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

Miaomiao Li; Arne Klungland; Bjørn Dalhus

doi:10.1007/978-1-0716-2477-7_15

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

Methods Mol Biol. 2022:2528:239-251. doi: 10.1007/978-1-0716-2477-7_15.

Authors

Miaomiao Li¹, Arne Klungland^{1

2}, Bjørn Dalhus^{3

4}

Affiliations

¹ Department of Microbiology, Oslo University Hospital, Rikshospitalet, Oslo, Norway.
² Department of Biosciences, University of Oslo, Blindern, Oslo, Norway.
³ Department of Microbiology, Oslo University Hospital, Rikshospitalet, Oslo, Norway. bjorn.dalhus@medisin.uio.no.
⁴ Department of Medical Biochemistry, Institute for Clinical Medicine, University of Oslo, Oslo, Norway. bjorn.dalhus@medisin.uio.no.

PMID: 35704195
DOI: 10.1007/978-1-0716-2477-7_15

Abstract

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.

Keywords: 6-methyladenine (m6A); Microscale thermophoresis (MST); RNA modification; RNA-binding; RNA:DNA hybrids (R-loop); YTHDF2.

MeSH terms

DNA / metabolism
Protein Binding
Proteins* / chemistry
RNA* / metabolism
Temperature

Substances

Proteins
RNA
DNA