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Guiando el diseño de moléculas sintéticas de unión al ADN con secuenciación masivamente paralela

Jordan L Meier¹, Abigail S Yu, Ian Korf

¹Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.

Journal of the American Chemical Society

|September 28, 2012

Resumen

Las poliamidas de pirrol-imidazol se unen al ADN con alta especificidad. La secuenciación de alto rendimiento revela reglas canónicas que predicen la unión, pero también identifica nuevos sitios, lo que permite el diseño personalizado de moléculas de unión al ADN para aplicaciones biológicas y nanotecnológicas.

Área de la Ciencia:

Biología Molecular Biología Molecular
La genómica es la genómica.
Biología Química Biología química.

Sus antecedentes:

Comprender la especificidad de la molécula de unión al ADN es crucial para las aplicaciones genómicas.
Las poliamidas de pirrolo-imidazol son una clase de moléculas diseñadas para la unión de ADN específica de la secuencia.
Se necesitan métodos de alto rendimiento para mapear sitios de unión a través de grandes bibliotecas de ADN.

Objetivo del estudio:

Para determinar la especificidad de unión al ADN de las poliamidas pirrol-imidazol utilizando un enfoque de alto rendimiento.
Para identificar sitios de enlace de alta afinidad canónicos y no canónicos.
Permitir el diseño racional de nuevas poliamidas con mayor especificidad.

Principales métodos:

Purificación de afinidad junto con secuenciación masiva paralela (secuenciación profunda).
Análisis de una biblioteca de secuencias de ADN de 10^12 miembros.
Diseño y ensayo de poliamidas de horquilla rediseñadas.

Principales resultados:

Las reglas de emparejamiento de bases canónicas predicen en gran medida la especificidad de unión al ADN de la poliamida.
Se identificaron sitios de unión de alta afinidad no anticipados en la orientación inversa para ciertas poliamidas.
Las poliamidas rediseñadas distinguen con éxito entre secuencias de ADN similares (5'-WCGCGW-3' y 5'-WGCGCW-3').

Conclusiones:

La secuenciación de alto rendimiento es una herramienta poderosa para caracterizar la especificidad de la molécula de unión al ADN.
Las poliamidas de pirrolo-imidazol se pueden diseñar racionalmente para una orientación precisa del ADN.
Este trabajo proporciona una base para el desarrollo de moléculas de unión específicas de secuencia para diversas aplicaciones.

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