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SiRA: un Aptamer de unión a la rodamina de silicio para imágenes de ARN de súper resolución de células vivas
Regina Wirth1, Peng Gao2,3, G Ulrich Nienhaus2,3,4,5
1Institute of Pharmacy and Molecular Biotechnology (IPMB) , Heidelberg University , 69120 Heidelberg , Germany.
Journal of the American Chemical Society
|April 16, 2019
Resumen
Los investigadores desarrollaron un nuevo aptamer de ARN (SiRA) que se une a los tintes de rodamina de silicio (SiR), lo que permite una emisión de luz roja lejana brillante y fotostable para las imágenes de ARN de células vivas. Este sistema ofrece una visualización mejorada del ARN en bacterias y células vivas utilizando microscopía de súper resolución.
Área de la Ciencia:
- Biología molecular
- La bioquímica
- La biofísica
Sus antecedentes:
- Los aptamers de ARN encendidos genéticamente son valiosos para la visualización de ARN de células vivas.
- Los pares de aptamer/ligand que emiten en el infrarrojo lejano y el infrarrojo cercano (NIR) son escasos.
- Las rodaminas de silicio (SiR) son fluoroforos fotostables y emisores de NIR con una alta permeabilidad celular.
Objetivo del estudio:
- Desarrollar un nuevo aptamer de ARN de iluminación para imágenes de fluorescencia de color rojo lejano/NIR.
- Para crear una herramienta de etiquetado de ARN versátil utilizando tintes de rodamina de silicio.
- Demostrar la aplicación de este sistema en imágenes de ARN de células vivas y microscopía de súper resolución.
Principales métodos:
- Selección in vitro de aptameros de ARN desde una biblioteca combinatoria.
- Análisis bioinformático, truncamiento y estudios mutacionales para identificar el aptamer mínimo (SiRA).
- Caracterización fotofísica y experimentos de imagen de células vivas, incluida la microscopia STED.
Principales resultados:
- Se identificó un aptamer mínimo de 50 nucleótidos, SiRA, con afinidad nanomolar para los SiR.
- SiRA exhibe una alta fotostabilidad, un rendimiento cuántico de 0,98 y un coeficiente de extinción de 86,000 M-1cm-1.
- Visualización exitosa de ARN en bacterias y ARNm en células vivas utilizando SiRA, incluyendo imágenes de súper resolución STED.
Conclusiones:
- El sistema SiRA-SiR es el aptamer de luz de color rojo lejano más brillante reportado hasta la fecha.
- Este trabajo es el primero en aplicar tintes SiR y espirociclado intramolecular para imágenes de ARN basadas en aptamer.
- El sistema SiRA tiene un potencial significativo para el avance de la investigación biológica a través de nuevas técnicas de etiquetado de ARN.
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