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Ligand Binding and Linkage00:49

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Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked.  In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence...
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Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
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Many proteins’ biological role depends on their interactions with their ligands, small molecules that bind to specific locations on the protein known as ligand-binding sites. Ligand-binding sites are often conserved among homologous proteins as these sites are critical for protein function.
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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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Riboswitches are non-coding mRNA domains that regulate the transcription and translation of downstream genes without the help of proteins. Riboswitches bind directly to a metabolite and can form unique stem-loop or hairpin structures in response to the amount of the metabolite present. They have two distinct regions – a metabolite-binding aptamer and an expression platform.
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A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates
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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
PubMed
Resumen
Este resumen es generado por máquina.

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.

Palabras clave:
modificación covalentequímica de ácidos nucleicossondaje de ARNfocalización de ARNriboswitch

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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.