A Quenched Size‐Expanded Nucleotide Reports Activity of the Leukemia Biomarker Terminal Deoxynucleotidyl Transferase (TdT)**

Abstract Terminal deoxynucleotidyl Transferase (TdT) is a template‐independent DNA polymerase that plays an essential role in the human adaptive immune system and is upregulated in several types of leukemia. It has therefore gained interest as a leukemia biomarker and potential therapeutic target. Herein, we describe a FRET‐quenched fluorogenic probe based on a size‐expanded deoxyadenosine that reports directly on TdT enzymatic activity. The probe enables real‐time 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‐lymphocyte cell extract and Jurkat cells using a simple fluorescence assay. Finally, employing the probe in a high‐throughput assay resulted in the identification of a non‐nucleoside TdT inhibitor.