Validation strategies for antibodies targeting modified ribonucleotides-猫眼课题宝

DOI：

10.1261/rna.076026.120

发表时间：

2020-07

期刊：

RNA

影响因子：

4.5

通讯作者：

中科院分区：

生物学3区

文献类型：

作者：

F. Weichmann;R. Hett;A. Schepers;Taku Ito-Kureha;A. Flatley;Kaouthar Slama;Florian D. Hastert;Nicholas B. Angstman;M. C. Cardoso;J. König;S. Huettelmaier;C. Dieterich;S. Canzar;M. Helm;V. Heissmeyer;R. Feederle;G. Meister

化学修饰几乎存在于所有RNA上，并影响其编码和非编码功能。mRNA上m6 A的鉴定及其在基因调控中的重要作用刺激了该领域研究mRNA上是否存在额外的修饰。事实上，检测到包括m1A、m5 C、m7 G、2′-OMe和m2 C在内的修饰。然而，由于它们的丰度很低，而且用于确证的工具往往没有很好的表征，它们的生理相关性在很大程度上仍然难以捉摸。经常使用靶向修饰的核苷酸的抗体，但其具有诸如低亲和力或特异性的限制。此外，它们并不总是被很好地表征，并且由于修饰的丰度低，特别是在mRNA上，生成的数据集可能类似于噪声而不是特定的修饰模式。因此，使用互补方法严格测试亲和力和特异性至关重要。在这里，我们提供了一个实验工具箱，允许在使用之前测试抗体性能。

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.

Validation strategies for antibodies targeting modified ribonucleotides

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