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Validation strategies for antibodies targeting modified ribonucleotides
RNA ( IF 4.5 ) Pub Date : 2020-07-07 , DOI: 10.1261/rna.076026.120
Franziska Weichmann , Robert Hett , Aloys Schepers , Taku Ito-Kureha , Andrew Flatley , Kaouthar Slama , Florian D. Hastert , Nicholas B. Angstman , M. Cristina Cardoso , Julian König , Stefan Hüttelmaier , Christoph Dieterich , Stefan Canzar , Mark Helm , Vigo Heissmeyer , Regina Feederle , Gunter Meister

Chemical modifications are found on almost all RNAs and affect their coding and non-coding 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 datasets 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 allow for testing antibody performance prior to their use.

中文翻译:

靶向修饰核糖核苷酸的抗体验证策略

几乎所有 RNA 上都存在化学修饰,并影响它们的编码和非编码功能。m6A 在 mRNA 上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 上是否存在其他修饰。实际上,检测到了包括 m1A、m5C、m7G、2'-OMe 和 Ψ 在内的修饰。然而,由于它们的丰度很低,并且用于证实它们的工具通常没有很好的特征,它们的生理相关性仍然难以捉摸。经常使用靶向修饰核苷酸的抗体,但具有局限性,例如低亲和力或特异性。此外,它们并不总是很好地表征,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。所以,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,允许在使用前测试抗体性能。
更新日期:2020-07-07
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