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It has therefore gained interest as a leukemia biomarker and potential therapeutic target. Herein, we describe a FRET\u2010quenched fluorogenic probe based on a size\u2010expanded deoxyadenosine that reports directly on TdT enzymatic activity. The probe enables real\u2010time detection of primer extension and de novo synthesis activity of TdT and displays selectivity over other polymerase and phosphatase enzymes. Importantly, TdT activity and its response to treatment with a promiscuous polymerase inhibitor could be monitored in human T\u2010lymphocyte cell extract and Jurkat cells using a simple fluorescence assay. Finally, employing the probe in a high\u2010throughput assay resulted in the identification of a non\u2010nucleoside TdT inhibitor.<\/jats:p>","DOI":"10.1002\/anie.202302796","type":"journal-article","created":{"date-parts":[[2023,3,7]],"date-time":"2023-03-07T07:15:48Z","timestamp":1678173348000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":18,"title":["A Quenched Size\u2010Expanded Nucleotide Reports Activity of the Leukemia Biomarker Terminal Deoxynucleotidyl Transferase (TdT)**"],"prefix":"10.1002","volume":"62","author":[{"given":"Mathijs J.","family":"Pals","sequence":"first","affiliation":[{"name":"Institute for Molecules and Materials Radboud University Nijmegen Heyendaalseweg 135 6525 AJ Nijmegen The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0257-2734","authenticated-orcid":false,"given":"Willem A.","family":"Velema","sequence":"additional","affiliation":[{"name":"Institute for Molecules and Materials Radboud University Nijmegen Heyendaalseweg 135 6525 AJ Nijmegen The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2023,3,27]]},"reference":[{"key":"e_1_2_7_1_1","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.201800658"},{"key":"e_1_2_7_2_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.8356451"},{"key":"e_1_2_7_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2006.04.013"},{"key":"e_1_2_7_4_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.83.6.1867"},{"key":"e_1_2_7_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/0145-2126(80)90043-0"},{"key":"e_1_2_7_6_1","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1201\/9781351071857-3","volume-title":"Enzymes of Nucleic Acid Synthesis and Modification: Volume\u20051: DNA Enzymes","author":"Coleman M. 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