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Authors
Abstract(s)
Nos últimos anos, a aplicação de DNA plasmídeo (pDNA) em terapia génica ou vacinas de DNA
tem-se revelado uma ferramenta com potencial aplicação no tratamento de inúmeras
doenças. Uma vez que esta abordagem recorre a vetores não-virais, é mais segura em termos
de ativação de oncogenes e apresenta uma diminuição de reações do sistema imunitário.
Deste modo, a indústria farmacêutica procura obter pDNA em maiores quantidades e isento
de contaminantes (RNA, DNA genómico, endotoxinas, proteínas). Uma técnica que permite
purificar o pDNA é a cromatografia de afinidade (AC). Esta metodologia baseia-se em
interações específicas entre ligandos e o pDNA, permitindo a eliminação de alguns passos de
purificação. A utilização de aminoácidos como ligandos de afinidade revelou ser uma
abordagem promissora para purificar ácidos nucleicos, pois baseia-se na bioespecificidade
aminoácido-nucleótido. A L-histidina e a L-arginina permitiram isolar a isoforma
superenrolada (sc) do pDNA, indicando a presença de interações específicas entre o pDNA e os
aminoácidos. Neste trabalho são apresentados os resultados de três ligandos, a L-tirosina e os
dipeptídeos L-tirosina-L-tirosina e L-tirosina-L-arginina. Os dipeptídeos foram sintetizados de
acordo com o método clássico de síntese química, que envolve a proteção do grupo amina e
do grupo carboxílico de cada aminoácido, e posterior formação da ligação peptídica e por fim
desproteção usando TFA (ácido trifluoracético). De seguida, os ligandos foram imobilizados na
Sepharose CL-6B através da ativação dos grupos epóxi com o reagente 1,4-butanediol
diglicidil éter. O suporte foi caracterizado por HR-MAS (Ressonância Magnética de Alta
Resolução de Ângulo Mágico) RMN. A técnica de STD-RMN (Diferença da Transferência de
saturação por ressonância magnética nuclear) permitiu identificar os grupos preferenciais dos
5´-mononucleótidos que interagem com os suportes Sepharose-L-tirosina, Sepharose-Ltirosina-
L-tirosina e Sepharose-L-tirosina-L-arginina. Utilizando a técnica de RPS (Ressonância
Plasmónica de Superfície), foi analisada a afinidade de cada um dos ligandos em estudo em
relação a cada uma das isoformas de pVAX1-LacZ a diferentes temperaturas (T=10°C e 25°C)
e usando diferentes tampões (Tris-HCl 10 mM e Hepes ácido 10 mM). Desta forma, a utilização
das técnicas STD-RMN e SPR permitiu fazer o screening das interações dos suportes-5’-
mononucleótidos e determinar a afinidade do complexo ligando: pDNA, para futura aplicação
em AC.
In the last years, the use of plasmid DNA (pDNA) in gene therapy or DNA vaccines has been proved to be a tool with potential application in the treatment of diseases. Since this approach uses non-viral vectors, it is safer in terms of oncogene activation and it presents a decrease of the immune system response. In this way, the pharmaceutical industry needs to obtain pDNA in larger quantities and free of contaminants (RNA, genomic DNA, endotoxins, proteins). A technique that allows purifying pDNA is affinity chromatography (AC). This method is based in specific interactions between ligands and the pDNA, allowing the elimination of some purification steps. The use of amino acids as affinity ligands has proven to be a promising approach to purify nucleic acids, since it is based on the amino acid-nucleotide biospecificity. L-histidine and L-arginine allowed isolating the supercoiled isoform (sc) of the pDNA, indicating the presence of specific interactions between the pDNA and the amino acids. In this work are presented the results of three ligands, L-tyrosine and the dipeptides L-tyrosine- L-tyrosine and L-tyrosine-L-arginine. The dipeptides were synthetized using chemical synthesis, which involves the protection of the amine group and the carboxylic group of each amino acid, the formation of the peptide bond and, finally, deprotection using TFA (trifluoroacetic) acid. Then, the ligands were immobilized in the Sepharose CL-6B through their activation by epoxide formation with 1,4-butanediol diglycidyl ether. The support was characterized by HR-MAS (High Resolution Magic Angle Spinning) NMR (nuclear magnetic resonance). The STD-NMR (saturation transfer difference- nuclear magnetic resonance) technique allowed identifying the preferential groups of the 5’ –mononucleotides that interact with the supports Sepharose-L-tyrosine, Sepharose-L-tyrosine-L-tyrosine and Sepharose-L-tyrosine-L-arginine supports. By the SPR (surface plasmon resonance) biosensor, the affinity towards the isoforms of pVAX1-LacZ is determined at temperatures 10°C and 25°C, and using different buffers (Tris-HCl 10 mM e Hepes acid 10 mM). Thus, STD-NMR and SPR techniques allow us to screening of the binding affinity of these ligands to plasmid isoforms for their future application in AC.
In the last years, the use of plasmid DNA (pDNA) in gene therapy or DNA vaccines has been proved to be a tool with potential application in the treatment of diseases. Since this approach uses non-viral vectors, it is safer in terms of oncogene activation and it presents a decrease of the immune system response. In this way, the pharmaceutical industry needs to obtain pDNA in larger quantities and free of contaminants (RNA, genomic DNA, endotoxins, proteins). A technique that allows purifying pDNA is affinity chromatography (AC). This method is based in specific interactions between ligands and the pDNA, allowing the elimination of some purification steps. The use of amino acids as affinity ligands has proven to be a promising approach to purify nucleic acids, since it is based on the amino acid-nucleotide biospecificity. L-histidine and L-arginine allowed isolating the supercoiled isoform (sc) of the pDNA, indicating the presence of specific interactions between the pDNA and the amino acids. In this work are presented the results of three ligands, L-tyrosine and the dipeptides L-tyrosine- L-tyrosine and L-tyrosine-L-arginine. The dipeptides were synthetized using chemical synthesis, which involves the protection of the amine group and the carboxylic group of each amino acid, the formation of the peptide bond and, finally, deprotection using TFA (trifluoroacetic) acid. Then, the ligands were immobilized in the Sepharose CL-6B through their activation by epoxide formation with 1,4-butanediol diglycidyl ether. The support was characterized by HR-MAS (High Resolution Magic Angle Spinning) NMR (nuclear magnetic resonance). The STD-NMR (saturation transfer difference- nuclear magnetic resonance) technique allowed identifying the preferential groups of the 5’ –mononucleotides that interact with the supports Sepharose-L-tyrosine, Sepharose-L-tyrosine-L-tyrosine and Sepharose-L-tyrosine-L-arginine supports. By the SPR (surface plasmon resonance) biosensor, the affinity towards the isoforms of pVAX1-LacZ is determined at temperatures 10°C and 25°C, and using different buffers (Tris-HCl 10 mM e Hepes acid 10 mM). Thus, STD-NMR and SPR techniques allow us to screening of the binding affinity of these ligands to plasmid isoforms for their future application in AC.
Description
Keywords
Affinity Chromatography L-Tyrosine L-Tyrosine-L-Arginine L-Tyrosine-L-Tyrosine Plasmid Dna Pvax1-Lacz Spr Std-Nmr