Non-covalent dyes in microscale thermophoresis for studying RNA ligand interactions and modifications

Microscale Thermophoresis (MST) is a powerful biophysical technique that measures the mobility of biomolecules in response to a temperature gradient, making it useful for investigating the interactions between biological molecules. This study presents a novel methodology for studying RNA-containing samples using non-covalent nucleic acid-sensitive dyes in MST. This mix-and-measure protocol uses non-covalent dyes, such as those from the Syto or Sybr series, which lead to the statistical binding of one fluorophore per RNA oligo showing key advantages over traditional covalent labelling approaches. This new approach has been successfully used to study the binding of ligands to RNA molecules (e.g., SAM- and PreQ1 riboswitches) and the identification of modifications (e.g., m6A) in short RNA oligos which can be written by the RNA methyltransferase METTL3/14. Time to shine a light on RNA: this protocol presents an innovative technique to study RNA using nucleic acid-sensitive dyes in MST.

Automated summary

Microscale Thermophoresis (MST) is a powerful biophysical technique that investigates the interactions between biomolecules by measuring their mobility in response to temperature gradient. This study presents a novel methodology for studying RNA-containing samples using non-covalent nucleic acid-sensitive dyes in MST. The mix-and-measure protocol using non-covalent dyes has advantages over traditional covalent labelling approaches, and has been successfully applied to study ligand binding to RNA molecules and identify modifications in short RNA oligos.