar estrutural e/ou funcionalmente essas metaloenzimas têm sido estudados nas últimas décadas, com o objetivo de elucidar o seu modo de ação.Neste trabalho foram sintetizados e caracterizados por análise elementar de CHN, espectroscopia eletrônica, eletroquímica, titulação potenciométrica e espectrofotométrica, EPR e magnetoquímica dois novos complexos binucleares de cobre(II) empregando-se os dois novos ligantes contendo átomos N,O-doadores: H2L1 e H3L2. Os complexos 1 - [Cu2(HL1)( -OAc)](ClO4)2 e 2 - [Cu2(HL2)( -OAc)](ClO4) tiveram suas estruturas cristalinas resolvidas por difratometria de raios-X. A partir dos dados estruturais, espectroscópicos, eletroquímicos e cinéticos, foi possível propor um ciclo catalítico tanto para a oxidação do 3,5-di-terc-butil-catecol, quanto para a hidrólise do bis(2,4-dinitrofenil)fosfato, mediada pelos complexos 1 e 2, podendo os mesmos serem considerados complexos promíscuos, modelos funcionais tanto para as catecol oxidases, quanto para as hidrolases/nucleases sintéticas.

This thesis describes the establishment of a precise gene targeting methodology in the silkworm Bombyx mori by technologies based on engineered endonucleases. Two classes of engineered endonucleases, ZFNs and full length TALENs were used for creating DSBs at specified sites in the colour marker genes (BmBlos2 and Bmwh3). Direct embryo microinjection of engineered nucleases mRNA were performed and let the nuclease proteins to disrupt the functions of these marker genes by creating DSBs and inducing error prone NHEJ mechanism. These experiments showed that both ZFNs and TALENs could be used for targeted gene disruption in silkworms.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-08-01T13:59:43Z No. of bitstreams: 1 JessykaFernandaSantiagoMonte_DISSERT.pdf: 1592894 bytes, checksum: 2e8d0cdaf53e251db5deb8479566e9dc (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-08-07T15:09:24Z (GMT) No. of bitstreams: 1 JessykaFernandaSantiagoMonte_DISSERT.pdf: 1592894 bytes, checksum: 2e8d0cdaf53e251db5deb8479566e9dc (MD5) / Made available in DSpace on 2017-08-07T15:09:24Z (GMT). No. of bitstreams: 1 JessykaFernandaSantiagoMonte_DISSERT.pdf: 1592894 bytes, checksum: 2e8d0cdaf53e251db5deb8479566e9dc (MD5) Previous issue date: 2017-05-29 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Estudos envolvendo estabilidade de plasm?deos tiveram in?cio h? pelo menos duas d?cadas e vem crescendo nos ?ltimos anos, desde que os pDNAs apresentaram um enorme potencial como vetores em terapia g?nica. O uso terap?utico desses vetores tem sido dificultado por quest?es de estabilidade, principalmente no que se refere ao processo de produ??o e purifica??o, armazenamento por longos per?odos e serem suscept?veis ? degrada??o por nucleases. Assim, ensaios que permitam analisar estes processos que levam a instabilidade do pDNA podem ser ferramentas importantes para sua compreens?o, associado a outras vari?veis, tais como temperaturas, tempo de armazenamento, tamanho do pDNA, presen?a de sequ?ncias procari?ticas e a??o nucle?sica, assim E. coli DH5-? competente foi produzida, transformada com os pDNAs estudados (pVAX1, pVAX1lacZ e MSPpVAX1), purificados e armazenados em diferentes temperaturas por um intervalo de tempo pr?-determinado e para estabelecer uma rela??o entre a estabilidade dos diferentes pDNAs e sua fun??o biol?gica enquanto vetores, estudou-se a resist?ncia da isoforma super-enrolada ? a??o da nucleases de soro em diferentes concentra??es e ao longo do tempo. Para tal, eletroforese em gel de agarose e transforma??o em E. coli com c?lculo da efici?ncia de transforma??o celular foram realizados. Foi observado ao longo do tempo que a integridade do pDNA super-enrolado foi perdida em fun??o do tempo e da temperatura de armazenamento, al?m disso, os pDNAs contendo sequ?ncias apenas procari?ticas se mostraram mais resistente a esses fatores quando comparado ao que possu?a sequ?ncia procari?tica, mostrando que h? influ?ncia desses fatores na estabilidade do pDNA. Em rela??o ? a??o nucle?sica, o maior plasm?deo foi mais acometido pela atividade dessas enzimas. Quanto a fun??o biol?gica, os ensaios de efici?ncia de transforma??o em E. coli indicaram que houve uma maior percentagem de c?lulas transformadas quando era utilizado plasm?deo na conforma??o super-enrolada. Verificou-se em todos os ensaios, que a isoforma super-enrolada era sempre mais eficiente que as demais isoformas, devendo-se esse fato possivelmente ? sua maior estabilidade citoplasm?tica e a difus?o mais r?pida desta isoforma em dire??o ao n?cleo. Assim esse trabalho mostrou a cin?tica de degrada??o, passo a passo, dos pDNAs estudados, mostrando que a perda da forma super-enrolada compromete a estabilidade dos pDNAs, afetando dessa forma a fun??o biol?gica dos mesmos, comprometendo sua utiliza??o em terapia g?nica e vacinas de DNA. / Studies involving plasmid stability have started for at least two decades and have been growing in recent years, since pDNAs have had enormous potential as vectors in gene therapy, however the therapeutic use of these vectors has been hampered by stability issues, especially in Refers to the process of production and purification, storage for long periods and being susceptible to degradation by nucleases. Thus, assays that allow the analysis of this degradation process can be important tools for its understanding, associated to other variables, such as temperature, storage time, pDNA size and nucleoside action. The competent E. coli DH5-? was produced, transformed with the pDNAs studied (pVAX1, pVAX1lacZ and MSPpVAX1), purified and stored at different temperatures for a predetermined time and to establish a relationship between the stability of the different pDNAs and their Biological function as vectors, the resistance of the supercoiled isoform to the action of serum nucleases at different concentrations and over time was studied. For this purpose, agarose gel electrophoresis and transformation in E. coli with calculation of cell transformation efficiency were performed. It was observed over time that the integrity of the supercoiled pDNA was lost as a function of time and storage temperature, in addition, pDNAs containing only prokaryotic sequences proved to be more resistant to these factors when compared to that having procaryotic sequence pDNA, showing that these factors influence the stability of pDNA. In relation to the nuclease action, the bigger plasmid was more affected by the activity of these enzymes. Regarding biological function, transformation efficiency assays in E. coli indicated that there was a higher percentage of transformed cells when plasmid was used in the supercoiled conformation. It was verified in all the tests that the supercoiled isoform was always more efficient than the other isoforms, possibly due to its greater cytoplasmic stability and the faster diffusion of this isoform towards the nucleus. Thus, this work showed the degradation kinetics, step by step, of the studied pDNAs, showing that the loss of supercoiled form compromises the stability of the pDNAs, thus affecting the biological function of the same, compromising their use in gene therapy and vaccines DNA.

No description available.

La famille des Arenaviridae contient des virus transmis par les rongeurs responsables notamment de graves fièvres hémorragiques dont la fièvre de Lassa qui infecte chaque année 300 000 personnes. L’absence de solutions thérapeutiques efficaces (vaccins et/ou antiviraux) en fait une priorité d’étude. Il est donc urgent d’identifier de nouvelles cibles thérapeutiques et des antiviraux spécifiques associés. Le génome des Arénavirus code pour deux nucléases possédant des fonctions cruciales pour la survie du virus. La protéine L possède un domaine endonucléase indispensable à la réplication virale réalisant le mécanisme de vol de coiffe. La protéine NP comporte un domaine exonucléase impliqué dans l’échappement au système immunitaire de l’hôte infecté. Ces deux enzymes possèdent un mécanisme d’action commun métal-dépendant de clivage de leur ARN substrat. Ce projet :1) se propose d’apporter la preuve de concept que ces deux enzymes sont de nouvelles cibles thérapeutiques pour combattre les Arénavirus.2) se base sur le criblage préalable d’une chimiothèque de 1ère génération sur l’endonucléase d’un Arénavirus et la découverte d’une famille de dicéto-­‐acides (DCAs) inhibitrice de son activité. Au cours de ce travail de thèse, une chimiothèque de 2nde génération de 22 composés a été synthétisée. Des voies de synthèses nouvelles et originales ont été développées. Les composés ont été évalués par des méthodes biophysiques pour leur capacité à se lier aux enzymes cibles, ont été évalués comme potentiels inhibiteurs dans des tests d’activité in vitro, et in cellula dans un test en mini-­‐génome. Certains composés se sont montrés de bons ligands, actifs in vitro et in cellula. / The Arenaviridae family comprises viruses transmitted by rodents responsible for several diseases such as hemorragic fevers among which Lassa fever infects 300,000 people each year. The current lack of effective vaccines and/or antivirals makes the development of therapeutic solutions of high priority. The genome of Arenaviruses encodes for two nucleases bearing crucial functions for virus survival. The L protein bears an endonuclease domain essential for viral replication through the cap-­‐snatching mechanism. The NP protein has an exonuclease domain involved in anti-­‐immune activity. Both enzymes share a common mode of action as they cleave their RNA substrate via a metal-­‐dependant (Mg2+) strategy. This work intends to :1) consider these two enzymes as novel Arenavirus therapeutic targets.2) take over a preliminary screening of a 1st generation of ligands on an Arenavirus endonuclease. Diketo-­‐acids (DKAs) have been discovered as potential inhibitors of its activity. To this end, a library of 2nd generation of 22 compounds was synthetized thanks to the development of an optimized synthesis pathway. Compounds were further evaluated by biophysical methods for their ability to bind therapeutic targets, as potential inhibitors in an in vitro assay and in cellula in a mini-­‐genome assay. Some of them are active in vitro and in cellula

The world of precision medicine was revolutionized by the discovery of CRISPR-Cas systems. In particular, the capabilities of the programmable nuclease Cas9 and its derivatives have unlocked a world in which applied genome engineering to cure human disease is a reality being pursued in patient clinical trials. Gene editing via the induction of programmable, site-specific double strand breaks (DSBs) has been revolutionary for the precision medicine field. However, there are many safety concerns centered on the induction of DSBs causing potential undesirable on- and off-target consequences, particularly for in vivo CRISPR applications. To circumvent these warranted concerns, many groups have attempted to repurpose recombinases or engineer new fusion systems to perform programmable genome engineering without the induction of DSBs. This dissertation will first highlight the development of recombinases for programmable DNA insertions over the course of decades, including efforts to evolve novel DNA recognition sequences, efforts to tether recombinases to programmable DNA-binding proteins, and the recent discovery of naturally occurring RNA-guided DNA transposition systems. This dissertation will then highlight the development of CRISPR-associated transposases (CASTs) as DSB-independent programmable mammalian gene editing tools capable of integrating large DNA cargos, as well as the future directions that may further enhance CAST activity in human cells. The works in this dissertation detail the initial efforts to engineer and optimize a new class of genome manipulation tools that were previously absent from the gene editing toolkit.