The nascent RNA labelling compound 5-ethynyl uridine (EU) integrates into DNA in some animals.

BACKGROUND: The detection of de novo synthesized mRNA transcripts is crucial for understanding the regulation of eukaryotic transcription. Using nucleoside or nucleotide analogues to label nascent RNA is potentially jeopardized by the ubiquitous presence of ribonucleotide reductase enzymes (RNRs) that can convert ribonucleotides into 2’-deoxyribonucleotides, the building blocks of DNA. Despite this challenge, the uridine analogue 5-ethynyl uridine (EU) has been commercialized and routinely used as specific label for nascent RNAs. Here, we employ confocal imaging, flow cytometry and biochemistry methods to study the specificity of EU to label RNA in six different animal species. RESULTS: We demonstrate that EU integrates as expected predominantly into RNA of human embryonic kidney cell line (HEK293T), the Drosophila wing disc and the comb jelly Mnemiopsis leidyi. In contrast, we found that EU predominantly labels DNA in the sea anemones Nematostella vectensis and Exaiptasia diaphana, and the polychaete Platynereis dumerilii. In Nematostella, we show that inhibiting RNR by hydroxyurea abolishes cell proliferation and the incorporation of EU into DNA. Alternative compounds for labelling nascent RNA, such as 5-ethynyl cytidine (EC), 5-ethynyl uridine triphosphate (EUTP) or 2-ethynyl adenosine (EA) show similarly low specificity for RNA in Nematostella. CONCLUSIONS: Our findings raise concerns about the specificity of ethynylated nucleosides and nucleotides, including EU, to label RNA in some animals. We therefore suggest good practice guidelines help identifying unintentional DNA labelling when using EU to label nascent RNA. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12860-025-00560-w.