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Selección de ligandos sitio-selectivos mediante perfilado mutacional para la focalización covalente de ARN

Phillip Yesley¹, Georgia Poulladofonou¹, Danny Incarnato²

¹Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, Nijmegen, 6525 AJ, Netherlands.

Angewandte Chemie (International ed. in English)

|December 24, 2025

Resumen

Los investigadores desarrollaron Covacil, una novedosa sonda covalente dirigida al ARN. Esta sonda modifica selectivamente el riboswitch FMN, lo que permite un análisis preciso del ARN dentro de muestras biológicas complejas.

Área de la Ciencia:

Biología química
Biología molecular
Terapéutica de ARN

Sus antecedentes:

Las sondas covalentes dirigidas al ARN son cruciales para estudiar la estructura y función del ARN.
El diseño de sondas selectivas para dianas de ARN específicas sigue siendo un desafío importante.
El riboswitch FMN es un elemento regulador clave en las bacterias.

Objetivo del estudio:

Desarrollar un enfoque ascendente, informado por la estructura, para el diseño de sondas covalentes dirigidas al ARN.
Crear una sonda covalente altamente base-selectiva dirigida al riboswitch FMN.
Validar la selectividad y aplicación de la sonda en entornos de ARN complejos.

Principales métodos:

Se utilizó el perfilado mutacional y una estrategia de diseño informada por la estructura.
Se sintetizó una biblioteca de sondas covalentes basada en el andamio Ribocil.
Se analizaron sondas contra aptámeros del riboswitch FMN de diversas especies bacterianas.
Se validó la selectividad y covalencia de la sonda mediante el etiquetado fotoafinidad competitivo y la espectrometría de masas.

Principales resultados:

Identificó Covacil, una sonda covalente altamente base-selectiva para el aptámero del riboswitch FMN.
Covacil logra la modificación covalente a bajas concentraciones micromolares en 10 minutos.
Demostró una selectividad de base >1.000 veces mayor en comparación con sondas no dirigidas.
Confirmó la reactividad selectiva de Covacil hacia el riboswitch FMN en muestras de ARN total.

Conclusiones:

El enfoque informado por la estructura desarrollado permite el diseño de sondas covalentes dirigidas al ARN potentes y selectivas.
Covacil representa una herramienta valiosa para estudiar la función del riboswitch FMN y la biología del ARN.
La reactividad base-selectiva de Covacil en ARN total destaca su potencial para el análisis transcriptómico.

Palabras clave:

modificación covalente química de ácidos nucleicos sondaje de ARN focalización de ARN riboswitch

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