Evaluación de la participación de Mg2+ en el mecanismo de catálisis de la dnazima 8-17
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2020
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es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
Las DNAzimas son hebras monocaternarias de ADN obtenidas en laboratorio
a través de un proceso llamado selección in vitro, las cuales son capaces de
catalizar diversas reacciones químicas, entre ellas la ruptura de ARN. En esta
reacción, la DNAzima acelera la transferencia interna del fosfodiéster,
utilizando un ion metálico como cofactor. Entre la gran variedad de enzimas
de ADN seleccionadas hasta la fecha, la DNAzima 8-17 ha sido, sin duda, la
más estudiada en términos catalíticos. Esta es una metaloenzima que tiene la
particularidad de ser activa con una serie de metales divalentes, pero que
muestra su mayor actividad en presencia de Pb
2+
. Sin embargo, la
participación específica del cofactor metálico en la catálisis no ha sido aún del
todo dilucidada, aunque en base a la evidencia colectada, una de las
posibilidades podría ser la existencia de un evento de transferencia de
protones que involucra al ion metálico hidratado. Por esta razón, este trabajo
propone evaluar la influencia del pH en el mecanismo de reacción de la
DNAzima 8-17, a través del estudio de perfiles de actividad (log (kobs) vs pH).
Para ello es necesario estudiar la actividad de la enzima en un amplio rango
de pH. No obstante, a altos valores de pH, la DNAzima tiene la particularidad
de tener constantes de velocidad elevadas que dificultan su determinación por
las técnicas disponibles. Para superar esta limitación, se plantea estudiar los
perfiles de actividad de un análogo menos activo de la DNAzima 8-17. De esta
manera, se compararán los perfiles de actividad de la DNAzima nativa y la
análoga en presencia del ion metálico divalente. Para esto se realizarán
ensayos de actividad a diferentes pHs con la DNAzima y un sustrato marcado
con la sonda fluorescente FAM en la terminación-5Ꞌ, bajo condiciones de
single-turnover. Las hebras correspondientes al sustrato y a los productos de
reacción serán separados mediante electroforesis, en un gel denaturante de
poliacrilamida al 20% (PAGE), la imagen obtenida será visualizada en un
G:box Syngene Imager System, y la cuantificación se realizará por medio del
software GeneTools Syngene. A partir de los datos de formación de producto
en el tiempo, se calcularán las kobs para cada reacción, a cada uno de los pH
bajo estudio. Estos datos cinéticos, permitirán evaluar la influencia del pH
sobre la actividad de la DNAzima 8-17.
DNAzymes are single strands of DNA obtained in the laboratory through a process called in vitro selection, which are capable of catalyzing various chemical reactions, including RNA breakdown. In this reaction, the DNAzyme accelerates the internal transfer of the phosphodiester, using a metal ion as a cofactor. Among the wide variety of DNA enzymes selected to date, the 8-17 DNAzyme has been the most studied in catalytic terms. The 8-17 is a metalloenzyme that has the particularity of being active with a series of divalent metal ions, but which shows its greatest activity in the presence of Pb2+ . However, the specific participation of the metal cofactor in catalysis has not yet been fully understood, although based on the evidence collected, one of the possibilities could be the existence of a proton transfer event that involves the hydrated metal ion. For this reason, this work proposes to evaluate the influence of pH on the reaction mechanism of the 8-17 DNAzyme, through the study of pH-activity profiles (log (kobs) vs pH). In this sense, it is necessary to study the activity of the enzyme in a wide range of pH. However, at high pH values, the 8-17 DNAzyme has the particularity of having high rate constants that make difficult their observation by the available techniques. To overcome this limitation, we consider studying the pH-activity profiles of a less active analogue of the 8-17 DNAzyme. Thus, the activity profiles of the native and analogous DNAzyme will be compared in the presence of the divalent metal ion. The experiments will be carried out by measuring the activity at different pHs with the DNAzyme and a substrate labeled with the fluorescent probe FAM at the 5Ꞌ-end, under single-turnover conditions. The strands corresponding to the substrate and the reaction products will be separated by electrophoresis, in a 20% polyacrylamide denaturant gel (PAGE), the obtained image will be visualized in a G:box Syngene Imager System, and the quantification will be carried out with the GeneTools Syngene software. Knowing the percentage of product over time, will allow the obtention of the kobs for each reaction, at each pH under study. The evaluation of the influence of pH on the activity of DNAzyme 8-17 will be achieved with the kinetic data.
DNAzymes are single strands of DNA obtained in the laboratory through a process called in vitro selection, which are capable of catalyzing various chemical reactions, including RNA breakdown. In this reaction, the DNAzyme accelerates the internal transfer of the phosphodiester, using a metal ion as a cofactor. Among the wide variety of DNA enzymes selected to date, the 8-17 DNAzyme has been the most studied in catalytic terms. The 8-17 is a metalloenzyme that has the particularity of being active with a series of divalent metal ions, but which shows its greatest activity in the presence of Pb2+ . However, the specific participation of the metal cofactor in catalysis has not yet been fully understood, although based on the evidence collected, one of the possibilities could be the existence of a proton transfer event that involves the hydrated metal ion. For this reason, this work proposes to evaluate the influence of pH on the reaction mechanism of the 8-17 DNAzyme, through the study of pH-activity profiles (log (kobs) vs pH). In this sense, it is necessary to study the activity of the enzyme in a wide range of pH. However, at high pH values, the 8-17 DNAzyme has the particularity of having high rate constants that make difficult their observation by the available techniques. To overcome this limitation, we consider studying the pH-activity profiles of a less active analogue of the 8-17 DNAzyme. Thus, the activity profiles of the native and analogous DNAzyme will be compared in the presence of the divalent metal ion. The experiments will be carried out by measuring the activity at different pHs with the DNAzyme and a substrate labeled with the fluorescent probe FAM at the 5Ꞌ-end, under single-turnover conditions. The strands corresponding to the substrate and the reaction products will be separated by electrophoresis, in a 20% polyacrylamide denaturant gel (PAGE), the obtained image will be visualized in a G:box Syngene Imager System, and the quantification will be carried out with the GeneTools Syngene software. Knowing the percentage of product over time, will allow the obtention of the kobs for each reaction, at each pH under study. The evaluation of the influence of pH on the activity of DNAzyme 8-17 will be achieved with the kinetic data.
Notas
Unidad de Investigación (Licenciado en Química)
Esta investigación fue financiada por Proyecto FONDECYT regular 1181438.
Los resultados fueron divulgados en modalidad poster en la XXIII Jornada de Química 2020, realizada en la ciudad de Puerto Varas, región de Los Lagos, Chile.
Esta investigación fue financiada por Proyecto FONDECYT regular 1181438.
Los resultados fueron divulgados en modalidad poster en la XXIII Jornada de Química 2020, realizada en la ciudad de Puerto Varas, región de Los Lagos, Chile.
Palabras clave
DNAzimas, Análisis