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 KD 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.
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References
Wienken CJ, Baaske P, Rothbauer U, Braun D, Duhr S (2010) Protein-binding assays in biological liquids using microscale thermophoresis. Nat Commun 1:100
Mueller AM, Breitsprecher D, Duhr S, Baaske P, Schubert T, Längst G (2017) MicroScale thermophoresis: a rapid and precise method to quantify protein-nucleic acid interactions in solution. Methods Mol Biol 1654:151–164
Jerabek-Willemsen M, Wienken CJ, Braun D, Baaske P, Duhr S (2011) Molecular interaction studies using microscale thermophoresis. Assay Drug Dev Technol 9:342–353
Flores JK, Kariawasam R, Gimenez AX, Helder S, Cubeddu L, Gamsjaeger R, Ataide SF (2015) Biophysical characterisation and quantification of nucleic acid-protein interactions: EMSA, MST and SPR. Curr Protein Pept Sci 16:727–734
Abakir A, Giles TC, Cristini A, Foster JM, Dai N, Starczak M, Rubio-Roldan A, Li M, Eleftheriou M, Crutchley J, Flatt L, Young L, Gaffney DJ, Denning C, Dalhus B, Emes RD, Gackowski D, Corrêa IR Jr, Garcia-Perez JL, Klungland A, Gromak N, Ruzov A (2020) N6-methyladenosine regulates the stability of RNA:DNA hybrids in human cells. Nat Genet 52:48–55
Desrosiers R, Friderici K, Rottman F (1974) Identification of methylated nucleosides in messenger RNA from Novikoff hepatoma cells. Proc Natl Acad Sci U S A 71:3971–3975
Jia G, Fu Y, Zhao X, Dai Q, Zheng G, Yang Y, Yi C, Lindahl T, Pan T, Yang YG, He C (2011) N6-Methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Chem Biol 7:885–887
Zheng G, Dahl JA, Niu Y, Fedorcsak P, Huang CM, Li CJ, Vågbø CB, Shi Y, Wang WL, Song SH, Lu Z, Bosmans RP, Dai Q, Hao YJ, Yang X, Zhao WM, Tong WM, Wang XJ, Bogdan F, Furu K, Fu Y, Jia G, Zhao X, Liu J, Krokan HE, Klungland A, Yang YG, He C (2013) ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol Cell 49:18–29
Wang X, Lu Z, Gomez A, Hon GC, Yue Y, Han D, Fu Y, Parisien M, Dai Q, Jia G, Ren B, Pan T, He C (2014) N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505:117–120
Fleming AM, Nguyen NLB, Burrows CJ (2019) Colocalization of m6A and G-quadruplex-forming sequences in viral RNA (HIV, Zika, hepatitis B, and SV40) suggests topological control of adenosine N 6-methylation. ACS Cent Sci 5:218–228
Hwang SY, Jung H, Mun S, Lee S, Park K, Baek SC, Moon HC, Kim H, Kim B, Choi Y, Go YH, Tang W, Choi J, Choi JK, Cha HJ, Park HY, Liang P, Kim VN, Han K, Ahn K (2021) L1 retrotransposons exploit RNA m6A modification as an evolutionary driving force. Nat Commun 12:880
Liu N, Dai Q, Zheng G, He C, Parisien M, Pan T (2015) N6-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions. Nature 518:560–564
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Li, M., Klungland, A., Dalhus, B. (2022). Studies on Protein–RNA:DNA Hybrid Interactions by Microscale Thermophoresis (MST). In: Aguilera, A., Ruzov, A. (eds) R-Loops . Methods in Molecular Biology, vol 2528. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2477-7_15
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DOI: https://doi.org/10.1007/978-1-0716-2477-7_15
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2476-0
Online ISBN: 978-1-0716-2477-7
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