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Dímeres intramoleculares: una nueva estrategia para el apagado de fluorescencia en sondas de oligonucleótidos de

Mary Katherine Johansson¹, Henk Fidder, Daren Dick

¹Biosearch Technologies, 81 Digital Drive, Novato, California 94949, USA. marykat@biosearchtech.com

Journal of the American Chemical Society

|June 13, 2002

Resumen

El apagado estático, no la transferencia de energía de resonancia de Förster (FRET), domina en las sondas de oligonucleótidos de doble etiqueta. Este hallazgo tiene un impacto en el diseño de sondas para ensayos genómicos al resaltar la formación de heterodimeros intramoleculares como un mecanismo clave de apagado.

Área de la Ciencia:

Biología Molecular Biología Molecular
La bioquímica es la bioquímica.
La genómica es la genómica.

Sus antecedentes:

Los ensayos genómicos utilizan con frecuencia sondas de oligonucleótidos profluorescentes con etiquetas de 5' fluoróforo y 3' quencher.
La transferencia de energía de resonancia Förster (FRET) es el supuesto mecanismo de apagado en las sondas sin tallo, lo que requiere la optimización de la superposición espectral.

Objetivo del estudio:

Para investigar los mecanismos de apagado en sondas de oligonucleótidos sin tallo y de doble etiqueta.
Para analizar la contribución del enfriamiento estático frente al FRET en sondas con diferentes fluoróforos.

Principales métodos:

Análisis comparativo de dos sondas de doble etiqueta (5'Cy3.5-beta-actin-3'BHQ1 y 5'FAM-beta-actin-3'BHQ1) en el estudio.
Las mediciones espectroscópicas incluyen espectros de absorción, rendimientos cuánticos de fluorescencia relativa y tiempos de vida de fluorescencia.

Principales resultados:

Se identificó el apagado estático como el mecanismo de apagado predominante en ambas sondas estudiadas.
El apagado estático fue particularmente dominante en la sonda 5'Cy3.5-beta-actin-3'BHQ1.
Los espectros de absorción indicaron la formación de un heterodimero intramolecular entre el fluoróforo y el apagador.

Conclusiones:

El apagado estático es un mecanismo significativo, a menudo dominante, en las sondas de oligonucleótidos de doble etiqueta sin tallo.
La formación de heterodímeros intramoleculares extinguidores de fluoróforo influye en la eficiencia de la sonda.
El diseño de sondas para aplicaciones genómicas debe considerar el apagado estático y la formación de heterodimeros, no solo FRET.

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