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Authors
Abstract(s)
As Terapias Moleculares, como a Terapia Génica e as Vacinas de DNA, estão a
criar um aumento da procura de grandes quantidades de DNA plasmídico (pDNA)
puro. Apesar de já estarem disponíveis vários métodos, baseados em protocolos da
biologia molecular, para a purificação de plasmídeos, a grande maioria não são
apropriados para o uso em larga escala. Para além disso, esses protocolos usam com
frequência reagentes tóxicos, os quais impedem a sua utilização para a purificação de produtos terapêuticos.
Já os métodos baseados na afinidade usados até agora, parecem ser bastante promissores, contudo a maior parte faz uso de macrobiomoléculas de preços elevados, que impedem a sua utilização extensiva.
Sabe-se que são várias as moléculas que ligam ao DNA com uma especificidade elevada, como certos antibióticos e agentes anticancerigenos. É o caso das moléculas
focadas neste estudo: a neomicina e a berberina. Desta forma, estes ligandos
representam uma boa alternativa para a aplicação a protocolos de purificação por
afinidade.
Tendo esse objectivo em mente, as afinidades de ligação entre as moléculas acima
mencionadas e um pDNA modelo (pVax1LacZ), foram avaliadas, recorrendo à técnica
de titulação fluorimétrica desenvolvida por Strothkamp. A titulação do pDNA com brometo de etídio (BrEt) foi seguida através da intensidade de fluorescência, na ausência e presença de diferentes concentrações dos ligandos em estudo. Aos dados obtidos foi aplicado o método de Scatchard, para assim se determinar o efeito de cada um dos compostos na ligação do BrEt ao pDNA. As constantes de ligação de cada composto foram depois calculadas para diferentes concentrações de cloreto de sódio. Os
resultados obtidos mostram que o antibiótico que apresenta a constante de afinidade
mais elevada é a berberina, a uma concentração de cloreto de sódio 1,0 M, podendo assim ser um ligando promissor para a purificação de pDNA por afinidade.
O suporte escolhido para os ensaios de cromatografia de afinidade foi o Epóxi –
(CH2)4 – Sepharose™, contudo a berberina não contém grupos hidroxílicos funcionais
para poder ser directamente imobilizada. Desta forma, foram realizados estudos
visando a clivagem do seu grupo metilenodióxido para a obtenção de um catecol, alcançando-se os melhores resultados através do uso de tricloreto de alumínio, como activador. Porém, em vez de um único composto, foi obtida uma mistura de quatro produtos, derivados da hidrólise dos grupos metóxido. Ainda assim, procedeu-se à
imobilização da mistura ao suporte e analisou-se a interacção do gel obtido com o
pDNA. Estudou-se também a influência da concentração de sal no perfil cromatográfico. Os resultados obtidos mostram que, para todas as concentrações de sal, a amostra foi imediatamente eluída após a injecção, sem qualquer retenção na coluna.
Este pode ter sido consequência de uma possível baixa densidade de ligandos na coluna ou ao facto da constante de afinidade calculada não representar a verdadeira afinidade existente entre a berberina e o pDNA. O facto de a derivatização ter sido realizada com quatro compostos diferentes, pode também ter sido responsável pela má retenção. Uma vez que esta foi a primeira vez que um antibiótico foi usado como ligando
para a purificação de pDNA, criou-se um campo de investigação promissor e pouco
explorado, que poderá melhorar e simplificar os protocolos já existentes, para além de,
num futuro próximo, poder ser usado para implementar esta abordagem
cromatográfica à purificação, em larga escala, de vectores plasmídicos para serem
usados nas terapias moleculares.
Molecular therapies such as gene therapy and DNA vaccines are creating an increasing demand for high quantities of purified plasmids. Although several methods based on molecular biology protocols are available for the purification of plasmids, most of them are not suitable to use in large scale. In addition they frequently use toxic reagents, which prevent their utilization for the purification of therapeutic products. Affinity methods appear to be very promising but currently most of them use expensive macromolecular biomolecules which prevent their wide application. It is known that several molecules, like certain antibiotics and anticancer agents, bind DNA with high specificity, molecules like the ones focused on this study: neomycin and berberine. In this way, these ligands represent a good choice for the application on affinity purification procedures. For this purpose, the binding affinities of the aboved-described DNA interacting compounds, to a model plasmid DNA (pVax1LacZ), were evaluated using the fluorimetric titration technique developed by Strothkamp. Titration of pDNA with ethidium bromide was followed by fluorescence intensity in the absence and presence of different concentrations of the compounds under study. From this data the Scatchard plot was employed to determine the effect of each compound in ethidium bromide binding. Affinity constants for each compound were then calculated for diferent sodium chloride concentrations. The results showed that berberine has the highest pDNA binding constant, using 1,0 M sodium chloride concentration, thus being a promising ligand for pDNA affinity purification. The chosen support for the affinity chromatography assays was the Epóxi – (CH2)4 – Sepharose™, since berberine does not have any hidroxilic group to be directly immobilised. In this way, studies to cleave the metilenodioxy group of berberine were performed and a cathecol was obtained using aluminium chloride as a catalyzer. However, instead of a single compound, it was obtained a mixture of four different cathecols, were the metoxy groups of berberine were also hydrolysed. However, the immobilization of the mixture compounds was performed and the interation between pDNA and the obtained chromatographic gel was analysed. The influence of the mobile phase composition on the chromatographic behaviour was also studied. The experimental chromatographic results showed an immediate elution of the sample after injection, without any retention on the column, for all salt concentrations tested. This may be due to a possible low ligand density in the column or the affinity constant calculated doesn’t represent the real affinity between berberine and pDNA. The fact that de derivatization was made with four different molecules may also be responsible for the poor retention. Since it was the first time that an antibiotic was used for pDNA purification, it opened a promising unexplored field that could be able to improve and simplify currently available protocols and could, in the near future, be used to implement this chromatographic approach in the large scale purification of plasmids vectors for molecular therapies.
Molecular therapies such as gene therapy and DNA vaccines are creating an increasing demand for high quantities of purified plasmids. Although several methods based on molecular biology protocols are available for the purification of plasmids, most of them are not suitable to use in large scale. In addition they frequently use toxic reagents, which prevent their utilization for the purification of therapeutic products. Affinity methods appear to be very promising but currently most of them use expensive macromolecular biomolecules which prevent their wide application. It is known that several molecules, like certain antibiotics and anticancer agents, bind DNA with high specificity, molecules like the ones focused on this study: neomycin and berberine. In this way, these ligands represent a good choice for the application on affinity purification procedures. For this purpose, the binding affinities of the aboved-described DNA interacting compounds, to a model plasmid DNA (pVax1LacZ), were evaluated using the fluorimetric titration technique developed by Strothkamp. Titration of pDNA with ethidium bromide was followed by fluorescence intensity in the absence and presence of different concentrations of the compounds under study. From this data the Scatchard plot was employed to determine the effect of each compound in ethidium bromide binding. Affinity constants for each compound were then calculated for diferent sodium chloride concentrations. The results showed that berberine has the highest pDNA binding constant, using 1,0 M sodium chloride concentration, thus being a promising ligand for pDNA affinity purification. The chosen support for the affinity chromatography assays was the Epóxi – (CH2)4 – Sepharose™, since berberine does not have any hidroxilic group to be directly immobilised. In this way, studies to cleave the metilenodioxy group of berberine were performed and a cathecol was obtained using aluminium chloride as a catalyzer. However, instead of a single compound, it was obtained a mixture of four different cathecols, were the metoxy groups of berberine were also hydrolysed. However, the immobilization of the mixture compounds was performed and the interation between pDNA and the obtained chromatographic gel was analysed. The influence of the mobile phase composition on the chromatographic behaviour was also studied. The experimental chromatographic results showed an immediate elution of the sample after injection, without any retention on the column, for all salt concentrations tested. This may be due to a possible low ligand density in the column or the affinity constant calculated doesn’t represent the real affinity between berberine and pDNA. The fact that de derivatization was made with four different molecules may also be responsible for the poor retention. Since it was the first time that an antibiotic was used for pDNA purification, it opened a promising unexplored field that could be able to improve and simplify currently available protocols and could, in the near future, be used to implement this chromatographic approach in the large scale purification of plasmids vectors for molecular therapies.
Description
Keywords
DNA plasmídico Berberina