Validation strategies for antibodies targeting modified ribonucleotides
- Franziska Weichmann1,
- Robert Hett1,
- Aloys Schepers2,
- Taku Ito-Kureha3,
- Andrew Flatley2,
- Kaouthar Slama4,
- Florian D. Hastert5,
- Nicholas B. Angstman6,
- M. Cristina Cardoso5,
- Julian König7,
- Stefan Hüttelmaier8,
- Christoph Dieterich9,
- Stefan Canzar6,
- Mark Helm4,
- Vigo Heissmeyer3,10,
- Regina Feederle2 and
- Gunter Meister1
- 1Regensburg Center for Biochemistry, Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany
- 2Institute for Diabetes and Obesity, Monoclonal Antibody Core Facility and Research Group, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), 85764 Neuherberg, Germany
- 3Institute for Immunology, Biomedical Center of the Ludwig-Maximilians-University München, 82152 Planegg-Martinsried, Germany
- 4Institute of Pharmacy and Biochemistry, Johannes-Gutenberg-Universität Mainz, 55128 Mainz, Germany
- 5Cell Biology and Epigenetics, Technische Universität Darmstadt, 64287 Darmstadt, Germany
- 6Gene Center, Ludwig-Maximilians-University Munich, 81377 Munich, Germany
- 7Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany
- 8Institute of Molecular Medicine, Section for Molecular Cell Biology, Faculty of Medicine, Martin Luther University Halle-Wittenberg, Charles Tanford Protein Centre, 06120 Halle, Germany
- 9Bioinformatics and Systems Cardiology, Klaus Tschira Institute for Integrative Computational Cardiology and Department of Internal Medicine III, University Hospital Heidelberg, 69120 Heidelberg, Germany
- 10Research Unit Molecular Immune Regulation, Helmholtz Zentrum München, Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH), 81377 Munich, Germany
- Corresponding author: gunter.meister{at}ur.de
Abstract
Chemical modifications are found on almost all RNAs and affect their coding and noncoding functions. The identification of m6A on mRNA and its important role in gene regulation stimulated the field to investigate whether additional modifications are present on mRNAs. Indeed, modifications including m1A, m5C, m7G, 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.
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Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna.076026.120.
- Received April 22, 2020.
- Accepted June 30, 2020.
This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
