Studies on Protein-RNA:DNA Hybrid Interactions by Microscale Thermophoresis (MST)

Microscale thermophoresis (MST) is a technology that allows for quantitative analysis of interactions between biomolecules with low sample consumption. MST uses localized temperature fields to measure the diffusion rates of the free and bound states of a fluorescently labeled protein, and to determine the dissociation constant K_D by fitting of the binding isotherm with a 1:1 binding model. Here, we describe the use of MST for quantitative analysis of the interaction of the N-terminal his-tagged 6-methyladenine (m⁶A) reader protein YTHDF2 with m⁶A modified and unmodified RNA, in single-strand configuration or with RNA:DNA hybrid substrates. The described protocol is also suitable for studies of interactions with proteins binding to double-stranded RNA or DNA substrates.