Validation strategies for antibodies targeting modified ribonucleotides.

M Cristina Cardoso, Gunter Meister, Regina Feederle, Vigo Heissmeyer, Mark Helm, Stefan Canzar, Christoph Dieterich, Stefan Hüttelmaier, Julian König, Franziska Weichmann, Nicholas B Angstman, Florian D Hastert, Kaouthar Slama, Andrew Flatley, Taku Ito-Kureha, Aloys Schepers, Robert Hett

Journal: RNA (New York, N.Y.) 2020;26(10):1489-1506

PMID: 32636310

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Abstract

Chemical modifications are found on almost all RNAs and affect their coding and noncoding functions. The identification of mA on mRNA and its important role in gene regulation stimulated the field to investigate whether additional modifications are present on mRNAs. Indeed, modifications including mA, mC, mG, 2'-OMe, and Ψ were detected. However, since their abundances are low and tools used for their corroboration are often not well characterized, their physiological relevance remains largely elusive. Antibodies targeting modified nucleotides are often used but have limitations such as low affinity or specificity. Moreover, they are not always well characterized and due to the low abundance of the modification, particularly on mRNAs, generated data sets might resemble noise rather than specific modification patterns. Therefore, it is critical that the affinity and specificity is rigorously tested using complementary approaches. Here, we provide an experimental toolbox that allows for testing antibody performance prior to their use.

Address: Regensburg Center for Biochemistry, Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany.; Institute for Diabetes and Obesity, Monoclonal Antibody Core Facility and Research Group, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), 85764 Neuherberg, Germany.; Institute for Immunology, Biomedical Center of the Ludwig-Maximilians-University München, 82152 Planegg-Martinsried, Germany.; Institute of Pharmacy and Biochemistry, Johannes-Gutenberg-Universität Mainz, 55128 Mainz, Germany.; Cell Biology and Epigenetics, Technische Universität Darmstadt, 64287 Darmstadt, Germany.; Gene Center, Ludwig-Maximilians-University Munich, 81377 Munich, Germany.; Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany.; Institute of Molecular Medicine, Section for Molecular Cell Biology, Faculty of Medicine, Martin Luther University Halle-Wittenberg, Charles Tanford Protein Centre, 06120 Halle, Germany.; Bioinformatics and Systems Cardiology, Klaus Tschira Institute for Integrative Computational Cardiology and Department of Internal Medicine III, University Hospital Heidelberg, 69120 Heidelberg, Germany.; Research Unit Molecular Immune Regulation, Helmholtz Zentrum München, Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH), 81377 Munich, Germany.

DOI: 10.1261/rna.076026.120

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MeSH Terms: Antibodies, Nucleotides, RNA, RNA, Messenger, Ribonucleotides